Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco’s Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium. The drug 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has long been used in vitro as an exercise mimetic due to its metabolism into the AMP analog ZMP following cellular uptake . TraditimTOR, in contrast to AMPK, responds to conditions of nutrient sufficiency and influences metabolism through two distinct complexes, mTORC1 and mTORC2 . mTORC1 After metabolic stress in skeletal muscle, prolonged metabolic effects linger. For example, insulin sensitivity is increased for hours after either exposure to AICAR or a set of 10 maximal skeletal muscle contractions . LikewisGiven the evidence of sustained metabolic signals after metabolic stress and the interplay between AICAR, mTOR, and AMPK, the goal of this study was to elucidate the prolonged changes in the phosphorylation of mTORC1 and mTORC2 substrates after the exposure of C2C12 cells to AICAR. We hypothesized that AICAR-mediated kinase activation results in a unique phosphorylation profile involving mTOR substrates despite the conflicting roles of AMPK and mTORC1. C1C12 mouse myoblasts were differentiated for 48 h and subsequently tested to mimic the effects of AICAR on myofibers in an in vitro model. Our results indicated that certain downstream substrates of mTORC1/2 are affected while others remain unchanged, suggesting that AICAR-mediated activation causes the heterogenous phosphorylation of mTOR substrates in skeletal muscle cells.Unless otherwise specified, reagents were purchased from Sigma . The following listed antibodies were purchased from Cell Signaling Technologies : p-AMPKα-thr172 (2531S), AMPKα (2532S), p-ULK1-ser555 (5869S), p-ULK1-ser757 (6888S), ULK1 (8054S), GAPDH, HRP-conjugated (8884S), p-S6K-thr389 (9205S), S6K (9202S), p-Akt-ser473 (9271S), Akt (9272S), p-PKCα-thr638 (9375S), PKCα (2056S), p-Akt substrate (6950S), p-GSK3β-ser9 (5558S), and GSK3β (9315S). The antibodies for pSGK1-ser422 (Cat# 44-1264G) and SGK1 (Cat# PA5-21147) were purchased from Invitrogen . DMEM and PBS were purchased from Thermo Fisher .2 and passaged every 48–72 h.The C2C12 mouse myoblast cells were purchased from ATCC . The cell culture procedures were generally as described by Somwar et al. , with sl2 environment for differentiation. Myoblast differentiation was verified by observation using a phase-contrast microscope , and the cells were in differentiation medium for 2–3 days before the experiments, which were initiated only after the cultures developed an elongated morphology associated with differentiation. Afterwards, the cells were incubated in DMEM or Krebs–Henseleit Bicarbonate (KHB) buffer (pH 7.2–7.4) [The C2C12 cells were grown to confluence in a 12-well plate before incubation in DMEM with 2% horse serum (Sigma) and 1% pen/strep for 24–48 h in a 37 ℃, 5% CO2, 1 mM of EDTA, 10 mM of sodium pyrophosphate, 100 mM of NaF, 2 mM of Na3VO4, 10 μg/mL of aprotinin, 10 μg/mL of leupeptin, 0.5 μg/mL of pepstatin, and 0.2 mM of PMSF) and centrifuged at 14,000 rpm for 10 min, and the supernatant was saved [After treatment, the cells were rinsed and incubated in fresh DMEM or KHB with 5 mM of glucose for 3 h. The cells were scraped in a lysis buffer containing phosphatase and protease inhibitors , as per the manufacturer’s directions. In each enzyme activity assay, the activity was measured using the linear portion of the curve of the absorbance change at 340 nm for NADPH. The reaction-specific rate was calculated by subtracting sample blanks from the samples containing substrate. The activity of glucose-6-phosphate dehydrogenase (G6PD), phosphofructokinase (PFK), and fructose-1,6-bisphosphatase (FBP1) was measured as in Passoneau and Lowry .+, 0.5 M of EDTA, and 0.02% BSA was used. The absorbance change was recorded before and after the addition of 2 mM of glucose-6-phosphate.For G6PD activity, a reagent medium containing 100 mM of glycine (pH 9.4), 0.5 mM of NADP2HPO4, 1 mM of DTT, 2 mM of MgCl2, 0.1 mM of NADH, 1 U/mL of aldolase, 5 U/mL of G6PDH, and 15 U/mL of triosephosphate isomerase was used. The absorbance change was recorded before and after the addition of 1 mM of fructose-6-phosphate.For PFK activity, a reagent medium containing 50 mM of Tris-HCl (pH 8.1), 1 mM of AMP, 10 mM of K2, 0.1 mM of NADP+, 0.25 U/mL of phosphoglucoisomerase, and 0.18 U/mL of G6PDH was used. The absorbance change was recorded before and after the addition of 0.4 mM of fructose-1,6-bisphosphate. For FBP1 activity, a reagent medium containing 50 mM of Tris-HCl (pH 7.0), 100 mM of EDTA, 0.05% BSA, 2 mM of DTT, 2 mM of MgClThe samples were run on 4–20% tris-glycine SDS-polyacrylamide gels  and transferred to 0.1 µm pore size nitrocellulose membranes for blotting. To determine the molecular weight of the target proteins, a pre-stained protein ladder  was loaded alongside the samples. The membranes were incubated overnight in a 1:1000 concentration of primary antibody in 1% milk in TRIS-buffed saline with tween (TBST). The membranes were then incubated in a 1:5000 concentration of secondary antibody for one hour before washing with TBST, followed by incubation with Western Lightning enhanced chemiluminescence reagent  and imaging . After the imaging, the membranes were stripped by washing with 0.2 N NaOH twice for five minutes each time, followed by washing with TBST for ten minutes twice each time.A Western blot densitometric analysis was performed using ImageJ software (NIH). The level of protein expression was calculated as a ratio of the band intensity for the phosphorylated protein to total protein, or to a loading control (GAPDH) as appropriate. The intensity values were normalized, with the control equal to 1.0.t-test was used. For the comparison of more than two groups, one- or two-way ANOVA was used. Fisher’s LSD was used for post hoc testing.Data are expressed as mean ± standard error of the mean (SEM). Statistical significance was tested in SPSS Statistics 25 (IBM) with an alpha value of 0.05. For the comparison of two groups, Student’s One hour of incubation of the C2C12 cells with the AMP analog AICAR was sufficient for increasing the AMPK phosphorylation at its activating thr172 site, which we validated through repeat experimentation A,B. ImmeIt has previously been reported that the activation of AMPK causes a decrease in the expression of glucose-6 phosphate dehydrogenase (G6PD) , as wellThe nutrient-dependent effects of prior exposure to AICAR shown in Given these findings, subsequent experiments were performed for cells that recovered in the presence of amino acids  versus their absence . When recovering in DMEM, prior AICAR exposure did not alter the phosphorylation of PKCα, SGK1, or ULK1 at their mTOR target sites D–F. CollPrior exposure to AICAR also caused an increase in the phosphorylation of Akt at ser-473, an mTORC2 target site. This effect appeared to be specific to Akt ser-473, as the phosphorylation of the mTORC2 targets PKCα thr-638 and SGK1 ser-422 was unchanged by prior exposure to AICAR . These rConsidering the immediate increase in ULK1-ser555 phosphorylation following the AICAR treatment C and proin lieu of mTOR. To address this possibility, we assessed the phosphorylation patterns after exposure to rapamycin, which inhibits mTORC1 but not mTORC2 [The data regarding the specific increase in phosphorylation at a subset of mTOR targets raised the question of whether other kinases could be acting at these sites t mTORC2 , and PP2t mTORC2 . As expeThe new information provided by this study shows that prior treatment with AICAR in differentiated C2C12 cells led to a selective increase in the phosphorylation of the mTORC1 target S6K and the mTORC2 target Akt, despite the phosphorylation of AMPK returning to baseline following recovery. Further, the C2C12 cell response to prior treatment with AICAR appeared to be dependent on the presence or absence of nutrients, in particular amino acids. It should be noted, however, that due to the density of the cells studied, most but not all of the myocytes were differentiated at the time of experimentation, and therefore the results described in this report do not exclusively refer to myotubes. Another caveat is that this was an in vitro study in which cell cultures were used to mimic myofibers. Although beyond the scope of this study, further ex vivo studies on harvested tissue lysates and sections would add additional perspectives to our findings.In a nutrient-replete state, such as after eating a carbohydrate-rich meal, the pancreas secretes insulin, which binds to insulin receptors on the cell membrane, activating insulin receptor substrate 1 (IRS1)-based signaling that activates phosphatidylinositol-3-kinase (PI3K) . PI3K geIn the current study, increased Akt substrate phosphorylation after prior exposure to AICAR G was assWhile mTORC2 aids in the Akt-induced phosphorylation of AS160 , mTORC1 Both mTOR complexes are similar, with the primary distinction being the presence of either Raptor (in mTORC1) or Rictor (in mTORC2) . They arAutophagy, a self-degradative cellular process, is highly upregulated in response to cell stress, the excess accumulation of proteins, or low nutrient resources . What re50 of 8 nM and is highly specific, its next-closest inhibition target being DNA-dependent protein kinases at 410 nM [PP242 inhibits mTOR activity by binding at the ATP-binding site, resulting in decreases in the activity of both mTOR complexes . Since tt 410 nM ,50,51. Ot 410 nM .In conclusion, prior exposure to the AMP analog AICAR caused a unique downstream phosphorylation profile in C2C12 cells immediately following and after a sustained recovery period. Under nutrient-replete conditions, prior exposure to AICAR caused a prolonged increase in the phosphorylation of the mTORC1 and mTORC2 targets, S6K and Akt, respectively. However, the phosphorylation of the other mTORC1 and mTORC2 targets, ULK1, SGK1, and PKCα, was unchanged. Thus, metabolic stress simulated by AICAR was sufficient to activate mTOR-regulated pathways, leading to protein synthesis (S6K) and the acquisition and storage of nutrients (Akt).


In spinal surgery, precise instrumentation is essential. This study aims to evaluate the accuracy of navigated, O-arm-controlled screw positioning in thoracic and lumbar spine instabilities.Posterior instrumentation procedures between 2010 and 2015 were retrospectively analyzed. Pedicle screws were placed using 3D rotational fluoroscopy and neuronavigation. Accuracy of screw placement was assessed using a 6-grade scoring system. In addition, screw length was analyzed in relation to the vertebral body diameter. Intra- and postoperative revision rates were recorded.Thoracic and lumbar spine surgery was performed in 285 patients. Of 1704 pedicle screws, 1621 (95.1%) showed excellent positioning in 3D rotational fluoroscopy imaging. The lateral rim of either pedicle or vertebral body was protruded in 25 (1.5%) and 28 screws (1.6%), while the midline of the vertebral body was crossed in 8 screws (0.5%). Furthermore, 11 screws each (0.6%) fulfilled the criteria of full lateral and medial displacement. The median relative screw length was 92.6%. Intraoperative revision resulted in excellent positioning in 58 of 71 screws. Follow-up surgery due to missed primary malposition had to be performed for two screws in the same patient. Postsurgical symptom relief was reported in 82.1% of patients, whereas neurological deterioration occurred in 8.9% of cases with neurological follow-up.Combination of neuronavigation and 3D rotational fluoroscopy control ensures excellent accuracy in pedicle screw positioning. As misplaced screws can be detected reliably and revised intraoperatively, repeated surgery for screw malposition is rarely required. The lifetime prevalence of back pain in Germany is 85.5%, with men and women over the age of 50 being particularly affected . Due to The purpose of this retrospective study was to evaluate the accuracy of pedicle screw positioning in navigated, O-arm-controlled posterior instrumentation for the thoracic und lumbar spine.n = 34) were also left out of the analysis.Retrospective data analysis was approved, and informed consent was waived by the local ethics committee. Information on patient history and surgical procedure was obtained from the clinical information system  and anonymized for further analysis. The evaluation of intraoperative imaging in terms of screw position and screw length was performed using an open-source DICOM viewer program (OsiriX Lite 8.0.1). For this study, we retrospectively analyzed data from patients who underwent dorsal spinal instrumentation with 3D fluoroscopic navigation  at the local neurosurgical clinic between June 2010 to June 2015. Treatment indication was based on spinal instability due to degenerative, traumatic, inflammatory or tumor-related conditions. Inclusion criteria included surgical treatment via a dorsal approach , surgery performed with an open or percutaneous technique and at least one rotational 3D fluoroscopy scan after dorsal instrumentation to evaluate the position of the screws. Patients who did not receive a rotational 3D scan were excluded from this study. Furthermore, screws that were not included in the field of view of the initial 3D fluoroscopy scan  opposite a large flat-panel detector . In 3D mode, the O-arm creates a series of projection images during a complete 360° rotation. Gantry rotation speed can be set to 30° per second in standard mode or 15° per second in high-definition mode with up to 400 or 750 images generated during a full 360° rotation. By integrating the navigation system  into the scanner setup, intraoperative imaging can be used directly for neuronavigation. This approach enables periprocedural display of entry points as well as identification of important neighboring structures.Qualitative evaluation of screw positioning for the thoracic and lumbar spine was performed using the 6-grade scoring system described by Zdichavsky et al., in which grade Ia represents an excellent position, whereas grades IIIa and IIIb are supposed to be surgically revised , 8. The t-tests were conducted to determine whether two normally distributed samples differ significantly. P values ≤ 0.05 were considered to indicate statistical significance.All data were transferred to a standard spreadsheet  for further processing. Statistical analysis was performed using dedicated software . Normal distribution was assessed with Kolmogorov–Smirnov tests. For normally distributed continuous variables, we report mean values and standard deviation, whereas absolute values and percentage distribution are displayed otherwise. Chi-square tests were applied to compare categorical data. To measure the effect size of the chi-square-test, Cramer-V was computed. Student’s Between June 2, 2010, and June 29, 2015, 285 patients  underwent 295 dorsal screw-rod instrumentations with 62 procedures performed on the thoracic and 233 on the lumbar spine. Lumbar vertebra 4 was stabilized most often (185 times) and thoracic vertebra 9 least often (14 times). Mean patient age at the time of surgery was 64.1 ± 12.6 years with almost 69% of patients over 60 years of age. Indication to perform surgical treatment was most frequently based on tumor-induced instability in the thoracic spine (46.8%) and degeneration-induced instability in the lumbar spine (76.8%). Table Of 1704 included screws, 1621 (95.1%) showed an excellent position (Ia) in the initial intraoperative imaging. Of the remaining pedicle screws, 25 (1.5%) protruded the lateral rim of the pedicle (Ib), 28 (1.6%) protruded the lateral margin of the vertebral body (IIa), 8 (0.5%) crossed the vertebra’s midline (IIb). Furthermore, 11 screws each (0.6%) fulfilled the criteria of full lateral (IIIa) or medial displacement (IIIb). Median screw length was 92.6% of the maximum diameter of the vertebral body with 1224 screws (71.8%) displaying “good” length in the first scan.After intraoperative revision, 58 of 71 screw positions were classified as Ia and one screw was classified as Ib. The other 12 revised screws were either not included in the field of view of the post-revision 3D scan or no further imaging was performed after intraoperative repositioning. Good relative length was ascertained in 1238 screws (72.7%). Screw position grading and relative length before and after intraoperative revision are summarized in Figs. At follow-up, 82.1% of patients declared total pain release or at least significant improvement of back and/or leg pain after surgery. No patient reported aggravating or new pain after surgery at the control examination. Neurological follow-up showed significant improvement or complete remission of symptoms in 70.8% of patients with neurological deficits. In contrast, 8.9% of patients with follow-up had new neurological symptoms that were not reported preoperatively.In this study, the accuracy of pedicle screw placement in the thoracic and lumbar spine was investigated using a combined approach of neuronavigation and intraoperative 3D rotational fluoroscopy. High precision of implant positioning was achieved in all spinal sections. With a total of 1738 screws placed, intraoperative revision was performed for 78 screws, whereas repeated surgery due to a missed malposition was necessary in just one patient.The classification system of Zdichavsky et al. represents a validated concept for determining the accuracy of pedicle screw placement in the thoracic and lumbar spine , 8. Our Revision surgery frequencies of up to 5.2% have been described in various studies on neuronavigated spinal surgery –22, whicPerdomo-Pantoja et al. showed in a recent meta-analysis on the accuracy of pedicle screw placement with different techniques that the highest accuracy results from CT navigation . NeverthSeveral limitations have to be addressed for this study. Since we performed a retrospective analysis, data quality regarding long-term outcome, neurological status and pain relief was inconsistent. Intraoperative revision rates of 4.2% were slightly higher than in comparable studies , 26. HowCombination of neuronavigation and 3D rotational fluoroscopy control ensures excellent accuracy in pedicle screw positioning. As misplaced screws can be detected reliably and revised intraoperatively, repeated surgery for screw malposition is rarely required.


Total immunoglobulin E (IgE) analysis is a common tool in allergy diagnosis. Suggested reference values for IgE are divergent and sometimes based on outdated assay methods. We aimed to validate the published reference values  shown in an Uppsala cohort from 1974 using Phadebas IgE PRIST, and the suggested clinical threshold of 100 kU/L (Zetterström and Johansson 1981).Immunoglobulin E was measured in two Uppsala cohorts from 1997 (Blood bank) and 2011 to 2013  using ImmunoCAP™ Total IgE. For the reference value calculations, exclusion criteria were atopy (both cohorts), doctor’s diagnosis of asthma and self-reported allergy  (only ECRHS III). Upper limit of normal was defined as mean + 2 standard deviations (SD) calculated using log-transformed values and back-transformation of the ULN prior to presentation. Common imputation methods for results below the assay range were evaluated.n = 63; imputation method range: 16.9–17.4 kU/L; ECRHS III, n = 113: 10.7–11.6 kU/L) and the overall mean ULN was 118 kU/L . The clinical sensitivity and specificity of the 100 kU/L IgE threshold were 37.8 and 94.3% for atopy, 34.9 and 89.5% for doctor’s diagnosis of asthma, and 24.5 and 97.3% for any self-reported allergy (ECRHS III).The average GM was 14.2 kU/L (Blood bank, The calculated ULN values were similar between the cohorts. We conclude that the total IgE reference values shown for Uppsala subjects from 1974 are still valid and suitable also for the ImmunoCAP Total IgE assay. The 100 kU/L threshold for total IgE had a low sensitivity but high specificity for atopy, asthma, and allergy. Measurement of immunoglobulin E (IgE) in serum and plasma  is commonly used as a complement to the investigation of sensitisation to allergens in patients with possible allergic disease. Total IgE is measured by laboratory assays, and is significantly elevated in patients with allergic asthma, hay fever or atopic eczema –3. HowevPhadebas IgE PRIST launched in 1976 was the leading total IgE assays in early attempts to establish reference values for IgE in healthy individuals . One of It has been argued that the reference range and the clinical threshold for total IgE recommended by Zetterström and Johansson is no more valid since their study is both old and performed with an assay not presently available. Indeed, more recent studies using the ImmunoCAP Total IgE assay have shown various values for the ULN, mostly at higher IgE levels , 5, 12. The aim of the present study was to validate the reference values reported by Zetterström and Johansson in 1981 by measuring IgE in two Uppsala cohorts collected in 1997 and 2011 to 2013 using the ImmunoCAP Total IgE assay. Moreover, the performance of the suggested and commonly used diagnostic threshold for total IgE at 100 kU/L was evaluated.Two sample cohorts from Uppsala were included. Venous serum samples were collected and prepared according to standard routines at the Uppsala University Hospital and were stored frozen (-20°C) until analysis. Besides total IgE measurement, all the samples were also analysed for the presence of IgE antibodies to common inhalant allergens. The first cohort from 1997 consisted of 95 serum samples from anonymous regular adult blood donors obtained from the blood bank of the Uppsala University Hospital (Blood bank cohort). They were considered as being healthy according to the routines of the blood bank, although having allergy without present symptoms were allowed for blood donors. The second cohort from 2011 to 2013 was from the European community respiratory health survey part III (ECRHS III) in Uppsala including 321 serum samples from adult subjects aged 40–66 years (ECRHS III cohort). Extensive clinical information was available for the subjects in the latter cohort based on self-reported questionnaire data and clinical examination. The ECRHS III study has been described more in detail elsewhere , and/or subjects with negative questionnaire data (only available for the ECRHS III cohort) for having a history of doctor’s diagnosis of asthma or any current allergic symptoms  were selected. Other potential causes of elevated serum IgE, for example parasitosis, were disregarded because of the very low incidence in the geographic area.Serum IgE was measured using the ImmunoCAP™ Total IgE assay with the measuring range of 2–5,000 kU/L . The Blood bank cohort was analysed at Pharmacia Diagnostics in Uppsala in 1997, and the ECRHS III cohort was analysed at Academic Medical Centre, University of Amsterdam, The Netherlands, in 2013–14.Serum IgE antibodies to a mix of common inhalant allergens were measured using ImmunoCAP Phadiatop™ (Thermo Fisher Scientific). Atopy was defined as having a positive Phadiatop test result (>0.35 Phadia arbitrary units per litre). The Phadiatop testing was performed at Pharmacia Diagnostics/Thermo Fisher Scientific, ImmunoDiagnostics in 1997 (Blood bank cohort) and 2019 (ECRHS III cohort).Geometric mean of total IgE and the lower limit normal (LLN) and ULN were calculated according to Zetterström and Johansson . LLN andDiagnostic performance of total IgE was evaluated from receiver operating characteristics (ROC) analysis where the area under curve (AUC) indicated the overall diagnostic performance in relation to the clinical parameter. The ROC curve was used to calculate the optimal IgE cut-off for each clinical parameter and was defined as the concentration corresponding to shortest distance from the ROC curve to the upper left corner. The diagnostic sensitivity and specificity of the established 100 kU/L total IgE threshold and the optimal IgE cut-offs were calculated.Statistical calculations were done using Prism v.8.1.2 .The use of the ECRHS III cohort was covered by ethical approvals by the Ethic Committee at the Medical Faculty of Uppsala University, Sweden (Dnr 1999/313 and 2010/068). Since the samples in the Blood bank cohort were from fully anonymous regular blood donors at the hospital (no tracking possible to subjects), no ethical approval was deemed required for the use of the samples in the present study in accordance with Swedish regulations.A total of 63 samples (66.3%) from the Blood bank cohort showed negative Phadiatop results and were included in the study as samples from non-atopic apparently healthy blood donors . The GM of total IgE of these blood donors was 17.4 kU/L and the ULN was 113 kU/L when using the BLQ 1 method for arbitrary value imputation of results below the LOQ of the assay (i.e. 2 kU/L was used for the BLQ results). The GM slightly decreased, and the ULN increased up to 130 kU/L, when using the BLQ 3 method (imputation with LLOQ/2 = 1 kU/L) because of an increased SD .In the ECRHS III cohort, 113 subjects (35.2%) were classified as being healthy non-atopic subjects  and were included in the reference value calculations . The GM of total serum IgE in this cohort was 10.7–11.6 kU/L dependent on which of the BLQ methods that was used . No diffThe overall GM and overall mean ULN obtained from the Blood bank cohort and ECRHS III cohort using the three BLQ methods were 14.2 kU/L and 118 kU/L, respectively.Having clinical information about allergic diseases in the ECRHS III Uppsala study, we studied the diagnostic performance of the commonly used 100 kU/L threshold for total IgE. The following three clinical parameters were studied: 1) Doctor’s diagnosis of asthma, 2) Any reported allergy , and 3) Atopy.About 35% of the subjects with asthma had total IgE >100 kU/L, and the clinical specificity of that threshold was 89.5% . The senROC analysis showed that total IgE as diagnostic marker performed best in identifying atopy with an AUC of 0.810 . The optIn the present study, reference values for total IgE in non-allergic apparently healthy subjects from Uppsala were established in two separate cohorts collected in 1997 (Blood bank cohort) and 2011 to 2013 (ECRHS III cohort). They were compared with the reference values obtained in Uppsala samples collected in 1974 and analysed using Phadebas IgE PRIST and reported by Zetterström and Johansson in 1981  is valid also today. In the ECRHS III Uppsala study, the 100 kU/L threshold for total IgE showed a high clinical specificity for atopy (positive Phadiatop test), doctor’s diagnosis of asthma, and any reported allergy-like symptoms, ranging from 89.5 to 97.3%. The sensitivity was considerably lower and did never exceed 40%. Lowering the threshold for total IgE to the individually calculated optimal cut-offs led to a sensitivity and specificity of 73.0 and 74.8% for atopy; but for doctor’s diagnosis of asthma and any allergy-like symptoms, the diagnostic accuracy was lower. Today it is generally recognised that measuring specific IgE antibodies has a higher diagnostic value than measuring total IgE in allergy considering the low diagnostic accuracy of the latter .A limitation of the present study is the long time between the original investigation by Zetterström and Johansson published in 1981, in which the Phadebas PRIST IgE assay was used, and the present study, where all analyses were done using ImmunoCAP. However, the comparison of the results from the two total IgE assays and over time was supported by the common calibration of the assays against international reference preparations for IgE , 19. BesTo summarise, the present study has validated the GM (13.2 kU/L) and ULN (114 kU/L) for total IgE reported by Zetterström and Johansson in 1981 for adult non-atopic apparently healthy individuals in Uppsala in a cohort from 1974. The present study showed an overall mean ULN of 118 kU/L for total IgE in serum from Uppsala subjects collected in 1997 and 2011 to 2013, and analysed using ImmunoCAP Total IgE. The previously suggested diagnostic threshold for total IgE (100 kU/L) as an aid in diagnosis of allergy was also evaluated and showed a high clinical specificity (89.5–97.3%) but a low sensitivity (<40%).


Alternaria alternata, Aspergillus flavus, Aspergillus ochraceus, Fusarium graminearum, and Fusarium verticillioides), and inhibitory activity on enzymes . All the tested compounds showed weak radical scavenging activity, while antifungal activity strongly depended on the tested concentration and fungal species. Biflavonoids, especially ginkgetin and isoginkgetin, proved to be potent acetylcholinesterase inhibitors, whereas monomeric flavonoids showed higher tyrosinase inhibitory activity than the tested 3′-8″-biflavones. Amentoflavone proved to be a potent α-amylase and α-glucosidase inhibitor, and in general, 3′-8″-biflavones showed a stronger inhibitory potential on these enzymes than their monomeric subunits. Thus, we can conclude that 3′-8″-dimerization enhanced acetylcholinesterase, α-amylase, and α-glucosidase activities, but the activity also depends on the number of hydroxyl and methoxy groups in the structure of the compound.Biflavonoids are dimeric forms of flavonoids that have recently gained importance as an effective new scaffold for drug discovery. In particular, 3′-8″-biflavones exhibit antiviral and antimicrobial activity and are promising molecules for the treatment of neurodegenerative and metabolic diseases as well as cancer therapies. In the present study, we directly compared 3′-8″-biflavones  and their monomeric subunits  and evaluated their radical scavenging activity (with DPPH), antifungal activity against mycotoxigenic fungi ( O-prenylation has also been found [O-methylquercetin. According to Boozari et al. [L-rhamnose, D-glucose, glucose–rhamnose, galactose, or arabinose [Flavonoids have been the focus of scientific attention for more than 40 years and 8000 different structures have been described to date . Howeveren found . The hyden found , the 2,3i et al. , the prei et al. . The samrabinose .A. alternata, A. flavus, A. ochraceus, F. graminearum, and F. verticillioides)  , and inhElectrophorus electricus (CAS 9000-81-1), 5,5′-dithiobis(2-nitrobenzoic acid) (CAS 69-78-3), 3,4-dihydroxy-L-phenylalanine (CAS 59-92-7), α-amylase from porcine pancreas (CAS 9000-90-2), 2,2-diphenyl-1-picrylhydrazyl (CAS 1896-66-4), 3,5-dinitrosalicylic acid (CAS 609-99-4), potassium sodium tartrate tetrahydrate (CAS 6381-59-5), starch (CAS 9005-84-9), tyrosinase from mushroom (CAS 9002-10-2), α-glucosidase from Saccharomyces cerevisiae (CAS 9001-42-7), 4-nitrophenyl α-D-glucopyranoside (CAS 3767-28-0), and RPMI-1640 medium were purchased from Sigma-Aldrich . Bilobetin (2) (CAS 521-32-4), isoginkgetin (3) (CAS 548-19-6), ginkgetin (4) (CAS 481-46-9), sciadopitysin (5) (CAS 521-34-6), genkwanin (7) (CAS 437-64-9), and acacetin (8) (CAS 480-44-4) were obtained from PhytoLab . Sodium phosphate, monobasic monohydrate (CAS 10049-21-5), Sodium phosphate, dibasic heptahydrate (CAS 7782-85-6), and dimethyl sulfoxide (CAS 67-68-5) were purchased from Thermo Fisher Scientific . Apigenin (6) (CAS 520-36-5) was purchased from Alfa Aesar  and S-acetylthiocholine iodide (CAS 1866-15-5) was purchased from Biosynth . Methanol (CAS 67-56-1) was obtained from Kemika  and sodium hydroxide (CAS 1310-73-2) was obtained from T.T.T. .Amentoflavone (1) (CAS 1617-53-4), 4-nitrophenyl α-D-glucopyranoside (CAS 3767-28-0), Tris base (CAS 77-86-1), acetylcholinesterase from DPPH scavenging activity was determined according to the method of Brand-Williams et al. . In the Alternaria alternata (WT), Aspergillus flavus (NRRL 3251), Aspergillus ochraceus (CBS 589.68), Fusarium graminearum (CBS 110.250), and Fusarium verticillioides (CBS 119.825)  . The comThe acetylcholinesterase inhibition assay was performed using the reaction-based assay of Ellman  with modThe tyrosinase inhibition assay was performed according to Jakimiuk et al. , with slThe α-amylase inhibition assay was performed according to Etsassala et al. , with slThe α-glucosidase inhibition assay was performed according to Tiwari et al. , with slp < 0.05.All analyses were performed in at least triplicate, and the results are expressed as mean ± standard deviation (SD). All statistical analyses were performed using PAST software (version 4.13) . One-wayHere, we studied five 3′-8″-biflavones  and their monomeric subunits , and their structures are shown in As shown in We measured antioxidant activity using the commonly used DPPH method, and the results are presented in Overall, all tested compounds showed very weak or no radical scavenging capacity, with no significant differences between them. The flavonoid quercetin, for example, showed 100% inhibition at the same concentration.In our experiment, we compared the antifungal activity against five fungi  at diffeF. graminearum, and the biflavonoids showed higher inhibition than the tested monomers. In the case of F. verticillioides, all biflavonoids and apigenin showed inhibition only at a concentration of 10 µg/mL, whereas at lower concentrations, they showed no inhibition or even slightly growth-promoting effects. In contrast, the antifungal activity against A. ochraceus was higher for all biflavonoids and genkwanin at lower concentrations and decreased with the increase in concentration. All biflavonoids showed antifungal activity against A. flavus, and the activity increased with concentration for most biflavonoids. Apigenin showed antifungal activity against A. flavus only at a concentration of 1 µg/mL, while genkwanin showed dose-dependent stimulatory effects on this fungus. In the case of A. alternata, antifungal activity was strongly concentration-dependent, but in general, biflavonoids showed weak activities, while apigenin and genkwanin showed no antifungal activity.As can be seen from the table, the antifungal activity depends on both the fungi used and the tested concentration. At the lowest concentration, 0.01 µg/mL, all the tested compounds showed significant inhibition only against Enzyme inhibition activities of the compounds at 100 μM are shown in 2− scavenging assay, the Cu2+ reducing assay, and the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay, and reported that the 3′,8″-dimerization increased the antioxidant capacity of flavonoids. From all these findings, we can conclude that the antioxidant activity of biflavones is still very poorly understood, and further studies should clarify their activity as antioxidants.Flavonoids are often considered antioxidants, but in reality, their antioxidant activity is highly dependent on the structure of individual flavonoids . This isFusarium generates a number of mycotoxins that can cause acute or chronic disease and, in some cases, death [F. graminearum, and biflavonoids showed higher inhibition than the monomers analyzed. When we directly compared amentoflavone with its monomeric subunits apigenin, we found that amentoflavone had a stronger inhibition against F. graminearum, which may indicate that the 3′-8″-dimerization increases the antifungal activity against this fungus. In the case of F. verticillioides, all biflavonoids and apigenin showed inhibition at a higher concentration (10 µg/mL). Krauze-Baranowska and Wiwart [F. culmorum, and at a concentration of 100 μM/mL, bilobetin, ginkgetin, and sciadopitysin showed 100% inhibition. It has also been reported that the extracts of Hypericum triquetrifolium have antifungal activity against Fusarium sp. [Hypericum sp. are known to contain biflavonoids [Fusarium sp. Other mycotoxigenic fungi we tested belong to Aspergillus sp., namely A. flavus and A. ochraceus. All the tested biflavonoids, as well as genkwanin, showed antifungal activity against A. ochraceus, which was higher at lower concentrations and decreased with the increase in concentration, whereas for A. flavus, activity increased with concentration for most biflavonoids. Previously, isoginkgetin was reported to have inhibitory activity against A. fumigatus [A. flavus but did not inhibit fungal growth at the concentration tested. They indicated that biflavonoids may be effective agents for controlling aflatoxin production without inhibiting growth. All the compounds we tested here showed weak or no inhibition against A. alternata at concentrations up to 10 μg/mL. Krauze-Baranowska and Wiwart [A. alternata, while the inhibition of amentoflavone, bilobetin, and sciadopitysin was 54%, 80%, and 59%, respectively.Monomeric flavonoids have been shown to be effective antifungal agents against a variety of pathogenic organisms through mechanisms that include the disruption of the plasma membrane; the induction of mitochondrial dysfunction; and the inhibition of cell wall formation, cell division, RNA and protein synthesis, and efflux-mediated pumping . Herein,s, death . At the d Wiwart  investigrium sp. , and Hypavonoids . Taken tumigatus . Gonçaleumigatus  reportedd Wiwart  investigGinkgo biloba L. [For the enzyme inhibitory activities of our compounds, we selected four enzymes that are involved in important metabolic functions. Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer’s disease and other dementias because the inhibition of AChE slows the hydrolysis of acetylcholine and increases choline levels, which improves cognitive function. Monomeric flavonoids have been recognized as AchE inhibitors for several decades . Accordiiloba L. ,28,38, ailoba L. . The inhiloba L. . Accordiiloba L. , amentofWe also investigated the inhibitory activity against α-amylase and α-glucosidase, the two enzymes involved in carbohydrate metabolism. The inhibition of these enzymes may help regulate blood glucose levels and prevent/control diabetes mellitus . AccordiA. alternata, A. flavus, A. ochraceus, F. graminearum, and F. verticillioides), and enzyme inhibition  of five 3′-8″-biflavones  and their monomeric subunits . All the tested compounds showed weak radical scavenging activity. The antifungal activity strongly depends on the fungi used and the concentration. At some concentrations, we even detected growth-promoting activity, as in the case of F. verticillioides. At the lowest concentration, 0.01 µg/mL, all the analyzed compounds showed significant inhibition against F. graminearum, and the biflavonoids showed higher inhibition than the tested monomers. The antifungal activity against A. ochraceus was higher for all biflavonoids and genkwanin at lower concentrations and decreased with the increase in concentration, while all biflavonoids showed antifungal activity against A. flavus, and the activity increased with concentration for most biflavonoids. Biflavonoids showed weak activities against A. alterata, while apigenin and genkwanin showed no antifungal activity. Isoginkgetin and ginkgetin showed the highest inhibition against acetylcholinesterase, while monomeric compounds showed higher inhibitory activity against tyrosinase. Amentoflavone proved to be a potent inhibitor of α-amylase and α-glucosidase, and the 3′-8″-biflavones tested showed higher inhibitory activity against α-glucosidase than their monomeric subunits. From all these results, we can conclude that the 3′-8″-dimerization affects the biological activity, such as the antifungal activity against F. graminearum, where the biflavonoids showed higher inhibitory activity than the tested monomers. Also, the inhibitory activity against α-amylase and α-glucosidase is enhanced by 3′-8″-dimerization but is also influenced by the number of methoxy and hydroxyl groups.In the present study, we evaluated the radical scavenging activity (with DPPH), antifungal activity against mycotoxigenic fungi (


Seasonal cycles within the marginal ice zones in polar regions include large shifts in temperature and salinity that strongly influence microbial abundance and physiology. However, the combined effects of concurrent temperature and salinity change on microbial community structure and biochemical composition during transitions between seawater and sea ice are not well understood. Coastal marine communities along the western Antarctic Peninsula were sampled and surface seawater was incubated at combinations of temperature and salinity mimicking the formation  and melting  of sea ice to evaluate how these factors may shape community composition and particulate metabolite pools during seasonal transitions. Bacterial and algal community structures were tightly coupled to each other and distinct across sea-ice, seawater, and sea-ice-meltwater field samples, with unique metabolite profiles in each habitat. During short-term (approximately 10-day) incubations of seawater microbial communities under different temperature and salinity conditions, community compositions changed minimally while metabolite pools shifted greatly, strongly accumulating compatible solutes like proline and glycine betaine under cold and salty conditions. Lower salinities reduced total metabolite concentrations in particulate matter, which may indicate a release of metabolites into the labile dissolved organic matter pool. Low salinity also increased acylcarnitine concentrations in particulate matter, suggesting a potential for fatty acid degradation and reduced nutritional value at the base of the food web during freshening. Our findings have consequences for food web dynamics, microbial interactions, and carbon cycling as polar regions undergo rapid climate change. Surface ocean temperatures along the western Antarctic Peninsula (WAP) have increased by >1 °C since the 1950s , 3. GlacWith global air temperatures expected to rise at least 1–3 °C over the next century , coastalTransitions between polar marine habitats are associated with shifts in light, nutrients, temperature, and salinity –22. HoweCompatible solutes, or osmolytes, are small organic molecules in a cell’s cytosol that maintain turgor pressure and stabilize enzymes; they also play other roles , 26. ForCompatible solutes contribute to the larger pool of intracellular metabolites. Although marine particulate organic matter is composed largely of macromolecules , small pHere, we characterize community structure and particulate metabolite profiles of bacterial and protist communities in sea-ice meltwater, seawater, and sea ice during austral spring along the WAP, recognizing that both the taxonomic composition of microbial communities and the chemical inventory of metabolites impact the flux of carbon and energy though polar food webs. We also examine the flexibility of metabolite pools in the surface seawater community under temperature and salinity conditions that mimic the formation and melt of sea ice. As polar oceans experience rapid warming, increased freshwater inputs, and shifts in seasonal ice formation and melt, this research serves to deepen our understanding of the potential responses of the microbial communities.Lawrence M. Gould using a Kovacs MARK II ice auger during LMG1810 (Cruise 10.7284/907972). For all ice cores, the bottom 5-cm sections were melted in the dark at 4 °C into prefiltered (0.2 μm) seawater to avoid osmotic shock and cell lysis. Final salinities of ice samples following this melt procedure are provided in Table Sea-ice meltwater, seawater, and sea-ice samples were collected near Palmer Station, Anvers Island, Antarctica in November of 2018 , we collected additional seawater for incubation experiments that simulated temperature(T)-salinity(S) conditions of sea-ice meltwater , ambient seawater , and sea ice . Triplicate 10-L polycarbonate carboys were used for each treatment. All samples were enriched with f/2 nutrients with silica  to prevea (Chl a), particulate organic carbon and nitrogen , DOC, particulate and dissolved extracellular polysaccharides , and major nutrients . All methods for the collection and processing of these samples are described in Supplementary information. Temperature, salinity, and photosynthetically active radiation (PAR) were measured using a digital thermometer, refractometer, and Walz US-SQS/L spherical quantum sensor with ULM-500 light meter, respectively.Field and incubation samples were processed for particulate metabolomics, DNA sequencing, chlorophyll Metabolites within the particulate pool were extracted from samples filters using a modified Bligh and Dyer extraction, separated via liquid chromatography with a Waters Acquity I-Class UPLC equipped with either a reversed phase or hydrophilic interaction liquid chromatography column, ionized with electrospray ionization with a Thermo Q-Exactive mass spectrometer, and data was acquired in full scan mode as modified from Boysen et al. (2018). Further details on metabolite extraction and LC-MS analysis are provided in Supplementary information.Metabolite peaks obtained from mass spectrometry were integrated using Skyline for small molecules . Full anAbsolute concentrations of 134 compounds were calculated or estimated from peak areas using commercially available standards run in the same batch as our samples, similar to previous work , 42, 44.All methods for DNA extraction, high throughput sequencing, and analysis for unique amplicon sequence variants (ASVs) are given in Supplementary information. The Inverse Simpson alpha diversity index was calculated using the phyloseq package in R  followinp values corrected for false discovery rate (FDR)  and Candidatus Thioglobus [SUP05]) during the onset of the phytoplankton bloom in situ  with distinct physicochemical properties showed unique community structures and taxonomic compositions Figs. –S3. Thisseawater –56. Commitu Fig. , as showitu Fig. .Microbial community composition is often sensitive to temperature and salinity in sea ice  and seawChaetoceros diatom with the flavobacteria Polaribacter and a Fragilariopsis diatom with the alteromonad Paraglaciecola). The specificity of co-occurrences  could reflect taxa-specific use of algal exudates by heterotrophic bacteria [Congruency between eukaryotic and prokaryotic community structure across all of our samples Fig.  indicatebacteria –73; it cParticulate community metabolomes were distinct across WAP meltwater, seawater, and sea ice Figs. D and 5, –1) similar to those measured in Arctic sea ice . Prolinylindrus  and Nitzecointei . DetermiMany other metabolites also increased in concentration under cold and salty conditions and decreased in warmer and fresher conditions Fig. , supportSeawater freshening left detectable imprints on organic matter across our samples, with the metabolomes of melt-influenced communities  more similar to each other than to higher salinity samples (seawater, SW_T-S, Sea ice_T-S; Fig. Freshening also drove reduced total metabolite concentrations, relative to POC and PN, and elevated C:N ratios Figs.  and 5. TAlthough our data suggest that fluctuations in particulate metabolite concentrations are unlikely to alter bulk stoichiometry, they may rapidly alter the composition of labile DOM during increasingly common freshening events. Many metabolites can be respired by the organisms that produce them , but oth2.In the present study, we show that the different marine habitats of sea ice, meltwater, and seawater harbor unique microbial communities distinct in both taxonomy and metabolite composition. Temperature and salinity change drove strong metabolome changes in these coastal WAP communities even in the absence of strong taxonomic change. Mechanistically, they exhibited a marked metabolic flexibility in response to rapid environmental change, the accumulation of compatible solutes under more wintry conditions, and the reverse depletion of those products with regulation of fatty acids under melt conditions. As model predictions in the WAP suggest earlier ice breakup and longer open water periods with warmer and fresher conditions, our results highlight that a subsequent restructuring of the microbial chemical inventory is possible, likely affecting the fate of organic matter and thus the balance between remineralization and sequestration of atmospheric COSupplemental MethodsSupplemental FiguresSupplemental Tables


Locking plates are commonly used in the treatment of comminuted metaphyseal distal femoral fractures. However, locking plates form a strong structure and promote asymmetrical callus formation, which is not conducive for rapid fracture healing and may increase fracture risk. To overcome this, we designed a micromotion-balancing fixation system based on locking plates.Six healthy pigs (Bama miniature pigs) were used to establish a model of bilateral comminuted distal femoral fracture (AO/ASIF: 33-C2). Standard drilling was performed on one of each pig’s hind limbs (control group), whereas eccentric drilling was performed on the other hind limb . Both femurs were fixed with a 3-hole locking compression plate using 5-mm-diameter screws. At 12 postoperative weeks, all pigs were euthanized and the femurs with compression plates were radiographically examined. The level of fracture healing and loosening/internal fixation failure were recorded. Bone mineral density, number of trabeculae, trabecular morphology, and calcification precipitations were assessed.All pigs survived, and the fractures healed. No complications related to fracture healing, such as infection and internal fixation failure, were noted. The bone mineral density of the near and far cortical calli, number of the near and far cortical callus trabeculae, and difference in bone mineral density between the near and far cortical calli in the experimental group were significantly higher than those in the control group (p < 0.01). However, the difference in the number of trabeculae between the near and far cortical calli was significantly lower in the experimental group than in the control group (p < 0.01).This newly designed system provides stable fixation for comminuted distal femoral fracture, increases the overall strain at the fracture site, and balances the strains at the near and far cortices to achieve uniform callus growth and fracture healing. The incidence of distal femoral fractures (DFFs) increases with age . TreatmeThe locking plate is the most common implant in the surgical treatment of DFFs. It forms a fixed angle on each screw hole, with each screw head fixed to the plate through a locking mechanism and an overall rigidity that can be increased by stabilizing the screw angle . BecauseFurthermore, excessive rigidity of the locking plate results in an unbalanced strain between the fracture ends and insufficient or uneven callus formation. To overcome these issues, far cortical locking (FCL) was developed , which pAdditionally, how the parameters of the screw and screw hole can be controlled to achieve the required micromotion remains unknown . The incTo further increase the range of axial motion of the cortex on the plate side, compensate for the lack of FCL design, promote uniform displacement between fracture pieces, and induce symmetrical callus formation, we previously designed a micromotion-balancing fixation system. This system uses the conventional locking plate and screw system with newly designed drill bit and sleeves. After drilling, the screw hole diameter on the far cortex is the same as the diameter of the ordinary locking screw to achieve occlusal fixation between the locking screw and far cortex. The screw hole on the near cortex is oval because it has the same lateral diameter as that on the far cortex but its longitudinal diameter is larger. The longitudinal diameter increases with the length of the screw and the fracture gap; therefore, the proximal end of the screw can move in the screw hole of the near cortex, and the direction of movement is parallel to the long axis of the femur. When the femur is subjected to a force parallel to its long axis, the distal end of the screw is bent, the femur is moved downward, and the proximal end of the screw moves in the screw hole. In other words, the displacement of the far cortex is based on the screw bending during compression, while that of the near cortex is due to the screw hole being larger than the screw thread. Thus, the cortical micromotion on both sides of the fracture end is always closely balanced, thereby promoting symmetrical callus growth.We sought to investigate the efficacy of our system in the treatment of comminuted metaphyseal DFFs in pigs to provide a reference for its clinical application. We hypothesized that our system would perform better at fracture healing than the standard system.In this study, the fracture gap was set at 1 cm (comminuted fracture). To achieve secondary healing, the strain at the far and near cortices should be controlled between 2 and 10% . We seleThe drill was divided into three parts: L1 (tip of the drill 2 mm]), L2 , and L3 (second segment of the drill [100 mm]). Additionally, d1 is the diameter of the first segment of the drill , whereas d2 is the diameter of the second section drill  , L2 We drilled the locking screws into the oval and circular screw holes at the near and far cortices, respectively. Because the oval screw holes were larger by 1 mm than the screws, the proximal part of the screws could move within the screw holes Fig. a. The diSix experimental pigs (Bama miniature pigs), weighing 20–25 kg, and the sexes were randomized. were raised by the Animal Experimental Center of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, China. The exclusion criteria used when selecting the animals were obvious claudication; X-ray revealing obvious knee joint trauma or femoral deformity; and basic diseases, such as infection, tumor, or other metabolic system diseases. Random standard drilling was performed on one of each pig’s hind limbs (control group), whereas eccentric drilling was performed on the other hind limb .2O. Fluid loss was substituted with continuous intravenous infusions of Ringer’s acetate and glucose solutions.Each pig received an intramuscular injection of ketamine and midazolam, followed by inhalation anesthesia with a mixture of isoflurane and oxygen. When a sufficient level of anesthesia was achieved, animals were intubated and anesthesia was maintained with isoflurane mixed with oxygen. After endotracheal intubation, the animals were mechanically normoventilated with a positive end-expiratory pressure of 5 cmHEach pig’s femur was operated on to construct a DFF model of a comminuted epiphyseal fracture (AO/ASIF:33-C2). Plates and screws were placed Fig.  and the Femoral frontal X-rays were taken at 12 postoperative weeks and, together with femoral specimens, were used to assess fracture healing. The bone calli from plate side and opposite side  were taken, and the specimens were scanned using micro-CT  to measure parameters, including bone mineral density (BMD) and number of trabeculae. Furthermore, histological observations were conducted through hematoxylin and eosin (HE) and von Kossa staining procedures.Data were processed using SPSS version 18.0 . The arithmetic mean values of micro-CT scan indexes (number of bone trabeculae and BMD) in both groups were calculated, and paired t-tests were performed. Statistical significance was set at p < 0.05.All six experimental pigs survived and had good appetite and activity. Skin wounds showed good healing without any complications, such as infection or internal fixation failure. Radiographs obtained at 12 postoperative weeks showed callus formation, unclear medullary cavity of the distal femur, and restored continuity of the cortex Fig. a. Gross 3 vs. 0.5357 ± 0.043 g/cm3; p < 0.01 [Fig. 3 vs. 0.7295 ± 0.020 g/cm3, p < 0.01 [Fig. Three-dimensional images of each specimen are shown in Fig. .01 Fig. a; 1.763 .01 Fig. b and far3 vs. 0.1938 ± 0.019 g/cm3, p < 0.01 [Fig. The difference in BMD and number of trabeculae between the near and far cortical calli was significantly lower in the experimental group than in the control group .Fig. 1Staining results indicated bone tissue formation at the fracture site on the plate side in both groups, recanalized Haversian canals, and several osteoblasts (dark blue after HE staining). The experimental group had thicker trabeculae and higher numbers of trabeculae, osteoblasts, and bone-like tissues than the control group. Furthermore, the arrangement of bone trabeculae and Haversian canals was neat and close in the experimental group, with obvious vascularization Fig. a, b. In Staining results indicated dense and large calcium deposits  in the callus specimens from the plate side in the experimental group Fig. a, whereaLocking plates are widely used in the treatment of DFFs. However, they form an overly stable structure that results in asymmetrical callus formation, which affects fracture healing , 6. BottWe expected that the strain at the far cortex of the fracture site would not significantly differ between the groups. However, we found that the BMD and number of trabeculae of the calli at the far cortex were significantly higher in the experimental group than in the control group, demonstrating that micromotion-balancing osteosynthesis can increase the overall strain at the fracture site. The difference in BMD and number of trabeculae between the calli of the near and far cortices was significantly smaller in the experimental group than in the control group, indicating that the system can help balance the strain at the near and far cortices, and promote uniform callus formation. Compared with the standard system, our system increased the strain at the near cortex, improved the overall structural rigidity, reduced the gap between the strains at the near and far cortices, and increased the load capacity. This will help patients with early weight-bearing exercise.This study has several limitations. First, animal experiments cannot fully simulate the complex biomechanical environment of the human body, which limits the conclusions of the study. Second, due to the limited length of the animals’ femurs, we only used 3-hole locking plates, which reduced the grouping. Thus, the effects of different plate lengths and different numbers and distributions of screws on the biomechanics of fracture could not be compared. Nevertheless, our system solves the problem of asymmetrical callus formation, while ensuring stability.Our micromotion-balancing fixation system provides a simpler and more economic approach for the treatment of fractures. When an axial load is applied, displacement of the far cortex occurs due to the bending of the screw under compression, while displacement of the near cortex occurs due to the screw hole being larger than the diameter of the screw threads. This system provides stable fixation for comminuted distal femoral fracture, increases the overall strain at the fracture site, and balances the strains at the near and far cortices, thus promoting uniform callus growth and fracture healing.


AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system with roles in autophagy and the cell cycle regulatory network. The intrinsic disorder of AMBRA1 has thus far precluded its structural determination. To solve this problem, we analyzed the dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). The HDX results indicated that AMBRA1 is a highly flexible protein and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase. Here, we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3.08 Å resolution. The structure shows that parts of the N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and increase cell cycle progression. Together, these results provide structural insights into the AMBRA1-ubiquitin ligase complex and suggest a mechanism by which AMBRA1 acts as a hub involved in various physiological processes. AMBRA1 functions as a substrate receptor for E3 ubiquitin ligase complexes to promote ubiquitination and degradation of Cyclin D. Here the authors demonstrated the structure of AMRBA1 in complex with DDB1. Disruption of intermolecular interactions caused deregulation of cell cycle and DNA damage repair. Ubiquitin is a highly conserved protein that is most commonly attached to lysine residues in target proteins through the sequential action of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3). First, E1 activates the C-terminal glycine of ubiquitin in an ATP-dependent reaction. The activated ubiquitin is then handed over to E2 and finally covalently attached to the substrate protein by E3 ubiquitin ligase. Proteins can be modified by the addition of a single ubiquitin (monoubiquitination) or a chain of multiple ubiquitin molecules (polyubiquitination), which signals the protein for degradation by proteosomes. Cullin-RING ligases (CRLs) are the largest E3 ligase family in eukaryotes and are organized by a scaffold protein Cullin and a catalytic RING subunit, RBX1 or RBX24. Damage-specific DNA binding protein 1 (DDB1) is a component of the Cullin4A/B-RING E3 ubiquitin ligase (CRL4) complex and functions as an adaptor protein between Cullin4A/B (CUL4A/B) and CUL4A-associated factors (DCAFs) to target substrates for ubiquitination. DDB1 has triple β-propeller  domains and can associate with diverse substrate receptors, which in turn recruit substrates.The two major intracellular protein degradation pathways in eukaryotic cells, autophagy and the ubiquitin‒proteasome system (UPS), are crucial to maintain cell homeostasis and the regulation of several cellular processes, including protein quality control, cell proliferation and apoptosis5. AMBRA1 can regulate autophagy at several stages. Under starvation conditions, AMBRA1 promotes the ubiquitination of the serine/threonine kinase ULK1 (Unc-51-like autophagy activating kinase-1) by the E3 ligase TNF receptor associated factor 6 (TRAF6). ULK1 is a component of the multi-subunit ULK complex and is essential for the induction of autophagy. Such ubiquitination results in ULK1 self-association, activation and autophagy initiation6. In nutrient-rich conditions, mTORC1  phosphorylates AMBRA1 on Ser-52, resulting in the termination of the autophagy response by preventing ULK1 ubiquitination7. In addition to promoting ULK1 ubiquitination, AMBRA1 functions as an E3 substrate receptor for Beclin1 ubiquitination during starvation-induced autophagy. Beclin1 is a component of the PI3KC3 (class III phosphatidylinositol 3-kinase) complex together with Vps34 lipid kinase responsible for generating PI3P (phosphatidylinositol 3-phosphate). In this process, AMBRA1 promotes the Lys-63 ubiquitination of Beclin1. This modification enhances the interaction of Beclin1 with PI3KC3 and promotes the activation of Vps34, which is required for autophagy induction8.AMBRA1 (activating molecule in Beclin-1 regulated autophagy) is one of the DCAF substrate receptors that plays a central role in the communication between autophagy, cell-cycle control and the UPSDDB1 E3 ligase complex. Loss of AMBRA1 leads to accumulation of Cyclin D1 and decreased sensitivity to CDK4/6 inhibitors, resulting in increased tumorigenic potential11. Despite the centrality of AMBRA1 in the intersection of autophagy, the UPS and cell growth, the structure and regulatory mechanisms are largely unknown. Here, we determined the cryo-EM structure of the human DDB1-AMBRA1WD40 complex, which revealed a split WD40 domain formed by the N- and C-terminal AMBRA1 regions. HDX-MS analysis showed that the binding of DDB1 stabilizes distinct AMBRA1 regions, allowing for structural determination. The N-terminal helix-loop-helix motif of AMBRA1 is essential for DDB1 binding, and point mutations within this region abolish their interaction, prevent the ubiquitination of Cyclin D1 in vitro and increase cell cycle progression. Together, these results provide structural insights into the AMBRA1-ubiquitin ligase complex and suggest a mechanism by which AMBRA1 targets proteins involved in essential cell cycles and autophagy pathways.Recently, AMBRA1 was identified as the major regulator of the stability of Cyclin D1, which is involved in regulating cell cycle progression. Cyclin D1 interacts with cyclin-dependent kinase 4 and 6 (CDK4/6) to drive cell proliferation by phosphorylating retinoblastoma protein Rb, which in turn dissociates from the E2F transcription factor, resulting in the expression of genes required for S-phase entry. AMBRA1 limits CDK4/6 activity by mediating ubiquitination and degradation of Cyclin D1 as part of the CRL412. Hydrogen deuterium exchange mass spectrometry (HDX-MS) can be used to study changes in protein dynamics, binding and folding as well as IDRs or regions with altered conformations between different states13. The accessibility of a backbone amide hydrogen is largely dependent on hydrogen bonding and local secondary structure, as well as solvent accessibility15. Stably folded secondary structure elements such as α-helices or β-sheets incorporate deuterium to a lower degree than interconnecting loops or unstructured regions.A detailed picture of the mechanism by which AMBRA1 engages CRL4 is needed to provide new insight into AMBRA1 regulation, the relationship between autophagy, the UPS pathway and tumor growth, and for the development of novel therapeutics. We therefore set out to dissect the molecular basis of AMBRA1 recruitment to CRL4. AMBRA1 has been found to contain a WD40 domain in the N-terminal region, while the rest of the protein is proposed to consist of intrinsically disordered regions (IDRs) due to a lack of recognizable protein domains Fig. 12. Hydro16. Therefore, the AMBRA1 N-terminal region alone was unlikely to exhibit a β-propeller architecture. Based on the AlphaFold2 prediction and recent in silico analysis, the N-terminal AMBRA1 is likely to form half of the WD40 domain (WD40-N), and it requires its C-terminal part (WD40-C) to complete the entire β-propeller, connecting by a long loop in between  due to its reduced deuteration levels and the predicted secondary structure consisting of β-strands  state before refolding occurs, resulting in two distinct mass envelopes21. The lower mass envelope represents the closed state, and the higher mass envelope represents the open state.To gain insight into the backbone flexibility of AMBRA1, purified full-length AMBRA1 was analyzed by HDX-MS Fig. . Experimnds Fig. . HDX-MS To identify the CRL4 binding region in AMBRA1, we co-transfected and reconstituted the AMBRA1-CRL4 complex Fig. . This puWD40, was designed for cryo-EM structural studies   Fig.  and a seNΔ39) completely abolished the interaction with DDB1, as demonstrated by an in vitro MBP pull-down assay , AMBRA1V10R/L13R/W14R, AMBRA1V10R/L13R/W14R/H929A , Simian virus 5 V (PDB: 2B5L) and CSA (PDB: 6FCV), suggesting a common molecular mechanism of DDB1 recognition  oligoribonucleotides were used to knockdown endogenous AMBRA1 in cells Fig. . AMBRA1-nts Fig. . The pernts Fig. . Previou762 Fig. . Re-expr5. The highly disordered nature and “split” domain organization of AMBRA1 have largely precluded its structural determination. Intrinsically disordered regions may enable AMBRA1 to perform conformational changes to associate with different interactors simultaneously. To date, AMBRA1 has been reported to recruit and scaffold numerous substrates. For example, residues 533-751 are the binding site for Beclin127, residues 1075-1077 and 1087-1089 are required for dynein light chain 1 (DLC1) interaction28, and residues S1043-L1052 are required for association with LC3 and induction of mitophagy29. Furthermore, the two PXP motifs (residues P275-R281 and P1206-R1212) were shown to interact with serine/threonine-protein phosphatase 2A (PP2A) and mediate dephosphorylation and degradation of the proto-oncogene c-Myc, resulting in inhibition of cell proliferation and tumorigenesis30. These reported regions are located at the IDRs linking WD40-N and WD40-C and the C-terminus. The N-terminal and C-terminal regions of AMBRA1 are involved in interactions with BCL-2, ULK1, ELONGIN B and PARKIN; however, the precise interaction sites are not known31. To gain insights into how these regions are organized relative to the AMBRA1 WD40 domain in the CRL4 structure, we superimposed the Alphafold2 model of AMBRA1, which retains all the loops, on our CRL4- AMBRA1 structural model , anti-Phospho-Histone H2A.X (Ser139) antibody was used at 1:1000 dilution , Fab2 Fragment (Alexa Fluor 647 Conjugate) was used at 1:1000 dilution , anti-β-Tubulin Mouse Monoclonal antibody was used at 1:5000 dilution , anti-c-Myc Mouse Monoclonal antibody was used at 1:2000 dilution , HRP Conjugated Goat anti-Mouse IgG was used at 1:2000 dilution , HRP Conjugated Goat anti-Rabbit IgG was used at 1:2000 dilution .6 tag in the C terminus. AMBRA1WD40 (residues 1-204/853-1044) was subcloned with N-terminal GST tag followed by a TEV cutting site, or with N-terminal GST tag followed by a TEV cutting site and a Twin-Strep tag in the C terminus. For Cyclin D1, it was cloned with N-terminal GST-TEV-myc tag. DDB1, Cullin4A, RBX1 and CDK4 were constructed with N-terminal Twin-Strep-FLAG (TSF) tag. The AMBRA1 mutants were generated by PCR-based site-directed mutagenesis approaches. For AMBRA1FL (residue M1-R1298), AMBRA1NΔ19 (residue A20-R1298), AMBRA1NΔ39 (residue M40-R1298), AMBRA1V10R/L13R/W14R, AMBRA1V10R/L13R/W14R/H929A and AMBRA1 single mutants used in the MBP pull down experiment, they were cloned as a N-terminal MBP tag and a Twin-Strep tag in the C terminus. DDB1 was also subcloned into pFastBac-dual vector with a His6 tag in the N terminus for insect cell expression. For in vivo cell experiments, taggless AMBRA1FL, AMBRA1NΔ19, AMBRA1NΔ39, AMBRA1V10R/L13R/W14R, AMBRA1V10R/L13R/W14R/H929A and AMBRA1 single mutants were cloned into pCAG vector. The primers used in this study are listed in Supplementary Table The gene for full length AMBRA1 (residue M1-R1298) was codon optimized. The genes encoding DDB1, Cullin4A, RBX1, Cyclin D1 and CDK4 were amplified from cDNA by PCR and subcloned into pCAG vectors with different tags using KpnI and XhoI cutting sites, respectively. For full length AMBRA1 used in in vitro experiments, it was constructed as a N-terminal MBP tag with a HisWD40 and N-terminal TSF DDB1 were co-expressed for structural studies, N-terminal GST tag human AMBRA1WD40 with C-terminal Twin-Strep tag was expressed for MS analysis, GST-Twin-Strep was expressed as negative control. N-terminal GST-TEV-myc tag human Cyclin D1 and N-terminal TSF tag CDK4 were co-expressed for in vitro ubiquitination experiment. Full length MBP tagged AMBRA1 with C-terminal His6 tag was co-expressed with TSF tagged Cullin4A-DDB1-RBX1 E3 ligase for HDX measurement. MBP tagged full length AMBRA1, AMBRA1NΔ19, AMBRA1NΔ39, AMBRA1V10R/L13R/W14R and AMBRA1V10R/L13R/W14R/H929A with a C-terminal Twin-Strep tag were also expressed individually in Expi293F cells for in vitro pull-down experiment. N-terminal His6 tag human DDB1 was expressed in sf9 cell.For mammalian cell expression, N-terminal GST tag human AMBRA16 cells/ml. Cells were harvested after 3 days and washed with 1× cold PBS.The Expi293F cells were grown in Union-293 medium and used for protein expression, with 1 mg DNA and 4 mg PEI per 1 liter of cells at density of 2 × 10WD40 and DDB1 co-expression, the cell pellet was lysed in lysis buffer , 1% Triton-X100 (v/v), 2 mM MgCl2, 5 mM beta-mercaptoethanol) with proteases inhibitors  for 20 min at 4 °C. After centrifugation at 39,191 × g for 30 min, GST beads were incubated with the cell supernatant at 4 °C for 2 h. The beads were washed three times with wash buffer . Bound proteins were eluted with wash buffer containing 50 mM reduced glutathione. After TEV digestion overnight, the elution was then subjected to Streptactin sepharose resin, washed three times with wash buffer and eluted with gel filtration buffer  supplemented with 5 mM desthiobiotin. The protein elution was further purified by Superdex 200 Increase 10/300 GL . Cyclin D1-CDK4 complex were purified in the same procedure as described above.For AMBRA12, 5 mM beta-mercaptoethanol) with proteases inhibitors  for 20 min at 4 °C. After centrifugation at 39,191 × g for 30 min, amylose beads were incubated with the cell supernatant at 4 °C for 1 h. The beads were washed three times with wash buffer  and eluted in wash buffer containing 10 mM maltose. The elution was further applied to Streptactin sepharose resin, washed three times and eluted with gel filtration buffer containing 5 mM desthiobiotin. AMBRA1NΔ19, AMBRA1NΔ39, AMBRA1V10R/L13R/W14R, AMBRA1V10R/L13R/W14R/H929A and AMBRA1 single mutants was purified in the same protocol.For full length AMBRA1 and Cullin4A-DDB1-RBX1 E3 ligase coexpression, the cell pellet was lysed in lysis buffer , 1% Triton-X100 (v/v), 2 mM MgCl6 cells/ml. Cells were harvested after 3 days and washed with 1× PBS. For DDB1 purification, the cell pellet was lysed in lysis buffer , 2 mM MgCl2, 5 mM Beta-mercaptoethanol) with protease inhibitors for 20 min at 4 °C. After centrifugation at 39,191 × g for 30 min, the supernatant was passed over Ni-NTA affinity beads. The beads were washed three times with wash buffer  and eluted with elution buffer . The protein elution was further purified by Superose 6 Increase 10/300 GL  in gel filtration buffer . The protein sample was flash frozen in liquid nitrogen and stored at −80 °C until use. All purification procedures were carried out at 4 °C.The sf9 insect cell was grown in ESF921 medium and infected with recombinant baculovirus (1:50 v/v ratio) at density of 2 × 10The proteins used in this study are shown in Supplementary Fig. g for 5 min to remove cells and large debris, transfer the supernatant into new sterilized tube, that’s P0 virus. To generate P1 virus, prepare 50 ml Sf9 cell with 2 × 106 cells/ml density, add 2 ml P0 virus into the cell, wait for 4–5 days and check the viability to drop to ~50–60%, Harvest the cell by centrifuge at 1455 × g for 15 min, Collect the supernatant, that’s P1 virus. Add FBS to a final concentration of 5% and store at 4 °C prevent from light. To generate P2 virus, follow the same procedure, the amount of P1 virus being used depending on the viral titers. For long-term storage, aliquot and store at −80 °C for later use.For insect cell expression, target gene was cloned into pFastBac-dual vector, then transformed into DH10Bac Competent cell. The Bacmid was extracted and used to transfect the insect cell using Cellfectin II Reagent (Gibco) in 6-well plate, according to the manufacturer’s instructions. 4 h after transfection, replace medium with fresh ESF921 medium containing 10% fetal bovine serum and 1% Antibiotic-Antimycotic solution. Cells were incubated at 27 °C for 4–5 days, then collect the supernatant and centrifuge at 1455 × 2O buffer  to a final volume of 100 µL. After different time periods of incubation at 25 °C, the reaction was quenched by addition of ice-cold quench solution  containing 2 M guanidinium hydrochloride and 0.2 M citric acid. This reaction was digested on an immobilized pepsin column inside a manual HDX system with the temperature maintained at 0 °C. Eluted peptides were desalted using chilled trap column  for 5 min at a flowrate of 200 μL/min and 0.1% formic acid as mobile phase. Subsequent peptide separation was performed on the chilled ACQUITY BEH C18 (2.1 × 50 mm) analytical column using a first gradient ranging from 9 to 45 % of buffer B (80% acetonitrile and 0.1% formic acid) for 10 min followed by a second gradient ranging from 45 to 99% of buffer B for 1 min, at an overall flow rate of 50 μl/min. Peptides were ionized via electrospray ionization and analyzed by Orbitrap Eclipse and QExactive HFX (Thermo Fisher) mass spectrometer. Non-deuterated samples peptide identification was performed via data dependent tandem MS/MS experiments and analyzed by Proteome Discoverer 2.5 (Thermo Fisher). Mass analysis of the peptide centroids was carried by HDExaminer v3.3 , followed by manual verification for each peptide. No corrections for back exchange that occurs during digestion and LC separation were applied. For peptides exhibiting a bimodal distribution each time point is fitted with two Gaussian peaks with different means and areas but with similar width into the spectra. Next, the fitted peak area parameter was used to calculate the relative amount of open state by taking the ratio of high and the sum of high and low mass subpopulation.H/D exchange was carried out by 10-fold dilution of 0.5 µM AMBRA1 or AMBRA1-CULLIN4-DDB1-RBX1 E3 ligase complex into a DWD40-DDB1 complex were prepared for cryo-EM data acquisition. The first one is the protein elution from strep beads while the other one is the peak elution from size exclusion column. For cryo-EM grid, 3 µL of 0.15–0.2 mg/ml protein sample were deposited onto freshly glow-discharged Quantifoil R1.2/1.3 Cu300 mesh grids and plunged into liquid ethane using a FEI Vitrobot Mark IV after blotting for 3 s with blot force 0 at 4 °C and 100% humidity. A total of 6147 movies were collected on a Titan Krios electron microscope operating at 300 kV equipped with Gantan K3 camera at a defocus of −1 µm to −1.8 µm in counting mode, corresponding to a pixel size of 0.85 Å. Automated image acquisition was performed using SerialEM with a 3 × 3 image shift pattern. Movies consists of 50 frames, with a total dose of 58.96 e−/ Å2, with a total exposure time of 3 s and a dose rate of 14.2 e−/pixel/sec. Imaging parameters for the dataset are summarized in Supplementary Table Two samples of human AMBRA140. CTF fitting and estimation were performed by patch CTF estimation. After particle picking with blob picker in the second dataset, 921,715 particles were picked and subjected to 2D classification. The good classes were selected as templates for the entire datasets, which generating 2,082,649 particles. The particles were cleaned up with iterative 2D classification and subjected for ab initio and non-uniform refinement, resulted in 3.0 Å resolution map. 780 K particles were imported into Relion 3.1 for 3D classification without alignment analysis. The map was postprocessed by deepEMhancer with tight target modes for model building41. All reported resolutions are based on the gold-standard FSC 0.143 criterion.The first dataset included 3456 movies and the another one has 2691 movies. For each dataset, the movies were firstly aligned using MotionCor2 wrapper in Relion 3 to correct the specimen movement and then imported into cryoSPARC v242. Figures were prepared using UCSF Chimera version 1.1543. The cryo-EM density map has been deposited in the Electron Microscopy Data Bank under accession code EMD-37752 and the coordinates have been deposited in the Protein Data Bank under accession number 8WQR.The coordinates for DDB1 (PDB: 2B5M) and AMBRA1 (identifier: AF-Q9C0C7-F1) downloaded from Alphafold2 Protein Structure Database were rigid body fitted separately into the density map using UCSF Chimera. Atomic coordinates were refined by iteratively performing Phenix real-space refinement and manual inspection and correction of the refined coordinates in Coot. To avoid overfitting, the map weight was set to 2 and secondary structure restraints were applied during automated real-space refinement. Model quality was assessed using MolProbity and the map-vs-model FSC by comparing the map-vs-model FSC with the FSC of the experimental cryo-EM density , 1.5 μM UBCH5C (Boston Biochem E2-627), 0.3 μM purified AMBRA1-DDB1-Cullin4-RBX1 complex, 20 μM HA-ubiquitin (Boston Biochem U-110), 0.16 μM myc-Cyclin D1-CDK4 complex and 10 mM MgATP solution (Boston Biochem B-20) in E3 ligase reaction buffer (Boston Biochem B-71). The reaction was incubated at 37 °C for 2 h and analysed by SDS-PAGE, followed by immunoblot. The experiment was repeated three times with similar result  next day, according to the manufacturer’s instructions.SiRNA oligoribonucleotides corresponding to the human AMBRA1 were ordered from Tsingke. AMBRA1 siRNA 1: 5′- GAGUAGAACUGCCGGAUAG−3′; AMBRA1 siRNA 2: 5′-CCACCCAUGUGAACCAUAA-3′; AMBRA1 siRNA 3: 5′-GCGGAGACAUGUCAGUAUC-3′; AMBRA1 siRNA 4: 5′-CUGAAUCGCUGUCGUGCUU-3′; For siRNA transfection, a total of 8 × 10g for 10 min to remove insoluble debris. Solubilized proteins were quantified by BCA Protein Assay Kit (Sangon Biotech), equal amounts of protein were mixed with loading buffer and incubated at 95 °C for 5 min. whole cell lysates were separated by SDS-PAGE and transferred to 0.45 µm Immuno-Blot PVDF membranes (BIO-RAD). Membranes were then blocked in 5% milk/TBST for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. For the detection of proteins, using the appropriate secondary antibodies conjugated to horseradish peroxidase  at 1:2000 dilution in TBST for 1 h at room temperature and visualizing with BeyoECL Plus (Beyotime). The images were acquired by Amersham Imager 680  next day, according to the manufacturer’s instructions. 24 h after siRNA transfection, transient plasmids transfections were performed using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer’s instructions. Cells were harvested 48 h after plasmids transfection. Cells were lysed in lysis buffer  plus protease inhibitors . Lysates were lysed for 30 min on ice, then centrifuged at 13,000 × 4 density per well, then follow the same procedure as for immunoblotting. For CHK1 inhibition, cells were treated with 100 nM of AZD7762 (MedChemExpress) for 24 h after plasmids transfection, cells were washed with cold PBS and fixed in 4% Paraformaldehyde (PFA) in PBS for 20 min at room temperature, then permeabilized with PBS/0.1% (v/v) Triton X-100 for 20 min and subjected to blocking with PBS/1% (w/v) BSA to block the cell for 1 h at room temperature. γH2AX antibody (CST) were diluted in PBS/0.01% (v/v) Triton X-100 at 1:1000 dilution, applied onto the coverslips at 4 °C overnight. Alexa647-conjugated secondary antibody was diluted in PBS/1% (w/v) BSA at 1:1000 dilution and incubated with the coverslips for 1 h at room temperature. Slides were mounted in Anti-Fade Mounting Medium with DAPI (Beyotime). Imaging was performed using a Zeiss LSM 900 confocal microscope with a Plan-Apochromat 40×/0.95 objective.For immunofluorescence, the human osteosarcoma U2OS cells were plated into the 12-well plate on coverslips at 5 × 10WD40 was co-expressed with a N-terminal TSF tag DDB1, the N-terminal GST-myc tag Cyclin D1 was co-expressed with a N-terminal TSF tag CDK4 in Expi293F cell. The complex was purified as described in the Protein expression and purification section. GST tag was remained for Cyclin D1-CDK4 complex. For GST pull down assay, 0.7 μM of AMBRA1WD40-DDB1 complex was incubated with GST-myc-Cyclin D1-CDK4 complex in 5 to 1 molecular ratio supplemented with 50 µL GST beads for 1 h. After washing with 500 µL of gel filtration buffer three times, the proteins were eluted with 100 µL of gel filtration buffer supplemented with 50 mM reduced glutathione and resolved by SDS–PAGE. The experiment was repeated three times with similar result  was incubated with 2.5 µM of purified DDB1 in 500 µL reaction volume supplemented with 50 µL amylose beads respectively. After 2  h incubation at 4 °C, the beads were washed three times with gel filtration buffer and eluted with 10 mM maltose containing buffer. The sample were further resolved by SDS–PAGE. The experiment was repeated three times with similar result  next day, according to the manufacturer’s instructions. 48 h after siRNA transfection, Transient plasmids transfections of Ambra1 WT and single mutants were performed using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer’s instructions. Cells were harvested 48 hrs after transfection. Using 0.25% Trypsin-EDTA (ThermoFisher Scientific) to detach the cell, collect the cell in 1.5 mL centrifuge tube by centrifuging at 1000 × g for 5 min. Discard the supernatant carefully, then adds ice cold PBS to re-suspend the cell. centrifuge at 1000 × g for 5 min. Remove the supernatant again and leave about 50 µL PBS. Tap the tube to avoid the clustering of the cell. Add 70% ice cold ethanol into the cell, gently pipette it upside down, leave in 4 °C to fix for 24 h. Centrifuge at 1000 × g for 5 min to discard the supernatant carefully on the next day, then adds ice cold PBS to re-suspend the cell. Centrifuge at 1000 × g for 5 min to collect the cell. Cells were then re-suspended with PI staining buffer containing RNaseA (Beyotime), incubate at 37 °C water bath for 30 min, prevent from the light. Then analysis the cell immediately by CytoFLEX S (Beckman). Data was analyzed by FlowJo 10.8.1.The human osteosarcoma U2OS cells were plated into 12-well plate at 8 × 104 cells were recorded for each condition. All cells were first gated by SSC area vs. FSC area, and single cells were gated by FSC width vs FSC height, followed by PI height vs PI area. G1 cells were PI positive with 2 N DNA content, and G2/M cells were PI positive with 4 N DNA content, S cells were PI positive between 2 N DNA content and 4 N DNA content. Cell cycle analysis function of FlowJo 10.8.1 is used to derive the cell cycle populations. Then the total cell cycle population of G1, G2, S phases were normalized to 100%. Data was then used to plot in GraphPad Prism 9.4.1 software. Statistical significance was determined by Tukey’s multiple comparisons of two-way ANOVA, P values are indicated in the graph, the value of p  <  0.05 was considered statistically significant. Graph bar was presented as mean values +/− SD.Each treatment has three replicates. For data analysis, around 2 × 10P values are indicated in the graph, the value of p < 0.05 was considered statistically significant. Graph bar was presented as mean values +/− SD.For Pan-γH2AX positive cells quantification, a minimum of three independent experiments were included in the representing graphs. For each group, total around 300 cells were manually counted by using ZEN (Blue edition) software, pan-γH2AX positive cells percentage were calculated. Data were analysed using GraphPad Prism 9.4.1 software. Statistical significance was determined by Tukey’s multiple comparisons of one-way ANOVA, Further information on research design is available in the Supplementary filePeer Review FileReporting SummarySource Data


A total of 88 proteins were significantly regulated. The largest change was observed in cystatin-SN, which was downregulated in MGD and correlated negatively with tear meniscus height. The downregulation of cystatin-SN was confirmed with targeted mass spectrometry by single reaction monitoring (SRM). Eighteen immunoglobulin components involved in B cell activation, phagocytosis, and complement activation were downregulated in MGD including Ig alpha-1 chain C region, immunoglobulin J chain, immunoglobulin heavy variable 3–15, and Ig mu chain C region. The changes in cystatin-SN and immunoglobulin chains are likely to result from the inflammatory changes related to tear film evaporation, and future studies may assess their association with the meibum quality. Meibomian gland dysfunction (MGD) is a highly prevalent condition and the most common cause of evaporative dry eye disease. Studying the proteome of MGD can result in important advances in the management of the condition. Here, we collected tear film samples from treatment naïve patients with MGD (n = 10) and age-matched controls (n = 11) with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography—tandem mass spectrometry. The proteins were considered differentially expressed if  Dry eye disease (DED) is generally associated with a significant reduction in physical and mental quality of life . DED mayThe conservative management of MGD includes warm compresses and lid hygiene, but medical treatments are emerging, including antibiotics, nonsteroidal and steroidal anti-inflammatory agents, fatty acid supplementation, and Demodex infestation . AntibioWhile changes in the lipid composition of the tear film is a well-established component in the etiology of MGD , systemaThe identification of tear film proteins associated with MGD can lead to significant therapeutic advances. The objective of proteome studies is to identify and quantify the entire set of proteins in a tissue or body fluid. With significant advances in sample processing, liquid chromatography, and mass spectrometry, modern proteomic techniques allow for in-depth large-scale protein analysis of the tear film ,8,9. HerA total of 2407 proteins were successfully identified after filtering . In totap = 0.0079) (p > 0.05). The proteins with the largest increases in MGD included polyadenylate-binding protein 1 (fold change = 2.67), dynactin subunit 1 (fold change = 2.50), and exportin-2 (fold change = 2.27).The largest downregulations were observed in the cystatin-SN (fold change = 0.23), apolipoprotein D (fold change = 0.30), and Ig mu chain C region (fold change = 0.46) . The str 0.0079) , in very 0.0079) , while cp = 0.047).Seven proteins correlated with NBUT. The proteins with positive correlations with NBUT included myosin regulatory light chain 12B, ubiquitin carboxyal-terminal hydrolase 47, hsc70-interacting proteins, and poly(rC)-binding protein 2. Proteins with negative correlations with NBUT included endoplasmic reticulum aminopeptidase 1, immunoglobulin heavy variable 3–15, and Ig mu chain C region . Myosin-MGD was associated with the regulation of proteins involved in the B cell receptor signaling pathway, the positive regulation of B cell activation, phagocytosis, and complement activation A. The grProteasome subunits also formed the largest cluster of the dataset , which cThe regulation of the proteins involved in deubiquitination was observed in MGD, including proteasome subunits and ubiquitin carboxyl-terminal hydrolase 14, ubiquitin carboxyl-terminal hydrolase 47, and ubiquitin carboxyl-terminal hydrolase isozyme L3 A,B. All Our study reports on tear film proteome changes in patients with MGD defined as MGD. The most pronounced change was observed in cystatin-SN. The downregulation of cystatin-SN was confirmed by targeted mass spectrometry, thereby confirming findings obtained with discovery-based proteomics. The downregulation of cystatin-SN has previously been reported in tear fluid samples from patients with fungal keratitis and ocular graft versus host disease ,11. CystOur study identified an extensive downregulation of 18 immunoglobulin components. There is only limited knowledge about the role of immunoglobulins in blepharitis, but our data indicate that immunoglobulins may play a major role in the molecular processes underlying the condition. Changes in immunoglobulins are likely to be involved in the inflammatory changes resulting from increased evaporation in MGD. Immunoglobulin heavy variable 3–15 and Ig mu chain C region correlated negatively with NBUT, indicating that changes in immunoglobulin components affect the quality of the tear film. Drew et al. 2+ = 2084.0556 Da. The lyophillized peptide was dissolved in 0.1% formic acid at 1 µg/µL. For each sample, 1 µg of generated tryptic peptides was injected together with 0.1 ng (48.04 × 10−15 mole) of the heavy cystatin-SN peptide. Samples were injected in duplicate. The heavy peptide peak area and the corresponding light peptide peak area were compared to calculate the molar amount of light peptide in each sample. By assuming the complete tryptic digestion of proteins in the samples, the molar amount of cystatin-SN was considered to be equal to the amount of light peptide generated. The amount of cystatin-SN protein per gram of total peptide in the samples was estimated by using the deduced molecular mass of cystatin-SN from Uniprot 16,388 Da. The technical duplicates had an average coefficient of variation at 2.3%.Single reaction monitoring (SRM) was performed using a TSQ Quantiva mass spectrometer  as previously described . The datIn conclusion, the proteomic analysis suggested a multifactorial pathogenesis in MGD. The downregulation of cystatin-SN and immunoglobulins is likely to be associated with alterations to the ocular surface immune response following increased evaporation. The decreased level of cystatin-SN is likely to render patients with blepharitis more susceptible to infection as cystatin-SN has antimicrobial features. MGD was associated with changes in the proteins involved in protein degradation, which may play a central role in the processes leading to increased evaporation.