Thaparocleidus wallagonius is a monogenean parasite and a fish-borne pathogen with a worldwide distribution. The genome for Thaparocleidus wallagonius is known. Therefore, it is of interest to report the DNA motif analysisdata in the 18S rDNA of Thaparocleidus wallagonius collected from the fish Wallago attu in India. This data forms a framework for an in-depth analysis of the parasite biology and development, immune evasion strategies, virulence and long-termsurvival within the definitive host. Thaparocleidus wallagonius (a monogenean parasite) isolated from the gills of Wallago attu at Lucknow, India (Bloch & Schneider) [Thaparocleidus wallagonius collected from the fish Wallago attu in India.Parasitic infections in fishes have been reported from different geographical regions worldwide. They aggravate a great threat to fish culture as fishes are important source of food in the developing countries .Consumphneider) . Genus Thneider) .It is khneider) .Genome hneider)  using knhneider) . TherefoThaparocleidus wallagonius was downloaded from the NCBI  database (https:/www.ncbi.nlm.nih.gov) with accession number KX364086.1.The DNA sequence for the 18S rDNA of The motifs were analyzed using the GLAM2 Web tool and TRANSFAC Motif tool as shown in Figure 0(see PDF).We found 6 motifs at 17 positions within the ribosomal genome of the parasite. rDNA is conserved and serves as a molecular machinery for protein synthesis. Data on motifs in the genome will help in blocking the activity of the parasite. Motif from positions13-16 and 124-127 had a role associated with Glycosylation serving as ASN_GLYCOSYLATION (N-glycosylation site) . Motif cAMP_PHOSPHO_SITE was found at positions 120-123 . Motif CK2_PHOSPHO-SITE (Casein Kinase IIphosphorylation site) was found on various positions i.e. from 26-29, 72-75, 149-152, 193-196, and 339-342 as shown in Figure 2. Motif MYRISTYL (N-myristoylation site) was found from positions 40-45, 268-273, and 276-281 . MotifPKC_PHOSPHO_SITE (Protein Kinase C phosphorylation site) was found at positions 65-67, 72-74, 145-147, and 293-295 . Motif TYR_PHOSPHO_SITE (Tyrosine kinase phosphorylation site) was also found at positions 84-92 and 225-231 .The role of these motifs has been widely studied in coding DNA till date. The coded functions includes protein folding and conformational stability, intracellular transport, antibiotic resistance and homeostasis processes, resistance to digestion by proteases,interaction with cell-surfaces, extracellular matrix proteins and other ligands, sexual commitment, inducing chemotaxis, DNA repair, cell cycle control, regulating the circadian rhythm and other cellular-processes, providing innate immune response,phosphorylation sites on human lamin and in nuclear lamina structural dynamics. This data is relevant in the understanding protein sequence, structure and function in drug discovery for the pathogen.We report the presence of sequence motifs such as AMIDATION, ASN-GLYCOSYLATION, CAMP_PHOSPHORYLATION, CK2_PHOSPHORYLATION, N-MYRISTOYLATION and PKC_PHOSPHORYLATION sites in the T. wallagonius genome for further consideration in the understanding proteinsequence, structure and function in drug discovery for the pathogen.


The deletion of the gene coding for poly(ADP-ribose) polymerase-1 (PARP1) or its pharmacological inhibition protects mice against cerebral ischemia and Parkinson's disease. In sharp contrast, PARP1 inhibitors are in clinical use for the eradication of vulnerable cancer cells. It appears that excessive PARP1 activation is involved in a specific cell death pathway called parthanatos, while inhibition of PARP1 in cancer cells amplifies DNA damage to a lethal level. Hence, PARP1 plays a context-dependent role in cell fate decisions. In addition, it appears that PARP1 plays an ambiguous role in organismal aging. Poly(AD+). This process results in the covalent attachment of PAR to multiple proteins. PAR then acts as a signal for the recruitment and the activation of other DNA repair enzymes [PARP1 is best known as is a DNA repair enzyme present in the nuclei of mammalian cells. In response to single-strand DNA breaks, PARP1 initiates the synthesis of poly(ADP-ribose) (PAR) chains from nicotinamide adenine dinucleotide  and the translocation of AIF from mitochondria to nuclei can be observed in skin lesions [Ted and Valina Dawson described a cell death modality relying on the overactivation of PARP1 and PAR-induced mitochondrial outer membrane permeabilization (MOMP) that they coined “parthanatos” . In this lesions . As an a lesions –29. Altoc which, once released from mitochondria, interacts with APAF1 to stimulate the formation of the apoptosome, which is a caspase-9 activation complex [+, which contributes to the lethal bioenergetic crisis that culminates in necrosis. In this context, the genetic or pharmacologic inhibition of PARP1 favors the apoptotic rather than necrotic demise of dying cells [Apoptosis usually involves two major hierarchically related events, namely (i) mitochondrial membrane permeabilization (MMP) and (ii) activation of specific set of proteases, which are called caspases. MMP may occur to variable proportions as mitochondrial inner membrane permeabilization (MIMP) and MOMP, leading to the arrest of oxidative phosphorylation and other essential metabolic functions, hence usually sealing the cell's fate , 31. MOM complex . Caspase complex . In thisng cells , 35. Thu+ [+ is an essential PARP1 substrate, but also due to the reduced binding of NAD+ to the protein deleted in breast cancer 1 (DBC1), which then engages in an inhibitory interaction with PARP1 [PARP1 is necessary for genomic stability, suggesting that its inhibition should derive genomic instability, which is one of the principal hallmarks of aging as well as of cancer. Indeed, transgenic PARP1 overexpression in basal skin keratinocytes from mice suppresses skin papilloma formation in a two-stage skin carcinogenesis protocol . In Dros+ . This reth PARP1 . AltogetCaenorhabditis elegans [, improves neurovascular and cognitive parameters in aging mice [In sharp contrast, there are also arguments in favor of a pro-aging activity of PARP1. Thus, pharmacological PARP1 inhibition rescues the short lifespan of hyperglycemic  elegans , improveing mice , and ameing mice . Conversing mice . The mecPharmacological inhibition of PARP1 may kill those cancer cells that are undergoing constant endogenous DNA damage due to defects in DNA repair enzymes as well as cells that are being exposed to DNA damaging irradiation or cytotoxicants. Paradoxically, PARP1 inhibition has potent cell death-inhibitory effects in other contexts, in particular against parthanatos, a non-apoptotic modality of cellular demise that relies on the enzymatic overactivation of PARP1 and that appears to be particularly prevalent in neurological diseases. In addition, PARP1 inhibition may accelerate or retard age-related pathologies, suggesting that PARP1 has an ambiguous role in aging as well. This consideration is important in view of the possible side effects of long-term treatments with PARP1 inhibitors.


She maintained normal renal functions with no lupus morbidity for 24 months, then rapidly progressed to end-stage kidney disease, and was then started on three to four sessions of regular hemodialysis per week.We report the case of an adolescent girl with frequent hospital admissions for severe eczematous skin rashes with recurrent epistaxis and chest infections. Investigations revealed persistent severely elevated serum total immunoglobulin E (IgE) levels but normal levels of other immunoglobulins, suggesting hyper-IgE syndrome. The first skin biopsy revealed superficial dermatophytic dermatitis (tinea corpora). Another biopsy performed after six months revealed a prominent basement membrane with dermal mucin, suggesting an underlying autoimmune disease. Her condition was complicated by proteinuria, hematuria, hypertension, and edema. A kidney biopsy revealed class IV lupus nephritis, according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS). Based on the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria, she was diagnosed with systemic lupus erythematosus (SLE). She was first administered with intravenous pulse methylprednisolone (600 mg/mHyper-IgE is known to be a marker of immune dysregulation as it facilitates the generation of immune complexes (ICs) that mediate lupus nephritis and juvenile SLE.Regardless of the different factors that are impacting the production of IgE, the present case illustrated that juvenile patients with SLE may have increased IgE levels, indicating that higher IgE levels might have a role in lupus pathogenesis and prognosis. The mechanisms regarding the increased levels of IgE in subjects with lupus need further investigation. Further studies are thus required to assess the incidence, prognosis, and possible new specific management for hyper-IgE in juvenile SLE. Systemic lupus erythematosus (SLE) is an autoimmune disease caused by autoantibodies and circulating immune complexes (ICs), in which the immune system attacks its own tissues. This causes inflammation and tissue damage in the affected organs .The hallmark of SLE is excessive production of antibodies to nuclear antigens (ANA), double-stranded DNA (dsDNA), ribonuclear proteins (RNP), Smith antigen (Sm), ribonuclear proteins (RNP), Ro (SSA), La (SSB), and some phospholipids; these antibodies accumulate over time. SLE pathogenesis is closely associated with the presence of dsDNA-specific immunoglobulin G (IgG) antibodies .However, kidney biopsies reveal kidney deposits comprising dsDNA IgG antibodies in numerous patients with SLE who develop glomerular nephritis . FurtherIgE is associated with immunity against various parasitic infections and is involved in mediating type I hypersensitivity in mast cells and basophils. It is considered the rarest immunoglobulin in blood, with serum levels of approximately 150 ng/mL in a healthy human body.The presence of IgE autoantibodies has been observed in some autoimmune diseases along with the prevalence of autoreactive IgE in SLE; however, its importance in the disease activity and pathologies remains unclear . PreviouCase historyWe report the case of an adolescent 11-year-old Burmese girl who presented to King Abdulaziz University Hospital with a diffuse, progressive, itching skin rash all over her body and scalp for seven months. The rash initially manifested on her back and abdomen, then progressed to the face and head, and subsequently to her entire body, including the palms and soles. It progressed to crusting and oozing clear fluid, associated with a three-week history of fever and bilateral eye swelling. She had a history of moderate unilateral epistaxis, which only required nasal packing in the emergency room; she had no other history of bleeding, petechia, or bruising. She was not on any specific therapy.She was delivered at full term through spontaneous vaginal delivery, and her perinatal history was irrelevant, with incomplete vaccinations because of social reasons. She was admitted to a hospital when she was four months old for bronchiolitis with secondary bacterial pneumonia. She was living with her non-consanguineous parents and her seven healthy siblings. The family belonged to a low socioeconomic status. There was no family history of rheumatological, immunological, renal, or any chronic illnesses.Physical examinationAn initial assessment revealed a low-grade fever (<38.5°C) but otherwise stable vital signs. She was not in distress and was well-hydrated with a normal capillary refill. Her weight was 23 kg (fifth centile), and her height was 125 cm (25th centile for age and sex). She was conscious, alert, and oriented. She was pale with erythematous maculopapular, non-blanchable, crusted, and pruritic skin rashes all over her body with scales including the palms and soles without any exudate. Her right eye was swollen and congested, but with normal eye movement.The remaining clinical examinations yielded unremarkable findings. Table Furthermore, the patient exhibited a positive immune profile for SLE, even though the characteristic clinical manifestations were absent. The ANA pattern was speckled with a moderate positive titer (1:640), 119.8 dsDNA, positive direct Coombs test (direct agglutination test) as IgG +1, weakly positive perinuclear antineutrophil cytoplasmic antibodies (p-ANCA), and negative c-ANCA. Thus, the immunology team was consulted. A primary immune deficiency was ruled out considering the time of presentation; however, partial innate immune defects and hyper-IgE syndrome were considered in her differential diagnoses. A genetic study using whole exome sequencing was performed, and the result was negative for possible variants responsible for hyper-IgE syndrome. Therefore, IgE and other immunoglobulin assays were performed. A definitive diagnosis of SLE was difficult during that time owing to the lack of clinical manifestations and negative lupus-specific serology.Hospital courseDuring her hospital stay, the skin rash was initially crusty and covered the entire body and scalp. Skin biopsy Figure  from theHer hemoglobin levels dropped, with pancytopenia Table . Skeleta2/dose twice daily), oral prednisolone (1 mg/kg/day), hydroxychloroquine (200 mg), omeprazole (20 mg), and enalapril (20 mg) once per day to treat hypertension associated with significant proteinuria.The patient required multiple admissions over the following year due to recurrent infections at 6, 12, and 18 months from her initial presentation; subsequent investigations at six months revealed strong positive ANA (1:1280) but negative anti-dsDNA. However, at 12 months, her investigations revealed persistently positive ANA, low C3 and C4, positive anti-Smith antibodies, and strong positive anti-dsDNA (Table 2 surface area daily) for three days, followed by oral prednisolone (40 mg/m2 surface area) daily, along with similar doses of mycophenolate mofetil and hydroxychloroquine, and three blood pressure lowering agents  at the maximum allowed doses.Subsequently, she exhibited generalized edema, anemia, heavy proteinuria, hematuria, and hypertension Table . She was2 for three consecutive days, followed by intravenous rituximab 375 mg/m2 together with cyclophosphamide 100 mg for two doses one week apart. She was maintained on full-dose oral daily prednisolone 60 mg/m2 with a maintenance dose of oral mycophenolate mofetil 600 mg/m2/dose twice daily. Unfortunately, her kidney function did not improve after four weeks of intensive management. The suggestion to repeat kidney biopsy was considered, but her lack of response to re-induction, immune suppression, and the original histopathological diagnosis of diffuse proliferative lupus nephritis deferred our decision to repeat the biopsy. She continued to receive regular hemodialysis three to four sessions per week, together with maintenance immunosuppressive treatment for persistently active SLE.She retained normal renal function for 24 months after the first presentation, but her kidney functions acutely deteriorated with generalized anasarca 12 months from the diagnosis of lupus nephritis. She was admitted through the emergency room to the PICU with a clinical picture of cardiorespiratory failure, hypertension, oxygen desaturation on room air, and acute rise in renal function profile with oliguric renal failure. X-ray chest and heart showed a picture of acute pulmonary edema with cardiomegaly. She was managed with positive pressure ventilation and urgent daily hemodialysis sessions. Re-induction of immune suppression for control lupus activity was done according to our hospital protocol with daily intravenous pulse methylprednisolone 500 mg/mIn this case, the diagnosis of SLE was delayed for one year based on the initial clinical picture and persistently high serum IgE levels, which indicated hyper-IgE syndrome. In addition to the initial negative lupus-specific immunological tests (anti-dsDNA), which were strongly positive after 12 months of initial presentation, the case was complicated simultaneously by active lupus nephritis. Kidney biopsy revealed class IV lupus nephritis based on ISN/RPS. The activity index was 6/12, and the chronicity index was 0/12 based on the modified National Institute of Health (NIH) scoring system [Limited studies have illustrated an association between juvenile SLE and elevated serum IgE concentrations, despite the existence of atopic symptoms or parasitic disease. Owing to the complexity of SLE pathogenesis, the reasons for these findings and their associations with the disease risk and characteristics remain unresolved .Previous studies have shown that elevated IgE production secondary to disturbance of immune tolerance could lead to polyclonal activation of B lymphocytes and aberrant Th2 responses along with the production of autoantibodies. Additionally, cells with the IgE isotype will be associated with the overproduction of interleukins (IL)-4, IL-5, IL-10, and IgE, which is primarily found in patients with parasitic infections and atopic diseases ,4.Furthermore, IgE overproduction has been reported in patients with partial T cell immunodeficiency and autoimmune diseases, which are usually associated with overactivity of the adaptive immune system ,5. ThesePrevious reports have demonstrated that elevated total IgE levels may be associated with a history of allergic and connective tissue disease owing to the release of vasoactive mediators from the basophils and mast cells. IgE is associated with an increase in vasopermeability, which may be significant in the deposition of circulating ICs in glomerulonephritis. However, some studies have assumed that the pathogenesis of connective tissue disease is not caused by an increased level of IgE ,9. HowevIgE deposition was not observed in the immunofluorescence study of the kidney biopsy from the current patient. However, IgE serum levels are reportedly increased in lupus patients with kidney involvement, with the detection of IgE immune complex deposition in kidney biopsies . MoreoveStudies in adult patients with SLE have identified an important correlation between disease activities as they have IgE levels at least two folds higher than those in patients with inactive disease. Moreover, IgE kidney deposits in patients with lupus patients correlate with a poor prognosis. This observation conflicts with that of previous studies of patients with SLE, which showed similar levels of IgE levels in subjects with or without nephritis .IgE levels inversely correlate with C4 levels, suggesting that the complement cascade is activated, and components are consumed. This was observed in the present case by the reduction of C4 level, which is a marker of disease activity, and the high IgE level, which is a purported marker of disease activity .Several studies have suggested the term “auto-allergy” to indicate the autoimmune processes that are mediated by IgE autoantibodies . There aInterestingly, high levels of serum IgE were noticed in children whose mothers have lupus disease, despite the existence of allergic disease in the mothers .Hyper-IgE is considered a marker of immune dysregulation and may be important in the generation of ICs that mediate juvenile SLE and lupus nephritis. Despite the various factors influencing IgE production, the present case demonstrated that juvenile patients with SLE may have higher IgE concentrations, suggesting that IgE overproduction could play a role in lupus pathogenesis.The specific mechanisms underlying the elevation of IgE levels in children with lupus remain to be clarified, and further studies are needed in this regard. Juvenile lupus nephritis with hyper-IgE may carry a worse prognosis; therefore, early detection is strongly recommended. Further studies are needed to assess the incidence, prognosis, and possible new specific management for hyper-IgE in juvenile SLE and to understand the causality and associations of hyper-IgE in juvenile SLE patients.


Mutated and unmutated IgE and IgG play different and partly opposing roles in allergy development, but the mechanisms controlling their relative production are incompletely understood. Here, we analyzed the IgE-response in murine food allergy. Deep sequencing of the complementary-determining region (CDR) repertoires indicated that an ongoing unmutated extrafollicular IgE response coexists with a germinal center response, even after long-lasting allergen challenges. Despite overall IgG1-dominance, a significant proportion of clonotypes contained several-fold more IgE than IgG1. Clonotypes with differential bias to either IgE or IgG1 showed distinct hypermutation and clonal expansion. Hypermutation rates were associated with different physiochemical binding properties of individual B-cell receptors (BCR). Increasing BCR signaling strength inhibited class switching from IgG1 to IgE in vitro, preferentially constraining IgE formation. These data indicate that antigen-binding properties of individual BCRs determine differential IgE hypermutation and IgE versus IgG1 production on the level of single B-cell clones. Membrane-bound Igs serve as the BCR and determine the antigen-specificity of the B-cell response3, with different Ig subclasses having distinct but overlapping signaling properties8. In their soluble form secreted by plasma cells, the different Ig classes have different effector functions and thus play distinct roles in immune protection and disease9. Although IgE has the shortest half-life and shows the lowest serum concentration of all Ig subclasses, it plays important roles for immune protection against helminths and has a key role in type-1 atopic disorders12. IgE production and serum levels seem to be limited by multiple mechanisms, including limited formation and/or survival of IgE+ B cells13. IgE expression requires an irreversible DNA rearrangement, called class switch recombination, that can occur in activated mature B cells14. Naive IgD+/IgM+ B cells can switch to IgE either directly, or sequentially through an intermediate IgG+ B-cell stage. Both processes involve directed deletional loop-out-mediated recombination between two highly repetitive switch regions, which generates short-lived switch circles that differ for direct and sequential switching16. Class switch does not affect the variable chains that contain the CDR responsible for antigen recognition16. Class switch can occur in both extrafollicular locations and in germinal centers. However, the extrafollicular response yields B-cell clones with comparably low hypermutation rates18.Human and murine B cells can produce a variety of different immunoglobulin (Ig) classes  using different constant regions of the heavy chain (CH) regions21. High-affinity clones are directly attracted to the plasma cell fate, while clones starting with lower affinities enter the germinal center reaction to become memory B cells22.Within germinal centers, activated B-cell clones proliferate and hypermutate their variable region genes, including the CDRs. Following selection of B-cell clones expressing antibodies of higher affinities, memory B cells, and plasma cells are released from germinal centers; these can persist for years to decades26. In different murine models and patients, persistent IgE production has been attributed either to the formation of long-lived IgE+ plasma cells28 or reactivation and class switching of IgG1+ memory B cells31. In humans, long-lived plasma cells seem to play a more prominent role for persistent IgE production than has been reported for most mouse models32. The generation of long-lived IgE+ plasma cells seems to require repeated antigen/allergen challenges27.Next-generation sequencing (NGS) of the variable region of the BCR repertoires has brought deep insights into these processes and their contribution to the pathogenesis of allergic and autoimmune diseases35.Not all IgE mediates allergic symptoms. Highly mutated, high-affinity IgE can activate mast cells and cause allergic anaphylaxis. In contrast, less mutated, low-affinity IgE does not activate mast cells and can prevent anaphylaxis by competing with high-affinity IgE for binding to Fcε receptors36, IgE and IgG1 (IgG4) have opposing roles in allergy development37. Allergen-induced crosslinking of IgE bound to the FcεRI on mast cells can induce type-1 hypersensitivity reactions. In contrast, allergen-specific IgGs can neutralize absorbed allergen, thereby inhibiting IgE-mediated diarrhea and anaphylaxis38. Therefore, the ratio between IgG and IgE production as well as the generation of poorly mutated versus hypermuted IgE can be relevant for allergy development, in humans and in mice.Although IgE, murine IgG1, and human IgG4 are all produced through T-cell-dependent type 2 immune responses involving interleukin (IL)-4 or IL-13Here, we studied the formation of unmutated and hypermutated IgE and IgG1, their repertoires, and their clonal relationships in a mouse model of food allergy to hen´s egg, using repeated allergen challenge over a relatively long period of time.39. Starting at week four, mice were examined for diarrhea and hypothermia in response to o.g. egg inoculation /egg yolk plasma (EYP) followed by repeated oral gavage (o.g.) with EW and EYPion Fig. . Dependiion Fig. . The devion Fig. . IgA andIn order to investigate the IgE and IgG1 repertoires, samples were taken from the intestine-draining mesenteric lymph nodes (mLN) and bone marrow (BM) of two naive, and five allergic mice that had developed a temperature drop ≥ 1 °C and diarrhea after seven weeks of repeated allergen challenges (13 o.g. egg inoculations). cDNA was generated from the individual samples, barcoded, and amplified, after which the BCR-repertoires encoding the antigen-binding VDJ-IgH-regions that were 5′ to Cε or Cγ1 sequences were analyzed by NGS Fig. .+ plasma cells within the BM is more tightly restricted than the accumulation of IgG1+ plasma cells in this tissue. This conclusion is consistent with results suggesting that IgE and IgG BCRs differently impact B-cell fate and the development of long-lived plasma cells41 and that IgG+ plasma cells have a better BM homing ability than IgE+ plasma cells42. While the majority of IgE and IgG1 clonotypes showed VDJ-hypermutation, VDJ-hypermutation was absent in approximately 7–12 % of IgE and IgG1 in mLN and BM clonotypes  or IgG1-biased .As expected from the overall excess of IgG1 compared to IgE sequences, most shared clonotypes showed a several-fold excess of IgG1 compared to IgE copies. However, other clones shared between IgE and IgG1 showed a strong bias towards IgE, containing 5 to 10-fold more IgE than IgG1 copies Fig. . Based oE and IgEG showed different levels of clonal expansion , IgEion Fig. . Mutatedons Fig. .E and IgEG clones represent a unique feature of our allergy model, the BCR repertoires of the IgE and IgG1 compartments in BM of two naive mice were analyzed. The number of distinct clonotypes was low, as expected for unimmunized mice 2 fragments. Anti-Ig kappa Ab stimulates both the IgM and IgD BCRs, which seem to be relevant for BCR activation by certain types of antigen7; and can also stimulate IgG1+ B cells.Next, we tested the possibility that differential BCR-signaling strength, which may mimic higher or lower affinities/avidities of BCRs for their cognate antigen, has an impact on the development of IgE versus IgG1. Therefore, we used a modified version of an advanced culture system that allows in vitro generation of germinal-center-like cells47. However, the decrease was greater for IgE+ cells and the ratio between IgE+ and IgG1+ cells dropped from approximately 0.14 in the absence of BCR stimulation, to below 0.03 at the highest concentration of stimulating antibodies. Therefore, we speculated that BCR stimulation could inhibit not only direct class switching from IgM to either IgG1 or IgE, but also the ability of IgM+ cells to switch sequentially to IgE via an IgG1+ intermediate, which could explain why IgE+ cells decreased more rapidly than IgG1+ cells. In line with this idea, sequential class switch-specific switch circles were reduced upon BCR stimulation 2 induced a dose-dependent calcium flux Fig. , indication Fig. . Of noteIn order to further address the idea that BCR signaling inhibits sequential class switching, IgG1+ cells were sorted from cultures and re-cultured in the presence of IL-4 and increasing amounts of anti-kappa Ig F(ab’)2. After one day, without BCR stimulation, between 12 and 22 % of the isolated IgG1+ cells had switched to IgE and lost IgG1 expression. Increasing BCR stimulation inhibited the appearance of IgE+ cells in a dose-dependent manner but increased the percent of IgG1+ cells back to nearly 100 % Fig. .47 by showing that increasing BCR signaling also effects sequential class switching of IgG1+ cells to IgE, eventually reducing the ratio of IgE/IgG1+ cells.These results extend the finding that BCR signaling inhibits class switch to IgG1 and IgE50, which could modulate the interaction of antigen-specific B cells with T follicular helper cells which provide IL-21. Therefore, we tested the effects of IL-21 in our culture system. IL-21 promoted the formation of IgG1+ cells, while inhibiting IgE, eventually shifting the ratios of IgE+ to IgG1+ cells from approximately 0.1 to above 0.2  were related to highly mutated IgG1 clonotypes Fig. . Hence, Together, our findings indicate that the physiochemical properties of individual BCRs affect their mutation rates and clonal selection and that the BCR affects the ratio of IgG1 to IgE formation through its individual antigen-signaling strength and soliciting T-cell help. This mechanism could bias high-affinity B cells, receiving strong antigen-induced BCR signaling and IL-21 from T follicular helper cells, to favor IgG1 over IgE production.Despite the great relevance of the production of mutated and unmutated IgE and IgG in allergy development, the mechanisms controlling the formation of these Ig subclasses are incompletely understood. Here we provide evidence that the physiochemical properties and signaling strength of BCRs control IgE and IgG1 formation and hypermutation on the level of individual B-cell clones.50. B cells with a high-affinity BCR may therefore receive stronger and/or prolonged help from T follicular helper cells, providing IL-21. Our results show that IL-21 can reduces the ratios of IgE to IgG1+ cells, at least in an in vitro model for the GC reaction. Based on these findings, we suggest a model in which strong antigen-signaling and optimized help from IL-21 producing T follicular helper cells bias high-affinity B cells to favor IgG1 over IgE production. Although our lineage tree analysis indicates that highly mutated IgG1 clones can still undergo class switch to IgE in vivo, the corresponding highly mutated IgE clones were very rare.In addition to the stronger direct inhibitory effects of antigen on B cells that express a high-affinity BCR, B cells with a high-affinity BCR can take up antigen very efficiently to present them to T cells, even at low antigen concentrations45. The data presented in this paper imply that IgE-BCRs with distinct physiochemical properties are differentially subjected to hypermutation and clonal selection. Particularly mutated IgEG showed a higher aliphatic index and hydrophobicity than unmutated IgEG in egg-immunized mice. In this context it is interesting that many allergens are partly hydrophobic52, and the interaction of some proteins with lipids is suspected to increase their allergenic properties53. Taking into account that IgEG clonotypes were much more abundant than IgEE clonotypes and that hypermutated IgE but not unmutated IgE are believed to contribute to allergic symptoms33, the formation of hypermutated IgEG might be most relevant for allergy development. If the hydrophobicity of allergens correlates with their capacity to promote the formation of hypermutated and highly pathogenic IgE remains to be elucidated.The antigen-binding priorities and polyreactivity of individual BCRs are determined by their physiochemical properties of the variable regionE and IgEG clonotypes also, on average, had lower PIs than their unmutated counterparts, indicating that the electric charge of an antigen/allergen could also have an impact on the relative production of these more or less allergenic IgE subtypes.Mutated IgE47. The Jabara paper also showed that the effects of BCR stimulation on IgE and IgG1 were not due to altered proliferation or apoptosis. Extending these results, we found that increased BCR signaling can also inhibit class switch from IgG1 to IgE, eventually increasing the IgG to IgE ratios. Although the BCR stimulation-induced changes were not dramatic, they were in the same range as the differences between IgG1 and IgE serum titers in mice showing diarrhea and anaphylaxis compared to mice having no symptoms. This suggests that the increase in IgG to IgE ratio induced by increased BCR signaling could be relevant for the development of allergic symptoms, particularly when acting in synergy with IL-21. Under pathophysiological conditions, such increased BCR signaling could be permitted by high-affinity BCRs or BCRs that recognize highly polyvalent antigens.Since allergen-specific IgG can inhibit IgE-mediated mast cell activation, the ratio between IgE and IgG is expected to be another factor relevant for allergy development. The finding that individual BCR clonotypes vary considerably in proportion of IgE and IgG1 indicates that the ratio between the two Ig classes is controlled on the level of single B-cell clones and is influenced by the properties of individual BCRs. In contrast to what we observed for the IgE hypermutation rates, we did not find evidence that the IgE to IgG ratios are affected by the physiochemical properties of the BCR. Instead, antigen-binding affinities and avidities seem to affect the formation of IgE versus IgG1. Under physiological conditions, these two factors determine the BCR signaling strength. In our experiments we used titrated amounts of anti-BCR antibodies to induce differential BCR signals. Our results are in accordance with the finding of Jabara and colleagues showing that increased BCR signaling inhibits class switch from IgM to IgG1 and IgE38, a mechanism which may be most efficient for IgGs that have the same fine specificity as their IgE counterparts. Therefore, it is possible that high-affinity BCR clones may have a lower IgE to IgG ratio which may compensate for their increased allergic potential, particularly when allergen concentration is low, but this hypothesis needs to be investigated further.That the ratios between IgE and IgGs are controlled on the level of single B-cell clones is a logical consequence, since IgGs can inhibit IgE-mediated anaphylaxis through neutralization of absorbed allergen10. In our model, long-lasting IgE production was induced by repeated allergen challenges, which might reflect a common condition for allergic patients. Despite the extensive rounds of allergen challenges, the IgE compartment still contained a considerable proportion of unmutated IgE clones which co-existed with hypermutated and highly selected IgE clonotypes. This indicates that an extrafollicular response persisted despite an effective germinal center reaction. We found this somehow surprising, because mutated, affinity-selected germinal center-derived clones might be expected to outcompete the unmutated clones. Nevertheless, the selection processes in B-cell responses involving multiple and complex antigens are still incompletely understood54. In this context it is interesting that there is emerging evidence that germinal center responses and an extrafollicular response also co-exist in another chronic immune response, the autoantibody response in systemic lupus erythematosus56. In that disease, the two pathways of Ab production are driven by distinct T helper subsets56. It remains to be elucidated whether the same mechanism is also relevant in IgE-mediated allergic diseases.While IgE production is normally short lived, even under conditions when IgG antibodies are maintained at high levels, it can persist in patients suffering from allergic diseases even without allergen re-exposureIn conclusion, our results imply that individual BCRs affect the overall nature and allergic potential of the IgE response. This finding may help to improve the efficiency of allergen-specific therapies through the use of allergens with altered physiochemical properties and/or binding valence.The animal experiments conducted in this study were done in strict accordance with the German regulations of the Society for Laboratory Animal Science (GVSOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). Protocols of the animal experiments performed at the University of Lübeck were approved by the respective local Committee on the Ethics of Animal Experiments of the state Schleswig-Holstein . Approval numbers: 22-39_2016-06-17_Manz, and 122-39(46-6/18)_Manz. Animal experiments performed at CCHMC were approved by the CCHMC IACUC .Female Balb/c mice were purchased from Charles River Laboratories  and maintained under pathogen-free housing conditions. Mice were housed according to standard operating procedures assuring mouse husbandry at the animal facilities of Cincinnati Children’s Hospital Medical Center and the University of Lübeck, with approval from the respective authorities. Animal caretakers regularly observed the mice for signs of disease, illness, or injury. If indicated, mice were observed by a veterinarian. After acclimatization period of at least one week, animal studies were performed with mice aged between 8–16 weeks at the start of the experiment.39. Briefly: EW and EY were separated and prepared as described. Mice were anesthetized by i.p. injection of 200 μL anesthetics , restrained and sensitized by intra-tracheal application of 40 µL EYP containing 50 µg EW. The procedure was repeated according to the sensitization schedules. For antigen (Ag) challenges, lyophilized EW was dissolved in sterile DPBS to a concentration of 500 mg/mL, then mixed with an equal volume of EYP. To assess diarrhea development, EW plus EYP was supplemented with food dye. Mice were intra-gastrically challenged by oral gavage with 300 µL of this mixture. Body temperature was measured by rectal thermometry (Physitemp). Potential confounders were minimized by co-housing the mice of all groups in shared cages and received identical treatments .Egg allergens were prepared and food allergy to hen’s egg was induced as described previouslyIn order to reduce pain, suffering, and distress for the experimental mice, and to minimize the effects of potential confounders on the experimental results such as the spontaneous development of unwanted/unexpected diseases, criteria for exclusion of experimental mice were defined a priori. Experimental mice were observed once a week for general health conditions, conspicuous behavior, injuries, scrubby coat, conspicuous excrement or urine, signs of pain, belly stiffness, and paralysis. Exclusion criteria and humane endpoints were set a priory according to a scoring system, including signs of disease, illness, and weight loss, as described in detail in the animal applications, approved by the authorities. (see above). Criteria for exclusion were the development of unexpected symptoms, indicating the development of unexpected diseases such as infections or tumors. No mice had to be excluded because of these criteria. The sample size was decided on the basis previous experiments. Blinding was performed by assigning individual mice a mouse tag number. After allocation of groups according to allergic symptoms, these mouse tags were anonymously used during subsequent analysis.To determine anti-EW and IgG1 levels, white Costar® 96-well plates were coated with 10 µg/mL EW in Tris/saline buffer (pH 7.2). For anti-EW-IgE levels plates were coated with 5 μg/mL anti-IgE mAb. Briefly, wells were washed and subsequently blocked with washing buffer containing 10% SuperBlock™(Thermo Scientific). Pooled serum samples of EW allergic mice were used as standard. Serum from naive mice were used as negative control. Standards  were serially diluted 1:2. After incubation with standards and diluted samples, wells were repeatedly washed and subsequently incubated with biotinylated anti-mouse IgG1 (Southern Biotech) or for the detection of EW-IgE incubated with biotinylated EW. Wells were then washed and incubated with Streptavidin-HRP (Thermo Scientific). Following further washing, substrate (Thermo Scientific) was added to the wells and responses were immediately measured with a Luminometer . Students performing the ELISA assays were not aware about the group allocation.Tissue samples were collected from BM and mLN samples, cut into small pieces (2 × 2 mm), transferred into tubes with RNAlater and incubated at 4 °C overnight, then stored at −20 °C in RNAlater solution (Qiagen). Later, RNA was isolated using the RNeasy mini Kit (Qiagen #74104). Reverse transcription of the RNA samples was performed using the Qiagen-One-Step-RT-PCR Kit (Qiagen). The custom-designed primers specific for murine IgG1 and IgE were synthesized by iRepertoire. PCR product was further amplified using generic primers provided in the iRepertoire kit that bind Illumina-adapters introduced by the primers used in PCR 1. For the subsequent amplification the Qiagen Multiplex PCR kit (Qiagen #206143) was used. A quality control was performed using an Agilent Bioanalyzer 2100 and an RNA 6000 Pico kit (Agilent). Based on the integrity of ribosomal RNA, RNA-integrity numbers (RIN) were determined . Only RNA samples with RIN of 6 or higher were used for NGS analyses.For preparation of the library for paired-end sequencing with the Illumina MiSeq system 5 μL aliquots of each sample at a concentration of 2 nM (diluted with Tris/HCl based on concentration determination as described above) were pooled and PhiX Control v3 was added to the library as an internal sequencing control. Approximately 1–1.5 million sequencing reads per sample of the BCR library were obtained.The quality of the NGS data was determined using FastQC. The criteria for sample exclusion were set a priory: samples showing a median quality score for any base of less than 20 units were excluded from further analysis. Due to insufficient cDNA quality, one BM sample of an allergic mouse and one BM sample of a naive mouse were excluded from NGS analysis.The data was de-multiplexed into the individual samples with ClonoCalc (153). The determination of clonotypes (unique CDR3 nucleotide sequences) was performed using MiXCR (168). The output data included a clone ID that was given by the program sorted by occurrence of this sequence starting at 1 for the most abundant clonotype, abundance of the clonotype , aligned V-, D-, J- and C-gene , CDR3 nucleotide and amino acid sequence, the quality score, and length of the CDR3-region. Additionally, to avoid artificial diversity due to PCR errors, all clonotype sequences that appeared only once were removed. Overlapping sequences between IgE and IgG1 were determined using InteractiVenn.57. The resultant IMGT/HighV-QUEST output file for IgE and IgG159 were used as input files in the IMGT/StatClonotype database to generate their corresponding IMGT/StatClonotype text files61. IgE and IgG1 V-REGION-nt-mutation-statistics.txt file from IMGT/HighV-QUEST and IMGT/StatClonotype.txt file from each mouse were used for further downstream analysis in an Excel VBA Macro (https://mega.nz/file/wwBgAZBD#yrZcvHVpLINE6GYmrEeRDSWiU6XpIeCqnlj425uvzhA). The VBA macro excludes non-productive clones, clones with one copy, clones without a conserved CDR3-IMGT anchors , and clones with no V, D, or J call. The macro defines a clone as a unique VDJ gene rearrangement with conserved anchors. From here, the macro further evaluates the shared and unique VDJ rearrangement between IgE and IgG1, the bias to IgE and IgG1 within the shared clones, the mutated and unmutated clones based on the presence or absence of mutation in the V gene.For analysis of clone sizes, identification of shared clonotpes and later specification of these clones, each mouse cDNA FASTA sequence from MIXCR or IRepertoire  was separated into IgE and IgG1 FASTA sequences. IgE and IgG1 FASTA files were uploaded to the international ImMunoGeneTics (IMGT) database62, and the lineage trees were generated using DNA maximum parsimony criterion. The resultant tree was viewed as a radiation tree with Mega 1163. In each mouse, the mutation table of the shared IgE and IgG1 clones was used to plot the mutation distribution in graph pad prism. Potential confounders are minimized by comparing the properties IgG vs IgE subtypes  in-between LN and BM samples from the same individual mouse, and in-between IgG vs IgE subtypes within the same sample, excluding cage effects and effects of differential treatment.For the analysis of the lineage trees, the V region nucleotide sequences from highly mutated IgE clones and its shared IgG1 counterparts were aligned by ClustalW64. At the start of the culture, B cells were stimulated with IL-4 together with various concentrations of polyclonal goat anti-mouse IgM F(ab′)2 fragments (Dianova), or anti-Ig kappa F(ab′)2 fragments (Sigma-Aldrich). To investigate the role of IL-21, recombinant murine IL-21 (ImmunoTools) was added at various concentrations at days 0 and 1. On day 4, B cells were isolated from the culture, stained for IgG1 (clone: RMG1-1), IgE (clone: RME-1), IgM (clone: RMM-1), IgD (clone: 11-26c.2a), CD138 (clone: 281-2), and CD19 , and analyzed by flow cytometry. Dead cells and feeder cells were excluded from the analysis using the Fixable Viability Dye eFluor 780 (eBioscience), and FSC-A versus FSC-H and SSC-W versus SSC-H gating. For analysis of sequential class switch from IgG1 to IgE, IgG1+ B cells were FACS sorted from 4 day cultures or directly from fresh spleens . Flow cytometry analysis was performed using an LSRII (BD). Data were analyzed using the Flow Jo 10.7.1 software. As a quality control for efficient B-cell stimulation, cell expansion was assessed under the microscope and/or by cell counting. Cultures with inefficient cell expansion, i.e. ≥ 3-fold expansion of B-cell numbers till day 4, were not used for analysis. In order to minimize potential confounders further, B cells isolated from individual mice were spitted and used to study the impact of various culture conditions. The outcome measures used to determine the sample size were the frequencies of IgE+ and IgG1+ cells. Sample sizes were planned on the basis of preliminary experiments. Since the flow cytometric analysis of the cell cultures is objective, analysis of the cell cultures was not blinded.B cells were isolated from single-cell suspensions of the spleen using the EasySep™ Mouse Pan-B-Cell Isolation Kit (Stem Cell) and co-cultured together with mitomycin C M0503 (Sigma-Aldrich) treated 40LB feeder cells, a CD40-ligand (CD40L)- and B-cell activating factor (BAFF)-transfected fibroblast cell line as describedp < 0.05, **p < 0.01, and ***p < 0.001. Data were plotted as mean ± SEM. A p value < 0.05 was considered significant.Data analysis was performed using GraphPad PRISM software, statistical tests are indicated in individual figure legends. NGS datasets were tested for normality using the D’Agostino & Pearson omnibus normality test. If appropriate, nonparametric tests were used and indicated in the respective figures. *Supplementary Figures and tables


The surface wear of the screws was analyzed. Finally, the stress distribution of the abutment screws was calculated by finite element analysis (FEA). The results showed that fluid contamination reduced the friction coefficient, increased the preload, decrease the settlement, improved resistance to screw loosening, and reduced wear on the thread surface. Appropriate antimicrobial lubrication may improve the anti-loosening performance of abutment screws and prevent excessive wear on the threaded surface.Screw loosening is one of the most common clinical problems of dental implants. Research on the influencing factors of screw loosening is very important to prevent screw loosening. The purpose of this in vitro study was to evaluate the influence of liquid contamination on the screw loosening. According to the contamination condition, forty-five abutment screws were divided into three groups (n = 15): no contamination, artificial saliva contamination, and mouthwash contamination. The preload and friction coefficient of the abutment screws were recorded. Then, the reverse torque values (RTVs) and settlement were measured after 3.0 × 10 One study showed that 26% of abutment screws need to be tightened again after the first year2. In another study, the loosening rate of abutment screws was 3.1–10.8% after 5 years3. In a clinical study of more than 15 years, the rate of abutment screw fracture was 3.5%4. Loose screws will not only cause implant failure but may also cause complications such as gum sensitivity, hyperplasia, and inflammation, which will seriously affect the patient’s daily life7.Biomechanical properties are a key factor in the success of dental implants. Despite the high success rate of implantation, some mechanical complications still exist. Abutment screws play a vital role in the connection between the implant and the abutment. Under the long-term influence of the mastication force, abutment screws will experience complications such as loosening or fatigue fracture8. It is generally believed that an increase in the preload helps to improve the stability of the implant-abutment interface11. When tightening the screw, 90% tightening torque is used to overcome friction, and the remaining 10% is converted into the preload12. Friction mainly occurs at the screw-abutment interface and screw-implant interface, and reducing the friction loss between these interfaces enables more torques to be converted into the preload13. After the screw is tightened, the preload will be reduced by 2 to 15% due to the settlement effect of the connection interface14. Although many methods of increasing the preload are used by reducing the surface friction coefficient, the results of screw loosening are different17. Therefore, high preload does not mean a good resistance to loosening. The reverse torque values (RTVs) reflects the maintenance of the preload under functional load, which shows the resistance to screw loosening18.The abutment screw is elastically deformed and elongated due to the torque during the tightening process, and a compression force is formed between the implant and the abutment, which is called the preload20. The friction coefficient between the interfaces changes after liquid contamination, which may affect the preload, thereby increasing the risk of screw loosening21. Nigro et al. found that a higher preload was produced under wet conditions  than under dry conditions22. However, according to Rathe et al., fluid contamination  did not produce higher preload values23. For the research of RTVs, Duarte et al. found that fluoridated artificial saliva can increase the RTVs24. Koosha et al. found that only chlorhexidine can increase RTVs, and saliva can decrease RTVs among different liquid contaminations25. However, according to Gumus et al., the RTVs of abutment screws decrease after chlorhexidine and saliva contamination26. Although some studies have been carried out on the influence of liquid contamination on the abutment screw loosening, there have been various results and no in-depth understanding of the mechanism of screw loosening. Therefore, a study of the screw loosening mechanism under liquid contamination is necessary to improve resistance to the abutment screw loosening. In addition, the research on the influence of fluid contamination on the abutment screw loosening under dynamic load conditions is very limited.In clinical practice, abutment screws will be contaminated by different fluids  due to surgical operationsThis in vitro study aimed to evaluate the influence of liquid contamination on the screw loosening under static and dynamic conditions. The relationship of preload, settlement, and screw loosening was obtained. The wear of the screw surface was analyzed. A finite element model was also established to obtain the equivalent stress and surface friction stress changes after screw liquid contamination.In this study, Morse taper connection dental implants (Ø4.3 × L11 mm), abutments (Ø4.5 × H6.0 mm), and abutment screws (Ø1.5 × L13 mm) were manufactured . Forty-five samples were divided into three groups according to the liquid contamination state of the screw (n = 15): control group (NC: no contamination), artificial saliva contamination (SC) group, and mouthwash contamination (MC) group. Artificial saliva was provided by Leagene Biotech. Co., Ltd. ; Mouthwash was obtained by Listerine . Before the test, the interior of the implant is filled with contaminated liquid through a pipette, and then the abutment and abutment screws were inserted.27 provided the formula for calculating the friction coefficient: (1) Tth is the thread torque; (2) Tc is the conical torque; and (3) T is the torques sum (Tth and Tc).m represents the pitch diameter (1.37 mm), L represents the pitch (0.35 mm), α represents the half angle of the thread (30°), F represents the preload measured by a sensor (N), μ represents the friction coefficient of the screw, D represents the outer head diameter (2.17 mm), d represents the inner head diameter (1.6 mm), β represents the cone angle (30°), and T represents the tightening torque (Nmm).According to ISO16042:2005 "Fastener Torque/Clamping Force Test", the preload  and friction coefficient (μ) of screws under three surface conditions were measured. A schematic of the preload test apparatus is shown in Fig. i) were recorded. According to Formula (i represents the RTVs (Ncm).After tightening the three groups of abutment screws to 32 Ncm, the screws were allowed to stand for 10 min, and the RTVs were measured. The average RTVs 16. After the 3.0 × 105 cycles test was completed, the RTVs (T3) were recorded. The liquid was added again after the T3 was tested. Then, the screw was retightened for the 6.0 × 105 cycles test, and the RTVs (T6) were recorded. In addition, the length of the implant system before and after loading was measured by a spiral micrometer, and the settlement was calculated by the length change. Five samples were measured in each group.The screw loosening dynamic test was performed according to standard ISO14801:2016After the dynamic loosening test was completed, the abutment screws were removed and ultrasonically cleaned to remove surface impurities. Scanning electron microscopy with energy dispersive spectroscopy (SEM–EDS)  was used to analyze the surface wear of the screws.The sample size was calculated using the pre-experimental method . Assuming a significance level of 0.05, an effect size of 0.5, and a statistical power of 80%, it is calculated that the required sample size is at least 42 (14 per group). Statistical analyses were performed with SPSS . Data were analyzed by ANOVA with Fisher LSD test, with P < 0.05 indicating significance.To reduce the calculation error, a 3D model of the same size as the implant component was built by the computer-aided design software , as shown in Fig. 29. The equivalent stress value and frictional stress value were used to assess the distribution of stress among the abutment screws16. The "frictional contact" was set for screw-implant and abutment-screw, and the "perfect bonding" was set for implant-abutment, implant-fixture, and abutment-crown, and "fixed" was defined for the fixture. According to the measurements performed as described in “Each part of the model and its material properties were imported into Ansys Workbench software . Ti6Al4V was used for the abutment and abutment screws, Ti was used for the implants, and stainless steel was used for the crowns and fixtures. The characteristics of the materials are shown in Table 1The preload and friction coefficient of the NC, SC, and MC groups under a torque of 32 Ncm are shown in Table i) and the torque loss rate (θ) in the three groups. There were significant differences between the MC and NC groups (P = 0.021), and no significant difference between SC and NC groups (P = 0.214) and the SC and MC groups (P = 0.207). Due to the torque loss during the tightening process, Ti was lower than the tightening torque. Ti in NC group was 22.82 ± 2.02 Ncm, indicating a 28.69% torque loss. Compared with the NC group, the SC and MC groups had a higher Ti and less torque loss, which were SC  and MC , respectively.Table 3 and T6) after cyclic loading are shown in Fig. 3 showed significant differences between the SC and NC groups (P = 0.036), the MC and NC groups (P = 0.001), and the SC and MC groups (P = 0.010). T6 showed significant differences between the MC and NC groups (P = 0.008) and the SC and MC groups (P = 0.046), and no significant difference between SC and NC groups (P = 0.202). The RTVs (T3 and T6) were lower than Ti in all groups after cyclic loading. The RTVs in the NC group were less than the SC and MC groups, and the MC group had the highest RTVs. Among the three groups, the T6 was greater than the T3.The RTVs  and the SC and MC groups (P = 0.048), and no significant difference between NC and SC groups (P = 0.217). After the 6.0 × 105 cycles test, settlement showed significant differences between the NC and SC groups (P = 0.003) and the NC and MC groups (P = 0.001), and no significant difference between SC and MC groups (P = 0.188). Among all groups, the NC group had the largest settlement, followed by SC group, and MC group had the smallest settlement.The settlement after cyclic loading are shown in Fig. Figure When the external load was 200 N, Fig. The present experiment investigated the influence of liquid contamination on the connection stability of the implant system. The results showed that the implant system after liquid contamination showed significant differences in the screw loosening and surface wear.11. The liquid-contaminated abutment screw produced a higher preload and a lower friction coefficient. Studies have shown that the probability of reaching the best range in a well-lubricated environment is 54%, while in a dry environment, it is only 0.02%30. During the tightening of the screw, sliding friction occurred between the screw thread and the internal thread of the implant, but this friction was not evenly distributed. The wet medium can eliminate most of the shear forces generated inside the thread31 and reduced the friction coefficient of the thread surface, which reduced the energy loss due to friction so that more tightening torque was converted into preload. Previous studies have also confirmed that a lower friction coefficient will lead to a higher preload33.The preload is generated by the tensile deformation during screw tightening, and its optimal range is 60% ~ 70% of the yield strength of the screw29. Therefore, the RTVs of liquid-contaminated screws before and after loading was evaluated. The Ti of the SC and MC groups that have been liquid-contaminated are larger than those of the NC group, consistent with previous studies25. This is because the high preload made the threaded connection tighter. In addition, microscopically, the thread surface was rough, and the embedment relaxation occurred under the state of force34. The liquid reduced the embedment relaxation of the thread surface, thereby reducing preload dropped22.Although liquid contamination increases the screw preload, it is more important that the preload remains stable rather than dropping rapidly5 cycles, T3 of the three groups decreased, which was lower than their respective Ti. The vibration generated by the external load intensified the slippage of the threaded contact surface, and the settlement effect occurred at the implant-abutment interface35, resulting in reduced preload and more torque loss. The two groups of liquid contamination showed better anti-loosening performance after dynamic cyclic loading. The larger the preload of the screw is, the smaller the fretting between the parts when subjected to external force, so that the torque is not easy to lose10. In addition, the abutment settlement was reduced due to the liquid inside the implant (Fig. 36. After a cyclic load of 6 × 105 cycles, T6 of the three groups has a certain increase, and the trend is similar to that of 3 × 105 cycles. This is because after 3 × 105 cycles, the originally rough contact surfaces were squeezed and smoothed out by each other, and more abutment settlement had occurred. When a cyclic load of 6 × 105 cycles was performed, the taper surfaces had less settlement, and thread surfaces were more tightly bonded, resulting in an increase in T637. Therefore, after a certain number of cycles, loosening and retightening helped prevent the screw from loosening38.After a cyclic load of 3 × 1026. The viscosity will affect the flow of liquid on the metal surface and affect preload and settlement39. Therefore, the RTVs were improved to varying degrees.In addition, there are differences in the lubricating effects of the two liquids. The RTVs of the MC group were higher than those of the SC group. The main factors affecting the lubrication effect of the two liquids are the viscosity, concentration, and composition of the liquid40. This can cause surface deformation, preload loss, and even loosening of screws. Effective liquid lubrication can protect the thread surface from excessive wear and prevent the aggravation of fretting damage26. The wear mainly occurred in the top area of the thread, mainly abrasive wear and oxidation wear, which was consistent with previous research41. Liquid lubrication increased the preload of the screw and reduced abutment settlement, thereby reducing the relative sliding of the threaded surface and surface wear42.In the process of the dynamic fatigue cycle, due to the existence of fretting, the screw surface will inevitably wear. The mechanism of wear may be abrasive wear, adhesive wear, oxidation wear, fatigue wear, etc.11. The increase in friction stress indicated that a greater friction force was generated between the contact surfaces, and relative sliding did not easily occur between the contact surfaces40. Therefore, the RTVs are greater, and the system stability will be improved. In addition, comparing the contact stress between screw-implant and screw-abutment, it was found that the friction stress in the contact area with the abutment was greater than the friction stress in the implant. The reason may be that the contact area of the abutment was small and there was a concentration of stress, which was not as good as the implant-screw threaded contact for dispersing the friction stress.The 3D-FEA results can explain the reason for the increase in the RTVs of the liquid-contaminated group. The equivalent stress and friction stress of the screw increased with increasing preload. This made the bonding between the contact surfaces closer, and the screw reached the appropriate stress valueThis study still has some limitations. For example, the FEA results are only under static conditions, which cannot reflect the dynamic screw loosening process. The types of contamination assessed are few and single, because screws are contaminated by multiple substances in the oral environment. Only screw loosening behavior was studied, but the effects of liquid contamination on aging and corrosion were not considered. Additionally, loading conditions in vivo are different from in vitro studies, so more complex loading states need to be tested. The Morse taper connection was used in this study, and other connected implant systems need to be studied in the future.In summary, liquid contamination on the abutment screw surface can increase the preload, reduce the friction coefficient, and reduce the settlement, thereby improving the resistance to loosening of the screw and reducing the wear of the screw surface. Therefore, antibacterial solutions or gels with lubricating properties may improve the long-term stability of implant systems.


The regulation of the mRNA decay activator Tristetraprolin (TTP) by the p38 mitogen-activated protein kinase (MAPK) pathway during the mammalian inflammatory response represents a paradigm for the control of mRNA turnover by signaling. TTP activity is regulated through multiple phosphorylation sites, including an evolutionary conserved serine in its CNOT1 Interacting Motif (CIM) whose phosphorylation disrupts an interaction with CNOT1 of the CCR4-NOT deadenylase complex. Here we present evidence that the TTP CIM recruits the CCR4-NOT deadenylase complex and activates mRNA degradation cooperatively with the conserved tryptophan residues of TTP, previously identified to interact with CNOT9. Surprisingly, the TTP CIM remains unphosphorylated and capable of promoting association with the CCR4-NOT complex and mRNA decay upon activation of p38-MAPK-activated kinase MK2, a well-established regulator of TTP activity. The CIM is instead targeted by other kinases including PKCα. These observations suggest that signaling pathways regulate TTP activity in a cooperative manner and that the p38 MAPK-MK2 kinase pathway relies on the activation of additional kinase pathway(s) to fully control TTP function. TTP is10–14–19–Recent attention has been turned toward this conserved C-terminal region of the TTP protein family. Studies examining the structural basis of TTP’s association with the central cytoplasmic deadenylase, the CCR4-NOT complex, revealed an interaction between the CCR4-NOT scaffold protein, CNOT1, and this C-terminal region of TTP, which was therefore named the CNOT1-Interacting Motif (CIM) , 22. SupTTP is a highly post-translationally regulated protein that has been shown to be a target of several kinase pathways. The most well-characterized of these is the p38 MAPK pathway, which is activated during the inflammatory response . A downs26–10–Another serine of TTP that undergoes phosphorylation is serine 316 (mouse numbering), an evolutionary conserved serine of the CIM motif. Phosphorylation of this residue has been shown to inhibit association of TTP-family proteins with CNOT1, suggesting that the association of the major deadenylase complex is repressed by phosphorylation , 29, 30.In this study, to better understand the significance of TTP co-factor interactions in promoting activation of mRNA decay and how these are regulated by phosphorylation, we monitored the combinatorial effects of phosphorylation site and co-factor interaction motif disruptions on TTP function. We find that the CIM acts cooperatively with the conserved tryptophans of TTP to recruit the CCR4-NOT complex and activate mRNA decay, but upon mutation of these motifs, TTP remains unimpaired in its interaction with decapping factors and partially active. Using a phospho-specific antibody, we find that phosphorylation of the TTP CIM occurs with kinetics similar to that of the MK2-phosphorylated TTP serine 178 in stimulated mouse fibroblasts and macrophage cell lines and reduces the ability of TTP to promote mRNA decay. Surprisingly, in the presence of active MK2 kinase the CIM remains unphosphorylated and TTP stays active primarily due to the activity of the CIM. These observations suggest that the CIM serves as an MK2-independent regulatory module of TTP and that the full range of TTP regulation requires cooperativity between kinase pathways.To establish a system for monitoring the interplay between the CIM and its phosphorylation with other known functional regions of TTP , we utilAlthough the TTP-CIM can promote mRNA decay on its own, other regions of TTP have been implicated in mRNA decay as well. We were therefore interested in understanding the importance of the CIM in promoting mRNA degradation in conjunction with other functional motifs of TTP. We first deleted the CIM from full-length TTP and from the carboxy-terminal domain (CTD) of TTP, each fused to the MS2 coat protein, and tested the effect on mRNA degradation. Consistent with previous reports , the tarTTP was previously reported to interact with another component of the CCR4-NOT complex, CNOT9, through its conserved tryptophan residues  (9). We Other regions of TTP that interact with mRNA repression factors are the tetraproline motifs, which are conserved among TTP orthologs and promote the association with the 4EHP-GYF2 translation repression complex , 33. MutHaving established the degree of cooperativity between the TTP CIM and other known functional motifs of TTP, we next turned to the significance of CIM phosphorylation. We generated and validated a phospho-specific antibody raised against the phosphorylated serine 316 residue of mouse TTP . ConsistThe TTP-CIM has been reported as a target of several kinase pathways , 29–31, 29–Other kinases that have been observed to phosphorylate the CIM of TTP-family proteins include p90 ribosomal S6 kinase 1 (RSK1) and Protein Kinase A (PKA) , 30, 35.PS;WA;ΔCIM triple mutant was compared to TTPPS;WA . We therefore wished to test whether CIM phosphorylation regulates TTP activity independently of MK2. We first tested the importance of CIM phosphorylation in the context of full-length TTP. As we observed earlier, TTP is phosphorylated at serine 316 in HeLa Tet-off cells independently of MK2 . MutatinFig. 6–. We therTo test the importance of the serine 316 residue in mRNA decay activated by the TTP-CIM alone, we mutated this residue to an alanine or a glutamate. MS2-tethered pulse-chase mRNA decay assays showed a minor increase in the degradation rate as a result of the serine to alanine mutation  and D, aThe regulation of TTP by the p38 MAPK-MK2 pathway during the inflammatory response is a well-established paradigm for the regulation of mRNA decay by signaling. In this study, we demonstrate that the highly conserved C-terminal CIM motif of TTP, which plays a key role in connecting TTP family proteins with the CCR4-NOT deadenylase complex central to mRNA decay, is regulated separately from the p38 MAPK-MK2 pathway. This conclusion is supported by our observations that, unlike TTP serine 178, the TTP CIM serine 316 remained phosphorylated in the presence of a p38 MAPK inhibitor and was not phosphorylated by constitutive active MK2 . MoreoveFig. 6Our finding that TTP remains highly active even when deletion of the TTP CIM and mutations in conserved tryptophans and tetraproline motifs disrupt associations with CCR4-NOT and 4EHP-GYF2 complexes  and 3, s40–The most surprising finding of this study was that the TTP CIM remains active and capable of promoting mRNA decay in the presence of active MK2, despite the p38 MAPK-MK2 pathway having been previously implicated in TTP CIM phosphorylation and inhibition of TTP deadenylase association , 43, 44.14–28–pcDNA3-FLAG-MS2 plasmids expressing FLAG-MS2-tagged TTP mutant proteins  were genHeLa Tet-off, HEK293T, RAW264.7, and NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. For experiments shown in Rabbit polyclonal anti-pS316-TTP antibody was generated with the synthesized oligo-peptide PRRLPIFNRI(p)SVSE . The antiserum was purified by affinity chromatography by first collecting antibody binding to a PRRLPIFNRI(p)SVSE-peptide column, and, after elution, collecting the flow-through from a subsequent unphosphorylated PRRLPIFNRISVSE-peptide column. Immunoblots were probed with the following antibodies at the indicated concentrations: rabbit polyclonal anti-FLAG , mouse monoclonal anti-FLAG M2 , rabbit polyclonal anti-DDX6 , rabbit polyclonal anti-GIGYF2 , mouse monoclonal anti-14-3-3 , rabbit polyclonal anti-TTP , rabbit polyclonal anti-CNOT1 , rabbit polyclonal anti-CNOT9 , mouse monoclonal anti-GAPDH , rabbit polyclonal anti-EDC4 ((g for 15 min at 4°C and supernatants were added to 50 μL of M2 anti-FLAG-agarose beads (Sigma) and nutated for 2 h at 4°C. Beads were washed 4 times with NET-2  and resuspended in 50 μL of 2x Laemmli sample lysis buffer . Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting. 0.5% of whole cell extracts and 20% of pull-down elutions were loaded for analyses.Co-immunoprecipitation assays were performed as previously described . Briefly20 at 80°C for 2 min followed by annealing at room temperature for 5 min. 7 μL of water, 2 μL of 10x RNase H Buffer  and 1 μL of 5 units/μL RNase H (NEB) were added and samples were incubated at 37°C for 30 min. RNA samples were extracted with phenol:chloroform (1:1), followed by ethanol precipitation, and analyzed by Northern blotting. Half-lives were determined as previously described  and β-globin_Control_R: TCTTCTGGGTGGCAGTGATG.Three micrograms of RNA, isolated using TRIzol (Invitrogen) according to the manufacturer’s protocol, were incubated with three units of RQ1 RNase-free DNase (Promega) at 37°C for 30 min. RNA was subsequently extracted with phenol:chloroform:isoamyl alcohol (50:49:1), ethanol precipitated, washed with 70% ethanol and dissolved in 10 μL of water . Two hundred nanograms of DNase-treated RNA was subsequently reverse transcribed using random hexamers with Superscript III reverse transcriptase according to the manufacturer’s protocol (Invitrogen). The corresponding cDNA generated was used for qPCR quantification using Fast SYBR green Master Mix (Applied Biosystems) on a StepOnePlus System (Applied Biosystems). qPCR primer-pair efficiency (E) was calculated by plotting log


Cassia tora, exhibits several biological activities, including the amelioration of memory impairment, prevention of breast cancer metastasis, and reduction of cartilage damage in osteoarthritis. We aimed to evaluate the inhibitory effects of obtusifolin and its analogs on CYP1A enzymes, which are responsible for activating procarcinogens, and investigate its inhibitory mechanism and chemopreventive effects. P450-selective substrates were incubated with human liver microsomes (HLMs) or recombinant CYP1A1 and CYP1A2 in the presence of obtusifolin and its four analogs. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. Molecular docking simulations were performed using the crystal structure of CYP1A2 to identify the critical interactions between anthraquinones and human CYP1A2. Obtusifolin potently and selectively inhibited CYP1A2-mediated phenacetin O-deethylation (POD) with a Ki value of 0.031 µM in a competitive inhibitory manner in HLMs, whereas it exhibited negligible inhibitory effect against other P450s (IC50 > 28.6 µM). Obtusifolin also inhibited CYP1A1- and CYP1A2-mediated POD and ethoxyresorufin O-deethylation with IC50 values of <0.57 µM when using recombinant enzymes. Our molecular docking models suggested that the high CYP1A2 inhibitory activity of obtusifolin may be attributed to the combination of hydrophobic interactions and hydrogen bonding. This is the first report of selective and potent inhibitory effects of obtusifolin against CYP1A, indicating their potential chemopreventive effects.Obtusifolin, a major anthraquinone component present in the seeds of  Cassiae semen (the seeds of Cassia obtusifolia L. and Cassia tora L.) is used as a traditional herbal medicine to protect the liver, improve vision, and treat photophobia in most regions of Asia, including Korea, China, and Japan [Cassiae semen [Cassiae semen [Cassia tora [nd Japan . Numerouae semen . Among tae semen ,3,4. Obtsia tora , preventsia tora , and redsia tora .O-deethylation (POD) activity with IC50 values of 0.5, 2.3, and 1.3 µM, respectively, in human liver microsomes (HLMs); at the same time, these exhibited negligible inhibition of CYP2C9, CYP2C19, CYP2D6, and CYP3A (IC50 > 9.6 µM) [Cassiae semen, on cytochrome P450 (P450) have not been investigated in HLMs to date.Anthraquinones are a class of natural phenolic compounds with pharmacological properties, including anticancer, anti-inflammatory, antimicrobial, and antioxidant activities . Several 9.6 µM) . HoweverIn humans, the CYP1A family is composed of two major isoforms, namely CYP1A1 and CYP1A2 . CYP1A e50 values of <0.50 µM [Cassiae seeds, is also a planar aromatic compound; however, enzyme inhibition studies assessing the effect of obtusifolin on CYP1A activity have not been attempted.CYP1A inhibitors share planar and compact structure with hydrophobic aromatic rings. For example, α-naphthoflavone (ANF), emodin, and 7-ethynylcoumarins, which are planar aromatic compounds, strongly inhibited CYP1A1 and CYP1A2 activities with IC<0.50 µM ,16,17. OCassiae semen, in HLMs. The difference in CYP1A2 inhibitory ability of the five phytochemicals was analyzed based on the interaction between the active site structure of the CYP1A2 enzyme and phytochemicals. An inhibition mechanism was identified for obtusifolin, which exhibited the most substantial inhibition of CYP1A2 activity; the inhibitory potential of obtusifolin against other nine P450 isoforms was also investigated to elucidate its selectivity in terms of CYP1A2 inhibition. In conclusion, we have demonstrated that obtusifolin acts as a selective CYP1A1 and CYP1A2 inhibitor in HLMs and also inhibits the activity of recombinant CYP1A enzymes, and is a promising chemopreventive candidate.Therefore, we evaluated the CYP1A2 inhibitory potential of obtusifolin, chrysophanol, emodin, physcion, and rubrofusarin, which are the major components of +), omeprazole, phenacetin, and trimipramine. Coumarin, midazolam, and tolbutamide were obtained from Toronto Research Chemicals . We purchased resorufin from Tokyo Chemical Industry . Pooled human liver microsomes  were obtained from XenoTech , and human recombinant CYP1A1 (rCYP1A1) and CYP1A2 (rCYP1A2) isoforms were obtained from SPMED . Chrysophanol (95.0% purity), emodin (94.3% purity), obtusifolin (95.4% purity), physcion (95.0% purity), and rubrofusarin (90.0% purity) : amodiaquine, bupropion, chlorzoxazone, dextromethorphan, 7-ethoxyresorufin, glucose-6-phosphate (G6P), G6P dehydrogenase (G6PDH), β-nicotinamide adenine dinucleotide phosphate (NADP purity)  were kinCassiae semen against CYP1A2-mediated phenacetin O-deethylase (POD) activity was evaluated using previously developed methods with minor modifications [+, 3.3 mM G6P, 1.0 U/mL G6PDH, and 3.3 mM magnesium chloride) was introduced after pre-incubation to initiate the reaction, and further incubated for 15 min. The reaction was quenched by the addition of 50 µL ice-cold acetonitrile containing internal standard . Aliquots of supernatants were analyzed by liquid chromatography–tandem mass spectrometry . All microsomal incubations were repeated three times.The inhibitory potential of five major bioactive compounds from ications . BrieflyCassiae semen (three anthraquinones and one naphthopyrone) were also docked by aligning to the obtusifolin-binding pose (“Align to substructure” option in DSC v19.1) to understand the structural and molecular interactions responsible for the variations in their inhibitory activity against CYP1A2. For the docked CYP1A2-ligand complexes, AutoDock VINA scores were calculated using the “score_only” option in Gnina 1.0. The molecular docking procedure was validated by re-docking α-naphthoflavone in the crystal structure; the rmsd value of the docked α-naphthoflavone and its crystal form was 0.3 Å, showing a high agreement  under accession number 2HI4 . The progreement . The fig50 shift approach was applied to assess the time-dependent inhibition of CYP1A2 by obtusifolin [The ICusifolin . For 30 The mechanism by which obtusifolin and α-naphthoflavone inhibit CYP1A2-mediated POD activity was studied in pHLMs or recombinant CYP1A2 (10 pmol/mL). Obtusifolin  or α-naphthoflavone  was added to the reaction mixtures, each containing varied concentration of phenacetin . The other conditions were similar to those used in the P450 inhibition study, as described earlier.The inhibitory potential of obtusifolin against the biotransformation of eight P450 probe substrates was examined using previously reported methods with slight changes ,22. The O-deethylation (EROD) activity of rCYP1A1 and rCYP1A2 enzymes (10 pmol/mL) were investigated. Obtusifolin  was added to the reaction mixture containing 7-ethoxyresorufin (2 μM). The other conditions, except for the incubation time (20 min), were similar to those used in the P450 inhibition study, as described earlier.The inhibitory effects of obtusifolin on the CYP1A-mediated ethoxyresorufin m/z 152 > 110 for acetaminophen (CYP1A2), m/z 214 > 186 for resorufin (CYP1A1 and CYP1A2), m/z 163 > 107 for 7-hydroxycoumarin (CYP2A6), m/z 256 > 238 for 6-hydroxybupropion (CYP2B6), m/z 328 > 283 for N-desethylamodiaquine (CYP2C8), m/z 287 > 89 for 5-hydroxytolbutamide (CYP2C9), m/z 362 > 214 for 4-hydroxyomeprazole (CYP2C9), m/z 258 > 157 for dextrorphan (CYP2D6), m/z 184 > 120 for 6-hydroxychlorzoxazone (CYP2E1), m/z 342 > 203 for 1′-hydroxymidazolam (CYP3A), and m/z 295 > 100 for trimipramine (IS) [Reverse phase column  and triple-quadrupole mass spectrometer  with an ultra high-performance LC system  analyze all the metabolites. Mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The elution condition was as follows: 0–0.5 min (B: 8%), 0.5–5 min (B: 8%→60%), 5–6 min (B: 60%), 6–6.1 min (B: 60%→8%), and 6.1–10 min (B: 8%) for the analysis of metabolites of the nine P450 substrates; and 0–1 min (B: 5%), 1–5 min (B: 5%→90%), and 5.1–7 min (B: 90%→5%) for the analysis of resorufin. Electrospray ionization was performed in the positive- and negative-ion modes at 4.0 and −3.5 kV, respectively. For each metabolite, quantitation was carried out in selected reaction monitoring (SRM) mode using the following precursor-to-product ion transition: ine (IS) .50 values. Based on Lineweaver–Burk double reciprocal plots, secondary plots of Lineweaver–Burk plots versus obtusifolin concentrations, and visual inspection of Dixon plots, the type of inhibition and apparent kinetic parameters for inhibitory activity (Ki) were estimated.WinNonlin software  was used to calculate ICKi value of 62 µM in rat liver microsomes [50 = 0.5 µM) than the activities of other P450 isoforms (IC50 > 9.6 µM) in HLMs [Cassiae semen in HLMs (50 < 2.2 µM) more strongly inhibited CYP1A2 activity than naphthopyrone (IC50 > 6 µM). Among the four anthraquinones, obtusifolin (IC50 = 0.19 µM) was found to be the strongest inhibitor of CYP1A2 activity (50 = 1.56 µM), isopimpinellin (IC50 = 0.46 µM), and trioxsalen (IC50 = 0.79 µM) [50 = 0.79 µM) of emodin against CYP1A2 in HLMs was observed to be similar to the previously reported value (IC50 = 0.50 µM) [50 = 1.99 µM) and physcion (IC50 = 2.16 µM) against CYP1A2 in HLMs were higher than those observed against rCYP1A2  [50 value of mollugin for CYP1A2-mediated POD activity in rCYP1A2 was five-fold higher than that in HLMs [In the previous study, Tang et al. (2009) reported that rhein, an anthraquinone in rhubarb root, inhibited CYP1A2-catalyzed POD activity with a crosomes . Liu et  in HLMs . Based o in HLMs . The amo in HLMs . Anthraqactivity . The inh0.79 µM) ,26. The 0.50 µM) . The inhIC50 = 2. µM againIC50 = 2. µM againctively) . For exa in HLMs .Cassiae semen and human CYP1A2 from the perspective of protein–ligand interactions and to clarify the mechanism behind the enhanced CYP1A2 inhibitory activity of obtusifolin compared with other anthraquinones, molecular docking simulations were performed using a previously reported crystal structure of CYP1A2 (PDB ID: 2HI4) [To explore the critical interactions between anthraquinones from D: 2HI4) . As showThe potential critical interactions between anthraquinones and CYP1A2 enzyme were comprehensively analyzed using the docking complex model. Obtusifolin binds to the CYP1A2 active site with the lowest docking score of −12.78 kcal/mol . As showConversely, emodin was the second-best CYP1A2 inhibitor among the examined compounds. Its hydroxyl group interaction with heme, along with hydrophobic contacts, contributed to a high CYP1A2 inhibitory activity . ChrysopAs observed in rubrofusarin and physcion, the presence of bulky methoxy groups in disadvantageous positions in the molecules may result in steric hindrance with Leu382, which could result in inefficient CYP1A2 inhibition . HoweverIn summary, the docking models suggest that the high inhibitory activity of obtusifolin against CYP1A2 primarily results due to the combination of extensive hydrophobic interactions with three Phe residues  via its methyl group and the non-classical hydrogen bond with Asp313 via the methoxy group . The met50 values of 0.19 μM, therefore, we sought to clarify the underlying mechanism of this inhibition. We measured the inhibition constant of α-naphthoflavone, a well-known CYP1A2 selective inhibitor, to validate our experimental system. α-Naphthoflavone inhibited CYP1A2-mediated POD activity with a Ki value of 0.0075 μM in HLMs. Our results with α-naphthoflavone are consistent with that of a previous study, which reported potent inhibition of CYP1A2 activity in HLMs by α-naphthoflavone with a Ki value of 0.01 μM using phenacetin as the CYP1A2 probe substrate [Ki value of 0.11 μM (2R = 0.997). The Dixon plot intersected above the X-axis, indicating that obtusifolin inhibited CYP1A2 in a competitive manner (Ki = 0.71 μM) [Ki = 1.2 μM) [Ki = 3.74 μM) [Ki = 0.01 μM), a well-known strong selective inhibitor of CYP1A2 [Ki value of 0.21 μM, which was similar to the value observed in HLMs  , isopimp 1.2 μM) , and mol3.74 μM) , but wasf CYP1A2 . Obtusif in HLMs .50 values of obtusifolin against CYP1A2. We found that obtusifolin showed time-independent inhibition of CYP1A2-mediated POD activity with an IC50 shift value of 1.46 . A chemical with an IC50 fold-shift decrease of <1.50 is considered a time-independent inhibitor [50 shift value of 0.83.In addition, several CYP1A2 inhibitors, including antofloxacin , furafylnhibitor . α-Naphtnhibitor , also exIn this study, we used in vitro experimental system such as human liver microsomes and recombinant P450 isoforms for enzyme inhibition studies. Since these systems have a limitation in that they can not reflect the exact intracellular concentration of the test compounds, it will be necessary to conduct enzyme inhibition studies using cultured cells, such as human hepatocytes.O-deethylation (IC50 = 0.19 µM). To evaluate whether obtusifolin selectively inhibits CYP1A2, we investigated its inhibitory activity against eight other P450 isoforms. Obtusifolin showed weak inhibition of CYP2C8 and CYP2C9, with IC50 values of 31.4 and 28.6 µM, respectively, and negligible inhibition (IC50 > 50 µM) of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP2E1, and CYP3A activities  at 0.5 µM concentration  [50 = 0.74 µM) [50 = 0.06 µM), a well-known strong inhibitor of CYP1A1 [50 = 0.06 µM) over rCYP1A2 (IC50 = 0.37 µM)   and curc0.74 µM) , which a0.37 µM) . Its selmax/Ki exceeded 1.0, and would be possible if it was between 0.1 and 1.0 [max/Ki were 7.82 and 4.91 from the data of pHLMs (Ki = 0.11 μM), suggesting that obtusifolin has possible drug interaction potential with CYP1A2 substrate drugs. Thus far, there have been no reports on the pharmacokinetics of obtusifolin in humans, therefore, it is difficult to estimate the drug interaction potential of obtusifolin for humans. However, rhein, which is one of the anthraquinone compounds like obtusifolin, has been reported to reach a maximum plasma concentration of 9.52 and 18.8 μM after oral dose of 50 and 100 mg, respectively, in humans [It was estimated that an in vivo interaction potential via the inhibition of P450 would likely occur if the ratio of inhibitor C and 1.0 . Based o and 1.0  and obtu and 1.0 , the resn humans . Therefon humans , olanzapn humans . TherefoCassiae semen on the activity of CYP1A2 isoform in HLMs. Among five compounds tested, obtusifolin potently inhibited CYP1A2-mediated POD activity, with Ki values lower than 0.5 µM in HLMs and rCYP1A2. Furthermore, obtusifolin selectively inhibited CYP1A1 and CYP1A2 enzymes; however, it had negligible inhibitory effects on other P450 isoforms, highlighting its potential chemopreventive effects. In conclusion, we confirmed the selective and potent inhibitory effects of obtusifolin against CYP1A enzymes in HLMs and recombinant CYP1A enzymes.In this study, we investigated the inhibitory potential of four anthraquinones and one naphthopyrone isolated from


Chronic rhinosinusitis (CRS) is an inflammatory disease that may require biological therapy. Omalizumab is an anti-IgE biologic that was recently approved by the FDA and Health Canada for use in severe CRS with nasal polyps (CRSwNP) recalcitrant to intranasal corticosteroids. Dosing is based on weight and pre-treatment serum IgE, with elevated levels of the latter being an indication for biologic treatment according to EPOS and EUFOREA guidelines. The goal of this study was to identify variables that predict IgE-high type 2 inflammation and serve as indicators for biologic treatment in CRS..Patients ≥ 19 yo diagnosed with CRS undergoing functional endoscopic sinus surgery were included retrospectively. Demographics, past medical history, preoperative blood work, Lund-Mackay (LM), Lund Kennedy (LK), and SNOT-22 scores were extracted. Descriptive statistics and binary logistic regression analyses were conducted. Model superiority was based on Nagelkerke R2 scores and receiver operating characteristic curvesSixty-five patients, average age 49.96 ± 13.59 years, were included. Sixty-one binary logistic regression models for elevated serum IgE were created. Among the top 3 models, the best model had sensitivity, specificity, positive predictive value and negative predictive values of 82.1, 69.2, 80.0, and 72.0. All performance measures except sensitivity exceeded the Canadian Biologics Guideline model. Serum eosinophils ≥ 300 cell/uL, CRSwNP and LM ≥ 17 increased the odds of elevated IgE.IgE-high type-2 inflammation can be predicted by a model that includes eosinophil ≥ 300 cell/uL, CRSwNP, LM ≥ 17, asthma diagnosis and SNOT-22 ≥ 40. Patients meeting these parameters have a high pretest probability for elevated IgE and would benefit from IgE serology to determine qualification for omalizumab. This could reduce unwarranted IgE serology in patients with CRSwNP but also target a patient population for further workup that will lead to optimization of resource allocation and improve healthcare equity in rural and remote areas within Canada. Chronic rhinosinusitis (CRS) is an upper airway inflammatory disease of the sinuses and nasal cavity that affects 5.2% of the Canadian population . This diCRS management utilizes pharmaceutical and surgical interventions to reduce symptoms and complications . A subseThese biological therapies seek to control the disease by mitigating the type 2 inflammatory response by targeting its respective cytokines and antibodies. Currently, biologic treatments have been and are being approved for the management of CRSwNP, which is phenotypically type 2 disease . These iThe cost-effectiveness of these biologics is important to consider, especially in Canada’s single payee healthcare system. The annual cost of dupilumab is $25,909 CAD for year one and $24,949 CAD for subsequent years . SimilarA baseline IgE level is an essential tool for dosing omalizumab. This may be a detriment to Canadian rural and remote hospitals which have access to basic blood work  but not IgE serology. The latter requires blood samples to be sent to the laboratory at the nearest tertiary centre. This distance can be considerable in Canada due to its population density of 3.9 people per square kilometer with 66% of the population and most tertiary centres being located within 100 km of the United States border . Shippinst, 2017 and December 31st, 2019. Patient data were collected and stored on University of British Columbia (UBC) Research Electronic Data Capture (REDCap) . Ad. Ad33]. Results from the binary logistic regression analysis for elevated serum IgE also provided insight into the performance of the top 3 models. Model 3 had the highest sensitivity (82.1%) and NPV (72%). Model 5 had the highest specificity (73.1%) and PPV (81.5%) Table . Model 1p = 0.004) was the only variable to significantly contribute to the model.Model 3  was determined to be the best model for predicting elevated serum IgE based on Nagelkerke R2 scores and AUC values. Amongst these 5 included variables only CRSwNP, Eosinophilia and Lund McKay increased the odds of elevated serum IgE with odds ratio values of 1.905, 7.477 and 2.038 respectively , patient weight (kg) and serum IgE levels are required to determine omalizumab dosing . The IgEThis algorithm could be seamlessly integrated into the care of patients with CRS who are recalcitrant to surgery and medical therapy as most variables are ordered preoperatively or during medical therapy . If the There could be additional benefits to this approach, however, more research is required to substantiate these assertions. One, understanding which patients have elevated serum IgE levels could indicate if they are to respond better or worse to omalizumab treatment. Phase 3 randomized studies–POLYP 1 and 2–prescribed omalizumab at doses that were based on weight (kg) and baseline IgE levels ranging from 72 to 3120 ng/mL. It is intuitive to believe that patients with elevated serum IgE are more likely to have a significant change in objective treatment outcomes due to the mechanism of action of omalizumab. Currently, there is one omalizumab study that demonstrated no significant impact of baseline serum IgE levels on the nasal polyp size and SNOT-22 score . HoweverFor instance, current Canadian biologic guidelines indicate that “patients with CRSwNP do not need another Type 2 inflammatory condition such as asthma to be considered for biologic therapy” . This stThis study had limitations. First, age was recorded in this study but not included in statistical analysis because it was difficult to dichotomize this variable into high and low due to the stratiform pattern that arises when graphing type 2 inflammatory marker concentration to age in patients with type 2 CRSwNP . This omCRS is a chronic and debilitating inflammatory disease that can greatly reduce a patient’s quality of life. IgE high type 2 CRS can be accurately predicted by a biomarker model consisting of eosinophils ≥ 300 cell/uL, CRSwNP, LM ≥ 17, comorbid asthma and SNOT-22 ≥ 40. Utilizing this model to triage IgE serology could improve resource allocation and reduce social costs. Additional high-quality research is required to determine if baseline serum IgE levels impact the efficacy of omalizumab.