Arachis hypogaea 2 (Ara h 2)-specific IgE is to date the best serologic marker to diagnose peanut allergy. Ara h 6 shares approximately 60% sequence identity and multiple epitopes with Ara h 2.Our aim was to assess the diagnostic utility and relative importance of Ara h 2 and Ara h 6 in peanut allergy.A cohort 100 of children was studied. The cohort included chidren who had peanut allergy, children who were sensitized to but tolerant of peanut, and children who were neither sensitized nor allergic to peanut. Levels of specific IgE to peanut and individual allergens were quantified by using ImmunoCAP. ImmunoCAP inhibition experiments and mast cell activation tests in response to both Ara h 2 and Ara h 6 were performed. Statistical analyses were done using SPSS version 14 and Prism version 7 software.P < .001), whereas Ara h 6–specific IgE binding was inhibited to a similar degree by Ara h 2 and Ara h 6 (P = .432). In the mast cell activation test, Ara h 2 induced significantly greater maximal reactivity (P = .001) and a lower half maximal effective concentration (P = .002) than did Ara h 6 when testing cosensitized individuals.Ara h 2–specific IgE and Ara h 6–specific IgE showed the greatest diagnostic accuracy for peanut allergy when compared with specific IgE to peanut and other peanut allergens. Most patients with peanut allergy were sensitized to both Ara h 2 and Ara h 6. Ara h 2 reduced Ara h 2–specific IgE binding more than Ara h 6 did (Ara h 2–specific IgE and Ara h 6–specific IgE provide the greatest accuracy to diagnose peanut allergy. Ara h 2 is the dominant conglutin in peanut allergy in the United Kingdom, despite a degree of cross-reactivity with Ara h 6.  Peanut allergy is a prominent IgE-mediated food allergy that has garnered substantial clinical attention owing to the severity of reactions and the increasing prevalence and rates of anaphylaxis.Arachis hypogaea 1 (Ara h 1) to Ara h 17, excluding only Ara h 4, which was initially established with unique nomenclature before being relegated as an Ara h 3 isoform (3.02).,Detection of allergen-specific IgE supports the diagnosis of peanut allergy; however, sensitization alone is not predictive. In the United Kingdom, 11.8% of school-aged children have detectable levels of peanut–specific IgE; however, only 2.6% of these children are genuinely allergic when challenged.,,,,Ara h 1, Ara h 2, and Ara h 3 were initially identified as the major peanut allergens, with 97% of patients with peanut allergy sensitized to at least 1 of these allergens.Given the sequential and structural similarities between Ara h 2 and Ara h 6, we aimed to assess the relative importance of Ara h 2 and Ara h 6 in peanut allergy, their possible cross-reactivity, and the utility of specific IgE to these allergens to diagnose peanut allergy.A/L or greater.A/L or greater. A total of 100 patients were selected purely on the basis of availability of serum samples of sufficient volume. The study and the use of samples were approved by the South East London Research Ethics Committee 2, and written informed consent was obtained from the parents of all of the children.Individuals who were (1) allergic to peanuts, (2) sensitized to but tolerant of peanut, or (3) neither sensitized nor allergic to peanuts and enrolled into the study “Diagnostic Markers of Clinical Allergy versus Sensitization to Peanut” were evaluated. Peanut allergy was determined by a positive oral food challenge (OFC) result, except in the case of patients who had a convincing clinical history of systemic allergic reactions within 1 year of sample drawing, together with a skin prick test (SPT) wheal size of 8 mm or greater and/or a peanut-specific IgE titer of 15 kUA/L were diluted with specific IgE diluent (Thermo Fisher) 1 in 10 and retested.Levels of specific IgE to peanut and peanut components Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 8, and Ara h 9 were quantified in patient plasma by using the ImmunoCAP assay  and the Phadia 100 analyzer according to the manufacturer’s instructions. Quantification of IgE to Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9 was performed at the time of recruitment and IgE to Ara h 6 at a later time, when the test became commercially available. Any values that indicated a specific IgE level greater than 100 kUThe ImmunoCAP ISAC assays (Thermo Fisher) were performed according to the manufacturer’s instructions. The arrays were visualized by using a LuxScan 10K scanner  and analyzed by using Phadia Microarray Image Analysis software.www.jacionline.org).For ImmunoCAP inhibition and the mast cell activation test (MAT), the peanut allergens Ara h 2 and Ara h 6 were purchased from Indoor Biotechnologies . Both Ara h 2 and Ara h 6 were purified natively from peanut kernels by using a combination of affinity and high-performance liquid chromotography. Neither allergen showed contamination with any other peanut allergens , and contamination with each other was minimal (≤0.0005% possible contamination of Ara h 2 with Ara h 6 and possible contamination ≤0.007% of Ara h 6 with Ara h 2 were reported)  or PBS. Patient plasma, which was reported to have a level of Ara h 2–specific IgE greater than 200 kUA/L (n = 2) in prior analyses, required an initial dilution by half in PBS before the aforementioned allergen/PBS preincubation step to ensure that results would be reported within the assays' measuring range. Preincubated samples were then assayed for Ara h2– and Ara h 6–specific IgE by using ImmunoCAP technology, as already described. Results were expressed as specific IgE concentrations (kUA/L) alongside the uninhibited (PBS) control.ImmunoCAP inhibition assays were performed to establish the dominant allergen in Ara h 2– and Ara h 6–dual-sensitized individuals. The samples used in Ara h 2 inhibition assays were selected according to the following criteria: dual sensitization to Ara h 2 and Ara h 6, Ara h 2–specific IgE of >40 kU,LAD2 cells, a human mast cell line that was originally isolated from the marrow of a patient suffering with mastocytosis and is named after the National Insitute of Health’s Laboratory of Allergic Diseases,U test and Kruskal-Wallis P test, respectively. The diagnostic performance of specific IgE to peanut extract and to allergen components was examined against the allergic status to peanut by using receiver operating characteristic (ROC) curve analyses. Optimal cutoff values were generated by determining the Youden index as follows: Sensitivity – (1 – Specificity). Statistical analyses were performed by using GraphPad Prism 7.0  and SPSS 25.0  software.Comparisons of unpaired and paired continuous variables were undertaken by using the Mann-Whitney A total of 100 children  were included in this study; they ranged in age from 5 months to 17 years, and 69 of them were male. The diagnosis of peanut allergy and determination of sensitization were carried out according to the criteria previously described.P = .019), Ara h 2 (P < .001), Ara h 3 (P = .046), and Ara h 6 (P < .001) were all significantly higher in children with peanut allergy than in individuals sensitized to but tolerant of peanut. Conversely, Ara h 1–, Ara h 8–, and Ara h 9–specific IgE were not able to discriminate between those individuals in this population who had peanut allergy and those who were sensitized to but tolerant of peanut . Following the initial analyses, optimal cutoffs for Ara h 2–specific IgE (0.28 kUA/L) and Ara h 6–specific IgE (0.32 kUA/L) were generated. They both reported 82% sensitivity, but the cutoff for Ara h 2–specific IgE had 94% specificity to diagnose peanut allergy and the cutoff for Ara h 6–specific IgE had 90% specificity.The diagnostic utility of each serologic marker was assessed by using ROC curve analyses . The areA-C, in the group of children sensitized to but tolerant of peanut, there was no significant difference between Ara h 2–specific IgE and Ara h 6–specific IgE levels (P = .084). However, for the group of those with peanut allergy, the levels of Ara h 2–specific IgE were significantly higher than the levels of Ara h 6–specific IgE (P < .001), suggesting Ara h 2 as the dominant allergen. This skewing of IgE sensitization toward Ara h 2 also appears to be true across the whole study population, in which the significant difference remains (P < .001) (see www.jacionline.org). Despite this, a strong correlation was observed between Ara h 2–specific IgE and Ara h 6–specific IgE levels (r = 0.8791), as seen in Considering the similarities in sequence and structure between Ara h 2 and Ara h 6 molecules, we wanted to assess which of the 2 allergens was dominant in our patient population. Most individuals sensitized to peanut were sensitized to both Ara h 2 and Ara h 6. For instance, Ara h 2 and Ara h 6 sensitization was seen in 61 of 90 individuals (68%) and 55 of 90 individuals (61%) sensitized to peanut, respectively, with 49 of 90 individuals (54%) reporting Ara h 2 and Ara h 6 cosensitization. As shown in www.jacionline.org). Of these, only 1 individual (patient 164) reported genuine monosensitization to Ara h 6 . Peanut allergy in this patient was confirmed by OFC. Subsequent investigations of this patient’s sample led to ImmunoCAP ISAC analyses, which, however, indicated sensitization to both Ara h 2 (0.6 ISAC standardized units) and Ara h 6 (0.8 ISAC standardized units) but not to any of the other 110 allergens present in that assay.In this study, 2 individuals (4%) from the group with peanut allergy (n = 50) showed detectable levels of specific IgE to Ara h 6 (≥0.1) but remained unsensitized for Ara h 2 (<0.1)  were sensitized to both Ara h 2 and Ara h 6, with 31 of 42 (74%) reporting higher levels of Ara h 2–specific IgE than Ara h 6–specific IgE. To test for possible cross-reactivity between Ara h 2 and Ara h 6 binding, we performed ImmunoCAP inhibition experiments examining binding to Ara h 2 and Ara h 6 by using samples from 15 dual-sensitized individuals with peanut allergy . In thesP < .001) or Ara h 6 (P < .001) was able to significantly block binding of IgE to Ara h 2, in comparison with the PBS controls (A). Notably, Ara h 2 preincubation led to a significantly greater inhibition of Ara h 2 binding than Ara h 6 (P < .001). Ara h 6 binding following preincubation with either Ara h 2 (P = .002) and Ara h 6 (P = .002) showed significant inhibition (B). However, preincubation with Ara h 2 inhibited IgE binding to the Ara h 6 ImmunoCAP to a degree similar to that with preincubation with Ara h 6. These data suggest that Ara h 2 is the dominant allergen in Ara h 2– and Ara h 6–dual-sensitized individuals.Inhibition of Ara h 2 binding assays demonstrated that preincubation of sera with Ara h 2  than Ara h 6 at 0.1 ng/mL (P = .033), 1 ng/mL (P < .001), 10 ng/mL (P = .005), 100 ng/mL (P = .013), and 1000 ng/mL (P < .001) of allergen (see A). Sensitivity and reactivity of the LAD2 cells in response to both allergens were also assessed. All half maximal effective concentration (EC50) values were calculated for both Ara h 2 and Ara h 6 dose responses, with Ara h 6 stimulation showing a significantly greater EC50 (P = .002) (see B). The maximal reactivity (greatest percentage of CD63+ activation) was significantly higher for Ara h 2 than for Ara h 6 in these dual-sensitized individuals (P < .01) (see C). Cosensitization to Ara h 2 and Ara h 6 was associated with a higher proportion of activated mast cells (see www.jacionline.org).MAT analyses showed that Ara h 2 was able to induce a significantly greater proportion of activated mast cells  see , B. The .01) see , C. CoseA/L confirms peanut allergy with 82% sensitivity and 90% specificity, with Ara h 2–specific IgE greater or equal to 0.26 kUA/L improving specificity slightly (94%) with the exact same sensitivity; thus, Ara h 2–specific IgE captures more cases with peanut allergy, and Ara h 6–specific IgE is rarely needed as an additional test for peanut allergy.Ara h 2 and Ara h 6 share sequence and structural similarities, and the majority of patients with peanut allergy are sensitized to Ara h 2. In this study, we confirmed that Ara h 6 is also a major peanut allergen in our patient population. Although most patients were cosensitized to both Ara h 2 and Ara h 6, levels of Ara h 6–specific IgE were generally lower in patients with peanut allergy, and the degree of IgE inhibition to both allergens was higher following preincubation with Ara h 2 than with Ara h 6, suggesting that Ara h 2 is the dominant allergen. Ara h 2 was a more efficient inducer of effector cell activation than Ara h 6 was. We evaluated the diagnostic utility of specific IgE to Ara h 6 in a large cohort of children being assessed for peanut allergy and generated optimal cutoffs for both Ara 6 and Ara h 2 that can be reliably used in the United Kingdom and other areas with similar populations of patients with peanut allergy. A level of specific IgE to Ara h 6 greater or equal than 0.32 kU.,,11,,To our knowledge, this is the largest study assessing the diagnostic performance of Ara h 6–specific IgE in the singleplex ImmunoCAP assay and the first in the United Kingdom. Ara h 2 has long been considered the best marker for peanut allergy in both adult and pediatric patients, and this was reflected in our populationA/L and showing (in a 1:1 dilution) levels below 20 kUA/L, but the majority of these samples showed levels very close to 20 kUA/L after dilution, and this variation would not have affected the results, as inhibition was expressed in relation to the respective PBS control, which was tested simultaneously. Inhibition experiments revealed Ara h 2 as the dominant inhibitory allergen, demonstrating an ability to reduce IgE binding to both Ara h 2 and Ara h 6 to a significantly greater degree than Ara h 6 did. This is consistent with previous data from our own laboratory and with data published by Bernard et al, in which additional linear epitope(s) that were identified on native Ara h 2, and not conserved within native Ara h 6, may offer unique binding sites.,Identifying the dominant 2S albumin between Ara h 2 and Ara h 6 is always likely to be complicated by the structural and/or binding similarities, with 41 shared epitopes previously identified between the 2 allergens.,4 to different peanut allergens, a recent study showed that changes in antibody titer in response to peanut AIT were predominantly to Ara h 2– and Ara h 6–specific IgE and IgG4, with significant increases in Ara h 2 and Ara h 6 IgG4/IgE ratios, highlighting the importance of these 2 allergens in peanut allergy, which is consistent with our findings.Ara h 2 is the dominant allergen and a more potent elicitor of mast cell activation than Ara h 6 is. Specific IgE to Ara h 2 is sufficient to diagnose peanut allergy in the majority of patients.Overall, our data showed that independent as well as cross-reactive sensitizations to Ara h 2 and Ara h 6 are present in dual-sensitized individuals but indicated that Ara h 2 is likely the dominant allergen in our population of United Kingdom–based children. Hence, we consider Ara h 2–specific IgE the most important diagnostic marker in peanut allergy even if Ara h 6–specific IgE levels may need to be determined in rare individual cases. As well as confirming Ara h 2 as the most dominant peanut allergen and demonstrating its superior diagnostic accuracy, our study may inform future allergen-specific immunotherapy (AIT) and other immunomodulatory treatments. Peanut AIT has shown potential as disease-modifying therapy, with large numbers of individuals undergoing treatment reporting reduced symptoms and increased levels of peanut-specific IgG4.


Pedicle screw loosening resulting from insufficient bone-screw interfacial holding power is not uncommon. The screw shape and thread profile are considered important factors of the screw fixation strength. This work investigated the difference in pullout strength between conical and cylindrical screws with three different thread designs. The effects of the thread profiles on the screw fixation strength of cannulated screws with or without cement augmentation in osteoporotic bone were also evaluated. Commercially available artificial standard L4 vertebrae and low-density polyurethane foam blocks were used as substitutes for healthy vertebrae and osteoporotic bones, respectively. The screw pullout strengths of nine screw systems were investigated (six in each). These systems included the combination of three different screw shapes  with three different thread profiles . Solid screws were designed for the cementless screw fixation of vertebrae using the standard samples, whereas cannulated screws were designed for the cemented screw fixation of osteoporotic bone using low-density test blocks. Following specimen preparation, a screw pullout test was conducted using a material test machine, and the maximal screw pullout strength was compared among the groups. This study demonstrated that, in healthy vertebrae, both the conical and dual-core/dual-thread designs can improve pullout strength. A combination of the conical and dual-core/dual-thread designs may achieve optimal postoperative screw stability. However, in osteoporotic bone, the thread profile have little impact on the screw fixation strength when pedicle screws are fixed with cement augmentation. Cement augmentation is the most important factor contributing to screw pullout fixation strength as compared to screw designs. Pedicle screw fixation has been widely used to treat spinal instability and other spinal disorders, including degenerative spinal diseases, scoliosis, vertebral fractures, spinal infection and metastatic spinal lesions –3. HowevNumerous studies have focused on different pedicle screw designs to prevent screw loosening. These designs include screws with an increased outer diameter or length , differeRegarding the design of the pedicle screw thread profile, most pedicle screws in current clinical application are designed with a single-thread profile . A closeIn osteoporotic bone, cannulated screws with cement fixation have been well described to enhance the screw holding power due to the infiltration of PMMA cement into the trabecular bone ,17,19–21The specific purposes of this study were as follows: 1) to investigate the difference in the pullout strength of solid pedicle screws designed with different thread profiles  and screw shapes  in healthy vertebrae, and 2) to test the influence of the thread profile  of cannulated screws combined with cement augmentation in osteoporotic bones.A total of nine screw designs (6 in each design) were examined in terms of mechanical performance in normal and osteoporotic vertebrae. Standard synthetic L4 vertebrae and low-density test blocks were used as the test objects to represent human normal vertebrae and osteoporotic bones, respectively.3, which provided a homogeneous and consistent material with a density in the range of that of human cancellous bone with severe osteoporosis [Commercially available synthetic L4 vertebrae  were used to model normal spinal vertebrae. The synthetic vertebrae were constructed solid foam cancellous cores with a density of 0.16g/cc, which provided a uniform and consistent morphometry similar to human vertebrae. In addition, a synthetic test block (Model #1522–507) was used to simulate severely osteoporotic bone. These synthetic test blocks were made with open-cell polyurethane foam with a density of 0.12 g/cmoporosis .For the screw pullout test in the normal vertebrae, six solid screw designs with combinations of two screw shapes  and three thread profiles  were employed. For the screw pullout test in the osteoporotic bone (test blocks), three cannulated screw designs with a cylindrical shape and the abovementioned thread profiles were employed.All screws have dimensions of 6.0 mm in diameter and 50 mm in length. The specific screw size was chosen to accommodate the geometry of an artificial standard L4 vertebrae into which the screw inserts. The main difference between the conical and cylindrical screws was the taper of the outer geometry from the hub to the screw tip. The cylindrical screws had a constant diameter (6.0 mm) from hub to tip; whereas the conical screws were tapered by 25%, from 6.0 mm at the hub to 4.5 mm at the tip. Both cylindrical and conical screws with the three different thread profiles, i.e., SFT, SCT and DC/DT. For the SFT profile, both the thread pitch and the thread depth were 0.75 mm along the entire length; for the SCT profile, both the thread pitch and the thread depth were 1.5 mm along the entire length; for the DC/DT profile, thread pitch and depth were 0.75 mm in the proximal 20-mm fine-thread region and 1.5 mm in the remaining distal 25-mm coarse-thread region, respectively. Additionally, both the SFT and SCT profiles were designed with a single-start along the entire length, whereas the DC/DT profile was designed with a single-start in the proximal coarse-thread region but two-starts in the fine-thread region . For theA total of 36 standard L4 vertebral specimens were divided into six test groups (6 specimens per group) based on different combinations of two screw shapes and three thread profiles. The method for the insertion of solid screws within standard L4 vertebral specimens was identical to that of our previous study . For allThe method for the insertion of cannulated screws with cement augmentation was identical to that described in our previous study . OsteoboFollowing screw insertion, the instrumented vertebral specimens were embedded in acrylate resin  to allow clamping for the pullout test. The posterior elements of the vertebra were not embedded. Each prepared specimen was then secured to a custom-made grip mounted on the platform of the testing machine . The pedicle screw was secured to a 10-mm cylindrical adapter with universal joint, and the cylindrical adapter was fixed to the upper wedge grip of the testing machine. Following the experimental setup of the specimen, a length-controlled pullout force with a constant crosshead rate of 5 mm/min was applied to the screw head . During Each prepared specimen was tested for failure on axial pullout using an Instron testing machine . The prepared test block was placed on an upper custom-made fixture capable of self-alignment to ensure coaxial alignment of pedicle screw with the pullout ram. The pedicle screw was connected to a cylindrical rod secured to the testing machine. After the specimens were mounted, the testing conditions were identical to that used in our previous study . All thep < 0.05.The magnitudes of the ultimate pullout force were statistically compared. Statistical software  was used to evaluate the effects of the screw design  and thread profiles  on the stability of spinal fixation. An ANOVA test with post hoc analyses was performed to evaluate the differences between groups. Differences were considered significant at Radiological images of the solid conical and cylindrical pedicle screws with the SFT, SCT and DC/DT profiles inserted into the standard vertebrae are shown in p < 0.05).The average maximal pullout strength of the inserted conical and cylindrical pedicle screws with different thread profiles is shown in p < 0.05) in the pullout strength compared to the SCT and SFT profiles, respectively. In contrast, for the cylindrical screws, the DC/DT profile provided 26% and 95% increases in the pullout strength compared to the SCT and SFT profiles, respectively (p < 0.05). Among the six solid screws, the conical screw with the DC/DT profile presented the highest pullout strength (p < 0.05).For the conical screws, the DC/DT profile provided 49% and 68% increases (p < 0.05). The average maximal pullout strengths of pedicle screws without cement augmentation were 45.05 ± 11.56 N, 91.74 ± 15.18 N, and 87.36 ± 22.36 N, respectively. However, no significant difference was found among cemented screws with the SFT, SCT, and DC/DT thread profiles (p > 0.05), suggesting that the thread profile has little impact on the screw fixation strength of pedicle screws fixed with cement in osteoporotic bone.Radiological images of cannulated screws with the SFT, SCT and DC/DT profiles inserted into test blocks are shown in Transpedicular screw instrumentation has been regarded as a successful surgical technique in the treatment of spinal disorders because it ensures a three-dimensional structure over the vertebral motion level, achieving a stiff stabilization. However, screw loosening may occur before spinal fusion takes place in the fixed segments and lead to surgical failure. A previous report indicated a higher failure rate of up to 11% for long-term pedicle screw instrumentation . Among vNumerous reports have indicated that pedicle screw anchoring power is associated with the screw shape, thread profile, screw pitch and screw diameter –14. Our Pedicle screws continue to be studied with different design parameters to improve their anchoring power. In terms of the dual-threaded screw, Brasiliense et al.  used humin vitro experiments. In our recent study [3 to compare the screw fixation stabilities of various perforated screws. They declared that test blocks with densities of 0.32, 0.24 and 0.16 g/cm3 can be used to mimic normal, osteopenic and osteoporotic bones, respectively. They concluded that screw fixation strength had significant correlations with bone density and thread design. Hashemi et al. [3 to mimic the porosity of normal and osteoporotic bones, respectively, to compare the pullout strength and insertion torque of pedicle screws with or without cement augmentation. Their report indicated a significantly positive correlation between screw pullout strength and insertion torque. Additionally, Patel et al. [3 to investigate if these commercial products are appropriate to simulate cadaveric osteoporotic cancellous bone. Their results suggested that PU foam blocks with densities of 0.32, 0.16 and 0.09 g/cm3 are suitable to be used as substitutes for normal, osteoporotic and very low density osteoporotic cancellous bone, respectively.Numerous biomechanical research studies using artificial standard vertebrae  ,33, and nt study  used poli et al.  used PU l et al.  conducteIn the present study, only one amount (3 ml) of PMMA cement was tested, adapted from the literature ,17,36. A3 [3 were chosen to simulate severely osteoporotic cancellous bone for the ease of cement injection. We believe that our experimental design with a combination of 0.12 g/cm3 polyurethane foam and 3 ml of PMMA volume provides a uniform platform for comparison of the fixation strengths of various pedicle screw designs.Human cancellous bone has a wide variety of density. It is generally reported that cancellous bone has densities ranging from 0.09 to 1.25 g/cmin vitro experiments have been extensively used to evaluate the efficacy of new screw designs that promise to enhance the screw stability [Screw pullout tests in tability ,33,34,36tability . In the in vivo are subjected to complex multiaxial loadings; subsequent investigations into the influence of other loading modes, such as lateral bending and dynamic fatigue, should be performed in the future. Nevertheless, in the present study, all experimental procedures were conducted to preserve uniformity and reproducibility. We believe that our results offer valuable information for orthopedists who perform pedicle screw instrumentation surgery.Our study has a number of limitations. First, synthetic composite bone specimens were used as substitutes for human bones. In particular, in the evaluation of cannulated screws inserted in osteoporotic bone, low-density test blocks were chosen to simulate osteoporotic bone rather than synthetic vertebrae because synthetic osteoporotic vertebrae cannot be accessed. This may have had an impact on the bone-screw interfacial strength because the complex geometry of actual vertebrae was not considered. However, the test blocks were made of uniform polyurethane foam, which reduces the influences of the variability of properties and the morphometry of cadaveric bones and provides an effective platform for comparison of the mechanical characteristics of various screw designs in a synthetic osteoporotic model ,15,17. Sp < 0.05). A combination of the conical and DC/DT designs may achieve optimal postoperative screw stabilization. In osteoporotic bone, regardless of screw profile , the strength was significantly higher with than without cement augmentation (p < 0.05). However, no significant difference was found among screws with the SFT, SCT, and DC/DT thread profiles (p > 0.05), suggesting that both the screw shape and thread profile have little impact on the screw fixation strength of pedicle screws fixed with cement in osteoporotic bone. Cement augmentation is the most important factor contributing to screw fixation strength in osteoporotic bone as compared to screw designs.In healthy vertebrae, both conical and DC/DT designs can enhance the bone-screw interfacial strength. Among the six solid screws, the conical screw with the DC/DT profile presented the highest pullout strength (S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


The recent advancements in spine fixation aid in the treatment of complex spinal pathologies. Both the iliac screw (IS) and the S2-alar-iliac (S2AI) screw provide adequate stability in the fixation of complex lumbosacral spine pathologies, leading to a significant increased rate of using these techniques in the daily practice of the spine surgeons. This study aims to analyze, describe, and compare the insertion and positioning parameters of the S2AI screw and IS techniques in children without spinal deformities.An observational retrospective study was conducted at a university hospital in 2018, with 25 computed tomography (CT) images selected continuously. Mann-Whitney-Shapiro-Wilk tests were performed. The reliability of the data was assessed using the intraclass correlation. The data were stratified by age group only for Pearson’s correlation analysis.The mean age was 11.7 years (4.5 SD). The mean IS length was 106.63 mm (4.59 SD). The mean length of the S2AI screw was 104.13 mm (4.22 SD). The mean skin distance from the IS entry point was 28.13 mm (4.27 SD) and that for the S2AI screw was 39.96 mm (4.54 SD).Through CT, the S2AI screw trajectory was observed to have a greater bone thickness and skin distance than the IS. There was a linear correlation between age and screw length for both techniques. A similar relationship was observed between skin distance and age for the S2AI screw technique. In children, the S2AI screw technique presents advantages such as greater cutaneous coverage and implant thickness than the IS technique. Because of the advances in spine fixation, several complex spinal pathologies can now be treated . Among tCompared to the former technique, the latter technique is advantageous with a greater ease of execution and increased mechanical resistance ,6. In coAnother recently developed spinopelvic fixation technique uses sacral and iliac points and is known as the S2-alar-iliac (S2AI) screw, which can even be used for minimally invasive applications -9. The aBoth the IS and the S2AI screws provide adequate stability in the fixation of complex pathologies of the lumbosacral spine, leading to a significant increased rate of using these techniques in the daily practice of spine surgeons .The aim of this study was to analyze computed tomography (CT) images of the lumbosacral area in patients without spinal deformities to determine the anatomical measurements for the insertion of screws into the pediatric pelvic spine.A retrospective analysis of lumbosacral CT images from 25 continuously selected patients who underwent elective procedures at a university hospital between January and December 2018 was performed to investigate abdominopelvic pathologies . The CT images and measurements were collected and analyzed using ISite enterprise software . Fine-cut images with bone windows were used to obtain the measurements.The inclusion criteria were as follows: lumbosacral CT images containing axial, sagittal, and coronal sections with the possibility of three-dimensional (3D) reconstruction; age between 3 and 18 years; and absence of bone fractures or bone tumors. The exclusion criteria were inappropriate images, incomplete records, and vertebra with congenital bone malformation or fusion defects. The study was approved by the institutional review board.The data were stored in an Excel for Mac spreadsheet . The data were entered and imported into SPSS 23 for Mac  for statistical analysis. Continuous data were described by the mean and 95% confidence interval (95% CI). Categorical data were described by the absolute frequency and respective categorical proportion. Inferential statistics were performed to compare the different sides of the body and sites in relation to the tomographic measurements. The data were tested for normality, and since no normal distribution was observed, a nonparametric paired comparison test, Mann-Whitney-Shapiro-Wilk test, was used. The intraclass correlation was used to evaluate the reliability of the data. A type I error up to 5% was accepted as a statistically significant difference. The data were stratified by age only for Pearson’s correlation analysis. Statistical analyses were performed by an independent statistician blinded to the data.The measurements were independently obtained by four examiners. The examiners were trained to perform the measurements prior to the study, with 10 cases selected only for training and were not included in the final sample. All examiners had the same training for all measurement evaluations, and the acceptable threshold variation was 2-3 degrees for angular measurements or 2-3 mm for linear measurements. The measures were standardized as follows: age was measured in years; sex was defined as male or female; thickness, length, and width were measured in millimeters; and angle was measured in degrees.Standardization of the IS trajectory measurements  and the anteroinferior iliac crest . The mean IS length was 106.63 mm (4.59 SD). The mean length of the S2AI screw was 104.13 mm (4.22 SD). The mean maximum bone thickness of the IS was 22.62 mm (0.65 SD) and that of the S2AI screw was 23.77 (0.73 SD). The mean minimum bone thickness of the IS was 14.02 mm (0.54 SD) and that of the S2AI screw was 18.18 mm (0.64 SD).The mean axial angle of the IS entry point was 24.06 (0.54 SD) and that of the S2AI screw was 31.96 (0.58 SD). The mean sagittal angle at the entry point of the IS was 29.86 (1.00 SD), and the S2AI was 30.50 (1.11 SD). The mean skin distance from the IS entry point was 28.13 mm (4.27 SD) and that of the S2AI screw was 39.96 mm (4.54 SD).Virtually all of the analyzed variables were significant different between the IS and S2AI screw, except the sagittal angle. The analysis is shown in The intraclass reliability analysis demonstrated an excellent reliability in all measurements among the examiners. The analysis is shown in p>0.05 but <0.10). However, the other data analyzed did not correlate with age  were higher than those of the S2AI screw. This can be justified by the difference in pelvic inclination and the volume between the upper and lower parts of the pelvic region or due to comparison in the differences between the expected trajectories without considering difficulties of execution between the techniques, generating values different than those seen in practice.Several authors have described similar results in the sagittal and axial angles . The scrAnother important consideration regarding the surgical and postoperative parameters is the distance of the implant from the skin. The IS had a mean distance of 28.13 mm, and the S2AI screw 39.97 mm. Thus, some of the fixation problems in the lumbosacral region are prominence of the synthesis material, minimal skin coverage, pelvic morphological change, possibility of failure of the implants, bone fracture, among others ,5,6. TheAlthough the data of this study and that from the literature are in excellent agreement, there are some numerical differences that can be explained by the different entry points for the two techniques, since even a few millimeters can significantly alter the final values . HoweverIn this study, possible biases may arise from the retrospective nature of the study and from using multiple examiners. However, the biases were minimized through prior training and the standardization of measurements, which can be observed from the excellent overall reliability of the measurements . DiffereThere were no significant differences between sex and laterality. Thus, the mean of the right and left sides was used to obtain the statistical power of the sample.Although it was not the focus of the study, the finding of the S2AI screw affecting pelvic balance should be emphasized, as this modifies the value of the S2AI screw ,15, and Through CT, the trajectory of the S2AI screw was observed to have a greater thickness and skin distance than the IS. A linear correlation between age and screw length was observed for both techniques. A similar relationship was found between age and skin distance for the S2AI screw technique. In the pediatric population, the S2AI screw technique presents advantages of greater cutaneous coverage and implant thickness than the IS technique.Tavares-Júnior MCM, Sanchez FB, Iturralde JDU, Fernandes RJR reviewed the literature, collected, and analyzed the data and wrote the manuscript. Marcon RM, Cristante AF and Barros-Filho TEP analyzed the data, final reviewed the literature, and were responsible for the project and manuscript. Leitaf OB designed the study, analyzed the data, final reviewed the literature, and was responsible for the project and manuscript.


Asthma is one of the most common respiratory diseases encountered in clinical practice. Although the vast majority of asthmatics can be adequately controlled with inhaled steroids and other preventer medications, a small proportion remain uncontrolled. Anti-IgE treatment with omalizumab has been proposed in patients as a preferred approach in step 5 asthma therapy according to GINA guidelines. Although therapy with this molecule is approved for patients with atopic asthma and pretreatment serum IgE levels of 30-1500 only, there have been a few reports of its efficacy in subjects outside this reference IgE range. We report the case of a middle-aged lady with severe corticosteroid-dependent asthma and low serum IgE levels who was successfully treated with 9 months of omalizumab therapy. She gained good asthma control and was tapered off steroid use by the fifth month of therapy with omalizumab. The case report stresses the need for further investigation into expanding the spectrum of omalizumab usage in asthma beyond the current IgE suitability range. The drug is approved for allergic asthmatics with a pretreatment total serum IgE level between 30 and 1500 IU/mL and body weight within a specified range, which is the range over which the drug can reduce enough free IgE to ensure a therapeutic effect. The suitability of the drug in severe asthmatics with IgE values < 30 IU/mL is not well defined although a few case reports suggest clinical benefits. , 5 We shA 58-year-old lady, nurse by profession, residing in the United States, presented to us in the second half of 2019. Her history was remarkable for systemic hypertension, dyslipidemia, and chronic persistent asthma. She denied any history of smoking or tobacco product use. She did not have any environmental or professional exposure to agents triggering her asthma. Her asthmatic symptoms dated back to late adolescence and had progressed in severity since then, with significant worsening over the last 3 years. She was steroid dependent for the last 15 months. Despite being on a maintenance dose of 10 mg of prednisolone per day, she used to experience nocturnal symptoms and needed salbutamol inhalations on an average 3-4 times a week. She used to get exacerbations once a month needing hiking up of prednisolone dose to 40 mg per day for 5-7 days. Her detailed evaluation was done in the United States. Her spirometry showed persistent obstructive ventilatory defect. Her compliance to medications was ascertained, inhaler technique was supervised, and medications were optimised. Thyroid abnormalities, gastroesophageal reflux, allergic rhinitis, and vocal cord dysfunction were ruled out by appropriate evaluations. She was obese (BMI 33) with no snoring or excessive daytime sleepiness measured by the Epworth scale. Cardiac function as measured by ECG and 2D ECHO were normal. CT chest showed no parenchymal shadows, bronchiectasis, or mucoid impaction. She had no features of anxiety or depression ascertained during a formal psychology evaluation. Her IgE levels were <10 mg/mL on multiple occasions in the United States as well as during her current evaluation. She was considered for therapy with biological agents  but therapy with this agent was not instituted as her IgE levels were below the range approved for therapy. She was having significant steroid adverse effects including truncal obesity and osteopenia.With this background, she presented to our outpatient department. Her blood counts revealed a normal total leucocyte count (7700 per cubic mm ) with 6 Omalizumab is a humanized monoclonal antibody with affinity for IgE molecule at its binding site of the high-affinity IgE receptor. Current GINA guidelines recommend omalizumab as a preferred therapy in step 5 care for patients with severe persistent allergic asthma after appropriate phenotype evaluation. Omalizumab is approved as an add-on therapy for moderate to severe allergic asthma in adults and children above 6 years of age. However, the drug is approved only for asthmatics with a pretreatment total serum IgE level between 30 and 1500 IU/mL. Administration in patients with a serum IgE level < 30 IU/L was discouraged, possibly because in the initial report of patients with bronchial hyperreactivity, none had such low IgE concentrations , 5, 7–10εRI expression on basophils and plasmacytoid dendritic cells was also noticed in this study [εRI activation by antiallergen, as well as by antiviral and antibacterial IgE. Omalizumab can neutralize this negative effect on innate immunity, and it can reduce the exacerbations induced by respiratory viruses as was noted in the PROSE study by Teach et al. [The success of omalizumab therapy in nonallergic patients with severe asthma has been linked to the “non-anticirculating IgE” effects of omalizumab. Excellent reviews have attempted to elucidate the mechanisms of action of this monoclonal antibody other than the anti-IgE effect . A 2-yeais study . Such exis study . The firh et al.  In this A study by Johansson et al.  suggestsIt may be prudent to restrict omalizumab administration criteria to atopic asthmatics within the currently mentioned IgE range till the time the potential mechanisms of non-IgE-mediated action are clearly substantiated. However, off label use in carefully selected patients being managed at experienced centres may be justified with informed consent from patients. It may be reasonable to expect that further basic and clinical research may expand the suitability criteria of omalizumab use in allergic states.We report the successful off label use of omalizumab in a lady with severe steroid-dependent asthma who had an IgE level of <10 IU/mL. Mechanisms of beneficial effects rendered by omalizumab in nonallergic asthmatics have been incompletely elucidated, and understanding in this arena is evolving. Studies reporting the efficacy of anti-IgE therapy in this subset of asthmatics are few, and the present article may be expected to add further impetus to exploring new horizons of omalizumab use in allergic states.


The term “pharmacological chaperone” was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein. We discussed a few papers to emphasize successes, stress limits, and indicate possible solutions.Pharmacological chaperones have ceased to be a niche category and have entered the clinical practice for some rare diseases caused primarily by protein instability. Many reviews exist to cover this subject with different points of view  all together . BesidesProtein targets of pharmacological chaperone can be diverse. Enzymes, in particular transferases, represent the most populated group (45%) followed by transporters (28%) and receptors (15%) .The proteins targeted by pharmacological chaperones can have different subcellular localization and, in the majority of the cases, are translocated across the endoplasmic reticulum .Pharmacological chaperones are low molecular weight chemical molecules and should not be confused with molecular chaperones that are proteins ,20. AccoIn principle, direct binding and stabilization should be tested on purified mutants. This is unpractical for mutants that have disulfide bridges and post-translational modifications such as those that are secreted, that are resident in membranes  or the endomembrane system  because they cannot be produced and purified with a high yield. They represent the majority of the cases because proteins that transit in the endoplasmic reticulum are the most frequent targets of pharmacological chaperones .However, although the effect of pharmacological chaperones is more evident on responsive mutants, it can be observed on the corresponding wild type proteins too . The enzyme cleaves globotriaosylceramide, generating galactose that is a product and a reversible inhibitor of the enzyme. There exist more than 400 pathological missense mutations, the majority of which reduce the stability of the protein but do not affect the active site [Ki is at least two or three orders of magnitude lower than that of galactose [Fabry disease is an X-linked lysosomal storage disorder due to mutations in the gene ive site ,32. In 1ive site  showed tive site  reportedive site ,36. The alactose . In 1999alactose . When DGGLA mutants, the HEK-293 cells are exposed to the drug, washed, and lysed. The amount of AGAL is quantified in the extracts by Western blot and standard enzymatic assays with a fluorogenic substrate. Almost 50% of the missense mutations tested so far are responsive to DGJ [Since the pioneering work by Fan and co-workers, the effect of DGJ has been tested on hundreds of missense mutations either on cells derived from patients or on transfected cells. To overcome the difficulty of obtaining lymphoblasts or fibroblasts from patients, Benjamin and co-workers  have deve to DGJ . HoweverDpH7 = 3.7 × 10−9 mol/L, KDpH5 = 6.0 × 10−9 mol/L) [DGJ is a potent inhibitor of AGAL, it binds and stabilizes the enzyme at the neutral pH as well as at acidic pH, (K9 mol/L) . It is u9 mol/L) . They tr9 mol/L) . The benDGJ was approved by FDA for the use of responsive mutations . An inteThe first reports on real clinical cases suggest that the beneficial long terms effects of DGJ should be evaluated carefully. In fact, a raise in AGAL residual activity has been observed in the leucocytes in patients of a Swiss cohort but regrettably, it did not correlate with a decrease in lyso-Gb3 concentration measured in dried blood spots .ab initio, the other ones, which are potentially responsive, must be tested individually. The situation is even more complex in cases where patients are composite heterozygous and hetero-oligomeric proteins can be formed [Pharmacological chaperones cannot be used by all patients affected by a given genetic disease. They act by binding specifically unstable folded  mutant proteins. Hence they are not useful in all the cases, e.g., in which the protein is absent because the gene is affected by a deletion, a stop gain mutation, a splicing mutation, or a mutation occurring in the regulatory regions. Frameshift or stop loss could be treated if the mutation caused the production of a protein with some few extra amino acids at the carboxy-terminus that is able to fold. Pharmacological chaperones are of no use if the active site is affected. Hence, some mutations can be excluded e formed . For some formed ), for ote formed ). For soe formed ,47 and Ge formed ,49). Fore formed . This tee formed . Successe formed ,40,51 ore formed ,52,53,54e formed .DGJ is able to stabilize a large proportion of missense mutations associated with Fabry disease and has been approved for the therapy and its long-term effects on the phenotype are still under verification. It might not be the ideal drug. However, certainly, it represents a good starting point. In the meantime, moving from the structure of DGJ, similar glycomimetic molecules have been proposed.Its enantiomer L-DGJ is 1000-fold less active than D-DGJ and its mode of action is different because it is a non-competitive inhibitor of AGAL. Interestingly the two sugars act synergistically .Iminosugars like DGJ are charged molecules with a negative octanol-water partition coefficient (Log Po/w) that indicates a low capacity to cross membranes . The mosp-methoxyphenyl and the fluorophenyl thiourea derivatives had a chaperoning effect superior to that of DGJ in cells.Druglikeness could be improved synthesizing DGJ-thioureas derivatives. DGJ is an iminosugar analog of galactose. Galactose, in turn, is the terminal part of the natural substrate globotriaosylceramide. Hence it could be expected that extending the structure of DGJ it would be possible to derive a more selective and potent pharmacological chaperone. Although DGJ-thioureas derivatives inhibited AGAL at neutral and at acidic pH similarly to DGJ, the authors  stated tDGJ-thioureas can be described as compounds in which thiourea bridges two moieties, a sugar-like part, which in chemical terms is known the glycone to a non-sugar-like part, which in chemical terms is defined as a non-glycon. Ortiz Mellet and co-workers  proposedKi is 5 times lower at pH 7 than at pH 5.6 and reduces the storage of the glucosylceramide in cells harboring a responsive mutation [pH-sensitive inhibitors can be found among molecules that do not look like substrates or products, screening large collections of small molecules. This was the case of ambroxol that is a mixed-type inhibitor and a stabilizer of lysosomal acid glucosylceramidase (Gaucher disease) and was found screening a library of 1040 FDA-approved compounds. It acts in the endoplasmic reticulum better than in the lysosome because its mutation . A similmutation . How commutation . On the 2-hybridation character takes the place of the amine-type endocyclic nitrogen found in iminosugars [Indeed most efforts to develop a pharmacological chaperone useful in clinical practice were concentrated on Gaucher disease . Isofagonosugars .Besides inhibitors, other ligands can act as pharmacological chaperones. It has been proposed for Fabry disease to look for allosteric ligands that stabilize missense unstable mutants but do not bind the active site and do not inhibit the activity. These molecules would be the ideal pharmacological chaperones. In a pilot study one molecule, 2,6-dithiopurine, was identified by in silico docking and tested by thermal shift assay before passing to tests on transfected cells . AnotherBesides allosteric non-inhibitory ligands, other types of molecules should be considered such as activators compounds. In this case, the example comes from PMM2-CDG a disorder caused by deficiencies in the gene encoding phosphomanno-mutase2. The enzyme catalyzes the conversion of mannose 6-phosphate into mannose 1-phosphate and uses as activator glucose 1,6 bisphosphate. The activator binds the active site but is not consumed. It stabilizes several pathological mutants ,71,72. OSo far we have described pharmacological chaperones. We now move to other small molecules, the so-called “chemical chaperones.” They have been proposed for the treatment of genetic diseases that are caused by unstable proteins too, but “chemical chaperones,” such as osmolites and hydrophobic compounds, do not bind their targets specifically. It has been proposed that osmolites such as betaine, sarcosine, glycerol, glycine, trimethylamine N-oxide increase the energy of the unfolded state of proteins. Hence pharmacological chaperones would raise the difference between the free energy of the folded and unfolded states by lowering the first one whereas osmolites would obtain the same effect mainly by raising the second one  Figure .β-synthase [Osmolytes are relatively aspecific and were proved active on different proteins such as cystathionine synthase , aspartysynthase , adenosisynthase , alpha-1synthase  in cellusynthase , cystathsynthase , alpha-1synthase , ABCC6 psynthase , ATP-binsynthase , parathysynthase , and Wilsynthase  and the synthase , transiesynthase , reducessynthase , and inhsynthase ,90 and ssynthase . Disentasynthase .Small molecules can stabilize missense mutants acting indirectly. This is the case of heat-shock protein inducers. The first example is provided by 4-PBA that could act as a hydrophobic chaperone as well as an inducer of hsp70. Other heat-shock response co-inducers have been described. Arimoclomol is an aromatic heteromonocyclic compound that underwent clinical trial phase 3 for Amyotrophic Lateral Sclerosis . It rescMost disease mutations destabilize the affected protein but do not affect the active site and mutant pathogenic proteins can be rescued. This goal can be achieved using small molecular weight molecules that can be administered orally and reach difficult districts such as the central nervous system. The approach is not limited to enzymopathies and has been transversally proposed for many diseases. Admittedly the pathway from tests in vitro to a clinical trial is long but the steps to be followed are clear since they are well established in traditional medicinal chemistry. On the contrary, other very promising approaches such as gene therapy are still at their infancy. Different classes of small molecules  can be cPharmacological chaperones play a special role among them. In fact, they are by far more specific than the others for a given protein. It is important to realize that most pharmacological chaperones tested so far are inhibitors or antagonists of their targets and that this occurred only for practical reasons. Special regimens must be envisaged to maximize their stabilizing activity and minimize their inhibitory activity. We might consider these molecules as first-generation drugs. They played an important role to prove the value of the approach but more effective molecules should be sought. If this was not feasible, we should consider combined therapies, using a specific pharmacological chaperone at a low dosage but potentiated by other types of small molecules. Unspecific drugs that promote protein folding and restore proteostasis can be useful for diverse diseases and can be found among approved or experimental drugs in the first place.


Complications in the form of esophageal injury, tracheal injury, injury to carotids, implant failure, loosening of screws, etc do occur after anterior cervical surgeries. Although intra-operative esophageal injuries are as such rare, there have been few reports of delayed esophageal perforation as well after anterior cervical surgeries. We report a very rare case of migration of missing screw from anterior cervical plate after anterior cervical corpectomy and plating, which had ultimately migrated down to colon and had to be removed via colonoscopy. Along with removal of migrated screw from colon, revision of failed anterior cervical surgery was done wherein plate and screws were removed with mesh cage left in-situ as it was snug-fit while pharyngeophageal perforation was explored and was found to be spontaneously healing, with addition of posterior Bohlman’s interspinous wiring for added stability. Migration of screw from the anterior cervical plate into the colon although very rare, should be always kept in mind and its potentially serious complications. We also conclude that particular attention should be given to elderly people with poor bony quality who have high chances of implant failure, along with attention to proper cage size, screw position and proper locking of the screw to further lessen the chances of implant failure. Hardware failure is the third most cause of esophageal perforation after intra-operative esophageal retraction and manipulation1. Although esophageal injuries usually occur intra-operatively, there have been reports of delayed esophageal perforation after anterior cervical surgeries which usually happen due to hardware failure or frictional injury due to the underlying implant. We report a very rare case of screw migration to the colon after anterior cervical plating which had to be removed via colonoscopy.Esophageal injury, tracheal injury, injury to carotids, implant failure, loosening of screws, etc are reported complications after anterior cervical surgeriesA 65-year-old male, without any comorbidities presented to us with complaints of weakness in all four limbs and bladder involvement for the past 18 months following a fall in the bathroom. The patient was diagnosed as degenerative cervical spondylotic myelopathy with OPLL and was associated with central cord syndrome. Initially after fall, he was suggested surgical treatment but he choose to go with conservative treatment. Even after one year of conservative treatment in the form of rehabilitation programme with gait training, balance exercises and proper counseling against fall prevention, patient had negligible improvement. After one year of injury, the patient was operated at another centre wherein anterior C5 cervical corpectomy with mesh cage, bone grafting, and anterior cervical plating was done.After surgery, although neurological status of the patient remained the same but was otherwise doing well, only to return after 1.5 months of primary surgery with mild neck pain which was insidious in onset, non-progressive in nature, aggravated by activity and relieved with rest with no diurnal variation and no associated systemic symptoms. An radiograph of the cervical spine done within three days of neck pain showed loosening and back out of upper screws of the anterior cervical plate . A totalA careful search for the missing screw was done by chest radiograph and CT chest where it was not found, followed by abdominal radiograph  and CT aUpper GI endoscopy was done to look for status of the upper GI tract. As screw had reached the colon and showed gradual movement on serial abdominal radiographs, we waited for its spontaneous expulsion with feces but it showed no significant movement over next 10 days and remained stuck to a single place on right side of colon which warranted colonoscopic removal to avoid perforation of the colon. On colonoscopic examination and screw removal, there was an evident erosion of superficial mucosa without any perforation of colon.After screw removal from colon, patient was planned for plate removal in view of possible irritation to surrounding structures including esophagus and additional posterior stabilization to maintain alignment of cervical spine. Anteriorly via Smith Robinson approach, the plate and screws were removed. As initial surgery was done on the left side with right sided screw migrating down the colon, we preferred the right side approach for repeat surgery so that pharyngeophageal perforation could be better examined from the right side. While removing, it was found that distal screws were holding well and bone strength seemed good. Pharynx showed healing perforation while the proximal portion of esophagus was also examined properly and could not find any erosion or perforation. Mesh cage was found snugly fit with fusion taking place between C4-C6. Posterior surgery was done by Bohlman’s interspinous wiring. Postoperatively patient was rehabilitated. Presently, patient is self-voiding, independent walker with the help of a stick and able to carry most of his activities of daily living. Patient was evaluated using JOA score (Japanese Orthopaedic Association Score) with Pre-operative score of 8/17 which improved to 13/17 at final follow-up of 1.5 years.2. Iatrogenic esophageal perforation can occur during the surgical approach due to inappropriate placement or displacement of sharp-toothed retractor blades into the esophagus. Any protruding bone or fixation device pressing on the pharyngoesophageal wall may lead to focal ischemia with necrosis, cellulitis, abscess, and perforation2. Esophageal perforations due to pressure caused by the metallic implant and its micro trauma has also been reported. Isolated reports of esophageal perforation due to screw migration have been reported. Fountas et al3 had a case of screw extrusion into the gastrointestinal tract 16 months after the primary surgery while Gazzeri et al4 reported a case of delayed screw migration into the gastrointestinal tract 11 years after the primary procedure. Migration of screw via esophagus can be asymptomatic, mildly symptomatic or can cause severe symptoms and even death. A case of fatal sudden airway obstruction due to plate failure and graft migration has also been reported.Esophageal perforation is a very rare complication associated with anterior cervical surgery and plating with incidence of 0.25-1.49% and high mortality rate of upto 20% which can go upto 50% if left untreatedet al5 reported a case of complete disappearance of anterior cervical plate and screws, which had eroded through the posterior wall of the esophagus and were expelled via the gastrointestinal tract.Fujibayashi Failure of plate screw construct may be due to the inadequate screw purchase, screw mal-position, poor bone quality, posterior ligamentous instability, or incomplete fusion. Most of the studies have concluded that the main predisposing factor in the development of screw or plate extrusion is the initial malposition of the screws. In our case, with distal screws having good hold precluding very poor bone quality, we presume that an initial mal-positioned screws or an inadequately sized MESH cage may have caused the failure.We conclude that particular attention should be given to the optimal position of the screw and ensure it is properly locked. Also, the quality of bone should be assessed pre-operatively and with poor quality bone, posterior fixation should be supplemented. If the anterior implant is failing, it should be tackled at the earliest to decrease the chances of severe complications. Chances of colonic perforation should be considered when a screw has migrated down from cervical spine to the colon and doesn’t show any movement in colon.


The mechanism behind its antidepressive action is unknown but could be linked to increased synaptogenesis and down-regulation of cerebral 5-HT2AR. Here, we investigate if a single psychedelic dose of psilocybin changes synaptic vesicle protein 2A (SV2A) and 5-HT2AR density in the pig brain. Twenty-four awake pigs received either 0.08 mg/kg psilocybin or saline intravenously. Twelve pigs (n = 6/intervention) were euthanized one day post-injection, while the remaining twelve pigs were euthanized seven days post-injection (n = 6/intervention). We performed autoradiography on hippocampus and prefrontal cortex (PFC) sections with [3H]UCB-J (SV2A), [3H]MDL100907 (5-HT2AR antagonist) and [3H]Cimbi-36 (5-HT2AR agonist). One day post psilocybin injection, we observed 4.42% higher hippocampal SV2A density and lowered hippocampal and PFC 5-HT2AR density (−15.21% to −50.19%). These differences were statistically significant in the hippocampus for all radioligands and in the PFC for [3H]Cimbi-36 only. Seven days post-intervention, there was still significantly higher SV2A density in the hippocampus (+9.24%) and the PFC (+6.10%), whereas there were no longer any differences in 5-HT2AR density. Our findings suggest that psilocybin causes increased persistent synaptogenesis and an acute decrease in 5-HT2AR density, which may play a role in psilocybin’s antidepressive effects.A single dose of psilocybin, a psychedelic and serotonin 2A receptor (5-HT MDL100907) and an agonist radioligand ([3H]Cimbi-36) for autoradiography.Using in vitro autoradiography, we measure SV2A and 5-HTthy pigs . We invethy pigs ,7. A folthy pigs . To inve3H]UCB-J autoradiography in the hippocampus and the PFC. Compared to the saline-treated group, psilocybin treatment was associated with 4.42% higher SV2A in the hippocampus (p < 0.0001) one day after psilocybin injection and 9.24% higher SV2A in the hippocampus (p = 0.024) seven days after psilocybin (p = 1), but seven days after psilocybin administration there was 6.10% higher SV2A in the PFC (p < 0.0001).ilocybin . One day2AR density as measured with the antagonist radioligand [3H]MDL100907 in hippocampus and PFC. One day after the intervention, hippocampal 5-HT2AR density (p < 0.0001) and PFC 5-HT2AR density was similar , and PFC 5-HT2AR density (p < 0.0001) in the psilocybin-treated group than in the saline-treated group. With [3H]Cimbi-36, similar to [3H]MDL100907, 5-HT2AR density was not significantly different in the hippocampus and the PFC seven days after the psilocybin intervention compared to saline. density  was 43.3 density  was 50.12AR density when measured with [3H]Cimbi-36 compared to [3H]MDL100907 one day after psilocybin intervention in the PFC. This difference of 41.26% was statistically significant at p = 0.033  γS binding stimulation mediated with DOI followed by immunoprecipitation with specific antibodies, while the complex is captured with protein A-polyvinyl toluene scintillation proximity assay. When this approach is made on post-mortem brain tissue from patients with schizophrenia, the canonical Gαq/11-protein pathway of 5-HT2ARs is found to be unaltered in the PFC, whereas the pro-hallucinogenic Gαi1-protein pathway is functionally overactive in the PFC Cimbi-36; 67% 5-HT2AR occupancy will elicit psychedelic effects in humans [Donovan et al.  identifin humans . Intraven humans .All animal experiments conformed to the European Commission’s Directive 2010/63/EU and the ARRIVE guidelines. The Danish Council of Animal Ethics had approved all procedures .3H]UCB-J . [3H]MDL100907  was used as an antagonist radioligand for 5-HT2AR and [3H]Cimbi-36  as an agonist radioligand for 5-HT2AR/5-HT2cR. Radio-Thin-Layered-Chromatography (R-TLC) was performed for all radioligands to measure the radiochemical purity (RCP) and integrity of the parent compound. The mobile phase for [3H]UCB-J R-TLC was Acetonitrile:Ammonium formate [25:75] . The mobile phase for [3H]MDL100907 R-TLC was Chloroform:Methanol:Ammonia solution [90:9:1]. The mobile phase for [3H]Cimbi-36 R-TLC was Chloroform:Methanol:Triethylamine [94:5:1]. [3H]UCB-J and [3H]MDL100907 had high RCP (96–98%) while [3H]Cimbi-36 had an RCP of 52–57%. Radioactivity was corrected for RCP of [3H]Cimbi-36 after TLC.Radioligands used for autoradiography included SV2A imaging with [3H]UCB-J or 0.01% ascorbic acid, 4 nM CaCl2 and 0.1% BSA for [3H]MDL100907 and [3H]Cimbi-36.Sections were thawed to room temperature for 30–45 min before prewashing twice for 10 min in 50 mM Tris-HCl pre-incubation buffer set to 7.4 pH containing 0.5% bovine serum albumin (BSA) for [3H]UCB-J in 50 mM Tris-HCl buffer containing 5 mM MgCl2, 2 mM EGTA and 0.5% BSA (pH 7.4) for 1 h. Incubation was terminated by three 5-min washes with 4 °C pre-incubation buffer followed by a rapid rinse in 4 °C deionized H2O (dH2O). For 5-HT2AR, sections were incubated in assay buffer containing 3 nM [3H]MDL100907 or 1 nM [3H]Cimbi-36 in 50 mM Tris-HCl containing 0.01% ascorbic acid, 4 nM CaCl2 and 0.1% BSA (pH 7.4) for 1 h. Incubation was terminated by two 10-min washes in ice-cold pre-incubation buffer followed by a rapid rinse in ice-cold dH2O.For SV2A, the sections were incubated in assay buffer containing 60 nM Cimbi-36 and [3H]MDL100907, was performed for the PFC using a permutation test (with 1000 permutations) on log-transformed values of treatment effect at one day and seven days (Equations (3) and (4)).The data were analyzed using R , while GraphPad Prism  was used for data visualization. Comparisons between group means (


In this study we analysed the acoustic properties and presence of haddock calls in the Arctic fjord Kongsfjorden  in one year. Data were collected with three autonomous acoustic recorders located in the inner, middle, and outer parts of the fjord. The fjord is characterized by a gradient of oceanographic conditions from the inner to the outer part, reflecting changes from Arctic to Atlantic waters. Haddock sounds were more abundant in the outer fjord than in the middle fjord, whereas they were absent at the inner site. Mainly at the open-water site, the call abundance exhibited strong periodicity and a correlation with the cycles of neap tide (15 days) in August, with a clear diel cycle (24 h) in September and October. This result suggests that in this extreme environment with 24 h of light during summer, haddock regulate their acoustic activity according to the main available oscillating external physical driver, such as tide during the polar summer, while when the alternation of light/dark starts, they shift the periodicity of their calls to a diel cycle. Calls were recorded outside the spawning period (from July to October), and their characteristics indicated non-reproductive communicative contests. By using a detailed sound analysis based on previous laboratory studies for the first time, we suggest that the monitored population contains mainly juveniles , showing the predominance of females in the middle fjord and juveniles at the open-water site. Melanogrammus aeglefinus) is a widely distributed fish belonging to the Gadidae family and represents an important target species for the fishery industry. It lives in the Atlantic and in the Barents Sea at a latitudinal range between 38° and 80° N, usually at depths between 80 and 200 m and at temperatures between 2 and 10 °C1. Haddock aggregate during the spawning period, i.e., in March to April in the deepest parts of the North Sea2 and up to early June off the coast of Norway3. Little is known about the habitat where haddock spawning occurs and which features promote fish aggregations. Usually spawning fish aggregate close to the seabed4 at depths of 400–500 m off the coast of Norway, but also in shallower waters in Vestfjord5 or in Balsfjord6. Reproductive maturity is reached at 4 years at a length of 40–60 cm1.The haddock  composed of two short pulses at low frequencies8. Sound production varies throughout the year, but it peaks during the reproductive season9, when the haddock produces a diversity of calls associated with particular behavioural contexts8. Studies performed in aquarium tanks revealed that the sounds are mostly produced by males during patrolling displays, which are both a territorial and a spawning behaviour in haddock9. Interindividual differences in the knock produced by patrolling males have been demonstrated, likely reflecting changes in the drumming muscles involved in sound production. As courtship proceeds, knocks are repeated at a faster rate culminating in a continuous hum10 and eventually leading to spawning. Outside the spawning season, M. aeglefinus calls are slightly different, composed mostly of two or more pulses of longer duration11.Similar to other gadoid species12 showed that the structure of the haddock sound unit changes according to fish maturity and is sex-specific. These differences are explained by sexual dimorphism in the generating mechanism, with drumming muscle size varying according to sex, age, maturity and season13.Sounds are also produced by females and by juveniles. Casaretto et al.6. The majority of the recorded sounds were very similar to those produced by patrolling males in the aquarium, and the presence of sounds with a fast repetition rate indicated that the fish were close to spawning. This confirmed the use of passive acoustic monitoring for studying the biology and distribution of the species. Neverthless, we are not aware of any published long-term studies on vocalizing Gadidae fish species in the sea. In addition, although captivity studies suggest the possibility of using haddock sound structure to infer the sex and maturity of emitters, similar work has not been applied to sea recordings.Haddock vocalizations recorded in captivity have been used for the identification of this species in the sea. At the head of the Balsfjord (Norway), reproductive haddock aggregations have been found by means of their vocalizations in several locations15, Georgia16, USA waters17, the Gulf of Mexico18, Australian waters19, and in Argentinean waters20. In these studies, it was commonly demonstrated that sound activity increases during the reproductive season, with vocal peaks during nighttime hours. Photoperiod and temperature are the principal natural cues triggering seasonal maturation and acoustic activity in fish, as observed infamily Scenidae21. In polar environments, however, the strength of the daily light–dark cycle is greatly reduced during summer, when there is almost continuous daylight. Few studies have investigated the presence of behavioural rhythmicity in the absence of light–dark alternation23, but variable behavioural responses to continuous polar lighting have been highlighted. In the small polar cod (Boreogadus saida), for example, diel vertical migration is exactly synchronized with the changing of day and night, and when the midnight sun starts in late May, this migration stops24; however, in another gadoid species, the burbot (Lota lota), the activity rhythms persist during the polar day or night26. Northward expansion of boreal Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) (in particular juvenile fish) into areas inhabited by the native polar cod (Boreogadus saida) has been observed only in the recent period of increased inflow of warmer Atlantic water to the western coast of Svalbard28, and no data on their rhythmicity have been reported. Thus far, seasonal patterns of haddock calls have been studied only in tanks with an alternating photoperiod9.Long-term studies investigating temporal patterns of fish calls are available for low-latitude geographical areas, such as the Mediterranean Sea29, making it the third most important fish species captured after the capelin  and the cod (Gadus morhua). In recent years, an increase in haddock captures was recorded. This can probably be partially attributed to a northward shift of some demersal species as waters have warmed, the sea ice extent has decreased30 and larger feeding areas have become available during the light season29.In the Svalbard Archipelago (Norway), the haddock represents an important species for the commercial fishery, with more than 239,000 tons caught between 1980 and 2013The main aims of this study are to (1) characterize haddock calls in the Arctic environment, (2) to evaluate the spatio-temporal distribution of the haddock by using its calls, (3) explore the correlations between haddock acoustic activity and external physical factors such as tide and solar elevation and (4) discuss the use of acoustic data for detecting fish age classes and sex and haddock behavioural patterns.A total of 7751 h of recordings from three sites that covered a period of one year (from April 2014 to March 2015) were analysed  for each month of acoustic activity  at the open-water and middle-fjord sites is reported in Fig.  for eachFigure In the whole period graph for the middle fjord Fig. , small pIn the best-fitting GAM  Table , Fig. 5,During July and October, a smaller number of haddock sounds were detected in the recordings see Fig. , and sevThe recorded sounds showed a clear double-pulsed low-frequency structure repeated in quick succession, similar to those recorded in the aquarium by Hawkins and Amorim (2000). Most of the recorded sounds were composed of short sequences (a few seconds) of irregularly spaced knocks Fig. . No callA significant difference between sounds recorded at the open-water and middle-fjord sites was highlighted for F1  and for pulse interval and the amplitude ratio  but not for F2  or sound duration . The mean values  of the considered parameters are reported in Fig. Table The median power spectral density of selected sounds (no = 451) is shown in Fig. 31.The recorded sounds confirmed the presence of haddock in the fjord due to their consistence with the sounds recorded in the laboratory. This result is consistent with that of a precevious study based on trawling and fishing gear12, presenting lower peak frequencies and a longer pulse interval 26. This indicates adaptive plasticity in timing cues for acoustic activity in Melanogrammus aeglefinus, possibly due to the recent presence of this species in the polar area or perhaps the cycle of its prey. According to Reneaud et al.28, the diets of co-occurring juvenile haddocks in fjords, open water, and sea ice around Svalbard are mainly based on zooplankton, i.e., appendicularians and Limacina spp., and the activity rhythms of the zooplankton have been documented in the polar area. for example, a recent study showed that during the polar night, vertical zooplankton migrations are driven by moonlight in synchrony with the altitude and phase of the moon, with a shift from solar-day to lunar-day vertical migration36.Our analysis revelead that haddock sounds are present in Kongsfjorden from July to October, i.e., outside the spawning period of this species. The temporal pattern at the open-water site, where we recorded the most haddock sounds, showed a gradient shift from long periodicity linked to the moon tide to shorter periodicity directly correlated with sunlight see Fig. . At the Erignathus barbatus, whose vocalisation rate varies significantly both daily and monthly37, showing a correlation with solar elevation when light and darkness turnover is occurs.In Kongsfjorden acoustic rhythms have also been reported also for the bearded seal 38. In contrast, the highest density of haddock sounds was recorded in the most “Atlantic” part of the fjord, the open-water site, which is influenced by water masses from the Fraim Strait belonging to the West Spitsbergen Current39. In the middle part of the fjord, which is probably characterized by conditions intermediate to those of the Arctic and Atlantic parts, the number of recorded sounds was significantly lower than that at the open-water site. This can be explained by the haddock being an Atlantic species that usually adapts well to temperatures up to 10 °C, although 0-group haddock fish (fish in the first year of their life) have been recorded above 80° N40 and a haddock spawning area has been reported in the Norwegian Sea between 60° and 75° N1. An effect of water depth on the distribution of fish (sounds have been recorded at 75 m at the open-water site and at 20 m at the middle-fjord site) cannot be excluded. However, the recording sites had different oceanographic conditions, and one, the middle-fjord site, presented la shallower depth (20 m versus 75 m for the other two sites). These differences, especially in depth, could affect detection among our recording sites due to differences in acoustic propagation among the sites. The detection range of a certain sound by a recorder depends on multiple factors, such as the sound source level (unknown for haddock sounds), background noise, bathymetry, hydrophone sensitivity, and software detector sensitivity. For this reason, in the future, the source level of haddock sounds should be assessed, and further studies in the natural environment should consider the detection range of the monitoring system to clarify the sampling volume at each recording site.Haddock sounds in the Arctic fjord Kongsfjorden were not homogeneously distributed at the three sites of recordings, and were characterized by an internal–external gradient corresponding to the gradient of Arctic–Atlantic oceanographic conditions. No vocalizations have been detected in the inner part of the fjord (glacier site), i.e., the most “Arctic” part, which is affected by the colder, fresh water coming from the run-off of glaciers and ice calving10 as well as recorded at sea35. As a consequence, we can conclude that the species is not using the Arctic fjord Kongsfjorden for spawning.Bacause they were recorded outside the reproductive season, the vocalizations reported here are not related to the breeding behaviour of the haddock. This is supported by the sound types, which lack the typical acoustic pattern associated with the patrolling behaviour  or continuous hums  that are produced during reproduction, as described in the captivity studies12. The relationships between physiological parameters and the resultant sound waveform were further described by Casaretto et al. (2016)12. On the other hand, clear overlap between males and females was evident here for the frequencies of the first (F1) and second (F2) pulses as well as for the pulse interval. These findings exclude a role of these sound features in sex recognition.For the first time, we applied sound analysis based on captivity studies, showing that the vocalizations recorded in the fjord are not mainly produced by mature haddock males. This conclusion is based on using the sound amplitude ratio as the most reliable variable for distinguishing the sex of the emitting fish: in the laboratory recordings, the amplitude ratio of females was always equal to or greater than 1, whereas it never exceeded 1 in males’ sound units. This sex difference was accompanied by sexual dimorphism in haddock drumming muscle mass, similar to that found in other gadiids: haddock males were found to have more well-developed drumming muscles than females of similar size throughout the whole year12, the sound units produced by immature fish consist of two pulses with similar amplitudes  and present higher peak frequencies than those of adults. The sounds recorded at sea, however, do not show clear intra-population differences in frequency values  of the juvenile callers may have played a role in the recorded sound structure. Poor development of the drumming muscle has been proven in haddock juveniles11, as in other juvenile stages of gadiids39; however, our knowledge about the ontogeny of fish sound production is poor and thus far limited to a few fish species38, so the effect of fish size on sound structure is difficult to fully evaluate. In any case, if we consider the aquarium values for the amplitude ratio of juvenile sounds  and our classification , it seems more plausible that a juvenile sound would be included in the ‘male-like’ category than a male sound would be included in the ‘juvenile-like’ category. This supports our analysis.In the present study, the amplitude ratio was slightly less efficient in distinguishing haddock fish maturity. According to the captivity studyues Fig. ; therefo31 captured using trammel and fyke nets and showed that only juvenile haddock  were present. Further studies are needed to fully confirm the results of our analysis.We conclude that the fjord is populated mainly by juvenile haddock. Further, the two recording sites seem to differ in their population abundance and composition, with predominantly females at the middle-fjord site and juveniles at the open-water site. These conclusions must be made while taking into account the technique used to obtain them. In passive acoustic monitoring, the sampled volume is limited to the detection range of the recorder system, which in this study could change with oceanographic conditions of the two sites. However, our results are in agreement with those of research on the Ichthyofauna of Kongsfjorden that quantitatively described the local shallow-water fish communityPassive acoustic monitoring (PAM) is a valid alternative to more expensive and logistically difficult techniques for monitoring the distribution of “new invasive species” in an area that is difficult to study, such asthe Arctic. This is also valid outside the reproductive season, as demonstrated by this work. Long-term PAM can provide important information on the relationship between soniferous species present in Svalbard  and their new adaptations to the different light/dark cycles in this changing environment as well as information on the sex and maturity of the individuals present.42. Actively calving marine-terminating glaciers supply icebergs and freshwater input, which, together with other physical factors, determine the local species composition39. Glacier fronts are upwelling areas for zooplankton43 and attract many top predators. However, climate-induced changes in oceanographic conditions are significantly altering Kongsfjorden habitats39. Tidewater glaciers in Kongsfjorden are retreating44, and recently, during the wintertime, sea ice formed only in the inmost parts of the fjord  with standard lengths between 6.0 and 19.0 cm (mode: 17.5 cm).Kongsfjorden, located at 79° N and 12° E along the western coast of Spitsbergen Island is a glacial fjord. Here, however, colder Arctic water masses mix with warmer Atlantic waters belonging to the West Spitsbergen CurrentData were collected year-round from April 2014 to March 2015 by using three autonomous passive acoustic recorders . The instruments were deployed at three different sites Fig. . The firThe hydrophones had a recording bandwidth of 8 Hz–150 kHz and a sensitivity of − 165 ± 4 dB re 1 V/μPa in the 25 Hz–10 kHz band. The recorders were located at a depth of 75 m for the glacier and open-water sites, and of 20 m for the middle-fjord site; they were 8 m from the bottom, anchored with a 35-kg ballast and kept vertical by a small subsurface buoy. Furthermore, to avoid any noise due to moving parts, the connections between the recorders, the acoustic release and the ballast, as well as those with the buoy, were composed of non-metallic ropes.We set the sampling frequency at 48 kHz with a resolution of 16 bits, and no pre-amplification or filtering was applied  during the recordings. Depending on the period of the year and the availability of logistic support at different sites, data were collected following different schedules with a daily duty cycle of 100%  or 30% (one day every three days). For each day sampled, we used a duty cycle of 50% (the first 30 min of every hour) , were analysed using two sets of Matlab code developed with the aim of detecting and characterizing all the haddock calls. The first code used the Teager Kaiser operatorTo achieve accurate measurements, a total of 451 sounds with a good signal/noise ratio and with a uniform temporal distribution  were used to analyse the sound structure of the recorded knocks, with 213 recorded at the middle-fjord site and 238 recorded at the open-water site. No haddock sounds were recorded at the glacier site. To avoid strong dependency of the measurements from a few individuals, for each file, we used a maximum of 10 signals.12, a second set of MATLAB code was used on the files resampled at 8 kHz to measure and check by an operator the temporal position and the correspondence amplitude of the peaks of the two pulses that composed each knock −1; (3) the frequency of the second pulse of a knock, calculated as f2 = 0.5(t7 − t6)−1; (4) difference in amplitude between the first and second pulses, calculated as (a7 − a6) (a3 − a2)−1; and (5) the pulse interval, i.e., the interval between the start of the first pulse and the start of the second, calculated as t6 − t2.Following the studies of Casaretto et al.ock Fig. . From th12, (1) in male sounds, the first pulse of the knock is higher in amplitude than the second pulse, resulting in an amplitude ratio value lower than 1 (0.6 ± 0.2 SD); (2) females produce sound units composed of two pulses, with the second pulse higher in amplitude than the first, resulting in an amplitude ratio value higher than 1 (1.6 ± 0.6 SD); and (3) immature fish show two pulses with similar amplitudes but different polarities, thus resulting in an amplitude ratio close to 1 (0.8 ± 0.3 SD). Accordingly, all the sounds with an amplitude ratio lower than 0.8 were assumed to be produced by haddock males , sounds with an amplitude ratio ranging between 0.8 and 1.1 were assumed to be produced by juveniles (juvenile-like sounds), and all the sounds with an amplitude ratio higher than 1.2 were assumed to be produced by haddock females .Differences in pulse amplitude can be considered indicative of the sex and maturity of the emitting fish. Following Casaretto et al. (2016)To evaluate any differences in these acoustic parameters between sites, the non-parametric Mann–Whitney U test was used.2/Hz) was calculated with Welch’s overlapped segment averaging estimator method , the power spectral density (PSD) . The down envelope tide (DE-Tide) was obtained using the MATLAB function Envelope (designed by L. Wang). DE-Tide represents the oscillation of tide amplitude . This oscillation occurs every 15 days, when the sun and the moon form a right angles.Tide tables for Kongsfjorden were obtained online  after inputting the geographical coordinates, metres above sea level  and UTC time.To explore temporal patterns in the presence and abundance of haddock sounds, we applied single-series spectrum analysis, considering only the period of occurrence of haddock sounds at each recording site.To evaluate differences in the abundance of haddock sounds between sites, the non-parametric Mann–Whitney U test was applied to all data set, considering each month of acoustic activity separately.47) were used to evaluate which predictors best explained the occurrence of haddock sounds, which were considered the response variable, over the months. Signals were tested as a function of the predictors hours , DE-Tide, and Solar Elevation angles as described above for each month, at both the open-water and middle-fjord sites. A zero-inflated Poisson regression distribution and an identity link function were chosen. Cyclic cubic regression splines were used for all the explanatory variables. All the analyses were performed in R48.Moreover, generalized additive models (GAM) (R “mgcv” package version 1.8–28; Wood