Although most patients who undergo transforaminal lumbar interbody fusion (TLIF) show favorable surgical results, some still have unfavorable results for various reasons. This study aimed to investigate the influence of differences in lumbar lordosis (LL) between the standing and supine positions (DiLL: supine LL–standing LL) on minimum 5-year surgical outcomes after short-segment TLIF. Ninety-one patients with lumbar degenerative disease who underwent short-segment TLIF (1–2 levels) were categorized based on preoperative differences in LL as DiLL (+) and DiLL (−). Comparison and correlation analyses were performed. The incidence of adjacent segment disease (ASD) by radiology (R-ASD) and symptomatic ASD (S-ASD), bony fusion rates, and pre- and postoperative clinical scores ; and Nakai’s score) were evaluated. Postoperatively, VAS for low back pain (LBP) in the sitting position, JOA scores for LBP, lower leg pain, intermittent claudication, ODI, and Nakai’s score were significantly worse in the DiLL (+) group than in the DiLL (−) group. DiLL values were significantly correlated with VAS for LBP, ODI, and Nakai’s score, postoperatively. Positive DiLL values were associated with poorer postoperative outcomes. DiLL is a simple and useful method for predicting mid-term outcomes after TLIF. Although most patients who undergo TLIF show favorable surgical results, some patients still have unfavorable results for various reasons such as residual low back and lower-extremity symptoms, adjacent segment disease (ASD), pseudoarthrosis, sagittal malalignment, and surgical complications8. Accordingly, several studies have been conducted to identify the predictive factors of clinical outcomes after TLIF12. Predictors of clinical outcomes after TLIFremain controversial; thus, further research is required to establish a consensus.Lumbar spinal fusion surgery is a common treatment for various pathologies of the lumbar spine. Transforaminal lumbar interbody fusion (TLIF) is a standard lumbar spinal fusion procedure used to treat degenerative lumbar diseases13. The report concluded that patients with higher preoperative DiLL values tended to show worse postoperative residual symptoms such as lower extremity symptoms, low back pain (LBP) upon standing, and gait disturbance 2 years after TLIF13. However, mid- to long-term postoperative clinical outcomes relative to the preoperative DiLL have not been reported. Therefore, we conducted a retrospective study to evaluate the minimum 5-year clinical outcomes for patients with lumbar degenerative disease who underwent short-segment TLIF. This study aimed to examine how preoperative DiLL influences the incidences of ASD, bony fusion rate, and preoperative and 5-year postoperative clinical outcomes.The preoperative postural difference in lumbar lordosis (LL) between the standing and supine positions (DiLL) has recently been reported to be correlated with short-term postoperative clinical outcomes after single-level TLIF9  were collected. All patients provided informed consent prior to the surgery. The clinical records of consecutive patients with lumbar degenerative disease including lumbar degenerative spondylolisthesis, lumbar foraminal stenosis, spondylolytic spondylolisthesis, lateral lumbar disc herniation, lumbar spinal stenosis, and lumbar facet joint cysts, who underwent short-segment TLIF9 Fig.  at one o15. Postoperative symptomatic ASD (S-ASD) was diagnosed when spinal canal stenosis, foraminal stenosis, disc herniation, or segmental kyphosis (> 5°) was present at segments adjacent to the operated segments and low back or lower leg symptoms were obviously caused by ASD. Patients were considered to have S-ASD when the symptoms persisted for at least three months and required additional treatment, including medications, epidural blockades, or subsequent surgeries. Bony fusion was evaluated using radiographs and computed tomography (CT) images 1 year postoperatively, and the evaluation was performed repeatedly until bony fusion was confirmed or until the final follow-up. Fusion was defined as (i) the presence of continuous trabecular bone formation through or outside the cages, (ii) < 3° movement on lateral flexion and extension radiographs, and (iii) the absence of radiolucent lines of > 50% of the implant19. In patients with two-level fusion, we considered that fusion was achieved when both levels achieved bony fusion. Clinical outcomes were assessed using the (1) visual analog scale (VAS) for LBP, lower-extremity pain and numbness between 0 (no pain) and 10 ; (2) our originally developed detailed VAS scoring system for LBP in motion, standing position, and sitting position20; (3) Japanese Orthopaedic Association (JOA) scores for LBP, lower-extremity pain, and intermittent claudication  of both cranial and caudal adjacent discs was evaluated at the 5-year follow-up. When there was slippage progression of > 3 mm, a posterior opening of > 5°, and a narrowing of the disc height of > 3 mm in comparison with preoperative flexion–extension lateral radiographs, patients were considered to have R-ASD, as described in previous studiesLumbopelvic parameters, such as LL (the angle between the superior endplates of L1 and S1) and pelvic incidence  were measured using preoperative lateral radiographs obtained with the patient in the standing position , and those with DiLL < 0° were defined as DiLL (−) Fig. . The patCorrelation analysis was performed between the preoperative DiLL and R-ASD, S-ASD, and bony fusion status at 1 and 5 years, postoperatively, and each clinical score to examine the association between preoperative DiLL and postoperative clinical outcomes. To exclude the influence of age, sex, BMI, presence of scoliosis, and the number of fused segments, multiple regression analysis was performed after appropriate adjustments.To examine the influence of DiLL on surgical outcomes in patients with or without PI-LL mismatch, patients were divided into two groups based on preoperative standing radiographs: mismatched (PI-LL > 10°) and matched (PI-LL ≤ 10°) subgroups. Clinical outcomes were compared between DiLL (+) and DiLL (−) patients in each group (mismatched and matched).Continuous data are presented as the mean ± standard deviation, and categorical variables are presented as numbers. The age, BMI, lumbopelvic parameters, VAS, and ODI of the two groups were compared using an unpaired t-test. Pearson’s chi-square test (or Yates’ chi-square test when any expected frequencies were < 5) was used to compare sex differences and percentages of patients with scoliosis, single-level fusion, R-ASD, S-ASD, and bony fusion rates. The Mann–Whitney U test was used to compare JOA and Nakai’s scores. To investigate the relative influence of preoperative DiLL on pre and postoperative clinical outcomes, such as VAS, JOA scores, ODI, and Nakai’s score, a multiple regression analysis was performed after adjustment for age, sex, BMI, presence of scoliosis, and number of fused segments, with DiLL as the independent variable and each clinical outcome as the dependent variable. Statistical significance was set at p < 0.05.The study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the institutional review board of Toho University Sakura Medical Center. (No. 2012–071).All patients provided informed consent prior to surgery.The DiLL (+) group included 48 patients and the DiLL (−) group included 43 patients Table . The DiLThere was no significant difference in the percentage of patients with postoperative R-ASD between the DiLL (+) and DiLL (−) groups. In the DiLL (+) group, 11 patients 22.9%) had postoperative S-ASD , similar results to the above-mentioned analysis were obtained regarding the difference in clinical outcomes between the DiLL (+) and DiLL (−) group Table . PatientThe correlation between the preoperative DiLL and postoperative outcomes was evaluated using multiple regression analysis Table . After aIn the PI-LL mismatched patients, postoperative S-ASD was more likely to occur in the DiLL (+) group (9/38 patients) than in the DiLL (−) group (0/14 patients); however, the difference was not significant  and DiLL (−) groups. In addition, no significant differences in VASs, JOA, ODI, and Nakai’s scores were found between the DiLL (+) and DiLL (−) groups Table .Mid-term clinical data of patients treated with short-segment TLIF were analyzed, and preoperative DiLL was found to be useful for predicting mid-term clinical outcomes. Our results suggest that patients with DiLL (+) tend to show worse postoperative outcomes,with regard to LBP, lower-extremity pain, and gait disturbance. This tendency was more proounced in patients with PI-LL mismatch. In addition, there is a possibility that patients with DiLL (+) more frequently experienced S-ASD after short-segment TLIF, although the difference was not statistically significant.23. Chevillotte et al. examined the lumbopelvic parameters of asymptomatic volunteers in standing and supine positions and reported that the mean LL values were greater in the standing position (54.8°) than in the supine position (50.2°)23. Park et al. examined patients with lumbar degenerative disease and found that the majority of them showed greater LL values in the standing position than in the supine position22. From these observations, the LL value in the standing position is typically slightly greater than that in the supine position, indicating that a normal DiLL is negative. Therefore, a positive DiLL value is considered abnormal. In the present study, we found that patients with DiLL (+) showed significantly smaller supine LL and standing LL and greater PI-LL and scoliotic curvature than patients with DiLL (−). However, the PI and lumbar flexibility were not significantly different between the two groups.Previously, a difference in LL depending on posture was reported24  or avoidance of pain-inducing postures26. Generally, neural element compression is relieved by lumbar flexion in patients with spinal stenosis. Thus, patients may develop a temporary sagittal plane deformity as compensation for neurogenic claudication28 These observations suggest that patients with positive DiLL have substantial dysfunction of the lumbar spine or severe neuropathic pain due to lumbar spinal stenosis. In contrast, this study revealed that the number of patients with scoliosis, a structural disorder of the lumbar spine, was significantly higher in the DiLL (+) group than in DiLL (−) group, suggesting that some patients in the DiLL (+) group had structural disorders. Functional and/or structural disorders may explain why the clinical outcomes after short-segment fusion surgery are expected to be unfavorable.Patients with a positive DiLL may have functional disorders in maintaining the lumbar lordotic angle in the standing position and may not be able to maintain a normal standing position because of the dysfunction of supportive spinal tissues10. Ohyama et al. reported that DiLL is associated with short-term surgical outcomes after TLIF13. Moreover, the results of this study revealed that the mid-term clinical outcomes were significantly correlated with DiLL. In addition, after excluding patients with scoliosis, this study confirmed that DiLL is associated with postoperative clinical outcomes29. The subgroup analysis in this study revealed that the difference between patients with DiLL (+) and those with DiLL (−) was more evident in patients with PI-LL mismatch (PI-LL > 10°). Our results suggest that a combination of DiLL (+) and PI-LL > 10° is a strong predictor of worse mid-term outcomes, bucause both DiLL and PI-LL are related to lumbopelvic alignment. However, the two parameters have different implications. PI-LL is a static factor whereas DiLL is a dynamic factor that may reflect patients’ lumbar spinal function. In patients treated with long-segment spinal fusion surgery of the thoracic and lumbar spine, the lumbar spine is completely fused after surgery and dynamic factors may not be related to postoperative outcomes. In these patients, surgical outcomes may only be related to static factors including PI-LL31. We believe that the dynamic factor DiLL has a significant influence on surgical outcomes after short-segment fusion as some segments are not fused. It is reasonable to assume that the functional status of the non-fused segments influences surgical outcomes. Therefore, surgical results after short-segment TLIF are associated with both DiLL and PI-LL.Previous studies have reported that a higher PI-LL value is associated with worse postoperative clinical outcomes and higher incidence of ASD after lumbar fusion surgery33; therefore, spine surgeons should carefully choose the shape of fusion cages to increase segmental lordosis at the operated level in the treatment of DiLL (+) mismatched patients with short segment fusion surgery. Additionally, this will be helpful when DiLL (+) mismatched patients require revision surgery, such as kyphosis correction, after short-segment fusion.Currently, we cannot conclude that DiLL (+) mismatched patients should be treated with long-segment fusion surgery. However, spine surgeons should be aware that unfavorable surgical outcomes are expected after short-segment fusion in DiLL (+) mismatched patients and should carefully evaluate whether long-segment fusion surgery is more appropriate in such patients. Surgical outcome after short-segment lumbar fusion are associated with postoperative restoration of segmental lordosis26. Our study results suggest that the pathology of DiLL (+) mismatched patients is different from that of DiLL (−) mismatched patient. Patients with DiLL (+) mismatch may have severe functional or neurological disorders worsened by lumbar extension, both of which may prevent them from maintaining LL in the standing position. Lumbar extension may be limited in patients with DiLL (−) mismatch due to structural factors such as facet joint contractures, spinous process impingements, or hyperostosis, but not due to functional disorders. The difference in the surgical results may be due to the difference in the pathologies of DiLL (+)and DiLL (−) mismatches. We believe that DiLL (+) mismatch is mainly due to functional factors and partly due to structural factors in some patients, whereas DiLL (−) mismatch is mainly due to structural factors. In this study, both R-ASD and S-ASD increased in DiLL (+) patients, although the difference was not statistically significant. The increase in ASD in the DiLL (+) group may be partly explained by the finding that the number of patients with scoliosis was higher in the DiLL (+) group. However, the DiLL (+) group showed a non-significant tendency towards a higher incidence of S-ASD (p = 0.091), even when the analysis was limited to patients without scoliosis. At this moment, the possibility that functional disorders of the lumbar spine may increase the incidence of ASD in patients with DiLL (+) cannot be ruled out. Regarding bony fusion, favorable fusion rate can be expected after short segment-TLIF, regardless of preoperative DiLL value, as well as PI-LL value.Patients with a PI-LL > 10° were considered to have lumbar sagittal spinal malalignment34, the two values could not be used in the multiple regression analysis to avoid multicollinearity. However, this study revealed that the difference in outcomes between patients with DiLL (+) and those with DiLL (−) was more evident in patients with PI-LL mismatches. These results indicate that the combination of DiLL and PI-LL is a strong predictor of the postoperative course after short-segment TLIF. Regarding scoliotic curvature, a comparison analysis between the DiLL (+) and DiLL (−) groups was performed after excluding patients with scoliotic curavature > 10°. It revealed significant differences in postoperative outcomes between the two groups. This result indicates that DiLL is an independent factor predictor of postoperative outcomes irrespective of scoliosis. Third, the number of patients included in the study was limited. However, this study successfully demonstrated a significant difference in clinical outcomes between the DiLL (+) and DiLL (−) groups. The incidence of postoperative S-ASD was higher in the DiLL (+) group; however, the difference was not statistically significant. Moreover, when performing comparative analysis by dividing patients into four groups by DiLL and PI-LL, the number of patients in each group was limited, suggesting that the analysis was underpowered. Future studies with more patients are warranted to more precisely investigate the incidence of S-ASD between the groups and the influence of a combination of DiLL and PI-LL on the postoperative outcomes. Fourth, there are no background data supporting the appropriateness of classifying patients into DiLL (+) and DiLL (−) groups because DiLL is a newly proposed lumbopelvic parameter13. In the future, further studies are needed to clarify the cut-off value for DiLL for classifying patients with normal or abnormal lumbar function.This study has several limitations. First, the sagittal vertical axis, an important factor influencing patient status, was not evaluated. However, whole-spine radiographs are not always obtained when patients are treated with short-segment TLIF. We believe that a simple factor that require no additional radiological examination is ideal for clinical use. Even when CT is not performed preoperatively, DiLL can be evaluated using supine radiography or magnetic resonance imaging. Therefore, DiLL is ideal for predicting postoperative outcomes. Second, there were significant differences in patient age, preoperative PI-LL, and scoliotic curvature between groups. Multiple regression analysis was performed to exclude the influence of age, sex, BMI, scoliosis, and number of fused segments and a significant correlation was found between DiLL and each clinical score. As DiLL and PI-LL were found to be significantly correlatedIn conclusion, higher DiLL values were correlated with poorer patient outcomes after TLIF. Poor outcomes were expected in DiLL (+) patients, particularly in those with preoperative PI-LL mismatch (PI-LL > 10°) Fig. . DiLL is


Intracardiac echocardiography (ICE) use can lead to early detection of periprocedural complications and may improve patient outcomes by providing real‐time visualization of catheter location and the treatment area during cardiac ablation (CA) for atrial fibrillation (AF).Examine complications and 12‐month healthcare use among patients with AF undergoing CA with versus without ICE use during the procedure in a real‐world setting.The 2015–2020 IBM MarketScan® Database was used to identify non‐elderly adults (age 18–64 years) undergoing CA for AF. Patients were classified into ICE/non‐ICE groups based on the presence or absence of ICE procedure codes. Patients in each group were matched on study covariates using propensity scores. Peri‐procedural complications, 12‐month cardiovascular (CV) or AF‐related inpatient admission, repeat CA, and cardioversion were compared using a Cox proportional hazard model.p < .001). The risk of complications was 50% lower with ICE use . For assessment of 12‐month healthcare utilization, 1250 patients were identified in each cohort after propensity matching. ICE use was associated with a 36% lower risk of 12‐month repeat ablation . No differences in CV‐ or AF‐related inpatient admission and cardioversion were observed.1371 patients were identified in each study cohort (ICE and non‐ICE) after propensity matching. Patients who had CA with ICE had a significantly lower rate of complications than those without (2.9% vs. 5.8%; Among patients with AF, the use of ICE during an ablation procedure was associated with lower incidence of complications and repeat ablation. AADantiarrhythmic drugAFatrial fibrillationCAcatheter ablationCDHPconsumer directed health planCHADS2VAScstands for congestive heart failure, hypertension, age≥75 years, diabetes mellitus, stroke or transient ischemic attack, vascular disease, age 65 to 74 years, sex categoryCIconfidence intervalCVcardiovascularEPOexclusive provider organizationHDHPhigh deductible health planHMOhealth maintenance organizationHRhazard ratioICEintracardiac echocardiographyIRBInstitutional Review BoardPOSpoint of servicePPOpreferred provider organizationSMDstandardized mean differenceUSUnited States1The incidence of atrial fibrillation (AF) continues to rise as the overall population ages.Recommended treatments for AF include antiarrhythmic drugs (AADs), and, for those who are refractory or intolerant to AADs, catheter ablation (CA).Though studies have demonstrated the incremental safety benefit associated with ICE use, a comprehensive assessment of both long‐term (12‐month) outcomes and safety end points in a large real‐world sample of patients are lacking. To address this gap in the literature, we performed analysis of a nationally representative administrative claims database to evaluate differences in complications and 12‐month AF‐related healthcare use among AF patients undergoing CA with versus without ICE in a contemporary cohort.22.1The 2015–2020 IBM MarketScan® Commercial Database,2.2Patients aged 18–64 years old undergoing CA for AF treatment were identified, with first such procedure considered as index procedure. Patients were required to be continuously enrolled for 12 months before the index procedure. Patients were excluded if, in the 12‐month pre‐index period, they had a CA procedure with a primary or secondary diagnosis of AF, had surgical ablation, left atrial appendage occlusion, valvular procedure, or atrioventricular node ablation.2.3The main independent variable of interest was the use of ICE during CA procedure defined by the presence or absence of ICE procedure codes during index ablation admission. Other study covariates included: age; sex; Elixhauser comorbidity score2.4Complications (assessed among patients with index ablation between 2016 and 2020), including acute coronary syndrome, ischemic stroke, hemorrhagic stroke, thromboembolism, transient ischemic attack, phrenic nerve damage, pericarditis, bleeding, vascular access complications, blood transfusion, laryngospasm, mandibular joint dislocation, vagal nerve injury, were assessed within 7 days post‐discharge. Cardiac perforation was assessed within 30 days and atrioesophageal fistula and severe pulmonary vein stenosis within 90 days. A composite of all complications was also included. Appendix Healthcare utilization was assessed within 1 year of CA . Utilization was assessed in the full 12 months after CA and also in the 4–12 months after CA following a standardized 90‐day “blanking period” to avoid capturing early recurrences per Heart Rhythm Society guidelines.2.5Bivariate tests comparing the ICE and non‐ICE groups on study variables were performed using Chi‐square tests of independence for categorical variables and analysis of variance tests for continuous variables. Patients who underwent CA with versus without ICE were matched on study covariates using propensity scores. Multivariable logistic regression was used to fit the propensity score model. Balance of study covariates was assessed using standardized mean difference (SMD). Time‐to‐event analyses were used, with patients right‐censored if they died, were lost to follow‐up (defined as gap of >30 days in enrollment), or reached the end of follow‐up time without an outcome of interest. Hazard ratios (HRs) from Cox regression models were used to assess the relationship between ICE use and study outcomes.p‐value < .05. We performed multiplicity adjustment by controlling for the false discovery rate using the Benjamini–Hochberg (BH) procedure. All analyses were conducted using R for Windows, version 4.0.2.The threshold for statistical significance was a 33.1p < .001, SMD = 0.120); however, after matching, no significant difference was observed . A significantly higher proportion of patients in the ICE cohort had prior AAD  and anti‐coagulant use  in the pre‐match cohort. After matching, no significant differences were observed among the ICE and non‐ICE cohorts on prior AAD  and anti‐coagulant use . Overall, no significant differences on study covariates were observed among the ICE and non‐ICE cohort after propensity matching.A total of 19 368 patients met the criteria for the assessment of complications, with 17 997 (92.9%) in the ICE group and 1371 (7.1%) in the non‐ICE group. Supporting Information: Table p < .001). The two groups did not differ significantly for any other complication. Overall, patients who had CA with ICE had a significantly lower rate of composite complication compared to those who had CA without ICE . Results from regression analysis showed that ICE patients had a 50% lower risk of composite complication end point compared to non‐ICE patients . Table Patients who underwent CA with ICE were less likely to experience acute coronary syndrome in the 7‐day period after the procedure compared to those who had CA without ICE  in the ICE group and 1250 (7.6%) in the non‐ICE group. Table p = .394; 0–12 months AF‐related inpatient admission rate: 4.9% vs. 6.0%, log‐rank p = .240). However, patients who had CA with ICE had a significantly lower rate of repeat ablation in the 12‐month postprocedure period compared to those who had CA without ICE . No differences were observed in the rate of electrical cardioversion . For all healthcare utilization outcomes, no differences were observed among the ICE and non‐ICE patients in the postblanking 4‐ to 12‐month period. Table Patients in the ICE and non‐ICE groups did not differ in rates of 12‐month inpatient admissions . The two groups did not differ significantly in the rate of repeat ablation for the postblanking 4‐ to 12‐month period , 40% (4/10) had the ICD‐10‐CM code I21.3 (representing “ST elevation MI [STEMI] of unspecified site”), 30% (3/10) had the code I21.4 (non‐ST elevation MI [NSTEMI]), and the remaining patients were identified based on diagnosis codes I20.0 (Unstable angina), I21.09 , I21.02 (ST elevation MI [STEMI] involving left anterior descending coronary artery), and I24.0 (acute coronary thrombosis not resulting in MI). Among the 38 patients in the non‐ICE cohort that experienced acute coronary syndrome, almost 45% (17/38) were identified based on diagnosis code I21.4 (non‐ST elevation MI [NSTEMI]), 18% (7/38) were identified based on diagnosis code I21.19 , 13% (5/38) were identified with code I20.0 (unstable angina), and the remaining were identified based on diagnosis codes I24.8 (other forms of acute ischemic heart disease), I21.29 (ST elevation MI [STEMI] involving other sites), I21.3 (ST elevation MI [STEMI] of unspecified site), I21.09 , I21.9 (acute MI [unspecified]), and I22.1 . Though rare, acute coronary syndrome represents a potential life‐threatening complication that could occur during CA due to injury to the vascular wall. Having tools such as ICE that allow target precision during ablation could alleviate the risk of coronary artery syndrome. The authors hypothesize such events as thromboembolism with resultant coronary thrombosis or alternatively inadvertent aortic puncture leading to thrombus formation and coronary event to be two complications that could be averted with appropriate visualization during CA. The fact that no significant differences were observed on any other specific complications of interest may partially reflect the inadequate power due to the limited sample size.Our finding that patients undergoing CA with ICE are at significantly lower risk for acute coronary syndrome is also notable. To better understand this difference, we examined the distribution of acute coronary syndrome diagnosis codes among the ICE and non‐ICE cohort. Among patients in the ICE cohort that had experienced acute coronary syndrome (p < .0001). No difference in AF‐related hospitalization rates  or cardioversion  was seen during this time among patients in the ICE and non‐ICE cohort. These differences are likely indicative of more accurate visualization of catheter‐tissue contact with ICE use, especially in areas of complex anatomy to ensure optimal lesion creation, which potentially reduces reconduction once the edema subsides within the blanking period. As in the current study, a Medicare fee‐for‐service claims study of AF ablation by Steinberg et al. found that ICE use was associated with a lower risk of repeat ablation .Differences in repeat ablation rates occurred with the inclusion of the blanking period suggesting potential differential in lesion quality in patients undergoing their CA with versus without ICE. To better understand this variation, we examined the difference in the rate of repeat ablation among the ICE and non‐ICE cohort during the blanking period (0–3 months). A significantly lower rate of repeat ablation was observed during the blanking period among patients in the ICE cohort as compared to those in the non‐ICE cohort . Given the use of claims data, coding errors, missing data, and reporting bias may also be present and could have influenced study results.6In our analysis of administrative claims data for non‐elderly adult patients with AF in the US, those who had an ablation procedure with ICE had significantly lower risk of having a complication and lower likelihood of needing a repeat ablation as compared to those who had the procedure without ICE. Our results suggest ICE use is associated with the significant clinical benefit of improved safety and effectiveness in AF ablation procedures. Further consideration should be given to the cost impact of these improved outcomes on overall AF management among patients undergoing ablation procedure.Supplementary information.Click here for additional data file.


Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs. Immunoglobulin E (IgE) antibodies have long been ascribed well-characterized roles in the pathogenesis of allergy and hypersensitivity ,2. In adSeparately, interest in IgE glycosylation has been recently reignited. Although heavily glycosylated, with 6 occupied N-linked Fc glycan sites on the constant regions in humans compared to IgG’s sole glycan site , limitedClearer insights into IgE glycosylation will enhance understanding and treatment of allergy and IgE-mediated atopic diseases and will help inform the development of novel antibody scaffolds aimed at filling the gaps in existing monoclonal antibodies for cancer therapy. Consequently, this creates a growing demand for the generation, purification, and characterization of recombinant IgEs with human Fc regions, an endeavor which has historically been highlighted to be an area requiring further research ,25. HencHerein, we sought to produce, purify, characterize and functionally evaluate matched native and exoglycosidase-generated sialic acid-deficient glyco-engineered versions of IgE. We first evaluated the IgE class-specific affinity matrix for its suitability for small-scale purifications, such as those utilized for glyco-engineering with glycosidase enzymes, versus a non-class specific method. We demonstrated the production and purification of native and glyco-engineered IgE–Neu-IgE, a sialic acid-deficient IgE, building on previous studies investigating sialylation as a contributor to the allergic functions of IgE ,28. We sTM IgE Affinity Matrix, herein referred to as CaptureSelect™) and assessed its suitability for small-scale purifications and downstream glyco-engineering.Using affinity matrices, we evaluated the purification of two recombinant IgE antibodies: the mouse/human chimeric antibody CSPG4-IgE recognizing the melanoma-associated antigen Chondroitin Sulfate Proteoglycan 4 and the humanized antibody HER2-IgE recognizing the breast cancer-associated antigen Human Epidermal Growth Factor Receptor 2. CSPG4-IgE was generated from stably transfected Expi293F cells as we described previously . Both anFirst, using IgE class-specific affinity matrix-isolated CSPG4-IgE, we evaluated the elution conditions able to generate the highest antibody yields from culture supernatants using Pierce™ Micro-Spin Columns . We assessed packing conditions using 100, 200, 300, or 400 μL of IgE class-specific matrix slurry in 500 μL spin columns A,B, lookWe next investigated the impact of different elution buffers on antibody yields returned following purification, using three elution buffers: Buffer 1, 50 mM sodium citrate and 150 mM NaCl (pH 3.5) (recommended by the manufacturer); Buffer 2, 0.1 M glycine (pH 2.3) ; and Buffer 3, 20 mM citric acid (pH 3.0), another common purification buffer, used for purification in our laboratory C,D. WhilUsing a 200 μL resin slurry volume, micro-spin columns and Buffer 1 for IgE purification, we contrasted the IgE class-specific matrix with the kappa light chain-specific matrix previously used for purification of IgEs in previous studies . We condWe next wished to generate glyco-engineered IgE, via glycosidase. Previous reports suggested that sialic acid glycans on IgE may participate in allergic functions . We therWe first wished to evaluate whether enzyme incubation conditions (in the absence of the enzyme), such as the low pH of the GlycoBuffer , could impact IgE interactions with its Fc receptors independently of the potential impact of neuraminidase-A. We therefore evaluated IgE binding to the high affinity FcεRI receptor expressed on RBL-SX38 cells and to the low affinity receptor CD23/FcεRII expressed on human B lymphoblastoid RPMI-8866 cells by flow cytometry. We observed that binding of antibodies at different concentrations to these cells was similar for CSPG4-IgE incubated with GlycoBuffer for 16 h at 37 °C and CSPG4-IgE incubated in PBS alone A,B. ThisWe then prepared and purified Neu-IgE using IgE class-specific affinity chromatography as described above. We confirmed removal of neuraminidase from the final preparation (Neu-IgE), as indicated by green arrows C. Next, We next evaluated the glycan profiles of matched native (Con-IgE) versus sialic acid-deficient (Neu-IgE) antibodies specific for CSPG4. This was conducted via release of the N-glycan structures from both Con-IgE and Neu-IgE using PNGase F, procainamide labelling of the released N-glycan structures and separation and detection of the antibody glycan profiles via high-performance liquid chromatography fluorogenic derivatization mass spectrometry HPLC-FD-MS . HPLC-FDAnalysis of the Mass Spectrometry (MS) profiles of Neu-IgE and Con-IgE resulted in a comprehensive list of predicted monosaccharide compositions and suggested glycan structures for each peak . MS analWe assessed whether Neu-IgE could recognize Fc receptor-expressing and target antigen-expressing cells via its Fc and Fab regions, respectively. The Fc-mediated properties of Neu-IgE were examined by investigating recognition of FcεRI expressing RBL-SX38 cells and recognition of CD23-expressing RMPI-8866 cells. Neu-IgE was able bind human FcεRI-expressing cells in a dose-dependent manner comparable to that of native CSPG4-IgE (Con-IgE) A. Neu-IgFor studying the roles of IgE in Allergy and AllergoOncology, as well as for the purpose of investigating potential therapeutic applications of IgE antibodies for the treatment of cancer, it is important to develop efficient and reproducible pipelines for the production of native and glyco-modified IgEs, and to evaluate the biophysical and biological characteristics of IgE glyco-variants. Previously, we reported the stable production of recombinant IgE in mammalian human embryonic kidney Expi293F) cells, which we exemplified using a melanoma-associated antigen (CSPG4)-specific IgE antibody 3F cells,. IgE proIt has long been known that differential glycosylation is common in IgG antibody isotypes, and that the presence or absence of certain glycan features can influence the functional capabilities of IgG ,33,34,35in planta glyco-engineered IgEs, in which we demonstrated the ability of the IgE glycan profile to modulate the binding to CD23-expressing  was gently resuspended, then 5 ml of resin transferred into the column. The column was fitted with an AC-10 Flow Adapter  to allow for a smaller resin bed. Prior to use, the column was flushed with at least 3 column volumes (CV) to equilibrate following constant bed height. Following resin equilibration with PBS, cell supernatants were diluted 1:1 with PBS and passed through the column at a rate of 5 ml/minute. Resin was then washed again with PBS. Captured IgE was eluted using 20 mM citric acid (pH 3.0) or 50 mM sodium citrate + 50 mM sodium chloride (pH 3.5)  was assembled according to manufacturers’ instructions and connected to a P-1 peristaltic pump . CaptureSelect(pH 3.5)  and neutPurification via light chain specific affinity chromatography was performed using HiTrap™ KappaSelect column  according to manufacturer’s instructions.For bulk purification, fractions containing antibody product were pooled to a maximum volume of 6 ml. 4 L of PBS was prepared, and samples transferred to Tube-O-DIALYZER Medi-50kDa dialysis tubes  floated in PBS on a magnetic plate with a stirrer and left to dialyze overnight at 4 °C. For fractions totaling less than 1 ml, fractions were transferred instead to Slide-A-Lyzer Dialysis Casettes   with PBS and dialyzed for 2 h at 4 °C. After 2 h, PBS was changed, then samples incubated overnight with stirring at 4 °C. Sample concentrations were measured via a spectrophotometer as described and concentrated if needed via Amicon Ultra-4 Centrifugal 50 Kda Filters . All samples were stored at 4 °C following purification and dialysis.g then washed for 3 CV with PBS. Bound IgE was eluted  for 2 h at 37 °C. Neuraminidase-A is a broad specificity exoglycosidase capable of cleaving both branched and linear terminal sialic acids with α2-3, α2-6, α2-8, and α2-9 linkages. Pierce™ Micro-Spin Columns  were packed with 100 μL CaptureSelect™ IgE Affinity Matrix and resin equilibrated with PBS. Columns were capped at the base, sample loaded to a maximum volume of 400 μL with PBS then capped and incubated for 30 mins at RT with end-over-end mixing. Columns were placed in 2 ml collection tubes and centrifuged for 1 min at 10,000× Antibody purification was confirmed using SDS-PAGE. Samples were mixed with Laemmli Buffer 4×  or boiled at 95 °C for 5 mins with Reducing Buffer  for reduw/v) sodium azide. The samples were filtered through a 0.2 μm filter immediately before the experiment. Size exclusion chromatography analysis was performed using a Superdex 200 column that had been previously equilibrated with PBS containing 0.1%  and stimulated for 1 h at 37 °C with either stimulation buffer alone or rabbit anti-IgE (1.5 μg/mL). For quantifying β-hexosaminidase, 25 μL culture supernatant was diluted 1:1 with stimulation buffer and transferred onto black 96-well plates containing 50 μL florigenic substrate per well . Plates were incubated for 1 h in the dark at 37 °C then the reaction quenched with 100 μL per well of 0.5 M Tris. Plates were read with a FLUOstar Omega Microplate Reader . Degranulation was expressed as a percentage of Triton X-100 release (100%).RBL-SX38 cell degranulation was measured by quantifying release of β-hexosaminidase, as described previously . Cells wp values of less than 0.05 were considered significant. Data were analyzed using GraphPad Prism 9 software. Error bars represent SDs or SEMs. Statistical significance of degranulation assays was calculated using 1-way ANOVA with the Tukey’s multiple comparisons test.


FKOOsmr) are insulin resistant and exhibit adipose tissue inflammation, suggesting that intact adipocyte OSM–OSMR signaling is necessary for maintaining adipose tissue health. How OSM affects specific adipocyte functions is still unclear. Here, we examined the effects of OSM on adipocyte lipolysis. We treated 3T3-L1 adipocytes with OSM, insulin, and/or inhibitors of SHC1 and ERK and measured glycerol release. We also measured phosphorylation of p66Shc, ERK, and insulin receptor substrate-1 (IRS1) and the expression of lipolysis-associated genes in OSM-exposed 3T3-L1 adipocytes and primary adipocytes from control and FKOOsmr mice. We found that OSM induces adipocyte lipolysis via a p66Shc-ERK pathway and inhibits the suppression of lipolysis by insulin. Further, OSM induces phosphorylation of inhibitory IRS1 residues. We conclude that OSM is a stimulator of lipolysis and inhibits adipocyte insulin response. Future studies will determine how these roles of OSM affect adipose tissue function in health and disease.Oncostatin M (OSM) is an immune cell-derived cytokine that is upregulated in adipose tissue in obesity. Upon binding its receptor (OSMR), OSM induces the phosphorylation of the p66 subunit of Src homology 2 domain-containing transforming protein 1 (SHC1), called p66Shc, and activates the extracellular signal-related kinase (ERK) pathway. Mice with adipocyte-specific OSMR deletion ( The primary function of adipose tissue is to store energy in the form of lipid, for later use by the body. Adipocytes mobilize lipids as free fatty acids (FFA) in settings where energy needs increase, such as fasting or exercise ,2,3. TheMany factors can stimulate lipolysis; adipose tissue cytokines, produced by immune cells, are major regulators of this process. The most well studied lipolytic cytokine in adipose tissue is tumor necrosis factor-alpha (TNFα), which exerts its lipolytic effects via several mechanisms ,4,6,7. TOncostatin M (OSM) is a member of the glycoprotein 130 (gp130)/interleukin-6 (IL6) family of cytokines which use the gp130 subunit in their receptor complexes ,13. The A distinguishing feature of OSM signaling is the recruitment of the p66 isoform of the adaptor protein, Src homology 2 domain-containing transforming protein 1 (SHC1), referred to as p66Shc, to tyrosine 861 (Y861) of OSMR ,22,23. TFKOOsmr mice) are insulin-resistant and have increased adipose tissue inflammation when compared to control mice (fl/flOsmr) [Our previous work in a mixed-background mouse model has demonstrated that mice lacking OSM signaling specifically in adipocytes (mrfl/fl) ,24. WhilIncreased adipocyte ERK activation was identified as a contributor to the increased basal lipolysis observed in obesity . This fiInsulin sensitivity is regulated in part by serine and threonine phosphorylation of the IRS1 and IRS2 proteins ,29. SpecThe phenomenon of OSM binding to promote p66Shc recruitment and subsequent ERK 1/2 activation has not been described in the adipocyte. We first assessed whether OSM promoted p66Shc phosphorylation in adipocytes, using the same cell lysates used to measure OSM-mediated IRS1 phosphorylation ), Lipe (encodes hormone-specific lipase (HSL)), G0s2 (inhibitor of ATGL activity), Abhd5 (encodes comparative gene identification-58 (CGI-58), an ATGL activator), Plin1 (encodes perilipin 1), and Hilpda (hypoxia-induced lipid droplet associated protein). OSM significantly decreased the expression of G0s2, Pnpla2, Lipe, and Plin1, while significantly increasing Hilpda expression; Abhd5 expression was not affected by OSM treatment . Conversely, insulin signaling induced by feeding inhibits lipolysis and enhances lipogenesis. Obese and insulin-resistant states are associated with elevated rates of basal lipolysis in the fed state, due in part to impaired insulin action in adipose tissue . ChronicImmune cell-derived cytokines can positively or negatively influence adipocyte function and, therefore, the overall function of adipose tissue. Adipose tissue inflammation, a hallmark of obesity and insulin resistance, contributes to aberrant lipolysis in an insulin-independent manner, particularly through the actions of TNFα and other inflammatory cytokines driving obesity-related adipose tissue inflammation ,9,10,11.The data presented here clearly demonstrate that OSM acts on adipocytes to: (1) stimulate lipolysis, (2) inhibit IRS1 phosphorylation, (3) stimulate p66Shc phosphorylation, and (4) induce ERK1/2 phosphorylation. Our findings using either antibody inhibition of OSMR or a MEK inhibitor suggest that OSM-induced lipolysis occurs via a p66Shc-ERK-OSMR mechanism. These findings are in line with the known ERK activating effects of OSM in other cell types ,21. Our Second, we have clearly demonstrated here that OSM induces p66Shc and ERK1/2 phosphorylation and that these events occur over similar times b. Our fiG0s2 [Finally, we show here that OSM stimulates lipolysis in a manner that depends, at least in part, on OSMR, p66Shc, and ERK1/2. However, the downstream events that trigger lipolysis transcriptionally, at the post-translational level, or both, remain unclear. In line with our gene expression findings in OSM-exposed adipocytes, ERK1/2 activation can result in decreased transcription of G0s2 ,40, whicG0s2 , but theFKOOsmr mouse are taken together is: if OSM induces adipocyte lipolysis—a seemingly harmful effect that promotes insulin resistance in obesity—how does adipocyte OSMR loss (and the loss of OSM-induced lipolysis) still result in adipose tissue inflammation and insulin resistance [Though we are the first to describe OSM effects on lipolysis, the effects of OSM on other aspects of lipid handling and insulin response have been described in both adipose tissue and the liver ,43,44,45sistance ,15,24?FKOOsmr mice [There are several possible explanations, and all will require intensive further study. First, the autocrine action of SDF1 on adipocytes is reported to induce inhibitory IRS1 phosphorylation and result in insulin resistance in mice . Given oFKO mice , so enhaFurther research regarding OSM and adipocyte lipid metabolism is required, but our present findings identifying OSM as a novel contributor to adipose tissue lipolysis provide valuable insight into the relationship between elevated lipolysis and insulin resistance.Murine 3T3-L1 preadipocytes were grown to two days post-confluence and differentiated into adipocytes as previously described ,15. SeveLipolysis assays were conducted on adipocytes grown in 12-well plates. Adipocytes were placed in an incubation medium with or without triacsin C  as described previously ,15,24. Mfl/flOsmr and FKOOsmr mice  were humanely euthanized and their fur was thoroughly moistened with 70% ethanol. Inguinal adipose tissue was quickly excised from each animal and placed into warm antibiotic-containing Hank’s balanced salt solution (HBSS). Under a laminar flow hood, tissues were pooled by genotype. Each pool was rinsed well in warmed HBSS + antibiotics then transferred to collagenase digestion buffer (1 mg/mL type I collagenase in HBSS supplemented with DNase I and magnesium chloride) [Isolation and culture of murine adipose-derived stem cells was performed using methods described in . Brieflyhloride)  and mincg at room temperature to separate the primary adipocytes and stromal vascular cell (SVC) pellet. The floating adipocytes and supernatant were aspirated and the SVC pellet was resuspended in 2 mL ammonium-chloride-potassium (ACK) lysis buffer, triturated 5–8 times, and incubated at room temperature for 3 min. 10 mL Dulbecco’s phosphate-buffered saline (DPBS) was added to neutralize the ACK lysis buffer, cells were centrifuged at 450× g at room temperature for 5 min and the supernatant aspirated. The pellet was then resuspended in 2 mL of stromal medium [2 for 24 h, rinsed, and placed in fresh stromal media.Minced tissues were placed into sterile 50 mL conical tubes and collagenase buffer volume increased to 10 mL. Tubes were placed into a 37 °C shaking water bath at 200 rpm until dissociated (approximately 1.5 h). Once dissociated, each tissue suspension was filtered through a sterile 100 μm filter into a fresh 50 mL tube and centrifuged for 5 min at 450× Two days after plating, cells were trypsinized and plated in 12-well plates at a density of ~7000 cells per well (five 12-well plates per genotype), and stromal media changed every other day. Five days after cells were placed into 12-well plates, at approximately 90% confluence, medium was changed to differentiation medium . Three dPpia (peptidyl prolyl isomerase A). Primers used were from Integrated DNA Technologies ; primer sequences appear in Total RNA was isolated from cell monolayers using the RNeasy Mini Kit  as previously described ,15,24. RIn these studies, we demonstrate for the first time that OSM stimulates lipolysis and increases inhibitory IRS1 phosphorylation in cultured adipocytes. Our data suggest that OSM-stimulated lipolysis is OSMR-dependent and occurs via a p66Shc-ERK1/2-mediated mechanism. We also show that adipocyte-specific loss of OSMR leads to upregulated expression of several key genes involved in lipolysis. We conclude from these results that OSM is a lipolysis inducer in vitro and deserves further study in the context of obesity and metabolic disease.


SLC7A1) plays a key role in the cellular uptake or export of l-arginine and some of its derivatives. This study investigated the effect of 113 chemically diverse and commonly used drugs (at 20 and 200 µM) on the CAT1-mediated cellular uptake of l-arginine, l-homoarginine, and asymmetric dimethylarginine (ADMA). Twenty-three (20%) of the tested substances showed weak inhibitory or stimulatory effects, but only verapamil showed consistent inhibitory effects on CAT1-mediated transport of all tested substrates.The cationic amino acid transporter 1 (CAT1/The online version contains supplementary material available at 10.1007/s00726-022-03156-2. SLC7A1) is a member of the solute carrier protein (SLC) superfamily, expressed in most human tissues  , where transport inhibition leads to potential drug–drug interactions and may cause changes in drug pharmacokinetics (Müller and Fromm 3H]‐l-arginine (43 Ci/mmol) was obtained from American Radiolabeled Chemicals, Inc , [3H]-l-homoarginine (6 Ci/mmol) was obtained from ViTrax Co , [3H]‐ADMA (25 Ci/mmol) was purchased from BIOTREND Chemikalien GmbH . Unlabeled substrates, library drugs, reagents, and solvents were purchased from commercial suppliers and used as received unless otherwise noted. Each of the pharmaceutical compounds was of analytical grade. The drugs used were selected based on a representative set of small molecule drugs previously shown suitable to investigate drug effects on organic cationic transporter 2 (OCT2)-mediated transport . To confirm the experimental setup, similar experiments in the presence of N-ethylmaleimide (NEM), a CAT-specific inhibitor (1000 µM)  = 100 – (V/V0* 100), where V and V0 are the net uptake rates (uptake into HEK-CAT1 cells—uptake into HEK-VC cells) with and without test drugs, respectively. Data are presented as the percent inhibition of the CAT1-mediated uptake of l-arginine, l-homoarginine, or ADMA.Uptake and inhibition assays using HEK-CAT1 and HEK-VC cells and l-arginine, l-homoarginine, or ADMA uptake between control and test drug. Data were presented as mean ± standard error of the mean. Graphs and statistical analysis were conducted using GraphPad Prism® 8.0 software (GraphPad Software Inc.).A one-way ANOVA followed by Dunnett’s test was used to determine the statistical significance differences in CAT1-mediated l-arginine uptake was moderately but significantly inhibited by nine drugs  at 20 µM (25–50% inhibition) and only fludrocortisone stimulated the transport by at least 30%. In contrast, the remaining drugs displayed biologically negligible inhibitory effects on CAT1-mediated l-arginine uptake. The same drugs were also tested against the transport of l-homoarginine (1 µM). Verapamil, prazosin, trospium, diflunisal, and norethindrone used at 20 μM, were the only drugs that significantly inhibited CAT1-mediated transport of l-homoarginine  showed a non-competitive pattern and verapamil an uncompetitive pattern [Figure supplement S4].A list of all tested substances together with their ATC code, CAS number, and their effect on CAT1-mediated transport is summarized in Table S1 [Table supplement S1]. As shown in Fig. l-arginine, l-homoarginine, and ADMA. Previous studies have shown that CAT1 preferentially accepts cationic amino acids such as l-arginine  is orders of magnitude higher than the maximum plasma concentrations of free (non-protein bound) verapamil (∼ 0.05 µM) . The observed drug-CAT1 inhibition, however, occurred at concentrations that are much higher than those commonly seen as plasma concentrations in the clinical use of these drugs, which makes any clinically relevant contribution of these drugs to CAT1 transport-related metabolic aberrations unlikely. Therefore, for some drugs, we may have missed possibly more profound inhibitory effects at concentrations exceeding those seen with normal therapeutic doses of the drugs. The example of verapamil indicates that we might have identified more, albeit weak, inhibitors using much higher concentrations. So far, there are insufficient data for computational analysis and in silico modeling of CAT1 inhibitors available to deduce or model the precise mechanism(s) of inhibition; only the crystal structure of a close homolog of the mammalian CATs is available Below is the link to the electronic supplementary material.


In addition, a heterologous booster vaccination (n = 50) of a protein subunit vaccine ZifiVax (ZF2001) significantly restored humoral immune responses and even showed an increasing response against wild-type, B.1.351, B.1.617.2, and B.1.1.529 than homologous inactivated vaccines. Our analysis of the humoral immune response elicited by the different vaccine regimens, including inhibiting antibodies, indicated that a booster, whether homologous or heterologous, could be essential for achieving greater efficacy against SARS-CoV-2.Safe and effective vaccines for Corona Virus Disease 2019 (COVID-19) can prevent the virus from infecting human populations and treat patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we discuss the inhibitory abilities of primary and booster vaccine-induced antibodies inhibitory ability toward the SARS-CoV-2 wild-type strain, as well as B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529. We confirmed these antibodies had the strongest inhibitory effects on the wild-type strain and cross-inhibition activities against other mutant strains after two inactivated vaccine doses. However, the B.1.351, B.1.617.2 and B.1.1.529 mutants exhibit antibody resistance in the vaccine serum. Antibodies induced by homologous inactivated vaccines (n = 92) presented more effective inhibition against tested SARS-CoV-2 strains ( The Corona Virus Disease 2019 (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, began in December 2019 and immediately threatened the people’s lives worldwide. As of 16 November 2022, a total of 632,953,782 confirmed cases of COVID-19, including 6,593,715 deaths, were reported globally. As of 8 November 2022, upwards of 12,885,748,541 vaccine doses were used for inoculation [The receptor-binding domain (RBD) of the spike protein, which exhibits immunodominance ,3, was aThe emergence of mutant strains poses a continuing threat to global public health. Vaccines must be able to develop a herd immunity barrier against SARS-CoV-2 infection. Anti-SARS-CoV-2 vaccines can be classified as inactivated vaccines , mRNA vaccines , viral vector vaccines , and recombinant protein subunit vaccines , etc. These vaccines elicit excellent neutralizing activity against the SARS-CoV-2 wild-type strain at the prescribed doses ,9,10. HoZifiVax  is a recombinant protein subunit vaccine whose antigen is a dimeric form of the receptor-binding domain (RBD) . This anWe performed an vitro experiment binding the antibodies with the SARS-CoV-2 spike protein RBD to block the RBD protein complex from forming with the ACE2 receptor. The detection of antibodies by blocking RBD-hACE2 binding has been positively correlated with neutralizing serum antibody titers . Our resSerum samples were collected and classified according to the time point of serum collection. The time points and information regarding the serum samples are displayed in In addition, we regularly collected samples from 32 volunteers at the following time points: day 14 and 21 following the first dose, day 7, 14, 21, 28, 58, 88, 118, and 180 after the second dose, and day 7 after the homologous CoronaVac booster. The 32 volunteers who received CoronaVac had neither SARS-CoV-2 infection history nor SARS-CoV-2 inhibiting antibodies prior to vaccination. All the vaccinated participants provided informed consent prior to data and specimen collection.Serum samples were collected in anticoagulant tubes and centrifuged at 5000 rpm for 10 min . We stored the samples in a laboratory serum biobank at −20 °C. On the day of detection, the frozen sample was thawed at 4 °C for one hour. The rethawed samples were subjected to eddy current treatment prior to analysis. The research program was consistent with the Declaration of Helsinki.2. Expi293F is a highly transfectable cell line that transiently expresses proteins. Cells are either grown in batches or supplemented in medium depending on the number of cells growing and the viability of transfected cells ≥98%. Recombinant plasmids with DNA sequences encoding the spike proteins RBD of the SARS-CoV-2 wild-type and mutant strains  from Hangzhou Quickgene Sci-Tech. Co., Ltd.  were transfected into Expi293F cells to obtain SARS-CoV-2 strains RBD recombination proteins, using the ExpiFectamine™ 293 transfection kit . The same procedure was used for hACE2 protein expression. The cell suspension was centrifuged at 10,000 rpm for 20 min in a refrigerated centrifuge and filtered through a 0.22 µm filter to obtain the supernatant on the seventh day after transfection. Expi293F cells  were cultured in suspension in Expi293F expression medium  at 37 °C with 8% COsample/ODnegative control) × 100. Our enzyme-linked immunoassay of competition test determined a negative cut-off value at 30% inhibition from testing over negative (n = 89) human serum  were precoated with hACE2 protein at 60 ng/well in 100 µL of 50 mM carbonate-bicarbonate coating buffer (pH 9.85) overnight at 4 °C for 24 h. Then, they were blocked with a blocking buffer at 4 °C for 48 h. After adding serum samples which was diluted into 50 µL of sample buffer to the hACE2-coated plates, wild-type-RBD-HRP diluted into 50 µL of enzyme buffer at optimal concentration was added into plates to detect antibodies inhibiting the ability of wild-type strain. We selected the corresponding RBD-HRP and optimal concentration according to different mutant strains to evaluate the antibody inhibition ability. We set the reaction system for 30 min at 37 °C. Unbound HRP-conjugated RBD was removed using three washes with phosphate-buffered saline, 0.05% Tween-20 (PBST). Then, 100 µL of tetramethylbenzidine  was added to the plates for 15 min in the dark at 37 °C. We added TMB stop solution to stop the reaction and used a microplate reader  to record the absorbance readings at 450 nm. This method was a suppression assay whose color intensity was inversely proportional to the level of inhibitory antibodies in the samples. The data were interpreted by calculating the percentage of RBD-HRP binding inhibition. The optimal concentration of RBD-HRP was determined according to the standard of negative control, and the OD value was 2.0 ± 0.2. Inhibition (%) = , inhibiting the RBD of SARS-CoV-2 wild-type strain or mutant strains  from binding with hACE2. The four time points included the third week (n = 68) following the first inactivated vaccination dose and the first (n = 201), third (n = 127), and fifth (n = 68) months following the second dose. Based on the comparison between groups, we study the cross-inhibition ability of antibodies in the serum of inactivated vaccines (CoronaVac/BBIBP-CorV) against wild-type and mutant strains of SARS-CoV-2.In addition, serum samples from 32 individuals who received two complete doses of CoronaVac were evaluated in detail at 10 time points. We analyzed the average inhibition rates of serum against the wild-type and mutant strains at each time point and trends in antibodies’ inhibition rates. We used our results to support research on the longitudinal kinetics and effective duration inhibiting antibodies in the serum. We also analyzed the rate of positive antibodies on day 180 following the second dose of vaccine.Twenty out of thirty-two volunteers received a single CoronaVac booster 6 months after their primary vaccination. By comparing the inhibition rates of serum antibodies on day 7 after the second does with the same time point of the booster, we determined whether the inhibitory effect on wild-type, B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529 increased significantly after the booster.The inhibitory abilities of serum antibodies against the wild strain, B.1.351, B.1.617.2, and B.1.1.529, after homologous and heterologous boosters were evaluated to investigate booster effectiveness. The samples studied involved serum samples from the first (n = 92) and second months (n = 61) after a homologous booster with the inactivated vaccine (CoronaVac/BBIBP-CorV) and from the first month (n = 50) following recombinant protein subunit vaccine (ZF2001) as a heterologous booster after inactivated vaccine-based injection. In addition, serum before receiving the booster vaccine was collected from 54 out of 92 volunteers who received a homologous booster vaccine. Our research methods and strategies are shown in p value was expressed in terms related to the alpha value. The significance value for all analyses was set to 0.05, and 0.01 was considered an extremely significant value. Brown–Forsythe, Welch ANOVA, and Games–Howell multiple comparison tests were used to determine the statistical significance between groups. A paired t-test was used to analyze the differences in serum antibodies between the seventh day following the primary two vaccinations and booster.We used GraphPad Prism 8.0.2 software  for data visualization. The The average inhibition rates of antibody induced by CoronaVac/BBIBP-CorV against wild-type and mutant strains were analyzed at four time points. The number of positive serum samples for wild-type strain: 36/68, B.1.1.7: 42/68, B.1.351: 11/68, P.1: 6/68, B.1.617.2: 34/68, and B.1.1.529: 18/68 at the third week following the first dose. The average antibody inhibition rates of serum collected at the third week after the first dose (n = 68) and at the first (n = 201), third (n = 127) and fifth (n = 68) months after the second dose of inactivated vaccine to wild-type, B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529 are shown in p < 0.0001), 65.04% (p < 0.0001), 69.99% (p < 0.0001), 58.97% (p < 0.0001), and 54.79% (p < 0.0001) after the first month of the second dose, respectively. Inactivated vaccines produced high-titer specific inhibitory antibodies targeting SARS-CoV-2 spike proteins. Inhibitory antibodies induced by CoronaVac/BBIBP-CorV exhibited the highest inhibition capacity against the SARS-CoV-2 wild-type and cross-inhibiting activities against SARS-CoV-2 mutant strains.The differences in inhibition rates of serum antibodies against each strain are shown in In the first month after the second dose, the average inhibition rates against B.1.351, B.1.617.2, and B.1.1.529 mutants decreased by 17.12% (CI: 13.53–20.71%), 23.20% (CI: 19.61–26.78%), and 27.37% (CI: 24.16–30.59%), with the wild-type strain as the control. Additionally, B.1.351, B.1.617.2, B.1.1.529 mutants decreased by 20.55% (CI: 16.71–24.39%), 21.15% (CI: 17.48–24.83%), 26.15% (CI: 22.80–29.50%), in the third month, respectively. The antibodies’ inhibition abilities were significantly reduced in the mutant strains B.1.351, B.1.617.2, and B.1.1.529, with high level against the wild-type strain. The average inhibition rates of serum antibodies against wild-type, B.1.1.7, B.1.351, P.1, B.1.617.2 and B.1.1.529 were 46.25%, 36.28%, 32.23%, 35.48%, 25.64%, and 24.58% during the fifth month after the second dose. The average inhibition of serum antibodies decreased to a limited cross-reactivity level for wild-type, B.1.1.7, B.1.351, and P.1 after five months. At the same time, it become undetectable for B.1.617.2 and B.1.1.529 .Serum inhibition curves and IC50s were generated from five volunteers (n = 5) who received inactivated vaccines  to evalup < 0.0001; B.1.1.529: p = 0.0031) sharply increased and reached their peak within 3 weeks following the second dose. The average inhibition rates of serum antibodies against SARS-CoV-2 wild-type, B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529 were 82.74%, 65.21%, 54.67%, 71.38%, 53.42%, and 43.26% at day 28 following the second dose, respectively. For wild-type, B.1.1.7, P.1, and B.1.1.529 strains, antibody inhibition rates lasted for about 60 days; however, the inhibitory level differed between the four strains. Subsequently, antibody inhibition rates were on the decline [To determine the persistence of inactivated vaccine antibodies’ inhibitory capacity, we followed 32 volunteers for 6 months after receiving two doses of CoronaVac vaccine. Serum samples were collected at certain intervals. The average inhibition rates of serum samples collected against wild-type and mutant strains are shown in  decline . We alsot-test was performed on the collected data , 21.55% (CI: 14.03–29.07%), 22.00% (CI: 12.78–31.22%), 12.03% (CI: 4.51–19.54%), 14.74% (CI: 5.12–24.28%) and 32.60% (CI:25.64–39.55%). The homologous booster significantly increased antibodies’ inhibitory activities for tested strains and enhanced the extensive cross-inhibition of SARS-CoV-2 mutant strains. It also had a high inhibition level of 90% against the wild-type strain. Meanwhile, average inhibition increased significantly with the booster, especially against B.1.1.529.In addition, serum samples before receiving the booster vaccine were collected from 54 out of 92 volunteers who received the homologous booster vaccine. The inhibition rates of serum antibodies against wild-type, B.1.351, B.1.617.2, and B.1.1.529 before and after homologous booster vaccination were compared to determine the change in humoral immune response after booster vaccination B. The avWe aimed to investigate the effectiveness of the recombinant protein subunit vaccine (ZF2001) as a heterologous booster for inhibiting hACE2 from binding with RBD of wild-type, B.1.351, B.1.617.2, and B.1.529 strains. We collected homologous inactivated/inactivated prime-boost individuals’ serum samples in the first (n = 92) and second (n = 61) months and inactivated/recombination protein submit vaccine (ZF2001) prime-boost samples (n = 50). The inhibitory abilities of antibodies against wild-type, B.1.351, B.1.617.2, and B.1.1.529 are evaluated in p = 0.002), B.1.351 (p = 0.0011), B.1.617.2 (p = 0.0016), and B.1.1.529 (p < 0.0001) also increased with respect to the homologous booster. Both homologous and heterologous booster vaccination exhibited the highest efficacy against the wild-type strain and showed extensive cross-inhibition of B.1.351, B.1.617.2, and B.1.1.529. Although the inhibitory effects of the serum following the booster vaccine on the B.1.351, B.1.617.2, and B.1.1.529 fell slightly, most of the inhibitory activity remained.The ZF2001 booster vaccine had an estimated 85.31% inhibition rate against B.1.351, 82.10% against B.1.617.2, and 81.27% against B.1.1.529. The inhibition rates of the ZF2001 booster serum on wild-type (p < 0.001), 5.77% (p = 0.0452), 7.22% (p = 0.0017), and 0.53% (p = 0.9558) in the second month against wild-type, B.1.351, B.1.617.2, and B.1.1.529, respectively, compared with the first month following the homologous booster. The inhibitory abilities of the serum antibodies in the second month following the homologous inactivated vaccine against wild-type, B.1.351, B.1.617.2, and B.1.529 were ≥69.12%. In addition, to evaluate the inhibiting activity of antibodies, we generated serum inhibition curves and IC50s from volunteers (n = 5) who received the inactivated homologous and heterologous (ZF2001) boosters for wild-type, B.1.351, B.1.617.2, and B.1.1.529 variants  following the reception of inactivated vaccines. We found that antibodies induced by the CoronaVac vaccine with the two primary doses inhibited the binding of hACE2 with SARS-CoV-2 strains. Protection from the first dose up until administration of the second dose was limited . We analAntibodies had the strongest inhibitory effects on the wild-type strain and extensive cross-inhibition activities with other mutant strains after receiving a second dose of inactivated vaccine (CoronaVac/BBIBP-CorV). We also noted that serum antibodies could inhibit the B.1.351 and B.1.617.2 mutants to a certain extent. However, this inhibition level could not be sustained for a longer period. Additionally, the antibodies’ ability to inhibit mutant B.1.1.529 was significantly reduced compared to the wild-type and other strains. B.1.1.529 strains exhibited significant inhibitory resistance, similar to B.1.351 and B.1.617.2 . This fiThe antibody concentration in the sixth month following the previous two doses of vaccine could not protect the human organism from infection with SARS-CoV-2 variants. Furthermore, rapid propagation of the B.1.617.2 and B.1.1.529 variants with antibodies inhibiting resistance forced many countries to consider the application of additional vaccine doses. Our research has highlighted the need for booster vaccinations when the inhibition activities of antibodies elicited by a two-dose inactivated vaccine decreased over time. Surprisingly, CoronaVac administered as a third dose significantly increased humoral immune response, thus reducing the possibility of infection by mutant strains . AdditioHomologous and heterologous booster vaccinations with inactivated vaccines are reported to be good candidates for curbing the pandemic . Our resThe limitations of this work were the lack of pseudovirus or real virus neutralization experiments regarding antibodies’ ability to neutralize live viruses. Numerous studies have demonstrated a close correlation between neutralization titers measured by surrogate virus neutralization tests and pseudovirus SARS-CoV-2 cultures ,23,42,43Finally, further studies are needed to investigate the persistence of antibodies elicited by the booster vaccine and clarify whether the antibodies’ binding, inhibition, and neutralization activities toward the mutant strains sufficiently protect against them.In summary, the main focus of our study was to assess the potential of vaccine-induced antibodies to inhibit the binding of hACE2 with circulating SARS-CoV-2 variants RBD. This study suggested that inhibitory antibodies elicited by homologous inactivated (BBIBP-CorV/CoronaVac) and heterologous recombinant protein subunit (ZF2001) vaccination boosters dramatically enhanced inhibitory abilities against B.1.351, B.1.617.2, and B.1.1.529 strains. Our results support the importance of enhancing immunity against epidemic strains for increasing vaccine effectiveness.


The gradual drying up of saltwater bodies creates habitats that are characterised by changing environmental conditions and might be available only for a subset of plants from the local flora. Using two terrestrial areas with different ages on the Caspian Coast as a chronosequence, we investigated factors including microtopography, ground water level and soil salinity that drive plant community succession after the retreat of the sea. Vegetation of the two key sites appearing after the retreat of the Caspian Sea about 365 and 1412 years ago were compared in terms of both evolutionary and ecological traits of plants. Both edaphic conditions and vegetation differed between the two sites with harsher edaphic conditions and more xerophytes on the elder site. Species that grew only in the ‘early’ site were dispersed across the phylogenetic tree, but their loss on the 'late' site was not random. Species that grew only on the 'late' site were phylogenetically clustered. On the level of microtopography, elevated spots were more densely populated in the ‘early’ site than lowered spots, but on the 'late' site the situation was opposite. The main edaphic factors that drive the difference in vegetation composition between the two sites are likely salinity and moisture. During environmental changes, different plant traits are important to survive and to appear in the community de novo. Microtopography is important for forming plant communities, and its role changes with time. In addition to the Aral16. The latest changes of the Caspian sea level lead to the formation of new dry land on its coasts19. New territories appear and are inhabited by plants. In the last 3000 years, the northern part of the Caspian Sea was under semiarid conditions20. Due to aridity, the salt accumulation was a typical process on the newly formed land. Salt-affected soils with high electrolyte content restrict the development of most plants except halophytes, which can survive. Much is still unknown about plant community succession caused by the reduction in groundwater level resulting from the drying up of the sea. As groundwater level continues to change, such a succession is likely to be highly dependent on changes in abiotic conditions that are not influenced by the community itself. In other words, it might be mostly allogenic. However, some mechanisms of autogenic (i.e. depending on community members) succession may also play a role. For example, large shrubs such as tamarisk might influence the growing conditions in their vicinity21, thus influencing the composition of community by facilitation or inhibition mechanisms22. Knowledge about the current changes and dynamics of vegetation might help to predict how the territory will look in the future and is critical for developing adaptation strategies to address the challenges posed by climate change and human activities on vulnerable and fragile semi-arid ecosystems23.Being a closed water body dependent on the inflow of the Volga and the Ural rivers, the Caspian Sea had multiple stages of regressions and transgressions in the past when the sea coast was found at elevations from − 150 to + 50 m above the sea level25.To understand the processes that drive changes in the structure of plant communities, a complex approach is needed that will consider edaphic factors, as well as ecological traits and the evolutionary history of plants that coexist or change each other during plant community succession28. The microhighs receive 4 times less amount of water than microlows, which leads to formation of sparse halophyte vegetation on the tops and dense and diverse vegetation on the bottoms. This phenomenon is well studied at the developed old-aged landscapes at the altitudes of + 0 + 50 m a.s.l. However, the studies on the influence of microtopography on the patchiness of vegetation at the early stages (last centuries) after the retreat of the sea are not well represented in the literature.The microtopography with the amplitude of about 30 cm has a strong influence on water relocation and, hence, vegetation in semiarid landscapes of the Northern Caspian Lowland31, and are located 1.6 km from each other. The names “early” and “late” reflect that the ‘early’ site is on earlier succession stage than the ‘late’ site. They were compared in terms of vegetation and edaphic factors to study temporal changes of the territory that newly appeared from under the sea in the semi-arid climate. We also compared the vegetation growing on different microtopographic positions (microhighs vs. microlows) both within each key site and between two key sites.In this work, two key sites of different ages were considered as proxy time points of evolution in the area after the Caspian Sea retreated. The two sites—‘early’ (Caspii-2 in our previous works) and ‘late’ (Caspii-1)—were freed from the sea about 293 and 1340 years ago, respectivelyDuring this work, we tested the hypothesis that on highly saline soils (solonchaks) of the Caspian Lowland, elevations are a harsher environment for plants than depressions in both macro- (i.e. different regressive phases) and microscale (i.e. elements of microtopography—microhighs and microlows).The Early and Late key sites were of 30 × 45 and 50 × 50 m size, respectively Table , Fig. S134.As can be judged from the age of soil organic carbon, the short-term flooding in the beginning of the nineteenth century did not impact the ‘late’ key site significantly. Notably, the entire coastal zone of the northern part of the Caspian Sea was repeatedly subjected to flooding associated with storm surges, for example, in 1952 and 1995, the absolute water levels reached about − 24 m for several hours. However, it did not have an influence on the general development of the region’s landscape. At the ‘late’ key site, the lower western and the higher eastern parts were clearly distinguished. The ‘late’ site partially occupied the western slope of the coastal bar formed in the 1880s29. At the ‘late’ site, a three-tier plant community consisting of a 0.5–1.0 m high Suaeda microphylla in the upper tier, a 0.2–0.5 m high Kalidium foliatum in the medium tier, and annual saltworts  with Frankenia hirsuta, Psylliostachys spersicicus and ephemeral plants in the lower herb’s tier occurred29. The vegetation of the ‘early’ site was a sparse degrading (deteriorating) community of a 1–1.5 m high tamarisk (Tamarix octandra mixed with some T. laxa) with annual saltworts , Frankenia hirsute, and Puccinellia gigantea in the lower tier with some ephemeral plants35. In the lower tier, two microgroups were clearly distinguished. Open habitats were occupied by sparse stunted halophytic plants . Under the crowns of the bushes, dense herb and grass microcommunities grew with a predominance of tall Puccinellia gigantea and a few Limonium caspium, L. scoparium, and Psylliostachys spicata, whereas saltworts and Frankenia were absent here.The soils of the ‘late’ site were more saline than soils in the ‘early’ siteIn autumn and spring, soils were sampled from 1-m deep boreholes at depths of 0–5, 5–10, 10–20, 20–30, 30–50, 50–70, and 70–100 cm. At the ‘late’ site, 40 auger holes were drilled at a distance of 2 m from each other along a 50-m long transect (26 auger holes) and on different elements of microtopography (14 auger holes)—microhighs and microlows Fig. . At the In autumn, vegetation was described in 2 × 2 m plots throughout the key sites along 50-m long  and 30-m long transects  located at a distance of 2–4 m from each other. The descriptions included the number and size of the dominant shrubs, plant species, total projective cover of the herb tier, and the portion of bare ground and plant litter. As the main description of 2 × 2 m plots took place in autumn, only 11 perennial species were found in the ‘early’ site and 15 in the ‘late’ site. In addition, in the spring, the species composition of both sites was studied to compile a list of flora, including not only perennials but also annuals and ephemer(oid)s. The vast majority of species was identified in the field. The formal identification of the plant material used in this study was conducted by Yulia D. Nukhimovskaya and Nina Yu. Stepanova. Voucher specimens were deposited in a publicly available herbarium of the Tsytsyn Main Botanical Garden of the Russian Academy of Sciences Table .36 based on the Takhtajan system37 during the field research and according to open-access continuously updated resources39 corresponded to the APG IV system40 to provide phylogenetic analysis. In subsequent work, autumn descriptions were used for analyses related to microtopography, species prevalence, and species co-occurrence. In phylogenetic analyses of the flora, full lists of flora present at the studied sites were used.The Latin names of plant species were given according to the S.K. Cherepanov listThe microtopography was measured using dynamic GSP (DGPS) Stonex-9 Plus equipment which allows sub-centimetre accuracy of measurements. The DGSP readings were taken with one meter interval. Maps of microtopography were calculated in SAGA GIS using Ordinary Kriging method. The resulting dataset for the ‘early’ site consisted of 86 microhighs and 225 microlows (microhighs had a relative height of 7–16 cm above the microlows), and the resulting dataset for the ‘late’ site consisted of 151 microhighs and 130 microlows (microhighs had a relative height of 17–40 cm above the microlows).In soil samples, the electrical conductivity (EC) was measured in the lab with a 1:2.5 soil-to-water ratio using a Hanna Combo 98130 conductivity meter. In reference soil pits, located at typical microhighs and microlows, the moisture and soil bulk density were measured using the gravimetric method. All measures of EC as well as species composition of 2 × 2 plots are available as Using published data, a list of 42 families from 22 orders of plants Table  that gro41. From either of the key sites, 27 of 35 species persisted in the phylogenetic tree of Angiospermae. Eight remaining species were added to the tree by adding their branches as basal polytomies within their genera or families with R scripts provided in41.Phylogenetic trees for Magnoliophyta species persistent in the ‘early’ or ‘late’ key sites and for families from the local flora were built based on a phylogenetic tree of Angiospermae from42.Taxonomic Distinctness Index based on presence-absence data was calculated as a mean pairwise taxonomic distance between species that were found in the community25—was calculated for species that were lost or acquired with time and compared with the null distributions for MPD as described in detail in our previous work43 and Supplementary text Considering the two key sites as a proxy of time points along the chronosequence of the new terrestrial area, we investigated whether species that have been lost or acquired in the ‘late’ key site in compare with the ‘early’ site are randomly distributed, clustered or overdispersed across the evolutionary tree. The mean pairwise distance (MPD)—a measure that reflects the extent of phylogenetic clusteringTo determine whether species that had been lost in the ‘late' key site in comparison with the ‘early’ site preferably belonged to particular plant families, for each of 10 families of the ‘early’ site, the hypergeometric probability of losing the same or a greater number of species than were actually lost was counted under the null hypothesis that species were lost randomly.To calculate the probability of four species acquired in the ‘late’ key site to belong to the same family Amaranthaceae by chance, the probability of each family to acquire a new species in the ‘late’ site was assumed to be proportional to its contribution to the species content of the ‘early’ site (fraction of the ‘early’ key site species belonging to this family). For Amaranthaceae, the hypergeometric probability of appearance of four new species or more was calculated under this null assumption.Comparison of means  between microhighs and microlows at both key sites was performed with the nonparametric Mann–Whitney U-test.44. In a code for calculation of the taxonomic diversity index, “separate” function of “tidyr” package45 was used. Scripts that were written for this study are provided in Supplementary Folder All statistical analyses were performed with basic R language47 and Amaranthaceae. The last one includes the largest number of halophytes50. The ‘late’ site had 9 species of Amaranthaceae and 6 species of Poaceae, whereas the ‘early’ site had 6 species of Amaranthaceae and 10 species of Poaceae. Regarding the remaining eight families, the key sites had 1–3 (rarely 4) species belonging to them.The most abundant families in the dry steppes and deserts of the Caspian Sea region were PoaceaeTen families from the ‘early’ key site belonged to six orders and were not significantly clustered on the evolutionary tree . Half of the ‘early’ key site families (5 of 10) were from the order Caryophyllales, making this order significantly overrepresented  among the pioneer vegetation during the draining of the Caspian Sea , pointing that their extinction during succession was not random.As salinity is considered the limiting factor for plant growth in the Caspian Sea coast, we tested whether differences in salinity between the two key sites may influence difference in their vegetation. Among several depth intervals see “”, the pe53.To better understand the differences between 24 species that persisted in the 'late’ site and 11 species that persisted in the ‘early’ site but were absent in the ‘late’ site, two ecological characteristics of these species  were compared in binary form (yes/no) by Fisher’s exact test Table . For sprLimonium scoparium occurred significantly more frequently in plots with large tamarisk bushes than in plots without large tamarisk  tamarisk bushes seemed to influence the microtopographic structure and controlled the local microclimate around them. Tamarisk might serve as a nurse plant for some species in local patches. Therefore, some species could have been lost in the ‘late’ site due to tamarisk extinction. Among 10 species found in the ‘early’ key site in autumn, only –6) for microlows (26%) than for microhighs (16%). However, in the ‘late’ site, the situation was reversed, and microlows had smaller uncovered area than microhighs . When comparing the two key sites, microlows had a similar percentage of bare ground in both key sites (p = 0.07), but microhighs had twice as large uncovered ground area in the ‘late’ site than in the ‘early’ key site , it may lead to wrong conclusions when some factors other than time have considerable influence on the differences between studied sites55. However, as our key sites are located less than in 2 km from each other, they experience similar influence of climatic factors and are available for colonization by the same plants. As the two key sites appeared from the sea with interval about 1000 years, their initial climatic conditions potentially might be different. However, the studied area has remained semi-arid for the 3000 years20. In this work, we were interested in transformation of young dried areas. Therefore, only two sites were considered. However, it will be interesting to consider the whole chronosequence covering 3000 years of the history of the Caspian Sea retreat in semi-arid conditions.As the Caspian Sea gradually retreats, lands of different ages forming a chronosequence are available for research. In this paper, the system of two key sites that appeared from the Caspian Sea 1412 and 365 years ago were considered as a proxy of two time points to estimate how local plant communities appear and transform while the sea is drying. Such a technique of space-for-time substitution is frequently used in comparative studies of territories with different level of disturbance or different times since disturbance56. The previous work on the ‘early’ and ‘late’ key sites showed a decrease in species richness in the latter in comparison with the former. Meanwhile, the communities of the key sites were similar in terms of the number of biomorphotypes and annual herbs29. Here, using taxonomic diversity index, we found that despite differences in species richness, phylogenetic diversity was similar between the two key sites.A decrease in species richness (number of species) as the sea retreats and the newly formed land becomes drier was previously recorded in studies of the Aral Sea region60.Our evolutionary analysis of species that were unique for either of the two key sites showed that species loss in the ‘late’ site in comparison with the ‘early’ site occurred irrespective of evolutionary relationships, whereas acquired species were phylogenetically clustered. Therefore, we suggest that different plant traits are important to survive and appear de novo in dynamic communities forming in the changing environment of the Caspian Sea coast. Traits that were important for staying in a community did not show a pronounced phylogenetic signal, and different plants may survive due to different combinations of traits. In contrast, the set of features important for coming into a community seems to be narrow and to have a strong phylogenetic signal on the evolutionary tree of the local flora. One may speculate that it is good to be a generalist to survive in changing conditions of the Caspian Sea coast, whereas it is necessary to be a specialist to break into the existing but stressed community. All species growing in the ‘late’ site but absent in the ‘early’ site belonged to the Amaranthaceae family, which is known to increase in proportion with the climate desertification in steppes21) may serve as a nurse plant for some other plant species. The increase in the role of positive plant-plant interactions was previously shown for stressed environments62.Our results suggest that microtopography has a noticeable impact on vegetation in both key sites. In the ‘early’ key site, the salinity of root-inhabited soil layers was lower for microhighs than for microlows, where the percentage of bare ground was greater contrary to our hypothesis. Therefore, for the ‘early’ key site, soil salinity was likely a determinant of better growing conditions at microhighs than at microlows. Additionally, big tamarisk bushes growing at microhighs of the ‘early’ site , which indicates a more arid environment65. Therefore, species growing at the ‘late’ key site tended to be more tolerant to water loss than species growing at the ‘early’ site.As the ability to live in deserts appeared to be important for growth in the ‘late’ site, we suggest that soil moisture determines the composition of the plant community in the ‘late’ site. Disappearing of tamarisk in the ‘late’ key site may also suggest this, as tamarisk was shown to drop out of the community with the decrease in groundwater level in tugay forests of Middle AsiaIn the studied system, plant preferences were complexly correlated with microtopography and area age. Our hypothesis suggesting that for the studied area, the environment for plants is better in elevated areas than in lowlands was confirmed on macrolevel. However, on the level of microtopography it was confirmed only for elder site, whereas for the ‘early’ site our results suggest the opposite.The phylogenetic distribution of species that were lost in the ‘late’ site in comparison with ‘early’ site was random, whereas species that appeared in the ‘late’ site but were absent in the ‘early’ site were highly clustered on a phylogenetic tree, suggesting that vulnerability to a changing environment depends on traits that do not show a phylogenetic signal, but the ability to appear in community de novo requires properties possessed by the Amaranthaceae family. Mosaic patches of vegetation had a pronounced correlation with microtopography. Edaphic conditions of microhighs and microlows changed with time, leading to changes in the spatial distribution of plants.Our work demonstrates the importance of considering both evolutionary and ecological plant traits in studying plant community succession at the Caspian Sea coast.Supplementary Information.


Antioxidative peptides that inhibit myeloperoxidase (MPO) enzyme activity can effectively defend against oxidative stress damage. The antioxidant peptides from tuna protein were produced using alcalase hydrolysis and purified by ultrafiltration and Sephadex G-15, and the fractions with the highest free radicals scavenging ability and oxygen radical absorbance capacity (ORAC) values were sequenced using HPLC–MS/MS. Fifty-five peptide sequences were identified, 53 of which were successfully docked into MPO. The representative peptide ACGSDGK had better antioxidant activity and inhibition of MPO chlorination and peroxidation than the reference peptide hLF1-11. The docking model further showed intense molecular interactions between ACGSDGK and MPO, including hydrogen bonds, charge, and salt bridge interactions, which occluded the active site and blocked the catalytic activity of MPO. These results suggested that the antioxidant peptide ACGSDGK has the potential to inhibit oxidative stress and alleviate inflammation in vivo because of its inhibitory effect on the MPO enzyme. Thunnus albacares) is the second most important species of tuna, accounting for approximately 30% of the global catch (FAO). Fish trade produces underutilized fish by-products including trimmings, heads, viscera, frames, skin, fins, and roe, which account for more than 60% of the total biomass -benzoic acid (APF), which is selectively cleaved by hypochlorite to produce fluorescein. The 4-aminobenzhydrazide was used as positive control. The MPO inhibitory activity was determined according to the kit instructions.p < 0.05 was considered as significant.All data were expressed as means ± standard deviation (SD) with at least three dependent experiment and analyzed by one-way analysis of variance (ANOVA) with the Bonferroni multiple range tests using IBM SPSS 22.0 software. In this study, two peptides, ACGSDGK and KFCSGHA, with potent inhibition activity of MPO were obtained from tuna protein hydrolysate using conventional antioxidant-guided fractionations and molecular docking methods. ACGSDGK showed a greater antioxidant activity and inhibition of chlorination and peroxidation of MPO than KFCSGHA and GRRRRSVQWCA. This activity may be due to the two regions of ACGSDGK; that is, the Lys at the N-terminus binds to the crevice of the MPO active site by electrostatic interactions with Asp94 and Asp98 on the A chain of MPO, and Cys forms a hydrogen bond and salt bridge with His336 and two hydrogen bonds with Arg239 at the catalytic active site of MPO. These results indicated that the antioxidant peptide ACGSDGK has the potential to inhibit oxidative stress and reduce inflammation in vivo. However, the antioxidant and anti-inflammatory effects of ACGSDGK should be further verified with animal experiments.