The purpose of this study was to analyze the feasibility and accuracy of a newly developed guide apparatus for the percutaneous retrograde lag screw fixation of posterior column of acetabular fractures. 3D pelvic models were reconstructed from the helical computed tomographic data of 33 adult patients using the Mimics 10.01 software. The virtual cylindrical implants were placed along a line passing through the central point of the ischial tuberosity and the midpoint between the most prominent point of anterior superior iliac spine and that of posterior superior iliac spine. Some anatomical parameters were then measured, based on which a guide apparatus was developed, and its safety and accuracy were experimentally validated with pelvic and cadaveric specimens. The screws were successfully placed in all of the 66 hemipelves. There was a significant difference between the male and female groups in the AB distance (156.26 ± 7.28 mm and 151.38 ± 8.11 mm), OI distance (139.53 ± 7.56 mm and 125.15 ± 11.17 mm), and diameter (12.19 ± 1.97 mm and 10.19 ± 2.14 mm) of the virtual cylindrical implants. This guide apparatus was proved effective for percutaneous retrograde lag screw fixation of posterior column acetabular fractures by the experiments with the pelvic and cadaveric specimens. Screw fixation of posterior column fractures via OI is safe and feasible. We designed a new percutaneous retrograde screw fixation guide apparatus to assist internal fixation of posterior column acetabular fracture. It was also reported to have a better biomechanical strength than internal fixation with a plate.Open reduction and internal fixation (ORIF) has proven to be effective in the treatment of posterior column acetabular fracture. However, because of the deep anatomical location of the acetabulum and its complex anatomical relationship with adjacent structures, the operation requires a long incision and wide exposure on the acetabulum, which is technically challenging. The intrinsic invasive nature of ORIF exposes patients to risks of hemorrhage and a number of postoperative complications, such as heterotopic ossification,–9 The advantage of this minimally invasive technique is to reduce bleeding and treatment costs. However, there is substantial evidence to suggest that percutaneous lag screw fixation increases the death caused by screw induced injury of large pelvic vessels, nerve or pelvic organ injuries, poor location or fixation, and screw fracture. The wider application of this approach is limited to a certain extent by the technical difficulties in determining the optimal entry point and direction, and the diameter of the screw, among others. The image-guided orthopedic surgery has the potential to be a widely used, minimally invasive, and intelligent option,,12 which in fact has been increasingly practiced by orthopedic surgeons.,14 Despite its therapeutic benefits, the application of this technology in most Chinese hospitals is limited, mainly because of the high cost of equipment and materials. Recently percutaneous lag screw fixation has gained popularity as a minimally invasive treatment of acetabular fractures, such as the sacroiliac fracture or dislocation, anterior or posterior column fracture, and iliac crescent fracture.In this study, the virtual 3D pelvic models were reconstructed using the Mimics 10.01 software and lag screw placement was simulated, based on which a guide apparatus was developed for percutaneous retrograde lag screw fixation of posterior column acetabular fracture, and its safety and accuracy were assessed with pelvic specimens.22.1The complete helical CT data were collected from 33 adult patients  without any bone lesions or anatomic abnormalities from November 2014 to November 2016. The study was approved by our hospital's institutional review board, and all the patients provided informed consent before participation. The CT scanning was performed using a GE Medical systems/Light speed 16 scanner under the conditions of 120 kV, slice thickness = 1.25 mm, and 512 × 512 matrix. The CT data were then imported in DICOM format into the Mimics 10.01 software . Firstly, the pelvic region was extracted from the CT images by the gray-level thresholding, then the bilateral pelvic boundaries were precisely extracted by the region segmentation, and finally the 3D pelvic models were reconstructed.2.2The 3D bilateral pelvic models were reconstructed using the Mimics 10.01 software , Oi distance , and diameter  of the virtual cylindrical implants between the male and female groups . The A′-end of the guide apparatus was fixed to the most prominent point of ASIS with kirschner pins, and the B′-end to that of PSIS, respectively. A sliding bar between the A′- and B′-end was used to measure the distance between ASIS and PSIS. The O′ bar was adjusted to the midpoint of AB′ and then fixed with a bolt. The guide apparatus was maintained stable throughout the surgical procedure with the help of an assistant. A kirschner pin was drilled into the ilium via the central point of the ischial tuberosity guided by the sleeve of the guide apparatus Fig. , and the2.5ASIS and PSIS are the bony projection of the pelvis that protrude at the front and back, which can be difficult to be precisely fixed with kirschner pins in real surgical practice. The location errors were evaluated with the 33 reconstructed models of the right hemipelves in the Mimics software. A plane of 250 × 150 mm was created by the most prominent points of ASIS and PSIS and the central point of the ischial tuberosity. A virtual cylinder of 6.5 mm in diameter was placed along the line connecting ASIS and PSIS as the lateral axis, and the line passing through the central point of the ischial tuberosity and the midpoint of the lateral axis was created as the medial axis. A sphere of 8 mm in diameter was placed at the cross point of the 2 axes. The medial axis of the implant was set close to the medial edge of the lateral axis, and moved from left-to-right or vice versa within the range of the sphere diameter (±4 mm), and the lateral edge was examined in a similar manner , 3 O’clock position , 6 O’clock position , and 9 O’clock position  of the central point of the ischial tuberosity  fresh frozen cadavers at the anatomy laboratory of the Southern Medical University. The soft tissues were completely thawed before the experiments. The cadavers were placed in a lateral position, and the surface locations of ASIS, PSIS, and ischial tuberosity were marked. A longitudinal incision of about 1.0 cm and 1.5 cm was made at the marked site of ASIS and PSIS, respectively, and then the subcutaneous soft tissues were stripped away to expose the most prominent point of ASIS and PSIS, to which the guide apparatus was fixed with a 2.0-mm kirschner pin. The AB length between ASIS and PSIS was determined by the transverse bar, and then the longitudinal bar was adjusted to the midpoint of AB′ and then fixed with a bolt. The guide apparatus was maintained stable throughout the surgical procedure with the help of an assistant. The lower limb on the operation side was maintained a position of hip extension and knee flexion. A incision of about 5.0 cm was made at the marked site of the ischial tuberosity, through which the sleeve of the longitudinal bar was inserted by about 2.5 mm to the central point of ischial tuberosity, then the longitudinal bar was locked by a bolt at the end of the bar. As the C-shaped arm showed that the ischial tuberosity and the oblique-iliac crest were accurately located, the kirschner pins were drilled by the electric drills. The accuracy of screw position was determined by postoperative x-ray images Fig. .2.8t test to assess differences between 2 groups. Between-group comparisons for all analyses were performed with analysis of variance. Significance was set at P < .05 for all tests.Data are presented as means standard ± deviation. Statistical analyses included a 2-tailed paired 3P < .05) . The results were rated as excellent in 15 cases (83%) and good in the rest 3 cases (17%), and the male specimens could accommodate a longer screw than the female specimens  Table .Among the 15 male hemipelves underwent screw fixation, the results were rated as excellent in all cases for screw fixation at the medial, anterior, and posterior side, whereas excellent in 3 20%) and good in 12 cases (80%) for that at the lateral side, respectively. Of the 18 female hemipelves underwent screw fixation, the results were rated as excellent in all cases for screw fixation at either medial or lateral side; excellent in 1 (6%), good in 8 (44%), and poor in 9 (50%) cases for that at the lateral side; and excellent in 5 (28%), good in 4 (22%), and poor in 9 (50%) cases for that at the posterior side, respectively. It showed that the entry point was preferably set at the medial side of ASIS and PSIS, or at a point deflected slightly to ASIS  approach include superior or inferior gluteal nerve injury, which would result in postoperative gait dysfunction. Neal et al Zhang et al previously proposed that the most prominent points of ASIS and PSIS were equidistant from a point at the ischial tuberosity, based on which they further proposed that the lag screws might be placed percutaneously through the perpendicular bisector of a line passing through the most prominent points of ASIS and PSIS. Based on Zhang's study, the 3D pelvic models were reconstructed using the Mimics 10.01 software and percutaneous retrograde screw fixation of posterior column acetabular fracture was simulated in this study. The results showed that a virtual cylindrical implant could be placed along the line passing through the central point of the ischial tuberosity and the midpoint between ASIS and PSIS. Adding a small incision did not expose the patient to a higher risk of complications.A previous study indicated that the maximum diameter of the virtual implant accommodated by the anterior or posterior column was much smaller than the smallest diameter of the columns; thus, the size of the screws used for percutaneous fixation of acetabular fractures should not be based solely on the measurement of cross-sectional diameter of the columns.The guide apparatus described in this study has several advantages. First, it consists of 2 halves of C-shaped frame joined together by a screw, as shown in Figure Traditional percutaneous retrograde screw fixation of posterior column fractures often requires repeated attempts to achieve normal reduction and fixation. It is difficult to put screws in precise positions, which will significantly increase the operation time. A major drawback associated with repeated operation is the bone injuries at a fixed position, which will have a detrimental effect on the adjustment of screw direction and the final fixation strength. CT-guided minimally invasive surgery, although accurate, has often been frustrated in many hospitals primarily because of the high cost. This new guide apparatus proposed in this study has the potential to become an alternative option, and proved to be effective by the Mimics simulation and experiments with pelvic and cadaveric specimens. It is safe and applicable to both male and female patients. It also has the advantages of high one-time success rate, long screw, simple structure, easy manipulation, small size, easy assembling, transportation, and sterilization. It allows for a minimal incision just large enough for the guide apparatus to be inserted, which as a result can reduce the pain of patient and reduce the hospitalization cost. The new guide apparatus is suitable for patients with acetabular posterior column fracture without displacement, or displacement ≤1 mm. Because it is difficult to palpate the ASIS, PSIS, and ischial tuberosities, the navigational apparatus is not recommended for use in obese patients.5A new guide apparatus was designed for percutaneous retrograde screw fixation of posterior column acetabular fracture using the Mimics software, which proved to be effective by the Mimics simulation and experiments with the pelvic and cadaveric specimens. In the next step, we will subject this guide apparatus to the clinical practice and assessment.Conceptualization: Shengjie Wang.Data curation: Jie Tang.Formal analysis: Yonghui Dong.Funding acquisition: Bing Wang.Investigation: Lu Lu.Methodology: Shifeng Song.Project administration: Pijun Zhang.Writing – original draft: Pijun Zhang.Writing – review & editing: Bing Wang.


Ratings surveillance is used in clinical trials to assure ratings reliability of site-based scores. One surveillance method employs audio-digital recordings of site-based clinician interviews to obtain remote, site-independent scores for assessment of paired scoring concordance and interview quality. We examined the utility of this surveillance strategy using paired site-independent scores derived from recorded site-based Montgomery-Asberg depression rating scale (MADRS) interviews obtained from patients with major depressive disorder (MDD) participating in 5 clinical trials.High correlations were noted between the 3736 paired site-based and site-independent scores across all visits. Some rater “outliers” were identified whose ratings performance improved following remediation. In 3 studies with available outcome data, the blinded remote ratings yielded a high predictive value (91.2%) for replicating treatment response rates.The magnitude of the total MADRS scores affected the directionality of paired scoring deviations in each of the 5 studies. Across all visits, site-based raters scored the more severe MADRS scores (≥30) higher than site-independent raters and the less severe MADRS scores (<20) lower than site-independent raters. Individual MADRS items were similarly affected by the directionality of symptom severity.This analysis affirms the utility of audio-digital recording of site-based interviews as a surveillance strategy for quality assurance (monitoring and remediation). In addition, the high predictive value of blinded remote ratings to replicate site-based treatment outcomes may be useful to affirm primary site-based results when there is a potential of functional unblinding. The use of remote ratings as a primary measure beyond its utility for quality assurance needs further exploration. Inter-rater scoring reliability can affect the power of the clinical trial to achieve signal detection . Given iRatings precision during clinical trials presumes that raters will conduct complete interviews in a consistent manner at each study visit regardless of time pressures or other exigencies. In an analysis of 63 published papers, it was noted that few reports examine or describe the reliability of the ratings that were conducted during the study . The useIn this report, we examined the utility of the audio-digital recording surveillance strategy from paired scoring data accumulated from 5 distinct clinical trials of major depressive disorder (MDD) that included 3736 site-based Montgomery-Asberg depression rating scale (MADRS) interviews .2Data for this ratings reliability analysis was obtained from 5 phase II or III clinical trials conducted between 2009 and 2017 as part of vendor grants awarded to Clintara LLC (or Bracket LLC) to conduct quality assurance/surveillance programs for ratings precision. The analysis was limited to double-blind, placebo-controlled trials that included patients with major depressive disorder (MDD) that used the MADRS as the primary efficacy measure and had obtained paired  site-independent scores based upon audio-digital recordings of site-based interviews ,17. All Clinicaltrials.gov as: NCT 01421134, 01500200, 02158533, 01912196, and 00739908. The work described in each study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. All patients provided written informed consent approved by an independent review board prior to participation in the studies.The 5 selected studies were registered in All site-based and site-independent raters participated in comprehensive rater training and certification programs for each study that included didactic presentations, observation of expert MADRS video interviews, and demonstration of MADRS scoring competency via inter-rater reliability (IRR) assessments of the MADRS video interviews. Site-based raters were also required to demonstrate interviewing competency skills via mock MADRS interviews using a structured interview guide for the MADRS ,23. AcroAs part of this program, the site-based raters were trained to conduct the MADRS interviews using an audio-digital recording pen. The pens simultaneously audio-recorded the MADRS interview and digitally copied accompanying written notes that were captured on specially manufactured source books. The recorded interviews were electronically forwarded to Clintara LLC  for random assignment to the site-independent raters who were blinded to the study visit, trial site, and site-based rater's scores. The site-independent raters generated their own paired  scores by listening to the audio recording and reading the site-based rater's accompanying digital notes that did not contain scores.The merged data from the 5 studies were examined to assess overall inter-rater reliability (IRR) and scoring concordance or deviations between the individual paired ratings. Scoring deviations were defined as the difference between the site-based score minus the site-independent score. Positive scoring deviations indicate that the site-based score was higher than the paired site-independent score. In addition, we examined the effect of the total MADRS score severity, study visit, and interview length on paired scoring deviations.The total sample was divided into five sub-groups based upon total site-based MADRS scores at any study visit  and four other sub-groups based upon the study visit itself .t-test and intra-class correlation (ICC) as appropriate to compare the site-based and site-independent scores relative to total and individual item MADRS scoring and timed interview length. The significance level was set at 5% for all tests in this analysis.Statistical analyses used Students’ 3There were 3736 MADRS “paired” scores available for site-based and site-independent ratings analyses.3.1As shown in The site-based MADRS scores were significantly higher than site-independent scores at the screen visit , but not significantly higher or lower at any other study visits.There were 249 paired interviews (6.7%) with total scoring deviations >6 points in either direction. The surveillance strategy identified site-based raters who were frequent “outliers” and provided telephone ratings remediation. The basis for most paired ratings discrepancies was usually a failure to apply ratings conventions or interviews that were simply too short to conduct a comprehensive assessment. Subsequent review of site-based rater performance following telephone remediation revealed greater paired scoring concordance in almost every case. In 3 instances, the raters were removed from the study because they were not remediable.3.2The magnitude of the total site-based MADRS score affected the paired scoring deviations. As shown in There were 136 paired MADRS scores  with >6 points positive scoring deviations  and 113 paired scores  with negative deviations. As noted above, the magnitude of the total MADRS scores affected the directionality of the scoring deviations. Thus, the mean total MADRS score was 31.8 ± 8.1 (SD) amongst the 136 paired scores with positive deviations and only 21.7 ± 8.0 amongst the 113 paired scores with negative scoring deviations .The magnitude of total MADRS scores affected mean paired scoring deviations across all visits. Higher total MADRS scores yielded more positive paired scoring deviations across all visits whereas lower scores tended to yield more negative scoring deviations regardless of the study visit .Fig. 4Me3.3The paired scores yielded a high ICC for each MADRS item with minimal scoring disagreement. However, the directionality of the paired scoring deviations of each of the 10 individual MADRS items was affected by the magnitude of the endorsed symptom severity. Individual item paired scoring differences were usually within one point of each other in either direction. For instance, the scoring difference was within one point in 96.4% of MADRS interviews for item 1 (reported sadness) of which 61.1% were exactly the same.Accurate scoring of MADRS item 2 (observed sadness) by listening to an audio recording is obviously limited by virtue of no visual observation. The structured interview guides used in these studies provided specific queries that generated useful information to facilitate scoring ,23. For We compared the use of the full MADRS scoring range (0–6) by site-based and site-independent raters. Site-independent raters used scores of 6 (most extreme) or 0 (no symptom endorsement) as often as site-based raters. For instance, site-based raters endorsed a score of 6 in 1.7% of interviews for MADRS item 1 versus 1.5% by site-independent raters and site-based raters endorsed a score of 0 in 13.3% of interviews versus 13.3% by site-independent raters.3.4Although most programs recorded 100% of MADRS interviews throughout a study, only a small percentage of MADRS scores were actually scored by site-independent raters at the endpoint of each study. Consequently, paired MADRS scores for both the baseline and study endpoint visits for the same subject were available from only a small sub-group of study subjects. Merging data from 3 of the studies in this analysis, 196 of the 215 site-independent score pairs (91.2%) correctly matched the response/non-response outcomes of the site-based raters with little variation between the 3 studies .3.5There were 1235 paired MADRS scores with reliable interview length data in this merged data sample. Recorded MADRS interview lengths range from 3:30 to 62:26 min (mean interview length = 17:37 ± 8:16 min). The mean interview length for the screen and baseline MADRS interviews was 21:17 ± 8:38 min and were significantly longer than the post-randomization and endpoint interviews (15:42 ± 7:22) due to the extent of symptom endorsement and the higher total MADRS scores at the early visits . The truncated scoring range at screen and baseline (due to the specified minimum eligibility criteria for each study) yielded a modest correlation between interview length and the site-based total MADRS score  in contrast to the post-randomization correlation  that included a broader MADRS scoring range from 0 to 52.In a previous report, we noted that site-based MADRS interviews conducted in ≤12 min yielded significantly greater paired scoring deviations than longer interviews at the screen visit . However, with one exception noted below, interview length was not associated with paired MADRS scoring deviations at the screen, baseline, or post-randomization visits in this larger analysis.In this analysis, we found that total MADRS scores ≥40 were affected by interview length at any visit. Thus, 5 of 10 paired screen interviews with MADRS scores ≥40 and interview lengths ≤12 min had >6 point scoring deviations (50%) in contrast to 9 of 52 interviews (17.3%) that were longer regardless of the study visit (Fisher exact test = 0.038). These ratings “outliers” were identified and remediated.4We examined the utility of audio-digital recordings as a surveillance strategy for quality assurance of site-based interviews from merged data from 5 clinical studies of MDD. Site-independent scoring of the audio-digital recordings of 3736 site-based MADRS interviews yielded highly reliable paired scores  with minimal scoring deviations. The high correlation found been site-based and remote scores based on audio recordings is consistent with the report of [The above findings from 5 different MDD studies affirm the utility of audio-digital recording of MADRS interviews as a quality assurance method to optimize site-based ratings precision. Of course, ratings reliability is contingent upon competent site-based interviews. This surveillance strategy reinforces competent ratings performance because raters are aware that their recorded interviews are subject to independent review and monitoring. Nonetheless, some raters conducted shorter, incomplete interviews or failed to correctly apply ratings conventions. We have found that telephone remediation of rater “outliers” who exceeded pre-specified paired concordance ranges contributes to improved ratings performance on subsequent MADRS interviews in most instances.Most of the 3736 MADRS interviews were complete and judged to be of good quality by the independent raters. The good interview quality may have been reinforced by the rater's awareness of the audio-digital recording surveillance strategy. Further, the actual interview length did not affect paired scoring concordance in most of 1235 timed MADRS interviews with the exception of short interviews (≤12 min) conducted in patients with MADRS scores ≥ 40 at any visit.The mean total site-based MADRS scores were significantly higher than the paired site-independent scores at the screen visit (p = 0.027). Although this significant paired scoring difference might reflect some site-based score inflation to meet study eligibility criteria, the difference was affected by the magnitude (severity) of the total MADRS score at the screen visit as well. In fact, symptom severity based on the magnitude of the total MADRS score was the primary driver of paired scoring concordance or deviations across all visits. High total site-based MADRS scores (≥40) generated significantly greater positive scoring deviations (site-based scores > site-independent scores) regardless of the study visit  in patients with generalized anxiety disorder (GAD), the centralized Ham-A scores were lower at the baseline visit and higher at the endpoint than the site-based scores . In anotIn summary, the current analysis of 3736 paired MADRS scores from 5 clinical studies affirms the utility of audio recording of site-based interviews as a surveillance strategy for site-independent quality assurance (monitoring and remediation). This method can effectively allay concerns about deceptive ratings practices [Dr. Targum participated in the design, implementation, and analysis of the original studies and conceived, analyzed, and wrote the current analysis reported in this manuscript. Mr. Catania assisted with the collection, collation, and analysis of the data and assisted with the preparation of the final manuscript.Dr. Targum is currently Scientific Director at Bracket Global LLC., and was previously at Clintara LLC during which time vendor grants were received to conduct quality assurance programs for the 5 specific studies in this analysis from Alkermes Inc., CeNeRx, Methylation Sciences Inc., and Sunovion Pharmaceuticals. Dr. Targum study has received no compensation or input from these sponsors for the analysis or preparation of this specific manuscript. He has also received consultation fees or vendor grants from Acadia Pharmaceuticals, AZ Therapies, Brain Cells Inc., Forum Pharmaceuticals, Functional Neuromodulation Inc., Intracellular Therapies, Inc., Johnson and Johnson PRD, Karuna Pharmaceuticals, Navitor Pharmaceuticals, Neurim Pharmaceuticals, Prana Biotechnology Ltd., Pfizer Inc., and Resilience Therapeutics.Christopher J. Catania is an employee of Bracket Global and has no other disclosures.Alkermes Inc., CeNeRx, Methylation Sciences Inc., and Sunovion Pharmaceuticals with additional support for the analyses from Bracket Global LLC . However, neither Bracket Global LLC nor any sponsor had any role in the analysis and/or interpretation of the data, the writing of this report, or the decision to submit the manuscript in its current form.Partial support for this study came from vendor grants to conduct quality assurance programs from


TMCT), composing rS and rF; thus, a novel chimeric infectious bronchitis-Newcastle disease (IB-ND) virus-like particles (VLPs) vaccine containing the rS, rF, and IBV M protein was constructed. Under the transmission electron microscope (TEM), VLPs possessing similar morphology to natural IBV were observed. To evaluate the immunogenicity of chimeric IB-ND VLPs, specific pathogen-free (SPF) chickens were immunized with three increasing doses . Results of ELISAs detecting IBV and NDV specific antibodies and IL-4 and IFN-γ T cell cytokines indicated that vaccination with chimeric IB-ND VLPs could efficiently induce humoral and cellular immune responses. In the challenge study, chimeric IB-ND VLPs (100 μg protein) provided 100% protection against IBV or NDV virulent challenge from death, and viral RNA levels in tissues and swabs were greatly reduced. Collectively, chimeric IB-ND VLPs are highly immunogenic and could provide complete protection from an IBV or NDV virulent challenge. Chimeric IB-ND VLPs are an appealing vaccine candidate and a promising vaccine platform bearing multivalent antigens.Infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) are two poultry pathogens seriously affecting the poultry industry. Here, IBV S1 and the ectodomain of NDV F proteins were separately linked with the trans-membrane and carboxy-terminal domain of IBV S protein (S Gammacoronavirus of the Coronaviridae family . The genome of IBV is about 27.6 kb in length, encoding fifteen non-structural proteins and four structural proteins: Spike glycoprotein (S), small membrane protein (E), membrane glycoprotein (M), and phosphorylated nucleocapsid protein (N). The M glycoprotein, the most abundant protein in the viral envelope, performs core functions in the process of coronavirus assembly and budding [Avulavirus under family Paramyxoviridae . NDV has a 15kb long genome comprising six genes which individually encode the nucleocapsid (N), matrix protein (M), phosphoprotein (P), fusion protein (F), haemagglutinin-neuraminidase protein (HN), and large polymerase protein (L) [ecto, aa1-499 of F protein of NDV) displayed on the viral envelope, a hydrophobic transmembrane domain , and a cytoplasmic domain  near the carboxyl terminus; similar to the IBV S protein, the TM/CT domain anchors the F protein in the surface of the virion [Avian infectious bronchitis (IB) and Newcastle disease (ND) are both common, highly contagious, and acute avian diseases and have been causing heavy losses in the poultry industry ,2. Infec budding ,4. Furth budding ,6. The S budding . The S p budding , while t budding ,9. ND istein (L) . F and Htein (L) . The F pe virion . F is the virion ,14,15,16Nowadays, IBV and NDV are controlled using live-attenuated vaccines and inactivated vaccines ,17. Liveecto and the IBV S1 protein were fused to the TM and CT domain of the IBV S protein (STMCT), forming the recombinant F (rF) and recombinant S (rS) protein, respectively, and chimeric infectious bronchitis-Newcastle disease (IB-ND) VLPs were constructed with these two recombinant proteins and IBV M proteins through the baculovirus system. Subsequently, the immunogenicity of the chimeric VLPs were evaluated as a vaccine in specific-pathogen-free (SPF) chickens.In this study, FThe IBV M41 and NDV La Sota and F48E9 strains were stored at −80 °C by Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Sichuan University, and propagated in 10-day-old embryonated SPF eggs  when used as previously described . Sf9 celTMCT were amplified with IBV M41 cDNA, and Fecto was amplified with NDV La Sota cDNA. S1 and Fecto were individually linked with STMCT by digestion-ligation, thus forming the rS and rF genes . Subsequently, cDNA was synthetized using the SuperScript III FirstStrand Synthesis System  following the manufacturer’s instructions. Briefly, the reverse transcription reaction mixture consists of 2 μL 5 × PrimeScript Buffer, 0.5 μL PrimeScript RT Enzyme Mix I, 0.5 μL Oligo dT Primer, 0.5 μL Random 6 mers, the RNA of the virus, and RNase Free dH2O, thus creating a final volume of 10 μL. S1 and SrF genes A.To make these three domains function naturally, a sequence encoding a short flexible peptide (GlyGlySerSer) was inserted at the fusion site. The sequences of primers used in this step are listed in pEASY®-T1 vector . Subsequently, rS and M were individually subcloned into the pFastBac1 plasmid  at the EcoR I/Hind III site, and rF was subcloned into pFastBac1 at the Sal I/Hind III site  to generate recombinant bacmids (rBMs). Positive clones were verified by blue/white selection and sequencing using M13 primers. rBM-rF, rBM-rS, and rBM-M were purified with the PureLinkTM HiPure Plasmid DNA Miniprep Kit  according to the manufacturer’s instructions, after which 2 μg of purified rBMs and 8 μL of Cellfectin® II Reagent were individually diluted in 100 μL Grace’s Medium. Afterwards, the diluted DNA and diluted Cellfectin® II were combined to make the rBMs packaged in lipidosome. After incubating for 10 min, the transfection mixture was added dropwise onto sf9 cells cultured in 6-well plate at a density of 8 × 105 per well. After incubating at 27 °C for 5 h, the transfection mixture was replaced by 2 mL of Sf-900TM II SMF . Signs of viral infection were observed 72 h post transfection. Cells were frozen-and-thawed twice to release the recombinant baculoviruses (rBVs). The culture medium was collected and centrifuged at 500 × g for 5 min to remove cell debris; thus, the first passages of rBV-rF, rBV-rS, and rBV-M were obtained. The titers of rBVs were determined by plaque assays according to the manual of baculovirus system .rS, rF, and M genes were individually cloned into the III site B. The re7–6 × 107 pfu/mL) at a multiplicity of infection (MOI) of 5. Seventy-two hours post-infection, cell pellets were lysed by RIPA , and the cytolysate—Together with supernatant culture media—Were analyzed by the western blot. Rabbit polyclonal sera against IBV was used as the primary antibody to detect rS and M, and rabbit anti-Newcastle disease polyclonal antibody  was used to detect rF. Anti-rabbit IgG HRP-linked antibody was used as the secondary antibody. The bands were visualized using the DAB Substrate kit  following the manufacturer’s instructions.To express target proteins, sf9 cells were separately infected by the third passage of rBV-rF, rBV-rS, and rBV-M  discontinuous sucrose gradient and ultracentrifuged at 80,000 × g for 5 h at 4 °C. Purified VLPs at the interface between 30% and 40% were collected. After purification, 297–423 μg of VLPs could be harvested from the 100 mL culture medium. SDS-PAGE was performed to analyze the VLPs samples. Proteins were stained with Coomassie brilliant blue (CBB). The total protein concentrations of VLPs, inactivated IBV, and inactivated NDV were determined by the Bradford Protein Assay Kit . The concentrations of rS and rF proteins in VLPs, S1 protein in inactivated IBV, and F protein in inactivated NDV were determined by the densitometry of CBB-stained SDS-PAGE gel with BSA as standard.For VLPs production, sf9 cells were co-infected with rBV-rF, rBV-rS, and rBV-M at a MOI of 5. After 96 h, the culture medium was collected and centrifuged at 4000 × w/v) phosphotungstic acid for 1 min, and then viewed under TEM .To observe the chimeric IB-ND VLPs, purified VLPs were loaded onto a carbon-coated copper grid for 5 min, negatively-stained with 2% (7.3 EID50) and inactivated NDV La Sota  with ISA206 adjuvant via the intramuscular route, respectively. M41 and La Sota strains were inactivated by formaldehyde with a final concentration of 0.2% at 37 °C for 24 h. Chickens in group 9 and 10 were mock control injected with culture medium from sf9 cells infected with wild-type baculovirus, and these two groups were set as the IBV or NDV infection control in the challenge study. On the 14th day-post-primary-vaccination (dpv), boost vaccinations were performed with the same program and doses as the primary vaccination. On the 28th dpv, chickens in odd groups were intranasally challenged with 106.7 EID50 IBV M41, and chickens in even groups were intranasally challenged with 106.4 EID50 NDV F48E9.In the first animal study, 100 7-day-old SPF chickens  were randomly divided into 10 groups, 10 chickens per group. Chickens in group 1 and 2 were immunized with VLPs containing 50 μg total of proteins (7.3 μg rS and 6.6 μg rF) per chicken. Chickens in group 3 and 4 were immunized with VLPs containing 75 μg total of proteins (10.9 μg rS and 10.0 μg rF) per chicken. Chickens in group 5 and 6 were immunized with VLPs containing 100 μg total of proteins (14.5 μg of rS and 13.3 μg of rF) per chicken. Chickens in group 7 and 8 were inoculated with inactivated IBV M41 . The immune and challenge programs were the same as that in the first study.In the second animal study to measure viral RNA levels in tissues, 40 7-day-old SPF chickens were randomly divided into 10 groups , surviving chickens were sacrificed to get tissue samples for the quantification of the replication of challenged viruses. Lungs, tracheas, spleens, kidneys, and small intestines were taken to determine IBV RNA levels; lungs, tracheas, spleens, brains, and small intestines were taken for NDV RNA level determinations.The animal experiments in this study were approved by the Animal Ethics Committee (ACE) of the College of Life Sciences, Sichuan University (license: SYXK-Chuan-2013-185). All experiment procedures and animal welfare standards strictly followed the guidelines of Animal Management at Sichuan University.IBV-specific antibodies in serum were detected by the Infectious Bronchitis Virus Antibody Test Kit ; NDV antibodies were detected by the Newcastle Disease Virus Antibody Test Kit . The concentrations of IL-4 and IFN-γ in serum were individually monitored using an ELISA Kit for Interleukin 4  and an ELISA Kit for Interferon Gamma  according to the manufacturer’s instructions.TM EvaGreen® Supermix . Primers were designed via Primer Premier version 6  according to the genome sequences of IBV M41 (GenBank accession No. AY851295.1) and NDV F48E9 (GenBank accession No. MG456905). Primer sequences were as follows: IB-sense, 5′-TCCAGAACCACCACCATT-3′; IB-antisense, 5′-TGTCACACTCCTCAGCAT-3′; ND-sense, 5′-CAGCGTCTTGACTTGTGGACAGAT-3′; and ND-antisense, 5′-ATGCCGACAGCGACTTCTTCATC-3′. All the qPCRs were carried out quadruplicately.To evaluate the viral RNA levels in tissues, 0.1 g of each tissue sample was used for RNA isolation. The swabs were suspended in 1 mL PBS; subsequently, 200 μL of supernatant from each sample was used to extract RNA for the evaluation of viral RNA levels in swabs. After reverse transcription , 1 μL of reverse transcription products  was used as template in absolute quantification real-time PCRs (qPCR) to determine IBV and NDV copy numbers using the SsoFastt-test with GraphPad Prism version 6 . Differences were considered to be significant at * p < 0.05 or ** p < 0.01.Statistical significance differences in serological and viral RNA level analyses between groups were evaluated by Student’s An about 90 kDa band and bands between 25–35 kDa were separately detected by anti-IBV sera in rBV-rS and rBV-M infected samples; an approximately 60 kDa band was detected by an anti-NDV antibody in the rBV-rF infected sample. The results showed that rS, rF, and M proteins could be expressed under current conditions A. In addPurified VLPs were analyzed by SDS-PAGE, and three distinct protein bands were observed: An about 90 kDa band corresponded to rS, a 60 kDa band corresponded to rF, and a 25–35 kDa band corresponded to M A.Under TEM, the morphology of chimeric IB-ND VLPs was observed, as shown in p < 0.01) (100 versus InM41/InLa Sota), no significant differences were observed.On the 7th, 14th, 21st, and 28th dpv, levels of IBV- and NDV-specific antibodies in serum were evaluated by indirect ELISAs. On the 14th dpv, very significantly higher mean IBV- and NDV-specific antibody titers in serum from immunized chickens than that from mock chickens were observed ( < 0.01) . The meap < 0.01). Furthermore, VLPs induced higher IL-4 levels than inactivated vaccines did, especially on the 28th dpv. IL-4 levels in serum of chickens from VLP groups were very significantly higher than that from the two inactivated vaccine groups (p < 0.01) (The ability of chimeric IB-ND VLPs in inducing cellular immune response was investigated by measuring the concentrations of IL-4 and IFN-γ in serum from immunized chickens on the 14th and 28th dpv. As  < 0.01) A.p < 0.01).As for IFN-γ, no significant levels of IFN-γ were detected in serum from inactivated vaccines-injected chickens compared with that in serum of chickens from Mock group, indicating that the two inactivated vaccines failed to induce IFN-γ production B. In con50 group including depression and huddling, and these 3 chickens died on the 5th, 6th, and 7th dpc, respectively. In the VLP75 group, only 1 chicken showed symptoms and died on the 5th dpc; chickens in the VLP100 and InM41 groups showed complete resistance to virulent IBV challenge, and no apparent symptoms were observed. In contrast, all chickens in the Mock group showed symptoms, and 8 of them showed severe depression. Finally, only 3 chickens survived ; on the 4th dpc, the mean IBV RNA levels in swabs from VLP75, VLP100, and InM41 were very significantly reduced compared with that from Mock group (p < 0.01). At the last three time points, the mean IBV RNA levels in swabs from all vaccinated groups were very significantly lower than that from the Mock group (p < 0.01). Furthermore, the mean IBV RNA levels in swabs from the VLP100 group were significantly lower than that of the InM41 group on the 6th, 8th, and 10th dpc (p < 0.01). As for NDV, the mean RNA level in swabs from the VLP50 group was significantly lower than that from the Mock group on the 2nd dpc. The mean NDV RNA levels in swabs from all vaccinated groups were very significantly lower than that from the Mock group on the 4th dpc; at the last three time points, no difference analyses between swabs from immunized chickens and Mock chickens were conducted, because all chickens died in the Mock group (100 group was significantly lower than that from the InLa Sota group (p < 0.05), and on the 10th dpc, the difference between the mean NDV RNA levels in swabs from VLP100 and InLa Sota was very significant (p < 0.01).Oral swabs were collected on the 2nd, 4th, 6th, 8th, and 10th dpc for viral RNA level monitoring. As ck group B. Furthep < 0.01). Furthermore, the mean IBV RNA level in tracheas of chickens from VLP100 was very significantly lower than that from InM41 (p < 0.01) , and the mean NDV RNA level in tracheas of chickens from the VLP100 group was significantly lower than that from the InLa Sota group  C. In theta group D.IBV S1 and NDV F proteins are both promising candidate antigens for the development of novel recombinant vaccines, and it has been proved that these two proteins are able to provide efficient protection against the IBV or NDV virulent challenge ,32,33. V100 induced comparable antibody levels as inactivated vaccines did (with adjuvant). In nonreplicating vaccines, adjuvants serve as critical components to elicit adequate immune response [To investigate whether chimeric IB-ND VLPs were efficient in inducing immune responses, purified VLPs were injected into chickens. Antibodies and cytokines in serum were measured. As for inducing humoral immune response, the chimeric IB-ND VLPs could effectively induce both IBV- and NDV-specific antibodies, and, even without adjuvant, VLPresponse ; VLPs, presponse . As for response . Due to response .100 compared with that from the InM41 group were observed on the 6th, 8th, and 10th dpc (p < 0.01)  and very significant (p < 0.01) on the 8th and 10th dpc. In tissues of challenged chickens, the mean IBV RNA level in tracheas and mean NDV titers in lungs, tracheas, and small intestines of chickens immunized with 100 μg of VLPs were lower than that of chickens immunized with inactivated vaccines (significant or very significant differences were observed). These results indicated that VLPs performed better than inactivated vaccines did. It is generally considered that the stronger humoral and cellular immunity resulted in less virus replication [100 were comparable to that from inactivated vaccine groups, so the lower virus titers in VLP100 groups are due to the stronger cellular immune responses induced by VLPs. In line with the results of the detection of cytokines in serum, VLPs were able to evoke both Th1- and Th2-type responses, while the inactivated vaccines only evoke Th2-type responses. Th1/Th2 mixed response is generally considered to be more preferable than a T2-type for preventing and treating viral infection [A quality vaccine should be able to prevent pathogen transmission. Our data showed that IBV shedding was efficiently suppressed in all vaccinated groups compared with the Mock group at all five time points, and NDV shedding was also efficiently suppressed on the 2nd and 4th dpc . In addi < 0.01) A; the melication . The antnfection ,38. In anfection , resultinfection . Due to In this study, chimeric IB-ND VLPs were constructed using the IBV M protein and two recombinant proteins, i.e., rS and rF. When used as a vaccine, chimeric IB-ND VLPs were able to efficiently induce humoral and cellular immunity responses, and, when compared with inactivated vaccines, chimeric IB-ND VLPs performed better in terms of inducing cellular response and suppressing virus replication. In conclusion, this study indicated that IB VLPs can be used as a molecular platform for the genetic fusion of heterologous antigens. This study also suggested the promise of chimeric IB-ND VLPs as an appealing vaccine candidate which could simultaneously prevent IBV and NDV.


We demonstrated that Celastrol effectively inhibited SW480 CRC cell proliferation. Two weeks of Celastrol gavage significantly inhibited the growth of xenografts in nude mice. A total of 69 candidate proteins were identified in the protein microarray experiment, including the most highly enriched protein Shoc2, which is a scaffold protein that modulates cell motility and metastasis through the ERK pathway. Celastrol significantly inhibited ERK1/2 phosphorylation in cell lines and xenograft tumors. Down-regulation of Shoc2 expression using Shoc2 siRNA also inhibited ERK1/2 phosphorylation. Furthermore, down-regulation of Shoc2 expression also significantly inhibited proliferation, colony formation, and migration functions of tumor cells. In addition, the LD0 of Celastrol by gavage is equal or more than 80 mg/kg in C57 male mice. In summary, we unraveled the anti-CRC function of Celastrol and confirmed for the first time that it inhibited the ERK1/2 pathway through binding to Shoc2.Colorectal cancer (CRC) is the third most commonly diagnosed cancer. Celastrol exhibits anti-tumor activities in a variety of cancers. However, the effect of Celastrol on human CRC and the underlying mechanisms still need to be elucidated. The present study aimed to use  Colorectal cancer (CRC) has the third highest incidence rate among cancers worldwide and is the second cause of cancer death ,2. CurreTripterygium wilfordii. Celastrol exerts various pharmacological and physiological activities in chronic inflammation, autoimmune diseases, and neurodegenerative diseases . B. B17]. BShoc2 was identified as a gene playing a positive role in mitogen-activated protein kinase (MAPK)/ERK pathway activation . Up-reguBecause we observed the reduction of ERKs phosphorylation by Celastrol, we investigated whether Shoc2 knockdown can reduce phosphorylation of ERKs. Immunoblotting analyses showed that expression of Shoc2 was significantly reduced by Shoc2 siRNA in SW480 cells . FurtherTo determine the effects of Shoc2 down-regulation on cell proliferation, CCK-8 assays were performed on the Shoc2 knockdown (Shoc2 siRNA) and the control siRNA. Shoc2 knockdown in SW480 cell line resulted in significant reduction in cell proliferation A. In add0 of Celastrol by gavage is more than 80 mg/kg  significantly decreased LoVo and HT-29 cells viability, but not FHC cells A. Acute 80 mg/kg B.in vitro and a xenograft tumor model in vivo. Regarding the study of mechanisms, we combined biotinylated Celastrol and the human protein microarray together to identify Celastrol-binding proteins. We confirmed 69 Celastrol-binding proteins through this method. Of these proteins, Shoc2 was significantly enriched. Celastrol inhibited the phosphorylation of the Shoc2 downstream protein ERK. In addition, down-regulation of Shoc2 expression inhibited ERK phosphorylation and significantly inhibited proliferation and migration of CRC cells. In summary, our study is the first to confirm that Celastrol exerts anti-tumor functions in human CRC at least partially by inhibiting ERK1/2 phosphorylation via its binding to Shoc2.In the present study, we demonstrate that Celastrol exerted anti-CRC functions in a CRC cell line Previous studies reported that Celastrol exhibited apoptosis-inducing function in cancer cells in both cell and animal models . Lu et aShoc2 is a conserved protein containing leucine-rich repeats. Shoc2 knockout in mice results in embryonic lethality . SHOC2 mIn summary, we comprehensively investigated the proteins directly binding to Celastrol. The confirmed Celastrol-binding proteins in the present study provided valuable resources. Our results elucidated the significant anti-CRC function of Celastrol, which was mediated by its inhibition of the Shoc2-ERK1/2 signaling pathway. This target might represent the general mechanism underlying the inhibitory function of Celastrol on cancer cells.


The three STY kinases were shown to phosphorylate the N-terminal sequences of some proteins we tested but exhibited different specificities. Interestingly, the N-terminal sequences of two mitochondrial ATP synthase beta subunit 1/3 (pF1β-1/3) could be phosphorylated by LE and RE but not by STY kinases, suggesting that there are uncharacterized presequence-phosphorylating kinases other than STY kinases present in RE and LE. Mitochondrial import studies showed that the import of RRL-synthesized pF1βs was impeded by the treatment of LE, and the addition of a short SSU transit peptide containing a phosphorylatable 14-3-3 binding site could enhance the import of LE-treated pF1βs. Our results suggested that the transit peptide of pSSU can compete with the presequences of pF1βs for an uncharacterized kinase(s) in leaf. Altogether, our data showed that phosphorylation of transit peptides/presequences are not uncommon for chloroplast-targeted and mitochondria-targeted proteins, albeit possibly differentially regulated.Phosphorylation of the transit peptides of nuclear-encoded preprotein is a well-known regulatory process of protein import in plant chloroplasts. In the Arabidopsis Protein Phosphorylation Site Database, 103 out of 802 mitochondrial proteins were found to contain one or more experimentally proven phosphorylation sites in their first 60 amino acid residues. Analysis of the N-terminal sequences of selected mitochondrial preproteins and their homologs from 64 plant species showed high conservation among phosphorylation sites. The ability of kinases from various sources including leaf extract (LE), root extract (RE), wheat germ lysate (WGL), and STY kinases to phosphorylate N-terminal sequences of several respiratory chain proteins were examined by  Chloroplasts and mitochondria are known as “energy” organelles because they supply energy to cells. Chloroplasts produce ATP and reducing power through photosynthesis; whereas mitochondria produce ATP through respiration . Both “ein vivo, indicating that the 14-3-3 guidance complex system is dispensable  was slower, but the import rate of the unphosphorylated RRL-pMORF3 was unaffected, suggesting that STY kinases and AtPAP2 might be involved in the import of pMORF3 into mitochondria by phosphorylating and dephosphorylating its presequences , WGL, and STY kinases. Therefore, phosphorylation of N-terminal sequences of nuclear-encoded mitochondrial preproteins is not uncommon in Arabidopsis. The N-terminal sequences of some mitochondrial preproteins can be phosphorylated by STY kinases  but two homologs of precursor mitochondrial ATP synthase subunit beta 1/3 (pF1β-1/3) are not phosphorylated by STY kinases but by unknown protein kinases in the LE. Interestingly, RE can phosphorylate the N-terminal sequences of these three pF1β homologs, but not the other mitochondrial preproteins we tested, including pMORF3. Our data showed that uncharacterized kinases other than STY kinases might play similar biological roles in modulating protein translocation into mitochondria and different kinases would have different substrate specificity. Like what was observed with pMORF3, the import of RRL-synthesized pF1β is impeded by the treatment of LE, and the inclusion of a short SSU transit peptide (pSSU) containing a phosphorylatable 14-3-3 binding site could enhance the import of LE-treated pF1β, possibly by competing for kinase phosphorylation. The inclusion of a short SSU transit peptide (pSSU-M31/34-S/A) containing a non-phosphorylatable 14-3-3 binding site did not enhance pF1β import, suggesting that the transit peptide of pSSU can compete with the presequence of pF1β for an uncharacterized kinase(s) in leaf and thus phosphorylation and dephosphorylation of transit peptide/presequence are not uncommon for proteins imported into chloroplasts and mitochondria.Here, from the Arabidopsis Protein Phosphorylation Site Database (PhosPhAt 4.0) , we foun1  according to the manufacturer’s instructions. The putative presequences of different precursor proteins were predicted from the WebLogo alignment, assuming that the mature sequences are highly conserved among the 64 species. Individual cDNA fragments encoding the 77 N-terminal residues of pF1β-1, 80 N-terminal residues of pF1β-2, 77 N-terminal residues of pF1β-3, 61 N-terminal residues of CAL2, 48 N-terminal residues of SDH1-2, 76 N-terminal residues of cytochrome C1, CytC1, 60 N-terminal residues of COX5b1, and 36 N-terminal residues of plant-specific subunit of the ATP17 were amplified by Platinum®Pfx DNA Polymerase  using primers listed in Supplementary Table E. coli was described previously in vitro mitochondrial import assay, the above pET21a vectors were used as templates for the generation of cDNAs containing attB sites and the PCR products were transferred into the pDONR201 vector  by Gateway BP reaction  according to the manufacturer’s instructions. The pDONR201 vector containing the fragment of interest was transferred into the pDEST14 vector (Life Technologies) by Gateway LR reaction  for in vitro protein translation.For overproduction of recombinant proteins in Arabidopsis thaliana ecotype Columbia-0 (WT) were sterilized with 20% (v/v) bleach and grown for 14 days on Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose under long-day conditions [16 h light (22°C)/8 h dark (18°C)] in a controlled-environment chamber. For the preparation of LE, WT seeds were grown for 10 days on MS medium. Similar size of seedlings were transferred to soil and grown until 28 days under long-day conditions in a controlled-environment chamber.For the mitochondrial import assay and preparation of RE, seeds of wild-type g for 10 min at 4°C. The supernatant was transferred to a new centrifuge tube and centrifuged at 12,000 × g for 20 min at 4°C. Next, the supernatant was further centrifuged at 100,000 × g for 1 h at 4°C by ultracentrifuge (Beckman). The final supernatant is the LE or RE.Twenty-eight-day-old WT leaves and 14-day-old WT root were ground with mortar and pestle to powder in liquid nitrogen. Grinding buffer  was added to the mortar and pestle and ground for 5 min. After that, the homogenate was filtered through four layers of cheesecloth and centrifuged at 350 × E. coli strain BL21 (DE3) pLysS. The overexpression and purification of recombinant proteins as well as in vitro kinase assay were performed as previously described by 2, 0.5 mM MnCl2, 2.5 μM ATP) containing 5 μCi of [γ-32P] ATP. All the reaction was terminated by Lammeli sample buffer and analyzed by 15% (v/v) SDS-PAGE gel and exposed to a Hyperfilm MP .pET expression vectors containing gene of interest were transformed into 2PO4, 1.0% (w/v) PVP-40, 1.0% (w/v) BSA, pH 7.5] on ice. The ground sample was filtered by four layers of miracloth and centrifuged at 2,450 × g for 5 min at 4°C and followed by 17,500 × g for 20 min at 4°C. The supernatant was discarded and the pellet was gently resuspended in wash buffer  and further centrifuged at 2,450 × g and 17,500 × g for 5 min and 20 min, respectively. Then, the crude mitochondria were resuspended in wash buffer and loaded onto PercollTM  containing 0–4.4% (w/v) PVP-40 gradient and centrifuged at 40,000 × g for 40 min at 4°C. A cloudy like band near the bottom of tube was transferred to new centrifuge tube and washed by wash buffer without BSA several times. Mitochondrial protein concentration was measured by Dye Reagent Concentrate Buffer (Bio-Rad).This method was previously performed by in vitro mitochondrial protein import assay was performed as described previously NT® T7 Coupled Wheat Germ Extract System  and TNT® T7 Coupled Reticulocyte Lysate System  were used to synthesize [35S]-Met-preproteins as described previously  mM BSA, 1 mM MgCl2, 1 mM methionine, 0.2 mM ADP, 0.75 mM ATP, 5 mM succinate, 5 mM DTT, 1 mM GTP, 1 mM NADH, pH 7.5] and 5 μl of [35S]Met-labeled preproteins either synthesized by TNT® T7 Coupled Wheat Germ Extract System  or TNT® T7 Coupled Reticulocyte Lysate System . For competition study, 5 μl of [35S] Met-labeled preproteins were pre-incubated with 5 μg LE and 0.5 mM synthetic peptide at 26°C for 2, 4 and 6 min in the reaction buffer  prior to performing the mitochondrial import assay. Phosphorylatable pSSU peptide  and non-phosphorylatable pSSU-M31/34-S/A peptide  derived from tobacco pSSU were synthesized by Peptide 2.0 Inc (United States). Unimported preprotein was digested by 8 μl proteinase K , and the PK treatment was stopped by adding 1 μl of 100 mM PMSF. After discarding the supernatant, the mitochondrial pellet was resuspended in Lammeli sample buffer and denatured at 95°C for 5 min. The reaction products were separated by SDS-PAGE gel and detected by autoradiography.The eviously . One imp35S]Met-labeled preproteins synthesized in RRL  were incubated with rotation for 1 h at RT in the presence of preimmune sera , anti-14-3-3  or anti-Hsp70 antibodies  in reaction buffer   . The antthaliana . The bouArabidopsis thaliana .Based on a large-scale protein phosphorylation study of thaliana , pMORF3 f pMORF3 . To examf pMORF3  was utilf pMORF3 . The mitf pMORF3 . From daf pMORF3 . These pf pMORF3 . Of thesf pMORF3  and the Table 1 were used to search for homologous sequences from the protein databases of 64 plant species . As shown in Figure 1, the red asterisks on sequence logos represented the positions of the experimentally proven phosphorylation sites. These results suggested that the majority of the phosphorylation sites are conserved among their homologous proteins in the 64 plant species we examined. The only phosphorylation site presented in Figure 1 that is not conserved is the Ser17 residue on the presequence of pF1β-2. This phosphorylation site is only found on pF1β-2 but not on pF1β-1/3 A study of 62 Arabidopsis, 52 rice, and 114 yeast mitochondrial proteins identified a conserved cleavage motif RX(F/Y/L)ˆ(S/A)(S/T) .A web server WebLogo (see footnote 2)  was used/A)(S/T) . The len/A)(S/T) . AlthougTogether with Hsp70, 14-3-3 proteins could bind to phosphorylated presequence of pMORF3 to form a complex . In thisTable 1 could be phosphorylated by STY kinases. In this experiment, pMORF3 was used as a positive control; presequence deletion mutant of pMORF3 (Δ1-50aaMORF3) was used as negative controls. First, recombinant proteins with the N-terminal sequences of selected mitochondrial proteins fused with the truncated MORF3 protein (Δ1-50aaMORF3) were expressed and purified from E. coli . By the in vitro kinase assay, STY8 was shown to phosphorylate the N-terminal sequences of SDH1-2 (1-48 a.a.), COX5b1 (1-60 a.a.) and ATP17 (1-36 a.a.) , whereas STY17 and STY46 phosphorylated only SDH1-2 (1-48 a.a.) and ATP17 (1-36 a.a.) . Interestingly, STY46 could also weakly phosphorylate F1β-2 (1-80 a.a.). The phosphorylation of these N-terminal sequences was generally weaker than that of pMORF3, which has three phosphorylation sites (T17/S20/T35) on its presequence. These results suggested that STY8, STY17, and STY46 have different but overlapping substrate specificities. These results corroborate with the different growth phenotypes of their T-DNA lines . Furthermore, the N-terminal sequences of these three pF1β homologous proteins and ATP17 (1-36 a.a.) could also be phosphorylated by LE and WGL (10 μg) but only weakly by RRL (65 μg) . In summary, N-terminal sequences of F1β-1 and F1β-3 were not phosphorylated by STY kinases but could be phosphorylated by unknown kinases in LE and RE, indicating that uncharacterized kinases other than STY kinases could have different substrate specificities and various presequence-phosphorylating kinases might play similar biological roles in modulating protein translocation into mitochondria. Recombinant SDH1-2 protein can be phosphorylated by 1 μg STY kinases  but not by 10 μg LE or RE , possibly because the amounts of STY kinases in LE or RE is not sufficient (much lower than 1 μg) to produce intense signals like pMORF3 and ATP17 in 20 min. Alternatively, STY kinases maybe inhibited by inhibitors in LE and RE. The uncharacterized kinases other than STY kinases in LE or RE may be very active in phosphorylating the N-terminal sequences of pF1βs, pMORF3 and ATP17, due to a higher abundance in LE or RE, and/or a higher specific enzyme activity.WGL and LE were previously reported to contain kinases that phosphorylate pMORF3 in the se assay . HoweverFigure 3), we tested whether pF1β-1 synthesized in RRL (RRL-pF1βs) could interact with Hsp70 and 14-3-3 before and after LE treatment by a co-immunoprecipitation assay. As shown in Figure 4, RRL-pF1β-1 could only bind to Hsp70 and 14-3-3 after LE treatment. In order to test if the first 77 residues of F1β-1 could bind to Hsp70 and 14-3-3, the N-terminal sequence of pF1β-1 was fused to a truncated MORF3 protein (1-77aaF1β-1 + mMORF3) and the fusion protein was synthesized in RRL. The fusion protein could only bind to Hsp70 and 14-3-3 after LE treatment . Since RRL-pMORF3-T17/S20/T35A does not bind to Hsp70 and 14-3-3 even after kinase treatment   share more than 97% sequence identity with each other and share about 82% a.a. sequence identity with NpF1β  . HoweverFigure 3). LE and WGL were also shown to phosphorylate chloroplast transit peptides, including tobacco pSSU , but this was partially relieved by the inclusion of SSU peptide (lanes 4 and 7). However, the import rates of LE-treated RRL-pF1β-1 (lanes 9 and 10) with or without pSSU peptides were indifferent when the phosphorylation period was extended to 6 min, suggesting that the majority of pSSU peptides have been phosphorylated at 6 min and lost the ability to compete for LE kinases.We have previously shown that LE and WGL could strongly phosphorylate pF1βs  was present, whereas the pSSU-M31/34-S/A peptide had no effect (lane 5). These data suggested that there was a competition for LE kinases between RRL-synthesized pF1βs and pSSU peptides.To obtain further evidence to show that there was a competition for LE kinases between RRL-synthesized pF1βs and pSSU peptides, a non-phosphorylatable mutant pSSU-M31/34-S/A peptide  containing two serine to alanine mutations (M) was used in this study . As showDuring the evolution of endosymbionts, the genomes of the ancient prokaryotic ancestors of chloroplasts and mitochondria have been greatly reduced. It is believed that many genes with no contribution to endosymbiosis have been discarded and thousands of functional genes beneficial to endosymbiosis have been transferred to the nucleus of plant cells. After transcription in the nucleus, their mRNAs are translated into proteins in cytosol which are then imported into chloroplasts and/or mitochondria through the main entry gates on the outer membranes, TOC and TOM complexes, respectively . Since bin vitro kinase assays to verify the mass spectrometry data . Our data showed that phosphorylation of N-terminal sequences is common to some of the nuclear-encoded mitochondrial preproteins . More importantly, different kinases have different substrate specificities. For example, some sequences can be phosphorylated by STY8 kinase but not by STY17 kinase . The three STY kinases are not the only kinases that can phosphorylate presequences in Arabidopsis. The N-terminal sequences of two homologs of Arabidopsis pF1β-1/3 are not phosphorylated by STY kinases but are phosphorylated by unknown kinases in LE and RE . The uncharacterized pF1β-phosphorylating kinases in RE and LE could be different kinases with different specificities, as the presequence of pMORF3 can be phosphorylated by LE but not by RE . In previous studies, it was reported that Arabidopsis LE and STY kinases cannot phosphorylate pF1β from Nicotiana plumbaginifolia  and substrate (N. plumbaginifolia) were from different species. To date, STY kinases are the only known kinase family which could phosphorylate both well-studied nuclear-encoded chloroplast preprotein (pSSU)   and miton (pSSU) . Meanwhin (pSSU) .Figure 6, the phosphorylatable pSSU peptide but not the non-phosphorylatable pSSU peptide could enhance import of LE-treated RRL-pF1β-1/2/3 into mitochondria, suggesting that there was a competition for LE kinases between RRL-synthesized pF1βs and pSSU peptides. Therefore, phosphorylation of transit peptides/presequences, an energy-consuming process, may serve a biological purpose in regulating and coordinating protein import into these two organelles. The presence of multiple kinases with different substrate specificities may allow selective phosphorylation under various physiological conditions. The kinases in different tissues  could therefore phosphorylate different preproteins and control the priority of protein import into plastids/mitochondria in different tissues , but the N-terminal sequence of pF1β-2 was less intensely phosphorylated by LE and RE kinases than the N-terminal sequences of pF1β-1/3 . The T-DNA lines of STY kinases exhibited different growth phenotypes, indicating that they cannot completely complement each other (The import rate of preproteins can be regulated by phosphorylation of their transit peptides (pSSU) or presequences (pMORF3) through formation of a guidance complex with 14-3-3 and Hsp70 . Complex tissues . There a tissues , their sch other . This coFigure 4). The binding of phosphorylated transit peptides/presequences to 14-3-3 protein may offer an additional regulatory mechanism. There are thirteen 14-3-3 genes in the Arabidopsis genome (TAIR). Pull-down assays and mass spectrometry showed that 14-3-3 isoforms chi (χ) and epsilon (𝜀) bind differentially to different but overlapped client proteins from developing Arabidopsis seeds , was shown to bind to the presequences of some mitochondrial proteins , 1994. MH. sapiens (mean charge of N-termini is 3.1) are rich in arginine but not serine (R > S), and those present in S. cerevisiae (mean charge of N-termini is 3.4) are rich in both arginine and serine residues (R = S)  and rich in arginine and serine residues (R = S), whereas the transit peptides of nuclear-encoded chloroplast proteins are less positively charged (mean charge of N-termini is 1.0) and rich in serine but not in arginine (S > T > R). In our study, serine and threonine are the major experimentally proven phosphorylated residues in Arabidopsis N-terminal sequences  . In A. tthan WGL , and thethan WGL .Figure 7). Phosphorylation of presequences has yet to be shown to affect protein import into mitochondria of animal or yeast. This design may have evolved in plants to coordinate protein import into chloroplasts and mitochondria.In yeast, more than 30 phosphorylation sites have been identified on the TOM complex . PhosphoAt5g40810; ATP17, At4G30010; AtPAP2, At1g13900; CAL2, At3g48680; COX5b1, At3g15640; F1β-1, At5g08670; F1β-2, At5g08680; F1β-3, At5g08690. MORF3, At3g06790; SDH1-2, At2g18450; SSU, At5g38430; STY8, At2g17700; STY17, At4g35708, and STY46, At4g38470.Sequence data from this work can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AtCytC1, BL designed the study. LN and JY produced overexpression constructs. SC carried out phylogenetic analysis. LK and YS carried out data-mining of experimentally proven phosphorylation sites and putative 14-3-3 binding sites on N-terminal sequences. Y-SL carried out all the other experiments and wrote the manuscript. SS provided guidance on this study, and all authors were involved in writing and/or revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Matricaria chamomilla L. contains antioxidant flavonoids that can have their bioactivity enhanced by enzymatic hydrolysis of specific glycosyl groups. This study implements an untargeted metabolomics approach based on ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry technique operating in MSE mode (UPLC-QTOF-MSE) and spectrophotometric analysis of chamomile aqueous infusions, before and after hydrolysis by hesperidinase and β-galactosidase. Several phenolic compounds were altered in the enzymatically treated infusion, with the majority being flavonoid derivatives of apigenin, esculetin, and quercetin. Although enzymatically modifying the infusion only led to a small increase in antioxidant activity (DPPH• method), its inhibitory effect on pancreatic lipase was of particular interest. The enzymatically treated infusion exhibited a greater inhibitory effect (EC50 of 35.6 µM) than unmodified infusion and kinetic analysis suggested mixed inhibition of pancreatic lipase. These results are of great relevance due to the potential of enzymatically treated functional foods in human health. Matricaria chamomilla L. (i.e. chamomile), native to southern and eastern Europe and widely found in Brazil, is one of the oldest known herbs of traditional medicine and belongs to the Asteraceae family. It contains a large group of therapeutically interesting and active compounds, the main classes being the flavonoids, sesquiterpenes, coumarins, and polyacetylenesMatricaria chamomilla L. plant and, evidently, its biological activity too. Chamomile is used mainly as an anti-inflammatory and antiseptic, but also has anticonvulsant, antispasmodic, and analgesic properties. In vitro and in vivo studies have found that M. chamomilla L. exhibits antifungal, antihypertensive, antiallergic, hypoglycaemic, analgesic, immunomodulatory, antiulcerogenic, hepatoprotective, chemopreventive, and anticancer propertiesPolyphenols are secondary metabolites affected by environmental and seasonal conditions and by the geographic origin of plants. In that light, a diverse polyphenolic fingerprint characterises the ,,,Natural flavonoids, predominantly found in their glycosylated form, are widely known for their antioxidant potentialThe hydrolysis of the glycosidic fractions of various natural flavonoids employing specific enzymes is an excellent way to modify the structure and improve the physicochemical and biological properties of flavonoidsl-rhamnosidase (EC 3.2.1.40) and β-d-glucosidase (3.2.1.21) activities resulting in rhamnose and glucose release, respectively, from a variety of conjugated flavonoidsAspergillus oryzae and exhibits a strong linkage specificity for β 1–4 linkages galactosyl residuesCommercial hesperidinase and β-galactosidase are widely employed in biotechnological deglycosylation processes producing the respective partially deglycosylated flavonoids or aglycones. Hesperidinase is a fungal enzyme that expresses both α-Matricaria chamomilla L. was carried out using the combination of these enzymes  and subsequently the metabolite profile, the antioxidant activity and the inhibitory effect on digestive enzymes of native and enzymatically modified infusions were evaluated. To elucidate the discriminating metabolic profile after the enzymatic hydrolysis of the chamomile infusion, an untargeted metabolomicsE was performed. This high resolution platform integrates full MS with MS/MS fragmentation for all precursor ions simultaneouslyIn the present work, the enzymatic hydrolysis of an aqueous infusion of Penicillium sp., β-galactosidase from Aspergillus oryzae, lipase from porcine pancreas (type II), α-amylase from Bacillus subtilis, α-glucosidase from Bacillus stearothermophilus, DPPH• , nitrophenyl-α-D-glucopyranoside, and p-nitrophenyl palmitate were purchased from Sigma-Aldrich .Hesperidinase  from The infusion was prepared by crushing dried plant material purchased from a local market in the city of Bragança Paulista, Brazil. A sample of plant material was accurately weighed, added to boiling distilled water (at a 1:10 w/v sample to solvent ratio) in a stainless steel pot and left at 25 °C for 15 min, then filtered under reduced pressure. Prior to analysis, the extraction solution was filtered thought a 0.20 µm nylon membrane filter into a HPLC vial.Matricaria chamomilla. To initiate the hydrolysis of the flavonoid glycosides, 1 ml of enzyme mixture solution prepared in 0.1 M acetate buffer pH 4.0 was added to the reaction mixture. The enzyme mixture used had equal parts of each up  to a final concentration of 0.02 mg/mL. Optimisation of hydrolysis conditions and incubation time using the enzyme combination was previously performedl-rhamnosidase (EC 3.2.1.40) and β-d-glucosidase (3.2.1.21) activities. One unit will liberate 1.0 µmole of reducing sugar (as glucose) from hesperidin per min at pH 3.8, at 40 °C. For β-galactosidase activity, one unit will hydrolyse 1.0 µmole of lactose per minute at pH 4.5, at 30 °C.The bioconversion reaction was carried out in screw-capped glass tubes with shaking (130 rpm) at controlled temperature (40 °C) for 8 h using 10 ml of aqueous infusion of E approach. Leucine encephalin  was used as a lock mass for accurate mass measurements and a 0.5 mmol/L sodium formate solution was used for instrument calibration.The analytical LC/MS experiments were performed on an ACQUITY FTN liquid chromatograph coupled to a XEVO-G2XSQTOF mass spectrometer . Data were acquired using MassLynx software. For liquid chromatography, an Acquity UPLC BEH C18  column was used. Water was used as mobile phase A, while acetonitrile was used as mobile phase B in a gradient elution mode. The flow rate was 0.4 ml/min at 45 °C and the gradient composition was 5% of B , 95% B (8.0 min), 95% B (8.5 min), 5% B , resulting in a 10 min analysis overall; the injection volume was 0.1 µL. For mass spectrometry, data were acquired in positive and negative modes. For positive mode, electrospray ionisation source parameters were set as follows: capillary voltage of 3000 V, sampling cone of 40 V, source temperature of 150 °C, desolvatation temperature of 450 °C, cone gas flow of 50 L/h, and desolvatation gas flow of 900 L/h. For negative mode: capillary voltage of 2500 V, sampling cone of 40 V, source temperature of 150 °C, desolvation temperature of 450 °C, cone gas flow of 50 L/h, and desolvation gas flow of 900 L/h. The acquisition scan range was from 100 to 1000 Da, and the data were acquired using the MSE-based putative identification results of LC–MS raw data prior to statistical analysis. PolyPhenols_PubChemID_v2013.24.11.01 database was used for identification with the following search parameters: precursor mass error ≤5 ppm, fragment tolerance ≤10 ppm. Fragmentation score, mass accuracy, and isotope similarity were considered for the identification of the molecules. To find any significant differences between the infusion before and after the enzymatic modification had been performed, univariate statistical analyses were applied using the MetaboAnalyst 4.0 web platform . Data were normalised by sum and pareto scaled before performing statistics. Fold change (FC), T-test, and Volcano plot methods were applied. Only features that fulfilled log2(FC) > 2, p values < .05 and FDR < 0.05 were considered significant. Solutions were analysed in quadruplicate.Progenesis QI (Waters) was used for peak detection, alignment, deconvolution, data filtering, ion annotation and MS• (0.02 mg/L) was added to 40 µL of aqueous infusion in acetate buffer 0.3 M, pH 3.8 or methanol, in the case of control. Flasks were incubated at 25 °C for 25 min and absorbance was determined at 517 nm. All assays were performed in triplicate. The scavenging capacity of the DPPH radical was calculated using the following equation:The antioxidant activity of samples was assessed on the basis of the scavenging activity of the stable 2, 2-diphenylpicrylhydrazyl free radical (DPPH•). Approximately, 250 µL of a methanolic solution of DPPHThe inhibitory effect of chamomile toward digestive enzymes was evaluated using both native infusion and the enzymatically modified infusion.The α-amylase activity was determined using starch as substrate, according to the methodology proposed by Kandra et al.α-glucosidase was tested according to a previously reported protocold-glucopyranoside as a substrate. Activity was assayed by using 50 µl of the chamomile infusion and 100 µl α-glucosidase solution (1.0 U/ml in 0.1 M phosphate buffer pH 7.0) incubated in a water bath at 25 °C for 10 min. After preincubation, 50 µl of substrate (5 mM p-nitrophenyl-α-d-glucopyranoside solution in 0.1 M phosphate buffer pH 7.0) were added to each tube at timed intervals. The reaction mixtures were incubated at 25 °C for 5 min. The absorbance readings were recorded at 405 nm and phosphate buffer at pH 7.0 was used as control.The inhibitory effect on 2 and 25 mM NaCl), 50 µL of the infusion, and 50 µL of 4 mmol/L p-nitrophenyl palmitate (0.05 mmol/L Tris − HCl buffer at pH 8.0) was incubated for 30 min. The reaction was stopped, transferring the tubes to an ice bath and adding 500 µL of 0.05 mmol/L Tris − HCl buffer at pH 8.0. The absorbance readings were recorded at 405 nm and Tris–HCl buffer (pH 8.0) was used as control. The concentration of inhibitors required for inhibiting 50% of the lipase activity under the assay conditions was defined as the EC50 value.The infusion was analysed for its ability to inhibit lipase activity using a modified method with p-nitrophenyl palmitate as substrate and porcine pancreatic lipase (PL)Acontrol is the absorbance value of the negative control and Asample is the absorbance value of the chamomile sample.All analyses were performed in triplicate and results were presented as percentage (%) of inhibition (I). The results were calculated using the following equation:Km, the inhibition constant, Ki, and the Vmax (maximum reaction rate), a series of substrate concentrations (200–800 mM) was tested in the assay system. Each analysis was performed without (control) and with the chamomile infusion as inhibitor (40 and 60 µl). Vmax and Km were calculated and the type of inhibition kinetics was identified using the SigmaPlot software . The Michaelis–Menten equation linearised by Lineweaver–Burk was used to determine Vmax and Km by plotting a graph, i.e. 1/V against 1/[substrate concentration], and estimated by the intercept and slope, respectively.In order to measure the Michaelis–Menten constant, p < .05 were considered significant.Statistical analyzes and graphs were done using GraphPad Prism 5.0 software. The data were expressed as mean ± standard deviation (SD) and the significance of difference was calculated using one-way ANOVA and Tukey as post-test of multiple comparison. Values of E analysis of the chamomile infusion enabled the putative identificationUHPLC-Q-TOF-MSE approach with the available metabolite database. A wide variety of secondary metabolites were altered with the enzymatic treatment and the main ones are shown in the The putative identities of statistically significant features of compounds from the enzymatic treatment of the infusion were investigated by comparing fragmentation pattern data acquired using an MSE that were altered with the enzymatic treatment and possibly involved in the antioxidant activity of chamomile infusion. Hydroxylated phenolic acids  and aglycones (hispidulin and hespertin) were increased; while glycosyl isoflavone forms  and other free phenolic acids (cinnamic and trans-cinnamic acids) were significantly reduced after enzymatic treatment. In addition, LC–MSE putatively identified rutinoside, galactoside, and neohesperidoside of quercetin and kampferol, which had a tendency to reduce due to enzymatic hydrolysis.d-glucoside), luteoloside (luteolin 7-O-glucoside) and rutin , were reduced, while their aglycones esculetin and luteolin were increased. The aglycone form of rutin (quercetin) has not been identified as a differential metabolite.In addition, umbelliferone, a natural product of the coumarin family was significantly increased after enzymatic treatment.● method was used to evaluate the hydrogen donating ability, and the antioxidant activity of the samples was expressed as the percentage of DPPH radical-scavenging activity (%). The enzymatic treatment led to an increase of approximately 12% in the antioxidant activity of enzymatically modified infusion in relation to the values observed for native chamomile infusion. A period longer than 8 h of enzymatic treatment showed no apparent change of antioxidant activity (date not shown).The DPPHp = .0008). The response of the modified flavonoids in inhibiting lipase activity is directly proportional to their concentrations and the EC50 value was 35.6 µM. With regard to the α-amylase assay, all infusions presented low inhibitory effect both before and after the enzymatic treatment.In the α-glucosidase inhibition assay, the modified infusion at 60 µM showed higher inhibitory activity than the native one . At the same concentration, lipase inhibition reached 73.6 ± 3.2%, while native infusion only showed 39.2 ± 5.7% of inhibition  plotted against 1/[S], where the intercept on the 1/Vo axis is equal to 1/Vmax and the intercept on the 1/[S] axis is equal to −1/Km led to the increase of aglycone flavonoid levels and of hydroxylated phenolic acids. Derivatives of cinnamic acids  and of benzoic acids (di and trihydroxybenzoic acids) tended to increase after enzymatic treatment compared to their contents in native infusion. Cinnamic acids, especially those with the phenolic hydroxyl group, are well-known antioxidants and are supposed to have several health benefits due to their strong free radical scavenging propertiesin vitro antioxidant activity than that of chlorogenic acidBased on the results obtained from UPLC-QTOF-MS• scavenging activity of chamomile aqueous infusion can be attributed in part to flavonoids and possibly to some other polyphenols with low molecular weights.The observed significant increase of esculetin, luteolin and apigenin aglycones in the treated infusion suggest that the hesperidinase was able to remove glucose from esculin (esculetin 6-O-glucoside), luteoloside (luteolin 7-O-glucoside), and apigenin derivatives (apigenin-7-O-glucoside and apigenin-7-apioglucoside). Incubation with the enzyme combination enabled efficient hydrolysis of rutinosides and hesperidoside from diosmin, quercetin and kaempferol. The DPPHApigenin, the most abundant flavenoid, is recognised as one of the most bioactive phenolic compounds in chamomile. In comparison to its bound forms, which include mostly apigenin-7-O-β-glucoside and various acylated forms, the aglycone is endowed with much higher bioactivity. Li et al.50 values of 64.1 and 42.5 µM, respectively, which were 10 and 5 times, respectively, more potent than its corresponding diglucoside (quercetin-3-O-β-glucopyranosyl-glucoside). This observation suggested that the increase in hydrophilicity by the extra glucose moiety in glycosylated quercetin dramatically reduced the inhibitory effectIsoquercetin showed α-glucosidase inhibition toward maltase and sucrase with ICChamomilla recutita inhibited lipase activity (86.6 ± 0.3%) in addition to having high antioxidant and anti-glycation capacities50 values have shown the potency of the natural compound, more valuable information can be obtained from the kinetics of inhibition by individual compounds or a mixture of compounds from the natural extract. The components of chamomile showed a non-competitive inhibition on glucosidase activity and a mixed inhibition on lipase activity  complex in a competitive manner and an enzyme–substrate–inhibitor (ESI) complex in a non-competitive way. Probably this result was due to the mixture of compounds found in the chamomile infusion containing compounds with both types of inhibition, as well as the high efficiency of the bioconversion reaction, which led to the conversion of active compounds to even more active metabolites that can act as enzyme inhibitors. Gholamhoseinian et al.Levisticum officinale can bind to the enzyme or ES complex, blocking the pancreatic lipase activity.In a recent study, the ethanolic extract of activity . When anin vitro, only a limited number of reports have diagnosed their mode of inhibition, showing, however, contradictory results. For instance, phenolics from the methanolic extract of finger millet seed coat showed strong inhibition towards glucosidase and pancreatic amylase with non-competitive inhibition50 value of 1.5 mg MT/mlThough a number of polyphenols have been reported as inhibitory to the action of digestive enzymes Km values for pancreatic lipase inhibition by quercetin were previously reported and varied from 2.15 ± 0.40 µMThat finding is in agreement with an earlier study that showed the inhibitory effect of flavonoids on pancreatic lipase activity and reported that the presence of sugar units in flavonoid structures reduces their inhibitory power so that an aglycone form, such as quercetin, is a more efficient inhibitor than the glycosylated form, rutinOn the other hand, Zhang et al.Matricaria chamomilla L. is considered a good source of functional polyphenolic compounds and the enzymatic treatment of flavonoids is known to improve bioactivity of such compounds due to removal of sugar molecules. In this study, we observed that around 30 putatively identified compounds were modified after treatment of chamomile infusion with hesperidinase and β-galactosidase. The infusion showed interesting results for the inhibition of digestive enzymes, mainly on pancreatic lipase. Thus, the enzymatic treatment of polyphenols can lead to bioactivity enhancement, showing the good potential of the enzymatically modified chamomile infusion as a functional food for human health benefits.