To evaluate the local effect of simvastatin (SVT) combined with deproteinized bovine bone (DBB) with hydroxyapatite/β-tricalcium phosphate biphasic ceramics (HA/TCP) and with collagen sponge (CS) on bone repair in critical size defects (CSDs) in rat calvaria.Forty-two 5-mm diameter CSDs were made bilaterally in the calvaria of 18 rats. The animals were allocated according to the type of biomaterial and associations used to fill the CSD. After 8 weeks, the animals were euthanized, and their calvaria were evaluated for repaired tissue composition using histologic and histometric analyses.In the histometric analysis, the use of SVT showed to increase bone formation in the CSDs when combined with all the bone substitutes tested in this study (p<0.05). Greater bone formation was observed in the groups with SVT compared to the groups without SVT.The use of SVT without the need for a vehicle and combined with a commercially available biomaterial may be a cheaper way to potentiate the formation of bone tissue without the need to produce new biomaterials. Therefore, SVT combined with DBB induced significantly greater new bone formation than did the other treatments. Although this type of graft has some of the best biological properties for bone formation 5 . These biomaterials have shown to be effective in increasing bone availability associated with maxillary sinus floor augmentation 4 , in alveolar ridge preservation following tooth extraction 6 and in lateral ridge augmentation 7 . However, in spite of these benefits, the use of strictly osteoconductive biomaterials has shown to reduce the formation of bone tissue in comparison to areas grafted with autogenous bone 8 , to reduce bone formation in non-critical size defects compared to those that were filled with clots 9 and to have worse implant osseointegration compared to implants placed in native bone 10 .Accordingly, osteoconductive biomaterials, such as deproteinized bovine bone (DBB) and hydroxyapatite/β-tricalcium phosphate biphasic ceramics (HA/TCP), have been indicated as alternative biomaterials to the use of autogenous bone grafts in bone grafting procedures 11 and with growth factors, such as rhBMP2 12 . However, these combinations still present limitations in terms of patient morbidity and a questionable cost-benefit ratio 12 . In addition, growth factors, such as rhBMP2, have been used in conjunction with collagen sponge (CS), which appears to be a more stable scaffold than osteoconductive biomaterials 13 despite leaving less space for bone formation during the healing of bone defects 14 . Thus, it is important to find alternative combinations to potentiate the formation of bone tissue in areas grafted with osteoconductive bone substitutes.Because of this, some combinations have been suggested to improve the biological properties of osteoconductive biomaterials, such as combining them with autogenous bone graft 16 . However, this drug has shown to have pleiotropic effects that are related to its anti-inflammatory and connective tissue-proliferating properties 18 . These effects have shown to benefit bone tissue formation both by inhibiting osteoclastogenesis through blocking the mavelonate pathway 19 and by stimulating osteoblastic activity through the increase in the expression of bone morphogenic protein (BMP2) 17 , vascular endothelial growth factor (VEGF) 20 and alkaline phosphatase 21 . These properties make SVT a good alternative as an osteoinductive agent that can improve the biological properties of osteoconductive biomaterials 20 .Simvastatin (SVT), for example, is a drug traditionally used in serum cholesterol control because it inhibits the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) 18 , increased bone formation when combined with a gelatin/nanohydroxyapatite scaffold in rabbit radio defects 20 and increased bone formation in critical size defects of rat calvaria when combined with a calcium sulphate scaffold, with SVT being slowly released by polyglycolic acid polymer microspheres 22 . However, other studies have shown that the combination with SVT did not promote increased bone formation. A recent clinical study that evaluated the application of a PLGA/HA/β-TCP with SVT scaffolds in maxillary third molar post-extraction sockets demonstrated a high rejection rate 23 . Moreover, a preclinical study showed that the use of SVT combined with β-TCP in critical size defects in rat calvaria at concentrations of 0.25 and 0.5 mg did not improve bone tissue formation 24 . Thus, it is clear that parameters, such as the type of scaffold or vehicle to be used in conjunction with SVT, as well as adequate dosages of this drug, still need to be determined 24 .The studies that combined the use of SVT with bone substitute biomaterials show contradictory results. Preclinical studies have shown that SVT increased bone formation in non-critical size defects in rabbit femur grafted with DBB and calcium sulphate Therefore, the objective of this study was to evaluate the combination of 0.1 mg SVT with different osteoconductive biomaterials  in terms of new bone formation in rat calvaria critical size defects (CSDs).ad libitum at room temperature with a 12-hour light/dark cycle (06:00 to 18:00 h). Female rats were used in this experiment because previous work from our research group showed very good response stability in the results obtained, although hormonal interferences were not considered 25 . The ARRIVE protocol guidelines was followed in this study.The study protocol was approved by the Committee for the Use of Animals in Research, University of Brasilia, Brasilia, DF, Brazil (protocol doc 7838/2015). Twenty-one female Wistar rats  were used in this study. The animals were housed in groups of five per cage in standard conditions with food and water ® Institute Geistlich, Zurique, Switzerland); HA/TCP group - CSD filled with HA/TCP ; CS group - CSD filled with CS ; DBB + S group - CSD filled with DBB associated with simvastatin (0.1 mg); HA/TCP + S group – CSD filled with HA/TCP associated with simvastatin (0.1 mg); and CS + S group – CSD filled with CS associated with simvastatin (0.1 mg). One side of the CSD was filled with the pure biomaterial while the other one was filled with the biomaterial combined with simvastatin.The animals were randomly allocated into six groups. The animals were allocated according to the type of biomaterial and associations used to fill the CSD. A total of 42 defects were created in 18 rats and were divided into 6 groups that included 6 defects in each group as follows: DBB group - CSD filled with DBB  and xylazine (10 mg/kg) by intramuscular injection. An antiseptic (povidone-iodine) was applied to the surgical sites, a skin incision was performed, and a flap was raised exposing the calvarial bone. Two critical-sized bone defects 5 mm in diameter just lateral to the sagittal plane were carefully prepared with a trephine bur under irrigation with saline solution and slight pressure to avoid damage to the dura mater. After filling the CSDs, the flaps were then sutured using 5-0 nylon suture. Aspirin (150 mg/kg) was given orally to the rats every 6 hours on the first day after surgery. The animals were observed daily for signs of inflammation. The animals were euthanized 8 weeks after the surgical procedure.After euthanasia, the calvarial bones were dissected, the soft tissues were carefully removed, and the specimens were then fixed in neutral 10% formalin for 24 hours. The specimens were then washed in water for 24 hours and decalcified with a solution of 50% formic acid and 20% sodium citrate for 30 days. The calvarial bones were divided in half longitudinally, and each half containing one treated defect was separately embedded in paraffin according to standard protocols. The embedded specimens were sectioned into 5 µm serial slices with a microtome. All sections were stained with haematoxylin and eosin for subsequent microscopic and histomorphometrical analyses.® software, and the area of the newly formed bone and the remaining biomaterial were calculated. Briefly, the total area was delineated on the captured digital images of the entire surgical defects as follows: two vertical lines were drawn on each side of the defect limited by the original cortical calvarial bone. These two vertical lines on each side were connected by two horizontal lines with one on top and another at the bottom, forming a rectangle containing the entire newly formed tissue. The area of this rectangle was considered to be the total area to be analysed. Then, the newly formed bone and the remaining biomaterial particles were selected, and the area was calculated as a percentage of the total area. This analysis was performed by a single blinded evaluator.Histological analysis was carried out with a light microscope under x20 and x200 magnification, and the morphology of the newly formed tissue in the bone defect area was examined. Tissue sections were screened under a light microscope, and the most central histological sections of each surgical defect were selected for the analyses. The histomorphometrical analysis was carried out using ImageScope vs . CS). A paired t-test was used to compare the scaffolds alone or combined with SVT. All the statistical tests were applied at a significance level of 95%.GraphPad Prism 6 software  was used to perform the statistical analysis. The histometric data had a normal distribution confirmed using the Shapiro-Wilk Test. For the inferential analysis, the histometric data were evaluated using a parametric test and one-way ANOVA complemented by the Tukey test to evaluate the different scaffold types  showed greater bone tissue formation than CSDs that were grafted with the same biomaterials alone.27 . Studies on periodontal disease in rats demonstrated that SVT has both a protective effect against the destructive inflammatory process 28 and is also capable of inducing the formation of bone tissue 30 . In fact, SVT has the potential to block osteoclastogenesis 19 . This effect is related to its action as a blocker of the mavelonate pathway, which, in addition to reducing cholesterol conversion, also reduces the formation of osteoclasts 28 . In addition, SVT has shown to reduce the expression of proinflammatory cytokines, such as IL-6, IL-8, TNFα and IL-1β 17 . Conversely, SVT-induced bone formation found in this study may be related to its effect on increasing the expression of BMP2, VEGF and alkaline phosphatase, which are biological markers of osteoblastic differentiation and function 30 .These results corroborate a range of studies that have demonstrated beneficial effects of SVT on bone tissue metabolism 23 . Some factors may influence these conflicting data regarding the use of SVT; one such factor is the route of administration. Studies in humans have shown that the systemic use of SVT mildly increases bone density in patients with osteoporosis. As high systemic doses of SVT have been related to myopathic and hepatic complications, the systemic use of this drug to improve bone tissue metabolism is not recommended 28 . For this reason, we decided to carry out this study using a local application of SVT combined with biomaterials.However, the real potential of using SVT clinically as an inducer of bone tissue formation is still highly questionable because not all studies have shown that this substance has a protective effect on bone tissue 24 . However, at the dose used, despite the increase in the inflammatory infiltrate, bone tissue formation still increased 24 , as observed in this study. SVT doses greater than 0.25 mg applied locally have shown to prevent bone formation, whereas this effect was also observed for doses less than 0.1 mg 24 . Thus, the dose used in this study is within the SVT dose range that induces bone tissue formation without the production of exacerbated inflammatory infiltrate 24 .In this study, the defects in which the biomaterials were used in combination with SVT had a greater degree of inflammation than did the groups in which the biomaterials were used alone. These findings corroborate findings from other studies showing that the use of SVT locally increases inflammatory infiltrate 22 . In fact, methylcellulose gel polymers and gelatin/nanohydroxyapatite allow the release of SVT in the medium for more than 15 days, which is the time that is essential for bone tissue formation 27 . In the present study, no vehicle was used to control the release of SVT, and no bone formation inhibition was observed in the groups applying the biomaterials combined with the drug. The use of a vehicle does not reduce the costs of the materials; however, it is possible that if SVT was attached to the biomaterials tested using a vehicle, the effects could have been even more positive.Another aspect to be discussed is the type of vehicle that may be associated with the use of SVT. The use of a vehicle allows for the controlled release of SVT in the medium for a greater amount of time 18 . A pre-clinical study evaluating the effect of SVT combined with DBB, calcium sulphate and CS on bone repair in rabbit tibial defects showed that DBB was the biomaterial that promoted the greatest bone tissue formation after 4 weeks 18 . Thus, as observed in the present study, the work of Papadimitriou et al . 18 , also demonstrated that the use of CS combined with SVT was the group the worst results regarding bone tissue formation. This was confirmed in a study that evaluated the effect of SVT combined with CS on the repair of critical size defects in rat calvaria; this study showed that this combination did not benefit bone tissue formation. It is likely that rapid rates of CS resorption will not allow the maintenance of the space required for the newly formed bone tissue 18 .In this study, DBB promoted the greatest bone tissue formation when combined with SVT or when used alone, followed by the HA/TCP biphasic ceramics. In addition, DBB was the biomaterial with the greatest amount of particle remnants, which shows that the maintenance of the space required for newly formed bone promoted by the presence of these particles on the defect may have been an important factor for bone tissue formation 20 . However, the use of SVT without the need for a vehicle and combined with a commercially available biomaterial may be a cheaper way to potentiate the formation of bone tissue without the need to produce new biomaterials.The results of this study must be interpreted with caution because of the study’s limitations. Although the use of 0.1 mg SVT benefited bone formation, it was not attached to the biomaterials using a vehicle, and it is possible that a more controlled release of SVT could benefit the formation of bone tissue over a longer period. The biomaterials used were either of slow resorption (DBB and HA/TCP) or very fast resorption (CS), and SVT has been shown to promote good results when combined with polymeric scaffolds that are likely to degrade at a more favourable rate that would improve the quality of the newly formed bone tissue The use of SVT, therefore, stimulated the formation of bone tissue regardless of the type of biomaterial it was combined with . Among the biomaterials evaluated, DBB resulted in the greatest potential for bone tissue formation despite being less resorbable than the other biomaterials.


E. coli small-conductance mechanosensitive channel (MscS) in nanodiscs (ND). They reveal a novel membrane-anchoring fold that plays a significant role in channel activation and establish a new location for the lipid bilayer, shifted ~14 Å from previous consensus placements. Two types of lipid densities are explicitly observed. A phospholipid that ‘hooks’ the top of each TM2-TM3 hairpin and likely plays a role in force sensing, and a bundle of acyl chains occluding the permeation path above the L105 cuff. These observations reshape our understanding of force-from-lipids gating in MscS and highlight the key role of allosteric interactions between TM segments and phospholipids bound to key dynamic components of the channel.Prokaryotic mechanosensitive (MS) channels open by sensing the physical state of the membrane. As such, lipid-protein interactions represent the defining molecular process underlying mechanotransduction. Here, we describe cryo-electron microscopy (cryo-EM) structures of the  In principle, all molecules are mechanosensitive. Remarkably, some have evolved as force transducers, where they participate in a variety of fundamental biological functions, including turgor control in plants, development and morphogenesis, touch, hearing, proprioception, as well as osmoregulation in bacteria . Many ofde facto standard in the search for the molecular principles underlying membrane force transduction. The E. coli MscS crystal structure in detergent  with a histidine tag at its N-terminal end (6xHis-MscS-ND) to a resolution of 3.1 Å . While tEnterobacteriales   ~1.4 Cα. PreviouTo evaluate the role of the anchor domain on MscS function, we designed a construct lacking the first 26 residues of the channel: Δ2–26 MscS (‘Cryst’), physically recapitulating the resolved regions crystal structure (2OAU) model, which has been the basis of many past experimental and computational insights . This code facto consensus had been reached where the bilayer interacted with the channel at the level of the resolved regions of the TM segments  , a de faents see . In factents see . We noteents see . The preents see  left is ents see , center.ents see .Given this location, the membrane not only interacts closely with the N terminal domain ,2A but aAs a quantitative evaluation of the bilayer interaction energetics, we carried out a potential of mean force (PMF) calculation where MscS-ND was moved along the Z-axis in relation to a fixed bilayer . CalculaE. coli membranes are composed mostly of PE, PG, and cardiolipin (E. coli polar lipids (at 10 Å resolution), suggesting that MscS-bilayer placement is not affected by alternative lipid compositions. The EM density in DDM . Indeed, binding of the hook lipids is stabilized by R88 in one subunit and Y27 from the neighboring subunit . Both reWe believe the pore and hook lipids were carried over from the cell membrane during purification and reconstitution, as we find evidence of similarly placed density in our DDM MscS map . FurtherPrevious studies of closed state water permeation in MscS  have shoThe new MscS structural features, membrane footprint and bound lipids all have important mechanistic consequences regarding MscS force transduction. For one, TM3b can no longer be considered an interfacial helix  as it isOur structure in nanodiscs reveals much of the anchor domain locked in the closed state. We suggest that the anchor domain must play a significant role in the mechanism of mechanotransduction in MscS. This is based on three primary observations. First, deletion of the anchor domain renders MscS unresponsive to tension, although it does not preclude its folding and oligomeric assembly ,4B; secoEarlier spectroscopic work  providesWe find that at least two general gating models are consistent with both the data provided by the present MscS-ND structure and the conformational changes observed crystallographically . In the The present results suggest that, for MscS, the energetic differences derived from tension changes in the plane of the lipid bilayer should be evaluated in the context of key lipids bound to mechanistically important regions of the channel (TM2 and TM3a). Functionally, MscS behaves as a lipo-protein complex, where the hook lipid may help transduce bilayer forces and the pore lipid is poised to influence ion and water fluxes at rest. These interactions, together with the revised location of the lipid-protein interface must be accounted for by mechanotransduction models which strong allosteric coupling between TM segments and the lipid-protein interface.E. coli MscS was expressed and purified as previously described (6 tag and a thrombin cleavage site on the N-termini. Rosetta 2 (Millipore Sigma) E. coli cells were transformed with MscS-pET28a vector and grown overnight in the presences of kanamycin and chloramphenicol. The cells were diluted 1:100 in LB medium and grown at 37°C to an OD600 of 0.8-1.0. Before induction, the cell culture was supplemented to a final concentration of 0.4% glycerol and allowed to cool to 26°C, and protein expression was induced with 0.8mM IPTG. The cells were grown for 4h at 26°C and were harvested, and either were frozen at -80°C for later use or immediately resuspended in PBS pH 7.4 (Sigma), 10% glycerol, protease inhibitors, and homogenized . The membranes were isolated via centrifugation at 100,000g for 30 min, and the pellet was resuspended in PBS and 10% glycerol. Solubilization was carried out in 1% Fos-Choline (Anatrace) 14 for 4-16h at 4°C. This resuspension was spun down at 100,000g for 30 min, and the supernatant supplemented with a final concentration of 5mM imidazole (Fisher) was incubated with cobalt resin(Clonetech) for 2-4h at 4 °C. The resin was washed with 20-bed volumes of 1 mM DDM(Anatrace), 10mM imidazole and 10% glycerol in PBS buffer. MscS was eluted in 1 mM DDM, 300mM imidazole, and 10% glycerol in PBS buffer. Unless explicitly stated MscS His, thrombin was added to cleave the his tag and incubated overnight. The final purification step was to run the protein on a Superdex 200 Increase 10/30 column  with 1 mM DDM and PBS buffer. The removal of glycerol is critical for EM grid preparation. The typical yield of MscS is about 5-8mg per liter of E. coli. For the MscS-Cryst construct, residues 2-26 residues were removed and subcloned into pQE70 and grown in MJF465 E. coli cells , a gift from Ian Booth . Nanodiscs were made by adding mix micelles to protein for 20 minutes on ice. MSP was added to the solution and incubated on ice for 5 minutes. The reconstitution mixture was incubated in activated bio beads (Biorad) overnight at 4°C. The detergent free mixture was run on a Superdex 200 Increase 10/30 column to separate the empty ND peak. The MscS ND peak was concentrated to ~2mg/ml and stored at 4°C.MscS nanodiscs (ND) were prepared following previously described protocol . Several-/A2 per frame for 50 frames. MscS ND EPL was collected on Titan Krios with a Falcon 3 detector in counting mode. MscS-Cryst ND POPC:POPG was collected on Talos Artica with a Falcon 3 detector in counting mode. Movies were acquired at 1e-/A2 per frame for 50 frames. Motion correction was performed using Motioncor2  to a final concentration of 0.01%. MscS and was applied twice, with a filter paper blotting between each application, onto Mesh 200 2/1 or Mesh 300 1.2/1.3 Quantifoli holey carbon grids and flash frozen in a Vitrobot (Thermofisher) set at 3 seconds with a force of 3 with 100% humidity at 22°C. MscS His ND POPC:POPG, MscS No His ND POPC:POPG, and MscS DDM were collected on a Titan Krios with a K2 detector in counting mode with a GIF energy filter using Latitude S (Thermofisher). Movies were acquired at 1etioncor2 , and K2 tioncor2 . Initialtioncor2  neural ntioncor2  for parttioncor2 , Chimerationcor2 , MDFF  for 2 hours at 25°C. The OD600 was measured and downshocks were performed by diluting cells 1:50 into a modified LB medium at 50mM NaCl and 1:100 was plated on standard LB agar plates overnight at 37°C. The colonies on the LB agar plates were imaged and counted and normalized by the OD600 readings. Additionally, to assess the expression of each mutant, a western blot was performed. The western blot of each MscS expressing mutant was from a pellet from the downshock experiment and resuspended in PBS and SDS to a final 1% solution. The lysate was then sonicated, ran on a 4-20% SDS-PAGE gel (Biorad), transferred to PVDF and probed with the Penta-HIS(Qiagen) primary and anti-mouse conjugated to Alexa 488 secondary.Downshock assays were performed from a modified protocol from what was previously described . MJF465 Enterobacteriales and Vibrionales MscS protein sequences were extracted from the complete proteomes in the NCBI Assembly database. From each proteome, only one protein showing the highest BLAST bit score    , and the (v.3.5)  (best-fi (v.3.5) . The rel.8.2.11) .The rate.8.2.11) . The seqebLogo 3 .Proto-liposomes were prepared using Dehydration Rehydration (D/R) method as fully described in previous studies . BrieflyCHARMM GUI was used to embed MscS structure into a POPC:POPG (4:1) bilayer mix (to mimic our nanodisc lipid composition) . Differehttps://github.com/Perozo-lab/PMF; copy archived at https://github.com/elifesciences-publications/PMF), which counts the number of hydrating water molecules that pass the central hydrophobic pore (i.e. L105) over the simulation period.Water molecules were counted for the last 10 ns of equilibration for each model. A custom TCL script was used  followed by 10 ns of production run (in the presence of harmonic force). Data were unbiased and combined using the weighed histogram analysis method using WHAM package  and curvature (C) were estimated by measuring the l and h values as following R=h2+C) and hydrophobic mismatch  at equilibrium , its insertion in the membrane causes symmetric curvature , as well as ordered lipid molecules seemingly bound along the periphery of the channel as well as inside the pore. These are important insights that will no doubt serve as the foundation for future studies of the mechanism of gating of MscS and related mechanosensitive channels.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by José D. Faraldo-Gomez as the Reviewing Editor andRichard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Roderick MacKinnon (Reviewer #2); Sergei Sukharev (Reviewer #3).Thank you for submitting your article "Molecular basis of force-from-lipidsgating in the mechanosensitive channel MscS" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers find that the central conclusions of the article are "well supported" and "convincing". By contrast, the computational examination of the mechanism of channel opening, and of the role that the bound lipids might play in this process, was perceived as too preliminary and inconclusive.Essential revisions:eLife. The reviewers are skeptical that these challenging questions can be addressed convincingly within the limited timeframe permitted to make revisions (2 months). Thus, we very strongly recommend that this element be removed from the article. The reviewers agree that this change will not diminish the value of the study, but rather the opposite. The authors are however encouraged to pursue this kind of studies in the future; some of the reviewers' comments are therefore provided below, as they might be useful to the authors:1) The reviewers agree that the computational section that examines the gating mechanism of the channel in response to a bilayer expansion and the potential role that bound lipids might have in this process is too preliminary and incomplete for publication in – As the data stands, it is concerning that the process induced is clearly far from equilibrium, as a very large perturbation in membrane morphology is created in a very short time. To establish statistical significance on the basis of this kind of data, the authors ought to carry out and analyze a large number of repeats; alternatively, the authors could change their simulation protocol so that the system remains close to equilibrium throughout the perturbation of the membrane.– The surface tension of the simulated membrane was increased from 50 mN/m at rest to 150 mN/m. The authors state that this is several times larger than the experimental value so that the opening can be observed in shorter times. But the lytic tension of ordinary lipid membranes is 10-15 mN/m, which means the tension in the simulation goes from about 5 times lytic to about 15 times lytic. What happens to the bilayer in the simulation? These simulated tensions deserve some careful thought, consideration and explanation for the reader.– Forcing MscS opening by applying 150 mN/m of tension within 10 ns is a regime that imposes unnatural force and time scales on the transition. The bilayer gets much thinner than can be expected under MscS gating tension; hence the lipid-derived forces are acting at very different z levels. The bilayer thins so much that in videos it looks like the outer monolayer reaches the level of the "anchor" lipids that used to be close to the midplane. Also, why so much (50 mN/m) tension at rest? POPE seems to be pretty well parameterized in the latest release of CHARMM. Did the bilayer over-compact too much at zero tension or was the structure collapsing as in some previous simulations? This should be explained.– The putative role of R88-associated lipids seems important and warrants a much more thorough MD study that would require several repetitions of channel opening under 'slow' regimes closer to equilibrium. Regarding ways to illustrate the conclusions, either cartoons should be presented  or a real and convincing MD trajectory, not both. We suggest that in the next paper the authors devote special effort to the MD part which appears unfinished in the present form.– The simulations do not seem to clearly reveal the role that either the 'hook lipids' or the 'pore lipids' might play in the mechanism of gating, or indeed whether they do play a role. For example, the authors propose that the pore lipids are a feature of the closed state, and that they must return the membrane upon channel opening. Simulations could in principle be used to evaluate the plausibility of this hypothesis, but the trajectories discussed in the manuscript are much too short to explore the dynamics of these lipids in a meaningful way. Similarly, much longer simulations (or simulations based on enhanced-sampling techniques) could be used to ascertain whether the sites occupied by lipid molecules in the cryo-EM structure indeed remain occupied in this closed state, as hypothesized, and to evaluate how this occupancy might depend on the membrane surface tension. In summary, this element of the study is promising but as it stands, it also appears inconclusive and not ready for publication.2) The hypothesis that lipids are naturally present inside the pore in the resting state and move out during gating seems to be far-fetched. The lipid action in K2P channels, for instance, appears to be feasible due to clear fenestrations in the channel wall; similarly, a possible action of the 'hook' lipids here in MscS can be envisioned because there is an exchange path with the lipid bilayer. There is no obvious path that would connect the pore lipids with the bilayer. In this case, the detachment from the hydrophobic wall would imply a complete solvation by water, which would be energetically costly and slow. Because the headgroups of these lipids have not been resolved in any of the structures, would it be possible that these densities are fatty acids that came as products of lipid degradation? Fatty acid exchange is much more feasible due to higher solubility.Given these concerns, we strongly recommend that the authors tone down their conclusions in regard to the observation of 'pore lipids' and that they discuss other plausible interpretations of the density signals. Otherwise, the authors are asked to provide an additional WT MscS structure obtained with a deliberate attempt to remove or minimize putative free-fatty acids, e.g. by incubating the nanodiscs with cyclodextrins or other type of fatty-acid absorbents. High-resolution structures might not be required to discern whether the densities inside the pore are still present or not.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by José D. Faraldo-Gómez as the Reviewing Editor and Richard Aldrich as the Senior Editor. The following individuals involved in review of your resubmission have agreed to reveal their identity: Roderick MacKinnon (Reviewer #2); Sergei Sukharev (Reviewer #3).Thank you for submitting the revised version of your article "Molecular basis of force-from-lipids gating in the mechanosensitive channel MscS" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife, pending the following revisions:The reviewers believe the most important issues addressed in the earlier evaluation have been addressed, and that the revised manuscript will be worthy of publication in The quantity defined as DGc in paragraph three of subsection “Continuum-Mean filed calculations of the free energy change” is not a free energy but a free-energy density, defined only locally at a point where the membrane curvature is C. The free energy that the authors seek should be an integral of this density over the area of membrane; the resulting values will be therefore considerably larger than the values currently plotted in Figure 3—figure supplement 3D. In addition, although it is valid as a first approximation to assume that at a given point  the curvature C is same in the both directions, C cannot be assumed to be constant everywhere – i.e. the value of the abovementioned integral must be finite. Thus, the statement that "Although the contribution of curvature in our PMF calculations are minimal compared to the effect of hydrophobic mismatch" is not evident based on the data provided. Essential revisions:1) The reviewers agree that the computational section that examines the gating mechanism of the channel in response to a bilayer expansion and the potential role that bound lipids might have in this process is too preliminary and incomplete for publication in eLife. The reviewers are skeptical that these challenging questions can be addressed convincingly within the limited timeframe permitted to make revisions (2 months). Thus, we very strongly recommend that this element be removed from the article. The reviewers agree that this change will not diminish the value of the study, but rather the opposite. The authors are however encouraged to pursue this kind of studies in the future; some of the reviewers' comments are therefore provided below, as they might be useful to the authors:Indeed. We had been aware that a surface tension of 150 mN/m (applied solely to better sample any putative transitions in MscS) led to a membrane deformation that was perhaps too sharp and fast (see below). The intention was to include those bilayer expansion calculations as placeholders for more precise and ongoing calculations closer to equilibrium conditions . These calculations (in replicates) started running before the manuscript was originally submitted. While the results are encouraging and reproducible, our intent is not to further delay publication of the present contribution. We agree with the reviewers that a dedicated computational study should be addressed in a separate publication. Therefore, we have removed this section of the manuscript.– As the data stands, it is concerning that the process induced is clearly far from equilibrium, as a very large perturbation in membrane morphology is created in a very short time. To establish statistical significance on the basis of this kind of data, the authors ought to carry out and analyze a large number of repeats; alternatively, the authors could change their simulation protocol so that the system remains close to equilibrium throughout the perturbation of the membrane.Agreed. These precise simulations (as mentioned above) are ongoing and shall be reported in a separate manuscript. Also, see below.– The surface tension of the simulated membrane was increased from 50 mN/m at rest to 150 mN/m. The authors state that this is several times larger than the experimental value so that the opening can be observed in shorter times. But the lytic tension of ordinary lipid membranes is 10-15 mN/m, which means the tension in the simulation goes from about 5 times lytic to about 15 times lytic. What happens to the bilayer in the simulation? These simulated tensions deserve some careful thought, consideration and explanation for the reader.– Forcing MscS opening by applying 150 mN/m of tension within 10 ns is a regime that imposes unnatural force and time scales on the transition. The bilayer gets much thinner than can be expected under MscS gating tension; hence the lipid-derived forces are acting at very different z levels. The bilayer thins so much that in videos it looks like the outer monolayer reaches the level of the "anchor" lipids that used to be close to the midplane. Also, why so much (50 mN/m) tension at rest? POPE seems to be pretty well parameterized in the latest release of CHARMM. Did the bilayer over-compact too much at zero tension or was the structure collapsing as in some previous simulations? This should be explained.These two comments touch on similar issues, so we combined our response here:zT ensemble (1-3) in which “target surface tension” refers to the area of the pressure profile of the lipid bilayer near the lipid-water interface , the core of the bilayer and 2) electrostatic repulsions between the headgroups , it means that the total additional force (membrane tension) that has been applied to our MD system is ~ 25 mN/m .Ultimately, however, we fully agree with the reviewers that the force in our MD simulation is still several times higher than the experimental range . This is given that the midpoint of activation of MscS is ~ 5-6 mN/m (references 5-7) or as pointed out by the reviewers the lytic tension of lipid bilayers are usually <15 mN/m. Nonetheless, this is a common issue with these types of MD simulations and is a ‘necessary evil’ to be able to capture noticeable gating transitions within limited computational time scales (~200-300 ns).– The putative role of R88-associated lipids seems important and warrants a much more thorough MD study that would require several repetitions of channel opening under 'slow' regimes closer to equilibrium. Regarding ways to illustrate the conclusions, either cartoons should be presented  or a real and convincing MD trajectory, not both. We suggest that in the next paper the authors devote special effort to the MD part which appears unfinished in the present form.Fully agree. Also, see response to comments above. We have eliminated the explicit trajectories and summarize our conclusion in cartoon form.– The simulations do not seem to clearly reveal the role that either the 'hook lipids' or the 'pore lipids' might play in the mechanism of gating, or indeed whether they do play a role. For example, the authors propose that the pore lipids are a feature of the closed state, and that they must return the membrane upon channel opening. Simulations could in principle be used to evaluate the plausibility of this hypothesis, but the trajectories discussed in the manuscript are much too short to explore the dynamics of these lipids in a meaningful way. Similarly, much longer simulations (or simulations based on enhanced-sampling techniques) could be used to ascertain whether the sites occupied by lipid molecules in the cryo-EM structure indeed remain occupied in this closed state, as hypothesized, and to evaluate how this occupancy might depend on the membrane surface tension. In summary, this element of the study is promising but as it stands, it also appears inconclusive and not ready for publication.We agree with the gist of these comments. Longer and targeted simulations will likely help discriminate between the different possible roles of the hook and pore lipids on MscS gating. As stated throughout this overall response to reviewers comments, the gating mechanism simulations are ongoing and shall be reported in a separate publication. Specifically, at identical external forces and computational conditions, we compared the channel opening with and without the presence of the hooked and pore lipids. We are also comparing all these scenarios with the model based on “previously believed” position of the bilayer systems with respect to the protein.We do however believe that the simple evaluation of the closed state water occupancy (with and without pore lipids) and its influence on “vapor lock” mechanism are still informative and will be somewhat toned down but remain in the manuscript. Also, see response below.2) The hypothesis that lipids are naturally present inside the pore in the resting state and move out during gating seems to be far-fetched. The lipid action in K2P channels, for instance, appears to be feasible due to clear fenestrations in the channel wall; similarly, a possible action of the 'hook' lipids here in MscS can be envisioned because there is an exchange path with the lipid bilayer. There is no obvious path that would connect the pore lipids with the bilayer. In this case, the detachment from the hydrophobic wall would imply a complete solvation by water, which would be energetically costly and slow. Because the headgroups of these lipids have not been resolved in any of the structures, would it be possible that these densities are fatty acids that came as products of lipid degradation? Fatty acid exchange is much more feasible due to higher solubility.Given these concerns, we strongly recommend that the authors tone down their conclusions in regard to the observation of 'pore lipids' and that they discuss other plausible interpretations of the density signals. Otherwise, the authors are asked to provide an additional WT MscS structure obtained with a deliberate attempt to remove or minimize putative free-fatty acids, e.g. by incubating the nanodiscs with cyclodextrins or other type of fatty-acid absorbents. High-resolution structures might not be required to discern whether the densities inside the pore are still present or not.We have indeed toned down our interpretation and expanded our discussion regarding the identity of the “pore lipids”. The density we observe supports the likely presence of some type of fatty acid chains in the pore. Indeed, we were able to fit a hexadecamer fatty acid within the density. As understood by the reviewers, likely due to dynamics/resolution we were unable to resolve a putative head group. We see one of two possibilities of where the head group can come from. Either the fatty acid chain could potentially thread through a gap between G104 residues, where MOLEonline (https://mole.upol.cz) supports the presence of a pathway in our structure ; alternaThe quantity defined as DGc in paragraph three of subsection “Continuum-Mean filed calculations of the free energy change” is not a free energy but a free-energy density, defined only locally at a point where the membrane curvature is C. The free energy that the authors seek should be an integral of this density over the area of membrane; the resulting values will be therefore considerably larger than the values currently plotted in Figure 3—figure supplement 3D. In addition, although it is valid as a first approximation to assume that at a given point  the curvature C is same in the both directions, C cannot be assumed to be constant everywhere – i.e. the value of the abovementioned integral must be finite. Thus, the statement that "Although the contribution of curvature in our PMF calculations are minimal compared to the effect of hydrophobic mismatch" is not evident based on the data provided.The reviewer is correct. We have now recalculated this free energy density as the integral of a series of concentric area rings (sed to reduce frame-to-frame-variability in the MD simulation run). We include that as a modified Figure 3—figure supplement 3 and a new Figure 3—figure supplement 4, with corresponding text and full methodological description in subsection “Continuum Mean-Field calculations of the free energy change”. The new calculations supports the original idea and demonstrates that “the contribution of free energy due to change in the curvature is still an order of magnitude smaller than that of the hydrophobic mismatch”.References[1] Feller, Scott E., and Richard W. Pastor. (1999) J Chem Phys 111: 1281-1287.[2] Marrink, S. J., and A. E. Mark. (2001) J Physical Chem B105: 6122-6127.[3] Gullingsrud, Justin, and Klaus Schulten. (2004) Biophys J 86: 3496-3509.[4] Bavi, Navid, et al. (2016) Nature Comm 7 (2016): 11984.[5] Akitake, Bradley, Andriy Anishkin, and Sergei Sukharev. (2005) JGP 125: 143-154.[6] Nomura et al. (2012) PNAS 109 (2012): 8770-8775.[7] Nomura et al. (2015) FASEB J 29: 4334-4345.


The CD4 (cluster of differentiation 4) counting method is used to measure the number of CD4+ T-lymphocytes per microliter of blood and to evaluate the timing of the initiation of antiretroviral therapy as well as the effectiveness of treatment in patients with human immunodeficiency virus. We developed a three-dimensional helical minichannel-based sample cartridge in which a thread-like microgroove formed in the cylindrical surface and configured a particle-positioning and imaging system equipped with a single DC (direct current) motor that can be controlled by a smartphone application. Confinement and enrichment of CD4 cells within a sharp focal depth along the helical minichannel is accomplished by spinning the cylindrical sample cartridge at high speed before acquiring cell images and thus CD4+ cells with weak fluorescence intensity can be detected even in a channel much deeper than existing two-dimensional flat chambers without an autofocusing module. By detecting more cells in a larger sample volume, the accuracy of the CD4 cell count is improved by a factor of 5.8 with a channel of 500 μm depth and the precision is enhanced by a factor of 1.5 with a coefficient of variation of 2.6%. The human immunodeficiency virus (HIV) causes infection and acquired immunodeficiency syndrome (AIDS) and destroy CD4+ T-lymphocytes in the blood . These TEarly diagnosis of HIV infection is considered potentially important from the viewpoint of public health and opportunity for treatment . TherefoTM  is a single-platform benchtop flow cytometer to enumerate the absolute number of CD4 cells and %CD4 values, where cell concentrations are calculated by adding fluorescent beads at a known concentration, allowing the volume-per-bead-detected to be calculated. However, this system still requires a skilled operator and regular maintenance and QC using calibration beads, thus lowering the test throughput. Partec GmbH  have launched CyFlow® miniPOC, which can measure absolute CD4+ counts and %CD4, making it useful for monitoring both adults and infants. PimaTM Analyzer —equipped with an LED (light-emitting diode) illuminator and multicolor CCD (charge-coupled device)—analyzes static images of fluorescently-labeled CD4 and CD3 cells in a whole blood sample. This instrument is a fixed-volume imaging cytometer that can generate a CD4+ count, but not a %CD4 readout, limiting this system for use for infant HIV monitoring 1/2 or as little as (RFA)(RAP) − [(1 − RFA2)(1 − RAP2)]1/2, but it cannot be outside this range of 0.912–0.99. This R2 value indicates that the correlation is reasonably high and the counting results agree. Here, RFA represents the R2 value between the FACSCalibur and the ADAM-II and RAP indicates the R2 value between the ADAM-II and the PIMA.For validation of the CD4 cell count measured with our system (Helios), we have compared our measurement with the commercially available CD4 cell counter, PIMA. Blood samples with low concentration CD4 cells that mimic blood from HIV/AIDS patients were made by diluting blood with phosphate buffered solution. Comparisons between the PIMA and Helios instruments shown in us study , we usedx- and y-directions to capture all areas of the chamber. As a result, the equipment size is large and complex software is required.HIV can weaken the immune system of humans and cause AIDS. Although a person infected with HIV can live only for 9–11 years, the progress of HIV can be controlled with proper treatment after early diagnosis. HIV/AIDS is one of the major causes of death in resource-limited regions, where diagnostic devices for detecting the disease need to be rapid, simple, portable and affordable . ExistinOur helical minichannel-based cell counting system overcomes these issues by employing a single DC motor to spin the sample cartridge, obviating the need for a translational stage. The system is controlled remotely using a smartphone over a Bluetooth connection, and its detection efficiency can be increased by confining cells to the top of the channel under the effect of centrifugal force by increasing the spin speed of the cartridge. We confirmed that cells with weak fluorescence intensity, such as fluorescently-labeled CD-expressing cells, can be concentrated within the focal plane, enabling detection without implementing autofocusing. Therefore, the additional cost of increasing the sensitivity is saved and the image acquisition rate can be increased. A sample preparation function can be added in future, as demonstrated by many research groups, for separation of particles based on size, detection of pathogenic bacteria and isolation of leukocytes from whole blood with the aid of spiral or helical microchannels ,26,27. SOur ultimate goal is to develop a helical minichannel-based rapid POC diagnostic system that enumerates CD4 T-cells with high accuracy and precision. To conform to the ASSURED criteria  set out by the WHO for microfluidic POC devices , we aim In the present study, we developed a method to enhance the detection efficiency by confining and enriching cells in the focal plane along the helical minichannel immediately before the detection step with fast spinning of the sample cartridge, rather than limiting the channel depth and sample volume as is done in conventional POC diagnostic devices. Confinement was highly effective in counting weakly fluorescent and low concentration cells such as CD4+ T-cells, improving the accuracy and precision greatly. If combined with a sample reaction module, we anticipate that a fully integrated POC system for counting various CD-expressing cells can be implemented successfully in near future. The integrated and automated system would provide a cost-effective and rapid solution for POC testing compared to existing technologies and also minimize human intervention, reducing laborious tasks and sample contamination.Confinement and enrichment of microparticles were investigated in stationary and spinning sample cartridges having a helical minichannel filled with 10% fluorescent beads and a 10% glycerol mixture, which has the same density as blood plasma. It was found that when the channel was stationary, no particle confinement was achieved; however, when it was spun at 1000–3000 rpm for 10–120 s, particles were observed to be confined to the top of the channel, facilitating detection of the enriched particles within the focal depth. We demonstrated the effect of spinning the cartridge using centrifugal force on particle confinement. Moreover, the forces acting on the particles were determined and the velocity and time required for the particles to move toward the top of the channel under centrifugal force were verified against the theoretical and the experimental values. Based on the results of fluorescent bead confinement, we applied the strategy to enumerate low concentration CD4 cells with weak fluorescence intensity. Since the weakly fluorescent CD4 cells were detected through confinement and enrichment even in the channel with a 500-μm depth, a large sample volume can be analyzed, thereby improving the accuracy of CD4 cell count by a factor of 5.8 and enhancing the precision by a factor of 1.5 with a coefficient of variation of 2.6%.


Among them, O2 has been the preferred oxidant due to its low cost and availability. However, due to the low stability of HMF and high processing cost to convert HMF to FDCA, researchers are studying the direct conversion of carbohydrates and biomass using both a single- and multi-phase approach for FDCA production. As there are issues arising from FDCA purification, much attention is now being paid to produce FDCA derivatives such as 2, 5-furandicarboxylic acid dimethyl ester (FDCDM) to circumvent these problems. Despite these technical barriers, what is pivotal to achieve in a cost-effective manner high yields of FDCA and derivatives, is the design of highly efficient, stable, and selective multi-functional catalysts. In this review, we summarize in detail the advances in the reaction chemistry, catalysts, and operating conditions for FDCA production from sugars and carbohydrates.Achieving the goal of living in a sustainable and greener society, will need the chemical industry to move away from petroleum-based refineries to bio-refineries. This aim can be achieved by using biomass as the feedstock to produce platform chemicals. A platform chemical, 2,5-furandicarboxylic acid (FDCA) has gained much attention in recent years because of its chemical attributes as it can be used to produce green polymers such polyethylene 2,5-furandicarboxylate (PEF) that is an alternative to polyethylene terephthalate (PET) produced from fossil fuel. Typically, 5-(hydroxymethyl)furfural (HMF), an intermediate product of the acid dehydration of sugars, can be used as a precursor for the production of FDCA, and this transformation reaction has been extensively studied using both homogeneous and heterogeneous catalysts in different reaction media such as basic, neutral, and acidic media. In addition to the use of catalysts, conversion of HMF to FDCA occurs in the presence of oxidants such as air, O The chemical industry is one of the dominant industries as chemicals are used in a variety of applications including in large scale production of fuels and fuel additives, polymeric materials and chemical intermediates for example, as hosting matrix for nanocomposites, nanocatalysts, anticorrosion coatings, membrane for energy unit, and so on Sheldon, , 2020. H2 emissions, reduced energy consumption, and lower cost since sugars are a relatively cheaper feedstock than HMF , derived from fossil fuels, are important platform materials utilized today for the production of a wide range of products , and other oxidants and further discusses the effect of noble metals, non-noble metals, and metal free catalysts with different supports. Moreover, the effect of neutral  and basic media and associated kinetics of these processes are also discussed with regards to the oxidation of HMF to FDCA. This review describes both the single-phase and multiphase reactions to directly convert sugars to FDCA, highlighting ways to effectively recover FDCA and strategies to minimize impurity contamination of FDCA. The review concludes by highlighting future areas of research to address issues around the competitiveness of FDCA production as a commodity chemical.The most extensively studied pathways for the oxidation of HMF to FDCA use either homogeneous or heterogeneous catalysis. Homogeneous catalysts have numerous drawbacks including low FDCA yield, high by-product formation as well as difficulties in catalyst separation and product purification. A recent review reports on advances in the catalytic synthesis of FDCA, wherein FDCA is produced from the oxidation of HMF, or directly from sugars by a one-step reaction process  was reacted with hydrogen bromide (HBr). However, this method was not commercially viable due to high cost of material, elevated working temperature, and long reaction time  or air, H2O2, and various other oxidants have been used in the oxidation of HMF to FDCA and are discussed in the following sections.Production of FDCA from HMF using heterogeneous catalysts is more advantageous than the use of homogeneous catalysts because of higher FDCA yield, easier separation and purification of FDCA and lower by-product yields. As such, extensive studies have been conducted on the heterogeneous catalytic oxidation of HMF to FDCA over various oxidizing agents. Oxidizing agents play significant roles in HMF oxidation by facilitating the oxidization on the catalyst surface , palladium (Pd), platinum (Pt) ruthenium (Ru), and rhodium (Rh) have been extensively used as the main heterogeneous catalysts for the aerobic oxidation of HMF to FDCA. Apart from the noble metal heterogeneous catalysts, transition metal supported catalysts, and metal free heterogeneous catalysts have also been used in the aerobic oxidation process of HMF to FDCA.Among the various type of oxidants, O2 pressure for 6 h at 23°C and observed relatively low yield (8%) with Au/C catalyst. This was due to the difficulty of Au to activate the alcoholic side chain of HFCA before the latter was oxidized to FDCA. However, at a higher concentration of a base  and a higher pressure of O2 the yield of FDCA increased, apparently due to the increase in the base concentration  with Au/AC catalyst. However, when the catalysts were protected with polyvinyl alcohol (PVA)  2 h at 100°C and 40 bar for total conversion of HMF, but a longer time of 4 h for the catalyst, prepared by cationic exchange of Ru on NaOCl oxidized activated carbon (Ruex/ACNaOCl). This is because, the surface of the carbon-treated NaOCl introduced some of the oxygenated groups , some may have broken down during carbonization, and the residual oxygen functionality impacted negatively on the reaction rate for the formation of FDCA. Further evidence of the carbon architecture and surface properties on catalytic activity was demonstrated by Niu et al.  of FDCA after 4 h at 90°C, in a Na2CO3 alkaline media at 1 bar O2 pressure  gave a high yield of FDCA at 94%. More so, at higher concentrations of PD-HMF more than 10 wt%, FDCA yield stabilized above 90%. It was concluded that the reactive formyl group of HMF can be stabilized by acetalization with 1,3-propanediol to produce a stable intermediate cyclic HMF-acetal that overcomes the thermal instability of HMF which inherently limits selective oxidation to FDCA.To improve catalyst recovery, reduce operating and catalyst costs, and recyclability, magnetic core-shell structured Fem et al.  because 2 by addition of Bi. The Pt/Ce0.8Bi0.2O2−δ gave improved FDCA yield of 98% working under 10 bar of O2 pressure at 23°C in NaOH, while only Pt/CeO2 gave only 20% of FDCA yield. Moreover, Pt/Ce0.8Bi0.2O2−δ catalyst showed excellent stability and reusability . Considering the reaction mechanism of HMF oxidation by Pt/Ce0.8Bi0.2O2−δ catalyst in alkaline media as shown in −) in the hydroxide groups of HMF. Then, oxygen vacancies and breakdown of transitional peroxides accelerated oxygen reduction in Bi-Ce complex, generating surface electrons that are continuously consumed, consequently safeguarding the catalytic efficiency even up to five times of recycling  and non-reducible oxides  over the oxidation of HMF to FDCA under 1 bar O2 pressure at 75°C in Na2CO3 media for 12 h . It was observed that Pt catalysts supported on reducible oxides gave poor FDCA yield around 2–8% while, non-reducible oxides gave above 90% FDCA yield  was studied by Wang et al. (3 and the abundant weak basic sites (OH− groups) present on the surface of HT. This catalyst can be reused five times with good recyclability and catalytic stability.Miao et al.  have inv Haruta, . An imprg et al. . They ob2 bimetal catalyst with Au/TiO2 and Cu/TiO2 monometallic catalysts and observed no FDCA formed with Cu/TiO2, 10% with Au/TiO2 and 30% with the alloy as shown in 2 bimetal catalyst also tested at various Au/Cu mole ratios, and the alloy of various composition showed improved catalytic activity compared to the monometallic catalysts. The reason for the high catalytic activity of the Au-Cu/TiO2 catalyst lies on the isolation of Au sites by alloying with Cu. As such, Au-Cu/TiO2 can be reused and recovered without significant leaching and agglomeration. A synergistic effect was observed for the bimetallic catalysts due to the derived benefit of improving the stability of the catalyst and resistance to poisoning . In this reaction, HFCA and FFCA observed as process intermediates and then oxidized to FDCA. For the Pt-Bi/C catalyst, oxidation of FFCA to FDCA is the rate-determining step. Incorporating Bi into the Pt/C catalyst increased the stability and catalyst life by removing the oxygen poisoning and Pt leaching likelihood. Apart from that, Gupta et al. 2, which is in situ prepared over the aerobic oxidation of HMF to FDCA in base free media. Among those catalysts, Ni0.9-Pd0.1/Mg(OH)2 resulted highest FDCA yield of 89% at 100°C. Improved FDCA production was obtained due to the activation of the hydroxyl group of HMF due to the basicity of support.Apart from the catalyst support, the alloying of noble metals as catalyst material has been evaluated for the aerobic oxidation of HMF to FDCA. The concept of alloying was considered because of the easiness of tuning the physical and chemical properties of the catalyst and their activity  in Na-HFCA and HFCA structures. As shown in 18O atoms combine with FDCA due to the reversible hydration of the aldehyde group in step 4 to the geminal diol. At the end of the reaction, complete HMF oxidation to FDCA could be observed by the incorporation of four oxygen atoms in FDCA instead of O2. From the isotope labeling, the authors inferred that the oxygen atoms came from water and not from the oxidants. However, oxidizing agents indirectly facilitate the oxidative production of FDCA from HMF, by releasing the electrons deposited on the catalyst surface . Pt-PVP catalyst has gained much attention as an oxidation catalyst in water because of its high oxidizing ability. Despite the potential of the Pt/PVP catalyst in terms of green environment protocol, it is not feasible economically because of its poor stability and recyclability.Even though, most of the noble metal incorporated catalysts are performed under aerobic oxidation of HMF to FDCA in basic media resulting in high yields of FDCA, when the environmental aspect is included in the equation, base-free oxidation systems are more desirable. Gupta et al. studied the reaction of Au/HT catalyst over the oxidation of HMF to FDCA at 95°C in water under 1 bar On et al.  have usen et al.  have synn et al.  have reph et al.  have exa18O atoms were confirmed by the mass spectra x catalyst only 19% FDCA yield with 99% of HMF conversion at 140°C under 1 bar air pressure after 24 h. The reaction was carried out at a high temperature of 140°C to minimize the influence of viscosity of the IL  and achieved 85% of FDCA yield at 100°C under 10 bar O2 pressure after 10 h in an aqueous reaction medium. The Fe-POP metal active site functions in a radical chain mechanism by thermal autoxidation of the organic material to peroxides. Wang S. et al. (3O4-CoOx), which has magnetic properties and observed 69% FDCA yield with 97% HMF conversion at 80°C after 12 h. A mixture of mixed oxides of Fe2O3 and ZrO2 has been used for the base free oxidation of HMF to FDCA using ionic liquids (ILs) as solvents and co-catalysts  catalyst and achieved 91% FDCA yield at 110°C temperature in KHCO3 media after 15 h. Structural analysis revealed that Mn4+ and Ce3+ ions functioned as active sites for HMF oxidation to FDCA. Zhang et al.  and Brφnsted (Co-O-H) acid sites of the catalyst.So far, base-free aerobic oxidation of non-noble metal catalysts has not received much attention. However, recently Gao et al.  reportedIn summary, recent work has shown that non-noble metal catalysts can have similar catalytic activity as noble metal catalysts in the aerial oxidation of HMF to FDCA. However, metal leaching, blockage of the active sites and changing the active phases during reactions are some of the adverse effects that affect the catalytic performance of non-noble metal catalysts in the oxidation of HMF to FDCA. From cost benefit point of view, further investigations should be conducted to design efficient non-noble metal catalyst that can provide economic routes to selectively produce FDCA.The high cost of using noble metal catalysts, and to a lesser extent transition metal catalyst, as well as metal ion leakage into the reaction medium (resulting in purification issues of the product) has hindered the development of industrial applications for FDCA production from HMF. Nguyen et al.  has deve2 is used as an oxidant in the oxidation reaction of HMF to FDCA. However, the need of high O2 pressure in the oxidation of HMF to FDCA raises safety issues. Therefore, H2O2 which produces O2 and water was tested in place of O2 in the oxidation of HMF to FDCA  to FDCA. The study found that the oxidation route of alcohol group in HMF is favored over the oxidation of the aldehyde group on the other side of the molecule.As a substitute for the noble metal catalyzed HMF oxidation reaction, Martínez-Vargas et al.  immobili2 and H2O2 in aspect of the oxidation reaction of HMF to FDCA. Because, H2O2 is a highly active oxidant and needs to be handled carefully, it is less favorable from an industrial aspect. The catalytic activity of O2 is relatively low compared to other oxidants  and O2 needs high pressure to facilitate the oxidation of HMF to FDCA which could cause issues in energy consumption and production costs. Gao et al. (4) showed the highest HMF conversion (96.5%) but the yield of FDCA was very low (3.9%). The reason for this could be the high oxidative property of NaIO4 causing the destruction of HMF furan rings , and smaller amounts of HFCA and FFCA  and due to the difficulty to obtain chloroperoxidase in the pure form from C. fumago is not suitable for the bioconversion of HMF to FDCA  which was identified to attack both the aldehyde, and alcohol groups was shown to convert HMF to 95% FDCA yield /H2O results rapid production of HMF from fructose  as co-solvent with water  cooperated sol-gel silica was used to convert fructose to FDCA, with 99% selectivity and 72% conversion obtained due to the synergistic effect of acidic and metal sites 3 was used in the process of converting glucose to FDCA in water/THF biphasic system and obtained a yield of 52% FDCA. After successful production of FDCA from fructose and glucose, conversion of biomass to FDCA was then studied with Jerusalem artichoke (JAT), selected because of its high inulin/fructose content, abundancy, and fast growing nature  and H2 catalyst used to oxidize the resulted solution in the first step and obtained overall FDCA yield of 29%.In the sucrose conversion to FDCA, 34% yield of HMF obtained after hydrolyzation and dehydration to HMF by using sulfuric acid in a tubular reactor at 200°C under 25 bar pressure  was used as phase I and dehydration of sugars occurred in this phase using Amberlyst-15 catalyst  has gained attention as FDCDM can be easily converted to FDCA by simple hydrolysis reaction after purification via vacuum distillation. In comparison to FDCA, when making polyesters, the use of diethyl or dimethyl esters of FDCA exhibit lower melt temperature and are normally more miscible in the corresponding diols. This is particularly the case for enzyme catalyzed polymerisations, such as the examples of work by Jiang et al. , 2016. P2 catalyst at 130°C in MeOH with the presence of 8% MeONa, where, MeONa was used to ensure the reaction proceeds to completion. Conversion of HMF to FDCDM projects similarities with HMF oxidation to FDCA in water over Au catalysts . However, the catalyst CoxOy-N@C shows low FDCDM selectivity (38% yield) but produces other products like 44% yield of 2,5-furandicarboxylic acid monomethyl ester (FDCMM) and 12% yield of 5-hydroxymethyl-2-furoic acid methyl ester (HMFM) at 100°C in 1 MPa O2 pressure after 12 h. Alternatively, K-OMS-2 was used as the strong oxidant to improve the alcohol group oxidation to aldehyde group and HMFM yield was decreased to 1% but FDCMM was still produced in yield of 41%.Taarning et al.  have repg et al.  have stuIn considering the oxidative esterification of HMF to FDCDM, apart from being able to produce purified FDCA, there is the option to conduct reactions in low amounts of base or base free media. Au has shown to have good catalytic effect and selectivity, but is not cheap. On the other hand, Co is a cheap catalyst with catalytic activity but exhibits low selectivity. Therefore, synthesizing cost-effective bimetallic catalysts consisting mainly of non-noble metal catalyst with high catalytic activity and selectivity should be considered in future research.CMF is a versatile chemical which has similar functionality and HMF and in some aspects, shows better properties than HMF. For example, the direct conversion of cellulosic biomass to CMF can be achieved in a high yield of 71–76% . Under microwave irradiation at 160°C in acetonitrile for 5 min, 54% of DFF was formed when the combination of PNO and copper(II) trifluoromethanesulfonate (Cu(OTf)2) was used. Mascal and his group was able to oxidized DFF to FDCC with 80% yield in the presence of tert-butyl hypochlorite (t-BuOCl). Compared to the FDCA, FDCC is soluble in most of the common solvents whereas, FDCA is only soluble in polar aprotic solvents like DMSO . Vicente et al.  have stuPolyethylene terephthalate and polyamides are important polymeric materials vital to the modern economy. Some industries have initiated production of bioplastics or green polymers from bio-chemicals which are derived from lignocellulosic biomass. Among them, bio-chemicals like ethylene glycol (EG) and FDCA have shown successful conversion to bioplastics. FDCA is mainly used to produce biobased polymers, and has also been used in the production of surfactants and polyesters (Jiang et al., Numerous researches have been conducted on the synthesis of FDCA from biomass-derived carbohydrates, are more preferable due to high conversion rate where initially carbohydrates are dehydrated to HMF and then subsequently oxidized to FDCA. This process can be achieved through chemical and biological catalytic conversion as well as electrochemical conversion pathways. However, chemical catalytic methods and high yield of FDCA (Sajid et al., 2 appears most feasible for the oxidation of HMF due to its availability and low cost. However, when O2 is used for the oxidation high pressures are generally required and that is the main disadvantage of using O2. Instead of O2, H2O2, and t-BuOOH have also been used as the oxidant in the oxidation of HMF. However, these oxidants have limited suitability for large scale production due to the explosive nature of peroxides.On the discussion of the oxidants, molecular OThe different reaction media was shown to affect reaction pathways, Au-based catalysts oxidized in alkaline media produced HFCA as an intermediate whereas, base-free oxidation proceeds via DFF route. This suggests the aldehyde group oxidation is much quicker than alcohol group oxidation in alkaline media. Also, isotope labeling confirmed that the reaction mechanism of base and base-free environment are similar. Water molecules in the reaction media were found to provide the oxygen source to convert HMF to FDCA and surprisingly not the oxidants. And, the role of molecular oxygen is to substitute electron deposition in the supported metals for the catalyst reimbursement. Even though significant researches have been carried out on the production of FDCA from various biomasses using different catalysts and techniques, there is a scarcity of market share for FDCA in the polymer market. Economically, HMF has similar or higher price than FDCA which raising the question of producing FDCA from high cost feedstocks like HMF. Therefore, extensive research has focused on the aspect of deriving commercial benefit synthesizing FDCA from cheaper feedstocks. Based on the information discussed in this review paper, the following directions are suggested for future research.Extensive studies on the effect of reaction media for the catalytic oxidation of HMF to FDCA will help in the selection of effective processes. Alkaline media provides higher yields compared to base-free but is economically and environmentally less feasible. Base-free media is the most preferable, however, generally suffers from low FDCA yield and selectivity in the HMF oxidation to FDCA. Therefore, more studies utilizing base-free catalytic oxidation to improve yield and selectivity of FDCA from HMF are imperative to addressing environmental issues.The direct conversion of lignocellulose biomass to FDCA in high yield is required for the competitive value for FDCA as a building block chemical for the production of green products. Furthermore, direct conversion to FDCA should address the problems related to HMF separation from the reaction media. Therefore, designing a catalyst with acid/base sites for carbohydrate dehydration to HMF in a hydrophilic environment and with metal sites for oxidation of HMF to FDCA in hydrophobic media could be effective in the production of FDCA with high yield and selectivity direct from lignocellulose biomass. Where, acid/base sites will adsorb carbohydrates and desorb HMF while metal sites will adsorb HMF and oxidize to FDCA. However, initially glucose has to be released from the lignocellulose biomass and isomerized to enable more efficient conversion to FDCA. Therefore, extensive researches need to be conducted to efficiently enable both release of glucose from the lignocellulose biomass and its isomerization to fructose.4, H2O2, and O2. Among them O2 has shown the ability to produce high yields of FDCA, however the major drawback is the high pressure usually required to complete the reaction. Therefore, finding a catalyst which can be used at low pressure to efficiently oxidize HMF to FDCA will provide much needed process benefit in terms of reduced energy.Various oxidants have been used to convert HMF to FDCA like KMnOVarious research has been conducted in the improvement of multifunctional groups and acid-base properties of ionic liquids and deep eutectic solvents which assist the catalytic reaction for the production of FDCA. To achieve the goal of direct conversion of lignocellulosic biomass to FDCA it will be advantageous using a solvent with strong catalytic activity. However, the major problem of using ionic liquids and deep eutectic solvents is their environmental impact. Future research should aim to address the environmental issue by developing these solvents from biomass.Extensive evaluation of the chemical kinetics and mechanisms for the conversion of HMF to FDCA and sugars to FDCA are required to allow more effective design of new catalysts with high catalytic activity, recyclable and that are economical for the direct conversion of lignocellulose biomass to FDCA.The enzymatic HMF conversion to FDCA should be further explored as it is achieved under mild conditions. McKenna et al.  have demCMF has similar chemical functionally as HMF. However, it is more versatile in some aspects than HMF due to its ability to directly couple with organometallics, and so a tendency toward nucleophilic substitution and high dissolving power. Therefore, conversion of CMF to FDCA is an emerging field which need extensive study so that an industrial application can be realized with lignocellulosic biomass as the feedstock.Finding other higher value applications of FDCA would help to both grow market demand and sustain further research and development. Polyesters and polyamides are important polymer applications that derive greater value than plastic packaging and also represent sustainable market size.When considering the large-scale production of FDCA, the process will need to be designed to address all technical, economic and environmental challenges. Additionally, energy implications associated with processing low feed loadings which are generally necessary to achieve high FDCA yield will also need to be addressed. This will require interdisciplinary research in material engineering, chemical engineering, and process design.AD, LA, LM, DR, JB, and WD drafting the manuscript. JB and WD coordinating and supervising. LA, LM, DR, JB, and WD supervision and editing. AD writing the original draft. 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.


The otolaryngology community has significant concerns regarding the spread of SARS-CoV-2 through droplet contamination and viral aerosolization during head and neck examinations and procedures. The objective of this study was to investigate the droplet and splatter contamination from common otologic procedures.Cadaver simulation series.Dedicated surgical laboratory.Two cadaver heads were prepped via bilateral middle cranial fossa approaches to the tegmen (n = 4). Fluorescein was instilled through a 4-mm burr hole drilled into the middle cranial fossa floor, and presence in the middle ear was confirmed via microscopic ear examination. Myringotomy with ventilation tube placement and mastoidectomy were performed, and the distribution and distance of resulting droplet splatter patterns were systematically evaluated.There were no fluorescein droplets or splatter contamination observed in the measured surgical field in any direction after myringotomy and insertion of ventilation tube. Gross contamination from the surgical site to 6 ft was noted after complete mastoidectomy, though, when performed in standard fashion.Our results show that there is no droplet generation during myringotomy with ventilation tube placement in an operating room setting. Mastoidectomy, however, showed gross contamination 3 to 6 ft away in all directions measured. Additionally, there was significantly more droplet and splatter generation to the left of the surgeon when measured at 1 and 3 ft as compared with all other measured directions. In the current climate, the safety of otolaryngology procedures is of particular concern, as current evidence suggests elevated risk due to close contact with upper respiratory mucosa, which harbors a high viral load.24The current global pandemic brought about by the novel coronavirus disease 2019 (COVID-19) has led to sweeping transformative change in the health care sector. US hospitals have essentially ceased all elective, nonurgent surgical cases in accordance with guidelines from the Centers for Disease Control and Prevention,5 which can travel >2 m and linger on contaminated surfaces for hours, if not days.6 This has led to significant concern for the transmission of the novel coronavirus due to aerosol-generating procedures.7 As a result, the American Academy of Otolaryngology–Head and Neck Surgery has issued a position statement to limit elective procedures requiring interaction with upper airway mucosal surfaces or those with increased risk of aerosolization, which may include otologic procedures such as myringotomy and mastoidectomy.9Viral transmission is thought to be primarily via respiratory droplets,10 and for droplet dispersion and aerosol generation with use of high-speed drills.11 This study seeks to investigate and clarify these risks by evaluating droplet dispersion patterns resulting from otologic procedures in a cadaver-simulated series.However, to our knowledge, no published literature exists to guide decision making on the safety of these common otologic procedures. This is an important area of investigation due to the potential for the middle ear and mastoid to harbor respiratory pathogensThe study was exempt from institutional review board because it involved the use of nonliving human cadaveric tissue specimens (IRB protocol 2004100753). The experiments in this study were all conducted in a dedicated surgical laboratory on 2 fresh-frozen cadaver head specimens prepared in identical fashion and placed in a standard position for the procedures.With the following technique, a middle cranial fossa (MCF) approach was performed bilaterally on both specimens to expose the floor of the MCF. A posteriorly based trapdoor incision approximately 6 × 8 cm was made superior to the auricle down to the calvarium, and then a 6 × 6–cm bone flap, centered above the temporal root of the zygoma, was fashioned with a 4-mm cutting burr. After the MCF floor was completely exposed, a 4-mm port was drilled into the middle ear through the tegmen.Fluorescein solution at a concentration of 1 mg/mL was created by mixing 500 mg of fluorescein  with 495 mL of sterile saline. The 1 mg/mL fluorescein solution was instilled with a 14-gauge angiocath through the port into the middle ear space . Three sets of nonabsorbent blue paper (183 cm [6 ft] × 50 cm [1.64 ft]) affixed to a rigid backing were placed 90° from each other in the following directions: (1) left of the surgeon, (2) anterior to the head or across from the surgeon, and (3) right of the surgeon Figure 2The following surgical procedures were performed systematically on each head: (1) left-sided myringotomy with insertion of a ventilation tube; (2) left-sided complete mastoidectomy, including entry into the mastoid antrum and exposure of the tegmen, sigmoid sinus, and lateral semicircular canal; (3) right-sided myringotomy with insertion of a ventilation tube; and (4) right-sided complete mastoidectomy. A Stryker S2 πDrive Drill with a 6-mm Multi Flute burr was utilized for each mastoidectomy procedure. Following each surgical procedure, the number and distance of the droplets and splatter on the nonabsorbent blue paper was evaluated and measured by the following technique. Transparent grid graphs (25 × 25 cm) were laid side-by-side at 1, 3, and 6 ft from the surgical site. The blue paper on the surgeon’s chest was removed and laid flat, and a grid was placed on it as well. The surgeon’s face shield was removed and laid flat, and blue paper with an overlying grid was placed underneath it.2 area containing any illuminated fluorescent spot or any gross contamination. Fluorescein did not penetrate the bone but was limited to the mucosa.Since fluorescein fluoresces yellow under ultraviolet light and blue paper does not, the evaluators used an ultraviolet light to visualize the droplets and splatter from each experimental condition. The evaluators then counted and recorded the number and distance of any 1-cmNo observable fluorescein droplets were noted in the measured surgical field in any direction after myringotomy and insertion of ventilation tube. Visible fluorescein contamination was noted only on surfaces in direct contact with surgical instruments. In contrast, gross contamination was measured 3 ft in all cardinal directions after every mastoidectomy. The number of droplets identified at 1 and 3 ft to the left of the surgeon was significantly greater than the number on the right of the surgeon or across from the surgeon. The right side of the surgeon had significantly more splatter and droplets than across at 1 ft Table 2.12 The primary mode of viral transmission of SARS-CoV-2 is believed to be through the spread of respiratory droplets, which has led to significant community spread of the disease.5 The potential for spread through opportunistic aerosolization during aerosol generation procedures is also a concern. Since the upper respiratory tract harbors a high viral load,3 otolaryngologists are vulnerable to SARS-CoV-2 transmission while performing head and neck procedures that utilize suction and powered instrumentation, such as the surgical drill, especially if they are doing so without appropriate protective personal equipment.4 With its connection to the nasopharynx through the eustachian tube, the middle ear can serve as a possible source of transmission for upper respiratory tract pathogens10 during routine otologic procedures, such as myringotomy and mastoidectomy. With the persistence of SARS-CoV-2 in the general population for the foreseeable future, we will need to navigate these risks as we resume elective surgical procedures and perform urgent operations on patients whose SARS-CoV-2 status is unknown or positive.COVID-19 has rapidly disseminated from the Hubei province of China across the globe, with over 3 million confirmed cases in 212 countries as of April 29, 2020.13 This is likely secondary to the nature of the operation, which involves high-speed drilling of the temporal bone under irrigation creating visible splatter from bone dust and irrigation droplets. Our study also demonstrated that significantly more droplet and splatter occur to the left of the surgeon, which corresponds to the direction of rotation of the drill. Those within 1 to 3 ft of the drill are at increased risk of exposure. In teaching institutions where multiple members of the team may be directly adjacent to the primary surgeon, this must be taken into account. While the drill is being operated, all steps should be taken to reduce the number of other people within a 3- to 6-ft radius.In conducting this cadaveric simulation study, we confirmed that performing a myringotomy with insertion of ventilation tube caused no droplet or splatter contamination. The potential for aerosolization remains, however, when suction is used across a mucosal surface. In contrast, a complete mastoidectomy performed in standard fashion resulted in gross contamination up to 6 ft from the surgical site, which was the farthest distance measured. Aerosol generation with surgical drills has been established in the orthopedic literature.Several limitations to this cadaveric simulation study deserve consideration. These procedures were not conducted in a normal adult clinic setting with an actively respiring patient. With stimulation of the external auditory canal during examination, patients can produce a cough reflex that may cause increased risk of viral transmission. Moreover, there was no assessment of aerosolization, either forced  or from drilling, in this experimental model. However, we believe that it is still vital to understand the quantity, quality, and range of droplet and splatter contamination involved during these common procedures, as respiratory droplets are considered to be the primary mode of SARS-CoV-2 transmission. Another limitation is that only droplets and splatter visible to the human eye were measured. Furthermore, instead of a complete 360° assessment, the design model allowed for measurements only in the cardinal directions surrounding the specimen.14 They reported that surgical visualization was not affected. Although a good step in the right direction for preventing the spread of gross contaminant, this methodology does not create an air-tight surgical field, and droplet or splatter contamination was not measured in any objective way. Further studies should be performed comparing different techniques to determine the extent of prevention of droplet contamination and aerosolization.In the context of the findings from this study, we believe that it is important to devise techniques to limit the spread of gross contamination from mastoid surgery. This will not be easily accomplished, because it is difficult to operate a microscope while wearing a face shield or powered air-purifying respiratory. Risks to the rest of the surgical staff and anesthesia team also are present in the operating room, which highlights that additional protective personal equipment is necessary for the surgical team, not just the operating surgeon. Carron et al recently published a simple technique involving the use of 2 readily available clear surgical drapes to control droplet and splatter contamination during mastoidectomy.It is essential to evaluate all procedures that have a risk of disrupting respiratory epithelium and spreading SARS-CoV-2. Our results indicate that there is no droplet generation during myringotomy with ventilation tube placement in an operating room setting. For mastoidectomy, however, gross contamination was visualized 3 to 6 ft away in all cardinal directions, and significantly more occurred on the left side of the surgeon when compared with the other sides, corresponding to the direction of drill rotation. It is critical to develop techniques to contain contamination as much as possible.