These studies suggest that redox modification of PKA-Cα differentially affects its activity toward different substrates. For instance, we found that diamide-mediated oxidation caused a marked decrease in PKA-Cα’s activity toward some substrates  while having little effect on others . In contrast, H2O2-dependent oxidation of PKA-Cα led to an increase in its activity toward each of the substrates at relatively low H2O2 concentrations, with differential effects at higher peroxide concentrations. Together, these studies offer novel insights into crosstalk between redox- and phosphorylation-dependent signaling pathways mediated by PKA. Likewise, since C199 is highly conserved among AGC kinase family members, they also lay the foundation for future studies designed to elucidate the role of redox-dependent modification of kinase substrate selection in physiological and pathological states.The cyclic AMP-dependent protein kinase (PKA) plays an essential role in the regulation of many important cellular processes and is dysregulated in several pervasive diseases, including diabetes, cardiovascular disease, and various neurodegenerative disorders. Previous studies suggest that the alpha isoform of the catalytic subunit of PKA (PKA-Cα) is oxidized on C199, both in vitro and in situ. However, the molecular consequences of these modifications on PKA-Cα’s substrate selection remain largely unexplored. C199 is located on the P + 1 loop within PKA-Cα’s active site, suggesting that redox modification may affect its kinase activity. Given the proximity of C199 to the substrate binding pocket, we hypothesized that oxidation could differentially alter PKA-Cα’s activity toward its substrates. To this end, we examined the effects of diamide- and H Though a great deal is known about how kinases function in cellular signal transduction, questions still remain about the mechanisms by which specificity is achieved in cellular signaling processes. For instance, a given kinase may drive one cellular process  under one condition while promoting a different set of outcomes under another condition . While the spatial and temporal regulation of kinase activity is believed to play an important role in determining when and where a given kinase is activated inside the cell, crosstalk with other signaling pathways also plays an important role in achieving signaling specificity. Recently, crosstalk between redox- and phosphorylation-dependent signaling pathways has been implicated in various pervasive diseases, including diabetes, cancer, cardiovascular disease, and several neurological disorders, including Alzheimer’s disease ,13,14,15i < 1 nM, the means by which they do so are quite different -[R/K]-X-[S/T]-X consensus phosphorylation motif recognized by PKA-Cα, Crosstide (GRPRTSSFAEG) contains a looser PKA consensus motif. Consistent with previous studies, pre-treatment of PKA-Cα with low  concentrations of diamide caused a modest decline in its activity toward Kemptide followed by a more pronounced decrease at higher concentrations of oxidant  followed by differential changes in activity toward each substrate at higher H2O2 concentrations. Strikingly, in contrast to diamide, PKA-Cα does not appear to form either intra- or intermolecular disulfide bonds under these conditions, as evidenced by non-reducing PAGE  is a versatile, label-free method that utilizes ligand-dependent changes in the refractive index at the surface of a gold-plated sensor surface to measure biophysical parameters, such as the binding affinity and association/dissociation kinetics characteristic of protein-protein interactions, in real-time [α-CREBtide interactions suggests that H2O2-dependent oxidation of PKA-Cα decreases its affinity for CREBtide  of 3.3 ± 1.0 µM, which is very similar to the reported KD of 3.9 µM for the PKA-Cα-CREBtide interaction [2O2 increased its KD,app to 7.2 ± 0.8 µM, corresponding to a 2.2-fold decrease in affinity. Kinetics analyses using SPR suggest that the altered affinity of PKA-Cα for CREBtide is due primarily to changes in the apparent off-rate  , koff,app increased over 2.9-fold following H2O2 treatment . This yielded a 2.2-fold change in KD,app, which is very similar to what was observed during the equilibrium binding experiments. Consistently, similar oxidation-induced changes in the affinity of PKA-Cα for full-length CREB fused to an N-terminal glutathione-S-transferase tag (GST-CREB) were also observed when binding was probed using steady-state kinetics analysis  . Indeed,analysis . For insα and the other peptides , we were not able to accurately measure PKA-Cα’s affinity for these substrates using SPR. Instead, we used steady-state kinetics analysis to investigate the biochemical basis for H2O2-dependent changes in PKA-Cα’s activity toward these substrates , the residue in the P-3 position  forms H-bonds with E127 in the hinge region, the backbone carbonyl of T51 in the N-lobe, and the 3′-OH of ATP in the active site cleft (RRTSSFAEG), the diamide titration curve looks very similar to that observed for Kemptide and CREBtide  before decreasing at higher peroxide concentrations. Because PKA-Cα oxidized by 5 µM H2O2 can be glutathionylated by subsequent treatment with GSH-biotin, it is likely that this ratio of H2O2-to-PKA-Cα leads to sulfenylation of the enzyme . Similarly, higher concentrations of H2O2 may lead to the formation of higher-order oxoforms, such as sulfinic acid and sulfonic acid, which could differentially affect PKA-Cα’s activity toward different substrates. This may account for the differences that we observed in the phosphorylation profiles of each substrate at the higher H2O2 concentrations. Interestingly, PKA activity has been shown to be modulated by redox-dependent signaling mechanisms during several key cellular processes [2O2 production leads to decreased rates of lipolysis. This decrease correlated with H2O2-dependent inhibition of PKA activity, as assessed by Kemptide phosphorylation [2O2-dependent inhibition of PKA in adipocytes is believed to be due to the formation of a disulfide bond between C199 in the catalytic subunit and C97 in the regulatory RII subunits expressed in adipocytes, the PKA catalytic subunit does not form a similar linkage with the RI subunits present in macrophages because C97 is not conserved in RI (though a disulfide may form between the two RI subunits in the type I holoenzymes) [Unlike diamide, oxidation of PKA-Cα by the physiological oxidant, He enzyme C. Sulfene enzyme B. At therosstide . In the rocesses ,39,40,41rylation . In contenzymes) . Interes2O2-dependent oxidation of several AGC family members, including PKA-Cα, altered their activity toward their cognate substrates [2O2-dependent oxidation of the kinase led to a dose-dependent decrease in its activity toward Kemptide. This is in contrast to the dose-dependent increase in PKA-Cα activity toward Kemptide that we observed during our H2O2 titration assays , the molar ratio of H2O2-to-PKA-Cα was 333,333:1 (100 µM H2O2:0.3 nM PKA-Cα). Meanwhile, we observed a maximal increase in PKA-Cα activity toward Kemptide at a 20:1 molar ratio (5 µM H2O2:250 nM PKA-Cα). Indeed, at higher H2O2 concentrations (corresponding to molar ratios between 60:1 and 160:1), we observed a ~15% decrease in PKA-Cα activity toward Kemptide, which is on par with the level of inhibition observed by Byrne et al. using 100 µM H2O2. This may suggest that PKA-Cα is initially sulfenylated at relatively low H2O2 concentrations, leading to an increase in activity toward Kemptide (and CREBtide and Crosstide in our assays) before undergoing hyperoxidation to sulfinic and/or sulfonic acid that may decrease its activity toward these substrates. At very high H2O2-to-PKA-Cα ratios , oxidation of C343 may promote intramolecular disulfide bond formation similar to what was observed following diamide treatment, inhibiting PKA-Cα’s activity toward Kemptide to a greater extent. Interestingly, even at the highest H2O2 concentrations used in our assays (corresponding to a 160:1 molar ratio), PKA-Cα-mediated phosphorylation of CREBtide remained elevated relative to the untreated control while its activity toward both Kemptide and Crosstide fell below baseline, underscoring the differences observed between substrates. Together, these data suggest that redox modification of PKA-Cα differentially alters its activity toward different substrates. In the future, it will be interesting to explore the molecular mechanisms underlying oxidation-induced changes in PKA-Cα-substrate interactions.In addition to its downstream substrates, PKA-Cα also recognizes key regulatory factors via redox-sensitive ligand-binding regions. For instance, interactions between PKA-Cα and regulatory factors, such as the endogenous PKA inhibitor, PKI, or the PKA regulatory subunits, RI and RII, are mediated by PKA-C’s P + 1 loop ,42,43,44bstrates . In the n assays A. This a


There are various internal fixation methods in treating lumbosacral spinal tuberculosis. The study compared the stability and stress distribution in surrounding tissues/implants, such as discs, endplates and screw-rod internal fixation system, etc. when applying three different lumbar internal fixation methods to treat lumbosacral spinal tuberculosis. A finite element model was constructed and validated. The spinal stability was restored using three methods: a titanium cage with lateral double screw-rod fixation (group 1), autologous bone with posterior double screw-rod fixation (group 2), and a titanium cage with posterior double screw-rod fixation (group 3). For comparison, group 4 represented the intact L3-S1 spine. Finally, a load was applied, and the ranges of motion and Von Mises stresses in the cortical endplates, screw-rod internal fixation system and cortical bone around the screws in the different groups were recorded and analyzed. All six ranges of motion  of the surgical segment were substantially lower in groups 1 (0.53° ~ 1.41°), 2 (0.68° ~ 1.54°) and 3 (0.55° ~ 0.64°) than in group 4 (4.48° ~ 10.12°). The maximum stress in the screw-rod internal fixation system was clearly higher in group 2 than in groups 1 and 3 under flexion, left/right lateral bending, and left/right rotation. However, in extension, group 1 had the highest maximum stress in the screw-rod internal fixation system. Group 2 had the lowest peak stresses in the cortical endplates in all directions. The peak stresses in the cortical bone around the screws were higher in group 1 and group 2 than in group 3 in all directions. Thus, titanium cage with posterior double screw-rod fixation has more advantages in immediate reconstruction of lumbosacral spinal stability and prevention of screw loosening. With the increase in the number of patients with acquired immunodeficiency and the widespread use of immunosuppressive drugs, the number of spinal tuberculosis patients is also increasing annually5. Delayed treatment of spinal tuberculosis may lead to vertebral bone destruction and collapse, kyphosis and even paralysis due to compression of the spinal cord or nerves8. Currently, drug therapy consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide is the preferred treatment for spinal tuberculosis. However, drug therapy cannot easily cure “closed lesions.” Moreover, 8% to 12% of patients have resistance to first-line antitubercular drugs4. Surgical treatment is still necessary for patients with spinal cord injury and spinal instability due to tuberculosis lesions10. The purpose of such operations is to completely remove the lesions, correct spinal deformities, relieve nerve compression, and improve the patient’s quality of life. Three main surgical strategies for treating lumbosacral spinal tuberculosis are the anterior-only approach, the posterior-only approach, and the combined anterior and posterior approach11. Although each approach has its own advantages and disadvantages, all three approaches have been widely used in clinics and have achieved satisfactory clinical efficacy for patients with lumbosacral spinal tuberculosis. Regardless of the surgical approach, the three most common internal fixation methods for restoring spinal stability after lesion removal are a titanium cage combined with lateral double screw-rod fixation12, autologous bone combined with posterior double screw-rod fixation13, and a titanium cage combined with posterior double screw-rod fixation14. However, clinical follow-up showed that the three internal fixation methods had complications such as prosthesis subsidence, screw loosening and titanium rod fracture, etc., and there is still a lack of relevant research on which internal bone fixation method has more advantages in mechanical properties. Therefore, to evaluate the influence of the three different internal fixation methods on the stability of the lumbosacral segment and the surrounding tissues during spinal reconstruction, we performed finite element (FE) analyses and compared the biomechanical properties of the three methods. The ranges of motion (ROMs) and Von Mises stresses in the discs, facet joints, cortical endplates, screw-rod internal fixation system and cortical bone around the screws in the different groups were compared. The results of this study will provide a useful reference for the clinical application of the three different internal fixation methods.Spinal tuberculosis, especially in the thoracic and lumbar segments, is a common type of extrapulmonary tuberculosis that accounts for approximately 50% of osteoarticular tuberculosis cases18, a healthy adult male volunteer  underwent a CT scan  of the L3-S1 spine. Then, the CT scan data were imported into Mimics  to generate a surface model of the vertebrae. In addition, 3-matic software  was used to construct solid models of the cortical shell (1125 Hounsfield Unit), cancellous bone (220 Hounsfield Unit), and intervertebral disc. Meshed models of the bony and ligamentous structures were constructed using Hypermesh . Abaqus  was used for material property definitions, model assembly, and FE analysis. Mesh refinement was executed for modeling accuracy until excellent monotonic convergence behavior with less than 5% difference in the L3-top displacement was achieved In this study, the boundary conditions described below were applied to the models, and convergence was tested for based on range of motion, bone strain, and disc stress. The size and density of the hexahedral elements was controlled manually, while the density of the tetrahedral elements was controlled by determining an appropriate average element side length.According to the modelling processes reported by some previous studies20. The intervertebral disc was divided into the nucleus pulposus and annulus fibrosus. The nucleus pulposus accounts for 30 ~ 40% of the intervertebral volume20. The nucleus pulposus was modelled as a linearly elastic solid element. The annulus fibrosus consisted of the annulus ground substance and fibres. Seven layers of annulus fibres were embedded into the annulus ground substance at an inclination of ± 30°20. The elastic modulus of the annulus fibres proportionally increased and varied from the innermost layer (360 MPa) to the outermost layer (550 MPa)21. In total, 7 ligaments were modelled: the anterior longitudinal ligament, posterior longitudinal ligament, ligamentum flavum, capsular ligament, interspinous ligament, supraspinous ligament, and intertransverse ligament. The element types and material properties used in the FE model  ranges of motion (ROMs) in response to 8.0 Nm loads were compared with the outcomes described in previous publications23.Figure 25. For all surgical models, the cage-end plate and screw-bone interfaces were defined as tied contacts to simulate internal fixation. See Table The FE models of the three lumbar internal fixation methods are shown in Fig. As shown in Fig. This study was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the Ethics Committee of Xi’an Jiaotong University. A computed tomography (CT) scan of the L3-S1 spine was obtained with the informed consent of volunteers, and the dissemination of relevant data was allowed for academic exchange.26. The intersegmental ROMs of the intact FE model at the L3-4 disc in the FLX, EXT, LLB, RLB, LT and RT directions were 4.5°, 2.8°, 3.4°, 3.2°, 2.3° and 2.1°, respectively. In contrast, the ROMs of the L4-5 disc in these directions were 4.9°, 3.7°, 3.6°, 3.5°, 2.3° and 2.1°, respectively. Moreover, the ROMs of the L5-S1 disc in these directions were 5.2°, 4.3°, 2.9°, 2.9°, 2.2° and 2.4°, respectively. Compared with the results of Shim's study26, the ROM of the L3-4 disc in our study was reduced by 9.5% in the RLB direction; the ROMs of the L4-5 disc in RLB and RT directions decreased by 7.0% and 4.0%, respectively; the ROMs of the L5-S1 disc in FLX, EXT and RT directions increased by 10.6%, 7.5% and 14.3%, respectively. However, The ROM results of the discs in our study in other directions were in agreement with the findings of Shim’s study. In addition, we compared the stress of the intervertebral disc of L3-4 and L4-5 with the relevant literature  in the FLX, EXT, LLB, RLB, LT and RT directions are shown in Table -S1 in thFigure Figure Figure The comparisons of the maximum Von Mises stresses of the L3-4 disc and facet joints of the three groups were shown in Fig. 27. Surgical interventions are required for patients with progressive neurological functional impairment, severe kyphosis, and massive cold abscesses and for those who have had no response to conservative treatment28. The three most common surgical strategies used for patients with spinal tuberculosis are a titanium cage combined with lateral double screw-rod fixation, autologous bone combined with posterior double screw-rod fixation, and a titanium cage combined with posterior double screw-rod fixation. However, it is not clear which internal fixation method can best restore the stability of the surgical site and reduce the incidence of prosthesis loosening and displacement. In this study, FE analyses were performed to compare the biomechanical properties of these three internal fixation methods, the results of which should provide a useful reference for their clinical application.Lumbosacral spinal tuberculosis accounts for 2–3% of all cases of spinal tuberculosis29, suggesting that all three methods of internal fixation can restore the stability of the surgical site after lesion removal. Further analysis showed that the ROMs of L4-S1 in group 3 were lower than those in group 2, indicating that under posterior double screw-rod fixation, a titanium cage could achieve better stability than autologous bone. Wu W et al.14 also compared titanium cage with autograft in the treatment of spinal tuberculosis, and found that titanium cage combined with posterior screw-rod fixation was more advantageous, which is consistent with our findings. Although group 1 and group 3 had similar ROMs at the surgical site in the LLB, RLB, LT and RT directions, the ROMs of L4-S1 in group 1 were higher than those in group 3 in the FLX and EXT directions, indicating that after titanium cage implantation, posterior double screw-rod fixation could achieve better stability in the FLX and EXT directions than lateral double screw-rod fixation. Reisener et al.30 reviewed the literature and found that titanium cage combined with posterior screw- rod fixation was more common than titanium cage combined with lateral double screw-rod fixation in the treatment of lumbosacral spinal diseases. In addition, Zheng et al.10 found that posterior screw-rod fixation was more advantageous than lateral double screw-rod fixation in rebuilding the stability of the lumbosacral spine. Thus, a titanium cage combined with posterior double screw-rod fixation can best restore the stability of the surgical site.This study showed that the ROMs of L4-S1 in the three internal fixation groups (groups 1–3) were substantially lower than those in group 4, which was consistent with the reduced ROMs at the surgical site after internal fixation14. However, the maximum Von Mises stresses in the screw-rod system in group 1 were higher in the FLX and EXT directions and lower in the LLB, RLB, LT and RT directions than those in group 3. This indicates that the two internal fixation methods have their own advantages in different movement directions. As lumbar FLX and EXT occur much more frequently in daily life than LLB/RLB and LT/RT, a titanium cage combined with posterior double screw-rod fixation seems to have more relevant advantages. Bian et al.11 also found through systematic review that the incidence of implant failure in posterior fixation was lower than that in anterior fixation, which is consistent with our findings.In all six directions, the maximum Von Mises stresses in the screw-rod internal fixation system in group 3 were substantially lower than those in group 2. This indicates that compared to anterior autologous bone implantation, anterior titanium cage implantation can reduce the stress in the posterior double screw-rod system and can reduce the risk of fracture of the screw-rod system to a certain extent. This is consistent with the research results of Wu et al.31 also found that traditional titanium cages had defects of high subsidence rate, which was closely related to high local stress, which also confirmed our research results. The maximum Von Mises stresses in the cortical endplates in group 1 were higher than those in group 3 in all directions of motion except the LLB direction. This indicates that after anterior titanium cage implantation, posterior double screw-rod fixation provides a greater stress reduction in the adjacent cortical endplates than lateral double screw-rod fixation, thereby reducing the incidence of titanium cage subsidence. Although the clinical follow-up study of Zhang et al.12 did not find obvious prosthesis displacement and subsidence in the treatment of lumbosacral spinal tuberculosis treated by anterior titanium cage combined with lateral double screw- rod fixation at the end of follow-up, this may be due to the short follow-up period.In this study, group 2 had the lowest peak Von Mises stresses in the cortical endplates in all six directions. One possible reason is that autologous bone has a larger contact area than titanium cage, which can better disperse the stress to a certain extent. The other reason may be that autogenous bone has a lower elastic modulus than a titanium cage and has a certain buffering function under load, which can reduce the pressure on the endplate to a certain extent. The lower the cortical endplate stresses are, the lower the frequency of implant subsidence in the future. Therefore, autologous bone grafting is more advantageous in preventing subsidence of the prosthesis. Wang et al.32 also found that posterior screw-rod fixation in the treatment of lumbosacral tuberculosis of the spine can better reconstruct the stability of the spine. Bezer et al.33 also had similar results. The maximum Von Mises stresses in the cortical bone around the screws in group 1 were higher than those in group 2 in the FLX, LT and RT directions but lower than those in the EXT, LLB and RLB directions. The stresses in the cortical bone around the screws in group 1 and group 2 have different advantages in different directions of motion, among which the former has more advantages in the FLX, LT and RT directions, which helps prevent screw loosening.In this study, the maximum Von Mises stresses in group 3 were the lowest among the three groups in all six directions, indicating that a titanium cage combined with posterior double screw-rod fixation could provide a greater stress reduction in the cortical bone around the screws than the other two internal fixation methods, thereby reducing the incidence of screw loosening after operation to a certain extent. Jiang et al. The results of this study showed that the maximum Von Mises stress of the adjacent disc in group 1 (L3-4 disc) in the EXT direction was slightly higher than that in group 2 and group 3, indicating that titanium cage combined with lateral double screw-rods fixation might be more likely to cause the degeneration of the adjacent disc in the EXT. The maximum Von Mises stress analysis of facet joints showed that the maximum stress of the L3-4 facet joints in group 1was slightly higher than that in group 2 and group 3 in the direction of LLB and RLB, indicating that the titanium cage combined with lateral double screw-rods fixation was more likely to cause the degeneration of adjacent facet joints during lateral bending. The maximum Von Mises stresses of L4-5 and L5-S1 facet joints of group 3 in all directions were zero, which indicated that the titanium cage combined with posterior double screw- rod fixation can obtain strong stability, so that the facet joints of the operative site did not produce local stress. Thus, group 3 was more advantageous in reconstructing surgical site stability and preventing adjacent disc and facet joint degeneration.This study has several limitations. In this study, FE modelling of the intact lumbosacral spine was based on CT data obtained from a young, healthy, male patient. However, these data might have neglected the influence of pathologies on the biomechanical performance of the lumbosacral spine. Second, in order to compare the biomechanical properties of the three surgical methods, we constructed the identical geometry of the lumbosacral spine. However, different surgical approaches differently affect spine lordosis and profile, which is not reflected in our study. Third, we adopted simplified loads to simulate the mechanical environment, which is somewhat different from the complex human body, and the research results still need to be further verified by in vivo studies. This FE analysis only constructed the lumbosacral segment tuberculosis model after L5 vertebral body resection but failed to cover the involvement of other vertebral bodies or multiple models. Hence, further studies are needed to analyse these specific situations.Titanium cage with posterior double screw-rod fixation has more advantages in reconstruction of lumbosacral spinal stability, prevention of screw loosening and alleviating degeneration of adjacent intervertebral discs and facet joints. Autologous bone fixation with posterior double screw-rod fixation has a higher risk of screw fracture and a lower incidence of prosthesis subsidence than the other two fixation methods. The titanium cage with the lateral double screw-rod fixation has better mechanical properties in some directions, but compared with the other two internal fixation methods do not show absolute advantages.


Elevated immunoglobulin E (IgE) is a hallmark of allergic diseases. However, high IgE levels also occur in a number of other infectious and noninfectious diseases. In most cases, elevated IgE levels indicate allergy, eczema, or chronic skin infection. Very high IgE levels are not uncommon in patients with active eczema but more often indicate monogenic atopic disorder or inborn errors of immunity with an atopic phenotype. We conducted a retrospective study of 385 children with suspected immune deficiency referred to the clinic over a 9-year period. Measurement of IgE, IgG, IgA, IgM, and IgG subclasses in blood samples revealed that nearly one-third of the patients had elevated serum IgE levels. Most of the cases with elevated IgE were children with underlying atopy—mainly atopic dermatitis and, to a lesser extent, bronchial asthma—whereas 40.12% (37 children) had no atopy at all. In the most severe cases (with extremely elevated IgE or severe dermatitis), we confirmed genetic mutations for underlying immunodeficiency. Our results indicate that allergic phenotype should not be underestimated and that children with more severe allergic disease should be evaluated for an underlying inborn error of immunity. If inborn error of immunity (IEI) is suspected, a comprehensive immunologic evaluation is required. Genetic testing helps identify the specific genetic abnormality, which provides important insight into the immunopathogenesis of the disease and accurate determination of optimal therapy. Elevated immunoglobulin E (IgE) is a hallmark of allergic diseases. However, high IgE levels are also found in a number of infectious diseases such as parasite infections, human immunodeficiency virus (HIV) infection, Mycobacterium tuberculosis, cytomegalovirus, Epstein–Barr virus, leprosy, and candidiasis. Inflammatory diseases such as eosinophilic granulomatosis with polyangiitis and Kawasaki disease are also characterized by elevated IgE levels. In addition, high IgE levels can be found in neoplasms such as Hodgkin’s lymphoma and IgE myeloma. Other diseases associated with elevated serum IgE levels include cystic fibrosis, nephrotic syndrome, bone marrow transplantation, graft-versus-host disease, and bullous pemphigoid. Tobacco smoking and the use of aztreonam or penicillin G may lead to an increase in total IgE levels . DistingIn a recent study, allergic comorbidities were found in almost 40% of patients with antibody immunodeficiency and in 20% of patients with combined immunodeficiency . We hypoWe performed a retrospective study that included 385 children (age range 3 months–17.9 years) referred to the clinic with suspected immunodeficiency. A 9-year period was covered—from January 2014 to December 2022. Most children were referred for evaluation because of recurrent respiratory infections. Interestingly, almost half of them had concomitant atopy, and other comorbidities included autoimmunity and malignancy. The following diagnostic tests were performed in each child—microbiological examination of throat and nasal swabs or sputum (if available), complete blood count, differential blood count, flow cytometric immunophenotyping with a wide range of monoclonal antibodies, and serum immunoglobulins and their subclasses—with an automated analyzer. Immunoglobulin levels were compared with age-adjusted normal reference ranges. In addition, antibody response to vaccines, evaluation of T-lymphocyte function, tests for autoantibodies, phagocyte activity, and genetic analysis for IEI were performed, as appropriate. Genetic analysis included whole-exome sequencing (Sanger method), examining all genes associated with a documented IEI. Immunological testing was performed when there was no evidence of current infection .p < α the null hypothesis is rejected.Statistical analysis of the data was performed using the Statistical Analysis Software Package (SPSS) version 26 (2021), IBM  and Excel version 2013, Microsoft . The analysis included descriptive statistics, tests to determine the normality of the distribution , tests to compare quantitative indicators in different groups (comparison of means), and correlation analysis . The significance level chosen was α = 0.05, i.e., for values of p = 0.81). Overall, the mean age of the group was 5.75 ± 3.85 years. Regarding family history, we found that 33% were burdened with allergic diseases, 17% with autoimmune diseases, and 7% with malignant diseases. Clinical characteristics are shown in Of the total of 385 children, 214 (56%) were boys. The boys were slightly younger than the girls, with a mean age of 5.49 ± 3.85 years vs. 6.08 ± 3.84, respectively, but the difference was not statistically significant . Of the remaining 365 children, elevated and highly elevated IgE levels were found in 105 (28.78%), whereas IgE levels were extremely low (<2.5 kU/L) or absent in 8 children (2.19%). Low IgA levels were found in 40 children (10.95%), whereas they were elevated in only 21 (5.75%). Low IgM levels were detected in 22 children (6.02%), whereas elevated levels were found in 31 (8.49%). For IgG, elevated or decreased levels were found in 15 children (4.1%). As for IgG subclasses, decreased IgG1 was found in 26 children (7.12%) and increased in 16 (4.38%), decreased IgG2 in 15 patients (4.1%) and increased in 34 (9.38%), decreased IgG3 in 11 children (3.01%) and increased in 30 (8.21%), and decreased IgG4 in 25 patients (6.84%) and increased in 41 (11.23%) . The refWe diagnosed primary immunodeficiency in 45 children (12.32%). Ten children were diagnosed with selective IgA deficiency (sIgAD), six with common variable immunodeficiency (CVID), three with X-linked agammaglobulinemia (XLA), three with hyper IgE syndrome (HIES), three with transient neutropenia (TrN), three with T-cell immunodeficiency (TCID), three children with selective IgG subclass deficiency, two with ataxia telangiectasia (AT), two with Di George syndrome, two with LAD deficiency, one with Netherton syndrome, one with STING -associated vasculopathy with onset in infancy (SAVI), one with C8 complement deficiency, one with DNA ligase IV (LIG4) syndrome, one with X-linked immunodeficiency with mutation in SD2S1A EBV–positive, one with Casteman disease (CD), one with CARD11 mutation, and one with FOXN1 immunodeficiency. p = 0.0062, Chi-square). As expected, only the group with extremely elevated IgE had patients with HIES, and we did not look for statistical differences in this comorbidity for the different groups but focused on differences in the frequency of patients with asthma, atopic dermatitis, allergic rhinitis, drug allergies, or no allergies according to IgE levels  or absent IgE levels—8 children, low IgE levels—47 children (below 30 IU/L), normal IgE levels—205 children, elevated IgE levels—92 children, and extremely elevated IgE levels (above 1000 U/L)—13 children. The children’s atopic comorbidities were distributed differently depending on the group, and the results were statistically significant  and the same number of children (10–22.22%) with low IgE, although 3 of them had extremely low IgE levels (6.66%), whereas the rest had normal IgE levels. Of the patients with extremely low IgE, one had CVID, one had AT, and one had selective IgG2 deficiency. The other seven children with low IgE included two with CVID, two with sIgAD, two with XLA, and one with TrN. In the group with elevated IgE, three children with HIES had extremely high IgE, and the others with high IgE included children with sIgAD, two children with TCID, the child with Netherton syndrome, the child with CARD11 mutations, and the child with SAVI. The small number of groups did not allow us to generate in-depth statistics. But, as in other reports, our CVID patients were more likely to be in the low IgE group, whereas those with TCID were more likely to have higher IgE. However, there were no statistically significant correlations between IgE levels and levels of other immunoglobulins.Of the 13 patients with extremely high IgE levels—above 1000 U/mL—we performed genetic testing, and only 3 had a confirmed HIES. Of the other 10, 5 patients were diagnosed with bronchial asthma and 5 with atopic dermatitis. The percentage of atopic comorbidities in this group was also high, but this came from the small numbers in this group, where two patients had a percentage of 15.38%.In the present study, we measured IgE, IgG, IgA, IgM, and IgG subclasses in blood samples from 385 children with suspected IEI. We found that almost one-third of our patients had elevated serum IgE levels. Most of the cases with elevated IgE were children with underlying atopy—mainly atopic dermatitis and, to a lesser extent, bronchial asthma—but 40.12% (37 children) had no atopy at all. At the same time, we found patients with allergic diseases and normal IgE levels, although it should be noted that our cohort of children was relatively young, with an average age of 5.75 years. Patients with IEI may have elevated IgE levels not only in HIES. Our results demonstrate once again that allergic phenotype should not be underestimated and that children with more severe allergic diseases should be evaluated for an underlying inborn error of immunity.Patients with IEI with atopic phenotype present with specific clinical manifestations and laboratory findings that must be carefully analyzed to identify the correct disorder. It is important to evaluate the presence or absence of a positive family history for primary immunodeficiencies and/or consanguinity, as well as the presence of prenatal and perinatal factors such as infections, smoking, e-cigarette use, alcohol, and drugs during pregnancy as risk factors for adverse birth outcomes.Many IEI disorders are inherited in an autosomal recessive manner. Therefore, individuals with autosomal recessive disorders and their families should be told that marriages between relatives increase the likelihood that children will be affected. There are several features that raise suspicion of immunodeficiency and suggest referral to a specialist for investigation. A detailed history, a thorough physical examination, and laboratory tests to assess immune system function are the first steps toward a correct diagnosis  7]..7].Immunodeficiencies associated with atopy include selective immunoglobulin A deficiency (sIgAD), common variable immunodeficiency (CVID), chronic granulomatous disease (CGD), and DiGeorge syndrome, as well as several other less common forms . As prevThe presence of atopic disease  may be a feature of CVID in children compared with CVID in adults. Absence of immunoglobulin E (IgE) is common in both children and adults, with 97% failing to detect allergen-specific IgE . In the It should be noted that, in our group, a relatively high percentage of patients with allergic rhinitis and asthma was found in the low or normal IgE group. There are several possible explanations for this, one of which is the age of our patients, with the average age being almost 6 years, and the fact that there were patients with viral-related asthma, which is not necessarily related to IgE . On the Studies have found an association between low serum IgE levels and levels of one or more immunoglobulin classes  below the age-appropriate reference range. Consistent with these reports, a recent literature review concluded that low IgE levels should be considered a clinical laboratory “flag” for screening for antibody deficiency . The revAn elevated IgE level (100 ≥ IgE < 1000 kU/L) indicates allergy, eczema, or chronic skin infection. A very elevated IgE level  is not uncommon in patients with active eczema but is suggestive of a monogenic atopy disorder or one of the hyper-IgE immunodeficiency syndromes in which extremely high total serum IgE levels  may be found. A very high IgE level  in a patient with recurrent bacterial or fungal infections and dermatitis would raise suspicion of a hyper-IgE syndrome and several other IEI-like signal transducer and activator of transcription 3 (STAT3), interleukin 21 receptor (IL21R), interleukin 6 receptor (IL6R), and interleukin 6 signal transducer (IL6ST) deficiencies; caspase recruitment domain family member 11 (CARD11) dominant negative defect; phosphoglucomutase 3 (PGM3) deficiency; Wiskott–Aldrich syndrome; Omenn syndrome; IPEX  syndrome; dedicator of cytokinesis 8 (DOCK8) deficiency; lipopolysaccharide-responsive-beige-like anchor (LRBA) deficiency; and a gain-of-function phenotype associated with enhanced phosphorylation and transcriptional activity of STAT6  .IgG subclasses are not routinely studied, although IgG subclass deficiency is a common finding among patient populations with recurrent sinopulmonary infections . IgG subRecent data suggest that IgG4 may be an important biomarker in cancer. IgG4 is the least abundant subclass of IgG in normal human serum but can reach elevated levels during chronic antigen exposure or inflammation. IgG4 are widely considered to be anti-inflammatory antibodies that play a positive and protective role in allergies and helminth infections. In allergies, IgG4 are responsible for neutralizing allergens and inhibiting IgE signaling and are associated with immune tolerance to allergens in allergen immunotherapy (AIT).On the other hand, in IgG4-related diseases such as sclerosing pancreatitis and autoimmune pancreatitis, a pathogenic role of IgG4 cannot yet be completely excluded. In contrast, the anti-inflammatory properties of IgG4 could have a negative impact in the context of cancer. Because tumors are characterized by chronic inflammation with prolonged exposure to tumor-associated antigens, elevated IgG4 has been demonstrated in extrahepatic cholangiocarcinomas, pancreatic cancers, melanomas, and glioblastomas. These tumor types are characterized by Th2-loaded expression of mediators known to stimulate B cells to produce IgG4. Tumor-specific IgG4 can act as a blocking antibody that competes with inflammatory antibodies such as IgE or IgG1 to bind to antigen or in the form of immune complexes that target the inhibitory receptor FcγRIIb to dampen inflammation. IgG4 antibodies in cancer may thus have a downside compared with their association with tolerance induction to allergens . ElevateAccording to epidemiological studies, atopic and allergic patients are less likely to develop certain types of cancer, such as lymphoma, neuroblastoma, pancreatic cancer, and melanoma . Mouse mThe overall risk of developing cancer in patients with IEI is less than twice that in the general population and is estimated to be between 4 and 25% . The incPatients with IEI are generally more likely to have cancer that has spread or is widespread at the time of diagnosis and therefore have a poorer prognosis. Patients with certain conditions, such as ataxia telangiectasia, Nijmegen breakage syndrome, and generalized variable immunodeficiency, should be informed of their radiosensitivity, which may increase the risk of developing malignancy. These patients should consult their immunologist before agreeing to any diagnostic or therapeutic intervention involving radiation suggested by other clinicians. Clinicians should weigh the risks and benefits of the procedure in the context of the underlying disease, the need for the procedure, replacement with nonradiation techniques , and, when possible, in vitro analysis of the patient’s radiation sensitivity .Advanced immunologic testing requires expertise to perform and interpret, is not widely available, and is often costly. Therefore, immunologic testing is best performed in a stepwise fashion, and referral to an immunologist should be made as early as possible. When the specific laboratory tests performed are inconclusive or it is difficult to classify the clinical phenotype and a definitive diagnosis cannot be made, genetic testing may be helpful. Gene panels, whole-exome sequencing, or whole-genome sequencing can be performed. Based on the genetic studies, overlaps were found between known atopy-related genes as described in the Human Gene Mutation Database and disease-causing genes of monogenic PIDs . IdentifIgE is known as a marker of allergy or parasitic infection, but based on previously published data and this study, we believe it is time to change the clinical approach and, in addition, screen these children for IEI. In cases of extremely high IgE levels, genetic testing is mandatory to confirm or exclude some of the HIES.IEI could be misdiagnosed based on the predominant clinical features of atopy. Without consideration of underlying IEI, some individuals remain undiagnosed, resulting in a high risk of morbidity and mortality. An underlying IEI with atopic phenotype should be considered, especially in severe cases of atopic disease with concurrent signs of autoimmunity and recurrent infections, unusual clinical course, and lack of response to classic treatment strategies. When IEI with atopic phenotype is suspected, a comprehensive immunologic evaluation is required, and genetic testing is essential to identifying the specific genetic abnormality. Good collaboration between pediatricians, allergists, and immunologists is necessary to correctly diagnose IEI and initiate optimal targeted therapy.Limitations of the study: There are fewer patients with particularly low and high IgE levels. Therefore, the correlation between atopic comorbidities and IgE levels may not reach statistical significance in this group.


Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class.Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria.Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from ε to ζ and that ζ, ε, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed. The ATP synthase is the terminal multiprotein complex of oxidative phosphorylation or photophosphorylation of most living beings. When the transmembrane ion gradient is partially or totally collapsed , the ATP synthase is thermodynamically prone to reverse its intrinsic rotation from clockwise  to counterclockwise (CCW) and, therefore, to hydrolyze ATP, thus becoming an ATP-driven primary ion pump. To prevent the futile and deleterious F1FO-ATPase activity, the ATP synthases of different organisms have acquired different inhibitory proteins or protein domains. Thus, nature designed, by evolution, several F1FO-ATPase inhibitory proteins, such as bacterial ε  (ζ) subunit of this PdF1FO-ATPase  . However, instead of showing a clear activation in their truncated Pd-εΔCT mutants, their results show that the stronger and more significant PdF1FO-ATPase activation occurred exclusively when ζ was deleted but not when only Pd-ε was truncated  . In contO-ATPase  exerts a1-ATPase . The key(Pd1222) , 2018b. oduction , 2018b. ructures . Recent runcated ; the detζ across the α-proteobacteria class. To this aim, we carried out bioinformatic, molecular biology, biochemical, and structural analyses of several ζ subunits in distinct bacterial families, including strictly free-living respiratory  or photosynthetic , facultative symbiotic , and strictly parasitic  α-proteobacteria. The results confirmed that the ζ subunit is a protein family (DUF 1476) essentially exclusive of the α-proteobacteria class. We confirmed the strong conservation of the functional N-terminal inhibitory domain of ζ and its divergence toward the C-terminus. We also carried out homologous or heterologous reconstitution of several ζ subunits into the respective α-proteobacterial F1 or F1FO-ATPases. The results showed that the evolution of ζ and the preservation or loss of its inhibitory function is in concordance with its bioenergetic requirement in different bacterial families, environments, and lifestyles. Finally, we resolved and correlated the NMR and AlphaFold (AF) structures of the ζ subunit from S. meliloti with its lack of inhibitory function. Taken together, the data show that bacterial ε, α-proteobacterial ζ, and mitochondrial IF1 emerged independently by convergent evolution as F1FO-ATPase inhibitors. These results also have important implications in the endosymbiotic evolution from α-proteobacteria to mitochondria and putative important future applications.Given its biological relevance, we studied here the evolution of P. denitrificans and the other non-photosynthetic α-proteobacteria were prepared as previously described , Rhizobium etli CFN42 (Re), or Sinorhizobium meliloti 1,023 (Sm) strains was purified from SBP or inverted membranes, as described previously (1-ATPase of Rhodobacter capsulatus from chromatophores. Solubilization of the F1FO-ATPase from SBP or chromatophores was carried out as described for the preparation of Blue Native Electrophoresis (BN-PAGE) with 2–4 mg/mg of protein of digitonin  from escribed , and chreviously . The F1 eviously . The samigitonin .P. denitrificans, R. etli, and S. meliloti F1-ATPase activities using a pyruvate kinase and lactate dehydrogenase coupled assay that follows the NADH oxidation, as described previously  ATPases to the reaction cells. Measurements were carried out using 0.15% LDAO as an ATPase activator. In the ATPase assays of chromatophores or SBP, we also added 0.03 μg/μL of rotenone and 5 mM of sodium cyanide. In chromatophores, the ATPase assays also included sulfite (≤2 mM) and 2 μM of FCCP.We measured the eviously . The reaζ subunits were PCR-amplified using genomic DNA of the strains P. denitrificans Pd1222, R. etli CFN42, S. meliloti 1,021, and Cereibacter sphaeroides 2.4.1 (formerly Rhodobacter sphaeroides) and designed primers (Rhodospirillum rubrum ATCC 11170 inserted into pET3a was purchased from GeneScript. The ζ amplicons were ligated into a pJET1.2 subcloning vector (Thermo Fisher Scientific), and competent cells of E. coli DH5α were transformed with this construction. The cloned ζ genes were confirmed by NdeI and BamHI double digestion and subsequently ligated into a pT7-7 plasmid, with the exception of ζ from C. sphaeroides (Cs-ζ), which was cloned into pET3a (E. coli BL21 (DE3) pLys S codon plus cells. The recombinant ζ subunits were overexpressed and purified as described previously (The recombinant  primers . The ζ gto pET3a . The pT7eviously .θλ is the observed ellipticity (degrees) at wavelength λ, d is the pathlength (cm), and c is the protein concentration (mg/ml). The spectra obtained were an average of three scans. The secondary structure content was calculated with the BeStSel online software (https://bestsel.elte.hu/index.php). See references in the link and also the work of Far UV CD measurements of Pd-ζ and Sm-ζ were carried out at a concentration of 0.07 mg/ml at 25°C on a Jasco J715 spectropolarimeter using a quartz cell with 0.1 cm pathlength. The protein samples were previously filtered and diluted in 25 mM of phosphate buffer with pH 8.0. The results were expressed as mean residue ellipticity at the wavelength (λ) given by1, F1FO, SBP, or chromatophores from each strain  were preincubated with the indicated concentrations of the recombinant Pd-ζ, Re-ζ, Sm-ζ, Cs-ζ, or Rr-ζ in the presence of 1 mM of sulfite and 1 mM of ATP and MgCl2 in a buffer containing 20 mM of Tris/HCl and 250 mM of sucrose at pH 8.0. The reconstituted samples were incubated for 20 min at room temperature; the ATPase activities of the samples were then measured by the coupled spectrophotometric method described previously.The F13C, 15N uniformly double-labeled Sm-ζ was overexpressed in E. coli BL21 cells transformed with the pT7-7/Sm-ζ plasmid. Cells were incubated in minimal M9 media at 37°C until they reached an absorbance of 0.7 at 600 nm. Afterward, 1 mM IPTG was added, and cells were incubated at 37°C overnight. The cells were then harvested by centrifugation, and the pellet was stored at −80°C until used. Incubations were performed with continuous shaking at 200 rpm. The M9 minimal media used had [13C6]-d-glucose (4 g/L) and (0.5 g/L) 15NH4Cl as sole carbon and nitrogen sources (Sigma-Aldrich), respectively. Purification of the 13C, 15N uniformly double-labeled Sm−ζ was carried out as described previously for the purification of the non-labeled recombinant ζ subunits.The ζ using a 3 kDa centricon, with 0.5 mM of DSS, 4.5 mM of NaN3, and 5% of D2O. Afterward, the sample was transferred into the NMR tube and then acquired in a Bruker AVANCE III HD 800 MHz spectrometer for a  HSQC (heteronuclear single quantum coherence) spectra -resolved, and 3D13C (aromatic)-resolved -NOESY experiments were recorded at 293 K with a mixing time of 120 m on a Bruker AVANCE III HD 800 MHz spectrometer. To avoid sample instability, 25% non-uniform sampling was used for each sidechain experiment. The final 20 conformers with the lowest residual CYANA target function values were then subjected to energy minimization. First, each NMR conformer was solvated with full atom TIP3P water containing Cl− and K+ ions at ∼0.15 M to mimic the physiological ionic strength. Then, energy minimization and calculation of the conformers were carried out using the GROMACS 2018  , which were also studied here (see the following section). In this ζ cladogram, some ζ sequences branched outside their respective orders, as in some Rhodobacterales and Rhizobiales, but these inconsistencies were corrected when rRNAs were used for the cladogram construction   (around αPATPsζ) , there aaeroides ), and infunction . Around function , there ation see , so thes1-ATPase from photosynthetic α-proteobacteria, closely related to P. denitrificans in the same Rhodobacterales order. The ATP synthases from some of these photosynthetic α-proteobacteria were functionally characterized (C. sphaeroides (Cs-ζ) bound to its CsF1FO-ATP synthase of chromatophores  should harbor the ζ-binding site at the INGECORE or αDPβDPγ interface , obtaining a functional F1-ATPase with the canonical α, β, γ, δ, and ε subunits and a sixth 11 kDa subunit migrating similar to Pd-ζ of Pd-F1 , Rhodopseudomonas palustris (Rp), Rhodovibrio salinarum (Rsa), Rhodospirillum rubrum (Rr), Rhodobacter capsulatus (Rc), and Phaeospirillum fulvum (Pf). The anti-β and anti-ζ Western blot analyses confirmed the presence of ζ in chromatophores of all of these photosynthetic α-proteobacteria, besides the presence of the catalytic ß subunit (P. denitrificans (first lane of R. rubrum (Rr-ζ) is one of the most distant to Pdζ  and Jannaschia sp. (Js-ζ) into the ATP synthase from photosynthetic α-proteobacteria. The latter is another marine α-proteobacteria of the Rhodobacterales order, also closely related to P. denitrificans (see 1-ATPase (1FO-ATPase of R. capsulatus (Rc-F1FO) solubilized from chromatophores. As expected, from their close proximity  to inhiC. sphaeroides (Cs-ζ)  to inhibit the PdF1FO-ATPase in PSB, which are in the μM range. Recently, three new AlphaFold (AF)-modeled structures of the Cs-ζ protein were uploaded, and the structure closest to our cloned Cs-ζ protein was 90% identical (AF-A0A3G6WKK1-F1-model-v4. pdb). We aligned this AF-Cs-ζ model  , inset al red in , inset wl red in , inset. l red in , inset. l red in . In summd so far , even hid so far . As we wJannaschia sp. (kindly donated by Prof. Mary Ann Moran) to estimate the appIC50 of Js-ζ for its own Js-ATP synthase. Nevertheless, it is evident that in free-living α-proteobacteria, the ζ subunit is a potent F1FO-ATPase inhibitor with nM or μM affinities for their respective or homologous free-living α-proteobacterial F1FO-ATPases. Although we were unable to clone the recombinant ζ from R. capsulatus (Rc-ζ) and to estimate its appIC50 to inhibit its own Rc-ATP synthase, we consider that Rc-ζ is likely a potent RcF1FO-ATPase inhibitor. This is because of the very close relationship of R. capsulatus with P. denitrificans, C. sphaeroides, and Jannaschia sp. (see Rhodospirillum rubrum (Rr-ζ). This α-proteobacterium belongs to the order Rhodospirillales but not to the Rhodobacterales  mechanism  to inhibit its own PdF1FO-ATPase complex in SBP .To assess the homologous inhibitory capacity of Rr-ζ on chromatophores from  subunit  and recoechanism . These A SBP see  and ≈1,9 SBP see  but alsoC. sphaeroides, and the more distant R. rubrum ζ subunits preserve their inhibitory function, although the latter exhibits a relatively much lower affinity. The data, thus, show a tendency to lose the inhibitory function of ζ in α-proteobacteria that are more distant from P. denitrificans.These observations indicate that the ζ subunit of photosynthetic free-living α-proteobacteria, such as P. denitrificans . Within these 22 structures, we found one with a higher identity to Rr-ζ named AF-A0A7V7E8W8-F1-model_v4. pdb. This structure, among others, was aligned to our model of Rr-ζ, and we found that the RMSD between this AF model and our Rr-ζ model was the smaller one (2.034 Å), as calculated in PyMol , but the inhibitory extended α-helical N-terminus that blocks the rotation of the central rotor of the PdF1 and PdF1FO-ATPases , S. meliloti (F1Sm), and M. nodulans (F1Mn), in addition to the presence of the control ß subunit (1 Pd). Although this might be due to the sequence diversity of the Rhizobiales ζ subunits, the lack of a clear ζ band intensity in the Coomassie-stained gels  and distantly  related to Pd-ζ, confirming, perhaps non-unexpectedly, the conservation of the inhibitory function of these ζ subunits. We, thus, turned our focus to the ζ subunits of symbiotic, pathogenic, and parasitic α-proteobacteria in order to define whether the inhibitory function of ζ is preserved in α-proteobacteria of different lifestyles, either closely or distantly related to green in , 9. The Pase see  and ZarcPase see  and the Pase see , 4A–C. WPase see , includi subunit . Howeverned gels  evidenceS. meliloti (Sm-ζ) and R. etli (Re-ζ) chromosomic DNA to determine the appIC50 of these Rhizobiales ζ subunits to inhibit their F1 and/or F1FO-ATPase complexes. The recombinant Sm-ζ and Re-ζ subunits were purified as described previously for Pd-ζ  spectroscopy, compared with Pd-ζ as the reference. As can be seen in 1-ATPase, which also showed more clearly the contents of α, β, γ, δ, and ε subunits better than other Rhizobial F1-ATPases (see 1-ATPase activity (11.24 ± 0.79 μmol/(min.*mg.pt.)) higher than that of PdF1Δζ (4.48 ± 1.21 μmol/min*mg pt), which is with the PdF1Δζ isolated from the ζ knockout mutant PdΔζ (1 (5.53 ± 0.03 μmol/(min*mg.pt.)) ATPases and similar to that of ReF1 (10.37 ± 2.57 μmol/(min.*mg.pt.)). Therefore, our SmF1 was an optimal preparation to assess the inhibitory function of Sm-ζ since it lacked most of its endogenous Sm-ζ and, thus, showed one of the highest F1-ATPase activities of the α-proteobacterial F1-ATPases isolated here. Somehow unexpectedly, we observed no inhibitory effect of the Sm-ζ whatsoever on its own SmF1-ATPase after reconstitution experiments with increasing concentrations to reach an excess of Sm-ζ up to 30 μg in the presence of MgATP  to make sure there was enough time to reveal any inhibitory function of Sm-ζ on SmF1, in case there were any  of Sm-ζ  estimated in the PdF1FO-ATPase of SBP with the same kinetic inhibition model and with appropriate statistical significance  with the recombinant Pd-ζ. After the custom reconstitution of Pd-ζ in the presence of MgATP into SmF1 and ReF1 ATPases, we could not observe any inhibitory function of the Pd-ζ whatsoever. Instead of repeating these experiments with negative results, we used a considerable excess of recombinant Pd-ζ, ≥100μg to confirm that there were no inhibitory effects of Pd-ζ on SmF1 and ReF1 ATPases , giving an α-helical content of 72.1%. This is in good agreement with the 75.7% of α-helix as obtained by our CD experiments  conformer of Sm-ζ into the extended N-terminal α-helical inhibitory conformer of Sm-ζ. We looked for structural insights that might explain this heterologous inhibitory effect of Sm-ζ, so we looked for the putative Sm-ζ structures modeled in AF. We found six structures of Sm-ζ predicted by the AF database, but these structures had different sequences and structures, originating from different S. meliloti strains , grown as an intracellular symbiotic host in human erythrocytes; since the latter lack mitochondria, this ensured the absence of putative contaminant mitochondrial F-ATPase (mtF-ATPase) present, for instance, in yeast that was used before as the W. pipientis host  to the inhibitory N-terminal extended α-helical structure (PDB_id 5DN6), we were able to simulate this disordered to α-helical extended inhibitory transition in the Pd-ζ subunit by MD analysis carried out at constant pH 8.0, suggesting that this transition could be reached spontaneously in solution  to compare it with that of Pd-ζ. The MD results clearly showed that the Sm-ζ was unable to undergo the transition from the compact non-inhibitory 5-α-helical bundle conformer , these PdF1-ATPase subunits induce the functional conformations of F1 subunits from other species  to rotate according to the newly described 3° × 120° rotation unique of the PdF1-ATPase and not according to the multiple rotary stepping in the other bacterial or mitochondrial F1-ATPases (1 individual subunits have the potential to induce other heterologous F1 subunits to adapt to their structure and catalytic rotary mechanism; thus, the PdF1 subunits work as robust dominant subunits (1-ATPase, but not the SmF1, is similarly able to induce the structural transition from the compact 5-α-helical non-inhibitory conformer of Sm-ζ (1 or PdF1FO-ATPase inhibition.In addition, we resolved and correlated the NMR structure of the Sm-ζ with its lack of inhibitory function, which is in concordance with its compact 5-α-helical non-inhibitory conformation , 8. We a ATPases , 8D. We -ATPases . This insubunits . It seem of Sm-ζ , 8B, int of Sm-ζ  to produ1 (1. Thus, we carried out MD analyses of the ζ subunit to shed light on the inhibitory and regulatory mechanisms of the ζ subunit. The MD analyses carried out here showed clearly that the inhibitory Pd-ζ subunit may experience, although sporadically, since it was observed in one of the 3 MD replications, a spontaneous transition from the N-terminus disordered conformation to the N-terminus extended α-helical conformation mimicking the inhibitory conformer of Pd-ζ bound productively to the PdF1 (1FO complexes (1-ATPase and indicates that a larger energetic barrier exists in Sm-ζ to achieve the transition from the compact non-inhibitory conformation to its N-terminal α-helical extended inhibitory conformer. Somehow, the PdF1-ATPase, but not the SmF1-ATPase, is able to overcome this energetic barrier due to its tendency to work as a dominant PdF1-ATPase to induce functional conformations on the reconstituted ATP synthases’ heterologous subunits from other species, adapted to the PdF1-ATPase subunits (MD analyses have been previously helpful in analyzing the inhibitory and regulatory mechanisms of the ε subunit from non-α-proteobacteria  and the 1 , describthe PdF1  or PdF1Fomplexes . Howeveromplexes . This issubunits , thus taevolutionary convergence of the mitochondrial IF1 as the endogenous inhibitor of the mtATPase, which is not homologous to the α-proteobacterial ζ subunit (1 eventually also promoted the mtATPase dimerization, oligomerization, and mitochondrial cristae formation, as we demonstrated previously (1 by convergent evolution and the concomitant formation of mitochondrial cristae (1 in α-proteobacteria and mitochondria, respectively, and their non-homologous character (see also the work of Our evolutionary results might have important implications in the endosymbiotic evolution from α-proteobacteria to mitochondria. In the case of the origin of mitochondria, it is strongly suggestive that the origin of mitochondria, as shown by rRNA evolution , indicat subunit . IF1 eveeviously . In a le


Ice protection techniques have attracted significant interest, notably in aerospace and wind energy applications. However, the current solutions are mostly costly and inconvenient due to energy-intensive and environmental concerns. One of the appealing strategies is the use of passive icephobicity, in the form of coatings, which is induced by means of several material strategies, such as hydrophobicity, surface texturing, surface elasticity, and the physical infusion of ice-depressing liquids, etc. In this review, surface-roughness-related icephobicity is critically discussed to understand the challenges and the role of roughness, especially on superhydrophobic surfaces. Surface roughness as an intrinsic, independent surface property for anti-icing and de-icing performance is also debated, and their interdependence is explained using the related physical mechanisms and thermodynamics of ice nucleation. Furthermore, the role of surface roughness in the case of elastomeric or low-modulus polymeric coatings, which typically instigate an easy release of ice, is examined. In addition to material-centric approaches, the influence of surface roughness in de-icing evaluation is also explored, and a comparative assessment is conducted to understand the testing sensitivity to various surface characteristics. This review exemplifies that surface roughness plays a crucial role in incorporating and maintaining icephobic performance and is intrinsically interlinked with other surface-induced icephobicity strategies, including superhydrophobicity and elastomeric surfaces. Furthermore, the de-icing evaluation methods also appear to be roughness sensitive in a certain range, indicating a dominant role of mechanically interlocked ice. Ice protection techniques have attracted increasing interest since 1950  owing to2 and NOX emissions and fuel consumption . H. HcosθreAnother assumption indicated in In order to find a better correlation, ice adhesion strength was plotted as a function of liquid tensile phenomena and the practical work of adhesion is shown in  minimal , and ice minimal , mitigat minimal .Susoff et al.  claimed Janjua et al.  plotted Kim et al.  noted thC is a constant and Apart from these comparisons, Golovin et al.  coined ation (7) .Golovin et al. plotted ice adhesion strength as a function of astomers b and elaA mixed-mode failure of ice cubes has been reported, i.e., when shear force is applied to remove the ice cubes, the ice does not move away completely, and up to 25% of the ice still adheres to the substrate area . It is aFor decades, the idea behind the use of superhydrophobic materials as icephobic materials has been widely studied, as a superhydrophobic surface offers a minimised contact with a water droplet, and a weak ice bond may be formed . A certaUnder low humidity conditions, superhydrophobic materials show excellent icephobic behaviours, even at a temperature of −20~−30 °C ,72,73. HIn our previous study , we condIn a follow-up study , we demod growth ,88,89.From a durability point of view, bulk ice shearing by means of shear force may alter the surface morphology and/or damage the material. Furthermore, the condensation of water in the void valleys forms ice crystals under high humidity conditions ,90. ThisThe hydrophobicity-induced icephobicity hypothesis is critically questioned in certain cases: (1) water droplets condense on surfaces and form a layer of fine ice particles, i.e., microfrost formation , (2) wheSurface roughness or asperities plays a detrimental role in the icephobicity of a surface. This could be in the form of overall surface roughness or imparting surface features that may aid to mitigate ice anchoring or encourage favourable wettability conditions to induce icephobic performance. First, overall surface roughness can be categorised into three ranges: (i) superhydrophobic roughness range of 50–100 μm ,99,100  that may promote the ice/surface interface bonding mechanisms . There iZou et al.  studied Susoff et al.  studied Hao et al.  found tha of 0.536 nm in the air. When the coating was submerged in the water, the surface became smoother with a Ra of 0.264 nm. The results indicated that a liquid-like layer was formed when the coating was submerged, and this thin layer would induce icephobicity by minimizing the interfacial contact area of the formed ice. For smoother surfaces, the results indicated a one order of magnitude reduction in ice adhesion, and for rougher surfaces, a three-fold increase in the magnitude of ice adhesion strength was reported in comparison to pristine substrates under similar conditions [Chen et al.  reportednditions .To further understand the role of surface roughness, the type of formed ice also matters when considering icephobicity. For example, impact glaze and rime ice form a hard bond with the texted surfaces, while bulk-formed glaze ice demonstrates a lower ice adhesion strength ,117. ImpIn our previous work , a systea in this section) also extends to Sa measurements, and a similar correlation with ice adhesion strength is reported [The data in reported ,121,122.Physical mechanisms such as electrostatic interaction, hydrogen bonding, and van der Waals forces are primarily responsible for ice adhesion on surfaces ,95,123. Jung et al. ,129,130 The samples, having a similar surface roughness but variable surface energies, showed a strong dependence on surface energy, and the surfaces with a lower surface energy demonstrated a longer freezing delay. The theory of classical heterogeneous ice nucleation states that surface roughness and wettability (hydrophobicity) are two main factors that decide the freezing probability, i.e., the rate of critical nuclei generation in the droplet . The depG is higher than that of heterogeneous nucleation [In terms of the thermodynamics for the homogeneous ice nucleation of a supercooled water droplet on a solid surface, the free energy barrier Δcleation . The thetion (8) :(8)rc=2·echanism .Therefore, it is reasonable to assume that the maximisation of the free energy barrier for the development of ice embryos (lower surface energy) and the minimisation of the effective interfacial contact area (smoother or lower surface roughness) of the supercooled droplet will significantly increase the icing/freezing delay. However, the formation of ice nuclei is not a simple function of the surface roughness . Thus, eD , and the Hamaker constant is U, bearing a value of 10~19 J. Typically, the adhesion strength of ice reduces and D increases with an increase in water contact angles. A fluoro-based polyhedral oligomeric silsesquioxane/poly(ethyl methacrylate) surface will have a depletion layer thickness in the order of 1 nm, and the highest intrinsic hydrophobicity values are measured on these surfaces [The interface between ice/water and superhydrophobic surfaces with low ice adhesion strength is dominated by van der Waals forces , and thesurfaces .Controlling roughness asperities to the nanolevel to annihilate ice nucleation before it grows heterogeneously is a sensible strategy for icephobic materials. Kim et al.  fabricatIn order to control the roughness asperities, samples with nanosize structures such as honeycombs, pillars, and brick patterns, etc., have been produced to study surface superhydrophobicity and ice freezing delay. Mishchenko et al.  prepareda ≈ 0.17 nm) hydrophobic surfaces with hierarchical structures. These freezing delays are some of the longest reported in anti-icing research to date [Eberle et al.  fabricat to date .Polymeric surfaces and coatings, including the use of SLIPSs, have been a popular choice of researchers for passive ice protection, and many reports have indicated a lower ice adhesion on polymeric coatings and surfaces ,137. LowAccording to the mechanism of hydrodynamic lubrication, the viscosity to thickness ratio of the aqueous film is directly proportional to the ice adhesion strength . The aboMemon et al.  examinedSLIPSs take advantage of polymer networks because of their capability of liquid incorporation and distribution, and supply the incorporated oil uniformly at their surface. The presence of a lubricating liquid layer between a rough surface structure and water could reduce the pinning effect of water droplets and lower ice adhesion strength. However, the concept of SLIPSs is not limited to polymeric surfaces; Yang et al.  proposedWp is the practical work of adhesion, Y is an experimental constant, and t is the thickness of the coating [Golovin et al.  showed t coating . To alteR is the universal gas constant, G is the shear modulus. The conservation of momentum in solid–solid contact articulates that at the interface there is no slip, and the velocity is zero. However, in the cases of soft or elastomeric surfaces, the polymeric chains are considerably mobile and produce slip conditions or non-zero velocity at the interface, and these kinds of behaviours are reported for rubbers [k is the Boltzmann constant, T is the temperature, a is the segmental length to detach a single chain, and f is the force needed [Assuming isotropy,  rubbers , adhesiv rubbers , and pol rubbers . This fee needed ,148. It cibility .According to the Griffith fracture criterion at the coating–ice interface, the square root of the composite modulus ess (τf) , and it To demonstrate the practicality of the concept, Golovin et al.  fabricatThe results present a good correlation between cross-link density and the measured strength of ice adhesion. Two important concepts could also be drawn: (1) extremely low ice adhesion strength can be obtained by using low cross-link density polymers, and (2) interfacial slippage can be deployed on a low-cross-link-density surface to further lower ice adhesion strength .Chen et al.  formulatA number of studies used polyurethane and polydimethylsiloxane as matrix materials and introduced various elasticity conditions, i.e., altering the cross-link density of polymers by varying the composition of the binder and polymer contents. We developed biomimetic glycerol-infused fibre-reinforced polyurethane (GIFRP) coatings as a solution to offer both icephobicity and durability , as depiWang et al.  mixed poWang et al.  reportedHowever, limited studies have been conducted on the durability of these coatings and surfaces, including liquid replenishment within the material matrix, and the material’s ability to maintain icephobic performance after significant microstructural damage. Furthermore, most studies conducted icing/de-icing cycles or mechanical tests, such as abrasion resistance or indentations, etc. ,159, whiThe comparability of icephobicity data is difficult due to the nonexistence of standardised icephobicity evaluation methods. This means that each research group acquired their data with their own parameters, testing setup, and procedures. This extends to variations in sample preparation, applied forces, interfacial contact areas, and measured magnitudes, etc. For example, in de-icing tests, ice adhesion strengths may vary by several orders of magnitude when measured using existing de-icing methods  and therThe majority of the de-icing evaluation includes mechanical tests (such as the push method) ,167,168,−5–10−3 s−1, whereas strain rates obtained using centrifuge methods are in the order of 10−6 s−1. This gives the idea that the centrifuge method has good accuracy in ice adhesion tests [Some approaches use moulds to grow the ice over the substrates using gravity, and some methods are based on the spray of supercooled water inside a centrifugal apparatus to mimic atmospheric conditions such as freezing rain (applicable on power lines) or supercooled droplet impinging (applicable on aeroplanes) . Howeveron tests .In actuality, the sample attached to a block of glaze ice was mounted on a rotor and spun with an increasing velocity. When the adhesion strength of ice on the surface was overcome by the centrifugal force originated by the rotation, the ice detached at a certain speed of rotation. These variables were recorded, and the shear strength to detach the ice was calculated as the adhesion strength between the ice and the specific surface . A typicThe ice adhesion strength measured using the force transducer method was reported by Jellinek et al. around three decades ago , and nowFirstly, empty columns are placed on a sample holder, and a specific volume of water is poured into the columns. Then, the test substrate is mounted on the base plate, and the arrangement is put upside-down on a Peltier plate to freeze the water on the test substrates. The temperature of the test substrate is measured by attaching a thermocouple near the sample area on the base plate .The ice adhesion strength is calculated by dividing the cross-sectional area of solid–water interaction with the maximum force measured at the time of ice detachment. Apart from custom-built equipment, commercially available equipment named a dynamic mechanical analyser (DMA) can also be deployed to measure ice adhesion strength, as shown in The zero cone method (a variant of shear lap joint tests) is one of the least used methods in the literature owing to its complex bulky setup and the need for a sophisticated mould. The apparatus consists of an outer cylindrical mould and an inner pin to hold the sample, as shown in As shear stress is strain rate dependent, the comparison of results just by their ice adhesion strength is not appropriate, and it is more reasonable to compare or put forward a factor with respect to the reference of pristine materials. It will give an idea of how many times the formulated materials reduced the ice adhesion strength in comparison to as-received or bare materials, and this factor is called the Adhesion Reduction Factor (ARF) . For exaives ARF  as(14)AC represents the retained icephobic performance after N cycles, and N tends to infinity, then the targeted adhesion reduction factor of the coating is Thus, from Equation (13), a higher factor of ARF means the coated surface has a lower adhesion strength . Janjua  ARF 1 C .The ice adhesion data also vary with the change in icing sample preparation, measured magnitude, applied force, and interfacial contact area ; howeverIn our previous work , we condThe breakage of ice adhered on a surface can be categorised into two distinctive fracture modes: adhesive (mode I) or cohesive (mode II). Different evaluation methods exert shearing forces that may not be uniformly distributed, especially in the case of microscopically rough surfaces, and this could jeopardise the accuracy of ice adhesion measurements. For instance, a microscopically rough surface is likely to induce a cohesive fracture due to a higher contact area and the mechanical interlocking of ice, and the probability of a pure adhesive fracture is lowered . FurtherIn the push and centrifugal methods, shear stresses mainly dominate across the interfacial contact area. However, the peeling stresses are concentrated at the edges ,190. ThiIdeally, a singular force promoting a complete adhesive fracture at the ice/surface interface may define the perfect de-icing evaluation method. The current de-icing methods may involve a combination of different ice shearing components, and the complete isolation of de-icing forces is not realistic. Among the methods discussed in this review, the tensile method offers one of the analytically straightforward stress conditions, consisting of a dominant peeling stress component. However, this makes the method susceptible to wetting conditions, and it is likely to induce a cohesive failure. On the other hand, the shear methods would be realistic as the shear forces could be used to mimic the forces present in potential de-icing applications. For instance, the largest stress component to remove ice by aerodynamic forces alone would be in shear .Passive icephobicity can be induced via a range of surface and design strategies, most notably via hydrophobicity or superhydrophobicity, surface roughness or texture, surface elasticity, and lubricating or infused liquids. The superhydrophobicity-induced icephobicity is well debated in the literature, and a reduced anchoring/contact area is the key strategy. However, superhydrophobicity is also the synergetic result of low surface energy and micrometric surface roughness, and it is vulnerable to microwater condensation or frost formation, enabling an enhanced mechanical interlocking with rough asperities. On the other hand, a smooth/textured surface morphology, with a surface roughness closer to the critical ice nuclei radius, is encouraged to favour homogenous ice formation or delay heterogeneous ice formation.Low-elastic-modulus polymeric surfaces show a degree of independence from surface energy and roughness, due to a mismatch in strain under stress as a result of having a large moduli difference with ice, thus inducing a lower ice adhesion strength. However, the formation of surface anomalies following mechanical damage may still lead to a mechanical anchoring of ice. The role of surface roughness also extends to icephobicity evaluation methods, and the shear-based methods  indicate a partial relationship with surface wettability over a certain range of surface roughness, which could be attributed to an increase in the true interfacial contact area. In essence, surface roughness is a deciding factor in inducing passive icephobicity and could sufficiently minimise ice anchoring points and/or delay ice formation. However, a synergetic effect of either low surface energy or a soft surface is crucial to induce low ice adhesion.


Loxops coccineus in the Hakalau Forest Unit of the Big Island National Wildlife Refuge Complex, Hawai‘i Island, USA. The soap film smoother predicted zero or near zero densities in the northern part of the domain and two hotspots . Along the boundary the soap film model predicted relatively high densities where ‘Ākepa occur in the adjacent forest and near zero elsewhere. The design-based and soap film abundance estimates were nearly identical. The width of the soap film confidence interval was 16.5% and 0.8% wider than the width of the TPRS smooth and design-based confidence intervals, respectively. The peaks in predicted densities along the boundary indicates leakage by the TPRS smooth. We provide a discussion of the statistical methods, biological findings and management implications of applying soap film smoothers to estimate forest bird population status.Birds are often obligate to specific habitats which can result in study areas with complex boundaries due to sudden changes in vegetation or other features. This can result in study areas with concave arcs or that include holes of unsuitable habitat such as lakes or agricultural fields. Spatial models used to produce species’ distribution and density estimates need to respect such boundaries to make informed decisions for species conservation and management. The soap film smoother is one model for complex study regions which controls the boundary behaviour, ensuring realistic values at the edges of the region. We apply the soap film smoother to account for boundary effects and compare it with thin plate regression spline (TPRS) smooth and design-based conventional distance sampling methods to produce abundance estimates from point-transect distance sampling collected data on Hawai‘i ‘Ākepa  This method captured spatial and temporal correlation in bird densities, thus reducing estimates of uncertainty. However, the penalized thin plate regression spline-based smoother used by leakage ;  leakage . The lea leakage .Conventional distance sampling combines model- and design-based methods. Model-based methods are used to estimate detection probability, allowing plot counts to be adjusted for undetected animals, and estimated plot densities are extrapolated using design-based methods to estimate abundance in the survey region . We can ‘Ākepa are an internationally and federally endangered Hawaiian honeycreeper  as a covariate in the spatial model. Sampling coverage was relatively uniform over the forest stratum thus minimizing extrapolation when making predictions. Managers and policy makers require population estimates at multiple spatial scales such as the species’ distribution, the distribution of suitable habitat or representative land-use, and political or management boundaries. As management plans develop there is increasing mismatch between the sampling extent or resolution and areas of interest, requiring extrapolation to unsampled areas. This can also occur with changes in political boundaries through land acquisition or designation for conservation . Here, wIn this article we model the 2002 ‘Ākepa forest stratum data, selecting the detection function, smoother and response distribution to estimate a density surface, and estimate population abundance and uncertainty. We compare the soap film smoother estimates to those of a TPRS smooth and design-based analysis. Finally, we identify how the soap film smoother handles extrapolation beyond the range of the data.Metrosideros polymorpha and koa Acacia koa with a mixture of native and non-native understory plants. Temperature averages 15 °C with annual variation <5 °C, and annual precipitation averages 2,500 mm with a maximum of 6,100 mm  on 14 transect lines following a systematic random design spanning the upper elevations of Hakalau. U.S. Fish and Wildlife, Big Island National Wildlife Refuge Complex, coordinated and conducted forest bird monitoring on the Hakalau Forest Unit. The distance among points is approximately 150 m with transects being either roughly 500 or 1,000 m apart . For thilocator  using the function locator  Fig. 3)locator (Distance (w where the estimated detection probability (using a preliminary detection function model) was about 0.1. Model selection for the detection function used AIC . Variance and 95% CI were calculated using analytic methods  smoother of the formR statistical language and code is provided in Distance abundance estimates expressed as Estimated coefficients cannot be directly compared between most spline models. Instead, we predicted density and uncertainty estimates and mapped the distribution of ‘Ākepa densities within the soap boundary on a grid with cells 200 m × 200 m. We also evaluated the model predictions at even distances along the boundary. Estimates of abundance and 95% confidence intervals (CI) were computed from the posterior distribution of the fitted soap model following methods described by 2.Using a preliminary detection function model, truncation was set at 58 m. The AIC for the hazard-rate detection function model was 11 units smaller than that of the half-normal model . The Craistogram . Inspectistogram . The shoOur soap film smoother explained 54.6% of the deviance in the data using 2.98 of 18 boundary degrees of freedom and 14.45 of 108 interior degrees of freedom, and the index to ensure adequate complexity was 1.1 . The negative binomial dispersion parameter was 10.55 indicating that the counts were over-dispersed.The main advantage of using the soap smooth is that there was no leakage across the pasture-forest boundary  within the domain and to model the domain boundary.The soap smooth model estimating the boundary reflects that ‘Ākepa densities may extend beyond the soap boundary. As seen in the ‘Ākepa densities, estimating density along the boundary is more realistic since it is not certain that the boundary value will be zero. Along the eastern and southern boundaries the coefficient indicates that the population extends beyond the boundary into the adjacent forest habitat and the boundary that poses no real physical barrier in the ‘Ākepa distribution. Fixing the boundary to zero instead of estimating it would force the smooth of the interior surface to decrease to zero at the boundary. This approach may be appropriate for island populations where birds are restricted to suitable habitat that is located within an inhospitable matrix. Examples of this include bird populations on Pacific islands such as Aguiguan in the Mariana Islands , Tau in R (mgcv (mgcv (The choice of whether to start with a TPRS or soap film smoother should take into consideration features of the study area and data that may affect predicted densities. Features such as a lake or a change in habitat along the study area may result in leakage. A general approach may be to start with a TPRS smoother and if leakage is identified switch to a soap film smoother. The peaks in predicted densities along the boundary indicate leakage by the TPRS smooth, and thus the soap film smoother provides more biologically realistic ‘Ākepa densities. The most common ecological applications of soap film smoothers are in marine ecosystems, with species such as fish , cetaceaR  package R (mgcv . LimitatR (mgcv . Moreovecv (mgcv  facilita--2. Similarly to the The hotspot in the southern part of the domain coincides with the locally abundant density estimates identified by Distance, soap, and TPRS models.Abundances of between 6,300 to 9,700 ‘Ākepa in the extended forest stratum boundary seems realistic for the 2002 survey in this high density ‘Ākepa population , 2016. SAlong the southern portion of the pasture-forest boundary ‘Ākepa occur in relatively large densities see . The comi.e., making predictions outside the range of the data, should be made with caution. The Distance and soap mean abundance estimates were nearly identical; differing by 21 birds. In our case, the soap estimated boundary densities near zero along the north boundary and down both the west and east boundaries in the top quarter of the domain. Within the domain in this region densities were also near zero with very little spatial variability, reflecting that no ‘Ākepa were detected in this region of Hakalau. The soap projected the near zero estimates into the unsampled north-western portions of the domain. Had there been even a few detections in the sampled area adjacent to the extended boundary the density surface would have been less smooth which would have resulted in more variable abundance estimates, as was seen along the south-eastern boundary (see Extending the extrapolation area to coincide with plausible management units, dary see . It is rdary see  and 4. Idary see . The mod10.7717/peerj.15558/supp-1Supplemental Information 1Click here for additional data file.