Mechanical complications from spinal fusion including implant loosening or junctional failure result in poor outcomes, particularly in osteoporotic patients. While the use of percutaneous vertebral augmentation with polymethylmethacrylate (PMMA) has been studied for augmentation of junctional levels to offset against kyphosis and failure, its deployment around existing loose screws or in failing surrounding bone as a salvage percutaneous procedure has been described in small case series and merits review.How effective and safe is the use of PMMA as a salvage procedure for mechanical complications in failed spinal fusion?.Systematic search of online databases for clinical studies using this technique.11 studies were identified, only consisting of two case reports and nine case series. Consistent improvements were observed in pre- to post-operative VAS and with sustained improvements at final follow-up. The extra- or para-pedicular approach was the most frequent access trajectory. Most studies cited difficulties with visibility on fluoroscopy, using navigation or oblique views as a solution for this.Percutaneous cementation at a failing screw-bone interface stabilises further micromotion with reductions in back pain. This rarely used technique is manifested by a low but increasing number of reported cases. The technique warrants further evaluation and is best performed within a multidisciplinary setting at a specialist centre. Notwithstanding that underlying pathology may not be addressed, awareness of this technique may allow an effective and safe salvage solution with minimal morbidity for older sicker patients. •Percutaneous cementation at a failing screw-bone interface arrests further micromotion with pain reductions.•This rarely used technique is manifested by a low but an increasing number of reported cases.•It warrants further evaluation but may allow an effective and safe salvage solution for older sicker patients. LooseniThe use of polymethylmethacrylate (PMMA) has expanded since its introduction by Galibert and Deramond in 1984 to include kyphoplasty and fenestrated pedicle screws with cement augmentation . Percuta22.1A systematic search of databases including Pubmed, MEDLINE, EMBASE and Cochrane library, Web of Science and OVID. Languages included English and French. The search words included: cement augmentation polymethylmethacrylate, vertebroplasty, pedicle screw loosening, instrumentation. A further search was conducted of papers citing or cited by relevant papers. Using the PRISMA guidelines , we sear2.2Studies were included in the analysis if the following inclusion criteria were met: (1) any clinical study design (2) study population: participants were patients, over 18 years of age with failed spinal fusion from a loosened pedicle screw; (3) who were treated with a salvage percutaneous cement augmentation at the level of the loosened pedicle screw(s); (4) clinical outcomes: pre- and post-operative visual analogue score (VAS) or patient global impression of change (PGIC). Studies that did not meet the above criteria were excluded from selection. Exclusion criteria were patients with pedicle screw loosening due to infection, patients who were treated with cement at the time of the original screw insertion, patients who underwent open salvage surgery or where the loose pedicle screw was removed and revised to a cemented screw.2.3Data were extracted independently by two authors, without blinding to the title and author affiliation. Relevant information was extracted from studies, included: (1) the title; (2) authors; (3) year of publication; (4) sample size; (5) type of intervention; (6) surgical approach; (7) duration of the follow-up; (8) clinical outcomes as reflected in the pre-, post- and follow-up VAS scores (9) cement dosage; (10) peri-operative complications and (11) bone cement leakage. We documented whether industry funding was received based on disclosures.2.4A total of 195 citations or papers were identified . 131 cit2.5The selection process of relevant studies is summarized in 2.6The overall assessment of the evidence displays a collection of low certainty evidence . There a2.7Cases performed by a radiologist were more likely to be CT and fluoroscopy guided  or with A latero- or extra-pedicular lumbar approach was taken along the path of the loosened screw, or parallel route to a sacral screw . In the PMMA was used in all cases and volume varied from 3 mls  to 12 mNone of the patients experienced vertebral cement augmentation-related complications such as neural element compression or cement embolization. Consistency of the cement was reported as low viscosity so as to allow flow around the screw  to high 2.8Length of stay varied from 1.2 days  to 7 day2.9One of the PJF patients demonstrated mild kyphosis correction on prone positioning which was sustained at 1 year . CT at o2.10Patients demonstrated decreases in VAS from pre-operatively to post-operatively ). Post-oFu et al. described two revision surgery cases . One pos2.11With adoption of the above research, the authors have used these techniques for salvage cases and have additional technical comments. Pre-operative CT analysis is crucial for planning. Guidance of the trocar is significantly more difficult than with vertebroplasty and as docking the trocar on a screw is difficult, a cross hairs  approach, as opposed to a triangulation approach is best for accurate trocar placement. The image intensifier will require oblique positioning, so that the projection of the rod is medial to the screw. Given the oblique trajectory, a long trocar (15–20 cm) may be required. A beveled trocar (as opposed to diamond head) will facilitate oblique docking where the bevel is facing away to stop sliding. Ensure that the bevel fully is inside the bone .If using a navigated or robotic system, it is crucial to ensure that the port diameter is not significantly larger than the trocar so as to reduce wander and leakage. A highly viscous cement is recommended, bearing in mind that under high insertion pressure, it will speed up the cure time. It is important to recognize tactile feedback from the cement and in particular to stop if there is a loss of resistance. Repeated lateral-projection fluoroscopy is needed with careful monitoring for back flow that may indicate intra-canal or intra-foraminal leakage. It is also worth combining cementation of loose screws with intra-discal cement placement , - cement discoplasty as previously reported, to confer additional stability and anterior column loading.3Percutaneous cement augmentation procedures have recently shown significant benefit in patients with degenerative spinal instability and in the presence of a vacuum disc phenomenon . This sy•Localized stress-strain mismatch with persistent motion, screw loosening, localized bone resorption and failure of fusion a and b.F•Segmental stress-strain mismatch at the end of a long construct, with loss of vertebral integrity, usually PJF a and b.FOsteoporosis is invariably present in these patients, thus medical treatments and appropriate rehabilitation should incorporate this. Two causative pathologies were evident from this series:Optimizing treatments thus rely on identifying causative pathologies requiring localized backfilling of the screw track in the former and approximating vertebroplasty in the latter  is a common complication of adult degenerative deformity and extension of instrumentation may be otherwise inevitable, all too frequently failing on a patient's expectations. Thus, PMMA insertion at the upper instrumented level (UIV) and UIV level-plus one, may be considered given the high morbidity alternatives a and b. Most conventional transpedicular instrumentation designs feature top-loading screws which means that revising a screw requires open surgery with removal of a significant proportion if not all of the rod. On revision, the “halo” of osteolysis around the screw threads or porosity of bone may be too extensive to accommodate a larger or longer screw. Other anatomical techniques include changing the screw trajectory (very difficult) or extending the instrumentation to another vertebral level (typifying a longer rod-working length) . The def, as opposed to the PJK/PJF mechanism of failure. Thus, sufficient backfilling of the screw cavity is important to achieve stability at the screw base and a transpedicular approach was mentioned by one study for S1 (The shape of the osteolytic “halo” has been described as ellipsoid at S1, greatest at the screw base and tapering off towards the screw tip , as oppoy for S1 . CriticaPMMA use in salvage cases is a recent phenomenon given the increasing prevalence of degenerative spinal deformity and greater levels of spinal fusion . This se4Developing a safe and feasible procedure that avoids high morbidity spinal surgery is a matter of great importance for healthcare providers, users and policy makers. While revision remains the gold standard, this systematic review identified that trialling percutaneous cementation for a failing screw-bone interface is worth consideration in selected cases, as an effective salvage procedure with minimal morbidity, appropriate for older or sicker patient and accepting that simply injecting PMMA does not address the underlying pathology. This rarely used technique is manifested by the low numbers of reported cases. This technique is within the remit of both the spine surgeon and interested interventional radiologists but is in its infancy and thus best performed within a multidisciplinary setting at a specialist center.The authors received no financial or material support for the research, authorship, and/or publication of this article.


In patients who undergo femoral fracture fixation with a cephalomedullary nail, the breakage of one or more of the distal interlocking screws is a well-described phenomenon. The presence of a broken interlocking screw in patients who require the removal of their cephalomedullary nail presents a unique challenge. The broken interlocking screw may be retrieved, or the screw may be retained if it is not engaged within the nail and the nail can safely be removed while leaving the broken screw fragment behind. We report a hip conversion arthroplasty case with a broken interlocking screw where the nail was removed with ease and the broken screw was assumed to have been left behind. Cerclage wires were placed for an apparent proximal femoral fracture. Postoperative X-rays demonstrated a large lucency tracking from the prior location of the distal interlocking screw to the calcar region. This finding made it evident that the broken screw had been retained in the nail and was dragged up the femur upon nail removal, causing a large gouge spanning the entire femur. In femoral fracture fixation with a cephalomedullary nail, distal interlocking screw breakage, also known as autodynamization, is a well-described phenomenon . SeveralA 69-year-old female with a remote history of a right subtrochanteric femur fracture treated with a long cephalomedullary nail  presented with symptomatic right hip osteoarthritis. Imaging demonstrated a healed fracture and autodynamization of the proximal-distal interlocking screw Figure .After a discussion of treatment options with the patient, she elected to proceed with conversion total hip arthroplasty. Using the patient’s existing incisions, a modified Hardinge approach was used to expose the hip joint. After the dislocation of the hip, it was reduced again, and attention was turned to the removal of the cephalomedullary nail. Through the proximal incision, the set screw was reversed, and the blade of the device was removed using the device-specific removal tools. Next, using the patient’s existing distal incisions , the intact distal screw and the lateral fragment of the proximal screw were removed with a screwdriver. The nail extraction tool was engaged, and the nail was gently removed. Because the nail was able to be removed without resistance and no screw fragments were revealed upon the nail’s removal, it was assumed that the broken screw fragment had disengaged and was retained in the distal femur.Next, the total hip arthroplasty was completed using a standard cementless acetabular component  and a fit-and-fill design proximally fitting a cementless femoral component . During femoral component implantation, a proximal femoral fracture was noted. After further exposure of the fracture line, it appeared to track distally, necessitating the placement of two cerclage wires above and below the lesser trochanter. Finally, the femoral component was impacted into place with a firm endpoint and no apparent fracture displacement.Postoperative X-rays demonstrated a total hip arthroplasty in the appropriate position with linear lucency tracking from the proximal screw hole to the calcar. In addition, the medial broken screw fragment was absent from the distal femoral region. With these findings, it was determined that the linear lucency was secondary to the medial aspect of the broken screw being retained within the nail and gouging the femoral canal as the nail was removed Figure .The patient’s immediate postoperative course was uncomplicated, and the patient was made touch-down weight-bearing. At the four-week postoperative visit, the patient presented walking with a cane and admitted non-compliance with the weight-bearing restriction. Radiographs obtained at this time demonstrated femoral stem subsidence of approximately 1.5 cm with no change in the radiographic linear lucency. The potential risks and benefits of the surgical and non-surgical options to address the implant subsidence were discussed, and a collective decision was made to proceed with compliant touch-down weight-bearing with the goal of achieving ingrowth in the subsided position. At each subsequent follow-up appointment, the radiographs revealed no evidence of further subsidence or failed ingrowth Figure .Lateral radiographs of the knee taken five years after surgery revealed complete healing of the femoral gouge Figure .The patient ultimately had a successful outcome with a Short Form 12-point physical outcome score of 43.1 and a Veterans RAND 12-point outcome score of 43.4.Hip conversion arthroplasty cases often pose substantial challenges related to hardware removal. Our case involved the removal of a cephalomedullary nail in the setting of a broken distal screw. Because the nail was removed without resistance, it was incorrectly assumed that the screw had disengaged from the nail and was retained in the distal femur. This resulted in an iatrogenic femoral gouge spanning the entire femur, which likely contributed to stem subsidence. In light of this complication, we recommend the removal of the broken medial fragment or fluoroscopic visualization of the distal screw prior to stem removal to confirm disengagement from the nail.Several previous studies have described different techniques for broken screw removal . Most meIf the complication had been recognized intraoperatively, the surgeon would need to consider fixation of the gouge and femoral stem fixation. Since the gouge was located on the medial aspect of the femur, a typical lateral plate would not achieve the goal of securing the stabilization of the gouge. Placing a plate anteriorly would have helped achieve this secure fixation, but would not be recommended due to the added surgical trauma and potential complications of securing a plate in this location. As such, the gouge would have been best secured with multiple cerclage wires spanning the line of the gouge or multiple anteroposterior screws distal to the femoral stem combined with multiple cerclage wires at the level of the femoral stem. For femoral stem fixation, due to the length of the gouge, a long-stemmed femoral component would not have been able to bypass it. Femoral stem options that could have been considered include a short conical stem, a tapered metaphysically engaging stem (as was used in this case), or a cemented stem. Although a cemented stem may have diminished the risk of stem subsidence, we would still recommend limited weight-bearing in the initial postoperative period to protect the fixation of the gouge.With proper pre-operative planning and the use of intraoperative fluoroscopy, potential complications related to broken interlocking screws can be prevented in total hip conversion arthroplasty. There are inherent risks to the retention and removal of broken distal interlocking screws. Surgeons should address the broken screws preoperatively, even if they intend to leave the fragment in place. The authors recommend thorough preoperative planning and the use of intraoperative fluoroscopy during the removal of a cephalomedullary nail, as this would reveal retained screw fragments and prompt early intervention to prevent complications.


Carbonic anhydrases (CAs) are a metalloenzyme family that have important roles in cellular processes including pH homeostasis and have been implicated in multiple pathological conditions. Small molecule inhibitors have been developed to target carbonic anhydrases, but the effects of post-translational modifications (PTMs) on the activity and inhibition profiles of these enzymes remain unclear. Here, we investigate the effects of phosphorylation, the most prevalent carbonic anhydrase PTM, on the activities and drug-binding affinities of human CAI and CAII, two heavily modified active isozymes. Using serine to glutamic acid (S > E) mutations to mimic the effect of phosphorylation, we demonstrate that phosphomimics at a single site can significantly increase or decrease the catalytic efficiencies of CAs, depending on both the position of the modification and the CA isoform. We also show that the S > E mutation at Ser50 of hCAII decreases the binding affinities of hCAII with well-characterized sulphonamide inhibitors including by over 800-fold for acetazolamide. Our findings suggest that CA phosphorylation may serve as a regulatory mechanism for enzymatic activity, and affect the binding affinity and specificity of small, drug and drug-like molecules. This work should motivate future studies examining the PTM-modification forms of CAs and their distributions, which should provide insights into CA physiopathological functions and facilitate the development of ‘modform-specific’ carbonic anhydrase inhibitors. Carbonic anhydrases (CAs) are a ubiquitous metalloenzyme superfamily that catalyse the reversible hydration of carbon dioxide. This process has a crucial role in several physiological functions including pH balance , respiraChlamydomonas reinhardtii  and reaction time (t) with consideration of product inhibition and/or substrate depletion was modified slightly to incorporate a delay in the measurement time (a)  such that KM + [S] ≃ KM. Such a condition is valid since the substrate concentration [S] used in the esterase assay was 0.3 mM at pH 8.0 while the reported KM towards pNPA is 2.92 mM and 15.4 mM at pH 7.8 for hCAII and hCAI, respectively [The catalytic efficiency (ectively .(2)v0=k® spin desalting column, Thermo Fisher Scientific). Nanoscale nanoelectrospray ionization emitters with tip inner diameters of ~680 nm were fabricated using a Flaming/Brown-type microcapillary puller  and coated with a mixture of gold and palladium by a sputter coater  as previously described [Human CAs were buffer-exchanged into 20 mM ammonium acetate (pH 6.8) by gel filtration columns (Zebaescribed . Native escribed .S30E/hCAIIS29E almost completely inhibit the esterase activity, while phosphomimics hCAIS49E/hCAIIS48E enhance the activity of hCAII but inhibit the activity of hCAI.Human carbonic anhydrases can be phosphorylated as intact and folded proteins. Potentially functional PTMs that may modulate enzymatic activity can be efficiently and accurately assessed by measuring enzyme activity directly in cell lysates with overexpressed hCA phosphomimics. The catalytic efficiencies obtained using this approach agree well with those obtained for fully purified hCAs. The significant increase or decrease in the catalytic activity of human carbonic anhydrases resulting from S > E phosphomimics are specific to both isoforms and modification sites. For example, phosphomimics at the homologous residue hCAI2 hydration reaction using a broader scope of inhibitors, such as sulfanilamide, metanilamide and other simple benzene-sulfonamides. Overall, these findings provide new insights into the regulatory mechanisms and functional effects of hCA post-translational modifications, with implications for the development of novel therapeutic approaches targeting this important enzyme family.This research suggests that phosphorylation may be a regulatory mechanism for hCAs, and identifying upstream kinases for hCAs and specific modforms of hCAs that are prevalent in vivo should be beneficial for understanding their pathophysiological functions. Furthermore, carbonic anhydrase phosphomimics can alter enzymatic activity and their inhibition profiles with sulphonamide small-molecule drugs, emphasizing the importance of investigating CA modforms for their pathological relevance and the development of modform-specific therapeutics. Future work will focus on identifying the specific modforms of hCAs in vivo and assessing the impact of the phosphorylation sites of hCAs on the enzyme kinetics of the CO


An active, bloom-associated prokaryotic community relies on these microalgae for their organic carbon requirements, however an analysis of the encoded metabolic pathways within them is lacking. Hence, our understanding of biogeochemical cycling within sea ice remains incomplete. Here, we generated metagenomic assembled genomes from the bottom of first-year sea ice in northwestern Hudson Bay, during a spring diatom bloom. We show that the prokaryotic community had the metabolic potential to degrade algal derived dimethylsulphoniopropionate and oxidise sulfur. Facultative anaerobic metabolisms, specifically, dissimilatory nitrate reduction and denitrification were also prevalent here, suggesting some sea ice prokaryotes are metabolically capable of adapting to fluctuating oxygen levels during algal bloom conditions. Such denitrification could be an important loss of fixed-N Nitzschia spp. having driven its release in previous studies of sea ice [Desulforhopalus (Deltaproteobacteria), have been found in Antarctic sea ice [Sea ice creates a dynamic habitat for microorganisms, controlled by fluctuations in light, salinity and temperature caused by seasonal extremes, combined with localised variability in atmosphere-ice-ocean gas and nutrient exchange. An area of particularly high seasonal productivity is the bottom centimetres of the ice, where an abundant microalgal community develops in spring, dominated by diatoms . This mi sea ice . Both EP sea ice , 4, 7, w sea ice , 9, alth sea ice , 11. Ind sea ice , and ana sea ice .n = 4) from northwestern Hudson Bay, to reveal a proportion of the prokaryotic community encoded for facultatively anaerobic processes. We also identified DMSP degradation and sulfur oxidation genes in dominant taxa, suggesting that algal derived DMSP is an important energy source for sea ice prokaryotes.In this study, we analyzed the prokaryotic communities associated with a diatom bloom in sea ice (−1). The community was dominated by pennate diatoms  and Entomoneis kjellmanii (Site F), which had a relative abundance of 63–82% of the community [Significant algal growth was documented in the bottom-ice at all sites along a transect to the ice flow edge  / Pseudomonadales (GTDB), Alteromonadales (NCBI) / Enterobacterales (GTDB), Flavobacteriales and Rhodobacterales Fig. , which tzobiales .Fig. 2SeFacultatively anaerobic Dissimilatory Nitrate Reduction (DNR) to ammonia and denitrification genes were prevalent in many abundant members of the prokaryotic community. Eight MAGs Fig.  encoded 2 fixation genes (RuBisCO), suggesting a chemoheterotrophic lifestyle. All Nitrincolaceae MAGs contained complete or partial pathways for sulfur oxidation, either via the soeABC enzyme complex, reverse-acting dissimilatory sulfate reduction (sat/aprAB) or the sox system, indicating that these organisms were all involved in sulfur cycling. Interestingly, members of the Thiomicrospirales were well represented by two partial MAGs . Thiomicrospirales are typical oxygen minimum zone taxa [Thioglobus spp. belonging to the SUP05 clade of chemoautotrophs, known to couple sulfite oxidation to nitrite reduction [Thioglobus sp. contained reverse-acting dissimilatory sulfate reduction genes (aprAB), whilst the other encoded a Sulfide:Quinone Oxidoreductase. Both encoded RuBisCO, which suggests a chemolithoautotrophic lifestyle, however, no dissimilatory nitrate reduction genes were identified, which may have been because of the incomplete nature of these MAGs (73–75% estimated by CheckM).The respiratory reduction for nitrate may also be coupled to the oxidation of reduced sulfur to support chemolithotrophic growth, evidence for this was found in three Rhodobacteraceae MAGs Fig. , one of eduction . From ouThis study highlights a heterogeneous sea ice prokaryotic community which is metabolically capable of sea ice algal DMSP degradation and associated sulfur oxidation, whilst also encoding for facultatively anaerobic metabolisms. These findings, combined with previous observations for anoxic conditions and denitrification, show that sea ice prokaryotic communities have the potential to maintain metabolic activity under fluctuating oxygen levels and influence nutrient cycles. Whilst we show genomic potential, we cannot comment on the activity of such processes. Further investigations are now needed to characterise the activity of these metabolic pathways in concert with the extent and variability of oxygen concentrations in sea ice. An understanding of sea ice denitrification is critical for our understanding of microbial production and nutrient limitation during algal blooms in this rapidly changing habitat.Supplementary TextSupplementary Table 1


Tightening and loosening of the prosthetic components of the implant are carried out with various screw designs. This study compared the rate of deformation of the abutment screw in two hexagonal and star screw head designs after consecutive tightening and loosening. P<0.05.  In this study, two fixtures were mounted vertically in die stone blocks using a surveyor. Then the corresponding abutment  was mounted on each fixture. In each fixture, six abutment screws from each manufacturer were used . The abutment screw head of the Dio-UF system is hexagonal, and the abutment screw head of the Dio-SM system is star-shaped. The samples were examined under a stereomicroscope (Nikon C-DS) at a magnification of×50. The image of each abutment screw head (6 abutment screws in each group) was prepared before the procedural steps and after 5, 10, and 15 times of tightening and loosening with 25-Ncm torque using a stereomicroscope. Then the differences in the surface areas of consecutive specimens  between hexagonal and star-shaped abutment screws were calculated. The study results were reported via descriptive statistical methods (mean±standard deviation). Statistical analysis was performed using SPSS 24, and the significance level was defined at P<0.05). In addition, there was a statistically significant difference between the area values and the number of tightening and loosening rounds separately in both screw types (P<0.001). Also, the surface areas of the head of both screws in all tightening and loosening rounds were significantly different (P<0.001).  Increasing the number of tightening and loosening rounds increased the screw head surface area in both hexagonal and star shapes. At all stages, the changes in the star-shaped screw head were greater than in the hexagonal screw. These changes were statistically significant at all stages ( Increasing the number of tightening and loosening rounds increased the screw head surface area in both hexagonal and star-shaped geometrical forms. Also, the extent of area changes at all stages in star-shaped screw heads was greater than in hexagonal screws. It is very important to replace missing or lost teeth. Failure to do so can cause esthetic and functional problems, speech disorders, and temporomandibular joint problems. Dental implants replace missing or dysfunctional teeth; they are surgically placed in the bone.4 In general, the reliability and stability of the implant and abutment connection is a necessary prerequisite for the success of dental implants.5 The connection of the abutment to the fixture significantly affects the success of the implant prosthesis. In the most common method of mechanical connections, screw abutments are used to connect the abutment to the implant.6 The coping screw is usually the weakest joint in the prosthetic chain. Any malalignment in the occlusion, cast adaptation, or force can lead to loosening or fracture of the screw during function. These problems can protect the body of the implant against more serious complications. When these problems occur in a splinted restoration, other implant abutments are exposed to the risk of overloading and more problems than the implant in question because a cantilever and force intensifier is created.9 Implant-abutment junction design can affect screw loosening, soft and hard tissue preservation, and leakage into the implant site.10 Fracture and loosening of the screw are related to the components, metal fatigue, intermittent movements during function, non-axial pressure, bone elasticity, and the amount of torque and preload.12 Screw fracture and loosening are among the most critical challenges of implant prosthetics that have been considered in research today.13 The excessive increase in torque can reduce the proportional limit in the screw and cause loosening and permanent deformation of the screw; it also decreases torque, which results in loosening and fatigue of the screw.15 Preload depends on various factors, such as the design of the screws and threads, the surface roughness, the materials used, and the applied torque. Adequate torque is vital for preventing screw loosening.16 In case of successive unscrewing of the screw, surface changes in the screw head‒screwdriver and abutment‒screw interfaces will be inevitable. Changes in the screw head due to fracture are a potential problem.17 proposed that the clinical expectation of a screw cap could be met by a maximum of 10 screw loosening rounds, after which the screw should be discarded, and a new screw should be used. Various screw designs have been proposed by implant manufacturers to solve this problem, including hex-slotted, star, and square-slotted hexagons. Guzaitis et al16 reported increased scratches during the placement of the square-shaped wrench over the hex-slotted shape. However, square-head screws performed better concerning fracture resistance and had a lower fracture rate than hexagonal screws. Kim et al2 reported no significant difference in the elastic deformation in the square and hexagonal designs after 10 times of tightening and loosening with 32-Ncm torque. De Paiva et al14 Clinically, the possibility of tightening and loosening the abutment screw in implant-based prostheses is significant.18 After loosening the screw, it is necessary to re-tighten or replace the abutment screw with another screw, but in some cases, more measures are required. Considering the high prevalence of screw loosening as a result of successive tightening and loosening of the abutment screw, and given the lack of sufficient data on the fracture of the screw head and the contradictory results in the existing studies, we aimed to compare the rate of deformation of the abutment screw in two hexagonal and star screw head designs in consecutive tightening and loosening. In this study, performed in the School of Dentistry, Tabriz University of Medical Sciences, two fixtures with a diameter of 4.1 mm and a length of 11.5 mm from two different types of an implant system, Dio-SM and Dio-UF , were used.® EP Corporation, Tokyo, Japan) made in a standardized metal mold measuring 50 × 20 × 20 mm. The gypsum blocks in which the fixtures were mounted were fixed on a table holder. Then on each fixture, the corresponding abutment  was mounted. Also, a 25-Ncm torque was applied with the abutment wrenches of Dio-SM and Dio-UF companies according to the manufacturers’ recommendations. Using a surveyor, the fixtures were then fixed vertically in die stone blocks . The abutment screw head of the Dio-UF system is hexagonal, and the abutment screw head of the Dio-SM system is star-shaped. The samples were examined under a stereomicroscope (Nikon C-DS) at a magnification of × 50. The image of each abutment screw head (6 abutment screws in each group) was prepared before starting the procedural steps and after 5, 10, and 15 times of tightening and loosening with 25-Ncm torque using a stereomicroscope.2 , and the surface areas of geometric shapes were calculated in mm2 -4. Then P < 0.05. The results were reported using descriptive statistical methods (mean ± standard deviation). First, the normality of data distribution was investigated to compare the difference in the area of the abutment head design in hexagons and stars. Then, repeated-measures ANOVA, Mauchly test, Bonferroni test, and t-test were used to analyze the differences between the groups. Finally, statistical analysis was performed using SPSS 24, and the significance level was defined at P < 0.001). An increase in the number of tightening and loosening rounds of the screws increased the surface area of the abutment screw head in both hexagonal and star geometric forms, increasing the wear in the hexagonal abutment screw from 0 to 15 rounds to 33.01 units and to 54.26 units in the star abutment screw. Also, in all the tightening and loosening rounds, the hexagon abutment screw surface area was less than that of the star-shaped abutment screw. The difference in the abutment screw surface areas between the two types of abutment screws in all the tightening and loosening rounds was statistically significant ( According to P < 0.001). Also, due to the significance of the results of the Mauchly test in both groups, the test for the sphericity assumption was used for comparisons. Repeated-measures ANOVA was used to compare the surface area of the hexagonal abutment screw design after 5, 10, and 15 rounds of tightening and loosening. For this purpose, this test was used separately in each type of abutment screw. According to the results, there was a statistically significant difference between surface area values in terms of the number of tightening and loosening rounds separately in each abutment screw design  from different types of an implant system (Dio-SM and Dio-UF) were used. Abutments related to each system were attached to the fixtures with the same diameter and length . To determine the effect of the number of consecutive tightening and loosening rounds on the deformation of the implant abutment screw, six abutment screws specific to each system in the hexagonal (related to Dio-UF) and star (related to Dio-SM) designs were used on the respective abutments. The surface area changes in the star design in all the stages were greater than those in the hexagonal screw. The difference in the surface area change in all the phases in the star design was significantly greater than that in the hexagonal head. Since the contact surface of the star-shaped screw head is larger than the hexagonal screw, the contact surface area of the wrench in the star screw head design is higher, and its fracture is more severe. It seems that the fracture of the star screw can accelerate rapidly as a result of use. Studies on the abutment screw changes are very limited.2 examined the resistance of square and hexagonal shapes in screw attachments to deformation and reported that the geometric shapes of screws did not affect their deformation. The difference in the results might be attributed to the methodology, the implant brands, and differences in the corresponding screw head designs. De Paiva et al16 found that the screw head design could affect the odds of fracture and deformation of the abutment head screw. Accordingly, the fracture in the head with a star design is more probable and more severe than the square head design, consistent with the present study. Kim et al19 examined changes in the screw abutment after repeated tightening and loosening rounds. There were several scratches near the screw head slot, possibly due to the repeated contact between the driver tip around the screw slot when the driver tip repeatedly tightened or loosened the screw. This was more noticeable in the square screw than in the hexagon. They also concluded that screws with a square design were better than hexagonal screws in the transmission and the effectiveness of screw tightening. According to this study, screws with a square slot design were more resistant to fracture or distortion than hexagonal screws. Kim et al According to the findings of the study in general:P < 0.001). By increasing the tightening and loosening rounds, the surface area of the abutment head increased in both hexagonal and star geometric forms. The results also showed a statistically significant difference between tightening and loosening rounds in both groups (The change in the surface area of the star design of the abutment screw in all the stages was more than the hexagonal screw of the abutment.  The highest increase in the surface area in both types of abutment screws was in 5‒10 rounds of tightening and loosening. This study was supported by a grant from the Dental and Periodontal Research Center, Tabriz University of Medical Sciences. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The authors declare that they have no competing interests related to authorship and/or publication of this work. The study protocol was approved by the Ethics Committee on Medical Research, Tabriz University of Medical Sciences. The authors received no financial support for the research, authorship, and/or publication of this article.


This study aimed to determine whether the positional relationship between the underside of the screw head and the surface of the alveolar bone could alter the stress on the two surfaces and affect the stability of implanted anchor screws. First, in order to confirm the extent of the gap between the mini-screw and the bone surface, a mini-screw was placed in the palate of rabbits and examined histologically. As a result, in the conventional screw implantation procedure, oral mucosa between the base of the screw head and the bone creates a spatial gap. Removal of the oral mucosa eliminates this gap. Then, we compared the positional difference of the screw in a contact and gap group by analyzing stress distribution on the bone and screw. Analysis using the finite element method showed that more stress was loaded on both the bone and screw in the gap group than in the contact group. Cortical bone thickness did not affect stress in either group. The effects of different load strengths were similar between groups. A surgical procedure in which mucosal coverings are removed so that implanted anchor mini-screws are in contact with the bone surface was found to reduce the stress load on both the bone and screw. This procedure can be used to prevent undesirable dislodgement of implanted mini-screws. Orthodontic anchor screws are an efficient and robust means of fixation in orthodontic treatment ,2,3,4. SOur animal care procedures and experimental protocols were approved by the Committee on the Ethics of Animal Experiments of Tsurumi University (permit number: 19A005). Surgical procedures were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize the suffering of our laboratory animals. Ten male Japanese white rabbits (2.5–3.0 kg) were obtained from Tokyo Laboratory Animals Science Co., Ltd. . They were fed a normal diet and maintained under a 12 h light–dark cycle at 22 °C.n = 10). In members of the group that underwent the conventional procedure (the penetration group), 2% lidocaine local anesthetic was administered, and the mini-screw was then implanted in the right palate by penetrating the oral mucosa without drilling. It was confirmed that the initial fixation was good (n = 10). The mini-screw used with both groups had a diameter of 1.6 mm and a length of 5 mm . The next day, maxillary tissue from around the mini-screw was obtained from each rabbit for histological analysis. The excised tissue was fixed with 10% neutral buffered formalin and embedded in methyl methacrylate (MMA) resin . Polished 40–50 µm-thick sections were made and stained with hematoxylin and eosin (H&E).Mini-screws were implanted in the palatal bones of two groups of Japanese white rabbits to compare implantation preceded by removal of the oral mucosa with the conventional procedure without removal of the mucosa. In the removal group, the rabbits were given local anesthetic of 2% lidocaine. A 3 mm-diameter biopsy punch  was then used to remove a circular piece of the left palatal mucosa corresponding to the size of the screw head from each rabbit. A mini-screw was implanted in the same place without drilling, and it was confirmed that the initial fixation was good , a computer-aided design support program. The cortical bone was simplified and simulated to a thickness of 1.0 mm or 2.0 mm, factoring in the buccal alveolar bone and palatal slope in which the mini-screw had been clinically placed. The shape of the mini-screw was set based on Jeil’s 16-JK-006 .The interface between the cortex and the cancellous bone was assumed to be fully bonded, that is, to have continuous elements sharing the same nodes along the interface. A node-to-node contact condition was modeled on the interface between the mini-screw and the bone block to imitate a stage without osseointegration. All materials in the model were homogeneous, isotropic, and linearly elastic. The mini-screw was assumed to be pure titanium, with Young’s modulus of 110 gigapascals (GPa) and Poisson’s ratio of 0.33 . For heax-axis perpendicular to its long axis to simulate orthodontic force. The nodal solution of the von Mises stress in the bone and the mini-screw were calculated for each model with the FEM program. To determine the loading effects, two force magnitudes were applied to mimic various clinical conditions. A loading force of 2.0 Newtons (N) was used to mimic the load in space closure using a NiTi coil spring or elastomeric chains. A load of 10.0 N was used to mimic orthopedic force, such as mini-screw-assisted rapid palatal expansion. The load direction was set perpendicular to the long axis of the mini-screw. The distance between the underside of the screw head and the bone surface was set to 0 µm for the removal group and 765.6 µm for the penetration group based on the results obtained earlier . Statistical analyses were performed using the Kruskal–Wallis H test and Scheffe’s test. The distances between the underside of the screw head and the bone surface in the removal and penetration group were compared. In the penetration group, there was an average gap of 765.6 µm. In the removal group, the underside of the screw head was in contact with the bone surface (0 µm) .The peak von Mises stress on the bone with a load of 10.0 N is shown in Since the mini-screw first came to be used as a fixation anchor in orthodontic treatment, various studies on the optimization of its implantation have been reported ,15,16,17First, to confirm the existence of such a gap, mini-screws were implanted in the palatal bones of rabbits, with and without the removal of the oral mucosa at the site of screw placement before implantation. We found that, with the mucosa left in place, there was an average gap of 765 µm between the underside of the screw head and the bone surface. In the rabbits with the excised mucosa, no gap was present. By removing the local mucous membrane, the screw can be implanted so that the underside of the screw head comes into full contact with the bone surface.The results of the FEM analysis are summarized in The stress on the screw itself in the removal group was suppressed to about 1/2–1/3 of that seen in the penetration group at both 2.0 N and 10.0 N. In the penetration group, the stress to the screw itself was as large as 100 MPa at 10.0 N. Considering these results, in treatments where a large load is expected, such as mini-screw-assisted rapid expansion, it may be possible to help prevent screws from falling out by using screws with thicker diameters or embedding deeper.The FEM analysis of the gap between the underside of the screw head and the bone surface was based on the results of an animal study, and the soft tissue thickness of human oral mucosa varies depending on the anatomical insertion site of the mini-screw ,30,31,32Indeed, the cause of the orthodontic anchor screw falling out is not limited to bone resorption. Screws sometimes fall out immediately after they are placed. The cause of these instances remains unclear. As previously reported, the cause may sometimes be infection or inflammation ,23,24,25We compared the conventional mini-screw implantation method with the new implantation method, in which the mucosa was removed. We have shown that the new method can reduce various forces on bones. However, these studies were conducted using animal experiments and are only stress analyses by FEM. We believe that more research is needed before this new technique can be applied to clinical practice as a better alternative to traditional methods. Specifically, we believe that prospective studies should be conducted in two groups of patients: traditional and new. We are preparing to conduct a trial in the near future.This study proposes a new procedure for implanting orthodontic anchor screws, in which the underside of the screw head is in direct contact with the bone surface, without mucosa between the two. This procedure helps to prevent excess stress on the surrounding bone and the loosening or falling out of screws.


To report the successful outcome with a single intravitreal ranibizumab injection in a 15-year-old boy with Bietti crystalline dystrophy (BCD) who developed a unilateral macular neovascularization (MNV) A retrospective case report A 15-year-old Caucasian boy with Bietti crystalline dystrophy was diagnosed to have a unilateral MNV a year after the initial examination with the help of multimodal imaging and he was treated with a single intravitreal ranibizumab injection. Five months later, visual acuity improved and anatomically a dry-looking macula was noted. MNV is among the rare macular complications of BCD. The present case is the third reported pediatric case with MNV in association with BCD and the first pediatric BCD patient who received a vascular endothelial growth factor inhibitor (anti-VEGF) agent. Bietti crystalline dystrophy (BCD) is a rare genetic disease characterized by the presence of retinal crystalline deposits mostly localized in the posterior pole and varying degrees of chorioretinal atrophy commencing at the central retina with or without accompanying perilimbal, subepithelial and anterior stromal corneal crystals , 2]. As. As2]. AA 15-year-old, otherwise healthy Caucasian boy was examined with mild visual complaints and very subtle night blindness in July 2020. On ophthalmological examination, visual acuity was 20/25 in both eyes with the correction of (–1.50 –0.50 α 90) in OD and (–1.00 –1.00 α 90) in OS on Snellen chart. Slit-lamp examination exhibited a clear cornea without any corneal crystals and clear lens OU. Fundus examination disclosed a normal looking optic disc and almost symmetrical widespread refractile white-yellow retinal deposits radiating 360° from posterior pole towards the equator bilaterally (Figure 1A/D Unfortunately, genetic analysis and electrophysiological tests could not be performed. Almost a year later, he experienced a sudden visual decrease in his left eye. This time, while the visual acuity of the right eye was unchanged, the best-corrected visual acuity was 20/100 in OS. The appearance of the right fundus was unchanged but there was a significant foveal retinal hemorrhage in OS (Figure 2A The therapeutic options were discussed with the parents and the patient, and intravitreal ranibizumab injection was administered without any complication under general anesthesia in OS. Two months later, left visual acuity improved to 20/25 and the intraretinal hemorrhage was almost completely cleaned up but the presence of MNV was still observable on OCT and OCT-A. Regular follow-ups were carried out and 15 months after the injection visual acuity of the left eye was 20/25 with no visual symptoms. OCT and OCT-A illustrated a very stable looking MNV with a relatively diminished lesion size (Figure 3A-D MNV is a rare clinical finding in children that can cause significant visual impairment if left untreated , 15]. P. P15]. PIntravitreal anti-VEGF administration is a viable treatment option in childhood MNVs despite being an off-label treatment and was previously reported in several retrospective clinical studies , 16], , . HoweverWe observed the occurrence of a unilateral MNV in a 15-year-old boy just a year after the initial diagnosis of BCD and were able to treat the MNV successfully with a single intravitreal ranibizumab injection.Though rare, macular neovascular membrane may compromise the vision further if left untreated in eyes with BCD. The present case is the third reported pediatric case with MNV in association with BCD and the first pediatric BCD patient who received anti-VEGF agent and did anatomically and functionally well after the treatment.0000-0003-2059-0696Mustafa Kayabasi: 0000-0002-6743-1142Ferdane Atas: 0000-0001-6848-7239Ali Osman Saatci: The authors declare that they have no competing interests.


The glaciers of North Greenland are hosting enough ice to raise sea level by 2.1 m, and have long considered to be stable. This part of Greenland is buttressed by the last remaining ice shelves of the ice sheet. Here, we show that since 1978, ice shelves in North Greenland have lost more than 35% of their total volume, three of them collapsing completely. For the floating ice shelves that remain we observe a widespread increase in ice shelf mass losses, that are dominated by enhanced basal melting rates. Between 2000 and 2020, there was a widespread increase in basal melt rates that closely follows a rise in the ocean temperature. These glaciers are showing a direct dynamical response to ice shelf changes with retreating grounding lines and increased ice discharge. These results suggest that, under future projections of ocean thermal forcing, basal melting rates will continue to rise or remain at high level, which may have dramatic consequences for the stability of Greenlandic glaciers. North Greenland ice shelves have lost more than a third of their masses, thinning dramatically from below due to increased ocean temperatures. In response, grounding lines have retreated and the amount of ice discharged into the ocean has increased. During the last forty years, mass losses from the ice sheet have increased from near-balance to a loss rate of 286 ± 20 Gt/yr in 2010–2018, with 66% being attributed to glacier dynamics and 34% to increased surface melt5. Recent studies have shown that the intrusion of warm Atlantic water was responsible for widespread enhanced calving rates at marine terminating glaciers around Greenland7. Mass losses increased more or less simultaneously in the northwest, southeast and centralwest part of the ice sheet during the 80s and the 90s8. However, glaciers in North Greenland only started to be out of balance after 20009, due to changes in the floating extension (ice shelves) of a couple of glaciers11. In 2018, the mass losses of these glaciers due to ice discharge remained however moderate compared to the other sectors of the Greenland ice sheet 4.The Greenland ice sheet has contributed 17.3% of the observed rise in sea level in the period 2006–2018, and has thus become the second largest contributor after ocean thermal expansion12. If the glaciers located in North Greenland lose the buttressing provided by ice shelves, the increase in discharge14 could rival the largest contributors to Greenland ice mass loss . Events such as the collapse of Zachariæ Isstrøm in 2003, the large calving event at Petermann in 2012 or the thinning of the 79 N ice shelf already triggered increase in dynamic mass losses16. Despite their fundamental buttressing role, there is to date no comprehensive overview of these ice shelves evolution, which hampers our ability to understand the processes leading to their weakening and collapse, and their relation with glacier mass changes. It is thus extremely important to define the timing and drivers of historical and current changes of ice shelves, as well as glacier response, in order to better predict the contribution of Greenland to sea level rise.Overall, 25% of the ice sheet area is drained through former or remaining ice shelves, which represents a sea level rise equivalent of 2.1 m17 (Methods).In this study, we provide a long-term and holistic view of ice shelf evolution in North Greenland. The eight ice shelves that are surveyed are the floating extensions of the following glaciers : Petermann , Steensby 1.4 cm), Ryder (13 cm), Ostenfeld (3.9 cm), Hagen Bræ (6.5 cm), 79 N (60 cm), Zachariæ Isstrøm  and Storstrømmen/Bistrup Bræ   to an average of 2.7 ± 0.6 km3 in 2009-present. The ice shelf of Zachariæ Isstrøm almost completely collapsed between 2003 and 20127, decreasing from 130 ± 1 km3 to less than 25 ± 1 km3  before slowing down to 543.5 ± 39.0 m/yr in 2020 . The general increase in water temperature for the period 2005–2016 corresponds to the calculated rise in basal melting at the GL of Petermann, Steensby and Ryder 4.Our results document a holistic overview of glacier-climate-ocean interaction in North Greenland. We are able to identify a widespread ongoing phase of weakening for the last remaining ice shelves of this sector. The GL are exposed to the warmest water layers and currently sitting on retrograde bed slopes. This makes them extremely vulnerable to unstable retreat and ice shelf collapse if ocean thermal forcing continues to rise, which is likely to be the case in the coming century32. Satellite altimetry from NASA’s Ice, Cloud and land Elevation Satellite missions (ICESat-1) were also used to document the evolution of ice shelf thickness between 2003 and 2009, after which the satellite was retired due to a laser failure. Between 2018 and present, we used satellite altimetry measurements from the recent ICESat-2 mission. Quarterly digital Elevation models  derived using Digital Globe imagery as part of the Greenland Ice Mapping Project were used between 2012 and 201634. We also used DEMs derived from Synthetic Aperture Radar using NASA’s GLacier and Land Ice Surface Topography Interferometer airborne (GLISTIN-A) which measured surface elevations in 2016-2019 around the periphery of the Greenland Ice Sheet using Ka-Band (8.4 mm wavelength) single-pass interferometry35. Note that the GIMP and GLISTIN-A DEM are co-registered to airborne altimetry data (see below).In this study we make an extensive use of all available surface elevation data to reconstruct a comprehensive yearly history of ice shelf thickness changes and basal melt rates between 2000 and present. Airborne altimetry measurements from NASA’s Operation Icebridge Airborne Topographic Mapper (ATM) and LVIS  were used to document ice shelf thickness changes from 1993 to 201636. These observations are aligned horizontally and vertically following the classical scheme from37, and using yearly mosaics of satellite altimeters as ground control points on land and ice38 36. We typically remove all pixel with correlation score below 85, and stack all elevation maps within a year into a composite mosaic. After stacking, the resulting DEM is again aligned horizontally and vertically to the relative reference surface elevation. Before stacking, we find an average standard deviation of the difference with satellite altimetry that is ranging between 6 and 9 m. We calculate the root mean square error between all ASTER DEMs and the latest version of the GIMP digital elevation model39. We removed outlier pixels with differences exceeding 200 m compared to the latest version of the GIMP v234. For elevations above 1000 m (the accumulation area), we additionally filtered pixels with differences over 75 m from the GIMP v2 DEM, considering the anticipated lower dh/dt values in this region. These threshold values were chosen arbitrarily through multiple rounds of iterative filtering tests.DEMs from ASTER imagery are generated at 30 m resolution between 2000 and present with the MMASTER processing chain, which is based on the MicMac photogrammetry softwareFor the GIMP, GLISTIN and ASTER DEM, we additionally calculate the yearly difference with the corresponding altimetry data on the ice shelf: if the average difference exceeds 1 m, the DEMs are shifted vertically, in order to be centered on zero. Overall, we find a mean standard deviation of the difference on ice shelves of 2 m on ASTER DEM (after stacking) and GLISTIN-A, and 0.8 m for GIMP Figs. S, S38. Fo3,40,and a water density of 1.028 g/cm3. Grounding line and ice front mapping determination is described below. Uncertainties in the ice volume are calculated by assuming an error in ice shelf area of 1 pixel at the ice front41, and the corresponding error in ice thickness using the error on the surface elevation (see above). We also consider an additional uncertainty of 1 m, for changes in thickness due to firn air content, as described in ref. 40. Mean firn air content in this region is typically <1 m, with change rates of less than 1 cm/yr33.Finally, mosaics of surface elevation for each ice shelf are assembled by merging all available elevation data from all available sensors, and by assigning higher priorities  to the best vertical accuracy . This ensures to have the most comprehensive spatial coverage and highest vertical accuracy on all ice shelves around Greenland. Ice shelf volume changes are calculated by converting the surface elevation of the ice shelf into ice thickness using the hydrostatic equilibrium equation within an ice shelf mask, with an ice density of 0.917 g/cm42. We measure the tide-induced vertical motion of ice using a Quadruple Differential SAR Interferometry approach (QDInSAR)43. Phase coherence is maintained in fast flowing regions by coregistering SLC data using speckle tracking45. We calculate interferograms with the phase difference between the co-registered SLCs. The grounding line position is obtained by differentiating two interferograms spanning the same time interval, after correcting for topography43. We use the GIMP v1 DEM time-tagged in 2007 to remove the topographic signal34, assuming that no changes in surface ice elevation has occurred in the time interval between the DEM and SAR data. For 2014–2021, we use Sentinel-1 (S1) data with a repeat cycle of 6 to 12 days. Phase jumps at burst boundaries were accounted for using the TOPS coregistration method44. We use the GIMP DEM v2 time-tagged in 2014 to correct for the topographic phase for S134. We map the inward limit of detection of vertical motion, where the glacier first lifts off its bed43. We processed around 1000 interferograms for Petermann, 600 for Ryder, 520 for Steensby and 500 for 79 N, which provide us with a good idea of the grounding zone (>80% is from Sentinel-1 imagery). We measure the distances of maximum grounding line retreat or advance relative to the earliest most retreated grounding line observation date. Example of grounding lines and interferograms are provided in Fig. SWe use Interferometric Synthetic Aperture Radar (InSAR) data from ESA’s Earth Remote Sensing radar satellite (ERS-1) acquired in 1992 with a 3-day revisit time, in 1995/1996 from the ERS-1/2 tandem mission, and in 2011 from a 3-day revisit time before the end of ERS-2 mission. Data are downloaded via the ESA Online Dissemination Service as single look complex (SLC) scenes and processed using the GAMMA Software46.We manually digitized yearly ice shelf frontal positions between 1990 and 2021 using summer imagery from NASA’s Landsat-1-8 optical satellite, ESA’s Sentinel-1/2 satellite. We used the Google Earth Engine Digitisation Tool (GEEDiT v1.012) developed by James M Lea at the University of Liverpool48. Between 1999 and 2012 we supplemented our Landsat-7 ice velocity record with repeat cycles ranging from 336 to 400 days. For L8, S2, and S1, sub-images of 32 × 32, 32 × 32, and 192 × 48 pixels are used, respectively. We calibrate our displacement maps by taking advantage of the ice velocity products from prior surveys in47. The final calibrated maps are resampled to 150 m posting in the north polar stereographic projection (EPSG:3413). The time series established is completed by historical measurements made from ERS-1/2, RADARSAT-1, ALOS/PALSAR, ENVISAT/ASAR, Landsat 4 to 7 and TerraSAR-X50.We monitor the evolution of the glacier dynamic state velocity by calculating the surface displacement from three different satellite sensors from images collected between 2013 and 2021. Two of them, ESA’s Sentinel-2 (S2) and NASA’s Landsat-8 (L8), are optical imagers and one, ESA’s Sentinel-1 (S1), is a synthetic aperture radar operating in C-band. We use persistent surface features or speckle to map ice displacements between two consecutive images. We calculate the normalized cross-correlations between the reference and search image chips using repeat cycles shorter than 30 days for Landsat-7/8 and Sentinel-2, and 12 days for Sentinel-12. The annual mosaics are assembled from the same observations as described earlier, using a strict procedure of spatial and temporal filtering. These data provides the most accurate ice velocities values with the best signal to noise ratio and lowest uncertainties in ice flow direction47. Strain rates were retrieved using the same methodology as described in51.In order to monitor changes in rates of ice deformation, we derive the evolution of the shear strain rate for 2000 and 2019 using annual ice velocity mosaics−3 and the ocean water density as 1.028 kg m−3.Changes in ice shelf thickness can be caused by 1) the rapid advection of ice, (2) surface mass balance, (3) firn air content, and (4) basal melting, can be summarized through Eq. , to appropriately account for changes in ice dynamics38. To avoid artifacts in ice velocity mosaic we smooth the ice flow observation using a 2.5 km rolling median filter38. Because the shear margin of glaciers is a region where surface flow velocity errors are the highest, we manually filter glacier boundaries to avoid artefacts in the thickness changes and ice flow divergence derivation (see below).Changes in ice thicknesses are determined using a Lagrangian framework, i.e., we track the evolution of every pixel in a given DEM to its downstream location in another DEM acquired later in time. We follow the work of ref. 52. To account for changes in thicknesses due to surface melting, we use the monthly version of the Modele Atmospheric Regional (MAR) at a resolution of 1 km53. Changes in surface mass balance are also calculated in a Lagrangian Framework using annual values of SMB. Mean firn air content in this region is negligible, hence we did not account for it in the melt rate calculation40 (see “Ice surface elevation and volume change section”).Changes in ice volume Dh/Dt are determined using yearly DEMs described above and covering the years 2000–2020. The ice shelf area dynamically evolves every year with changing ice front and grounding lines. We calculate a dense time series of basal melt rates using all possible combinations of DEMs since 2000, with time difference between the DEM sources spanning from 2 year  to 6 years (best SNR). Several tests were conducted with DEMs separated by 1 year, but the SNR  was insufficient for use in long-term melt rate trend interpretations. Prior to the calculation of the Dh/Dt term, we corrected every DEM over vertically induced tidal motion using the pyTMD toolbox, which reads outputs from the AOTIM-5 inverse tide model54. This was calculated considering Fig. 5 from ref. 54 and the SMB values <−3 mWE which are the ranges with the largest discrepancies between observations and regional climate models. An arbitrary and conservative error of 100% is applied on the ice flow divergence. We use the related yearly errors on the DEMs that are used for calculating the changes in ice thickness (see Section “Ice surface elevation and volume change”)We calculate changes in ice thickness due to the advection using the ice flow divergence based on yearly maps of surface flow velocity as described previously. Finally basal melt rate maps are smoothed using a rolling median of 450 m. For each pair of DEMs, we use the central date to reference the basal melt map. For all ice shelves, we stacked all available melt maps within a year to produce  most accurate time series of basal melting. The evolution of the melt history is plotted using an hovmöller diagram Fig. , which i12, that can reach hundreds of meters, we decided to use estimates from4 for the period 1990–2017 and an updated version of the calculation of8. For 2017–2022, we evaluate changes in ice discharge based on the PROMICE solid ice discharge data product, calculating ice flux through gates located approximately 5 km upstream of the grounding line  using an input-output approach17. In addition to the in-situ observation, we also document the spatial variation of ocean thermal forcing using the Arctic ocean physics reanalysis  produced by the Nansen Environmental and Remote Sensing Center, Norway, and distributed by the Copernicus Marine Environment Monitoring Service. The AOPR captures the trends in the CTD in-situ measurements and allows us to interpret changes in ocean temperature with a finer temporal scale. The ocean and sea-ice model assimilate CTD profiles as well as remotely sensed data such as sea surface temperature, sea-ice concentration and sea surface height, for the North Atlantic Ocean and Arctic between 1992 and 202056. The reanalysis is provided on a horizontal grid cell size of 12.5 km × 12.5 km and 40 vertical levels, and monthly outputs are used to calculate yearly ocean thermal forcing . In North Greenland, grounding line depth, sub-ice shelf, and fjord bathymetry are highly uncertain and results from rough interpolation  spanning 1960 and 2019, combined with CTD from NASA’s Ocean melting Greenland campaign from 2016 to 2021on Fig. S. Furtheron Fig. S. In ordeon Fig. S. For eacon Fig. S, we extron Fig. S. We use 53) forced by the ERA5 reanalysis57. The simulations were run over Greenland at a resolution of 11 km, then statistically downscaled at a resolution of 1 km.We investigate changes in surface mass balance using a simulation from the Modèle Atmosphérique Régional (MAR-v3.12Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Arachis hypogaea) allergen powder-dnfp  is an FDA-approved treatment to desensitize peanut allergic participants.Oral immunotherapy (OIT) with peanut (n = 20) enrolled in phase 3 PTAH OIT clinical trials.Here we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants  synthesized on microarray slides.Statistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered.At a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut.  Overview of methods used in the analysis of samples from the PALISADE study. The risk of life-threatening allergic reactions has substantial negative impacts on quality of life for nearly 10% of adults , while c2.2.1.n = 20) during the Aimmune Therapeutics funded peanut allergy oral immunology clinical studies, ARC001 (n = 15) receiving PTAH and the Placebo group (n = 5) receiving oat flour (Avena sativa). Sera were collected prior to pOIT trials and after 12 months of treatment. Participants were removed from trials if they had anaphylactic reactions to peanut. Baseline and exit maximum tolerated dose (MTD) of peanut were administered to participants before and after pOIT and ranged from 3 to 1,000 mg, and all participants undergoing the trial passed this oral food challenge. Baseline and exit skin prick tests (SPTs) were administered to gauge IgE reactivity via mean wheal diameter (MWD) measurements at pre and post timepoints  to detect IgE binding measured by Cy3-green (532 nm). Slides were then washed and incubated with 160 ul of anti-human IgG4 (rabbit), incubated for 30 min at RT, washed, and incubated with anti-rabbit Alexaflour-red for 30 min. Slides were scanned for IgG4 signals at 635 nm with a GenePix-4000B scanner.Microarrays were utilized for detection of participant sera IgE and IgG4 binding to linear peanut peptides. Synthetic 15-mer linear peptides offset by 5 amino acids that represented the entire amino acid sequences of Ara h 1, 2, 3, 5, 6, 7, 8, 9, 10, and 11  were com2.3.GenePix GPR output files containing IgE and IgG4 signal intensities were processed with the Limma package v3.52.4 using R v4.2.1 and RStudio v2022.07.0. Median feature pixel intensities at 532 nm (IgE) and 635 nm (IgG4) were background corrected using the backgroundCorrect.matrix function in Limma  and were base 2 logarithm transformed, and the median for each background-corrected peptide triplicate was taken. Cyclic-Loess normalization for each peptide was performed between all possible pairs of arrays for 3 iterations, with a loess smoothing window span of 0.3 using the normalizeBetweenArrays function .Median peptide fluorescence signal-to-noise ratios (SNRs) across microarray chip lots were used to determine major reactive IgE and IgG4 epitopes. Positive IgE or IgG4 binding was identified as a median SNR >3, which is based on previous chicken sera controls having SNR values of 3. Peptides with positive IgE or IgG4 binding by at least 50% of the sera  were considered major epitopes . Peptide2.4.t-tests of IgE/IgG4 binding intensity ratios (IgE/IgG4) were performed in R v4.2.1  for each 15-mer linear peptide from pre- to post-pOIT. Prior to conducting the paired t-tests, the approximate normality of peptide binding intensity distributions were assessed with quantile-quantile plots  correction  and imposing an adjusted p value threshold <0.05. Peptides with significant adjusted p values were considered to have undergone a significant IgE/IgG4 ratio increase or decrease over the course of the study if their mean difference was >1 or <1, respectively. Cohen's d effect size for paired samples  comparisons of per-peptide IgE/IgG4 were performed with linear regression models (LM) using the lm function in R: rrection . Contrasrrection . Baselinrrection . Linear rrection , and pre2.5.Correlation matrices were constructed from normalized log2 IgE and IgG4 intensities for pre- and post-pOIT, with pairwise Pearson correlation performed between pre- and post-pOIT matrices via the correlate function in the corrr R package v0.4.4  (paramet2.6.n = 42) sera from a separate peanut sublingual oral immunotherapy (pSLIT) study utilizing identical microarray-bound peptides and a pre/post experimental design with comparable treatment durations (n = 2) and phase 3 (n = 15) participant pOIT IgE/IgG4 were used as the validation (unseen) data set. The pSLIT and PALISADE GPR outputs were processed together with the Limma package v3.52.4 using R v4.2.1, as described in Methods section z-scores of per-peptide IgE/IgG4 (features) and pre/post timepoints (labels) were used for supervised binary classifiers using the Python machine learning libraries PyCaret v2.3.10 (n = 120), train and test sets were sampled from the pSLIT dataset , and all base classification estimators available in the SciKit-Learn v0.23.2 library were trained and evaluated with Stratified KFold cross validation (splits = 10) on the training set. The top 4 models were selected based on accuracy, and hyperparameter tuning was performed for these models with a Random Grid Search with 50 iterations using Stratified KFold cross validation (splits = 10) with F1 score as the optimization metric. The original models were used in downstream analysis if they outperformed their tuned versions. Ensemble methods were performed via bagging  immunoassays  were used to measure pre- and post-pOIT participant sera IgE binding to purified intact Ara h 1, 2, 3, 6, 8, 9 proteins. IgE signal intensity levels in ISAC Standardized Units (ISU-E) with an operating range of 0.3–100 ISU-E were generated by Phadia Microarray Image Analysis software v1.2 . IgE intensities were log2 transformed [log2(x + 1)]. Wilcoxon signed rank tests were used to compare IgE binding intensity distributions from pre to post for each protein using the wilcox.test function in the R stats package v.4.2.1 . Spearman correlation matrices were constructed using the cor function in the R stats package v4.2.1, and correlation plots were made using the corrplot v0.92 package in R.3.3.1.p = 0.018), Ara h 3 (p = 0.0087), and Ara h 6 (p = 0.00073), with IgE binding exhibiting an overall decrease in the study population after pOIT (p = 0.15). The total IgE binding intensity to Ara h 8 and Ara h 9 was much lower compared to Ara h 1, 2, 3, and 6. Ara h 8 binding increased for two and decreased for two participants, however all other participants did not exhibit notable IgE binding shifts to this protein and pre-to-post distributions were not significant (p = 1)  . Similar3.2.t-tests, Ara h 1 had 1.6% of peptides exhibiting significantly increased IgE/IgG4 (increasing IgE and/or decreasing IgG4) from pre- to post-pOIT in the Active group, while 0.8% of peptides had significant shifts towards decreased IgE/IgG4 (indicating increasing IgG4 and/or decreasing IgE binding)  , Table 2binding) .t-tests, while no significant changes were observed in the Placebo group . Using a more liberal adjusted p value cutoff of <0.15 to adjust for small library size, known IgE epitope regions (p = 0.1), 14 (p = 0.1), 16 (p = 0.059), 17 (p = 0.046), and 27 (p = 0.1) were shown to have lower IgE/IgG4 in the Active group vs. Placebo group at the end of pOIT.For the major peanut allergen Ara h 2 (Ara h 2.0201), 21.2% of the 15-mer peptides exhibited increased IgE/IgG4% and 3% exhibited decreased IgE/IgG4 over the 12-month pOIT period within the Active group based on paired bo group . Peptide, and IV  39), an, ant-tes had 11% of peptides undergo IgE/IgG4 increases and 13% undergo IgE/IgG4 decreases in the Active group, with no significant changes in the Placebo group . Within Peanut profilin Ara h 5 is an allergen linked to pollen-associated peanut allergy . No peptt-tests   . The Aras (>0.8) ]. The kns (>0.8) . Ara h 2s (>0.8) . Ara h 3s (>0.8) . Ara h 5s (>0.8) . This mas (>0.8) . Ara h 1s (>0.8) .We trained an ERT classifier to predict participant desensitization to peanut  and used4.in vivo state of a participant's allergen sensitivity  IgE/IgG4 comparisons  versus Ara h 2 peptide 17 (p = 0.046), this does support the finding that these peptides were important for predicting desensitization since they showed significantly lower IgE/IgG4 (increased IgG4 and/or decreased IgE) in the Active treatment group, which underwent pOIT and became desensitized, vs. the Placebo group, which did not. While machine learning algorithms can be very powerful tools, the limited number of participant sera obtained for the PALISADE study and necessity of training the model on a larger pSLIT dataset may be responsible for the identification and some inconsistencies in important peptides compared to paired t-tests. Future studies will require larger amounts of training data to increase model performance.This study utilized an ERT binary classifier to predict participant desensitization to peanut and we used Gini Impurity feature importance to identify the most important peptide features for this particular model's classification task . This meparisons . Ara h 2Increasing our understanding of which epitopes shift from IgE to IgG4 binding over the course of oral immunotherapy is vital towards developing a framework for identifying successful pOIT treatments, as well as improving potential peptide-based diagnostic methods for food allergy. It is also important to note that the binding of IgE and IgG4 to extracts, components, or allergen peptides can diverge and that each level of binding may contribute important information regarding the state of immune modulation and subject response to types and duration of treatments. Future studies with more timepoints and clinical data regarding other allergies will aid in determining long-term shifts in IgE and IgG4 epitope binding once participants reach a maintenance phase. This study contributes to our growing knowledge of peanut allergy dynamics over the course of oral immunotherapy, demonstrates which epitopes shift for participants taking PTAH, and provides an initial framework for identifying critical epitopes that determine if peanut-allergic participants have reached desensitization after treatment.


For this aim, GST was purified from C. tarichi Pallas liver with 37.36% yield and 29.304 EU/mg specific activity using the chromatographic method. The Vmax values of liver GST were determined for CDNB and GSH to be 1.245 and 0.562 EU/mL, respectively, and the Km values were found 0.89 and 0.06 mM, respectively, using the Lineweaver–Burk plot. The effects of the metal ions at different concentrations on in vitro GST activity were studied. The IC50 values were determined for Cu+2, Cd+2, Ag+, and Co+2 as 0.163, 0.235, 0.00021, and 0.446 mM, respectively. The Ki constants were determined as 0.049 ± 0.009, 0.117 ± 0.031, 0.002 ± 0.0007, and 0.893 ± 0.3 mM, respectively. Ag+ showed the best inhibitory effect among the studied metal ions. Cd2+, Cu2+ and Co2+ showed a competitive inhibition mechanism, while Ag+ was noncompetitive.Glutathione-S-transferase (GSTs) is a multifunctional enzyme that provides homeostasis by catalyzing the first step of the formation of mercapturic acid, the end product in detoxification metabolism. They can prevent reactive electrophilic compounds from harming the body by covalently binding the same type of compounds to each other. In this study, we determined the in vitro inhibitory effects of metal ions such as Cu Analysis of the data and drawing of graphs were realized using GraphPad Prism ver. 8 for Mac . The results were exhibited as mean ± standard error of the mean . Differences between data sets were considered statistically significant when the p-value was less than 0.05.ICch metal –32. The rk plots –35. For GSTs play a key role in the metabolism and synthesis of endogenous and exogenous compounds that include an electrophilic carbon, sulfur, or nitrogen atom. Their substrates contain arene oxides, α,β-unsaturated carbonyls, halogenonitrobenzenes, and quinones. During oxidative stress, GSTs have also been situated to intercede signal transduction and reduce the impacts of oxygen toxicity –28. To tC. tarichi Pallas liver with 37.36% yield and 29.304 EU/mg specific activity . They reported that Ag+, Cu2+, Cd2+, and Zn2+ showed inhibitory effects, but Cr2+, Fe3+, and Pb2+ did not show any impact on enzyme activity. The inhibitory effect order was determined as Cu2+>Ag+>Cd2+>Zn2+. The enzyme was inhibited noncompetitively by these metals.There are a few studies about interactions between GST and metal ions in the literature. For instance, in our previous study, GST was purified from Lake Van fish gills, and the effects of Cdy effect . In anoty effect  purifiedy effect . These rmax, and Km. The specific activity of the enzyme was found higher than those determined in previous studies. The inhibitory effects of various heavy metals on GST enzyme activity were also studied. Findings in the relevant literature and our findings have showed that living organisms have varying levels of susceptibility to metal ions. These metal ions, which give rise to inhibition in GST enzyme activity on micromolar levels, may cause toxic effects in fish.Consequently, in this study, we purified GST from Lake Van fish liver and studied various characteristics such as the optimum pH, temperature, ionic strength, stable pH, V


The serum anti-IBV antibody titer was significantly higher in challenged groups compared to non-challenged groups. The IBV genome load was significantly lower in the VC group than NVC group in oropharyngeal and cloacal swabs and in bursa of Fabricius (BF) and cecal tonsils (CT). The histopathological lesion scores were significantly lower in VC group than NVC group in BF and CT. These findings suggest that the presence of maternal antibody in chicks could provide some degree of protection against IBV infection in BF and CT.Infectious bronchitis virus (IBV) causes infectious bronchitis disease in chickens. IBV primarily infects the upper respiratory tract and then disseminates to other body systems including gastrointestinal, reproductive, and urinary systems. Unlike original IBV serotypes, the novel IBV variants target lymphoid organs, but information on this is scarce. In this study, we aim to evaluate the impact of the presence of maternal antibodies on IBV infection in primary and secondary lymphoid organs. Maternal antibody free, specific pathogen free (SPF) hens were divided into vaccinated and non-vaccinated groups. The progeny male chicks from these hens were divided into four groups; vaccinated challenged (VC), non-vaccinated challenged (NVC), vaccinated non-challenged (VNC), and non-vaccinated non-challenged (NVNC). The challenge groups were given 1 × 10 Infectious bronchitis (IB) is a contagious viral disease caused by a gamma coronavirus known as infectious bronchitis virus (IBV) . IB is oAlthough IBV primarily infects the upper respiratory tract, it can show tropism to other tissues depending on the strain of the virus. Although the IBV DMV/1639 variant was first reported as a nephropathogenic strain , in receMaternal antibodies are transferred from vaccinated hens to their progeny via the yolk and can be detected in the serum and respiratory mucus of newly hatched chicks ,19. MoreThere is a scarcity of knowledge regarding the role of maternal antibodies against IBV infection in the chickens, and there are little to no studies on IBV infection in lymphoid organs. Furthermore, the limited literature on the role of maternal antibodies against IBV infection in hatched chicks is focused on determining if the presence of the maternal antibodies provide protection against IBV infection in the respiratory tract ,19,20. IGallus gallus domesticus) were purchased from the Canadian Food Inspection Agency (CFIA), Ottawa, Ontario, Canada and were raised for 30 weeks of age until egg production in the Veterinary Science Research Station (VSRS) at the Spy Hill campus, University of Calgary. The young chicks for the experiment were obtained by hatching the eggs collected from these White Leghorn SPF hens by incubating at Health Research Innovation Center (HRIC) 2C53, Foothills campus, University of Calgary. The hatched male chicks were housed in the VSRS at the Spy Hill campus, University of Calgary. The ethical approval for this proposed work was obtained from the Health Science Animal Care Committee (HSACC) of the University of Calgary (Protocol number: AC19-0011). All the experiments were conducted in negative pressure rooms.The Canadian IBV DMV/1639 strain used in this study, designated as IBV/Ck/Can/17-036989, was isolated, propagated, and titrated as previously described ,7. Ninetn = 91) were divided into two groups, named as vaccinated  and non-vaccinated controls . The birds in the vaccinated group were vaccinated with live attenuated Mass vaccine  at 2 weeks of age and Mass and Conn IB vaccine  at 5 and 9 weeks of age. Finally, at 16 weeks of age, Newcastle Disease (ND)-Bronchitis Vaccine-Salmonella Enteritidis Bacterin, Mass type, killed virus vaccine  was given to the vaccinated pullets. At 30 weeks of age, eggs were collected from vaccinated and non-vaccinated hens, incubated, and hatched to obtain progenies. Day-old male chicks were randomly divided into four groups (n = 16), named as vaccinated challenged (VC), non-vaccinated challenged (NVC), vaccinated non-challenged (VNC), and non-vaccinated non-challenged (NVNC). Serum samples were collected from all groups at the day of hatching before the viral challenge. The VC and NVC groups were challenged with 100 μL of IBV DMV/1639 strain at a dose of 1 × 106 embryo infectious dose (EID)50 by the oculo-nasal route. The VNC and NVNC groups were mock challenged with 100 μL of phosphate buffered saline (PBS)  for extraction of ribonucleic acid (RNA). In addition, samples of all the collected tissues were fixed in 10% neutral buffered formalin  for histopathology evaluation and immunohistochemistry assay. Swabs and tissues collected for RNA extraction were stored at −80 °C until further processing, and tissue samples in 10% neutral buffered formalin were stored at room temperature.The White Leghorn SPF chickens (ne (PBS) . At 3 anA commercial ELISA kit  was used to measure the titer of maternal antibodies against IBV in serum at a serum dilution of 1:500 according to the manufacturer’s instructions. The antibody titers were calculated with the formula provided by IDEXX, where sample to positive ratio was calculated and titers above 396 (cut-off) were considered positive.® reagent  and total RNA was extracted from tissue samples using Trizol reagent  according to manufacturer’s guidelines. Following this, the amount of extracted RNA was quantified using a Nanodrop 1000 spectrophotometer  at 260 nm wavelength. Finally, 1000 ng of RNA from swab samples and 2000 ng of RNA from tissue samples were used to synthesize cDNA using RT random primers high-capacity cDNA reverse transcriptase kit  according to the manufacturer’s instructions.The OP and CL swab samples were vortexed and 250 μL of the sample was used to extract the RNA using Trizol LS® Green Master Mix  with a final reaction volume of 20 µL using cDNA as a template. Each reaction volume consisted of 10 µL of SYBR Green master mix, 100 ng of respective cDNA sample, 0.5 µL of forward primer, 0.5 µL of reverse primer, and molecular biology grade water. All these qPCR assays were carried out using a CFX 96-c1000 Thermocycler  and the thermal cycling conditions were initial denaturation at 95 °C for 20 s, followed by 40 cycles of amplification at 95 °C for 3 s, and annealing at 60 °C for 30 s. The primers targeted the conserved IBV nucleocapsid (N) gene where the forward primer and reverse primer sequences were 5′GACGGAGGACCTGATGGTAA-3′ and 5′CCCTTCTTCTGCTGATCCTG-3′, respectively [The qPCR assay was performed using Fast SYBRectively .2O2 solution in methanol for 10 min at room temperature to block the endogenous peroxidase activity. Subsequently, the tissue sections were microwaved at 850 W for 15 min with a 10 mM citrate buffer at pH 6.0 to unmask the viral epitopes. The tissue sections were then incubated overnight at 4 °C in a humidified chamber with mouse primary anti-IBV nucleoprotein antibody  diluted 1:400 in PBS. Following this, the tissue sections were incubated with goat anti-mouse IgG (H + L) secondary antibody , for 30 min at room temperature. Then, ABC peroxidase kit and 3,3’-diaminobenzidine (DAB) substrate solution  were used according to the manufacturer’s instructions to detect the antibody binding. All the above incubation steps were followed by 5 min of washing with Tris-buffered saline (TBS). Henceforth the slides were counterstained with haematoxylin  for 8 min, and bluing was achieved by running under tap water for 30 min. The dehydration and cleaning were conducted in an ascending series of alcohol and xylene, respectively. Finally, the tissue sections were cover-slipped using a mounting solution .All the collected tissue samples were examined using immunohistochemistry technique to identify the presence and localization of the IBV nucleoprotein. Immunohistochemistry was performed following the methods described in the past literature . InitialThe tissues fixed in 10% neutral buffered formalin were submitted to the Diagnostic Services Unit (DSU) at the University of Calgary, Faculty of Veterinary Medicine for obtaining paraffin embedded tissue blocks and hematoxylin-eosin (H&E) stained and unstained tissue sections on positively-charged slides . The stained tissue sections were examined under the microscope  and scored following the methods previously described with some alteration . There wp < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. All the statistical analyses were performed with GraphPad Prism 9.2.0 Software , and this software was also used to generate graphs.The mean IBV antibody titer in the serum of pre-challenged birds were compared using unpaired t test while mean antibody titers in post-challenged birds at 3 and 7 dpi were compared using one-way ANOVA followed by Tukey’s multiple comparisons test. IBV genome loads in OP swabs, CL swabs, and tissues were compared using one-way ANOVA followed by Tukey’s multiple comparisons test. The significant differences of histopathological lesion scores between the four groups were analyzed using two-way ANOVA followed by Tukey’s multiple comparison test. The statistical significance between the groups were denoted with different number of asterisks in the graphs, where *, **, ***, and **** implies significance when p < 0.0001; p < 0.001; p < 0.01; The antibody titer against IBV in the progeny chicks from vaccinated hens (V) (mean antibody titer = 2529.71) was significantly higher than that in progeny chicks from non-vaccinated hens (NV) (mean antibody titer = 0)  groups at 3 and 7 dpi in all target organs . In contp < 0.05; No histopathological lesions were detected in thymus and spleen of any experimental groups, and no histopathological lesions were detected in the NVNC and VNC groups in any target organs . There wThere was not any significant difference between the NVC and VC groups in lung score at both 3 and 7 dpi b. The lep < 0.05; The VC group showed a significant decrease in lesion scores at 3 and 7 dpi compared to the NVC group in kidney  was recorded at 7 dpi between these groups  compared to chicks from non-vaccinated hens (the NVC and NVNC groups) at 3 and 7 dpi, and, correspondingly, the IBV genome load in OP swabs of chicks from the VC group was significantly lower compared to chicks from the NVC group at 3 and 7 dpi. In addition, the IBV shedding observed in CL swabs was significantly lower in the VC group compared to the NVC group at 3 dpi. All these data suggest that the presence of maternal antibodies in serum could provide some degree of protection against IBV infection and replication in young chicks. The study by Mockett et al. in 1987, where eggs from vaccinated hens were hatched, and 1-day-old and one-week-old chicks were challenged with IBV strain VF69-149 (Mass serotype) also reported that the presence of maternal antibodies in serum provided protection against challenge from IBV, as chicks with maternal antibody had a lower percentage of mortality . The pasIBV viral genome was detected in all of the tested organs  of IBV challenged birds. A few studies have previously reported the presence of IBV genome in the primary lymphoid organs BF ,12,13 anTo further confirm the replication of IBV in lymphoid organs and the impact of the presence of maternal antibodies against IBV replication in lymphoid organs, immunohistochemistry technique was performed in order to demonstrate the expression of IBV nucleoproteins in infected tissues. Similar to the IBV genome loads, a remarkable amount of IBV nucleoprotein was detected in the lymphoid organs BF and CT at 3 and 7 dpi. In both BF and CT, at 3 dpi IBV nucleoprotein was detected in all the birds (100%) of the NVC group while only 87% of the birds (7/8 birds) were positive in the VC group. Hence, it could be suggested that the presence of maternal antibodies in the VC group provides a certain extent of protection against IBV replication in BF and CT at 3 dpi. In addition, in the thymus and spleen, none of the birds in the VC group were positive for IBV nucleoprotein, while 12.5% (1/8 birds) and 25% (2/8 birds) of the birds in the NVC group were recorded as IBV nucleoprotein positive in the thymus and spleen, respectively. Moreover, IBV nucleoproteins were detected in the trachea of both the VC and the NVC groups at 3 and 7 dpi. The expression of IBV nucleoproteins in lungs were detected at both 3 and 7 dpi in the NVC group, whilst it was detected only at 3 dpi in the VC group. The absence of IBV nucleoprotein and, thus, the absence of IBV replication in the thymus and spleen of the VC group could be due to the presence of maternal antibodies in the VC group. There are no previous studies in the literature that report on the impact of maternal antibodies against IBV replication in lymphoid organs by analyzing the presence of IBV nucleoprotein. Hence, these results provide valuable primary information for the formulation of future studies to determine the effect of a maternal antibodies against IBV replication in primary and secondary lymphoid organs. However, further quantitative studies are necessary to confirm that the presence of maternal antibodies negatively impact IBV replication in primary (BF and thymus) and secondary (CT and spleen) lymphoid organs as well as the potential impact on the cellular immune system. Furthermore, although we detected IBV genome in all of the birds in the primary (BF and thymus) and secondary (CT and spleen) lymphoid organs at both 3 and 7 dpi, not all of the birds were positive for IBV nucleoprotein in these tissues. The discrepancy in the detection of IBV genome load and IBV nucleoprotein in the primary and secondary lymphoid organs is not surprising, as qPCR detects both replicating and non-replicating IBV while the immunohistochemistry technique detects only IBV antigen expressed by the replicating virus . MoreoveThere is limited literature reporting the presence of IBV-induced gross and microscopic lesions in primary (BF and thymus) and secondary (CT and spleen) lymphoid organs. IBV induced microscopic lesions have been previously described in BF ,12,13 anOne limitation of this study is that it does not include the viral tropism and pathological damage in HG due to IBV DMV/1639 challenge in the birds. In future, studies assessing the viral tropism and the pathogenicity of the IBV DMV/1639 strain in HG could be performed. Further studies could be performed in order to determine and compare the cross protection provided by homologous and heterologous vaccine strains. It would be of great importance to determine and compare the immunological protective effect of DMV/1639 inactivated vaccine with Mass and Mass and Conn vaccines through cross-challenge experiments in adult hens and their progenies due to the protective effect of maternal antibodies. In addition, a study comparing the tissue tropism and pathogenicity between DMV/1639 strain to Mass and Conn strains could also be undertaken in the future.In summary, we observed that the IBV antibody titer in serum was significantly higher in chicks from vaccinated hens compared to chicks from non-vaccinated hens, and, correspondingly, that the viral shedding in OP and CL swabs was significantly lower in chicks from vaccinated hens (VC) compared to chicks from non-vaccinated hens (NVC). In addition, we found that the IBV genome loads, and the histopathological lesions were significantly lower in chicks from the VC group compared to chicks from the NVC group in BF and CT. Although we still need further studies to determine a direct correlation between maternal antibody and its impact on IBV infection in lymphoid organs, our study provides preliminary data to suggest that the presence of maternal antibodies in chicks does provide some degree of protection against IBV infection in BF and CT up to 7 days post hatching.