Global, population-wide oscillations in models of cyclic dominance may result in the collapse of biodiversity due to the accidental extinction of one species in the loop. Previous research has shown that such oscillations can emerge if the interaction network has small-world properties, and more generally, because of long-range interactions among individuals or because of mobility. But although these features are all common in nature, global oscillations are rarely observed in actual biological systems. This begets the question what is the missing ingredient that would prevent local oscillations to synchronize across the population to form global oscillations. Here we show that, although heterogeneous species-specific invasion rates fail to have a noticeable impact on species coexistence, randomness in site-specific invasion rates successfully hinders the emergence of global oscillations and thus preserves biodiversity. Our model takes into account that the environment is often not uniform but rather spatially heterogeneous, which may influence the success of microscopic dynamics locally. This prevents the synchronization of locally emerging oscillations, and ultimately results in a phenomenon where one type of randomness is used to mitigate the adverse effects of other types of randomness in the system. Models of cyclic dominance are traditionally employed to study biodiversity in biologically inspired settings12345674141520An important result of research involving the rock-paper-scissors game is that the introduction of randomness into the interaction network results in global oscillations2225262728631Interestingly, however, although long-range interactions and small-world networks abound in nature, and although mobility is an inherent part to virtually all animal groups, global oscillations are rarely observed in actual biological systems. It is thus warranted to search for universal features in models of cyclic dominance that work in the opposite way of the aforementioned types of randomness. The questions is, what is the missing ingredient that would prevent local oscillations to synchronize across the population to form global oscillations? Preceding research has already provided some possible answers. For instance Peltomäki and Alava observed that global oscillations do not occur if the total number of players is conserved34228363738394041424344454647484950515253545556575859Here we wish to extend the scope of this research by considering a partly overlooked property, namely the consideration of site-specific heterogeneous invasion rates. Importantly, we wish to emphasize an important distinction to species-specific heterogeneous invasion rates, which have been considered intensively before. In the latter case, different pairs of species are characterized by different invasion rates, but these differences are then applied uniformly across the population. In case of spatially variable invasion rates, these could be site-specific, and hence particular pairs of species may have different invasion rates even though they are of the same type. Such a setup has many analogies in real life, ranging from differing resources, quality or quantity wise, to variations in the environment, all of which can significantly influence the local success rate of the governing microscopic dynamics.Notably, this kind of heterogeneity was already studied in a two-species Lotka-Volterra-like systemQ exceeds a threshold. If we then assume that species-specific invasion rates are heterogeneous, say δ0 = 0.6, δ1 = 0.8, and δ2 = 1 (here δi denotes the invasion rates of Si → Si+l transition where i runs from 0 to 2 in a cyclic manner), it can be observed that nothing really changes. In fact, the threshold in Q remains much the same, and the order parameter A (the area of the limit cycle in the ternary diagram) reaches the same close to l plateau it does when these invasion rates are homogenous. Further along this line, we can even adopt invasion rates that are chosen uniformly at random from the unit interval at each particular instance of the games. More precisely, we still keep the original Si → Si+l direction of invasion, but the strength of the invasion rate δi is chosen randomly in each particular case. But no matter the fact that this rather drastically modifies the microscopic dynamics, the presence of shortcuts will still trigger global oscillations , the global oscillations never emerge , while for v > 0 the fraction of sites having wj = 0.1, and thus the level of heterogeneity, increases. At the other extreme, for v = 1, we of course again obtain a homogeneous population where everybody has wj = 0.1, but we do not explore this option since it is practically identical, albeit the evolutionary process is much slower. By introducing heterogeneity into the system gradually, we can monitor how it influences the stationary state.As demonstrated previously34Qand P values that practically ensure an optimal support for local oscillations to synchronize across the population into global oscillations. Moreover, it can be observed that both transitions to the oscillation-free state are continuous. In other words, there does not exist a sharp drop in the value of A at a particular value of v. Instead, the suppression of global oscillations is gradual as the level of site-specific invasion heterogeneity in the population increases.In wj = 1. Accordingly, we have a fraction v = 0.1 of nodes, which instead of wj = 1 have the invasion rate wj = 1 − Δ. Here Δ becomes the free parameter, which for zero returns the traditional rock-paper-scissors game with homogeneous invasion rates, while for Δ > 0 the distance in the peaks of the discrete double-peaked distribution, and thus the level of heterogeneity in the population, increases. Representative results obtained with this approach are shown in v. In the former case, when Δ is small, the slight heterogeneity has no particular influence on the stationary state and global oscillations persist well beyond Δ = 0.3 for annealed randomness and Δ = 0.6 for quenched randomness. But if the difference reaches a sufficiently large value, global oscillations disappear in much the same gradual way as observed before in Similar in spirit, another way to introduce invasion heterogeneity gradually into the population is to use a fixed fraction of nodes with a lower invasion rate, but vary the difference to To sum up our observations thus far, different versions of the same concept reveal that spatially quenched heterogeneity in site-specific invasion rates is capable to effectively suppress global oscillations that would otherwise be brought about by either annealed or quenched randomness in the interaction network. However, there is yet another possible source or large-amplitude global oscillations in the population, namely mobility. As is well-known, mobility can give rise to global oscillation that jeopardizes biodiversityLastly, we thus verify if heterogeneity in site-specific invasion rates is able to suppress global oscillations brought about by mobility. As results in 64We have studied the impact of site-specific heterogeneous invasion rates on the emergence of global oscillations in the spatial rock-paper-scissors game. We have first confirmed that species-specific heterogeneous invasion rates, either fixed or varying over time, fail to disrupt the synchronization of locally emerging oscillations into a global oscillatory state on a regular small-world network. On the contrary, we have then demonstrated that site-specific heterogeneous invasion rates, determined once at the start of the game, successfully hinder the emergence of global oscillations and thus preserves biodiversity. We have shown this conclusion to be valid independently of the properties of the distribution that determines the invasion heterogeneity, specifically demonstrating the failure of coordination for uniformly and double-peak distributed site-specific invasion rates. Moreover, our research has revealed that quenched site-specific heterogeneous invasion rates preserve biodiversity regardless of the type of randomness that would be responsible for the emerge of global oscillations. In particular, we have considered quenched and annealed randomness in the interaction network, as well as mobility. Regardless of the type of randomness that would promote local oscillations to synchronize across the population to form global oscillations, site-specific heterogeneous invasion rates were always found to be extremely effective in suppressing the emergence of global oscillations. Drawing from the colloquial expression used to refer to alcohol that is consumed with the aim of lessening the effects of previous alcohol consumption, the introduction of randomness in the form of site-specific heterogeneous invasion rates lessens, in fact fully suppresses, the effects of other types of randomness – hence the “hair of the dog” phenomenon.Our setup takes into account heterogeneities that are inherently present in virtually all uncontrolled environments, ranging from bacterial films to plant communities. Examples include qualitative and quantitative variations in the availability of nutrients, local differences in the habitat, or any other factors that are likely to influence the local success rate of the governing dynamics1234There is certainly no perfect spatial system where microscopic processes would unfold identically across the whole population. These imperfections are elegantly modeled by the heterogeneous host matrix that stores the individual invasion rates of each player. As we have shown, the coordination of species evolution is highly sensitive on such kind of heterogeneities when they are fixed in space. Ultimately, this prevents the synchronization of locally emerging oscillations, and gives rise to a “hair of the dog”-like phenomenon, where one type of randomness is used to mitigate the adverse effects of other types of randomness. We hope that these theoretical explorations will help us to better understand the rare emergence of global oscillations in nature, as well as inspire further research, both experimental and theoretical, along similar lines.L × L square lattice with periodic boundary conditions, where each site j is initially randomly populated by one of the three competing species. For convenience, we introduce the Si → Si+l notation, where i runs from 0 to 2 in a cyclic manner. Hence, species S0 (for example paper) invades species S1 (rock), while species S1 invades species S2 (scissors), which in turn invades species S0 to close the loop of dominance. The evolution of species proceeds in agreement with a random sequential update, where during a full Monte Carlo step (MCS) we have chosen every site once on average and a neighbor randomly. In case of different players the invasion was executed according to the rock-scissors-paper rule with probability λ.The spatial rock-paper-scissors game evolves on a λ = 1. Species-specific heterogeneous invasion rates can be introduced through the parameter λ = δi, which is simply the probability for the Si → Si+l invasion to occur when given a chance. The values δ0, δ1 and δ2 can be determined once at the start of the game, or they can be chosen uniformly at random from the unit interval at each particular instance of the game. On the other hand, site-specific heterogeneous invasion rates, which we denote as wj, apply to each site j in particular, and determine the probability that a neighbor will be successful when trying to invade player j according to the Si → Si+l rule. This rule can be considered as “prey-dependent” because the w value at the prey’s position determines the probability of invasion. As an alternative rule, we can consider the w value of predator’s position that determines the invasion probability. Lastly, we can assume that the w values of both the predator and prey’s positions influence the invasion rate via their wj · wj′ product. While the time dependence of the evolution will be different in the mentioned three cases but the qualitative behavior is robust. Therefore we restrict ourself to the first mentioned “prey-dependent” rule.In the simplest, traditional version of the game, all invasion rates between species are equal to wj, we also consider site-specific heterogeneous invasion rates drawn from a discrete double-peaked distribution, where a fraction v of sites have wj = 1 − Δ, while the remaining 1 − v have wj = 1.These invasion rates are determined once at the start of the game and can be drawn uniformly at random from the unit interval, or from any other distribution. Here, in addition to uniformly distributed Q of the links that form the square lattice whilst preserving the degree z = 4 of each site. This is done only once at the start of the game. This procedure returns regular small-world networks for small values of Q and a regular random network in the Q → 1 limitP, while with probability 1 − P the invasion is restricted to a randomly selected nearest neighborP = 1, while for P = 0 only short-range invasions as allowed by the original square lattice are possible.To test the impact of site-specific heterogeneous invasion rates under different circumstances, we consider interaction randomness in the form of both quenched and annealed randomness. Quenched randomness is introduced by randomly rewiring a fraction σ. Oppositely, with probability 1 − σ, the dominant species in the pair invades the other in agreement with the rules or the rock-paper-scissors game. The parameter σ hence determines the intensity of mobility while the number of players is conserved. Technically, however, the strategy exchange between neighboring players is determined not only by the level of mobility σ, but it also depends on the individual wj and wr values characterizing the neighboring sites j and r. In this way, we can consider the fact that different sites may be differently sensitive to the change of strategy, and the success of mutual change is then practically determined by the site that is more reluctant to change its state. Accordingly, when the strategy exchange is supposed to be executed, then this happens only with the probability wjr that is equal to the smaller of wj and wr values .We also consider the impact of site-specific heterogeneous invasion rates in the presence of mobility. The latter is implemented so that during each instance of the game we choose a nearest-neighbor pair randomly where players exchange their positions with probability A, which is defined as the area of the limit cycle in the ternary diagramL × L = 4.106 sites, which was large enough to avoid accidental fixations when the amplitude of oscillations was large, and which allowed an accurate determination of strategy concentrations that are valid in the large population size limit. Naturally, the relaxation time depends sensitively on the model parameters and the system size, but 105 MCS was long enough even for the slowest evolution that we have encountered during this study.Global oscillations are characterized with the order parameter How to cite this article: Szolnoki, A. and Perc, M. Biodiversity in models of cyclic dominance is preserved by heterogeneity in site-specific invasion rates. Sci. Rep.6, 38608; doi: 10.1038/srep38608 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The exposure to CCR5 (CC chemokine receptor 5) specific natural antibodies in vitro produces a Class B β-arrestin2-dependent CCR5 retention with the aid of ERK1, due to the formation of a CCR5 signalosome, which remains stable for at least 48 h. Considering that β-arrestins and MAPKs are receptive to environmental signals, their signal complexes could be one of the key junction for GPCRs internalization related signal transduction. Here, we demonstrate that, in T cells, the phosphorylation status of either CCR5 receptor or ERK1 protein is necessary to drive the internalized receptor into the early endosomes, forming the CCR5 signalosome. In particular, our data show that β-arrestin2/ERK1 complex is a relevant transducer in the CCR5 signaling pathway. Understanding the mechanism of CCR5 regulation is essential for many inflammatory disorders, tumorigenesis and viral infection such as HIV. Chemokine receptors belong to seven-transmembrane proteins , which play a fundamental role in a multiplicity of developmental and chemotactic events ,2.2+) and their relative target proteins, including protein kinase A (PKA), protein kinase C (PKC) and the related substrates. This in turn results in regulation of several cellular functions, via heterotrimeric G proteins, and also G protein-dependent and -independent activation of mitogen activated protein kinases (MAPKs), including the extracellular signal-regulated kinase (ERK) cascade [After stimulation, these receptors activate a restricted number of effectors , which consequently influence the intracellular concentrations of second messengers , a member of the superfamily of 7TMRs, has been identified as the receptor for chemokines such as MIP-1α , MIP-1β , RANTES  .Natural antibodies, directed to the first external loop of CCR5 (ECL1), have been found in HIV infected Long Term Non-Progressor subjects (LTNPs) and are able to induce a long lasting internalization (48 h) of the receptor, which inhibits HIV entry . Even moAs β-arrestins and MAPKs are receptive to environmental signals, their signal complex could be one of the key regulatory points of signal transduction related to GPCR internalization. Our data demonstrate that the phosphorylation status of the major players of the CCR5 signalosome, in the cellular environment, is responsible for a stable CCR5 sequestration.+ T cells either in vitro  supplemented with 10% FCS (Lonza), 2 mM l-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin and with 3 μg/mL of blasticidin .R5-SupT1-transducted cell lines, engineered and kindly provided by H. Garg, are CCR5-expressing cell lines mentioned as SupT1-R5 clones L23 (expressing low level of CCR5 at cell surface) and M10 (expressing medium level of CCR5 at cell surface) as previously reported [ous work ) or ex vous work ). The ceAnti-p-CKR-5(E11/19), which specifically recognizes phosphoserine at position 349 (a specific GRK phosphorylation site) , anti-CK+ T-cell count persistently higher than 500 cells/μL of blood (mean 725 cells/μL). The group included 2 women and 3 men ranging in age between 35 and 52 years. The serum samples from these subjects contain antibodies that specifically recognized ECL1 of CCR5, induced complete receptor internalization after 48 h of incubation to either primary CD4+ T cells or T cell lines, blocked HIV infection and transcytosis, which mimics mucosal transmission [The pool of serum samples from 5 LTNPs positive for anti-CCR5 natural antibodies (CCR5 Ab Pos) included in this study  have been previously characterized and described . Brieflysmission . A pool smission ,11.The investigations were approved by the Institutional review board named “Comitato Etico della Fondazione San Raffaele del Monte Tabor”, Milan, Italy, (May 2002). A written informed consent was signed by all subjects and all methods were performed in accordance with the relevant Italian guidelines and regulations.The internalization assay was performed accordingly with our experimental conditions previously published ,12. Brie2, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40) in the presence of complete protease inhibitor cocktail . Samples were resolved by SDS–PAGE and transferred to nitrocellulose membranes . After blocking in 5% non-fat milk, the membranes were incubated overnight at 4 °C with the appropriate primary antibody and then probed, at room temperature for 1 h, with HRP-tagged secondary antibodies. The chemiluminescent signals were detected by ECL method . Comparative analysis of the bands was performed by quantitative densitometry and concomitant use of the TINA software . The normalization was done per each band based on the density of GAPDH signal per each line.The cells were lysed in RIPA lysis buffer . The protein samples were precleared with 50% of protein-A slurry for 16 h. Immunoprecipitations were performed with the indicated antibodies pre-adsorbed on protein A-Sepharose beads (Amersham Pharmacia Biotech AB) for 2 h at 4 °C [The immunoprecipitation assay was carried out as previously described ,11,14. BChemically synthesized siRNAs sequences  specific for human β-arrestin1 and -2 and GAPDH (NSsiRNA) were nucleofected using an Amaxa Nucleofector Device (Lonza) in accordance with the manufacturer’s instructions. The cells were then seeded onto 24 multi-well plates for 5 h and treated in accordance with the experimental procedure described in our previous works ,11,14.t-test was used and Prism version 5.0a  was used to analyze the data. p-values: * stands for p ≤ 0.05, ** stands for p ≤ 0.01, *** stands for p ≤ 0.001 and **** stands for p ≤ 0.0001.The two-tailed Student’s It is well demonstrated that natural antibodies to CCR5 receptor (CCR5 Ab Pos), detected in the sera of LTNPs, induce a long-lasting internalization (48 h) with the recruitment of β-arrestin2 and ERK1 proteins ,10,11. ATaking into account that cellular signaling pathways trigger protein–protein interactions mediated by phosphorylation and dephosphorylation events , here we+ T lymphocytes, as previously reported [The experiments were carried out in the R5-SupT1-L23 T lymphoblastoid cell line that shows an activation status and a CCR5 cell surface level comparable to human CD4reported . We usedreported ,10,11; aTo this end, cells were pre-treated with 50 nM staurosporine, a broad spectrum protein kinases inhibitor, and then stimulated for 30 min with CCR5 Ab Pos, CCR5 Ab Neg and with RANTES or not. The first group of samples was collected after 30 min of stimulation and it is used as a control of the classical short term kinetics of CCR5 internalization (30 min) described for its natural ligands, such as RANTES ,17,18,19To evaluate the CCR5 localization at single cell level, we performed an immunofluorescence analysis by using a specific antibody, which binds the external regions of CCR5 (amino acids 66–250).p ≤ 0.001) in comparison with the respective controls. As expected, the pre-treatment with staurosporine statistically significantly reduced the percentage of punctuate cells (p ≤ 0.001 for RANTES and p ≤ 0.01 for CCR5 Ab Pos) and the number of CCR5 puncta per cells (p ≤ 0.01) upon stimulation with both ligands tested  the percentage of cytoplasmic CCR5 puncta structures, as expected. In addition, the presence of staurosporine still determined a reduction both in the percentage of CCR5 positive cells (p ≤ 0.0001) and in the number of positive specific CCR5 puncta per cell (p ≤ 0.01), in the cells stimulated with CCR5 Ab Pos  in the cells stimulated with RANTES  is relevant for the cytoplasmic accumulation of CCR5 as shown by the reduction in the CCR5 retention in all the samples treated with staurosporine.To verify if the inhibition of protein kinases activity could be a restriction factor for the CCR5 signalosome formation, we analyzed the early temporal association of the internalized receptor CCR5 with the endogenous ERK1 and β-arrestin1/2 by immunoprecipitation assay, after pre-treatment or not with staurosporine, upon stimulation with RANTES and CCR5 Ab Pos, as described above. As agonist binding to CCR5 triggers the clustering of the receptor in clathrin-coated pits, which are delivered to the early endosomes ,19, we aThe experiment was performed in R5-SupT1 M10 cell line that has higher levels of CCR5 expression than R5-SupT1 L23 [In detail, p ≤ 0.0001), which is notable to induce the signalosome formation at this time point, and after CCR5 Ab Pos stimulation as well.Interestingly, at 30 min, the staurosporine treatment, which inhibits protein kinases activity, strongly reduced the basal level interaction of β-arrestin1/2 and ERK1 with CCR5; its presence also statistically significantly inhibited their interaction after RANTES stimulation  in the cells incubated with CCR5 Ab Pos compared to the non-treated control ones, at 150 min. Furthermore, the inhibition of protein kinases activity statistically significantly affected the accumulation of Rab5 protein, both at 30 min (p ≤ 0.01) and 150 min (p ≤ 0.0001), upon stimulation with CCR5 Ab Pos. As expected, the cells stimulated with RANTES and treated with staurosporine showed a statistically significant decrease of β-arrestin1/2 (p ≤ 0.01) and Rab5 (p ≤ 0.001) at 30 min only.A parallel Western blot analysis of the lysates was done to prove that the staurosporine treatment can also affect the accumulation of the proteins involved in the CCR5 signalosome complex. As shown in These findings suggest that, after internalization, the CCR5 complex is driven to early endosomes directly but the inhibition of protein kinases cascade deeply alters the protein–protein interaction among the CCR5 signalosome in a ligand-independent manner.It has been published that the binding of natural ligands to CCR5 induces the phosphorylation of four distinct C-terminal serine residues mediated by GPCR kinase (GRK) and protein kinase C (PKC) . Based oConsidering that CCR5 Ab Pos induce the CCR5 signalosome formation starting at 150 min, with the involvement of GRK-mediated phosphorylation and consequent interaction with β-arrestin1/2, we investigated the effects of staurosporine inhibition on the regulation of the core components of the CCR5 signaling pathway activation. Thus, after 1 h of treatment with staurosporine inhibitor, the R5-SupT1-L23 cells were incubated with CCR5 Ab Pos and relative controls and then harvested at 150 min .p ≤ 0.001) and ERK1 (p ≤ 0.0001), as well as the accumulation of the non-phosphorylated form of these proteins (p ≤ 0.001) in the cells incubated with CCR5 Ab Pos compared with the non treated controls. No activity was observed in cells either treated or untreated with CCR5 Ab Neg and RANTES respectively  and Rab5 (p ≤ 0.001) proteins as well , incubated with CCR5 Ab Pos and the relative controls and then harvested at 150 min, as described in our previous works ,14.The immunoblot showed that β-arrestin1/2 siRNAs duplexes specifically reduced protein expression levels a.We performed an immunoprecipitation assay, in the cells nucleofected or not with siRNAs specific for β-arrestin1 and -2, to determine the possible interaction between the endogenous Rab5 protein and the internalized CCR5 receptor. At 150 min, Rab5 had a weak basal level interaction with the receptor and it was strongly engaged to CCR5 upon stimulation with CCR5 Ab Pos in the cells nucleofected with non-specific targeting siRNA (NS-siRNA) b. MoreovThese data confirm that the CCR5 stimulation with CCR5 Ab Pos requires a β-arrestin2-dependent endocytosis and its depletion prevents the formation of the CCR5 signalosome into the early endosomes.Human CC chemokine receptor 5 (CCR5), a member of seven-transmembrane proteins , is the main coreceptor for the entry of R5 tropic strain of HIV-1, in association with CD4 . BindingReceptor interaction with β-arrestin triggers many conformational changes of the β-arrestin itself, which activate various kinases leading to specific signaling outcomes and physiological responses. The type of the phosphorylation barcode on cytoplasmic domains of GPCRs in association with the ubiquitination status of β-arrestin determines the stability of activated GPCR–β-arrestin complexes and their co-trafficking to endosomes .On the other hand, β-arrestins are able to induce intracellular signal transduction mediated by protein–protein interactions with several proteins, including MAPKs ,23,24,25The endocytic system recruits a very dynamic network of vesicles and endosomes, and the evolution of this network is important to understand the intracellular trafficking. Rab GTPases are specifically localized into various intracellular organelles ,27,28; iAntibodies (IgG and IgA) direct to ECL1 of CCR5 have been found in ESNs  as well + T lymphocytes [Anti ECL1-CCR5 antibodies induce a stable and long-lasting downregulation of CCR5 with the involvement of ERK1-based pathway , startinphocytes , and thiphocytes , even thThe endocytic mechanism CCR5 Ab Pos mediated could be strictly related to their specific binding site, as natural CCR5 antibodies recognize a conformational epitope within the first extramembrane loop (ECL1) . ConversThere are many published pieces evidence indicating that β-arrestins and MAPKs generally regulate downstream signaling pathways of different type of receptors and their signal complex could be relevant in the cross-communication integrating nodes . HoweverAs published data have demonstrated that protein kinases inhibitors are able to block the interaction between β-arrestins and ERKs, here we demonstrate that the staurosporine (pan-kinases inhibitor) statistically significantly reduces the internalization with consequent cytoplasmic accumulation of CCR5 in a ligand-independent manner . MoreoveIn addition, it was recently observed that the binding of natural ligands to CCR5 triggers the phosphorylation of four distinct C-terminal serine residues mediated by GPCR kinase (GRK) and protein kinase C (PKC) . In partOf note, it is well known that, following receptor endocytosis mediated by stimulation with RANTES and its analogs, CCR5 accumulates in perinuclear recycling endosomes and return back unphosphorylated to the plasma membrane with a short term kinetics. In addition, CCR5 does not significantly colocalize with late endosomal and lysosomal markers . MoreoveFinally, the data on the interaction between CCR5 and Rab5, after silencing of either β-arrestin1 or β-arrestin2 in the presence of CCR5 Abs, highlighted that CCR5-Rab5 complex formation was evident only if β-arrestin2 was functional ; this reTo summarize, upon stimulation with anti CCR5 antibodies, CCR5 is usually sequestered into complexes together with β-arrestin2 and ERK1, and driven to early endosomes. These events determine the formation of a stable CCR5 signalosome (150 min) , panel AInterestingly, the block of the protein kinases phosphorylation mediated by staurosporine treatment, that has an effect on both CCR5 and ERK1 proteins, abrogates the capture of the CCR5 signalosome into the early endosomes , panel BUnderstanding the mechanisms for CCR5 signaling pathway is relevant to understand many aspects of cell and cancer biology as well as immunology, considering the involvement of this receptor not only in HIV-1 infection but also in many inflammatory disorders, including rheumatoid arthritis, multiple sclerosis  and tumo


A surge in interest in cortical bone trajectory (CBT), first described by Santoni in 2009, may be a result of its numerous advantages, including reduced surgical incision length and lateral dissection, limited disruption of the facet joints, and decreased blood loss. In addition, CBT offers improved screw pullout strength and the ability to perform hybrid constructs with pedicle screws using minimally invasive approaches. However, one of the main limitations of the technique involves the small screw size, which limits the potential for long-segment constructs. We describe a technique involving a more in-line anatomical trajectory, allowing for larger screw diameters. A feasibility study using a cadaveric model was performed and evaluated. Moreover, a focused review of the literature on the use of CBT was performed. Screw entry points are located along the inferomedial aspect of the facet and angled superolaterally. The use of this technique allows for the placement of larger screws (4.5 to 6.5 mm diameter) without pedicle breaches along with the alignment of screw heads from L1 to S1. In addition, the technique can be performed using stereotactic navigation or fluoroscopy. A direct, more in-line technique allows for larger screws to be placed using CBT. This technique can be combined with minimally invasive approaches. The potential advantages of the CBT technique support its use as a probable alternative to traditional pedicle screw fixation techniques. First described in 2009 by Santoni et al., cortical bone trajectory (CBT) is a relatively new surgical approach used to reduce morbidity from spinal lumbar fixation and fusion . As provIndications and ContraindicationsThe indications for using CBT are similar to those for using traditional transpedicular screws Table . These iPreoperative PreparationStandard preoperative preparation can be performed similarly to the workup for traditional pedicle screws. An evaluation of computerized tomography (CT) scans to assess bone anatomy and quality, magnetic resonance imaging (MRI) to evaluate the ligamentous integrity and thecal sac compression, and upright X-rays to visualize dynamic instability and spinopelvic parameters can be performed. Preoperative anesthetic preparation is standard, although the expected reduction in operative time and EBL may be considerations in patients with a higher American Society of Anesthesia (ASA) physical status classification, where pedicle screws may not be possible. Similarly, the use of cortical screws for patients with osteoporosis or adjacent segment disease can be specifically considered.Equipment NeedsMultiple manufacturers are available to provide cortical screw instrumentation. In this study, K2M  was utilized for the cadaveric and case studies Figure . BecauseTechniqueThe patient is placed prone on a standard spine frame with imaging/navigation capabilities using standard positioning, preparation, and draping Figures -3. A midThe entry point is defined by the medial border of the superior articular process of the vertebra to be instrumented and the location on the pars interarticularis approximately 35 mm inferior to the inferior border of the facet joint complex. This is generally approximated by the inferior margin of the transverse process . A clock face can be imagined around the facet with the left pedicle entry at the 5 o’clock position and the right pedicle at the 7 o’clock position.The high-speed burr is now used to open the cortex to a depth of approximately 2 mm. The drill with the 3.0-mm drill bit is then used to develop the screw tract. The drill is directed from the entry point approximately 25° cephalad and 8° laterally. Visualizing the clock face again, this is approximately toward the 10 o’clock and 2 o’clock positions for the left and right screws, respectively.A tap that is 1.0 mm smaller than the intended screw is used to develop threads for the full length of the screw. The tap is advanced to a depth of at least 30 mm. Care is taken to remain within the bony borders. The desired endpoint is defined by the lateral border of the superior endplate, anterior to the midpoint of the vertebral body when viewed from a lateral position. Specialized taps that disengage can be left within the screw tract and used to distract the disc space for discectomy and preparation for an interbody fusion device. Alternatively, a marker  can be placed in each screw tract to demarcate the tract while performing laminectomies and/or disc space preparation. The spinous processes can be removed before tapping and screw placement if they inhibit the technique.CBT can potentially be applied safely from any lower thoracic level  to the fLaminectomy and Interbody FusionThe screws are not placed until the final stages of the posterior procedure, as the surgeon’s access to the canal and disc space may be impeded by the limited exposure and the screw head. This may be a potential disadvantage of cortical screws compared to pedicle screws.Any posterior decompression or interbody fusion procedures may be completed at this point. Care should be taken to preserve the region of the pars for the CBT screw, to avoid weakening the bone and creating the risk of screw fracture. Particular attention should be paid to the removal of the inferior articulating process. Care should be taken to limit potential fractures or the extension of the bone removal, which would encroach into the cannulated pars interarticularis-pedicle junction. Mindful dissection of the medial-lateral aspect of the lamina-pars interarticularis junction is also needed. Maintaining the cortical surface surrounding the cannulated pars interarticularis/pedicle will maintain the greater strength of the larger screw. After the posterior decompression and interbody grafts have been placed, the appropriate screws are placed with the screw inserted to the depth of the thread. Overtightening of the screw, resulting in strain on the cortical bone as well as the impingement of the screw head, causing an inability to reposition, should be avoided.Thought should be given to the choice of interbody fusion devices to be used. If placed from a posterior approach, transforaminal devices can create a challenge because of the limited lateral exposure used for CBT. This may require the removal of the medial two-thirds of the inferior and superior articulating processes. Oblique anterior and paramedian devices may also be used.The decortication of the remaining posterior arch and the disruption of the internal facet joints of the levels to be fused are completed. If a wide facetectomy is required during the decompression, the decortication of the lateral aspect of the superior articulating process along with the medial aspect of the upper instrumented vertebrae provides a less invasive approach and provides for robust fusion. Rods are placed and screws locked. Bone graft is applied, and the wound is closed in the appropriate fashion.CBT PositionIn contrast to the traditional pedicle screw that starts at the junction of the transverse process and the lateral pars interarticularis, the CBT screw begins at the medial position of the pars. Its trajectory is angled superolaterally compared with medialized pedicle screws. Prior studies have suggested lateral angles of 89° and cephalad angles of 25° -5. An anAdvantages and Disadvantages of CBT ScrewsSeveral distinct advantages of the CBT screw exist over traditional pedicle screws Table . The traDisadvantages can include a difficulty in performing decompression and interbody fusion with limited dissection or obstructing screw heads. Distinct techniques from pedicle screws, such as placing screws after decompression, may need to be employed. There is also a potential for misaligned screw heads requiring the use of off-set connectors, especially during the placement of hybrid constructs linking cortical and pedicle screws. Finally, the use of CBT screws for long-segment fusion constructs remains to be further explored. Biomechanical data shows improved pullout strength and multiaxial toggling for CBT; however, these results may not necessarily relate to patients or account for patient-specific factors.The use of a more in-line, anatomical approach may allow for larger-size cortical screws expanding the range of lumbar fusion possibilities and hybrid constructs. Here, techniques for such an approach are presented along with a focused review of the literature. Cephalad and lateralized angles are provided for potential use at any position on the lumbar spine. The advantages of CBT involve reduced surgical dissection, lower blood loss, and potential combinations using hybrid techniques with pedicle screws. Further study regarding the efficacy of this technique is required in patients in order to establish whether the modification of the technique can truly improve patient outcomes.


Hurricane Patricia in 2015 was the strongest Pacific hurricane to make landfall in Mexico. Although Patricia fortuitously spared major cities, it reminded us of the threat tropical cyclones (TCs) pose in the eastern North Pacific (ENP) and the importance of improving our understanding and prediction of ENP TCs. Patricia’s intensity and the active 2015 ENP hurricane season have been partially attributed to the strong El Niño in 2015, however there is still a lack of fundamental understanding of the relationship between El Niño-Southern Oscillation (ENSO) and ENP TCs. Here, we demonstrate that ENSO drives intrabasin variability of ENP TCs, with enhanced (reduced) TC frequency in the western portion of the ENP during El Niño (La Niña), but reduced (enhanced) TC frequency in the eastern nearshore area, where landfalling TCs preferentially form. This intrabasin difference is primarily driven by the Central American Gap Winds (CAGW), which intensify (weaken) during El Niño (La Niña), producing low-level anticyclonic (cyclonic) relative vorticity anomalies and thus an unfavorable (favorable) environment for TC genesis. These findings shed new light on the dynamics linking ENP TC activity to ENSO, and highlight the importance of improving CAGW representation in models to make skillful seasonal forecasts of ENP TCs. TC landfalls on the Pacific coast of Central America and Mexico are relatively infrequent due to the tendency for westward/northwestward TC trajectories. However, those TCs that do make landfall can bring catastrophic flooding, especially when TC-associated winds and moisture interact with mountainous orography5. For example, significant coastal flooding was triggered by Hurricanes Pauline in 1997 and Patricia in 2015. The latter is the strongest hurricane on record for the Western Hemisphere and inflicted an estimated $460 million USD damage across Mexico and Southern Texas7. Observed historical TC tracks in the ENP indicate that all landfalling TCs formed east of 116°W  of the ENP has both outstanding scientific motivation and profound socioeconomic implications.The ENP basin 9, and is a critical predictor in seasonal TC forecasts11 During El Niño, vertical wind shear over the ENP is reduced in response to anomalously warm eastern-central Equatorial Pacific sea surface temperature (SST), which tends to favor above-normal ENP TC activity12. Anomalous eastern Pacific ocean heat content during El Niño can also affect TC intensity13. However, whether there is a statistically robust relationship between ENSO and observed TC activity over the ENP has been called into question14. By dividing the ENP into Western Development Region (WDR) and EDR, recent studies16 found that only TCs that originated in the WDR are robustly correlated with ENSO , with more frequent and intense TCs observed during El Niño than La Niña. Locations of TC genesis and dissipation within the ENP are also characterized by a westward shift during El Niño compared to neutral ENSO and La Niña17. Such a TC response in the WDR has been attributed to changes in SST and mid-tropospheric relative humidity during ENSO18. However, ENSO’s impact on TCs in the EDR, which have a much higher probability to make landfall along the Pacific coast of Central America, has not been thoroughly investigated. There is currently no consensus on whether and how ENSO can exert an influence on TCs in the EDR.El Niño-Southern Oscillation (ENSO) is the most dominant climate mode on interannual timescales19, to relate ENP TC variability with ENSO indices16. However, few studies explored the influence of ENSO on the spatial distribution of TCs within the ENP, and the associated mechanism. As will be demonstrated below, a distinctive signature of ENSO’s impact on ENP TCs is an east-west spatial variation in TC activity. Such a feature cannot be understood using area-integrated analyses, which average out the opposite changes of TC activity in different parts of the basin. To address this issue, we carried out analyses focusing on ENSO’s impact on the spatial distribution of TCs within the ENP. We note that the boundary separating the EDR and WDR in our study is set at 116°W, following a previous study8. However, our analyses do not include Central Pacific TCs between 180° and 140°W, because the focus of this study is on the eastern part of the ENP near the coastline where mountain influences are potentially greater. Thus our WDR extends only to 140°W, rather than to the dateline8.Many previous studies on ENSO’s impact on ENP TCs used area-integrated measures, such as TC counts, TC days and the power dissipation index (PDI)We first made a composite of TC genesis location and track density for all El Niño and La Niña events during the period from 1970 to 2015 and compared the results to the climatological genesis location and track density for this period. 13 El Niño and 11 La Niña hurricane seasons were identified based on the 75th and the 25th percentiles of the Niño3.4 index  during El Niño (La Niña). These changes are similar in magnitude to those observed in the Caribbean and Atlantic during ENSO10. This ENSO-related ENP TC activity change in the nearshore area is also consistent with a previous study20.Similar spatial variability of ENP TCs, but opposite in sign, occurs during La Niña, with an expected decrease of TC activity in the WDR and an increase in the EDR Fig. . These f21; details in Methods) using the National Centers for Environmental Prediction (NCEP) Climate Forecast System Reanalysis  and its extension, the Climate Forecast System version 2 . We chose CFSR and CFSv2 because they produce a more realistic representation of fine-scale atmospheric circulation features over the EDR24 than other reanalysis products. However, owing to the shorter record length of CFSR and CFSv2 data, only 11 El Niño and 7 La Niña hurricane seasons were identified during the 1979–2015 period. But, the TC activity response to El Niño and La Niña is insensitive to changes in the record length  . Over much of the ENP, the El Niño-induced changes in vertical wind shear and hurricane maximum potential intensity provide more favorable conditions for tropical cyclogenesis  GPI anomalies during El Niño (La Niña) show a well-defined structure extending westward from the Sierra Madre topographic gap areas, especially near the Isthmus of Tehuantepec , and the depth of these gaps can approach ~1000 meters vertically , along with the corresponding SST. Both CAGW at TT and PP are clearly visible in the climatology, but the northerly TT jet is stronger and more effectively cools local SSTs. The CAGW at TT follows anticyclonic arcs downwind from the coast. In contrast, the CAGW at PP is more zonal because of the orientation of topographic gaps and the influence of steady trade winds. ENSO modulates the strength of the CAGW31, with intensified winds observed at both TT and PP during El Niño, due to an increased frequency of cold frontal intrusions31 and a southward shift of the Intertropical Convergence Zone (ITCZ)24.Figure Because of its narrow structure and strength, the CAGW plays a dominant role in determining low-level relative vorticity in the EDR, which is a key factor in influencing environmental favorability for tropical cyclogenesis. Figure 35. The southern flank of the PP jet, on the other hand, is characterized by a cyclonic (positive) relative vorticity anomaly. During La Niña, however, the reduced CAGW at TT and PP  basin-wide ENP TC activity during El Niño (La Niña) caused by changes in vertical wind shear, SST, and mid-tropospheric relative humidity is incomplete. The ENP TC response to ENSO is characterized by intrabasin variability, with enhanced TC activity confined to only the WDR and reduced TC activity in the EDR during El Niño, and the opposite during La Niña. Furthermore, this study identifies a topographically locked feature – CAGW– as a primary player in regulating TC activity in the EDR through low-level relative vorticity changes. As all TCs that make landfall onto the Pacific coast of Central America and Mexico form in the EDR, the results presented here have important implications for improving seasonal forecast skill of ENP TCs and their impacts. It points to the necessity of properly resolving and representing the CAGW and its response to ENSO in TC forecast models.7. These types of synoptic CAGW events are especially effective in triggering tropical cyclogenesis when they interact with the monsoon trough37. Understanding synoptic CAGW events as a mechanism of tropical cyclogenesis is an active research area, and more studies are clearly needed to elucidate the underlying dynamical processes and the distinction between their roles in synoptic versus seasonal and longer timescales.We note that the overall reduction of TC activity over the EDR in response to El Niño does not exclude occurrences of individual extreme hurricanes in the region during El Niño. Indeed, Hurricane Patricia in 2015 and Hurricane Pauline in 1997, both among the strongest hurricanes to make landfall over Mexico, occurred during strong El Niño years. Individual hurricane genesis can be initiated by short-lived synoptic events that may not contribute significantly to the mean environment for TC genesis. Hurricane Patricia, for example, appeared to be initiated by a synoptic-scale CAGW strengthening event near TT that provided an injection of cyclonic relative vorticity on the cyclonic shear side and spurred the formation of the initial tropical depressionst through November 30th (hereafter ‘hurricane season’), which accounts for more than 95% (792 out of 832) of the total number of TCs during 1970–2015. Note that our analysis period is slightly different from the ENP hurricane season defined by the National Hurricane Center (NHC), which runs from May 15th through November 30th. Since the number of TCs during our research period covers more than 95% of total number, it is unlikely that the results are sensitive to this difference in hurricane season definition.In this study, all composite and diagnostic analyses focus on the period from June 138 to define ENSO events. The seasonal mean values above the 75th (below the 25th) percentile are chosen as El Niño (La Niña) hurricane seasons. During the period from 1970–2015, 13 El Niño hurricane seasons  and 11 La Niña hurricane seasons  are identified  ×2° (longitude) square box during each hurricane season. To reduce noise and better capture principal anomaly patterns of TC locations, track density is then smoothed by averaging the surrounding eight points. Genesis density has a similar definition, except only the number of tropical cyclogenesis events  in each 2° × 2° square box are counted. Genesis density is also smoothed in the same way as track density.The World Meteorological Organization (WMO) subset of International Best Track Archive for Climate Stewardship (IBTrACS-WMO)40 is also applied to determine the statistical significance level of both genesis and track density. The El Niño composite anomaly is constructed with 13 years randomly chosen from among a total of 46 years. This re-sample process is then repeated 10,000 times to obtain a 95% and 90% confidence interval. The same process is also applied for the La Niña composite anomaly, but by choosing 11 years out of 46-year climatology.In addition to the Student’s t-test, the bootstrap technique22) and its extension, the NCEP Climate Forecast System version 2 (CFSv2)23, are used for TC environmental condition analyses. Both of these datasets are at a high spatial resolution of 0.5° × 0.5°. The CFSR was completed over a 32-year period from January 1979 to March 2011, and then the dataset was extended to the present as CFSv2. To better represent TC environmental conditions, we use 6-hourly rather than monthly mean reanalysis data. 11 El Niño  and 7 La Niña  hurricane seasons are identified during the entire 37-year period from 1979 to 2015 and are used to diagnose TC environmental condition changes during El Niño and La Niña events.To reveal the underlying physical mechanism of intrabasin ENP TC variability, the National Centers for Environmental Prediction (NCEP) Climate Forecast System Reanalysis (CFSR21 (GPI), which is defined as:The environmental favorability for tropical cyclogenesis is assessed by analyzing the genesis potential indexη is absolute vorticity at 850 hPa (s−1), H is relative humidity at 700 hPa (%), Vshear is the magnitude of vertical wind shear between 850 and 200 hPa (m s−1). Vpot is hurricane maximum potential intensity , defined as:TS is SST (K) and To is outflow air temperature at tropopause (K).CP is heat capacity of air,Ck and Cd are the surface exchange coefficients of enthalpy and the drag coefficient (dimensionless), respectively. θ*s and θs are surface saturation equivalent potential temperature and boundary layer equivalent potential temperature (K). The application of PI theory is discussed online (http://wind.mit.edu/~emanuel/holem/node3.html). FORTRAN and MATLAB subroutines are available online (ftp://texmex.mit.edu/pub/emanuel/TCMAX/). To estimate the primary contributions that lead to the ENSO-induced GPI anomalies, the GPI is also recalculated by interannually varying just one term while setting the other three terms to values of the long-term climatology45.where 30. This dataset is at a horizontal resolution of 0.25° × 0.25°, based on an improved Ku-band Geophysical Model Function (GMF) to optimize wind speed retrievals, especially in high wind speed areas. QuikSCAT ocean surface wind is available from 19 July 1999 to 23 November 2009, and is widely used in CAGW observational studies46.The ocean surface wind vector is derived from the National Aeronautics and Space Administration’s (NASA’s) Quick Scatterometer (QuikSCAT) Version-4 (V4) wind productq is specific humidity (kg kg−1), u and v represent the standard horizontal wind components (m s−1), p is air pressure (Pa), and g is gravitational acceleration (m s−2). Therefore qV has the units of kg kg−1 m s−1. Since the units of dp/g, Pa/(m s−2) can equally convert to kg m−2, the term ‘mass weighted’ is used for vertical integration. The vertically-integrated moisture flux is in units of kg m−1 s−1. To convert to a column of water, we assume a constant water density of 103 kg m−3, so the units of the vertically integrated moisture flux can be written as mm m s−1.By vector identity, vertically integrated horizontal moisture flux can be written as:https://rda.ucar.edu/] and National Climatic Data Center (NCDC) repository [https://www.ncdc.noaa.gov/].The datasets generated during and/or analysed during the current study are available in the University Corporation for Atmospheric Research (UCAR) repository [Supplementary Information


Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels organized as pentameric complexes expressed predominantly on the plasma membrane . One of Acetylcholine, an endogenous agonist of muscle nAChRs, binds at the interface between the extracellular domains of the receptor subunits, while the ion channel of the receptor is composed of the transmembrane segments of the subunits. Therefore, some path for information transmission between the distant parts of the protein complex (extracellular and transmembrane domains) should exist. Muscle nAChR activity is based on the allosteric transitions between different functional states . One sucIn principle, antagonists or channel blockers can be used to treat “excessively active” receptors. Most muscle nAChR antagonists work as strong muscle relaxants suppressing the muscle tonus. Application of classic muscle relaxants requires skilled anesthesiology personnel and could hardly ever be used in therapy under “slow-channel” myasthenic conditions. However, there are two therapeutics which are used for such purposes now, namely quinidine ,9 and flZyzzia sponge of Histodermella genus and showed moderate (IC50 ~ 3 μM) topoisomerase I inhibiting, immunomodulatory and cytotoxic activities at concentrations ranging from about 1 μM to about 70 μM  gain-of-function mutation found in patients with SCCMS. The Gly153 residue is situated in the loop B near the conservative Trp149 residue crucial for nAChR activation  Figure B. It als2+ accumulation in the endplates -epibatidine) competition. It has already been mentioned that MG shows cytotoxicity at 1–70 μM on different types of cells -αBgt, makulavamine G and d-tubocurarine (at varied concentrations) were pre-incubated for 3 h at room temperature with Torpedo californica electric organ membranes  in 50 μL of binding buffer . After that, [125I]-αBgt was added to the membranes to a final concentration of 0.1 nM and the mixtures were additionally incubated for 5 min. The binding was stopped by rapid filtration on GF/C filters (Whatman) pre-soaked in 0.25% polyethylenimine, the unbound radioactivity having been removed from the filters by washout (3 × 3 mL) with a binding buffer. Non-specific binding was determined in all cases using 3 h pre-incubation with 30 μM α-cobratoxin. The results were analyzed using ORIGIN 7.5  fitting to a one-site dose-response curve by Equation: % response = 100/{1 + ([compound]/IC50)n}, where IC50 is the concentration at which 50% of the binding sites are inhibited and n is the Hill coefficient.Fluorescent ligand competition assay. Neuro2a cells transiently expressing α1β3γ2 GABAAR were washed with the external buffer . Cells were then pre-incubated with 50 μM Makulavamine G for 15 min. Then, 50 μL of 100 nM Alexa Fluor 546 α-Ctx conjugate  was added to the cells for 20 min. To estimate the level of non-specific fluorescence intensity, simultaneous experiments with 3 μM α-Ctx was performed. Afterward, the cells were washed three times with a two-fold excess of extracellular solution. By epifluorescent microscope IX71  three fields in each experimental plate well were chosen in bright field illumination in an unbiased manner and pictures were taken with an appropriate filter combination. The fluorescent image analysis was done on 8-bit gray value pictures in TIFF format [F format  and ImagF format  open sou2+ riseFluorescent detection of cytoplasmic Ca. Calcium imaging was performed following Reference [50)n}, where EC50 is the half-maximal response and n is the Hill coefficient. Negative controls were run in the presence of 5 μM α-Ctx.eference . BrieflyModeling of ion channel kinetics. Ion channels were modeled in the QUB Express software [2+-containing solution at 22 °C. Possibilities of MG binding to different states of the receptor (from 1 to 5) were explored. Each possibility was studied with four increasing simulated acetylcholine concentrations. We searched the models which would reproduce the following features of the MG muscle nAChR inhibition: un-competitive mode of inhibition (4 (open di-liganded) or 5 (desensitized) and not the other states can explain un-competitiveness or increased desensitization rate (see software . Constansoftware  for the hibition A and an hibition A. Bindinrate see C.Site-directed mutagenesis. Mutation G153S in mouse α1 nAChR cDNA (pRBG4-vector) was created using mutagenic primers 5′-CTATGACAGCTCTGTGGTGGC-3′ and 5′-CACAGAGCTGTCATAGGTCCAG-3′, and Phusion Hot Start DNA-polymerase . Polymerase chain reaction was performed at the following conditions: 98 °C for 1 min, 25 cycles of 98 °C for 10–30 s, 55–70 °C for 1 min, 72 °C for 3–4 min, and extended at 72 °C for 7 min. Then the PCR products were digested with the DpnI restriction enzyme (New England Biolabs) for elimination and the parental DNA and XL1-Blue competent cells (Evrogen) were transformed with the digested PCR products. The mutant mouse α1 nAChR cDNA was sequenced to confirm the mutation of interest (Evrogen).Isolation and purification of Makaluvamine G. Makaluvamine G was isolated and purified from the Australian collection of Zyzzya fuliginosa as described in reference [Histodermella sp. [eference . Earlierella sp. .


These results suggest that a lipid emulsion reverses toxic-dose bupivacaine-induced vasodilation during sodium orthovanadate-induced contraction via the activation of a pathway involving either tyrosine kinase, JNK, Rho-kinase and MYPT1 or tyrosine kinase, PLC γ-1 and ERK, and this reversal is associated with the lipid solubility of the local anesthetic and the induction of calcium sensitization.The goal of this in vitro study was to examine the effect of a lipid emulsion on toxic-dose bupivacaine-induced vasodilation in a model of tyrosine phosphatase inhibitor sodium orthovanadate-induced contraction in endothelium-denuded rat aortae and to elucidate the associated cellular mechanism. The effect of a lipid emulsion on vasodilation induced by a toxic dose of a local anesthetic during sodium orthovanadate-induced contraction was examined. In addition, the effects of various inhibitors, either bupivacaine alone or a lipid emulsion plus bupivacaine, on protein kinase phosphorylation induced by sodium orthovanadate in rat aortic vascular smooth muscle cells was examined. A lipid emulsion reversed the vasodilation induced by bupivacaine during sodium orthovanadate-induced contraction. The lipid emulsion attenuated the bupivacaine-mediated inhibition of the sodium orthovanadate-induced phosphorylation of protein tyrosine, c-Jun NH Lipid emulsions are widely used to mitigate systemic toxicity induced by local anesthetics or lipid-soluble non-local anesthetic drugs . In agreTyrosine kinase-induced protein tyrosine phosphorylation in vascular smooth muscle contributes to contraction by both calcium sensitization, which is mediated by the regulation of phospholipase C and D and mitogen-activated protein kinase (MAPK) activity, and the regulation of voltage-operated calcium channels . SeveralVasodilation induced by anesthetics is mediated by either decreased calcium sensitization or reduced intracellular calcium concentrations . However−3 M), lidocaine (3 × 10−3 M) and mepivacaine (10−2 M) produced vasodilation in isolated endothelium-denuded rat aortae precontracted with sodium orthovanadate (10−3 M) , whereas a high concentration (1.5% to 2%) of the lipid emulsion alone slightly reversed mepivacaine-induced vasodilation (p < 0.05). In addition, naloxone (10−5 M) had no effect on the lipid emulsion-mediated reversal of bupivacaine-induced vasodilation , the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (10−5 and 3 × 10−5 M) and the Rho-kinase inhibitor Y-27632 (10−6 and 3 × 10−6 M) greatly inhibited sodium orthovanadate (10−3 M)-induced contraction (p < 0.001 versus dimethyl sulfoxide (DMSO) or time-matched control). In addition, the phospholipase C (PLC) γ-1 inhibitor U-73122 (10−5 to 10−4 M) and the extracellular signal-regulated kinase (ERK) inhibitor PD98059 (3 × 10−5 and 10−4 M) attenuated sodium orthovanadate (10−3 M)-induced contraction (p < 0.01 versus DMSO).Bupivacaine (10(10−3 M) . The lipdilation . The magdilation . Lipid e−3 M) induced protein tyrosine and PLC γ-1 phosphorylation in rat aortic vascular smooth muscle cells (RAVSMCs) (p < 0.001 versus control). Genistein (10−4 M) and bupivacaine (10−3 M) attenuated the protein tyrosine and PLC γ-1 phosphorylation induced by sodium orthovanadate , whereas the lipid emulsion (0.5% to 2%) attenuated the bupivacaine-mediated inhibition of sodium orthovanadate-induced protein tyrosine and PLC γ-1 phosphorylation (p < 0.001 versus bupivacaine plus sodium orthovanadate). Sodium orthovanadate (10−3 M) also induced JNK phosphorylation (p < 0.001 versus control). Genistein (10−4 M), U-73122 (8 × 10−6 M), SP600125 (5 × 10−5 M) and bupivacaine (10−3 M) attenuated the induction of JNK phosphorylation by sodium orthovanadate , whereas the lipid emulsion (1%) attenuated the bupivacaine-mediated inhibition of sodium orthovanadate-induced JNK phosphorylation (p < 0.001 versus bupivacaine plus sodium orthovanadate). Moreover, sodium orthovanadate (10−3 M) induced ERK phosphorylation (p < 0.001 versus control). Genistein (10−4 M), U-73122 (8 × 10−6 M), PD98059 (3 × 10−5 M) and bupivacaine (10−3 M) attenuated the ERK phosphorylation induced by sodium orthovanadate , whereas the lipid emulsion (1%) attenuated the bupivacaine-mediated inhibition of sodium orthovanadate-induced ERK phosphorylation (p < 0.001 versus bupivacaine plus sodium orthovanadate). In addition, sodium orthovanadate (10−3 M) induced MYPT1 phosphorylation (p < 0.001 versus control). Genistein (10−4 M), SP600125 (5 × 10−5 M), Y-27632 (3 × 10−6 M) and bupivacaine (10−3 M) attenuated the MYPT1 phosphorylation induced by sodium orthovanadate , whereas the lipid emulsion (1%) attenuated the bupivacaine-mediated inhibition of sodium orthovanadate-induced MYPT1 phosphorylation (p < 0.001 versus bupivacaine plus sodium orthovanadate). Finally, sodium orthovanadate (10−3 M) induced caldesmon phosphorylation (p < 0.001 versus control), whereas genistein (10−4 M), U-731225 (8 × 10−6 M), SP600125 (5 × 10−5 M) and PD98059 (3 × 10−5 M) attenuated the caldesmon phosphorylation induced by sodium orthovanadate .Sodium orthovanadate (10−4 to 10−3 M) inhibited the contraction and intracellular calcium level ([Ca2+]i) induced by sodium orthovanadate (10−3 M) . However, the magnitude of the bupivacaine-induced vasodilation was greater than that of the bupivacaine-induced [Ca2+]i decrease during sodium orthovanadate-evoked contraction (p < 0.001 at 10−4 to 10−3 M bupivacaine).Bupivacaine  a lipid emulsion reversed the vasodilation induced by bupivacaine; (2) genistein, SP600125 and Y-27632 greatly attenuated sodium orthovanadate-induced contraction; (3) a lipid emulsion attenuated the bupivacaine-mediated inhibition of protein tyrosine, JNK, MYPT1, PLC γ-1 and ERK phosphorylation induced by sodium orthovanadate; and (4) bupivacaine-induced vasodilation was greater than the bupivacaine-induced reduction in i during sodium orthovanadate-induced contraction, suggesting that the bupivacaine-induced reduction in calcium sensitization is mediated by the inhibition of sodium orthovanadate-induced MYPT1 phosphorylation : bupivacaine [27.5], lidocaine [2.9], and mepivacaine [0.8]) ) . Howevere [0.8]) ,22. A hie [0.8]) . Howevere [0.8]) . The cure [0.8]) . Second,e [0.8]) . In addie [0.8]) ,27,28. Fe [0.8]) ,29. Endoe [0.8]) ,31. Howevanadate ,31. Thirvanadate . These ftoxicity .All experimental procedures and protocols  were approved by the Institutional Animal Care and Use Committee at Gyeongsang National University and Catholic Kwandong University. All experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals prepared by the Institute for Laboratory Animal Research.2 and 5% CO2 to maintain pH values from 7.35 to 7.45. A 3.0-g resting tension was used to equilibrate the rings for 120 min, and the bath solution was changed every 30 min. A 25-gauge needle was inserted into the aortic lumen, and all the aortic rings were rubbed for several seconds to remove the endothelium. To confirm the endothelial denudation, we added phenylephrine (10−8 M) to the organ bath with endothelium-denuded aortic rings, and the phenylephrine produced a sustained and stable contraction. Next, we added acetylcholine (10−5 M) to the organ bath containing aortic rings with phenylephrine-induced contraction, and endothelial denudation was confirmed by observing less than 15% acetylcholine-induced relaxation from phenylephrine-induced contraction. After returning the resting tension to baseline by exchanging the Krebs solution containing phenylephrine with fresh Krebs solution, the following experiments were performed. A single ring was used to obtain dose-response curves induced by lipid emulsion  or various inhibitors. Sodium orthovanadate-induced contraction is attenuated by endothelial nitric oxide i and tension were performed as previously reported by our laboratory [N = 20) were sacrificed by intraperitoneal injection of sodium pentobarbital (50 mg/mL) followed by exsanguination. The descending thoracic aorta was isolated and dissected free from the surrounding connective tissue and fat under microscopic guidance and was placed in Krebs solution . [Ca2+]i was measured using the fluorescent Ca2+ indicator fura-2. Muscle strips were treated with acetoxymethyl ester of fura-2  in the presence of 0.02% Cremophor EL for 5–6 h at room temperature. Fura-2-loaded muscle strips were then transferred to a temperature-controlled 7-mL organ bath in a fluorometer  and rinsed with fresh Krebs solution at 37 °C for 20 min to remove unhydrolyzed fura-2/AM. Isometric contraction of the smooth muscle was recorded using a force-displacement transducer . The muscle strips were illuminated alternately (48 Hz) with excitation wavelengths of 340 and 380 nm. The light emitted from the muscle strips (F340 and F380) was collected by a photomultiplier through a 500-nm filter, and the ratio of F340/F380 was used as a measure of [Ca2+]i. The absolute [Ca2+]i was not calculated in the current experiment because the dissociation constant of the fluorescence indicator for Ca2+ in the cytosol may differ from that measured under in vitro conditions [−3 M) were considered to be 100% of the [Ca2+]i and contraction, respectively. Muscle tension and F340/F380 ratios were recorded with PowerLab/400 using the Chart program (ADInstruments). Muscle strips were placed under an initial 3.0-g resting tension. When both the [Ca2+]i and contraction evoked by sodium orthovanadate (10−3 M) reached a sustained level, bupivacaine (10−5 to 10−3 M) was cumulatively added to the organ bath.The loading of fura-2 and the simultaneous measurement of i and tension induced by bupivacaine are expressed as the percentage of [Ca2+]i and tension induced by sodium orthovanadate, respectively. For these data, a normality test was performed. As effect size and previous similar experiments were not available, we used the resource equation method to calculate the sample size in each group [p-values less than 0.05 were considered statistically significant.The values are shown as the means ± SD. Vascular responses (vasodilation or vasoconstriction) induced by local anesthetics , lipid emulsion and various inhibitors  during sodium orthovanadate-induced contraction are expressed as the percentage of maximal sodium orthovanadate-induced contraction. The simultaneously measured values of [Cach group . Accordich group . p-valueIn conclusion, lipid emulsion reversed toxic-dose bupivacaine-induced vasodilation via the activation of a pathway involving either tyrosine kinase, JNK, Rho-kinase and MYPT1 or tyrosine kinase, PLC γ-1 and ERK during sodium orthovanadate-induced protein tyrosine phosphorylation-mediated contraction . This re


Schoenoplectus acutus and Schoenoplectus americanus. We simulated sea‐level rise across existing marsh elevations and those not currently found to reflect potential future sea‐level rise conditions in two tidal wetlands differing in salinity. Plants were grown individually and together at five tidal elevations, the lowest simulating an 80‐cm increase in sea level, and harvested to assess differences in biomass after one growing season. Inundation time, salinity, sulfides, and redox potential were measured concurrently. As predicted, increasing inundation reduced biomass of the species commonly found at higher marsh elevations, with little effect on the species found along channel margins. The presence of neighbors reduced total biomass of both species, particularly at the highest elevation; facilitation did not occur at any elevation. Contrary to predictions, we documented the competitive superiority of the stress tolerator under increased inundation, which was not predicted by the stress gradient hypothesis. Multifactor manipulation experiments addressing plant response to accelerated climate change are integral to creating a more realistic, valuable, and needed assessment of potential ecosystem response. Our results point to the important and unpredicted synergies between physical stressors, which are predicted to increase in intensity with climate change, and competitive forces on biomass as stresses increase.Climate change impacts, such as accelerated sea‐level rise, will affect stress gradients, yet impacts on competition/stress tolerance trade‐offs and shifts in distributions are unclear. Ecosystems with strong stress gradients, such as estuaries, allow for space‐for‐time substitutions of stress factors and can give insight into future climate‐related shifts in both resource and nonresource stresses. We tested the stress gradient hypothesis and examined the effect of increased inundation stress and biotic interactions on growth and survival of two congeneric wetland sedges,  The SGH posits that biotic interactions are driven by facilitation under conditions of high abiotic stresses, such as temperature, water availability, or inundation, and that competition drives interactions under more benign conditions  without competition, S. acutus would perform better than its congener, S. americanus, under increased inundation stress; (2) S. americanus would have a competitive advantage under conditions of lower inundation stress; and (3) when grown together, S. acutus would facilitate the growth of its congener under the greatest inundation stress  owing to its potential to aerate anoxic soil through its rich aerenchymatous tissue  on plant growth and survival under field conditions using experimental planters called “marsh organs” Morris, , which a22.1We conducted the experiment within two historic brackish tidal wetlands: Browns Island  and Rush Ranch Open Space Preserve . Both sites experience mixed semidiurnal tides. Water salinity fluctuates seasonally, with the lowest and highest salinities found in the early spring and early fall, respectively, and the magnitude depends on winter precipitation, snow pack, and river flow  Volkart ex Schinz & R. Keller (Olney's bulrush) forms solid stands across mid‐ and high marshes. Stems are 0.3–1.8 m tall, and rhizomes are 0.5–2 cm wide, forming both clumps and runners  Á. Löve & D. Löve var. occidentalis (S. Watson) S.G. Sm. (hardstem tule) grows along tidal channel, river, and lake margins and forms stands of erect 1.5–3‐m‐tall stems. Rhizomes are 1.5–4 cm wide and grow linearly with few branches  were coAs noted in previous marsh organ experiments . No pore water or Eh measurements were taken in August.In April 2010, rhizomes of both species were collected from multiple locations within a 5 m diameter at the fresher site, washed, and grown in fresh water in a glasshouse. We chose to collect rhizomes from the fresher site to (1) control for maternal effects that could differ across site ; and (2) to use plants that predominantly experience freshwater conditions. No genetic analyses were conducted on the source material. In February 2011, all rhizomes and shoots were clipped to a standard length and weighed, and rhizomes were planted in the marsh organs at both sites in early March. Because we were focused on the effect of each species on the other, rather than comparing the relative importance of intra‐ and interspecific competition, we chose to use an additive design for our planting; one rhizome of each species was planted individually, and one rhizome of each species was planted together to examine the role of biotic interactions. Every month from April to September 2011, all stems were measured, and total stem length and stem density were calculated. Pore water salinity, pore water sulfides, and redox potential were collected monthly during low tides at both sites within the same week. Channel water level stations were installed at both sites and recorded water salinity and depth relative to meters NAVD88 every 15 min. The time inundated was calculated for each marsh organ elevation at both sites between March and September, and common tidal summaries  were computed. In one randomly selected pipe in every row of every organ, pore water was collected 15 cm deep. Salinity was measured, and 2–5 mL of pore water was mixed immediately with a sulfur antioxidant buffer solution in a vacuum‐evacuated vial. Sulfide concentrations were measured in the laboratory and compared against a standard curve. Every month at each wetland, one organ was randomly chosen to collect redox measurements, Aboveground biomass was removed between September 26 and 30 at the fresher site and October 10 and 13 at the saltier site; the difference in the timing of removal was due to high tides restricting access to all marsh organ elevations. All aboveground growth had stopped by the time of removal, and all biomass from a given organ was removed on the same day. Intact marsh organ tubes containing belowground biomass were removed between October 26 and 28 at Browns Island and October 31 and November 4 at Rush Ranch. Aboveground biomass was washed, sorted by species and live and dead shoots, dried at 70°C until a constant weight was obtained , and weighed. Belowground biomass was removed from the pipes, washed thoroughly of all sediment over a 2‐mm screen, sorted by species, roots, and rhizomes, dried at 70°C until a constant weight , and weighed.2.5Eh over time were analyzed using a repeated measures ANOVA (rmANOVA). Salinity and sulfides were square root transformed. A simple linear regression was run to test for effects of initial wet biomass on total harvested plant biomass. To address our first hypothesis at each site, differences in aboveground, belowground, total biomass, live‐to‐dead biomass ratio, and root‐to‐shoot ratio between species and among elevations were analyzed using a two‐way ANOVA, and all variables were square root transformed except for the live‐to‐dead biomass ratio, which was log‐transformed. We ran the same analysis with the same transformation to assess differences in the same biomass metrics of plants grown together. To address our second and third hypotheses, the natural log response ratio . Differences in lnRR among species at each elevation and site were analyzed using an ANOVA with planned comparisons.Values <0 indicate competition, whereas values >0 indicate facilitation. The lnRR was calculated for total biomass of both species within each organ row, and the treatment effects of elevation and site were analyzed using a one‐way 33.1P < 0.004 for all comparisons); the top two elevations did not differ in inundation time between sites (P > 0.90 for both comparisons). The depth of inundation was greater at the saltier site than at the fresher site by an average of 11 cm, and the tidal amplitude also was greater .Inundation duration increased significantly with decreasing elevation, and the effect differed by site . The bottom three elevations at the fresher site were inundated longer than at the saltier site . Both pore water sulfide concentrations and Eh varied over time, but there was no consistent or significant temporal trend or trend with elevation or species . Combining data across elevations, sulfide concentrations were higher at the saltier than at fresher site, but only marginally (P = 0.066).As expected, salinity stress was consistently higher at the saltier site than at the fresher site ; salinity increased over time, but only varied significantly among elevations in September . All 3.2S. americanus  or for S. acutus at the fresher site . There was a significant relationship for S. acutus at the saltier site; however, initial biomass explained very little variation in the final total biomass .There was no significant relationship between the initial biomass and total harvested biomass for 3.3S. americanus more than S. acutus at both sites  except that the top two and subsequent lower two elevations did not differ significantly (P > 0.2). At the fresher site, total biomass of S. acutus at the lowest elevation was significantly less than its biomass at all other elevations (P < 0.03); otherwise, the effect was negligible (P > 0.7). At the saltier site, S. acutus biomass did not differ across the top three or bottom three elevations (P > 0.4), but biomass was greater in the top two elevations than in the bottom two (P < 0.02). Similar effects of inundation were detected for both above‐ and belowground biomass, individually, and with average stem density and total stem length . The end of season live‐to‐dead biomass ratio was not different across elevations or between species at the fresher site; however, the ratio increased significantly with increasing elevation and was greater for S. acutus than for S. americanus . The root‐to‐shoot ratio was lower for S. americanus than for S. acutus at the fresher site but not at the saltier site and tended to be lower at the lower elevations compared to the highest two at both sites .When grown alone, inundation reduced biomass of S. americanus had a greater reduction in biomass than S. acutus with increased inundation at both sites . Total biomass of S. acutus did not differ significantly across elevations within either site when grown together (P > 0.2). Similar inundation effects were observed with above‐ and belowground biomass, individually, and with total stem length and average stem density . The end of season live‐to‐dead biomass ratio was greater for S. acutus than for S. americanus at both sites and tended to increase with increasing elevation at both sites, although the pattern was not strong . There were no differences detected for root‐to‐shoot ratio between species or across elevations at either site .When grown together, inundation effects were similar to those when grown alone; 3.4Schoenoplectus acutus was affected more negatively by the presence of its congener at the highest elevation at the fresher site compared to any other site/elevation combination  at the top two elevations at the saltier site, it never was outcompeted by S. americanus at any elevation . However, when it was removed from the analysis, a significant negative effect of competition was detected , and S. americanus performed worse than its congener , supporting our first hypothesis. The general trend was consistent between wetlands with different salinity regimes, but the magnitude of biomass reduction for S. americanus was greater at the saltier site. Regardless of the presence of its congener and salinity, the low marsh species, S. acutus, tolerated greater inundation, growing at elevations 80 cm lower than its current average marsh distribution. Biomass of the marsh plain species, S. americanus, was greatly reduced when grown with increased inundation and decreased even more when grown with S. acutus; surviving plants had only 7% of the biomass at the lowest elevation compared to the highest elevation where it had the highest biomass . The elevation range in the experimental marsh organs was greater than the observed distribution of either species at both sites, which supports the short‐term resilience of both species to predicted increases in SLR. However, longer duration experiments are needed to assess whether these species will survive increased inundation over multiple years.Our first objective was to document individual species’ responses to simulated SLR under field conditions. This experiment instantaneously increased inundation depths between 0.2 and 0.9 m relative to current average plant elevations, depths that are within the lower range of 2100 predictions of 0.4–1.8 m increases , even though the difference in channel water salinity was only 3‰. Although we cannot say this conclusively due to the absence of replicate sites within each salinity range, we likely can attribute the reduced biomass to increased salinity because other environmental factors largely did not differ significantly between sites (Table S2). The depth of inundation was greater at the saltier site (Table S1), but the inundation duration was greater at the fresher site , which we would argue has a stronger influence of soil biogeochemical processes and plant response ; our data support this hypothesis but only under the most benign conditions tested: the top elevation at the fresher site (Figures S. americanus was grown at inundation levels that were much greater than at its current marsh distribution; therefore, any competitive advantage observed within its normal tidal elevations could have been overwhelmed by inundation stress. Under the two highest inundation levels at the fresher site, S. americanus had a reduction in biomass with little observed effect of competitive or facilitative interactions (Figure We predicted that the marsh plain species,  Figures . Within s Figure . Greiners Figure  documentS. americanus was competitively inferior to the stress tolerator, S. acutus, across most inundation levels (Figure An unexpected result not predicted by the stress gradient hypothesis was that, at the saltier site, s Figure . Furthers Figure . A meta‐s Figure  on the ss Figure , was not4.3As suggested by our data, predicting climate change effects likely will not be as straightforward as offered by the stress gradient hypothesis. The direct effects are clear and followed what was predicted based on existing distributions/tolerances; however, the indirect effects of climate change on species interactions are more complicated and not predictable. In our case, we documented no facilitation by the stress tolerator under the greatest simulated SLR and demonstrated that its presence was more deleterious for the other species. Regardless of species interactions, we documented that the low marsh species could grow at elevations that were 80 cm lower than its current average elevation, which indicates its high inundation tolerance and potential to persist under conditions predicted by increased sea levels. Although species diversity may be reduced across the marsh as a whole, our results suggest that these wetlands in theory could remain vegetated in light of increased submergence.Multifactor manipulation experiments addressing plant response to accelerated climate change are uncommon and often are expensive to run, yet provide a more realistic, valuable, and needed assessment of how systems might respond (Dieleman et al., The implications of reduced biomass and lack of facilitative effects for marsh sustainability under increased SLR are significant. We observed a reduction in biomass for both species examined that was amplified with an increase in salinity of just 3‰. This reduction implies that there will be a decrease in the organic matter contribution to marsh accretion that could compound the loss of elevation and inundation stress within the marsh, especially for freshwater and brackish marshes that have organic rich soils Craft, . WetlandNone declared. Click here for additional data file.


The anterior iliac crest (AIC) and proximal tibia (PT) are common donor sites for autologous bone graft harvesting. We compared pain levels at these harvest sites on 1 day, 5 days, 2 weeks, 4 weeks, and 8 weeks post-harvest.We retrospectively reviewed 18 patients undergoing autologous bone grafting surgery at a level I trauma center between June 2013 and October 2014. Ten grafts were harvested from the AIC group and eight from the PT group. A standard visual analog scale (VAS) was used to rate pain at the harvest sites on postoperative day (POD) 1, 5, 14, 28, and 56 and at the recipient site on POD 1.p = 0.474), gender (p = 1.00), incidence of harvest site morbidity (p = 1.00), and average VAS at the recipient site on POD 1 (p = 0.471). VAS at the harvest site on POD 1, 5, and 14 confirmed statistically that pain was more severe in the AIC group than in the PT group (p < 0.001). However, no significant difference was observed on POD 28 and 56 between both groups. Pain was significantly less on POD 1 in the PT group at the harvest site than at the recipient site (p < 0.001).There were no statistically significant differences between both groups in age (The PT is a suitable harvest site, producing statistically less pain for at least two postoperative weeks than the AIC. Besides, patients report less postoperative pain at the PT harvest site than at the recipient site. Orthopedic surgeons use bone grafts to augment bone healing, perform arthrodesis, treat nonunions, lengthen bones, or fill defects. Autogenous bone is considered as the best origin for bone grafts, with inherent osteogenic, osteoconductive, and osteoinductive properties . The iliThe anterior iliac crest (AIC) and the PT are common sources of bone graft harvesting for surgeries on the ipsilateral lower extremity. Major complication rates at the donor site from 2.4 to 6.2% have been reported in studies of iliac crest graft, resulting in repeat surgery, readmission, prolonged hospital stay, or long-term disability –11. HoweTo the best of our knowledge, there are no comparable studies evaluating donor site morbidities among patients undergoing bone graft harvesting from the AIC or PT. Furthermore, results from reports evaluating harvesting techniques and methodologies for accessing outcomes are conflicting , 9, 10. We retrospectively reviewed 30 patients undergoing lower limb autologous bone graft surgery in a level I trauma center between June 2013 and October 2014. Indications in our study for lower limb autologous bone graft surgery included fracture malunion, nonunion, and bony defect after tumor excision. We included patients whose bone grafts were harvested from either the ipsilateral AIC or the lateral aspect of the ipsilateral PT and who underwent regular postoperative follow-up over 2 years. All surgeries were performed by a single orthopedic surgeon, and all patients were evaluated by an independent observer. Patient exclusion criteria were as follows: (1) age < 20 years, (2) missing pain severity data assessed using visual analogue scale (VAS) during postoperative follow-up, (3) history of bone graft surgery before June 2013, (4) cognition impairment, or (5) paresthesia of the lower limbs. The final study cohort comprised 18 patients. The age, gender, surgical indication, and complications of each patient were recorded entirely. This retrospective study was approved by the Institutional Review Board (IRB) of Kaohsiung General Veterans Hospital (IRB No.:VGHKS17-CT11-04).Grafts were harvested from the AIC in 10 patients (AIC group) and from the lateral aspect of the PT in eight patients (PT group).In the AIC group, bone was harvested from the iliac tubercle region approximately 5 cm posterior to the anterior superior iliac spine. A 5-cm skin incision parallel to the iliac crest was made in line from posterior to anterior superior iliac spine to avoid lateral femoral cutaneous nerve injury. Careful dissection with preservation of the tissue planes, elevation of the iliacus muscle, and harvest of inner table graft from the ilium using the curettage technique were performed subsequently to facilitate the closure , 14–16.In the PT group, an incision was made on the lateral aspect of the PT, just below Gerdy’s tubercle, as the entry point into the PT for harvesting Fig. . CarefulPostoperative treatment during hospitalization in both groups was standardized, including change of dressing with sterilized normal saline, cold packing, and oral analgesics. Analgesics were prescribed for 1 week after discharge. Outpatient follow-up was performed regularly for at least 2 years after the surgery.The VAS rating was completed by each patient. Patients were required to rate the pain at the harvest sites using a standard VAS on postoperative day 1 (POD 1), 5 days (POD 5), 2 weeks (POD 14), 4 weeks (POD 28), and 8 weeks (POD 56) and at the recipient site on POD 1. The VAS rating ranged from 0 (no pain) to 10 (worst pain ever).U test was used to compare continuous variables for abnormal distribution in this study. Fisher’s exact test was used to compare the categorical variables. The level of significance was set at p < 0.05. Values are represented as mean ± standard deviation.Statistical analysis was conducted using the Statistical Package for the Social Sciences version 22.0 . Data were analyzed using the Shapiro–Wilk test to determine the normality of the tested variables for the small case number of each group. The Mann–Whitney p = 0.474) and gender (p = 1.00). The mean ages for the AIC group and the PT group were 49.7 and 47.4 years, respectively.There was no significant difference in patient demographic and clinical characteristics between the two groups in age (p = 1.00). In the AIC group, only one case of harvest site fracture with hematoma was identified. In the PT group, no donor site morbidity was noted. In addition, there were no other donor site complications such as wound infections, deep hematoma formation, tibial plateau fracture, joint perforation, gait disturbance, and nerve injury.Moreover, there was no difference between the incidence of harvest site morbidity in the AIC and PT groups (p = 0.471).There was no significant difference in the average VAS rating of pain severity at the recipient site on POD 1 between the AIC and PT groups (p < 0.001)  Table .Table 1Dp = 0.598); however, in the PT group, the average VAS rating on POD 1 for the harvest site (1.5 ± 0.5) was significantly lesser than that at the recipient site (5.3 ± 0.9) (p < 0.001)  and harvest site 5.2 ± 1.0) ( ± 1.0 (pThe most common donor sites for harvesting include the iliac crest, greater trochanter, and PT. The choice of bone graft harvest site depends on the surgeons’ preference and the patient’s injured limb. However, different techniques have led to various complications and morbidity associated with the donor site in the past, necessitating the evaluation of the overall efficacy of the procedure. We must account for the quantity, quality, and morbidity of different harvest sites, especially regarding pain from the patient’s perspective.Mauffrey et al. conducted a prospective in vivo quantification study that indicated no significant difference in the volumetric amount of cancellous bone available for harvest between the PT site and the AIC site . Chiodo Although the iliac crest is the most common donor site for autogenous cancellous bone graft, it has associated donor site morbidity as high as 7% , 10, 21.Techniques for iliac cortex harvesting offer improvement in cosmetics and donor site pain . A retroBaumhauer et al. compared patient-reported outcomes of acute and persistent pain at 1 year after foot and ankle surgery, in which bone graft harvest site pain at 3 weeks showed more significant pain in the iliac crest than proximal tibia and calcaneus, respectively . In our The issue about the bone graft volume was ever evaluated by the syringe according to certain literatures. In our study, we harvested the bone graft as much as possible during surgery. Even the graft volume from proximal tibia was larger than the iliac crest, the proximal tibia donor site gave less complication than the iliac crest, according to the report of Salawu et al. , and theLimitations in our study included the retrospective study design, the limited sample size for each group, comparative analysis of the quantity of graft, the detailed consumption of analgesics, functional scale, clinical outcomes, and the bias of the patient’s subjective tolerance to pain. In addition, there was no evaluation of graft volume and quality or of the mechanical factors resulting from the implant choice for surgery that may influence the VAS rating at the recipient site and bone union.To summarize, the PT and AIC are optimal donor sites for autologous bone grafting. However, harvesting from the PT decreased pain at the donor site for at least two postoperative weeks. In addition, patients appeared to experience lesser pain at the PT donor site than at the recipient site on postoperative day 1.