Background: The effects of cannulated screws made of polyetheretherketone (PEEK) on the biomechanical properties of the vertebral body during vertebra-pediculoplasty remain unclear. This study aimed to investigate whether PEEK screws have the potential to replace titanium alloy screws.Methods: The surgical model of two different materials of screws was constructed using the finite element method. The biomechanical effects of the two models on the vertebral body under different working conditions were compared.Results: ① The peak von Mises stress of PEEK screws was significantly lower than that of titanium screws, with a reduction ranging from 52% to 80%. ② The von Mises stress values for the injured T12 spine were similar for both materials. Additionally, the segmental range of motion and intervertebral disc pressure showed no significant difference between the two materials.Conclusion: PEEK screws demonstrated advantages over titanium screws and may serve as a viable alternative for screw materials in vertebra-pediculoplasty. Vertebra-Pediculoplasty has emerged as a novel treatment approach for managing split and delayed osteoporotic vertebral fractures that were at risk of cement dislocation . It addrTo overcome these limitations, this study proposed the use of polyether-ether-ketone (PEEK) material as an alternative to titanium alloy screws. PEEK has been extensively studied as an orthopedic implant material since the 1990 s . It has in vitro experiments  analysis served as a valuable tool for assessing the biomechanical parameters of vertebral columns , and severiments . TherefoThe CT data of a healthy 30-year-old male were imported into Mimics software  to initially create a geometric model of the thoracolumbar spine (T11-L1). The thoracolumbar spine model was then imported into 3-Matic  for individual processing of each vertebral body, resulting in a more accurate model structure in Geomagic software . The model was further processed in HyperMesh software  for meshing, material property assignment, and assembly. Finally, the model was imported into Abaqus software  for calculations and analysis . The elaThe hollow lateral screw geometry was created in SolidWorks and imported into HyperMesh to assemble it with the vertebral body. The vertebral body-screw connection was simulated using a “binding” constraint, completing the vertebral screw fusion model. Two postoperative models of different materials (titanium and PEEK) for screw placement into the vertebral body were established based on vertebra-pediculoplasty. The cannulated screws used were 6.5 mm in diameter and 50 mm long. During the operation, bone cement was injected into the vertebral body through the hollow screw, and it diffused around the side hole of the screw, wrapping the screw evenly in a cylindrical shape . Each caA follower load of 500 N was applied to the upper surface of the T11 vertebral body to simulate physiological compressive loading. A moment load of 7.5 N m was applied to the T11 vertebral body to simulate forward flexion, back extension, lateral bending, and axial rotational motion. During loading, all degrees of freedom of the lower surface of the L1 vertebral body were constrained. .The maximum von Mises stresses of the screws and the injured T12 vertebral structure were compared for each model with different materials under various gestures such as flexion, extension, left bending, right bending, left rotation, and right rotation. Additionally, the segmental range of motion and intervertebral disc pressure were also evaluated.The range of motion (ROM) of the vertebral body was calculated under different postures. The ROMs of the T11-T12 segments were found to be 7.4°, 8.9°, and 4.6° for flexion and extension, lateral bending, and axial rotation, respectively. Similarly, the ROMs of the T12-L1 segments were 7.2°, 8.7°, and 3.8° for the corresponding postures. These results were compared with previous experimental data, and they were consistent with the findings reported in the literature .In the six models, the peak von Mises stress of the PEEK screws was 17.52 MPa, 9.125 MPa, 16.66 MPa, 8.48 MPa, 14.94 MPa, and 17.8 MPa. On the other hand, the peak stress of the titanium alloy screws was 89.03 MPa, 29.93 MPa, 69.06 MPa, 31.37 MPa, 90.88 MPa, and 37.48 MPa e.g., . Upon coThere were no significant differences in the maximum stress of the T12 cortical bone in the injured vertebra when using PEEK screws compared to titanium alloy screws under the six different states. Similarly, no noticeable differences were found in the maximum stress of the T12 cancellous bone or the analysis of the endplates e.g. . FurtherDue to its unique properties, PEEK has gained significant interest in bone implant research. The use of PEEK in orthopedic screws offers a promising avenue for exploration. In this study, we conducted a simulation-based analysis to assess the potential of PEEK as a substitute for the conventional titanium alloy used in screw fabrication. PEEK is a semi-crystalline polymer with excellent properties such as high modulus, melting point, processing performance, and strength . Its elaRecent efforts have focused on optimizing the mechanical and biological properties of PEEK through various methods such as 3D printing, coating, chemical modification, and the introduction of bioactive or antibacterial substances . These mIn recent years, numerous studies have explored the factors that influence screw stability, including screw diameter, shape, length, thread shape, implantation method, angle, and combination . The curBy comparing the von Mises stress of screws made from different materials under different vertebral body motions, our analysis reveals that PEEK screws have a significant advantage in reducing peak stress compared to titanium alloy screws. Specifically, the range of reduction observed in the von Mises stress with PEEK screws ranges between 52% and 80%. The observed reduction in peak stress indicates that the use of PEEK screws may lead to reduce the incidence of screw loosening, thereby establishing its potential as a promising alternative material. Some experiments have confirmed the idea that PEEK screws have a low risk of loosening. Richard Lintner et al  conducteWhen assessing the risk of screw fracture, it is important to consider the ratio of peak stress to yield stress rather than focusing solely on stress magnitude. The ratio of peak stress to yield stress for PEEK screws ranged from 8% to 17%, while for titanium screws, it fell within the range of 3%–12% . The higher percentage for PEEK screws suggests a potential escalation of breakage risk e.g., , consistPrevious investigations have examined the load transmission properties of titanium alloy posterior screw rod systems and PEEK screws in posterolateral lumbar fixation. These studies indicated that the titanium alloy system can transmit approximately 67% of the axial compressive load, while the natural upright state can bear only about 20% of the load . PEEK scNext, we analyze the finite element results of the vertebral structure. The von Mises stress of the vertebral body, as well as the segmental Range of Motion and Intervertebral Disc Pressure, were obtained through finite element analysis under various conditions. The results show that the stability of the vertebral body remains largely unaffected. Similar findings have been reported in related investigations. Nomidis et al  conducteSeveral limitations should be considered when interpreting the results of this study. Firstly, the finite element model used is based on theoretical numerical simulations and may not fully capture the complexity of the human spine system, as it does not account for factors such as cyclic loads and the influence of muscles. Secondly, the thoracic and lumbar spine models used are limited to a single subject, and the number of models is small, which may limit the generalizability of the findings. Lastly, this study represents a preliminary exploration of finite element analysis. Further research and exploration are necessary to establish a solid foundation for the long-term development of PEEK material in lumbar spine repair.In conclusion, PEEK screws demonstrate comparable efficacy to titanium alloy screws in providing segmental stability post-surgery. Additionally, PEEK screws facilitated the prevention of loosening, which was a great clinical advantage. Moreover, the radiolucent nature of PEEK screws facilitates postoperative imaging without interfering with radiation therapy. Thus, the PEEK or PEEK composite material may emerge as a viable alternative for screw materials in clinical practice. .


Community and rural hospitals often lack resources for infectious diseases (ID) specialists. Tele-antimicrobial stewardship programs (TASPs) have emerged as a method for health systems to meet regulatory requirements. We analyzed the impact of a fully remote TASP at two small community hospitals in partnership with an academic medical center over a two-year period.A TASP, co-led by an ID physician and ID pharmacist, was implemented at a 21-bed hospital in Princeton, MN, and 49-bed hospital in Wyoming, MN, in August 2020. Figure 1 outlines daily TASP workflows in which the ID physician and ID pharmacist were located at an academic medical center. Frequently encountered restricted antimicrobial agents included vancomycin , piperacillin-tazobactam, cefepime, meropenem, ertapenem, micafungin, and remdesivir. Antimicrobial stewardship interventions were tracked monthly. Restricted antimicrobial days of therapy per 1000 patient days (DOT/1000 PD) mean averages two years pre- and post-implementation were compared. Annual antimicrobial expenditures were followed.For the first two TASP years (8/1/2020 to 7/31/2022), a total of 789 antimicrobial interventions were made with 85.6% being accepted. Each site boasted similar TASP intervention acceptance rates, 85% for the Princeton hospital and 86% for Wyoming . Restricted antimicrobial use trended down from 141.97 to 113.97 DOT/1000 PD at Princeton. A smaller decrease from 106.3 to 103.12 DOT/1000 PD was seen at Wyoming. Annual antimicrobial costs per total patient days also decreased . Princeton hospital’s annual antimicrobial expenditures per total patient days fell from $18.89 in 2019 (pre-implementation) to $6.64. Wyoming showed a reduction in antimicrobial costs per total patient days by decreasing from $11.20 to $5.36.A fully remote TASP in partnership with an academic medical center for two small community hospitals resulted in a rapid increase in the rates of accepted interventions. These high rates were sustained over two years. Restricted antimicrobial use and antimicrobial costs trended down. Consistent communication with tele-hospital physicians and pharmacists helped establish trusting relationships.All Authors: No reported disclosures


Without these tools, it is difficult to advance research in this field.Immediate hypersensitivity reactions to peanuts are a considerable public health concern due to the acute and severe IgE mediated reactions. To conduct research on the pathogenesis and therapeutics of peanut allergies, it is imperative to have mouse anti-crude peanut extract (CPE) IgE monoclonal antibodies (mAbs) for both In this study, four hybridomas producing anti-CPE IgE mAbs were developed and the IgE mAbs were validated using immune-blot analysis, Sandwich ELISA, Indirect ELISA, a cell-based assay using RBL-2H3 cells, and footpad type I hypersensitivity reaction studies in mice.in-vitro and in-vivo peanut allergy studies, as they induce allergic reactions with sensitization alone in mice. These novel anti-Ara h1 and Ara h 3 IgE mAbs, in combination with the detailed protocols outlined in this article, offer valuable guidance for studying acute allergic reactions involving mast cells across various platforms. With some considerations, the IgE mAbs can significantly advance peanut allergy research.The results indicate that two of the four mAbs can be effectively used for both   Specifications tableDescription of protocolThe peanut allergy, an IgE-mediated food allergy, is a major public health concern, particularly in westernized countries. Food allergies affect 10 % of the US population, with over 6 million people, or 2 % of the population, experiencing peanut allergies Seventeen potentially peanut allergens have been identified Mouse peanut allergy models have been used to study the pathogenesis of the peanut allergy and to develop new therapeutics. The models can be induced by the administration of crude peanut extract (CPE) or each purified Ara allergen and evaluated for the humoral immune responses such as serum anti-IgE and IgG antibodies against the allergens, cytokines levels associated T-cell mediated immune responses, as well as changes in body temperature, and clinical signs of anaphylaxis. These changes observed in the disease models are useful for studying the efficacy of treatments in preventing allergic reactions There are several protocols for developing mouse peanut allergy models, such as a combination of an aluminum hydroxide adjuvant and oral administration In the development of peanut allergies, anti-allergen IgE antibodies play a crucial role in inducing allergic and anaphylaxis reactions by activating mast cells (MCs) in-vitro and in-vivo studies. These novel anti-CPE IgE mAbs, along with the protocols outlined in this article, offer valuable guidance for studying allergic reactions across various platforms, thereby advancing peanut allergy research.Although IgE monoclonal antibodies (mAbs) against various antigens have been developed for MCs assays Crude peanut extracts (CPE) were prepared by modifications of a published protocol Six-week-old female BALB/c mice were purchased from Envigo  and housed under specific pathogen-free (SPF) conditions. To obtain sufficient mice for somatic cell hybridization, a group of five mice were housed together in a cage, and each mouse was sensitized by intragastric gavage with 5 mg of CPE and 10 µg of cholera toxin  in 0.5 ml of Phosphate Buffered Saline, pH 7.4 (PBS) per mouse on day 0 and again on day 7. Three weeks after the initial sensitization, the mice were fasted overnight and challenged with 10 mg CPE in 0.5 ml PBS, given in two doses separately with 30-40 minutes in between, through intragastric gavage 5 cells/well) and cultured in DMEM medium supplemented with 15 % fetal calf serum (FCS) containing hypoxanthine, aminopterin and thymidine in a humidified atmosphere of 7 % CO2 in air at 37°C. All plates were screened for anti-CPE IgE antibody-producing hybridomas by ELISA kits , according to the manufacturer's instructions. Hybridomas producing anti-CPE IgE mAbs were cloned through the limiting dilution method Monoclonal antibodies (mAbs) were produced through the technique of somatic cell hybridization. Briefly, BALB/c mice received a booster injection intravenously with 10 µsg CPE dissolved in 0.1 ml PBS, after intragastric sensitization and CPE challenge. Three days after the booster injection, the mice were sacrificed, and their spleens were removed to prepare single cell suspensions in DMEM medium. These spleen cells were then fused with SP2/0-Ag4 cells at a ratio of 5:1 using 50 % polyethylene glycol (MW.4000). The fused cells were plated onto 96-well flat-bottomed tissue culture plates  following the manufacturer's instructions.96-well high-binding ELISA plates  were coated with 1 µg/well in PBS at 4°C overnight. After washing the plates with PBS containing 0.05 % Tween 20 (PBST), the plates were blocked with PBS containing 1 % bovine serum albumin  (BSA/PBS) for 1 hour at RT. After washing the plates with PBST, anti-CPE IgE mAbs diluted with BSA/PBS were added to the plates and incubated for 2 hours at RT. After washing the plates with PBST, biotin-labeled anti-mouse IgE monoclonal antibodies (Chondrex) diluted in 0.05M Tris-buffered saline buffer, pH 8.0 containing 2 % Casein  and 0.05 % Tween 20 (Casein/TBS) were added to the plates and incubated for 1 hour at RT. After washing the plates with PBST, HRP-labeled streptavidin (Chondrex) in Casein/TBS was added to the plates and incubated at RT for 30 minutes. After washing the plates with PBST, the color was developed at RT for 25 minutes by adding 100 µl of 3,3′,5,5′-tetramethylbenzidine solution . The enzymatic reaction was stopped by adding 50 µl of 2 N sulfuric acid and optical density (OD) values were measured at 450 nm with a reference at 630 nm CPE proteins were separated in a 10 % or 12 % SDS gel with or without 0.05 % 2-Mercaptoethanol (Sigma). To assess protein separation, the gel was stained with Coomassie brilliant blue R-250 (Sigma) according to the manufacturer's instructions. The separated proteins were transferred to a nitrocellulose membrane . The membrane was then blocked with Casein/TBS and incubated with diluted anti-CPE mAbs at 4°C overnight. After washing the membrane with PBST, it was incubated with biotin-labeled anti-mouse IgE monoclonal antibodies (Chondrex) in Casein/TBS at RT for 2 hours. After washing the membrane with PBST, it was then incubated with HRP-labeled streptavidin (Chondrex) in Casein/TBS at RT for 30 minutes. The protein bands were visualized by adding TMB for immuno-blot (Thermo Fisher).A mAb affinity column was prepared following the procedures described by Sterogene Bioseparations . To prepare the column, mouse anti-CPE IgE mAb 2G11G7 was dissolved in 3 ml PBS at a concentration of 2 mg/ml and incubated with 1 ml ALD-Actigel and ALD-coupling solution overnight at 4°C. Unbound sites on the antibody-bound gel were blocked by incubating the gel with 0.1M Tris and ALD-coupling buffer, followed by washing with 0.1M TBS at pH 8.0. CPE in 0.1M TBS at pH 8.0 was then passed through the column and any unbound proteins were washed out with the TBS buffer. The bound fraction was eluted with 0.1M glycine-HCl buffer at pH 2.6 and then dialyzed against 0.1M TBS at pH 8.0.5 cells/ml in 0.2 ml fresh medium and plated into 96-well flat bottom tissue culture plates (Greiner). The cells were allowed to adhere for at least 3 hours. Afterwards, they were incubated with 0.2 ml fresh medium containing various concentrations of the anti-CPE IgE mAbs for 16 hours. The cells were then washed twice with PBS and challenged with 0.2 ml of various concentrations of CPE in Tyrode's buffer . After incubating at 37°C for 1 hour, 0.1 ml of the medium was transferred to a 96-well plate (Greiner). 40 µl 1.3 mg/ml p-nitrophenyl N-acetyl-D-glucos-aminide in citrate buffer, pH 4.5 were added, and the plate was incubated at 37°C for 1 hour. The reaction was stopped by adding 45 µl of 0.5M NaOH to each well, and OD values of the formed p-nitrophenolate were measured at 410 nm. The absorption was converted into the percentage of total cellular beta-hexosaminidase content by comparison with the absorption produced by the cells which received 1 % Triton X-100 (Sigma) in Tyrode's buffer RBL-2H3 (CRL-2256) cells were purchased from the American Type Culture Collection . The cells were cultured in DMEM with 15 % FCS and scraped to prepare a single-cell suspension. The cells were then resuspended at 2×10As a preliminary study to test the biological activities of these IgE mAbs, groups of three BALB/c mice were housed together in a cage and each mouse received 1 mg of mAbs by intravascular (IV) injection. After 24 hours, the mice were challenged with 0.1 mg of CPE or PBS by intradermal (ID) injection at their footpads. The thickness of the footpad (in millimeters) was measured using a Loop Handle Dial Thickness Gauge .After somatic cell hybridization, four hybridomas, clones 6E10C12, 2G11G7, 6B7B10, and 5E7B12 were determined to produce IgE mAbs against CPE, by screening using sandwich ELISAs employing anti-mouse IgE antibody-coated plates and biotinylated CPE as a tracer. Each IgE mAb was purified by ion-exchange chromatography from culture medium collected in a bioreactor system. In the sandwich ELISA, the four IgE mAbs reacted with biotinylated CPE dose-dependently . 6E10C12Mouse anti-CPE IgE mAbs: 6E10C12 (Circle), 2G11G7 (Triangle), 6B7B10 (Square), and 5E7B12 (Diamond), diluted with 0.1M Tris-buffered saline pH 7.5 containing 1 % BSA were added to wells coated with 10 µg anti-mouse IgE mAb, and incubated at RT for 2 hours. After washing the plates with PBST, biotinylated CPE diluted with Casein/TBS was added and incubated at RT for 1 hour. After washing the plates with PBST, avidin-peroxidase diluted with the casein buffer was added and incubated at RT for 30 minutes. After washing plates, the antibody binding was visualized with TMB at RT for 25 minutes and OD values were measured at 450 nm with a reference at 630 nm. Data is expressed as the mean ± standard deviation.Then, the four IgE mAbs were evaluated by indirect ELISAs that consisted of CPE coated plates and anti-mouse IgE secondary antibodies. In the indirect ELISA, 2G11G7 and 6B7B10 demonstrated higher dose-dependent reactivities against CPE coated on the plate at between 31.5 – 2000 ng/ml. However, 6E10C12 showed lower reactivities and 5E7B12 failed to react with CPE coated on the plates in the same antibody concentration range .Fig. 2DoAnti-CPE IgE mAbs: 6E10C12 (Circle), 2G11G7 (Triangle), 6B7B10 (Square), and 5E7B12 (Diamond) diluted with 0.1M Tris-buffered saline pH 7.5 containing 1 % BSA were added to wells coated with 1 µg CPE and incubated at RT for 2 hours. After washing the plates with PBST, biotinylated anti-mouse IgE antibodies diluted with Casein/TBS was added and incubated at RT for 1 hour. After washing the plates with PBST, avidin-peroxidase diluted with Casein buffer was added and incubated at RT for 30 minutes. After washing plates, the antibody binding was visualized with TMB at RT for 25 minutes and OD values were measured at 450 nm with a reference at 630 nm. Data is expressed as the mean ± standard deviation.The protein profile of CPE was analyzed using SDS-PAGE under both reducing and non-reducing conditions. As expected, the separation of CPE antigens resulted in the detection of multiple protein bands that corresponded to Ara h 1, Ara h 2, and Ara h 3 proteins. These proteins were expected to have molecular weights of approximately 66-69 kDa, 17-19 kDa, and 57-62 kDa, respectively, when analyzed under non-reducing conditions Of the four mAbs, 2G11G7 was selected for further investigation to verify its antigen. An immuno-affinity column was prepared using 2G11G7, and CPE was passed through the column to obtain the bound fraction. The fraction was analyzed by SDS-PAGE under reducing and non-reducing conditions. Under non-reducing conditions, the fraction showed bands at 65 and 60 kDa, which disappeared under reducing conditions. In contrast, major bands at 42, 37, and 20 kDa were observed under reducing conditions . N-termiImmuno-blot; 2: 6E10C12, 3: 2G11G7, 4: 6B7B10, and 5: 5E7B12B. SDS-PAGE analysis of 2G11G7 affinity purified samples under 6: non-reducing conditions and 7: reducing conditions.The biological activities of the four IgE mAbs were evaluated by beta-hexosaminidase release from RBL-2H3 cells in degranulation assays. The RBL-2H3 cells were sensitized with serial dilutions (0.128 - 2000 ng/ml) of the IgE mAbs and were then challenged with 2 µg/ml CPE. A typical prozone effect was observed with a bell-shaped dose–response curve. With 2G11G7 and 6E10C12, the degranulation peaked at 80 ng/ml of IgE mAbs, ranging from approximately 20 % to 60 %. At concentrations of 0.128 ng/ml IgE mAbs, beta-hexosaminidase release approached background levels. With 6B7B10, the degranulation peaked at between 3.2 and 80 ng/ml of IgE mAb, ranging from approximately 3 to 15 %. Interestingly 5E7B12 failed to degranulate RBL-2H3 cells .Fig. 4RB6 cells/well in a 96-well plate for 3 hours, and then treated with anti-CPE IgE mAbs: 6E10C12 (Circle), 2G11G7 (Triangle), 6B7B10 (Square), and 5E7B12 (Diamond) at 37°C for 16 hours. After washing cells with PBS two times, 2 µg/ml CPE in Tyrode's buffer was added at 0.2 ml/well and then were incubated at 37°C for 1 hour. 0.1 ml medium from each well was transferred to a 96-well plate and assayed for beta-hexosaminidase activity. The degranulation of RBL-2H3 cells was expressed as a ratio compared with 100 % degranulation of the cells which received 1 % Triton-X in Tyrode's buffer. Data is expressed as the mean ± standard deviation.RBL-2H3 cells were cultured in 0.1 ml DMEM containing 15 % FBS at 10The dose dependency of CPE concentration for sensitization of RBL-2H3 cells was evaluated with the three responding anti-CPE IgE mAbs. Sensitizing with CPE at 100 ng/ml, both 2G11G7 and 6E10C12, induced degranulation dose-dependently from 500 to 8000 ng/ml. For 6B7B10, CPE sensitization at 20 ng/ml 6B7B10 showed degranulation between 200 and 8000 ng/ml .Fig. 5DoThree anti-CPE IgE mAbs were evaluated for mouse footpad type I hypersensitivity. Each mouse was given an IV injection with 1 mg of each IgE mAb, followed by a CPE injection in the footpad. Mice given 2G11G7 and 6B7B10 IgE mAbs developed paw swelling, 1.10 +/- 0.42 mm and 1.25 +/- 0.78 mm respectively, that peaked at 1-2 hours after the CPE injection and resolved to base levels within 6 hours. However, 6E10B10 failed to induce paw swelling. In contrast, mice that received anti-CPE IgE mAbs and PBS did not exhibit any paw swelling .Fig. 6FoThe footpad thickness of mice after IV administration of 1 mg of mAbs: 6E10C12 (Circle), 2G11G7 (Triangle), 6B7B10 (Square), followed by ID administration of 0.1 mg CPE (Solid line) or PBS (Dashed line) at the footpad. Data is expressed as the mean ± standard deviation.The dose-dependency of footpad type I hypersensitivity induced by mAb 2G11G7 was evaluated. Mice given CPE and 0.1, 0.3, or 1 mg of mAb 2G11G7 developed paw swelling, 1.30 +/- 0.43 mm, 1.60 +/- 0.13 mm, and 2.07 +/- 0.10 mm, respectively. The paw swelling lasted for 6 hours at all doses and resolved to base levels within 8 hours after CPE injection at the footpad. In contrast, mice that received IgE mAbs and PBS did not exhibit any paw swelling .Fig. 7DoIn the present study, we established four mouse hybridoma clones that produce IgE mAbs against peanut antigens. The IgE mAbs showed different reactivity patterns in two ELISA systems: the sandwich ELISA and indirect ELISA  and 2. IThe IgE mAbs recognized their antigens in immuno-blot analysis under non-reducing conditions, indicating that they recognize linear epitopes that remain stable even under heat denaturation at 100°C . Based oAra h 1 and Ara h 3 exist in homotrimer (and hexamer) in physiological conditions that were used for oral immunization. Their monomeric or trimeric form and native or denatured conditions may affect mAb reactivities by exposing their epitopes in the antigen. Since there are discrepancy in indirect ELISA using CPE and purified allergens and immuno-blot, further investigations will be needed to understand these discrepancies among assays. 2G11G7 likely binds to the Ara h 3 protein as shown in immune-blot analysis under non-reducing conditions . We specThe biological activities of the IgE mAbs were evaluated by measuring the degranulation of RBL-2H3 cells. The results showed a bell-shaped dose–response curve, with three mAbs capable of degranulating the cells. Specifically, 2G11G7 and 6E10C12 had peak degranulation levels of 60 % at 80 ng/ml, while 6B7B10 peaked at 15 % at 12.5 ng/ml. This bell-shaped reaction has been reported previously with an anti-Dinitrophenyl (DNP) IgE antibody In degranulation assays, a high concentration of CPE demonstrated partial RBL-2H3 cell degranulation (data not shown). As a result, a CPE concentration of 2 µg/ml was chosen, as it showed low self-activation and effectively degranulated RBL-2H3 cells in the presence of IgE. The degranulation of RBL-2H3 cells by CPE alone may be a result of the inherent presence of Lipopolysaccharides (LPS) in CPE in-vivoIn type I hypersensitivity reaction studies, the development of footpad swelling was observed in mice who received 2G11G7 and 6B7B10, but not in those who received 6E10C12. These findings revealed an inconsistency with the results from the RBL-2H3 cell degranulation assays in which 6E10C12 was able to degranulate RBL-2H3 cells but failed to induce type I hypersensitivity in mice. It is possible that 6E10C12 has a higher affinity for rat IgE receptors, but lower affinity for mouse IgE receptors, despite all four hybridomas being generated using mouse spleen cells and Sp2/0-Ag14 myeloma cells. Thus, it may be crucial to use mouse MCs with 6E10C12 to determine its ability to degranulate mouse MCs and induce delayed type I hypersensitivity in-vivo studies. In conclusion, 2G11G7 is the most suitable IgE antibody for studying CPE-related allergic reactions in in-vitro and in-vivo assays. 6E10C12 can be used as a negative control in in-vivo studies, while 5E7B12 works as a negative control in mast cell degranulation assays and indirect ELISAs.It Is important to note that while 6B7B10 had a lower activation rate of only 15 % in cell degranulation assays, its peak activation concentration was lower than that of the other mAbs. Interestingly, 6B7B10 was able to induce a similar level of footpad type I hypersensitivity as 2G11G7. These results indicate that it is necessary to consider the total activation capacity by determining both the optimal concentration and the activation rate when evaluating the biological activities of these mAbs in Despite all the IgE mAbs showing positive results in immuno-blot analysis, three out of the four mAbs failed to bind to heat denatured CPE in indirect and sandwich ELISAs, as well as failing RBL-2H3 cell degranulation assays with heat-denatured CPE (data not shown). Additionally, it is worth noting that upon heating, Ara h 1 and Ara h 3 may form aggregates, which can decrease their solubility and alter their structure in-vitro and in-vivo peanut allergy studies. These mAbs can induce acute allergic reactions with only sensitization in mice, making them useful for evaluating therapeutic methods related to mast cell activation. The use of these mAbs eliminates the need for lengthy immunization protocols, streamlining the process of allergic studies. These novel anti-CPE IgE mAbs, along with the protocols outlined in this article, offer valuable guidance for studying allergic reactions across various platforms, thereby advancing peanut allergy research.A summary of the applications of the IgE mAbs are shown in in-vitro studies, although ELISA is effective for validating IgE mAbs to Ara allergens, assay results must be considered with their epitope, sensitivities and specificities. Furthermore, when assessing biological activities of mouse IgE mAbs to mouse or rat MCs, variations may arise by affinities between IgE mAbs and IgE receptors affecting to cell-based assays (or ex-vivo assays) and in-vivo assays. In in-vivo studies, this protocol has limited efficacy in studying the interaction between IgE and MCs during the allergic acute phase, as IgE may only remain in tissues in a short period. For the investigation of T-cell responses and chronic phases of allergic reactions, conventional oral immunization methods using CPE or allergens must be used. Additionally, there are discrepancies between mouse and human allergic reactions depending on mouse strains and environmental factors However, we must pay attention to some considerations. In Nonetheless, the use of mAbs ensures high reproducibility compared to serum or polyclonal antibodies, as they do not exhibit batch-to-batch variations. Although effective utilization of IgE mAbs should take these considerations and limitations into account the IgE mAbs can still serve as valuable tools, especially in specific studies like inhibitor screening in MC activation.Takaki Waritani: Conceptualization, Methodology, Investigation, Data curation, Writing – original draft. Sidney Lomax: Investigation. Dawn Cutler: Investigation. Jessica Chang: Validation, Writing – review & editing.The authors declare that they have no competing interests.


Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae. Neisseria gonorrhoeae. According to the Centers for Disease Control and Prevention (www.cdc.gov (accessed on 2 February 2023)) N. gonorrhoeae represents an urgent threat because of its high drug resistance and the potential to become a serious worldwide threat. There are no vaccines against this microorganism, and since it progressively develops resistance to antibiotics, research on new targets has become critical.Gonorrhea is a sexually transmitted disease caused by the obligate human pathogen Lactobacillus sp.) and intracellular sources. N. gonorrhoeae has many mechanisms to detoxify ROS )g PROPKA . The finhttps://www.ddl.unimi.it/vegaol/propka.htm (accessed on 2 February 2023)) [Ka values using the coordinates from the crystal structure as input.PropKa (y 2023))  was alsoNgBCCP is a highly conserved enzyme in N. gonorrhoeae strains, anchored to the outer membrane. This dihemic enzyme, unlike other classical BCCPs, only dimerizes at high protein concentration and in the presence of added calcium ions. This can be explained by the NgBCCP dimer interface, which, in comparison to other classical BCCPs, is less hydrophobic and has fewer inter-subunit interactions in its N-terminal domain. Its location in vivo, anchored to the outer membrane, and the interface properties suggest that the dimer assembly does not need to be as strong as for other enzymes that freely diffuse in the periplasm.NgBCCP P heme is that it remains partially as a high-spin species at cryogenic temperatures. This reflects changes in the surroundings of the P heme cavity and calcium site that affect the pKa of the solvent-derived molecule coordinating P heme iron, although comparison of the available BCCP structures showed no significant differences. A dynamic monomer–dimer equilibrium could cause significant changes at the P heme, namely, in the conserved loop region, carrying the P heme distal histidine ligand. In fact, this loop is not stabilized at the dimer interface by the conserved tryptophan π-stacking motif between monomers found in other classical BCCPs.One unique spectroscopic feature of the NgBCCP catalytic activity mainly by competitive inhibition, as they bind directly to the P heme. This was demonstrated for the first time in an azide-inhibited BCCP structure in which the active site was blocked by this ligand and therefore prevented substrate binding.The P heme cavity is accessible to its substrate but also to other exogenous ligands, specifically cyanide, which has high affinity for the active site. Furthermore, the binding of cyanide, imidazole, and azide inhibits NgBCCP structure can be part of an intermediate step of the catalytic cycle.The proposed catalytic mechanism based on the catalytic intermediate species and on the kinetic data suggests that the two waters in the P heme pocket of the NgBCCP is conserved in N. gonorrhoeae, a human pathogenic bacterium, but absent in human cells and accessible in the outer membrane, making this enzyme a good candidate for immunization. Thus, the analysis of these structural data and further studies with additional exogenous ligands and/or compounds with functional groups present in the inhibitors studied here should be pursued to develop new drugs with higher affinity and specificity.


We present a series of 1-km spatial resolution rebound (isobase) surfaces based on publicly distributed predictions obtained from the glacio-isostatic adjustment models known as ICE-5G (VM2 L90), ICE-6G_C (VM5a) and ICE-7G_NA (VM7). Our objective is to provide readily accessible tools for a broad range of geological and paleoenvironmental studies, and to facilitate direct comparison between models’ predictions and field-based observations. Rebound surfaces were interpolated at the scale of North American ice sheets  and for each time increment of the models . The assessment of the interpolations indicates that the rebound surfaces have an overall vertical accuracy of ∼0.4 m compared to original ICE-xG outputs. These rebound surfaces were combined with the GEBCO 2021 present-day elevation grid to reconstruct the paleotopography for each time increment of the models and are all presented as raster files that can be easily integrated into geographical information systems. The resulting datasets therefore provide a unique support for geological, paleoenvironmental and archeological studies. However, the predictions of these models have yet to be fully integrated into empirical research due to the gap that exists between the modelling and observational (field-based) studies. Indeed, the geological  and GIA modelling communities often operate separately, which can inhibit full-scale analyses of field-based datasets, lead to incomplete interpretations, and thus introduce some uncertainty into the models. This lack of synergy could be explained in part by model outputs that are commonly provided in formats that are often difficult to integrate into geoscience studies, while presenting a spatial resolution that is often too low to be used in paleotopographic reconstructions that generally derive from finer-scale field-based investigations. The development of easily accessible formats of model outputs would facilitate direct comparisons with geological data and lead to a better assessment of model performance. At the same time, such outputs should provide specific targets for geoscientists to collect data and thus generate new constraints with an increased precision that would in turn result in more robust boundary conditions for models.Our understanding of the evolution of the Earth system generally relies on observational data that provide basic boundary conditions for the development of complex numerical models. The ability of these models to faithfully reproduce past and current changes in the different components of the Earth system ultimately allows for simulations of future changes. As such, glacial isostatic adjustment (GIA) models represent unique tools to gain unparalleled insights about the intricate interactions between the cryosphere, the solid Earth and climate, as demonstrated by detailed analysis of past changes in relative sea level (RSL) and in the projections of the anticipated changes to come 5, ICE-5G (VM2)7, ICE_6G_C and D (VM5a)10 and ICE-7G_NA (VM7)13. Other relevant contributions with an equally good fit to observations also include the predictions generated by different combinations of Earth-ice models and parametrization approaches known as the ANU LW-6 (E-6)15, GLAC-1D16, Laur1617 and NAICE models18. Here we focus on the ICE-xG (VMy) since these models are extensively validated, in addition to be constantly improved (or refined) to fit new global field-based and observational constraints. Applications of the ICE-xG (VMy) predictions are numerous and include past/present-day ice-sheet19 and paleoclimate modelling20, transient simulations of the last deglaciation21, simulations of RSL changes and drainage pathways during critical time intervals of the last deglaciation23, paleotopographic reconstructions of Beringia24, governing parameters of ice stream dynamics , reconstructions of glacial lakes28, calibration of 10Be production sites and corrections of surface exposure ages , evaluation of North American plate angular velocity30, and evaluation of the response of hydrocarbon reservoirs to the GIA-induced vertical motion31. One of the key predictions made by the combination of ICE-xG (VMy) models consists in the postglacial rebound (PGR) component of the GIA – i.e., the viscoelastic deformation of the Earth’s surface in response to variations in surface loading generated by ice sheets growth and decay , which causes the crust to rebound in regions formerly covered by or adjacent to ice sheets, and subside beneath ocean basins. In North America, the observed PGR is mainly the result of the Laurentide Ice Sheet (LIS) deglaciation after it reached its maximum thickness and extent at the Last Glacial Maximum 33. Information about the present-day uplift rates and their spatial distribution are mostly provided by Global Positioning System (GPS) stations, while past loading histories close to the former ice dispersal centers (domes) are inferred using relative sea-level curves in coastal regions  and by deformation (tilt) of former glacial lake strandlines further inland . However, the scarcity of direct geomorphological indicators of crustal deformation at the continental scale, combined with the low number and varying resolution of geochronological constraints in the core regions of the LIS limit the inference of accurate PGR patterns .In the last decades, significant advances in the understanding of the history of global sea-level variations linked to the growth and decay of the North American ice sheets during the last glacial-deglacial cycle have been made by GIA inversion-based numerical models integrating ice thickness/loading history and Earth’s radial viscosity models – such as the series of ICE-xG (VMy) models: ICE-3G (VM1)4), which requires the interpolation of continuous (isobase) raster grid surfaces. Such high-resolution rebound surfaces combined with accurate elevation data and ice-margin histories have the potential to significantly improve paleotopographic reconstructions, which form key elements in understanding the role of meltwater released by the changes in geometry of the LIS in climate perturbations of the last deglaciation .A common challenge to all GIA models like the ICE-xG (VMy) resides in the integration of their predictions in a Geographic Information System (GIS) to facilitate paleotopographic reconstructions at a much higher resolution than their native degree-scale resolution. In fact, the development of such reconstructions primarily resides in the capacity of extracting absolute values for the time-dependent evolution of the depth of crustal depression beneath the ice masses, i.e., the subglacial paleotopography 9 and ICE-5G (VM2)7 versions, which have an equally or better fit to data in some regions. The main objective of this reconstruction is to provide accessible tools for geoscientists working in a wide array of fields  and to allow direct comparisons between the models’ predictions and the geological observations constraining these models39.Here, we derive 1-km resolution interpolated rebound (isobase) surfaces along with ice-free paleotopographic reconstructions of the glaciated North America since the LGM, based on the depth of the land deformation predicted by the three most recent iterations of the ICE-xG (VMy) models. The outputs include the predictions made by the ICE-7G_NA (VM7)www.atmosp.physics.utoronto.ca/~peltier/data.php (last accessed 2022-12-19) in their native NetCDF format at a 1 × 1 degree global grid and were imported in ArcMap using the Make NetCDF Feature layer function. The layers were then converted to points shapefiles, each comprising the models’ predicted variables provided in the NetCDF files. Since different reference frame definitions were used through the evolution of the ICE-xG models41, and as they might not be supported in common GIS software’s, all the datasets were referenced in ArcMap using the World Geodetic System 1984 (WGS84) horizontal reference frame. For the reconstructions of rebound (isobase) surfaces, the vertical datum (or reference geoid surface) was kept unspecified as the land deformation computed for each time increment is the difference in elevation between two surfaces relative to the same vertical reference frame, therefore providing absolute deformation values (or difference in orthometric heights) in meters. Before proceeding with the interpolation process at the scale of the North American continent, all data points were projected using a common projection , which is appropriate to preserve both area and length, and minimize distortion when interpolating continuous surfaces with evenly distributed point-data .The ICE-5G (VM2 L90), ICE-6G_C (VM5a) and ICE-7G_NA (VM7) datasets were downloaded from Data_points; see the Data Records section) were selected between 35.5°-89.5°N and 45°-165°W , the ice thickness (sftgit), and the ice mask (sftgif), the latter corresponding to the LIS, Cordilleran Ice Sheet (CIS) and Innuitian Ice Sheet (IIS) extent since LGM44. The first step to compute the land deformation of the land surface at X ka is to define the present-day (ice-free) topography, which will be used as the reference surface. The present-day topography is obtained by subtracting the ice thickness values at 0 ka from the orography at 0 ka. The same step is then repeated for each time increment of the model, i.e., by subtracting the ice thickness at X ka from the orography at X ka, yielding the subglacial paleotopography at X ka. The land deformation corresponds to the difference between the present-day (ice-free) topography at 0 ka and the (ice-free) paleotopography at X ka.The ICE-5G (VM2 L90) dataset (hereafter referred to as ICE-5G) is composed of 3 variables: the surface altitude/orography (stgit) and orography (orog), but also contains the topography, which includes the bathymetry and the ice thickness (Topo), the topography difference from present (Topo_Diff), the land area fraction (sftgif) and ice area fraction (sftlf), the latter two again corresponding to the ice sheet extents depicted in the Dyke et al. (refs. 44) reconstruction of the last deglaciation. The calculation of the land depression from the ICE-6G model is slightly more complex as the model is tuned to predict the presence of ice shelves that needs to be accounted for prior to the calculation. Where the ice margin is floating, the orog variable, as for the continental ice masses, gives the ice sheet surface elevation whereas the Topo variable corresponds to the ocean depth. Then, by selecting points where orog > 0 and the Topo ≤ 0, ice-shelves can be singled out. In this case, the surface loading is associated with the thickness of the water column below the floating ice mass instead of being associated with the ice mass itself. Therefore, under ice-shelves, the ice-free topography is directly given by the Topo variable at either 0 ka or X ka. Otherwise, for grounded continental ice masses, the ice-free topography at 0 ka or X ka is calculated by subtracting the ice thickness (stgit) from the topography (Topo). The land deformation corresponds to the difference between the grounded ice/ice-shelves free topography at 0 ka and the grounded ice/ice-shelves free topography at X ka, which is equivalent to the Topo_Diff variable for the non-glaciated/deglaciated areas.The ICE-6G_C (VM5a) dataset (hereafter referred to as ICE-6G) contains the same variables as the ICE-5G model, i.e., ice thickness (stgit), the topography (Topo) and the topography difference from present (Topo_Diff), but the orography variable (orog) that was used to target the ice-shelves with the ICE-6G model is absent from the ICE-7G outputs. Since the geometry of the ice sheet margins employed in the ICE-7G is also based on the deglaciation isochrones from Dyke et al. (refs. 44), it is possible to obtain the information needed to identify the land-based vs. marine/glaciolacustrine-terminating ice masses. As for the ICE-6G model, where the ice is grounded , the orography variable can be derived without difficulty as it is equal to the Topo variable. However, where the Topo < 0 and stgit > 0, and where the ice could be floating, we used the ICE-6G predictions to derive a ratio for the height of ice above the water surface vs. the ice thickness  and combined the result with the ICE-7G predicted ice thicknesses to compute the orography. Then, using the same basic conditions as for the ICE-6G model , we were able to reveal most of the ice shelves and ultimately compute the ice-free land deformation for all the points.Despite that the ICE-7G_NA (VM7) model (hereafter referred to as ICE-7G) represents the most recent and up-to-date iteration of the models, the computation of the land deformation at the scale of glaciated North America since LGM is complicated by the lack of information on ice shelves in the ICE-7G dataset. The predicted variables consist in the ice thickness  and the robustness of the results obtained with the spline interpolator , we chose to resolve this issue by manually removing the problematic points prior to the interpolation of the surfaces.A raster grid corresponding to the predicted isostatically-depressed (isobase) land surface was computed for each time increment. This was carried out in ArcMap using a spline algorithm to interpolate at a 1-km spatial resolution continuous surfaces between the projected 1 × 1 degree point-data grid. Other interpolation methods such as Natural Neighbours, Inverse Distance Weighted, Kriging and Topo To Raster were also tested. However, all were outperformed by the Spline algorithm, which is fitting a smooth surface with minimum curvature through the input points. Figure 45; hereafter referred to as GEBCO) to reconstruct the paleotopography and the paleobathymetry of the glaciated North America  that offers a full coverage at a spatial resolution of 15 arc seconds (∼500 m pixel size at the equator) of the land and seafloor topography, as well as the Greenland sub-ice topography derived from the IceBridge BedMachine Greenland . In the ocean, the GEBCO grid has an estimated absolute vertical accuracy  of 180 m nearshore and 150 m in the deep ocean46, while on land the absolute vertical accuracy is unspecified. The horizontal reference frame of the GEBCO is the same as the isobase surfaces  and the elevations refer to geoidal heights provided by the Earth Gravitational Model 1996 (EGM96) reference frame. Since the isobase surfaces were interpolated at a 1-km spatial resolution, the GEBCO was resampled at the same grid size using bilinear interpolation before subtracting the isobase surface from the modern topography to reconstruct the paleotopography of glaciated North America . Consequently, the elevation of the paleo-digital elevation models  is referenced to the WGS84/EGM96 geoid.We used the GEBCO 2021 grid  and are included in a set of geodatabases . Data_points contain the original model variables converted from the NetCDF files for each time step (see Methods section for a complete listing of the variables) to which were appended the 0 ka variables  and the computed parameters such as the orography  and land deformation (GIA); Contours are 100 m contours of the interpolated land depression; Deformation are the interpolated land depression rasters; and Paleo_subice are the PaleoDEM rasters. The ICE-7G dataset also include the GDB Data_points_used which contains the files generated following the manual removal the problematic points  prior to the interpolation of the surfaces. No raster were generated for 0 ka since the deformation is equal to 0 for each model.Our methodological approach yielded a series of 132 rebound surfaces and associated paleoDEMs raster files  deriving from interpolations at a 1-km spatial resolution for the area covered by the former North American ice sheets , for time increments ranging from 500 to 1,000 yrs for the interval covering the LGM (26-21 ka BP) to present-day. The output files for each model are available on PANGAEA . This was accomplished by first extracting the interpolated values at each point location , then by computing the difference between the reference and the interpolated values to obtain the elevation errors (Tables The best interpolation results were obtained through an iterative process using the regularized spline method (0 weight and 15 points) in ArcGIS. We assessed the overall vertical accuracy associated with this interpolator by selecting four time slices  for each model . The models’ predictions at each point location correspond to the reference  against which the interpolated values must be compared to  for the ICE-5G and left-skewed (negative) for the ICE-6G. For both models, the excess of kurtosis (>3) observed for all time slices reflects the presence of slightly heavier tails than observed with a normal distribution , thus close to normal distributions with the mean error being around zero and the standard deviation (SD) and the RMSE being identicalion Fig. . For theion Fig. . The effion Fig. , 2.Fig. th percentile error (Tables 54). For the ICE-5G model, these values correspond to 0.06 m (5 ka), 0.22 m (10 ka), 0.36 m (15 ka) and 0.36 m (20 ka). For the ICE-6G, these values are 0.06 m (5 ka), 0.20 m (10 ka), 0.33 m (15 ka) and 0.33 (20 ka) and for the ICE-7G model they correspond to 0.05 m (5 ka), 0.20 m (10 ka), 0.32 m (15 ka) and 0.33 (20 ka). The vertical accuracy of all the interpolated paleo-surfaces is therefore confidently determined at ∼0.4 m using both metrics.Overall, for all the selected time slices, the distributions of errors are centered on 0 m (unbiased), symmetrical and approximating normal distributions. All three models show a greater dispersion of the data towards older time slices Fig. , which ir Tables , 2 (e.g.56). While this ∼1 m must be added to the overall vertical uncertainties of ∼0.4 m resulting from the interpolation process  and as such, each projected/interpolated rebound surfaces can be considered as exact with respect to the predictions.Furthermore, the generalization of the datum used to reference each individual ICE-xG dataset in a GIS  and the selection of the appropriate projection  involve a transformation from the WGS84 to the NAD83 datum that results in coordinates differences of ∼1-2 m horizontally and ∼1 m vertically  that can be directly imported in ArcGIS. With routine raster operations, users will be able to modify the projection, adjust the spatial resolution and subtract the isobase raster grids from present-day high-resolution digital elevation models  to rapidly generate high-resolution PaleoDEMs that can be used to support quantitative studies . The resampling of the isobase surfaces to higher (or lower) resolutions is not expected to significantly alter the overall ∼1.4 m vertical accuracy associated with the GIS referencing/projecting and interpolation process given the very low complexity level of the terrain involved , in particular at the scale of the models native 1-degree resolution .All the 1-km resolution interpolated rebound surfaces produced in this study are available under the form of individual raster files , but with the amplitude of the Earth’s crust deformation being ∼1/3 of the ice thickness anomalies . These differences between the models’ predictions highlight the improvements between each model iterations (beyond the viscosity profiles used), notably with the addition of space-geodetic constraints between the ICE-5G and ICE-6G, and the regional optimization of the model parameters between the ICE-6G and ICE-7G models. This has led to a more accurate prediction of the deformation along the border between the Alberta and the British Columbia and over the James Bay area, in addition to a significant reduction of the deformation in central Canada and in the Keewatin sector of the LIS, which are better aligned with the predictions made by other models such as the LAUR1659 and the NAICE18.Figure 9) or predicted by the ANU model60. Although there are major differences in ice loading histories between the ICE-5G and the ICE-7G models – which cause significant differences in terms of predicted land deformation in the core regions of the LIS –, the ICE-5G interpolated surfaces are still adequate in peripheral regions of the LIS  and should still be considered to test the validity of the model against geological observations.The resulting deformation is dominated by a double bull’s-eye pattern where the extrema straddles Hudson Bay – a pattern very similar to what is obtained from GRACE observations  models generally fit the observations within 2 sigma confidence limits . Inland, the observed present-day tilt of former glacial lake strandlines can provide this type of information, although at present, the scarcity of high-resolution records combined with the limited geochronological constraints increase uncertainties. The highest level of uncertainties occurs in the interior regions of the LIS  where ice margin geometries are poorly defined and the timing of ice retreat loosely pegged in the deglaciation framework, which translates into a significant decrease in the accuracy of any type of ice thickness reconstructions. Since the same ice extent history44 is used in the different ICE-xG models to infer ice thicknesses, it is advised that any intended paleotopographic reconstructions targeting regions where limited information exist minimally consider the uncertainty associated with the ice extent history - ultimately propagated in the computed land deformation -, by integrating the predictions of ± 2 time increments before 18 ka and to ± 1 time increment after 18 ka .These differences between the models’ predictions clearly show the direct influence of the inferred ice-sheet loading history  used to tune the models. Most of the ‘direct’ information documenting the temporal evolution of the ice thickness of the North American ice sheets derives from reservoir-corrected 61) in association with predicted land deformations to evaluate key aspects of the LIS deglaciation such as the routing of the meltwaters. This can be achieved by flooding the topography using GIS hydrology algorithms to better document the redistribution of meltwater between ice sheets and the ocean domain . Such methodology has been used effectively to facilitate correlation of scattered and discontinuous shorelines mapped using LiDAR-based DEMs in the eastern Lake Agassiz-Ojibway basin to establish lake stages28, and to evaluate the configuration the areal extent of Lake Agassiz-Ojibway prior to its final drainage64. These studies show how different sources of geological information comprised by ICE-xG prediction-based paleotopographic reconstructions can eventually contribute to produce realistic estimates of the meltwater volumes involved in certain drainage events of the last deglaciation, a key parameter in evaluating the impact of freshwater forcings that presumably triggered abrupt climate changes during the early Holocene.Accordingly, accounting for the uncertainty of the ice extent history provides a range of plausible ice margin configurations and associated land deformations for a given time interval. This permits paleotopographic reconstructions integrating different ice margin configurations (e.g., ref.


Background and Aims: Gut smooth muscle dysfunctions contribute to symptoms such as abdominal cramping, diarrhea, and constipation in inflammatory bowel disease (IBD). The mechanisms for muscle dysfunctions are incompletely understood. We tested the hypothesis that mechanical stress plays a role in muscle dysfunction in a rat model of Crohn’s-like colitis where inflammatory stenosis leads to mechanical distention in the pre-inflammation site.Methods: Crohn’s-like colitis was induced by intracolonic instillation of TNBS (65 mg/kg) in Sprague-Dawley rats. Control rats were instilled with saline. The rats were fed with either regular solid food or exclusively liquid diet. Rats were euthanized by day 7.Results: When rats were fed with solid food, TNBS treatment induced localized transmural inflammation with stenosis in the instillation site and marked distention with no inflammation in the pre-inflammation site of the colon. Smooth muscle contractility was suppressed, and expression of cyclo-oxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2) were increased not only in the inflammation site but also in the pre-inflammation site. Liquid diet treatment, mimicking exclusive enteral nutrition, completely released mechanical distention, eliminated COX-2 expression and PGE2 production, and improved smooth muscle contractility especially in the pre-inflammation site. When rats were administered with COX-2 inhibitor NS-398 , smooth muscle contractility was restored in the pre-inflammation site and significantly improved in the inflammation site.Conclusion: Colonic smooth muscle contractility is significantly impaired in stenotic Crohn’s-like colitis rats not only in the inflammation site, but in the distended pre-inflammation site. Mechanical stress-induced expression of COX-2 plays a critical role in smooth muscle dysfunction in the pre-inflammation site in Crohn’s-like colitis rats. Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), represents a group of chronic gut inflammatory disorders with remitting and relapsing features that lead to significant disruption in bowel function and patient quality of life . IBD prePatients with IBD suffer from symptoms such as abdominal cramping, diarrhea, and rectal bleeding during active inflammation or relapses. Previous investigations have shown that gut smooth muscle dysfunctions account for cramping, diarrhea, and constipation . Those sThe gastrointestinal (GI) tract is subjected constantly to mechanical stress as intraluminal contents  pass through it . During 2, which mediates inflammatory responses and has profound effects on gut smooth muscle function (Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for converting arachidonic acid to prostaglandins (PG), such as PGEfunction . COX-2 ifunction . We hypofunction , was foufunction . FurtherAll the animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch in Galveston, Texas. Experiments were performed in accordance to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, United States.ad libitum with regular pellet foods  unless specified otherwise. The average daily intake of the pellet food for a control rat is nearly 20 g with ∼70 kcal . Rats were euthanized 7 days after saline or TNBS treatments. Body weight was documented over 7-day course. Inflammation was scored 1 to 3 in each rat according to criteria described previously  were used in the study. Rat model of CD-like colitis was induced by intracolonic instillation of TNBS  as described previously . Prior teviously .in vivo inhibition of COX-2 activity, control and TNBS-instilled rats were treated with COX-2 inhibitor NS-398  daily at 5 mg/kg intraperitoneally (ip) in 250 µL of 20% DMSO starting on the day inflammation was induced  ad libitum . Although the rodent liquid diet is not identical as Ensure liquid diet in terms of nutrient contents, it had very similar effect as Ensure liquid diet in eliminating mechanical distention in the colon. We decided to use Ensure liquid diet in the study, as it has been used in the EEN treatment for CD patients and in eliminating mechanical distention in rats . After cleansing, the colon was opened along the mesenteric border, and pinned flat in a Petri dish with Sylgard base . DiffereImmunohistochemistry staining of COX-2 was performed as described previously , on formT (ΔΔCT) method with endogenous reference 18S rRNA  as the normalizer.Total RNA was extracted from tissues using the Qiagen RNeasy kit . One microgram of total RNA was reverse transcribed by using the SuperScript III First-Strand Synthesis System  . Real-ti2 was measured with the PGE2 enzyme immunoassay kit from Cayman Chemical  by following the manufacturer’s protocols  supplemented with protease inhibitors for protein extraction. PGErotocols .–6 to 10–2 M) in the muscle bath. ACh doses were tested one by one incrementally with the lowest dose first and the highest dose last with an interval of 2–3 min between each dose of ACh. The ACh-evoked contractile response was quantified as the increase of AUC during 2 min after addition of each ACh dose to the bath over the baseline AUC during 2 min before the addition of the lowest dose of ACh. The tissue dry weight of each muscle strip was used to normalize the contractile activities.Freshly obtained colon segments were opened along the mesenteric border, cleansed, and pinned flat in a Petri dish with Sylgard base in carbogenated Krebs solution. The muscularis externa layer was collected for muscle bath experiments. The smooth muscle strips (3 mm × 10 mm) were mounted along the circular muscle orientation in individual muscle baths  filled with 10 mL of carbogenated Krebs solution at 37°C. The contractile activity was recorded as previously described  with Grap-value of ≤0.05 was considered statistically significant.All data points are expressed as means ± SEM. Statistical analysis was performed by analysis of variance with non-repeated measures (by Student-Newman-Keuls test) for comparisons of multiple groups and Student’s t-test for comparisons of two groups. A When rats were fed with regular pellet food, intracolonic instillation of TNBS led to localized transmural inflammation with stenosis in the instillation site (site I) and lumen distention in the site proximal to inflammation (site P). Though mechanically distended, site P did not show visible inflammation. Interestingly, the colonic site distal to inflammation (Site D) showed neither distention nor inflammation . However−6 ∼ 10−2 M)) was dramatically suppressed in the I-site compared to the corresponding site of control rats (p < 0.05 vs. sham control. N = 5 each group)  in the colitis rats compared to sham controls when they were fed with regular solid food . We founh group) . This reh group) . More imh group) . Howeverh group) . The sitLiquid diet treatment was found to release fecal retention in constipation . In the We then determined the site-specific changes of muscle contractility in colitis when rats were fed exclusively with liquid diet and mechanical distention was eliminated in the colitis rats. Our results showed that muscle contractility was significantly improved in the I-site and largely normal in the P-site in inflammation when the rats were fed with liquid diet . Interesp < 0.05 vs. sham control. N = 4 or 5) . Furthermore, COX-2 mRNA expression was increased significantly also in the mechanically distended P-site [16.6 (±4.3)-fold] -fold] . However3)-fold] . This da3)-fold] .2 is among the most potent prostaglandins derived from COX-2 and found to suppress colonic smooth muscle contractility in rats  but also in the P-site  of the colitis colon, compared to sham colon   (PGE in rats . We next3 pg/μg) . Howevered rats) .P = 0.02 vs. sham. N = 4 or 5 rats in each group). However, when rats were administered with NS-398, TNBS colitis rats did not demonstrate any significant body weight decrease compared to sham control rats (p > 0.05 between sham/vehicle and sham/NS-398). We also assessed inflammation in the rats. NS-398 showed a trend to reduce inflammation in TNBS colitis rats (p = 0.064 vs. TNBS colitis with vehicle administration. N = 4 or 5) (p < 0.01 vs. sham control.)  or vehicle control was administered in sham and colitis rats daily. Rats were fed with regular pellet food and euthanized 7 days later. The body weight was decreased significantly over the 7-day course in TNBS-induced colitis rats when treated with vehicle (rol rats . NS-398  4 or 5) . Howeverontrol.) .p < 0.01 vs. sham control. N = 5 each group) . However, with NS-398 administration, the baseline contractile activity in the P-site of TNBS colitis rats was not different from sham control (p > 0.05 vs. sham control. N = 5) . The baseline activity of the I-site was still significantly reduced compared to sham control (p < 0.01) even with NS-398 treatment. However, NS-398 treatment improved contractile activity in the I-site compared to the same site with vehicle treatment (p = 0.03) . Representative traces of colonic baseline contractile activity were shown for each group (p > 0.05 between sham/vehicle and sham/NS-398).The baseline contractile activity of colonic muscle strips was significantly decreased in TNBS colitis rats in both P-site and I-site in the DMSO vehicle treatment group (ch group . NS-398 –6 to 10–2 M) in both the I-site and the P-site   in muscle contractility at the distended P-site for all ACh concentrations, indicating that COX-2 may play a critical role in smooth muscle dysfunction in the pre-inflammation site . NS-398 treatment also partially improved muscle function in the inflammation site (I-site), as smooth muscle contractility at the I-site was not significantly different between sham and TNBS colitis rats for all ACh concentrations, except that of ACh 10−2 M  . Compared to vehicle treatment, NS-398 administration decreased the contractile response of control colons to ACh 10−4 and 10−3 (M) . Representative traces of smooth muscle contractile response to ACh doses were shown for each group  and significantly decreased PGE2 level at I-site  compared to the I-site level in rats with no NS-398 treatment .We also studied muscle contractile response to cholinergic agonist ACh and found that in the TNBS-treated colitis rats given a DMSO vehicle control, there was a significant reduction of muscle contractility in response to all ACh concentrations  phosphorylation . We do not know exactly what accounts for these changes. However, although NS-398 is considered a specific COX-2 inhibitor, its possible effects on COX-1 or other non-specific targets and associated changes of various prostaglandins may have complicated effect on smooth muscle contractions especially high-amplitude contractions as that induced by high doses of ACh. Nevertheless, we used the same doses of NS-398 (5 mg/kg) and vehicle control (DMSO) for sham and colitis rats to minimize any non-specific effect of NS-398 on muscle contractility.Our results showed that inhibition of COX-2 activity with NS-398 administration not only improved colonic smooth muscle function, but also blocked body weight decreases in colitis rats. Interestingly, NS-398 treatment did not significantly reduce inflammation. This data indicates that improved gut smooth muscle function and motility during gut inflammation may contribute to the wellbeing of the animals with colitis. We noticed that compared to vehicle treatment, NS-398 administration did not affect baseline contractile activity of the control colon but decreased the contractile response to ACh 10in vivo, we focused our effort on the pre-inflammation site in the present study. Our study suggests that mechanical stress, via mechano-transcription of mechanosensitive molecules such as COX-2, may play a crucial role in smooth muscle dysfunction in CD.It is noteworthy that mechanical stress is commonly encountered in gut inflammation. Inflammatory infiltration, edema, tissue deformation and fibrosis in the inflamed tissue  are all The current goal of treatment in CD is to induce prolonged remission. Corticosteroids, immune modulators, and biologic agents are all found to be effective in reducing inflammation . UnfortuLiquid diet, such as EEN for the management of Crohn’s disease, may affect gut microbiota composition and diversity . It was 2 production are increased not only in the inflammation site, but also in the distended pre-inflammation site. Liquid diet treatment mimicking EEN regimen released lumen distention, eliminated COX-2 expression and PGE2 production, and improved smooth muscle contractility. Administration with COX-2 inhibitor NS-398 significantly improved colonic smooth muscle contractility in the pre-inflammation and inflammation sites in colitis rats. These results suggest that mechano-sensitive expression of COX-2 plays a critical role in colonic smooth muscle dysfunction in Crohn’s-like colitis rats. Our study indicates that the benefits of EEN in CD may depends on its effect to release mechanical stress in the gut.Taken together, we found in the stenotic Crohn’s-like colitis model that colonic smooth muscle contractility is suppressed, and COX-2 expression and PGE


Impairment of development, migration, or function of inhibitory interneurons are key features of numerous circuit-based neurological disorders, such as epilepsy. From a therapeutic perspective, symptomatic treatment of these disorders often relies upon drugs or deep brain stimulation approaches to provide a general enhancement of GABA-mediated inhibition. A more effective strategy to target these pathological circuits and potentially provide true disease-modifying therapy, would be to selectively add new inhibitory interneurons into these circuits. One such strategy, using embryonic medial ganglionic (MGE) progenitor cells as a source of a unique sub-population of interneurons, has already proven effective as a cell transplantation therapy in a variety of preclinical models of neurological disorders, especially in mouse models of acquired epilepsy. Here we will discuss the evolution of this interneuron-based transplantation therapy in acquired epilepsy models, with an emphasis on the recent adaptation of MGE progenitor cells for xenotransplantation into larger mammals. Interneurons mediate inhibitory synaptic transmission, brain oscillations and cortical processing . AlteratPreclinical transplantation studies using murine embryonic progenitors from medial ganglionic eminence (MGE) consistently demonstrate robust abilities to (i) migrate, differentiate and integrate into host circuits as inhibitory interneurons , (ii) main vitro, such as fast-spiking and regular-spiking firing properties in current-clamp recordings  and somatostatin-expressing (SOM) cells . Over thcordings , and reccordings . Importacordings . Enhancecordings . As expecordings  and optocordings . Moreovecordings . The conMice provide unique advantages for development of MGE-derived transplantation protocols as a potential disease modifying therapy for epilepsy. First, our understanding of interneuron origins, sub-populations and functions derive from a wealth of rodent publications. Second, a variety of rodent epilepsy models are already available and easily accessible. As discussed, anatomical, immunohistochemical and functional properties of transplanted murine MGE-derived interneurons are now established . As are Potential physiological incompatibility and ethical issues that come with murine progenitor cells limit the translation of this approach to a larger mammal. While some would argue that human induced pluripotent stem cells (hiPSC) could solve these issues, published hiPSC protocols do not convincingly report data for PV-and SOM-expressing neurons with the ability to migrate widely and functionally integrate as mature interneurons following transplantation. Despite Nkx2.1, Lhx6, and Dlx2, indicative of an MGE lineage   . Fourth,r (vGAT) . Note, cWhile additional characterization of porcine MGE progenitors awaits, a highly successful N-of-1 trial was recently reported for a refractory, domoic-acid poisoned epileptic California sea-lion playfully named Cronutt . Domoic-While precise mechanism(s) by which interneuron dysfunction contributes to epilepsy  remains an active area of investigation, an aspirational treatment goal is a “no seizures, no side effects” outcome. Although this goal remains elusive, patient and animal model data converge on a hypothesis that such a treatment could involve enhancement of GABA-mediated synaptic inhibition. Transplanted embryonic MGE progenitors have consistently been shown to migrate and functionally integrate in host circuits to enhance synaptic inhibition in a manner resembling that possible with native interneurons. Properties of interneurons derived from murine and porcine MGE progenitors make them ideal candidates for a cell transplantation treatment . Indeed,JR wrote the first draft of the manuscript. SB wrote and reviewed the final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by NIH/NINDS grant R01 NS-071785–13 to SB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Staphylococcus aureus is an important and common microbial cause of SSI. Nasal carriage of S. aureus has been shown to be an important determinant for the development of SSI, and interventions aimed at eradicating preoperative nasal carriage are associated with a reduced risk of infection. Yet, it is not entirely clear how the nasally residing S. aureus causes SSI at distant body sites. In this commentary, we describe our view on how S. aureus can be transported from the nares to the incision site during surgery. In addition, we shed light on the implications of our view for infection prevention research.Surgical site infections (SSIs) are important healthcare-associated infections, leading to increased morbidity and mortality, healthcare costs, and prolonged hospital stays.  SSIs are caused by a variety of microorganisms, depending on the type of surgery that is performed. In clean surgical procedures, Staphylococcus aureus is a common microbial cause of SSI.S. aureus is a commensal microorganism that is present on the skin and mucosal surfaces of humans and different types of animals. Population-based cross-sectional studies have shown that approximately 20–30% of the human population is colonized with S. aureus, and longitudinal studies have demonstrated that at least three S. aureus carriage patterns can be distinguished in a healthy population: persistent carriers, intermittent carriers, and noncarriers. The primary body site associated with S. aureus carriage is the nares of the nose, although S. aureus also colonizes other body sites, including the throat, axilla, and perineal area.Surgical site infections (SSIs) are important healthcare-associated infections, leading to increased morbidity and mortality, healthcare costs, and prolonged hospital stays.S. aureus colonization of the patient, particularly nasal S. aureus colonization, and the occurrence of S. aureus infections, including S. aureus SSI. In addition, studies have shown that eradication of S. aureus colonization by using decolonization treatments, including antiseptic body washes and nasal mupirocin, leads to a substantial decrease in the incidence of S. aureus SSIs and other staphylococcal infections. Furthermore, studies that employed genetic characterization of staphylococcal samples, derived from S. aureus colonized (including the nose) and infected body sites from the same individuals, have shown that a large proportion of infecting S. aureus shares strain type with colonizing S. aureus, suggesting an endogenous origin. For instance, the ASPIRE-SSI study , which was a prospective cohort study conducted at 33 sites in 10 European countries and that assessed the incidence and etiological factors for S. aureus SSI and bloodstream infections, found that 83% of S. aureus SSI in S. aureus carriers had probably an endogenous origin . Based on these findings, the question arises of how colonizing S. aureus lead to infections at other body sites. Answering this question is crucial to further guide interventions aimed at reducing the incidence and burden of S. aureus SSI.A substantial body of evidence has shown that there is a significant association between S. aureus carriage site is the nares of the nose. In theory, there are several mechanisms through which S. aureus can spread from the nose to the site of infection. With regard to SSI, the bacterial transmission from the nares to the surgical site can occur via one of four routes (or a combination of these routes): (1) direct contact through contaminated skin or mucosal surfaces; (2) indirect contact involving contaminated operating room (OR) equipment or instrumentation; (3) hematogenous transmission from the colonized body site to the surgical site; and (4) airborne transmission via the air within the OR.As stated earlier, the most common S. aureus, and the surgical wound becomes infected when or shortly after the incision is made. It has been shown that the skin of nasal carriers is frequently colonized with S. aureus originating from the reservoir in the nose. So, it is safe to assume that nasal carriage leads to skin carriage and that this can subsequently cause infection at distant body sites. However, it is standard practice to use antiseptic agents at the surgical site prior to surgery, which should prevent the development of infection via this route.This occurs when the skin covering the surgical site is colonized by S. aureus originating from the nose of the patient, come into contact with the surgical site. However, considering the high level of infection control and especially aseptic conditions during surgery, this transmission route is considered unlikely as a cause of S. aureus SSI.This can occur when for instance medical equipment or OR personnel that are contaminated with S. aureus enters the bloodstream while the patient is undergoing surgery. For instance, S. aureus may enter the blood circulation following injury of the oropharyngeal mucosal surface during mechanical intubation. Incidental S. aureus bacteremia may cause secondary infection of the surgical site. While this is theoretically possible, it is common practice to treat patients perioperatively with antibiotic prophylaxis consisting of broad-spectrum antibiotics with good activity against S. aureus. Therefore, this is probably also not a major transmission route in regard to S. aureus SSI.This can occur when  In addition, compared to S. aureus carriers with lower bacterial loads of colonizing S. aureus, patients who are more heavily colonized with S. aureus are known to spread more S. aureus to their surroundings. Therefore, airborne transmission could be an important source of infection in S. aureus SSI.This can occur when pathogens are carried to the surgical site by small airborne particles, such as droplet nuclei. These pathogen-loaded particles arise during procedures related to the surgery, for instance during inhalation anesthesia, are disseminated by air currents in the OR, and can remain airborne for extended periods of time. Earlier studies have shown that improper OR ventilation can lead to displacement of airborne particles carrying bacteria to the surgical site.S. aureus from the nares to the surgical wound, even though we cannot exclude the other options. There is no question that the filtered air that is introduced into the OR is clean and does not contain S. aureus. However, there are many factors in the OR that can disturb the airflow during surgery, including OR lights, frequent door openings, patient warming devices, and people. These disturbances cause air turbulence, and this can subsequently lead to contaminated particles being transported to the incision site. As an example, this online video shows how particles, that are produced near the head of a patient, are transported to the incision site (ref: https://youtu.be/yq-hVBjgZEk). There are also experimental data showing that direct ventilation of the operative site with a mobile laminar airflow unit, which is not influenced by lights etc., does significantly decrease the bacterial contamination of the surgical site.Considering the abovementioned arguments, we argue that airborne transmission is currently the most likely route of transmission of  which is consistent with results from earlier reviews. Whilst this is true, it is important to note that the majority of these studies were conducted with national surveillance and registry data, which often lack comprehensive confounder data. In addition, these studies were not conducted with S. aureus carriers specifically, which may have diluted the effect.However, the literature is still ambiguous with regard to the role of airborne transmission in the etiology of SSI. The studies conducted to date assessing the relationship between different air flow types in the OR and SSIs have yielded contradicting results. In addition, a recent meta-analysis found no evidence that laminar flow (which is considered the standard of care in modern surgery) is associated with a lower occurrence of SSI compared to conventional turbulent airflow,S. aureus SSI in S. aureus carriers. Current guidelines, including the WHO guidelines, recommend the use of perioperative decolonization in combination with skin disinfection prior to surgery in nasal carriers of S. aureus undergoing high-risk surgical procedures. While this approach is proven effective and targets the reservoir of the infection, it does not take into consideration the route of transmission. As a matter of fact, current guidelines do not account for airborne transmission at all as a possible transmission route in the etiology of SSI. Therefore, we believe that more research is needed to elucidate the role of airborne transmission in the development of S. aureus SSI. For instance, modeling studies assessing the airflow dynamics in the OR under different conditions, or trials including S. aureus carriers specifically and assessing the association between different air flow modalities in the OR and the occurrence of S. aureus SSI, could inform us about the importance of airborne transmission in the etiology of S. aureus SSI. With this knowledge, we will hopefully be better suited to develop more effective antistaphylococcal preventive interventions.Based on the above, we conclude that the current evidence does not completely rule out airborne transmission as a major player in the etiology of