WDR5-interaction (WIN) site, have been proposed to selectively kill MLL-rearranged malignancies via an epigenetic mechanism. We discovered potent WIN site inhibitors and found that they kill MLL cancer cells not through changes in histone methylation, but by displacing WDR5 from chromatin at protein synthesis genes, choking the translational capacity of these cells, and inducing death via a nucleolar stress response. The mechanism of action of WIN site inhibitors reveals new aspects of WDR5 function and forecasts broad therapeutic utility as anti-cancer agents.WDR5 is a component of multiple epigenetic regulatory complexes, including the mixed lineage leukemia (MLL)/SET complexes that deposit histone H3 lysine 4 methylation. Inhibitors of an arginine-binding cavity in WDR5, known as the  Comment on: Aho ER, Wang J, Gogliotti RD, et al. Displacement of WDR5 from chromatin by a WIN site inhibitor with picomolar affinity. Cell Rep. 2019;26:2916.e13-2928.e13. doi:10.1016/j.celrep.2019.02.047. PubMed PMID: 30865883. PubMed Central PMCID: PMC6448596. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448596/1 The idea that epigenetic proteins are druggable targets has rapidly moved from concept to reality, and it is not unreasonable to suggest that we are approaching a time where cancer epigenomes can be selectively reprogrammed to reverse the tumorigenic state.Growing awareness of the importance of epigenetic processes in cancer has fueled interest in the idea that targeting the proteins that read, write, and erase epigenetic marks could have therapeutic benefit against a range of malignancies. Two classes of epigenetic drugs have already been approved by the US Food and Drug Administration (FDA) for cancer therapy, more have progressed to late-stage clinical trials, and even more are in preclinical development.2 For epigenetic processes that are less well understood than DNA methylation, the degree of difficulty in anticipating exactly what a specific inhibitor will do, or what it can be used to treat, is much higher. If, however, a good target is picked and good inhibitors become available, the process of drug discovery itself can hone understanding of epigenetic players and vulnerabilities considerably. Case in point: WDR5.Given the battle that any new drug faces making in it to the clinic, reaching an era of true epigenomic cancer therapy will require a steady stream of new targets, coupled with creative approaches toward their pharmacological inhibition. The last 20 years has seen an explosion in our knowledge of epigenetic players and their mechanisms, and there is no shortage of targets that, on face value, could serve as effective focal points for drug discovery programs. However, this does not mean that epigenetic therapies are as simple as picking targets, finding ways to drug them, and then watch as predictable changes in the malignant epigenome unfold. Even for FDA-approved drugs like DNA methylation inhibitors, for example, it is hard to find compelling evidence that clinical efficacy is directly tied to alterations in DNA methylation status.WDR5-interaction (WIN) site of WDR5.3 These inhibitors were discovered with the intent of driving changes in histone H3 methylation that would cause certain leukemia cells to differentiate or die. As that project unfolded, however, and potent chemical probes against the WIN site became available, we learned that their mechanism of action was very different from what we expected. Here, we describe how WIN site inhibitors act in leukemia cells, reflect on what this tells us about the functions of WDR5, and speculate on what the utility of this new class of inhibitors may be.We recently described a set of potent small molecule inhibitors against the 4 Its best-studied role is within the mixed lineage leukemia (MLL)/SET histone methyltransferases (HMT), multi-subunit enzymes that lay down histone H3 lysine 4 (H3 K4) di- and tri-methylation (me2 and me3) at transcriptionally active sites in the genome. The MLL/SET complexes contain a common module made up of WDR5, RBBP5, ASH2 L, and DPY30, usually referred to as “WRAD,” that assembles with one of six catalytic MLL/SET proteins . All six MLL/SET family members depend on association with WRAD for full enzymatic activity, and all six carry a conserved WIN motif consensus (“ARA”) that engages a deep arginine-binding pocket on WDR5 called the WIN site.5 Interestingly, although WIN site binding is a common feature of MLL/SET proteins, MLL1 preferentially depends on this interaction for its enzymatic activity.7 It is this preference, together with the potential druggability of the WIN cavity, that first gave rise to the notion that WIN site inhibitors could be discovered that would block MLL1-mediated HMT activity.WDR5 is a highly conserved WD40-repeat containing protein that has multiple functions in the nucleus, including scaffolding the assembly of chromatin regulatory complexes linked to gene activation.MLL1 locus to any one of ~70 different partner genes.8 For common translocations, this fusion creates a hybrid transcription factor that drives malignancy, in part, by reactivating expression of developmentally important genes such as the HOX loci. Importantly, although hybrid MLL1 proteins lose HMT activity, the untranslocated MLL1 allele is usually retained in MLL patients, leading to the idea that wild-type MLL1 is essential for tumorigenicity in this context. Logically, if MLL1 is required for leukemogenesis in MLL-fusion cancers, and if its function depends on binding the WIN site of WDR5, then WIN site blockade could be used to specifically inhibit MLL cell viability.Early on, the obvious center stage for WIN site inhibitors was MLL, so-called because leukemic blasts from these patients express cell surface markers associated with both the lymphoblastic and myeloid lineages. MLL is predominantly a pediatric malignancy, and children diagnosed with MLL have dismal outcomes, creating an urgent need for new therapeutic options. MLL cancers are typified by translocations involving chromosome 11q23, which result in fusion of the 9 became the first to reduce this idea to practice when they described MM-401, a cell-permeable peptidomimetic that binds the WDR5 WIN site with high affinity (Kd ~1 nM). As a tool compound, MM-401 comports with all expectations. It selectively inhibits MLL1-mediated HMT activity in vitro. It preferentially blocks proliferation of MLL-rearranged leukemia cells in culture. And it induces the expected decreases in H3K4 methylation at the HOX genes—decreases that ultimately starve MLL-fusion cells of the vital HOX gene products they need to maintain the malignant and stem-like state. The ability of MM-401 to selectively block MLL cancer cell survival, and its compliance with all of the anticipated mechanisms of action, forecast a promising future for drug-like WIN site inhibitors in the treatment of MLL-fusion cancers.In 2014, Cao et al10 Two of these molecules, C3 (Kd ~1 nM) and C6 (Kd ~100 pM) were used as chemical probes to explore the biology of WDR5 and the therapeutic potential of WIN site blockade. In in vitro HMT assays, C3 and C6 are potent inhibitors of MLL1-mediated HMT activity (the IC50 for C6 in this case is ~20 nM), and are at least 250 times more effective at inhibiting MLL1 than any of the other MLL/SET family affiliates. Moreover, when paneled against more than a dozen human and mouse cell lines, both compounds show preferential inhibition of leukemia cells bearing the most common MLL-fusions, MLL–AF4 and MLL–AF9. The biochemical and cell-based selectively of these WIN site inhibitors is thus entirely as predicted. The mechanism of action of these compounds in MLL-fusion cells, on the other hand, is not.We used fragment-based screening and structure-based design to discover small molecules that bind tightly and selectively to the WIN site of WDR5.MLL1 is not required for survival of MLL-fusion leukemia cells.11 A second distinction was that we cast aside the assumption that epigenetic inhibitors necessarily act over a long time frame, and can require multiple rounds of cell division to erase or dilute relevant marks on DNA or histones.12 Performing mechanism of action studies days after compound treatment, as done for MM-401, makes it impossible to tease apart cause from effect, and is problematic for modifications like H3K4 methylation, which can change as a result of changes in transcription, direct or indirect. Instead, we measured changes in chromatin-centric processes within minutes and hours of inhibitor treatment, allowing us to monitor primary responses and watch as these unfold to induce cell death.It is worth emphasizing that our approach to mechanism of action studies was different to that taken with MM-401. One important distinction was our assumption that targets for WIN site inhibitors would be genes that are bound by WDR5, and not necessarily those we expect to be regulated by vestigial MLL1 complexes. This distinction turned out to be important, as work from Patricia Ernst’s group subsequently demonstrated that, contrary to expectation, wild-type HOX genes. Instead, we found that WDR5 associates with a small (~150) but polarized group of genes connected to protein synthesis. This cohort of protein synthesis genes (PSGs) includes about half of the ribosomal protein genes (RPGs), as well as those encoding nucleolar RNAs and translation initiation factors. We saw that WIN site inhibitors rapidly and comprehensively displace WDR5 from chromatin, resulting in changes in the transcription of a subset of WDR5-bound PSGs that can be detected as early as 15 minutes after inhibitor treatment—changes that are not associated with a decrease in histone H3K4 methylation. Consistent with diminished PSG expression, MV4:11 cells display decreased translational capacity within a day of C6 treatment, and this is associated with activation of a nucleolar stress response; a failsafe mechanism that induces p53-dependent cell killing when the supply of ribosome subunits cannot meet the cellular demand. Accordingly, WIN site inhibitors activate p53 in MLL-fusion cells, and the response of these cells to C3 and C6 is stimulated by presence of an intact p53 pathway. Thus, despite our best expectations and the empirical sensitivity of MLL-fusion cells to WIN site blockade, the action of these inhibitors occurs through rapid and persistent decreases in PSG transcription , and were surprised to see that it is not bound to any of the classic developmental loci, including cription , and see13 Within PSGs, the majority of these peaks occur just downstream of the transcription start site, and they are highly conserved in disparate cell types,3 suggesting that both the binding of WDR5 to these genes, and its specific placement within them, is a precise and stably-determined event. Given the pattern of binding, it is entirely possible that placement of WDR5 on chromatin is driven by a sequence-specific DNA binding protein. None of the dozen or so known WIN site binders4 have been reported to recruit WDR5 to chromatin, and none of them share this explicit pattern of chromatin binding. If there is a WIN-carrying protein that recruits WDR5 to chromatin, however, it is awaiting discovery, and it is going to have to be discovered the hard way, as there are many thousands of proteins encoded in the human genome that carry an ARA (or similar) sequence. Future experiments to define the full repertoire of WIN site binders, coupled with a detailed exploration of the role of DNA elements in positioning WDR5 at PSGs, are needed to identify the missing piece of this puzzle.Before elaborating on the clinical implications of our work, it is worth taking some time to discuss what our study tells us about WDR5 itself. The most obvious new information we learned is that stable association of WDR5 with chromatin is mediated by the WIN site. There are many ways this could occur, the simplest of which would be if WDR5 binds to a WIN motif in a chromatin-resident protein . WDR5 ha4—and its frequent overexpression in cancer—the ability of a protein like WDR5 to couple epigenetic regulation to enhanced biomass accumulation could have real functional significance, allowing new patterns of gene activity to be met, in lock-step, with commensurate changes in protein synthesis capacity. However, delving deeper into the connection, this simple idea starts to get more complicated. Of the 80 RPGs, only ~40 are WDR5 targets.3 If the overt biological role of WDR5 is to enhance protein synthesis capacity by coordinating ribosome protein gene expression, it is unlikely to do so by stimulating production of half of the ribosomal proteins. So what does the biased control of a subset of RPGs by WDR5 mean?Another conspicuous finding from this work is the connection between WDR5 and PSGs, which appears to be a conserved aspect of the chromatin-binding profile of WDR5. Given its importance in development13 MYC promotes transcription of almost all ribosome subunits, as well as many ribosome biogenesis factors.14 As MYC levels rise in a nascent cancer cell, the avidity provided by WDR5 could cause a preferential activation of WDR5-bound RPGs, resulting in an excess of these subunits. Alternatively, if binding of WDR5 to these RPGs is a signal-responsive event, and that signal is not received at the time that MYC is induced, this could result in selective activation of the non-WDR5-bound RPGs. In either case, the net effect would be an imbalance in the stoichiometry of ribosome protein subunits entering the nucleolus and induction of p53 via the nucleolar stress response. Given the high rate of flux through the ribosome assembly pathway, such a process could serve as a sensitive counting mechanism for MYC protein levels, and an efficient nuclear trigger to activate p53 at the first signs of aberrant MYC expression.We suggest that the significance of this phenomenon lies not in coordination of ribosomal protein expression, but in the imbalance of ribosome protein subunits it could promote in response to oncogenic threat. We have previously reported that the oncoprotein transcription factor MYC is recruited to chromatin through its interaction with WDR5 (which is mediated via a surface of WDR5 distinct from the WIN site).9 and every indication is that WIN site inhibitors could be developed into effective treatments for MLL cancers. But the precise molecular underpinnings of this sensitivity remain unknown. The mechanism of action we describe for WIN site inhibitors is distinct from all expectations, and there is no direct point of intersection between MLL-fusion oncoproteins and transcriptional processes controlled by WDR5. On one hand, this is an unsatisfying realization, given the motivation behind discovery of these inhibitors. On the other hand, disconnecting the mechanism of action of WIN site inhibitors from MLL-fusion oncoprotein opens a world of potential clinical use for WIN site inhibitors that was not previously imaginable.A strong empirical case can be made that MLL-rearranged leukemia cells are sensitive to WIN site blockade,15 WIN site inhibitors, which ultimately restrict the supply or alter the balance of ribosome protein subunits, can be thought of as a type of nucleolar-targeted therapy, and should be expected to have similar or at least overlapping anti-tumor activities. Importantly, however, because WIN site inhibitors act through a fundamentally different mechanism than other nucleolar-targeted drugs, we also expect them to have different properties that could be clinically beneficial, including different on-target toxicities, different mechanisms of resistance, and different patterns of drug synergy. Having a new way to choke translation in malignant cells is thus an important addition to this arsenal of anti-cancer agents.The most general prospective utility for WIN site inhibitors relates to their ability to inhibit PSG expression, as enhanced protein synthesis is a prominent recurring feature of cancer. As far back as the 19th century, it was reported that nucleoli are often engorged in cancer cells, which we now recognize to be a result of ribosome biogenesis hyperactivity. We now also know that oncoproteins such as MYC and RAS—and MLL-fusions—drive accelerated biomass accumulation as part of their oncogenic programs, and we appreciate that aneuploidy, together with relentless cell division, create an enhanced demand for protein synthesis in malignant cells that likely pushes proteostatic checkpoints to the brink of collapse. As a result, there is excitement in developing new drugs that impair protein synthesis, and good reason to believe that nucleolar-targeted therapies will be powerful anti-cancer approaches.3 Half of all cancers retain wild-type p53, and having a new way to reactivate or induce p53 in cancer cells can be viewed as a therapeutic positive. Of course, half of all cancers also lack wild-type p53, raising the question of whether WIN site blockade will be ineffective in this context. Here, thinking about WIN site inhibitors as nucleolar targeted therapies is again useful. The overwhelming precedent from other therapeutic approaches targeting protein synthesis is that cancer cells have both p53-dependent and -independent responses to nucleolar stress, and exactly how they respond to nucleolar stress is determined by the specific pathways that remain intact in the cell. Clearly, we have a lot to learn about the mechanisms through which decreased PSG expression can trigger cell death in different contexts, but the beaten path laid out by other nucleolar-targeted therapies gives confidence that WIN site inhibition can be applied to treat cancers with wild-type, mutant, or even missing p53.The mechanism of cell killing we report with WIN site inhibitors in MLL-fusion leukemia cells is largely p53 dependent.13 and it’s not a stretch to predict that if WIN site inhibitors displace WDR5 from target genes, MYC will be displaced in the process. Although our recent work demonstrated that the number of target genes controlled by WDR5 in MV4:11 cells is rather small,3 their biological clustering—together with the importance of enhanced protein synthesis as part of the core tumorigenic program of MYC—forecast that WIN site inhibitors should be able to effectively block this crucial aspect of MYC function. One third of all cancer deaths are due to inappropriate MYC expression or activity, and if WIN site inhibitors can be applied in this context, their impact could be profound.In addition, we posit that WIN site inhibitors will also be effective against MYC driven tumors. WDR5 recruits MYC to chromatin,4 If cancers exist that uniquely depend on the histone marks laid down by MLL1- or NSL-containing complexes, this decidedly non-epigenetic tale of WIN site inhibition may take a profoundly epigenetic turn.Finally, it is worth emphasizing that, despite everything we learned, and all the surprises along the way, WIN site compounds are potent and selective inhibitors of MLL1-mediated HMT activity. They may also inhibit other enzymatic complexes that involve WDR5, such as the NSL histone acetyl-transferase.


Iliosacral screw fixation is a popular method for the management of posterior pelvic ring fractures or dislocations, providing adequate biomechanical stability. Our aim in this study was to describe the use of a new patient-specific external template to guide the insertion of iliosacral screws and to evaluate the efficacy and safety of this technique compared with the conventional fluoroscopy-guided technique.This was a retrospective study of patients with incomplete or complete posterior pelvic ring disruptions who required iliosacral screw fixation. For analysis, patients were divided into two groups: the external template group (37 screws in 22 patients) and the conventional group (28 screws in 18 patients). The operative time per screw, radiation exposure time and the rate of screw perforation (accuracy) were compared between groups. In the external template group, the difference between the actual and planned iliosacral screw position was also compared.2, with 4 cases of screw perforation. In the external template group, the average operative time per screw was 17.9 ± 4.7 min, with an average radiation exposure dose of 742.8 ± 230.6 cGy/cm2 and 1 case of screw perforation. In the template group, the mean deviation distance between the actual and planned screw position was 2.75 ± 1.0 mm at the tip, 1.83 ± 0.67 mm in the nerve root tunnel zone and 1.52 ± 0.48 mm at the entry point, with a mean deviation angle of 1.73 ± 0.80°.In the conventional group, the average operative time per screw was 39.7 ± 10.6 min, with an average radiation exposure dose of 1904.0 ± 844.5 cGy/cmThe external template provides an accurate and safe navigation tool for percutaneous iliosacral screw insertion that could decrease the operative time and radiation exposure. Iliosacral (IS) screw fixation is a popular method for the management of posterior pelvic ring fractures or dislocations, providing adequate biomechanical stability . The claThe use of computer-assisted navigation systems has been advocated to lower the risk of screw malposition, and of associated neurovascular injury, as well as to decrease exposure to radiation during IS screw insertion –7. AlthoIndividualized internal templates, based on three-dimensional (3D) image reconstruction and reverse engineering, provide an alternative to computer-guidance for the placement of a K-wire to guide the accurate insertion of the IS screw Fig. . AlthougEmergent external fixation is widely used for the primary management of pelvic fractures. The external fixator pins, which are inserted deep into the iliac crest, could provide a simple geometric surface and solid cornerstone for mounting an external template. Therefore, we developed a low-profile 3-dimensional (3D) printed external template that can be firmly attached to the external fixator pins, overcoming the abovementioned disadvantages of internal templates, to guide IS screw insertion Fig. . The aimThe study group consisted of 40 patients who were treated at Wuhan Union Hospital, between January 2016 and September 2017, for traumatic incomplete or complete disruptions of the posterior pelvic ring  using IS screws . The radIS screw placement, using an external template, was performed in 22 patients (template group), including 11 men and 11 women, 51.7 ± 15.2 years of age . All 22 of these patients had undergone external fixation of the pelvic fracture in the emergency department at the time of admission. The conventional fluoroscopy-guided technique of IS screw insertion was used in 18 patients , including 10 men and 8 women, 50.1 ± 13.7 years of age . The distribution of the cause and classification of injuries for both groups is summarized in Table Two partially threaded Schanz 5 mm pins (inserted into the iliac crest for emergent fixation at the time of admission) were used as marker pins, as well as provided a stable mounting base for the external template. Consistent with the conventional technique of external fixation of the pelvis, the tips of the two pins were directed in convergent fashion toward the thick bony area above the acetabulum to obtain a firm insertion. Of note, a supra-acetabular pin, which is more commonly used for external fixation of the pelvis than iliac crest screws, was inappropriate for our purpose as it provides inadequate rotational stability to the mounted external template. Moreover, the supra-acetabular pin occupies the supra-acetabular corridor, which is needed to mount one of the pins of the pelvic reduction frame , 13 duriThereafter, all patients in the template group underwent computed tomography (CT) imaging of the pelvis for both trauma assessment and template planning, with additional CT imaging avoided to minimize patients’ radiation exposure. CT data were saved in the Digital Imaging and Communications in Medicine (DICOM) format and used subsequently for design of the template.The Schanz pins inserted as part of the external fixation of the pelvis were retained in situ until definitive surgery.trim and curve software, to connect the marker pins and the IS screws. The template design was also exported into the STL format for use with the 3-matic software  to complete the virtual template through Boolean calculation  were imported into Mimics 10.01  software for 3D reconstruction. In the 3D model of the pelvis, the trajectory was planned for placement of a virtual screw (7.0 mm diameter) into the S1 or S2 vertebra along the midline of the osseous corridor, with absence of penetration of the cortex confirmed on CT imaging along the three anatomical planes . The placement of another virtual screw into either the injured hemipelvis or contralateral hemipelvis, depending on the type of posterior pelvic ring injury, was also planned. The 3D model  was then exported into an STL format for use with the Geomagic Studio image-processing software . A virtual template, using the skin model as a substrate, was then designed using the ion Fig. . The datFor patients who did not require fracture reduction, the template was designed to mount on the pins inserted in the iliac crest of the injured hemipelvis, as per the usual method Fig. . For patIn our clinical setting, the cost for the design and printing of the template is approximately 250 dollars, requiring about 2 h for the design and more than 10 h for the printing. We cooperate with our affiliated 3D digital orthopaedic center, with a delay between CT imaging and definite surgery of 48–72 h, which takes into account the sterilization process and transportation. This cooperation obviates the need for the hospital to purchase this equipment.Patients were placed in the supine position on a radiolucent operation table, and surgery was performed under general anesthesia. A fluoroscopic C-arm was placed on the side opposite to the surgeon. The external pelvic fixation frame was removed, retaining the two marker pins to mount the template. The sacral fracture or dislocation was reduced using the pelvic reduction frame described by Lefaivre et al.  Fig. 2b2b. Then,The same procedure was followed for the conventional group, up to the insertion of the K-wire. Specifically, once the sacral fracture or dislocation was reduced, a K-wire was inserted under C-arm fluoroscopy guidance, using lateral, inlet and outlet views. Adjustments in the position of the K-wire under fluoroscopy were made until the correct position was confirmed. A 6.5-mm cannulated screw was then inserted, following the K-wire.t-test, the chi-squared test, and Fisher’s exact test, as appropriate for the data type and distribution. All analyses were performed using SPSS .Postoperative pelvic radiographs and CT images were reviewed. Two observers independently evaluated the position of the screw from postoperative CT images, using the following grading criteria, as previously described : grade 0p < 0.001). The radiation exposure dose was lower for the template (742.8 ± 230.6 cGy/cm2) than conventional (1904.0 ± 844.5 cGy/cm2) group . No incidence of neurovascular injury was identified among cases of screw perforation  in the template group (22 patients) and 28 screws  in the conventional group (18 patients). The quality of the reduction was not significantly different between the two groups , 1.8 ± 0.6 mm (nerve root zone) and 1.5 ± 0.5 mm (entrance point), with a mean deviation angle of 1.8 ± 0.8°.Use of our novel external template to guide K-wire insertion, and subsequent IS screw placement, provided high accuracy, with a shorter operative time and lower radiation exposure than the conventional fluoroscopy-guided technique. Moreover, despite the conventional use of inlet, outlet and lateral fluoroscopy views to guide percutaneous IS insertion, violation of the cortex remains an inherent problem of IS screw implantation –20, with2), for both patients and the medical staff, compared to the conventional technique (at 1904.0 ± 844.5 cGy/cm2).Radiation exposure is a significant concern for percutaneous IS screw insertion. Excessive amounts of radiation can cause muscle weakness, hair loss, cataracts, and even cancer . ComputeThe accuracy of IS screw insertion has traditionally been evaluated using the penetration grades . In our p = 0.049). This longer delay for the template group, however, did not negatively influence the quality of the fracture reduction, which was comparable between the two groups. Of note, all definitive surgeries of both groups were performed within 14 days of the trauma, and the use of the pelvic reduction frame for closed reduction [The delay in definitive surgery because of the time required for the design and printing of the template is a limitation of our technique. The mean time from injury to surgery for the template group in our study was 5.4 days, compared to 4.1 days for the conventional group (eduction  partly oPin site infection, which is a common complication of external fixation , 25, is In summary, the external template is an effective tool for percutaneous IS screw insertion, decreasing radiation exposure for both surgeon and patients and avoiding neurovascular injury caused by screw malposition. Considering the high accuracy in IS screw placement that we achieved using an external template, we propose that this technique could potentially be used for other percutaneous insertions of screws in pelvic or acetabular surgery Fig. . Further


Thirty L4 Sawbones were designed to represent intact pedicles, semi-pedicles, and nonpedicles and were prepared for screw insertion. Three sizes of polyaxial screws  were independently and randomly distributed into the intact-pedicle group , the semi-pedicle group , or the nonpedicle group . The experiments were conducted across a minimum of five trials for each of the chosen screw sizes. We then analyzed the results of the imaging, pullout testing, and embedded bone volume. Any fractures or defects of the vertebrae caused by screw insertion were excluded from the study. The appropriate screw trajectory and insertional depth were confirmed using axial and sagittal X-ray imaging prior to screw pullout testing. A pullout strength of only 41% to 45% for the SP group and 29% to 39% for the NP group was retained following a broken pedicle. The use of longer or larger-diameter screws appears to be an effective salvaging procedure for the NP group (p < 0.05). The embedded bone volume percentage analysis indicated that, compared to the IP group, 68% to 76% of effective bone embedded into the screw threads in the SP group, and 58% to 65% in the NP group. There was no direct correlation between the pullout strength and the embedded bone volume; however, less effective embedded bone volume was associated with lower pullout strength. This study describes the evolution of the well-established screw pullout test being applied to the broken pedicle Sawbone testing model. The pedicle plays an important role in determining the pullout strength of a screw. However, a salvage procedure that utilizes a longer or larger-diameter screw might be a reliable clinical approach to address broken pedicles.Pedicles are often broken when screws are inserted into hard pedicles with small diameters or when the diameter of the screw itself is inadequate. However, there is a lack of biomechanical literature that addresses screw loosening as a result of broken pedicles or the resulting salvage of those screws. We performed a novel  The use of a pedicle screw as a spinal implant was first described by Boucher  in the 1Thirty L4 Sawbones  that were specifically designed to be radio-opaque under fluoroscopy were prepared for screw insertion. The Sawbones have an inner cancellous core and an outer cortex layer, which simulate human spine vertebrae with normal bone properties and morphometry. The specimens were designed to represent either an intact pedicle, a semi-pedicle, or a nonpedicle, illustrated in Axial and sagittal views were examined via X-ray for all specimens prior to the pullout test to confirm an appropriate screw trajectory and insertion depth. The vertebrae were also examined thoroughly to rule out any fractures or defects caused by screw insertion.Each of the 30 Sawbones was potted in metal boxes using specific epoxy resins . Judicious potting was performed to ensure that the cement did not come into contact with any portion of the pedicle screw. The prepared specimens were mounted onto a material testing machine  to conduct axial pullout tests with the screws . The polThe bone volume embedded into the screw thread was quantified by analyzing the radiographic image of the inserted screw with image analysis software , and is defined as follows:ectively . To invep < 0.05.The measurements for the semi-pedicle and nonpedicle groups of each screw size were collected in five trials. Moreover, measurements for the intact-pedicle groups of each screw size were collected in ten trials, and expressed as the mean ± standard deviation (SD). Statistical software  was used to analyze the pull out strength and embedded bone volume of all specimens. All of the measurements were collected for 30 vertebrae and expressed as the mean ± standard deviation (SD). An ANOVA test with post hoc analyses was performed to evaluate the differences between groups. Differences were considered to be significant at An appropriate screw trajectory and insertional depth was confirmed using axial and sagittal X-ray imaging prior to pullout testing Figs  and 7. IThe 6.0 mm × 45 mm, 6.0 mm × 50 mm, and 6.5 mm × 45 mm screws had mean pullout strength values of 1054.88 ± 231.9 N, 1071.25 ± 267.19 N, and 1019.52 ± 198.68 N, respectively, in the IP group; 436.2 ± 57.7 N, 469.69 ± 52.76 N, and 457.98 ± 110.61 N, respectively, in the SP group; and 314.89 ± 42.82 N, 424.76 ± 43.90 N, and 405.63 ± 52.23 N, respectively, in the NP group  (p < 0.05). Higher pullout strengths were also measured in the semi-pedicle groups than in the nonpedicle groups for all sizes (p < 0.05). The mean maximum pullout force of the specimens treated with longer (6.0 mm × 50 mm) or larger-diameter (6.5 mm × 45 mm) screws presented as percentages of the standard (6.0 mm × 45 mm) screw is illustrated in The pullout strengths of the various sized screws were not significantly different from each other in the intact pedicle and semi-pedicle groups. In contrast, the pullout strengths of the longer and larger-diameter screws were significantly higher in the nonpedicle group ( < 0.05) . Signifi3, 1008.62 ± 46.6 mm3, and 925.34 ± 50.04 mm3, respectively, in the IP group; 605.71 ± 30.53 mm3, 801.88 ± 40.62 mm3, and 662.59 ± 40.51 mm3, respectively, in the SP group; and 496.71 ± 22.94 mm3, 662.06 ± 30.58 mm3 and 529.12 ± 38.01 mm3, respectively, in NP group  or partial (SP group) pedicle removal was only 29%-39% and 41%-45% of the intact pedicle, respectively , and eveThis study presents an evolution of the well-established screw pullout test using the broken pedicle Sawbone testing model. The pedicle plays an important role in contributing to screw pullout strength. However, once the pedicle is broken, a change to a longer or larger-diameter screw might be necessary. Our image quantification data of embedded bone volume were consistent with the biomechanical testing results, which indicated that relative pullout strength might be converted from the embedded bone volume observed in a standard homogeneous spinal bone surrogate.S1 Files(PDF)Click here for additional data file.


Pedicle screws are commonly used for posterior stabilization of the spine. When used in deformed or degenerated segments, the pedicle screws are often not fully inserted into the bone, but instead the threaded portion is exposed by 1 or 2 threads to accommodate rod placement and ensure alignment between the tulip of the screw and the rod. However, broken pedicle screws have been reported with the use of this method. The aim of this study was to determine how the fatigue life of the screw is affected by not fully inserting the screw into the bone. Spinal constructs were evaluated in accordance with ASTM F1717. The following three screw positions were subjected to compression bending fatigue loading; (i) pedicle screw fully inserted in the test block with no threads exposed (EXP-T0), (ii) pedicle screw inserted with one thread exposed outside the test block (EXP-T1), (iii) pedicle screw inserted with two threads exposed outside the test block (EXP-T2). Corresponding finite element models FEM-T0, FEM-T1 and FEM-T2 were also constructed and subjected to the same axial loading as the experimental groups to analyze the stress distribution in the pedicle screws and rods. The results showed that under a 190 N axial load, the EXP-T0 group survived the full 5 million cycles, the EXP-T1 group failed at 3.7 million cycles on average and the EXP-T2 groups failed at 1.0 million cycles on average, while the fatigue strength of both the EXP-T1 and EXP-T2 groups was 170 N. The constructs failed through fracture of the pedicle screw. In comparison to the FEM-T0 model, the maximum von Mises stress on the pedicle screw in the FEM-T1 and FEM-T2 models increased by 39% and 58%, respectively. In conclusion, this study demonstrated a drastic decrease in the fatigue life of pedicle screws when they are not full inserted into the plastic block. Traditional rigid pedicle screw-rod systems are widely used in the treatment of spinal diseases as they offer immobilization, stabilization and mechanical support to the adjacent vertebrae as an adjunct to spinal fusion procedures. On average, the lumbar region moves through approximately 3 million cycles per year ,2, and fImproper device design is the main factor to cause the fatigue failure. However, under the same material properties and device design, fatigue life of pedicle screws is more likely to decrease due to improper selection, orientation or position of the screw ,10. WhenIn this study, a commercial spinal implant system was subjected to cyclic loading in accordance with ASTM F1717. The aim was to determine how the fatigue life of the pedicle screws was affected by varying the unsupported screw length.Fatigue testing was performed using the compression dynamic bending test and loading conditions detailed in ASTM F1717 . A DepuyThree finite element models  were created to replicate the experimental fatigue test setup detailed above ; (i) scrThe model was validated by demonstrating that the stiffness of entire model (31.95 N/mm) was within the range of experimental data (31.84~32.48 N/mm) as shown in The results of the dynamic compression bending test showed that the EXP-T0 group had a fatigue strength of 190 N, while both the EXP-T1 and EXP-T2 groups had a fatigue strength of 170 N . The impThe maximum von Mises stress on the screws appeared at the region where the screws entered the UHMWPE blocks . In the The goal of posterior spinal fixation is to reconstruct the compromised columns within a spinal motion segment to provide temporary stabilization until bone fusion is achieved. Failure of posterior spinal fixation typically occurs as a result of high-energy impact injuries or metal fatigue from repetitive spinal movements. The primary source of mechanical stress on the fixation screws is from daily physical activities , and theThe fatigue strength of both the EXP-T1 and EXP-T2 constructs was found to be less than EXP-T0, but all constructs failed in the same manner, through breakage of the pedicle screw. These results were consistent with the FEM calculations. For all FEM models, the maximum von Mises stress on the screw was greater than on the rod, which is consistent with literature ,9,14–16.Under a 190 N cyclic load, the EXP-T2 constructs failed after approximately 1,000,000 cycles. However, screwing in by a further one thread  allowed the construct to survive up to 3,700,000 cycles, while fully inserting the screw threads (EXP-T0 group with an unsupported screw length of 3.6 mm) allowed the construct to survive the full 5,000,000 cycles. Under a 220 N cyclic load, the average fatigue life of each group decreased drastically in comparison to the groups loaded by 170 N or 190 N, with the EXP-T2 group suffering the greatest decrease. The length of the lever arm  of the bending moment acting on the screw is a critical factor influencing the fatigue life, with this study showing that the maximum von-Mises stress increased with each incremental increase in the length of the unsupported screw .The results of this study suggest that increasing the length of the unsupported screw is a potentially dangerous practice and should be avoided if possible. It can greatly reduce the fatigue strength and fatigue life of the screw. Generally, the non-thread part of screw is large enough for polyaxial head movement. Hence, to maintain the fatigue strength of a pedicle screw, it is recommended to fully insert the screw threads into the pedicle. ASTM F1717 does not specify a suitable length for unsupported screws for posterior spinal fixation and, as such, the reduction in fatigue strength may not be a widely recognized risk in clinical settings as well as, in the preclinical evaluation of standard constructs.There are some limitations to this study that should be noted. The vertebrectomy model used was constructed in accordance with ASTM F1717 and represents a ‘worst case’ condition where the anterior spine is missing and all the load applied on the posterior fixation construct. The complex loading conditions in the human spine have not been fully considered in ASTM F1717 and so the results of this study may not directly predict in vivo performance. ASTM F1717 is typically used to compare different component designs or surgical techniques in terms of the relative mechanical parameters ,13,26,27The fatigue life and strength of the screw were greatly reduced when the unsupported screw length was increased. In conditions where there is insufficient support for the anterior column, in order to avoid early fracture of the pedicle screw, it is recommended that pedicle screws are always fully inserted into the bone leaving no threads exposed. It is also recommended that manufacturers include appropriate warnings on labelling advising that the fatigue strength of the screws may be reduced if the unsupported length of the screw is increased.


Microcystis aeruginosa LTPNA08 (MG7XX). Porcine AMP inhibition—a model for human AMP—activity was spectrophotometrically measured by the formation of p-nitroanilide from L-leucine-p-nitroanilide substrate by AMP. AMP inhibition by MG770 exhibited comparable inhibition levels to amastatin , while MG756 was slightly less potent . Molecular modelling suggests a potential binding mode, based on the interaction with the Zn2+ cofactor, where MG770′s extra methyl group contributes to the disturbance of the Zn2+ cofactor complex and highlights the importance of hydrophobicity for the site.Aminopeptidase M (AMP) inhibition is of interest for several diseases, such as highly vascularized cancer types. AMP can be inhibited by linear pentapeptides isolated from  The aminopeptidase M  are extracellular anchored proteins, composed of a transmembrane domain which anchors it to the cell, and an ectodomain with approximately 930 amino acid residues, and heavily glycosylated . AMP is N-terminal domains (I–III) being evolutionary conserved -3-methylamino-2-hydroxy-decanoic-acid and HTy defines homo-tyrosine, the configuration of all other amino acids was based on previous reports ,36,37,38+ or Cl− adjusted to neutralize the overall system charge). The system was treated in a cubic box with periodic boundary conditions specifying the shape and the size of the box as 13 Å distance from the box edges to any atom of the protein. We used a time step of 1 fs, the short-range coulombic interactions were treated using a cut-off value of 9.0 Å using the short-range method, while the smooth particle mesh Ewald method (PME) handled long-range coulombic interactions [Molecular dynamics (MD) simulation was carried out using Desmond  with theractions .Initially, the relaxation of the system was performed using steepest descent and the limited-memory Broyden-Fletcher-Goldfarb-Shanno algorithms in a hybrid manner. The simulation was performed under the NPT ensemble for 5 ns implementing the Berendsen thermostat and barostat methods. A constant temperature of 310 K was maintained throughout the simulation using the Nose-Hoover thermostat algorithm and Martyna-Tobias-Klein Barostat algorithm to maintain 1 atm of pressure, respectively. After minimization and relaxation of the system, we proceeded with a single production step of 100 ns. The representative structure was selected by clustering the structures from the RSMD values, using 1 Å as a cut-off , small R2+ interaction site. Microginin inhibitory activity could also be attributed to additional with another hydrophobic portion pocket composed by Phe467 and Phe893.The results of the present study clearly indicate the aminopeptidase M inhibitory activity of microginins 756 and 770 on in vitro assays in the same range as the known inhibitor amastatin. Molecular modelling, by the means of docking and molecular dynamics analysis of the active site, suggests a binding mode where the extra methyl group of MG770 improves hydrophobicity near the Zn


Ki constants ranging from 9.4 μM to 6.4 mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional π–π interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.We investigated the influence of bronchodilating β2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with   At present, more than 56 mutations of the human BCHE gene have been identified16,The transfer of nervous impulses via acetylcholine (ACh) depends on a series of well-tuned receptors, enzymes that take part in the synthesis and hydrolysis of ACh and a series of transporters2,β-Adrenergic agonists are potent bronchodilators widely used in the management of bronchial asthma. These agents interact with β2-adrenergic receptors on the smooth muscle of bronchial tissue relieving bronchospasms and reducing airway resistance. Beside the bronchial tissue β-adrenergic agonists also act on other tissues where signals to other cells are regulated by acetylcholine, like smooth muscles of the vascular system, intestines and uterus. Recent studies have shown that oral intake of salbutamol and ephedrine appeared to be effective supplement treatments in severe cases of acetylcholine receptor (AChR)21R)-enantiomer of salbutamol, which is approximately 80 times more potent as a β-adrenoceptor agonist than (S)-enantiomer and the administration of pure (R)-salbutamol improved therapeutic activity and reduced side effectsin vivo results were obtained with enantiomers of terbutaline7,9,27,R)- over (S)-bambuterol7,27,Among the various chiral drugs, one enantiomer is often more active than another in treating a medical condition. An example is the -epinephrine, were purchased  in the form of salts and converted to hydrochloride salts . All of the chemicals, reagents and solvents for the preparation of resorcinol, catechol and saligenin derivatives were purchased from commercial sources and used without further purification. Acetylthiocholine iodide (ATCh), propionylthiocholine iodide (PTCh) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich. All of the inhibitors and substrates were dissolved in water, while DTNB was dissolved in 0.1 M sodium phosphate buffer (pH 7.4). Only salmeterol was dissolved in ethanol due to its low solubility, while all further dilutions were made in water.Racemic epinephrine, isoetharine, fenoterol and terbutaline, as well as . BChE variants were confirmed earlier as described previously30,Racemic salbutamol and salmeterol were synthesised starting from methyl 5-acetylsalicylate (Sigma-Aldrich) according to literature procedures described previously32R)-terbutaline were prepared from their racemic form using diastereomeric salt crystallization method32,R)-terbutaline and (R)-salbutamol precursor was (+)-O,O′-di-p-toluoyl-d-tartaric acid and (−)-O,O′-di-p-toluoyl-l-tartaric acid for a precursor for (S)-salbutamol. Enantiomeric excess (ee) for the obtained enantiomers was determined using a brush-type chiral stationary phase previously synthesised in our laboratory (CSP 1)S)-salbutamol, (R)-terbutaline and (R)-salbutamol, respectively.The enantiomers of salbutamol and -fenoterol, -fenoterol, (R)-salmeterol and (S)-salmeterol, respectively. The absolute configuration of enantiomers was determined by comparison of the elution order of these compounds on AD and OJ columns with literature data36,Supplementary).Enantiomers of fenoterol and salmeterol were resolved using semipreparative chiral chromatography. The columns used were CHIRALLICA PST-4  and CHIRALLICA PST-2  for fenoterol and salmeterol, respectively. The obtained ee were 98%, 96%, 97% and 86% for  by Ellman method at 412 nmK(I) was evaluated from the impact of substrate concentrations [S] on the degree of inhibition applying the Hunter–Downs equation17,Ki,app is an apparent enzyme-reversible inhibitor complex dissociation constant obtained at a given substrate concentration [S] in the presence of inhibitor concentration [I] and calculated from the enzyme activities v0 and vi measured without inhibitor and in the presence of inhibitor concentration [I], respectivelyK(I) is the enzyme-reversible inhibitor complex dissociation constant and can stand for a complex formed at the catalytic site or for a complex formed at the peripheral site of the enzyme. K(S) is the enzyme-substrate dissociation constant that could be approximated to the Michaelis constant or to the enzyme-substrate dissociation constant when two molecules of the substrate are bound to the enzyme.The dissociation constant for an enzyme–inhibitor complex ,The docking of the studied compounds was performed using the Accelrys DiscoveryStudio 2.1 CDOCKER docking protocol and CHARMm force field. For the structural model of AChE, we used a crystal structure deposited in the Protein Data Bank, PDB ID 1B419meta position in the resorcinol group, and in the ortho position in the catechol group. In the saligenin group, the hydroxyl group and the hydroxymethyl group were in the ortho position. Derivatives of each group differed in the size of the hydroxyaminoethyl chain attached to the benzene ring.The tested bronchodilating β2-agonists were divided into three groups according to the disposition of their hydroxyl groups on the benzene ring, resembling the structural feature of resorcinol, catechol and saligenin . The hydK(I)) ranging between 30 μM and 6.4 mM  constants of resorcinol, catechol and saligenin derivatives were very similar, indicating that the inhibition potency of the tested bronchodilating β2 agonists towards human AChE does not depend on the disposition of hydroxyl groups on the benzene ring. However, it seems that the inhibition potency of the tested β2-agonists depends on the size of the hydroxyaminoethyl chain attached to the benzene ring. For example, in the resorcinol group, metaproterenol and terbutaline differ only in the end of hydroxyaminoethyl chain  exhibiting very similar inhibition, while the enlargement of the hydroxyaminoethyl chain with the hydroxybenzyl group, as it is a fenoterol, caused a fivefold increase in inhibition potency. In the catechol group, an elongation of the hydroxyaminoethyl chain from methyl to isopropyl lead to a 2-fold increase in inhibition potency of isoproterenol compared to epinephrine. Additional substitution on the hydroxyaminoethyl chain with the ethyl group, as in isoetharine, caused a further 12-fold higher inhibition potency compared to isoproterenol. The largest, 67-fold, increase in inhibition potency was observed in the saligenin group between salbutamol and salmeterol, where the largest difference in the size of hydroxyaminoethyl chain was. Additionally, when compounds with identic hydroxyaminoethyl chain but different disposition of hydroxyl group on benzene ring (metaproterenol vs. isoproterenol) were compared, no difference in inhibition potency was observed. Similarly two times higher inhibition potency of salbutamol, compared to terbutaline, can be attributed to the substitution of one hydroxyl group on benzene ring with the hydroxymethyl group. From all of the tested bronchodilating β2-agonists, salmeterol was singled out as the most potent inhibitor with an inhibition potency similar to that of flavonoids quercetin and myricetinRanges of The inhibition potency correlated well with the docking results expressed here as the interaction energy of the bronchodilating β2-agonists − AChE complex . The higK(I) constants ranging between 9.8 μM and 3.8 mM  constants for AChE and BChE. Generally, all of the compounds were more potent inhibitors of BChE than of AChE. The most selective inhibitor was fenoterol, followed by salbutamol and terbutaline that inhibit usual BChE 36, 28 and 27 times more potently than AChE, respectively. It is worthy to mention that these β2-agonists are currently the most frequently used bronchodilators. Catechol derivatives were the least selective inhibitors, out of which epinephrine showed the lowest selectivity. Salmeterol, the most potent inhibitor of all of the tested cholinesterases, displayed only a two times higher potency towards BChE than toward AChE. In view of the BChE polymorphism, inhibition of fluoride-resistant BChE is similarly selective as that of usual BChE, while no selectivity in the inhibition of the atypical variant was determined.Due to a difference in residues lining the active site of AChE and BChE, these two enzymes have different reactivity toward substrates, inhibitors and ligands9K(I) constants determined for the (R) and (S) enantiomers of fenoterol, salbutamol and salmeterol (K(I) constants obtained for racemate with those for the (R) enantiomer (R)-terbutaline over its racemate, respectively. The lack of cholinesterase stereoselectivity was probably the consequence of interactions with residues that did not involve the chiral centre of the tested compounds. Indeed, the molecular modelling of complexes demonstrated that the stabilisation of the compounds into the active site of the enzymes was based on other interactions (The tested compounds are chiral molecules  and the lmeterol . For terantiomer . IrrespeDue to the important and versatile functions of cholinesterases in organisms, it is important to define the inhibition potency of compounds that reduce the level of their hydrolytic activity, especially for those in use as drugs. All of the tested β2-agonists are in use as bronchodilators, except isoetharine whose use was discontinued. Our results have shown that the compounds are reversible cholinesterase inhibitors that reduce human cholinesterase activity and therefore can affect their roles in an organism. Cholinesterases had the lowest affinity towards epinephrine, the hormone of adrenal glands and neurons that plays an important role in the organism during acute stress response. Interestingly, AChE also displayed low affinity towards terbutaline and salbutamol, compounds that are active components of the most commonly used bronchodilators. It should also be mentioned that the tested β2-agonists showed the highest inhibition potency towards usual BChE among the three variants of BChE. Moreover, our results show that the racemates of these compounds in terms of cholinesterase inhibition are safe for use since there was no stereoselectivity exhibited in the interaction with cholinesterases.


Streptomyces melanosporofaciens, which catalyzes the formation of cycloocta-9-en-7-ol, a precursor to the next-generation anti-inflammatory drug cyclooctatin. In this work, we present evidence for the significant role of the active site's residues in CotB2 on the reaction energetics using quantum mechanical calculations in an active site cluster model. The results revealed the significant effect of the active site residues on the relative electronic energy of the intermediates and transition state structures with respect to gas phase data. A detailed understanding of the role of the enzyme environment on the CotB2 reaction cascade can provide important information towards a biosynthetic strategy for cyclooctatin and the biomanufacturing of related terpene structures.Terpene cyclases are responsible for the initial cyclization cascade in the multistep synthesis of a large number of terpenes. CotB2 is a diterpene cyclase from  Enzymes catalyze numerous complex biochemical reactions in different cellular compartments –2. More The first crystal structure of a monoterpene cyclase  was repo14Diterpenes are generated from the universal aliphatic substrate geranyl geranyl pyrophosphate (GGPP) . In vitrS. melanosporofaciens, which catalyzes the formation of cyclooctat-9-en-7-ol, representing the first committed step in the biosynthesis of the next-generation anti-inflammatory drug cyclooctatin. The intracellular target of cyclooctatin is an as of yet uncharacterized lysophospholipase, which is involved in early steps of the inflammatory signaling cascade [CotB2 is a bacterial diterpene cyclase from  cascade –40. In tA. Intermediate A undergoes a 1,5-hydride shift, forming intermediate B. A subsequent cyclization forms intermediate C. Intermediate C generates intermediate E via one of two possible pathways: either a direct 1,3-hydride shift or an indirect pathway involving two 1,2-hydride shifts. Theoretical investigations by Hong and Tantillo suggested that the indirect transformation from intermediate C to E is energetically favored and might be biosynthetically relevant [G forms via a 1,5-hydride shift from C6 to C10 to generate a homoallylic cation, and the formation of intermediate H occurs due to cyclization to yield a cyclopropyl ring. Intermediate I forms due to isomeric formation of a cyclopropylcarbinyl cation, as shown by isotope labeling [H and I [Meguro and co-workers  establisrelevant . This firelevant , who perlabeling . QM calc[H and I –39. FinaAlthough gas phase calculations shed light on the reactivity of the isolated species and provided crucial mechanistic insights, the biorelevant mechanism cannot be fully understood without taking into account the enzyme–solvent environment. A common problem when studying these enzymes is the lack of high-resolution crystal structures that are biologically relevant, i.e., that have a ligand bound in a reactive configuration and have a fully closed active site. Recently, a crystal structure of the CotB2 enzyme that met these criteria was published . In the The energy profiles for both gas phase (orange) and for the active site model (blue) reactions were characterized by a sequential decreasing pattern . The insA was stabilized through π–cation interaction with W186, while B was stabilized due to dipole–cation interactions of the allylic carbocation at C6–C7–C8 with N103, T106, and I181. These variations in interactions resulted in an energy difference of 14.2 kcal/mol, favoring B, and the barrier was reduced by 2.8 kcal/mol  and likely contributed to a slight destabilizing effect in the active site model. This was in spite of interactions between cation D and N103, T106, F107, and F149. Nonetheless, the energy barrier to form E was higher in the active site model than in the gas phase. An elevated energy barrier was also observed for the formation of G . This may be explained by the loss of interactions between G and the pyrophosphate moiety as the cation moved further away, deeper into the hydrophobic part of the pocket. A distinct carbocation G was not observed in the enzyme model. Instead, a cation resembling H, with a C8–C10 bond that was already partly formed, was observed. This carbocation was more stable in the active site model than G in the gas phase by almost 9 kcal/mol. Hence, in the enzyme model, cation G was not a stable species, and instead, H was formed spontaneously. The energy for the transformation of E to H was very similar in the gas phase and in the enzyme. Carbocation H formed interactions with N103, W186, and especially with I181. The relative energy difference between H and I was also similar in the gas phase and in the enzyme model. However, the activation energy was higher by 3.0 kcal/mol in the active site model, possibly due to steric effects. I was stabilized via interactions with N103, I181, and W186, which likely made similar stabilizing contributions as in cation H.Carbocation kcal/mol . Anotherkcal/mol . The enelized it . Moreoveas phase . Moreove as well . The dihractions –46. In tIt is well established that the inherent reactivity of carbocations , as well36In this work, we compared the energy profiles of the terpene cyclase CotB2 reaction obtained in the gas phase and using an active site model. The calculations used identical QM methods, facilitating a direct comparison. We presented evidence for the important role played by the active site residues in CotB2 on the reaction energetics in an active site cluster model, suggesting that reaction control in terpene synthase is obtained via a combination of inherent reactivity, initial substrate folding, and enzyme environmental effects. Specifically, the results using the active site model revealed the significant effect that the active site residues have on the relative electronic energy of the intermediates and TS structures in comparison with gas phase data due to ionic, π–cation, and dipole–cation interactions. A detailed understanding of the role of the enzyme environment on the reaction cascade in CotB2 can provide important information to derive a synthetic strategy for cyclooctatin and related terpene manufacturing. Future studies using hybrid quantum mechanics and molecular mechanics techniques to model the enzyme reaction in a complete enzyme environment will allow careful evaluation of the usefulness of such active site theozyme models.All calculations were carried out with Gaussian 16 . GeometrA main limitation of the current cluster modeling approach was freezing of the active site residues, which did not allow any accommodation of the active site to the evolving reaction intermediates. Flexible residues were not considered due to the possible perturbation of the active site contour and the rapid fluctuation of the total electronic energy as a function of amino acid residue geometry. An additional obvious limitation were medium and long-range nonbonded interactions beyond the active site cage considered here. These effects could be considerable and will be scrutinized in future work.The Cartesian coordinates of all species are reported in File 1Cartesian coordinates for all species.


Boreogadus saida). The data-driven biophysical model of polar cod early life stages assembled here predicts a strong mechanistic link between survival and variation in ice cover and temperature, suggesting imminent recruitment collapse should the observed ice-reduction and heating continue. Backtracking of drifting eggs and larvae from observations also demonstrates a northward retreat of one of two clearly defined spawning assemblages, possibly in response to warming. With annual to decadal ice-predictions under development the mechanistic physical-biological links presented here represent a powerful tool for making long-term predictions for the propagation of polar cod stocks.The Arctic amplification of global warming is causing the Arctic-Atlantic ice edge to retreat at unprecedented rates. Here we show how variability and change in sea ice cover in the Barents Sea, the largest shelf sea of the Arctic, affect the population dynamics of a keystone species of the ice-associated food web, the polar cod ( Mats Huserbråten et al. apply a biophysical model approach to estimate spawning locations for polar cod for the years 1990–2017. They incorporate ocean current models, Arctic ice cover, and temperature data and observe a northward retreat of spawning assemblages and predict recruitment populations will collapse if heating and ice reduction trends continue. With a warmer and increasingly ice-free Arctic Ocean, the border between the Boreal and Arctic biomes is predicted to move north6. While the present warming has allowed an opportunistic northwards expansion of Boreal species into the northern Barents Sea Arctic ecosystem7, little attention has been given to the effect of the recent ice retreat and variability on the ice-associated fish community. A keystone species in the ice-associated Arctic marine food web is the polar cod, one of few species linking the lower (i.e. zooplankton) and higher  trophic levels10. The polar cod is endemic to the Arctic and their early life history is strongly adapted to the presence of ice: from spawning of eggs under the ice11; the ability of eggs to develop in sub-freezing temperatures12; larvae feeding on zooplankton specific to the seasonal ice-melt-water blooms14; and low mortality of larvae in the close to freezing temperatures typical of Arctic water masses15. At the same time observations made by Soviet-era researchers in the early 1960s11 remain the most complete descriptions of polar cod spawning along the Eurasian shelf, with a historical stronghold in spawning activity in the south-eastern corner of the Barents Sea  and a suggested spawning east of Svalbard. The Barents Sea polar cod stock have been monitored annually by a joint Norwegian-Russian survey since 198616, and the total stock biomass (TSB) has varied vastly in the past three decades, between a minimum of 127,000 t in 1990, up to a maximum of 1,941,000 t in 2006 yet with no clear trend17. The spatial distribution and abundance of 0-group polar cod (~6 months old) has also varied considerably in this period18, suggesting high recruitment variability both in space and time.The Arctic Ocean winter sea ice cover has steadily declined since the 1970s, and the majority of this reduction has been observed in the Barents Sea18. The two main factors driving the observed variability in ice cover in the Barents Sea is the inflow of warm Atlantic water from the west and the inflow of cold, less saline Arctic water from the north and east20. The inflow of Atlantic water from the west and Arctic water from the northeast set up a strong temperature and salinity front across the entire Barents Sea, here termed the Polar Front22 , and subsequently an objective search algorithm identified drift trajectories that intersected the 0-group observations of the autumn survey. The coupled biophysical model was simulated over 28 spawning seasons (1990–2017) and was compared to the catch of 0-group polar cod in 8302 pelagic trawls distributed throughout the Barents Sea. The mechanistic interpretation of the scores from the objective search algorithm is twofold: first the backtracking from observations indicates spawning at a given release point for a given year ; and second the frequency of links between spawning location and the observed 0-group abundance reflects larval survival during the drift phase and indicates supply of recruits from a given spawning location . We report a positive relationship between year class strength of polar cod to the inter-annual variability of ice cover, and a negative effect of maximum summer temperatures encountered in late larval phase, where a common driver is the heating of the system. We also observed a clear northward retreat of one the two identified spawning assemblages towards the end of the study period that may also be a response to warming. Understanding the physical-biological interactions during recent decades of both varying and changing ice conditions allow predictions of future development of the polar cod stock in the Barents Sea.The objective search algorithm revealed two clearly separated maxima in spawning location probability, with one spawning assemblage in the Pechora Sea and one east of Svalbard. Moreover, the location of the two main spawning areas identified coincided with the high probability of 0-group occurrence downstream to the two areas  explaining 40% of the variation in 0-group presence/absence, Fig. p < 0.001, see Fig. p < 0.001), confirming the Pechora Sea spawning assemblage as the main supplier of recruits to the Barents Sea stock monitored during autumn surveys. Recruitment east of Svalbard had no correlation with either ice cover, temperature, or TSB at any lag, indicating other effects at play than those tested within the scope of this study. A linear regression of maximum ice cover over the study period showed a weak, yet significant yearly decrease of 1.1% . Moreover, the 10-day mean-filtered maximum temperature encountered by larvae in late summer was significantly different between the two main spawning areas , with maximum temperatures encountered in the eastern Barents Sea on average between 2.1 °C and 3.6 °C degrees warmer (95% CI). The average yearly maximum temperature encountered in the Pechora/eastern Barents Sea was 5.7 °C but in extreme years more than 9 °C (95% CI: 2.4–9.0 °C), well beyond the thermal tolerance limit of the larvae . A linear regression model of eastern Barents Sea recruitment strength as function of covariates Barents Sea ice cover, maximum temperature encountered by larvae, and TSB, explained 72% of the variation in recruitment strength . Ice and temperature explained the majority of the variance, and a smaller portion explained by TSB p = 0.045).Recruitment strength in the eastern Barents Sea/Pechora Sea had a significant correlation with maximum yearly ice cover  C. hyperboreus, both advected from the west with the warmer Atlantic water masses, and all three Calanus species having exceptionally high energy content29. The ample food supply available to the later larval stages in the Atlantic-influenced water masses found in the south-eastern parts of the Barents Sea thus represents a near ideal nursery habitat. At the same time, the high summer temperatures sometimes encountered there may also be associated with high risk by pushing larvae beyond thermal thresholds15. For example, the seven warmest years in terms of maximum summer temperatures encountered , resulted in the five years of lowest recruitment. However, due to the collinearity of ice cover and high temperatures encountered in south-eastern Barents Sea during drift in late summer, where a common driver is the wind-driven advection of warmer water masses from the west20, we were not able to disentangle which of the two identified mechanisms that affected recruitment in the Barents Sea the most.We suggest two plausible mechanisms driving the large recruitment variability observed in the south-eastern parts of the Barents Sea, mediated in turn by both heat and ice. First, the presence/absence of ice likely causes a “match/mis-match” scenario31. However, due to the strong topographic steering of the incoming Atlantic water masses away from the northern parts of the Barents Sea  we estimated the total mass of polar cod found in echo-sounder transects and pelagic trawls throughout the Barents Sea17, identical to the method used for estimating capelin  stock size in the Barents Sea40.The 0-group polar cod data were sampled on annual surveys run between late August and early October in the period 1990 and 2017, covering almost the entire Barents Sea within a regular grid of ~65 km. At each station the upper water layer (0–60 m) was sampled by three pelagic trawls with a 20 × 20 m opening, keeping the headlines at 0 m, 20 m, and 40 m. The pelagic trawls were towed at a speed of 3 knots over a time interval of 10 min, corresponding to a tow length of 0.5 nautical miles (≈0.93 km). If dense concentrations of fish appeared on the echo-sounder deeper than 40 m, additional tows were performed at 60 and 80 m. During the study period of 27 years 8302 of these depth-integrated trawl hauls were done. Due to the selectivity of the gearhttp://myroms.org), a free-surface, hydrostatic, primitive equation ocean general circulation model42. The ROMS model was run with a horizontal resolution of 4 × 4 km in an orthogonal, curvilinear grid covering parts of the North Atlantic and all the Nordic and Barents seas  in the study area was based on the Regional Ocean Modeling System  across the entire Barents Sea shelf shallower than 400 m that had been covered by an ice concentration of more than 15% in the period 1990–2017 , maximum temperature encountered by larvae (10-day mean-filtered over 100 larvae released from the most likely spawning area for a given year), and estimated TSB. This regression model was fitted separately for the north-western  and south-eastern (Pechora Sea) spawning areas as implemented in the base R-package “stats”49. In the model selection phase, we applied a stepwise model selection scheme with the initial inclusion of all relevant variables, where only the variables deemed significant was included in the final model. Due to the high degree of collinearity between some of the variables, we also did a variance partitioning analysis to disentangle the separate and/or common effects of the variables50.A probabilistic map of 0-group polar cod presence was calculated by using a two-dimensional binomial GAM smoother, based on the geographical coordinates of pelagic trawls, presence-absence of polar cod larvae in the pelagic trawls, and using the logit-link function as implemented in R-package “mgvc”Further information on research design is available in the Reporting SummaryPeer Review File


Inhibition of the respiratory chain complex I plays a key role in the pathogenesis of metformin-induced lactic acidosis. In a work recently published in this journal, a novel cell-permeable succinate prodrug (NV118) increased in vitro mitochondrial oxygen consumption coupled with energy production and decreased lactate production in intact human platelets intoxicated with metformin. This result was interpreted in light of a “bypass” strategy. NV118 entered platelets and released succinate in their cytoplasm; succinate in turn donated electrons to complex II and thus reactivated the flow of electrons to the distal part of the respiratory chain independent of complex I. Herein, I will (1) comment on these findings; (2) highlight the potential therapeutic application of succinate in other critical conditions accompanied by complex I inhibition, including sepsis, traumatic brain injury, and inherited neurological disorders; and (3) examine the most important issues that remain to be solved to transfer these observations to the bedside. Metformin is the drug of choice for adults with type 2 diabetes . It is tMetformin is a safe drug when correctly used in properly selected subjects . NonetheBeing a cationic compound, metformin accumulates in mitochondria driven by the (negative) membrane potential of these organelles. There, and depending on dose, it can inhibit the respiratory chain complex I . At micr2) adenine dinucleotides that transfer electrons to complexes I and II, respectively. Electrons then flow through complexes III and IV, transported via ubiquinone and cytochrome c, and finally reduce oxygen to water. Electron transfer through complexes I, III, and IV generates a proton-motive force across the inner mitochondrial membrane that is used by complex V (the ATP synthase) to generate ATP [2. Therefore, most of electrons normally enter the respiratory chain through complex I, missing out complex II.Mitochondria are the “powerhouse” of the cell. They generate energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation. The electron transport (or respiratory) chain consists of enzyme complexes and carrier molecules associated with the inner mitochondrial membrane. Oxidation of nutrients is coupled with reduction of nicotinamide (NADH) and flavin , and correcting life-threatening acid-base alterations. Mortality is 20–30% .In a recent issue of this journal, Dr. Piel and colleagues report their findings in vitro on the use of a novel cell-permeable succinate prodrug NV118) or methylene blue in intact human platelets exposed to toxic doses of metformin (10–50 mmol/L) [8 or meth2), iron-sulphur centers, and then to ubiquinone (that is transiently reduced to ubiquinol). In turn, ubiquinol transfers electrons to complex III [Succinate is an intermediate of the Krebs cycle. It is produced in the mitochondrial matrix from succinyl-CoA by the succinyl-CoA synthase; it is then oxidized to fumarate by the succinate dehydrogenase, a subunit of complex II of the electron transport chain. Complex II consists of four subunits. Two are hydrophilic and project into the matrix: they contain the succinate-binding site. The other two are hydrophobic and are embedded in the inner mitochondrial membrane: they contain the ubiquinone-binding site. The succinate-binding site is connected to the ubiquinone-binding site by a chain of redox centers including the FAD and other iron-sulphur clusters. When succinate is oxidized to fumarate by the succinate dehydrogenase, as part of the Krebs cycle, electrons are sequentially transferred to the FAD  but not with succinate , 15. Thietformin .As part of a larger research project , my collSeveral other critical diseases associated with predominant or selective inhibition of complex I may benefit from the use of succinate. For example, infusion of succinate prevented the decrease in liver ATP content and prolonged survival time in two different animal models of sepsis , 18. My Several issues related to the use of succinate as a drug remain to be properly addressed. Herein, I will focus on three of them, which I consider most important. First, many mammalian membranes are impermeable to succinate  and someComplex I is by far the most complex component of the respiratory chain. This can be the reason why it is much more vulnerable to dysfunction or damage than other complexes, including complex II. Succinate may be a substrate to support the Krebs cycle and ATP generation by oxidative phosphorylation when mitochondrial function is impaired, as it transfers electrons to the respiratory chain downstream complex I. Several issues still need to be addressed, including those related to how to administer succinate in vivo. Therapeutic implications are not limited to metformin intoxication; they extend to several other critical conditions accompanied by some degree of mitochondrial dysfunction. As long as it means bypassing a defective complex I to reactivate energy production, taking a shortcut should not be regarded as cheating.


Background: Atopic eczema is a common childhood disease associated with high IgE and eosinophilia. We characterized the clinical features associated with hyper-IgE (defined as IgE > 2000 IU/L) in eczema. Methods: Nottingham Eczema Severity Score (NESS), family and personal history of atopy, skin prick test (SPT) for common food and aeroallergens, highest serum IgE ever and eosinophil counts were evaluated in 330 children eczema patients. Childhood-NESS (NESS performed at <10 years of age) and adolescent-NESS (NESS performed at >10 years of age) were further analyzed. Results: IgE correlated with NESS  and eosinophil percentage . Compared with IgE ≤ 2000IU/L (n = 167), patients with hyper-IgE (n = 163) were associated with male gender (p = 0.002); paternal atopy (p = 0.026); personal history of atopic rhinitis (p = 0.016); asthma (p < 0.001); dietary avoidance (p < 0.001); use of wet wrap (p < 0.001); traditional Chinese medicine use ; immunomodulant use ; skin prick sensitization by dust mites (p < 0.001), cats (p = 0.012), dogs (p = 0.018), food (p = 0.002); eosinophilia (p < 0.001); more severe disease during childhood (p < 0.0001) and during adolescence (p < 0.0001), but not onset age of eczema or maternal atopy. Logistic regression showed that hyper-IgE was associated with personal history of asthma (exp(B) = 5.12, p = 0.002) and eczema severity during childhood and adolescence (p < 0.001). For patients <10 years of age, dust mite sensitization (p = 0.008) was associated with hyper-IgE. For patients >10years of age, food allergen sensitization was associated with hyper-IgE (p = 0.008). Conclusions: Hyper-IgE is independently associated with asthma, more severe atopy and more severe eczema during childhood and adolescence. IgE > 2000 IU/L may be a tool to aid prognostication of this chronic relapsing dermatologic disease and its progression to asthma. Childhood eczema is a chronic distressing disease ,4,5,6,7.D. pteronysissnus and D. farina, cat, dog, cockroach, grass pollen), highest serum total IgE ever (and age of measurement), and highest eosinophil counts ever were analyzed. Hyper-IgE is defined as IgE above 2000 IU/L. In case of missing data, hard copy, electronic medical records and the investigator’s own database were accessed. In a subset of patients whose first visits were prior to 10 years of age, childhood-NESS (NESS performed at <10 years of age) were recorded. Adolescent-NESS (NESS performed at age 10 years) were also recorded if patients presented after 10 years of age or were followed up beyond 10 years of age. Fisher’s Exact test for categorical variables were performed. For continuous variables, Mann-Whitney U tests and Krustal-Wallis one-way ANOVA were used. Logistic regression were analyzed using SPSS Statistics 20 . Probability values less than 0.05 were considered significant. This is a review of clinical data of patients followed at our outpatient clinic over the years. The ethics application was obtained from the University Ethics committee for this review.The clinical data of Chinese patients with atopic eczema (AE) followed consecutively at the pediatric dermatology clinic of a university hospital were reviewed. Eczema was diagnosed according to the U.K. Working Party’s Diagnostic Criteria for Atopic Dermatitis . Diseasep = 0.001 (two-tailed)) and eosinophil percentage . Compared with IgE ≤ 2000 IU/L (n = 167), patients with hyper-IgE (n = 163) were associated with male gender (p = 0.002); paternal atopy (p = 0.026); personal history of atopic rhinitis (p = 0.016); asthma (p < 0.001); dietary avoidance (p < 0.001); use of wet wrapping (p < 0.001); traditional Chinese medicine use ; immunomodulant use ; skin prick sensitization by dust mites (p < 0.001), cats (p = 0.012), dogs (p = 0.018), food (p = 0.002); eosinophilia (p < 0.001); more severe disease during childhood (p < 0.0001) and during adolescence (p < 0.0001), but not onset age of eczema or maternal atopy . Using skin prick dust mite sensitization, food allergen sensitization, personal history of allergic rhinitis, personal history of asthma, and the NESS, logistic regression showed that hyper-IgE was associated with a personal history of asthma (exp(B) = 5.12, p = 0.002) and eczema severity during childhood and adolescence (p < 0.001). For patients < 10 years of age (n = 122), dust mite sensitization (p = 0.008) was associated with hyper-IgE. For patients > 10 years of age (n = 270), food allergen sensitization was associated with hyper-IgE (p = 0.008). IgE correlated with NESS ) and eosinophil percentage .Spearman correlation showed IgE also correlated with disease severity . In a prospective correlational study of 117 patients, we demonstrated that the IgE level correlates significantly with various objective clinical scores (including Scoring Atopic Dermatitis (SCORAD)) and chemokine markers of AD [c eczema ,21. In trs of AD . Furtherrs of AD . Total srs of AD , but Samrs of AD . HoweverNew studies proposed a novel concept of atopic eczema/dermatitis syndrome (AEDs) that is classified into being intrinsic and extrinsic. The extrinsic form of AEDs is the IgE-mediated form and it is associated with respiratory allergies and high serum IgE levels . This clet al. demonstrated that the prognostically unfavorable factors influencing eczema were, in order of importance, severe (widespread) dermatitis in childhood, family history of AE, associated allergic rhinitis and/or bronchial asthma , female sex and early age at onset. [et al. followed 121 children with infantile eczema and found persistence into adult life occurring in 63%, with 32% having a chronic continuous course. Prognostically unfavorable factors were early-onset severe disease within the first six months of life and very high serum IgE levels [Over the years, many studies have tried to look into the associating factors of eczema severity and disease persistence. Roth and colleagues reported 50 years ago that AE persisted into adult life in approximately 70% of patients with severe AE and in 60% of those with milder disease.  A familyt onset. . WuthricE levels . Our preE levels ,31.Hyper-IgE and asthma. The theory of “Atopic March” favors the consideration of eczema as a systemic disease, and indicates that many children with AE go on to develop asthma and allergic rhinitis as their eczema improves with time [ith time ,5,6,32. ith time . Our patS. aureus infection [We used NESS instead of other common scores for the determination of disease severity and eczema chronicity over a 12-month interval ,18,19. Snfection ,34,35. Nnfection .Documentation of disease severity (NESS) before and after 10 years of age was not available in some of the patients, precluding the ability of studying the subjects in a case control manner. Also, IgE levels were not checked at the same time point in the disease in all patients because many were managed elsewhere before referral to our tertiary centre. Ideally disease severity and IgE should be assessed at the time of onset and serially to document progress during toddlerhood, childhood, adolescence and adulthood in a large prospective cohort. Furthermore, in a tertiary hospital setting, the population of patients encountered and followed is likely to be biased towards more severe disease . Hence, In conclusion, hyper-IgE in patients with eczema is independently associated with asthma, more severe atopy and more severe eczema during early childhood and adolescence. IgE > 2000 IU/L may be a tool to aid prognostication of this chronic relapsing dermatological disease and its progression to other atopic disorders.