Camellia sinensis and auxins play promotive effects on this process. Nowadays, the mechanism of auxin-induced AR formation in tea cuttings is widely studied. However, a lack of global view of the underlying mechanism has largely inhibited further studies. In this paper, recent advances including endogenous hormone changes, nitric oxide (NO) and hydrogen peroxide (H2O2) signals, secondary metabolism, cell wall reconstruction, and mechanisms involved in auxin signaling are reviewed. A further time course analysis of transcriptome changes in tea cuttings during AR formation is also suggested to deepen our understanding. The purpose of this paper is to offer an overview on the most recent developments especially on those key aspects affected by auxins and that play important roles in AR formation in tea plants.Adventitious root (AR) formation is essential for the successful propagation of  Camellia sinensis (L.) O. Kuntze. (Family: Theaceae), is one of the most popular and consumed beverages worldwide [Tea, the product of orldwide . It possorldwide , obesityorldwide , and carorldwide . Moreoveorldwide ,5, albinorldwide , and higorldwide  have beeArabidopsis may initiate from xylem pole pericycle cells in both intact and de-rooted hypocotyls, while the ARs of rice may initiate from the peripheral vascular cylinder. The formation of ARs is a strictly controlled developmental process, which generally includes the following three steps: (1) Cell specification and reprogramming, in which differentiated cells acquire new cell fate programs leading to the specification of AR founder cells; (2) initiation, in which the AR founder cells undergo successive cell divisions leading to the formation of primordia; and (3) primordia emergence and outgrowth. Slow or insufficient rooting leads to the death of whole plants [Vegetative propagation is widely used in forestry. Among vegetative propagation methods, including budding, grafting, layering, and cutting, the method using nodal cuttings from good genotypes is the most efficient and economical way to produce large numbers of homogeneous plants . Usuallye plants . Therebye plants . Phytohoe plants . Auxins,e plants . The rece plants ,14,15,16Recent studies in different plants suggest that complex interactions of many hormones occur during AR formation, and auxin is likely to take part in almost all of the interactions. In addition, the interaction of different hormones regulates the successive stages of AR development, including the induction, initiation, and expression of AR formation . Such inExternal application of auxins firstly affects the balance of endogenous hormones, which leads to changes in the plant developmental process ,19. It hCytokinin is another important hormone affected by external auxin application and associated with AR formation in tea cuttings. The low ratio of auxin vs. cytokinin levels will lead to weak rooting . ExternaANTHRANILATE SYNTHASE ALPHA 1/WEAK ETHYLENE INSENSITIVE 2 (ASA1/WEI2) and ANTHRANILATE SYNTHASE BETA 1/WEAK ETHYLENE INSENSITIVE 7 (ASB1/WEI7) involved in auxin synthesis also take part in the regulation of ET signal [RUB-CONJUGATING ENZYME1 (RCE1) gene still retains the high IAA content of superroot2-1 (sur2-1) [sur2 indicates that the cross regulation of auxin and ET take part in the AR formation process. The AR formation regulated by the interactions between auxin and ET in tea plants remains unreported. Whether ET is important for AR formation in tea plants still remains unclear. Therefore, further research in this field is crucial for tea scientists.Ethylene (ET) was shown to positively regulate AR formation, which was probably through the modulation of auxin transport. It has been reported that ET can promote AR development in cuttings of different plant species . MoreoveT signal . ET may (sur2-1) . The isoArabidopsis [bushy mutants were much shorter than their counterparts in the “Micro-Tom” background, which were correlated with the internal BR contents [Brassinosteroid (BR) signaling might also play a key role in AR formation. Both auxins and BRs are interdependent and show overlapped functions in cell expansion and proliferation in bidopsis . Furtherbidopsis ,32. In tcontents . In tea contents ,34. Howecontents . However2O2) are considered as key signals involved in auxin-induced AR formation in plant species [2O2 [2O2 are able to mimic the effect of auxins in AR formation in herbaceous plants [2O2 in AR formation in woody plants. Therefore, we used the fluorescent probe diaminofluorescein diacetate (DAF-2DA) and 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) to in situ detect NO and H2O2 in IBA-induced AR formation in tea cuttings [2O2 signals were not directly induced by exogenous IBA application, as they were not detected before the initiation phase of tea cuttings. The bursts of NO and H2O2 were only observed in the rooting parts of tea cuttings irrespective of IBA treatment. Furthermore, our previous experiment found exogenous sodium nitroprusside (NO-donor) and H2O2 did not show any promotive effect on AR formation, but lead to plant death in tea cuttings. These results illustrate that NO and H2O2 participate in AR formation in tea cuttings, but their roles might not be that important as compared with those in herbaceous plants. Both signals play important roles in the initial and expression phases, but not the induction phase for tea cuttings [2O2 on AR formation of herbaceous plants might be correlated with their short induction phase [2O2 might not be involved in that preparation stage. Therefore, that might explain why exogenous application of them did not show any obvious effects on AR formation.Both nitric oxide (NO) and hydrogen peroxide  involved in the mevalonate (MVA) pathway was induced by IBA [HMG1 gene in the MEP pathway is closely associated with pigment formation, and plants treated with inhibitors of MEP pathway exhibit albino phenotypes [Besides flavonoids, isoprenoid biosynthesis was also found to be affected by IBA in tea cuttings . The 2-Cd by IBA . The MEPd by IBA . The MEPd by IBA . While td by IBA . The HMGenotypes . On the enotypes . The genCell walls acting as exoskeleton structures surrounding certain cells undergo lots of changes during AR formation. The degradation, loosening, and stretching of cell walls are essential for cell elongation and division, which are the prerequisite for AR initiation ,48. CellCellulose microfibrils are the largest cell wall polysaccharides. They are much more ordered than any other component of the primary cell wall, which provide the stiffness and load-bearing properties . HemicelWALLS ARE THIN 1 (WAT1) genes also perform similar functions. For example, the expression of WAT1 is preferentially associated with vascular tissues, including developing xylem vessels and fibers. The knock-out of WAT1 results in a defect of cell elongation [WAT1 [Moreover, the cell wall contains various structural proteins, which control its growth and expansion. For example, EXTENSINs are well-characterized proteins involved in controlling wall expansion. They are able to disrupt hydrogen bonds between cellulose microfibrils and facilitate cell wall loosening . Cell waongation . During ongation . Our preon [WAT1 . The actFurthermore, for hardwood cuttings, the degree of lignification is negatively correlated with the rooting ability . A similSUR1 and SUR2, lead to the overproduction of auxin [AUXIN RESPONSE FACTORs  controls the AR formation [ARF6 and ARF8 increase in the vascular tissue of the hypocotyl, whereas ARF17 expression decreases [In model plants, some genes, such as of auxin . The rooof auxin . A balanormation . In addiecreases . In tea A. thaliana, AR initiation is also controlled by auxin, which includes four main cell fate transition steps [WUSCHEL-RELATED HOMEOBOX11 and 12 (WOX11/12) by auxin and results in the formation of root founder cells from regeneration-competent cells [WOX5/7 and LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) by WOX11/12 and leads to the formation of root primordium cells from root founder cells [LBD16 is gradually decreased in the meristem and many meristematic genes, like SHOOT ROOT (SHR) and SCARECROW (SCR) participate in this process [NAC1  transcription factor might be involved in modulating the cellular environment to affect the emergence of AR tips [As a key model plant, recent studies in Arabidopsis could offer us a general framework of auxin-mediated AR formation, which will also shed new light on future studies of AR formation in tea cuttings. In on steps ,62. The nt cells ,63,64. Ter cells ,65,66. T process . The fou AR tips . The ide2O2 signals were only detected in the initial and expression phases, but not the induction phase, indicating that they only play important roles in the later stages of AR formation in tea cuttings. The roles of other pathways, like polar transport of auxin and auxin signaling in AR formation, and key genes involved in cell fate transitions still remain elusive. Some directions for future study were also suggested, which would facilitate our understanding of AR formation in tea cuttings.In summary, auxins significantly improve the AR initiation of tea cuttings. The potential mechanisms involved in auxin-induced AR formation in tea cuttings were shown in


Self-administered foam rolling (SAFR) is an effective massage technique often used in sport and rehabilitation settings to improve range of motion (ROM) without impairing the strength performance. However, the effects of unilateral SAFR on contralateral non-intervened muscle’s rate of force development (RFD) are unknown. Therefore, the purpose of this investigation was to examine the acute effects of unilateral hamstrings SAFR on the contralateral limb flexibility, the isometric strength, and the RFD parameters.Thirty-four subjects (21 women) completed two separate randomly sequenced experimental visits, during which the control (rested for 10 min) or ten, 30-second SAFR were performed with the dominant hamstring muscle group. Before (Pre) and after (Post) the interventions, the contralateral hip flexion passive ROM, the maximal explosive isometric strength of the contralateral knee flexors with the corresponding prime mover muscles’ surface electromyographic (EMG) amplitude were measured. Separate two-way (time ×intervention) repeated measures analyses of variance (ANOVAs) were used to examine the potential changes of the dependent variables.p < 0.001; d = 0.22). No change was found for the contralateral isometric strength or the maximal EMG amplitude. For the RFD parameters, the percent changes of the RFDs for the first 50, 100, and 200 ms of the maximal explosive isometric contraction were −31.2%, −16.8%, and −10.1%, respectively, following the unilateral SAFR, relative to the control condition. In addition, the decrement of the first 50-ms RFD reached statistical significance .The SAFR significantly improved the contralateral limb ROM  of the maximal explosive contraction. Foam rolling is a type of massage technique that has been extensively used both in athletic and rehabilitation settings, mainly due to its portability and ease of use. Over the last decade, the increasing number of research studies have focused on the examinations of performing self-administered foam rolling (SAFR) as a pre-exercise warmup and/or a post-exercise recovery strategy. These research studies have examined the short-term effects  and the In addition to the aforementioned effects that SAFR directly imposes on the local intervened muscle group, potential changes in musculoskeletal functions on the contralateral homologous or non-related heterologous  muscle group were also observed more recently. For example, Recent work suggests that foam rolling can also have nonlocal effects potentially influencing muscle mechanical sensitivity. For example, high-intensity foam rolling or rolling massage may decrease the contralateral or nonlocal muscle mechanical sensitivity , thereby0−50; RFD0−100; and RFD0−200) of the contralateral hamstring muscle groups. This study is based on a project where other parts of the data was published previously  might have played a role influencing the contralateral non-intervened limb muscles. As shown in 0−200 and the peak force of the contralateral muscle group would not likely to be statistically different following the unilateral SAFR. This work will build upon previous work by explicating the effects of high volume, long duration SAFR on musculoskeletal functions. The potential practical applications of this research may not be suitable for athletic performance, but may be of great importance for clinical practice  participated in this investigation. All subjects were healthy and physically active . In addition, all subjects were familiar with foam rolling, and they all had the experience of performing foam rolling exercises previously. Prior to any experimental testing, each subject completed an informed consent and a pre-exercise health and exercise status questionnaire, which indicated no current or recent neuromuscular or musculoskeletal disorders in the lower body. During the consenting process, the subjects were instructed to maintain their normal habits in terms of dietary intake, hydration status, and sleep during the investigation. In addition, they were refrained from performing any upper or lower body resistance exercise at least 72 h prior to each testing session. All experimental procedures for this investigation were approved by the University Institutional Review Board .Thirty-four adults  and isometric strength, as well as the hamstring muscles biceps femoris (BF) and semitendinosus (SEMI) EMG amplitude before (Pre) and immediately after (Post) the unilateral SAFR. After the first visit served as the familiarization to practice generating explosive isometric force and the SAFR protocol, the following experimental visits were conducted with a randomized order, during which the SAFR and control conditions were delivered. Between visits, a minimum of 24 h of rest was provided. All measurements were taken from the subject’s non-dominant (NONDOM) leg, and the interventions were performed on the subject’s dominant (DOM) leg. Five subjects  were left-footed, based on the leg dominance test .This investigation used a within-subjects randomized crossover design to examine the potential crossover effects of unilateral hamstring SAFR. Specifically, dependent variables included contralateral hip flexion passive ROM, knee flexion RFD parameters , and NONDOM knee flexor isometric strength testing (Pre-MVIC). Following the baseline tests, either the control or SAFR intervention was performed. During the control visit, the subjects lay down on the same medical bed for ten minutes. During the SAFR visit, the subjects performed the same protocol as described in ® Bubble® inclinometer  placed on the NONDOM knee cap, a member of the research staff grasped the ankle of the DOM leg and raised it slowly to the point where the subjects first felt tension from the hamstring muscle group. The value from the inclinometer was then recorded as the NONDOM hip flexion passive ROM. To ensure measurement consistency, the researcher used a Sharpie pen to mark the location where the inclinometer was placed, and for subsequent measurements, the researcher placed the inclinometer in the exact same spot to perform this measurement. In addition, extra care was taken to ensure the NONDOM knee was kept straight during this procedure. At least three trials with 15-s rest between trials were performed to establish the NONDOM hip flexion passive ROM. If the values from any two trials differed more than two degrees, then extra trials would be conducted. The average of the three closest trials was then calculated and recorded as the NONDOM hip flexion passive ROM.The straight leg raise test was used to measure the NONDOM hip flexion passive ROM in the supine position. With a BaselineFollowing the measurement of NONDOM hip flexion passive ROM, the subjects were instructed to lie down on the same medical bed with the prone position. With both ankles hanging off the edge of the medical bed, the subjects kept the knee joints straight and relaxed. The research staff then put a cuff around the NONDOM ankle and connected the cuff to one end of a force transducer , with the other end of the transducer attached to a wooden platform mounted on the floor. Before testing the NONDOM knee flexor MVC strength, the subjects were instructed to perform three isometric contractions at about 50% of the perceived maximal effort to warmup. Specifically, they were told to “squeeze as fast as possible”, as they practiced during the familiarization session. The subjects then performed three, 3-s MVICs of the NONDOM knee flexors with 2 min of recovery between the contractions. During each MVIC, the research staffs provided a verbal countdown “three, two, one, pull” to the subject, with specific emphasis on “pull as fast as possible and hard”, based on the instruction recommendation by 15 Ω, DE 2.1 Single Differential Surface EMG Sensor; Delsys, Inc., Natick, MA, USA; 10 mm interelectrode distance) placed on the NONDOM BF as well as the SEMI muscles, based on the electrode placement recommendations from the SENIAM project , 0–100 (RFD0−100), and 0–200 (RFD0−200) ms relative to the onset of the contraction , and stored in a laboratory computer  for further analyses. The EMG signals were preamplified  and went through a 4th-order Butterworth filter with the bandpass set at 20–450 Hz. A custom-built LabVIEW  program was used to analyze the RFD parameters, and the peak force along with its EMG amplitude. For each MVIC, the peak force output was determined from the highest mean 500-ms portion of the force plateau during the contraction of the 3-s MVIC. The isometric strength was determined by the highest peak force output among all three MVICs. The EMG amplitude was then calculated as the root-mean-square (rms) of the same 500-ms window corresponding to the peak force of the contraction. To determine the RFD during the MVICs, we first determined the force onset point at which the force signal exceeded the baseline by 2% of the baseline-to-peak value . The RFDtraction . The RFDTest-retest reliability was calculated across the pre-values from two experimental visits by determining the intraclass correlation coefficient  using Model “3,1” . In addid /pooled Standard Deviation (SDpooled), where SDpooled = square root [(SD12+ SD22)/2] , with 0.2, 0.6, and 1.2 as the thresholds for small, medium, and large effect sizes, respectively, based on Hopkins’ interpretations for the magnitude of effect size  residuals were checked and confirmed using the Shapiro–Wilk test. Separate two-way (time [Pre vs. Post] × intervention [Control vs. SAFR]) repeated measures ANOVAs were performed to examine potential changes of all the dependent variables before and after the different interventions. When appropriate, the follow-up tests included paired t-tests with Bonferroni corrections (if there was an interaction). All statistical tests were conducted using statistical software  with alpha set at 0.05. In addition, effect sizes Cohen’s d  were calect size . Lastly,ect size  were genect size .0−50, RFD0−100, RFD0−200, and contralateral BF and SEMI muscle maximal EMG amplitude).F = 27.371, p < 0.001). The follow-up paired samples t-tests indicated that the NONDOM hip flexion passive ROM significantly increased following the SAFR , but not following the control . In addition, the Post-ROM value were significantly higher following the SAFR than that following the control .The results from the two-way repeated measures ANOVA indicated that there was a statistically significant time × intervention interaction  = 3.784, p = 0.061) nor main effects (time and intervention) for the isometric strength of the NONDOM knee flexors. In addition, no interactions  = 0.038, p = 0.847; SEMI: F  = 0.162, p = 0.690) as well as main effects (time and intervention) were found for the EMG amplitude of both BF and SEMI muscles.The two-way ANOVA showed neither an interaction  = 5.834, p = 0.022). The follow-up paired samples t-tests indicated that the RFD0−50 value significantly decreased following the SAFR , but not following the control . In addition, the RFD0−50 was significantly lower following the SAFR than that following the control .For the RFD0−100 and RFD0−200, the results from the two-way ANOVAs did not show any time × intervention interactions  = 3.152, p = 0.086; RFD0−200: F  = 0.872, p = 0.358). For both RFD0−50 significantly decreased following the unilateral SAFR. Relative to the control condition, the SAFR imposed a small treatment effect (d = 0.42) on the decrement of the RFD0−50. In addition, the estimation plots with each paired mean difference (between deltaControl and deltaSAFR) suggest that the mean differences for deltaControl and deltaSAFR were below zero (mean deltaControl was less than mean deltaSAFR) for both RFD0−100 and RFD0−200  of one or some of the above-mentioned neural factors, induced by the unilateral SAFR.With the RFDThe next obvious question is how ten, 30-s unilateral SAFR could influence the central nervous system, causing a change on the contralateral muscle RFD. In addition, another important question is where in the entire corticospinal pathway that this crossover could occur. As mentioned, repetitive high-intensity foam rolling or rolling massage exert mechanical pressure on skin, muscle, and fascia, primarily influencing mechanoreceptors and nociceptors , such as0−200 for the ipsilateral, but not for the contralateral homologous muscle. An obvious difference between the current study and Cavanaugh et al. is the different interventions (foam rolling vs. rolling massage). However, review articles . Thus, contralateral athletic performance such as the explosive power does not necessarily decrease following a typical SAFR with a shorter duration. Instead, our results may specifically be important for areas of physical therapy and rehabilitation, as patients may undergo longer duration of interventions.A bout of ten, 30-s unilateral hamstring SAFR intervention improved the contralateral hip flexion passive ROM without altering the contralateral isometric strength performance and the prime mover muscles’ maximal EMG amplitudes. However, the contralateral muscle group’s ability to generate explosive force was impaired. In addition, the magnitudes of the impairments seemed to be phase-dependent (with greater impairment observed in the early phase such as the first 50 ms than those in the later ones such as the 100 and 200 ms). These results show the evidence of contralateral crossover effects of unilateral SAFR.10.7717/peerj.7028/supp-1Dataset S1Range of motion (ROM) raw data before (Pre) and after (Post) the interventions (control vs. SAFR)Click here for additional data file.10.7717/peerj.7028/supp-2Dataset S2Biceps femoris (BF) EMG amolitude raw data before (Pre) and after (Post) the interventions (control vs. SAFR)Click here for additional data file.10.7717/peerj.7028/supp-3Dataset S3Semitendinosus (SEMI) EMG amolitude raw data before (Pre) and after (Post) the interventions (control vs. SAFR)Click here for additional data file.10.7717/peerj.7028/supp-4Dataset S4Raw data for isometric strength data before (Pre) and after (Post) the interventions (control vs. SAFR)Click here for additional data file.10.7717/peerj.7028/supp-5Dataset S5Rate of force development (RFD) raw data before (Pre) and after (Post) the interventions (control vs. SAFR)Click here for additional data file.10.7717/peerj.7028/supp-6Dataset S6Subject information Click here for additional data file.10.7717/peerj.7028/supp-7Supplemental Information 1Pre-exercise Health Status Questionnaire.Click here for additional data file.


KDM6B mRNA expression is downregulated in poorly differentiated high-risk neuroblastomas and upregulated in differentiated tumors, and high KDM6B expression is prognostic for better survival in neuroblastoma patients. In neuroblastoma cell lines, KDM6B depletion promotes cell proliferation, whereas KDM6B overexpression induces neuronal differentiation and inhibits cell proliferation and tumorgenicity. Mechanistically, KDM6B epigenetically activates the transcription of neuronal genes by removing the repressive chromatin marker histone H3 lysine 27 trimethylation. In addition, we show that KDM6B functions downstream of the retinoic acid-HOXC9 axis in inducing neuroblastoma cell differentiation: KDM6B expression is upregulated by retinoic acid via HOXC9, and KDM6B is required for HOXC9-induced neuroblastoma cell differentiation. Finally, we present evidence that KDM6B interacts with HOXC9 to target neuronal genes for epigenetic activation. These findings identify a KDM6B-dependent epigenetic mechanism in the control of neuroblastoma cell differentiation, providing a rationale for reducing histone H3 lysine 27 trimethylation as a strategy for enhancing differentiation-based therapy in high-risk neuroblastoma.Induction of differentiation is a therapeutic strategy in high-risk neuroblastoma, a childhood cancer of the sympathetic nervous system. Neuroblastoma differentiation requires transcriptional upregulation of neuronal genes. How this process is regulated at epigenetic levels is not well understood. Here we report that the histone H3 lysine 27 demethylase KDM6B is an epigenetic activator of neuroblastoma cell differentiation.  Approximately half of all neuroblastoma cases are classified as high risk5, which have an overall survival rate <50%, even after intensive, multimodal therapy7. High-risk neuroblastomas are predominantly Schwannian stroma-poor, undifferentiated or poorly differentiated tumors8, and induction of differentiation by agents, such as all trans-retinoic acid (RA)10, has been exploited as a treatment strategy in high-risk neuroblastoma14. A better understanding of the molecular basis of neuroblastoma differentiation may suggest new therapeutic targets or strategies for enhancing the efficacy of differentiation-based therapy for high-risk neuroblastoma patients.Neuroblastoma is a common pediatric cancer of the sympathetic nervous system derived from the neural crest cell16. For example, trimethylation of histone H3 at lysine 27 (H3K27me3) is required for the transcriptional repression of development genes and the maintenance of pluripotency in embryonic stem cells (ESCs)18. Interestingly, recent studies have provided evidence for an important role of the H3K27-specific methyltransferase EZH2 in neuroblastoma pathogenesis. It has been reported that high EZH2 expression is prognostic for poor clinical outcome in neuroblastoma patients and that EZH2-catalyzed H3K27me3 is responsible for transcriptional repression of several neuroblastoma tumor-suppressor genes, including CASZ1 GLU, RUNX3, and NGFR19. In addition, Ezh2 activity has been shown to be critical for the growth of mouse neuroblastoma spheres in culture and for tumor development in the TH-MYCN mouse model20. In line with these findings, a more recent study has revealed a crucial role of EZH2 in blocking neuroblastoma cell differentiation21.Studies of normal stem cell differentiation have demonstrated a pivotal role of epigenetic regulation via histone lysine methylation in the differentiation process23, we reasoned that histone lysine demethylases (KDMs) that antagonize the activity of EZH2 by removing H3K27me3 might have an onco-suppressor function in neuroblastoma. The KDM6B family of demethylases KDM6A and KDM6B, also commonly known as UTX and JMJD3, respectively, are responsible for the removal of H3K27me324. Our investigation provides evidence in support of this model, revealing an anti-tumorigenic activity of KDM6B in neuroblastoma cells by inducing neuronal differentiation.Since histone lysine methylation levels are determined by the balance between the activities of histone lysine methyltransferases and demethylasesTH-MYCN mouse25, an animal model of high-risk neuroblastoma with MYCN amplification29. To assess the functions of KDMs in determining neuroblastoma differentiation states, we examined the microarray gene expression profiling data from three independent lines of sphere-forming cells (stem cell state) in comparison with their parental primary tumors (differentiated state)25. Strikingly, Kdm6b expression was selectively downregulated in sphere-forming cells compared to primary tumor cells, as no significant changes were observed in the expression levels of other demethylase genes examined 31. We found that lower KDM6B expression is significantly associated with reduced event-free survival of neuroblastoma patients -C and SMS-KCNR) and non-MYCN-amplified (SH-SY5Y and SK-N-AS) human neuroblastoma cell lines  lentiviral constructs to reduce KDM6B expression Fig. , which hKDM6B in a cohort of 498 neuroblastoma patients (the SEQC dataset)30 , glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 3 (GFRA3), and RET  analysis of the genes that are co-expressed with wth Fig. . We furtRET Fig. . In addiRET Fig. . It is kNEFM as a model system. Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) revealed that KDM6B overexpression in BE(2)-C cells led to a marked increase in KDM6B levels at the promoter of NEFM -C cells with HOXC9 overexpression, ChIP-qPCR analysis detected significant levels of HOXC9 associated with the promoter of KDM6B, particularly around the potential HOX-binding sequence TAATTG starting at 1232 bases upstream of the transcription start site (+1)  Fig. . We foun39. Since HOXC9 activates KDM6B transcription, we asked whether KDM6B upregulation is required for HOXC9-induced differentiation. We used shRNA lentiviral constructs to reduce KDM6B mRNA and protein expression in neuroblastoma cell lines with inducible HOXC9 expression -C_tetoff-myc-HOXC9 cells cultured in the absence of doxycycline underwent neuronal differentiation, showing growth arrest and extensive neurite outgrowth -C cells overexpressing KDM6B. As shown above Fig. , KDM6B oter Fig. . This inter Fig. . Moreoveter Fig. . Togetheter Fig. .Fig. 7MoKDM6B is downregulated in neuroblastoma stem-like cells and in poorly differentiated, high-risk neuroblastoma tumors. Moreover, higher KDM6B expression is correlated with higher expression of genes that promote neuronal differentiation and is prognostic for better survival in neuroblastoma patients. Finally, in a panel of neuroblastoma cell lines, we show that KDM6B depletion enhanced cell proliferation, whereas KDM6B overexpression induced neuronal differentiation and inhibited the proliferation and tumorigenicity of neuroblastoma cells. Collectively, these findings suggest an onco-suppressor function of KDM6B in neuroblastoma pathogenesis by promoting differentiation. Our findings, together with those from recent studies on EZH221, indicate that modulation of the H3K27 methylation status is a key epigenetic event in neuroblastoma development.In this report, we present evidence for KDM6B being an epigenetic activator of neuroblastoma cell differentiation. We show that 12, which, in combination with bone marrow transplant, can significantly improve the event-free survival of high-risk neuroblastoma patients14. However, resistance to RA presents a major barrier to successful RA-based therapy12. Identification of downstream mediators of RA action may suggest new strategies to bypass resistance to RA or to enhance its therapeutic effect. We recently identified an RA-HOXD8-HOXC9 axis in driving neuroblastoma cell differentiation:40 RA induces HOXD8, which, in turn, transcriptionally activates HOXC9 expression. Knockdown of either HOXD8 or HOXC9 expression confers resistance to RA-induced differentiation and forced expression of HOXD8 or HOXC9 can recapitulate the differentiation phenotype induced by RA. Data from the current study reveal an additional step in this molecular pathway, identifying KDM6B as a component of the axis downstream of HOXC9 , with 0.1% DMSO as vehicle control. Phase contrast images were captured using an EVOS digital inverted microscope  or an Axio Observer microscope and AxioVision software .Neuroblastoma cell lines BE(2)-C (CRL-2268), IMR32 (CCL-127), SH-SY5Y (CRL-2266), SK-N-AS (CRL-2137), and SK-N-DZ (CRL-2149) were obtained from ATCC , LA1-55n from Sigma-Aldrich , and SMS-KCNR from the Childhood Cancer Repository at Texas Tech University Health Sciences Center. IMR32, SH-SY5Y, SK-N-AS, and SK-N-DZ were cultured in DMEM (HyClone SH30022); BE(2)-C in DME/F-12 1:1 (HyClone SH30023); and LA1-55n and SMS-KCNR in RPMI 1640 (HyClone SH30027) from Thermo Fisher Scientific . All media were supplemented with 10% FBS . Mouse neuroblastoma sphere-forming cell lines were established as described previously51 were obtained from Addgene  and verified by DNA sequencing. Lentiviral shRNA constructs targeting human KDM6B  were obtained from Sigma-Aldrich. Lentiviral constructs expressing shRNA to HOXC9 have been described previously38. BE(2)-C and SK-N-DZ cell lines with inducible expression of Myc-tagged HOXC9 in the absence of doxycycline (Doxy) were established as previously described38. Retroviruses were produced in 293FT cells using the packaging plasmids pHDM-G and pMD.MLVogp, and lentiviruses were produced in 293FT cells using the packaging plasmids pLP1, pLP2, and pLP/VSVG (Thermo Fisher Scientific).Retroviral constructs for expressing human KDM6B  and its demethylase-defective mutant KDM6B-H1390A Proteins (20–40 µg) were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with rabbit anti-HA tag , rabbit anti-KDM6B , mouse anti-NEFM  or mouse anti-α-tubulin . Histones were extracted using the EpiQuik total histone extraction kit  and analyzed by immunoblotting using mouse anti-histone H3 , rabbit anti-H3K27me3  or rabbit anti-H3K4me3 . Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies. Proteins were visualized using a Clarity Western ECL kit . For visualization using the Odyssey system , goat anti-mouse IRDye 800 or 680 and anti-rabbit IRDye 800 or 680 from LI-COR were used as secondary antibodies.2 SYBR green/Fluorescein PCR master mix  on an iQ5 real-time PCR system (Bio-Rad) with primers against various genes , and reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen). qRT-PCR was performed using a RTes Table . All pri52. Cross-linked chromatin was sheared by sonication and immunoprecipitated using ChIP grade rabbit anti-HA tag , rabbit anti-histone H3 , rabbit anti-H3K27me3 , mouse anti-Myc tag  or control mouse or rabbit IgG (Santa Cruz Biotechnology). For qPCR, two independent ChIP samples were analyzed, and each sample was assayed in triplicate using primers that cover the promoter, coding and/or 3′ untranslated regions of KDM6B and NEFM  or mouse IgG . After washing with extraction buffer, the beads were suspended in standard SDS sample buffer and analyzed by immunoblotting using rabbit anti-KDM6B , mouse anti-Myc tag or Horseradish peroxidase-conjugated goat anti-mouse IgG .Co-IP was performed with a Dynabeads Co-IP kit  using the detergent lysis method with extraction buffer containing 150 mM NaCl and 10 µg/ml DNase I. BE(2)-C_tetoff-myc-HOXC9 cells were cultured in the absence of doxycycline for 6 days. Extracts from approximately 1 × 106 neuroblastoma cells in 200 µl serum-free DMEM were injected subcutaneously into flanks of 6-week-old female NOD.SCID/NCr mice . Approximately 4 weeks after injection, tumors were removed and weighed. All animal experiments were pre-approved by the Institutional Animal Care and Use Committee of Medical College of Georgia, Augusta University.Approximately 5 × 1031. All analyses were conducted online using R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl), and the resulting figures and p value were downloaded.Patient data used in this study were described previouslyn values in the corresponding figure legends). All quantitative data showed apparent normal distribution and equal variance. Statistical significance was estimated using Student’s t-test  for qRT-PCR and ChIP-qPCR data, and two-way ANOVA for cell growth data. Statistical analyses were conducted using Prism v7.0d for Mac .Quantitative data are presented as mean ± s.d. Cell growth, qRT-PCR, and ChIP-qPCR results are representative of at least two independent experiments with each experiment being conducted in three or four technical replicates (Supplementary MaterialsSupplementary Table 1Supplementary Table 2


In mouse fibrosarcoma, VEGFR2/NRP1 trans‐complexes correlated with reduced tumor vessel branching and reduced tumor cell proliferation. Pancreatic ductal adenocarcinoma (PDAC) strongly expressed NRP1 on both tumor cells and endothelial cells, in contrast to other common cancer forms. Using proximity ligation assay, VEGFR2/NRP1 trans‐complexes were identified in human PDAC tumor tissue, and its presence was associated with reduced tumor vessel branching, reduced tumor cell proliferation, and improved patient survival after adjusting for other known survival predictors. We conclude that VEGFR2/NRP1 trans‐complex formation is an independent predictor of PDAC patient survival. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Unstable and dysfunctional tumor vasculature promotes cancer progression and spread. Signal transduction by the pro‐angiogenic vascular endothelial growth factor (VEGF) receptor‐2 (VEGFR2) is modulated by VEGFA‐dependent complex formation with neuropilin 1 (NRP1). NRP1 expressed on tumor cells can form VEGFR2/NRP1  Aberrant angiogenesis affects the progression and dedifferentiation of cancer Neuropilin 1 (NRP1) is a broadly expressed transmembrane glycoprotein lacking intrinsic enzymatic activity. NRP1 influences angiogenesis by forming a ternary complex with VEGFA and VEGFR2, thereby potentiating the bioactivity of VEGFR2, resulting in increased signaling and enhanced endothelial cell migration cis) compared with arrested signaling when the two molecules are expressed on adjacent endothelial (VEGFR2) and tumor (NRP1) cells (denoted trans). We showed that trans‐complex formation in these mouse tumor models results in reduced tumor angiogenesis, correlating with suppressed tumor initiation trans‐complex formation was identified at low density in human gastric cancer (GAC) and at high density in PDAC. The presence of VEGR2/NRP1 trans‐complexes in PDAC, but not in GAC tumors, was associated with decreased vessel formation, reduced cancer cell proliferation, and improved patient survival. Thus, although an overall high level of NRP1 expression in cancer is associated with an unfavorable prognosis trans‐complex formation in human PDAC.In a recent study, we used mouse tumor models expressing NRP1 or not, to establish the concept of a distinct, productive signaling pattern induced when VEGFR2 and NRP1 are co‐expressed on endothelial cells  tumor tissue microarrays (TMAs) in this study (denoted HPA‐TMA) was covered by the HPA ethical permit  and an ethical permit granted to the investigators . All patient samples were anonymized.Also included were pancreatic tumor tissues collected from patients undergoing Whipple resection at Oslo University Hospital, here denoted the ‘nine‐patient PDAC cohort’. Patient diagnosis was concluded after histological evaluation by a pathologist. PDAC samples were staged according to the International Union Against Cancer classification . Consent documents and study protocols were approved by the Regional Committee for Medical and Health Research Ethics, Norway , and were in compliance with the Declaration of Helsinki, 1975.A TMA of PDAC patient samples from 75 individuals who underwent surgery at the Umeå University Hospital between 1990 and 2009 was used for clinical analysis (here denoted the Umeå‐TMA). All individuals participating in this study provided their written informed consent. The study was conducted in accordance with the ethical standards of the Declaration of Helsinki, 1975 and was approved by the regional research ethics board of northern Sweden (Dnr 09‐175 M/2009–1378‐1331).T241:EV (empty vector) or T241:NRP1 cells suspended in Matrigel were injected subcutaneously into the right and left flanks of C57Bl/6 wild‐type mice in situ hybridization (ISH), immunohistochemical (IHC) staining, immunofluorescent (IF) staining, RNAscope® ISH , and proximity ligation assay (PLA) consisted of 20 different cancer types. Of these, 17 cancer types with duplicate samples from 12 patients and triplicate samples from 44 healthy tissues were analyzed in this study  The HPA‐TMA used for The Umeå‐TMA was constructed using cores of 1 mm diameter selected by an experienced pathologist and placed on recipient blocks using a TMA Grand Master machine . Three cores were included from each primary tumor and one to three cores from metastatic lymph nodes, if present. The cores were coded and randomly placed on the recipient blocks. Clinical data were retrieved from hospital charts. The observers were blinded for the clinical information during analysis of tissue staining.NRP1 and KDR (gene symbol for human VEGFR2) expression was scored on a four‐grade scale: 0 = no detectable signal; 1 = weak; 2 = moderate; and 3 = strong signal. Images were scored by one author, blind to the patient identity and clinical parameters.Whole‐slide scanned images, with 40× objective, of the ISH and IHC arrays were acquired using an Aperio ScanScope CS Slide Scanner system . ISH signal intensity was scored using a three‐grade scale: 0 = no detectable signal; 1 = weak; and 2 = moderate. The distribution of staining in tumor cells and blood vessels was evaluated, and discrimination between structures was based on morphology. RNAscope ISH to detect trans were defined as complexes in proximity to the endothelium (one nucleus away). Complexes that were overlapping with the endothelial staining were not included in the scoring. Occurrence of complexes in trans was scored on a four‐grade scale of 0–3, where 0 indicates not present; 1, one to two PLA signals per cell; 2, three to four PLA signals per cell; and 3, more than four PLA signals per cell. PLA complexes were scored blindly by two authors independently; in cases of a difference in scoring, images were re‐examined to reach consensus.VEGFR2/NRP1 PLA complexes in in vivo, we previously generated a T241 fibrosarcoma cell line stably expressing murine NRP1 (T241:NRP1) by lentivirus transduction. In parallel, T241 tumor cells, which lack endogenous expression of NRP1, were transduced with empty virus (EV) as a control cis) and between tumor cells and endothelial cells (trans), thus creating a cis + trans condition. In T241:EV tumors, VEGFR2/NRP1 complexes were established only on endothelial cells (cis condition), which expressed both VEGFR2 and NRP1 endogenously , analyzing duplicate tissue samples from 17 different types of cancer with 12 patients per disease NRP1 expression was seen in less than 50% of the samples. Only pancreatic adenocarcinoma (PDAC) and gastric adenocarcinoma (GAC) showed the presence of NRP1 transcripts in endothelial cells in more than 50% of the biopsies , were used on the HPA‐TMA and on a separate nine‐patient PDAC cohort, along with positive (human cyclophilin B) and negative  controls  of both GAC  and PDAC  in the HPA‐TMA. Both NRP1 and KDR were more abundantly expressed in PDAC than in GAC tumors. NRP1 expression was present in both endothelial cells and surrounding tumor cells. Most tumors expressing NRP1 also displayed KDR transcripts in endothelial cells  consisting of 75 PDAC patients with known survival outcome. The PLA result was scored as trans or no‐trans    pathway High tumor vessel density correlates directly with an increased risk for liver metastasis and decreased overall survival in PDAC patients s Figure . Such VENRP1 also interacts with ligands and receptors apart from VEGF/VEGFR2, such as semaphorins, fibroblast growth factors, and platelet‐derived growth factors, influencing their downstream signaling in different cell types in situ PLA) were studied in the intact tumor tissue. While transcript and protein levels may not strictly correlate To identify the expression pattern of NRP1, transcripts , proteins (IHC), as well as protein complexes  dominate over cis‐complexes . The eventual outcome results in suppressed or augmented angiogenesis, respectively, which may influence the patient's prognosis. In agreement with the data presented here, VEGF‐dependent vessel branching has previously been shown to require VEGFR2/NRP1 cis‐complex formation The use of  Figures , and 5. trans‐complex‐containing samples  rather than the overall NRP1 expression level, as the former parameter correlates with reduced vessel density, reduced tumor proliferation, and increased patient survival.NRP1 is expressed in a wide range of non‐tumor cells that may be present in a tumor, including endothelial cells, fibroblasts, and bone marrow‐derived macrophages and immune cells The potential benefit of blocking NRP1 function has been tested using neutralizing anti‐NRP1 antibodies on human cancer‐xenografted mice. The anti‐NRP1 antibodies showed additive effects in slowing tumor growth when combined with anti‐VEGFA antibodies cis‐complexes within the endothelium, promoting angiogenesis, which would be blocked by bevacizumab. In contrast, in tumors with a high degree of trans‐complexes, anti‐VEGF treatment would potentially interrupt the complexes and unleash VEGFR2 signaling, thereby promoting tumor angiogenesis.Anti‐angiogenic therapy has been established preferentially in combination with chemotherapy for a number of cancers trans‐complexes correlate with reduction of several vessel parameters and tumor proliferation, as well as increased patient survival. It would be highly relevant to continue studies of this interaction in a larger cohort of PDAC patients, and also to investigate if the same effect of VEGFR2/NRP1 trans‐complexes can be observed in other cancer types not explored in detail here. Another important question is whether the trans‐group of PDAC patients shows a therapy response distinct from that of patients lacking trans‐complexes. We propose that the VEGFR2/NRP1 complex status could be of significant value as a prognostic marker and a potential predictive marker of anti‐angiogenic therapy in PDAC and possibly other cancer types.To our knowledge, this is the first time that complexes between VEGFR2 and NRP1, and their correlation with vessel and tumor cell parameters, have been described in human cancer. We have shown that EM and LCW conceived the project. EM, ES, CT, VT, SK, TS, and LCW planned the experiments. EM, ES, CT, and SK performed experiments. EM and ES analyzed and evaluated the ISH, IH, IF, and PLA data. CLB, MS, DÖ, and OF provided critical tumor samples. EM, ES, and LCW wrote the manuscript. All authors went through and accepted the text and figures.Supplementary materials and methodsSupplementary figure legendsFigure S1. GAC and PDAC transcript expressionFigure S2. Antibody validation and proximity ligation assay (PLA) technical and biological controlsFigure S3. Vessel parameters and tumor proliferation in GAC tumorsAppendix S1. Supplementary materials and methodsClick here for additional data file.Supplementary figure legendsClick here for additional data file.Figure S1. GAC and PDAC transcript expression.  Sense probe for neuropilin 1 (NRP1) shows no binding in gastric adenocarcinoma (GAC) (A) and pancreatic adenocarcinoma (PDAC) (B) tumor samples, confirming the specific binding of the corresponding antisense probes . Statistical analysis using Student's t‐test, presented as mean ±SD. ***p < 0.005, stomach n = 35, pancreas n = 38.Click here for additional data file.Figure S2. Antibody validation and proximity ligation assay (PLA) technical and biological controls. (A) In situ PLA on porcine aortic endothelial (PAE) cells lacking vascular endothelial growth factor (VEGF) receptor‐2 (VEGFR2) and NRP1 expression , or with expression of both receptors  treated with 75 ng/ml VEGFA. Leftmost column shows in situ PLA with primary antibodies against VEGFR2 and NRP1 and corresponding secondary antibodies. Middle column shows in situ PLA with VEGFR2 primary antibody and two appropriate secondary antibodies. Rightmost column shows in situ PLA with NRP1 primary antibody and two appropriate secondary antibodies. (B–E) In situ PLA technical controls on PDAC tissue. (B) In situ PLA (red dots) with VEGFR2 primary antibody and two appropriate secondary antibodies. (C) In situ PLA with NRP1 primary antibody and two appropriate secondary antibodies.  Negative controls with VEGFR2 (D) or NRP1 (E) primary antibody omitted, confirming the specificity of the PLA reaction. Counterstained for CD34 (green) and Hoechst 33342 (blue). Scale bars = 10 μm. (F) Representative immunofluorescence images of sectioned paraffin‐embedded PAE cells lacking  or expressing NRP1 (green) and VEGFR2 (red) , counterstained for Hoechst 33342 (blue). Scale bar = 50 μm. (G) Representative images of PDAC tumors stained for NRP1  and VEGFR2  merged with CD34 (cyan) and Hoechst 33342 (blue) (right column) scored as low NRP1 (upper panel) and high NRP1 (lower panel). Scale bar = 200 μm. (H) Analysis of NRP1 immunofluorescence score in PDAC patients classified as trans or no‐trans. 0 = no expression; 1 = low expression; and 2 = high expression. Statistical analysis using Mann–Whitney's test, presented as mean ±SD. ***p < 0.005. trans n = 8; no‐trans n = 10 tumor samples.Click here for additional data file.Figure S3. Vessel parameters and tumor proliferation in GAC tumors. (A–E) Vessel parameters and tumor proliferation in GAC tumors from the Human Protein Atlas tumor microarray (HPA‐TMA). (A) Total vessel area and (B) vessel number in no‐trans and trans groups. (C) Area of individual vessel in no‐trans and trans samples and (D) vessel branches per individual vessel area. (E) Tumor cell proliferation by Ki67 staining in GAC no‐trans and trans groups. Statistical analysis using Student's t‐test, presented as mean ±SD. trans n = 8; no‐trans n = 12 tumor samples.Click here for additional data file.


Oryza sativa L.) is well adapted to partial submergence rendering it a suitable crop plant to understand flooding tolerance. Formation of adventitious roots (ARs), that support or replace the main root system, is a characteristic response to flooding. In rice, AR emergence is induced by ethylene and in the dark where roots grow upward. We used the synthetic auxins 2,4-D and α-NAA, and the auxin transport inhibitor naphthylphtalamic acid (NPA) to study emergence, growth rate and growth angle of ARs. While α-NAA had no effect, NPA and 2,4-D reduced the root elongation rate and the angle with a stronger effect on root angle in the dark than in the light. Furthermore, NPA delayed emergence of AR primordia suggesting that efflux carrier-mediated auxin transport is required for all aspects of directed AR growth. Expression analysis using OsPIN:GUS reporter lines revealed that OsPIN1b and OsPIN1c promoters were active in the stele and root cap in accord with their predicted role in acropetal auxin transport. OsPIN2 was expressed at the root tip and was reduced in the presence of NPA. Auxin activity, detected with DR5:VENUS, increased in primordia following growth induction. By contrast, auxin activity was high in epidermal cells above primordia and declined following growth induction suggesting that auxin levels are antagonistically regulated in AR primordia and in epidermal cells above AR primordia suggesting that auxin signaling contributes to the coordinated processes of epidermal cell death and AR emergence.Flooding is a severe limitation for crop production worldwide. Unlike other crop plants, rice ( Lycopersicon esculentum) (Catya illinoinensis)  primordia form at each node during normal development that emerge during submergence to replace the original dysfunctional root system . AR formulentum)  and pecainensis) . ARs areinensis) . The horinensis) . It is hSolanum dulcamara, the gaseous hormone ethylene that accumulates in submerged tissues due to a reduced diffusion rate in water, promotes AR growth . Their degradation allows transcription of ARFs which regulate auxin-responsive genes including most Aux/IAA genes, hence providing a negative feedback loop (OsIAA26 expression whereby the OsIAA26 protein stability is controlled by the auxin-dependent E3 ubiquitin ligase Soil-surface Rooting 1 (SOR1). This study hence revealed a molecular link between ethylene and auxin signaling in rice root development.Unlike growth of nodal ARs, growth of crown roots in rice seedlings is inhibited by ethylene. Inhibition of root growth by ethylene and auxin has been reported for Arabidopsis where ethylene and auxin pathways interact . A recen studied . The E3 ack loop . In ricein planta is DR5-based auxin-inducible reporters. The DR5 promoter contains several ARF binding sites resulting in reporter gene induction in the presence of auxin  and OsGNOM1 show reduced crown root initiation and delayed lateral root differentiation  and two auxin analogs, 2,4-dichlorophenoxyacetic acid  and 1-naphthalene acetic acid (α-NAA). Rice stem sections were treated with NPA, 2,4-D or α-NAA in the dark . DarknesDR5:VENUS auxin reporter line  of epidermal cells was suggested to facilitate AR emergence . EpidermThe growth angle contributes substantially to AR system architecture. It is controlled by red and blue light signaling . Upward OsPIN2 in epidermal cells above AR primordia and subsequent depletion of auxin in these cells. Polar auxin transport furthermore controls AR elongation and determines growth direction. Taken together, local auxin activities controlled by polar auxin transport through OsPIN efflux carriers is a major determinant of AR system architecture in rice.Adventitious root emergence is controlled by polar auxin transport likely through activation of the auxin efflux carrier gene Oryza sativa L., Pin Gaew 56) were originally obtained from the International Rice Research Institute (Philippines). The OsPIN:GUS lines in the rice japonica cultivar Nipponbare were generously donated by Prof. Chuanzao Mao . The DR5:VENUS line of the japonica rice cultivar 9522 was generously provided by Prof. Zheng Yuan . The VENUS protein is directed to the nucleus for better detection and resolution (-2s-1) at 27°C and 8 h dark at 19°C with 70% relative humidity for 12–14 weeks (-2s-1) for the times indicated or exposed to light in the presence of the ethylene-releasing compound ethephon.The seeds of the deepwater rice variety PG56  and the angle measured with ImageJ (Fiji). Subsequently, roots were excised to determine the root length with a ruler.Evans Blue staining was used for epidermal cell death determination and to better visualize AR penetration at the rice stem. Five mm of the node were cut and stained in a 2% (w/v) Evans Blue solution for 3 min followed by two washing steps in tap water. Root penetration was determined using a light microscope  and the percentage was calculated based on the total number of AR initials that was taken as 100%. For Lugols staining, 1 cm of the root tip was isolated and stained with Lugols’ solution for 1 min. Subsequently, the samples were washed once in distilled water. The starch-containing statoliths were visualized and photographed using a binocular .4[Fe(CN)6], 2 mM K3[Fe(CN)6], and 2 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide [Duchefa], pH 7.5) was added to each sample. Samples were kept in a vacuum chamber for 10 min followed by shaking at 37°C in the dark . The staining buffer was removed and samples were incubated in 70% ethanol to remove chlorophyll. GUS staining was visualized using a binocular .Histochemical β-glucuronidase (GUS) staining was performed according to DR5:VENUS samples were taken with a Leica SP5 confocal laser scanning microscope (CLSM). For an overview, a 5 mm segment containing an AR primordium and the surrounding nodal tissue was excised from the node. Root primordia and the epidermis were isolated and prepared on a slide with a drop of 10% (w/v) glycerol for imaging. Imaging of VENUS was done at 510–550 nm and bright field was regarded as a control. ARs that were transferred from dark to light were marked on the upper and lower sides to allow for proper orientation of the root in CLSM.Fluorescence images of P-value was set to P < 0.05. Comparison of means was analyzed for statistical difference with an analysis of variance (ANOVA) (Tukey test) or a two sample t-test.Statistical analysis was performed using Minitab. The CL and MS designed the project. CL performed the experiments. MS wrote the manuscript with contributions from CL.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.


Fabry disease (FD) is an X-linked inherited storage disorder caused by deficiency of lysosomal alpha-Galactosidase A. Here we describe new retinal findings in patients with FD assessed by Spectral domain optical coherence tomography (SD-OCT) and their possible clinical relevance.54 eyes of 27 FD patients and 54 eyes of 27 control subjects were included. The ophthalmic examination included visual acuity testing, tonometry, slit lamp and fundus examination. SD-OCT imaging of the macula was performed in all subjects. Central retinal thickness and retinal nerve fiber layer analysis were quantified. Vessel tortuosity was obtained by a subjective scoring and mathematically calculated. Inner retinal hyperreflective foci (HRF) were quantified, clinically graded and correlated with a biomarker of Fabry disease (lyso-Gb3).p < 0,001). In addition, the vessel tortuosity was remarkably increased in FD patients compared to control persons and correlated significantly with lyso-G3 levels (p = 0.005). A further subanalysis revealed significantly higher HRF and vessel tortuosity scores in male patients with the classic FD phenotype.In comparison to an age-matched control group, a significant amount of HRF was identified in macular SD-OCT images in FD patients. These HRF were localized within the inner retinal layers. Furthermore, lyso-Gb3 levels correlated significantly with the quantitative evaluation of HRF (The observational, cross sectional, comparative study describes novel intraretinal findings in patients with FD. We were able to identify suspicious HRF within the inner retinal layers. These findings were not accompanied by functional limitations, as visual acuity remained unchanged. However, HRF correlated well with lyso-Gb3, a degradation product of the accumulating protein Gb3 and might potentially indicate Gb3 accumulation within the highly metabolic and densely vascularized macula. Fabry disease (FD) is an X-linked multisystemic lysosomal storage disorder characterized by decreased or deficient activity of the enzyme α-Galactosidase A. This results in progressive deposition of sphingolipids within a multitude of cells and organs. The prevalence of FD is estimated between 1/40.000 and 1/117.000 [FD patients present with a wide spectrum of phenotypes – from mono-or oligosymptomatic cases to severe multi-organ involvement . Severe,Early systemic manifestations of FD include superficial vascular lesions (angiokeratomas), intermittent episodic pain sensation in the extremities (acroparesthesia), proteinuria, hypohidrosis, heat/cold and exercise intolerance as well as gastrointestinal symptoms –16. As tThe availability of enzyme replacement therapy (ERT) with agalsidase alfa and beta has effectively reduced mortality and morbidity in patients with FD over the last decades –27.Ocular manifestations occur most superficially as “cornea verticillata” (CV), a vortex keratopathy representing the most commonly reported feature of ophthalmologic disease in FD , 29. ConMonitoring of organ dysfunction and damage in a lysosomal storage disorder such as FD plays a pivotal role to estimate prognosis and planning of specific and symptomatic therapy. The eye and its vascular status represent an easily accessible end organ, where rapid and noninvasive imaging methodologies can be applied.The provided study reveals novel insight into retinovascular and intraretinal features of FD, with special emphasis on the herein first described inner retinal hyperreflective foci (HRF), which can be apictured by spectral-domain optical coherence tomography (SD-OCT). Subjective as well as objective methods were applied to describe retinovascular and intraretinal features to implement the latter into clinical practice.The study was approved by the medical ethics committee of the Ärztekammer Hamburg, Germany and followed the tenets of the declaration of Helsinki for research involving human subjects. Informed consent was obtained from all participants of the study.54 eyes of 27 patients with genetically confirmed FD and an age matched control group of 54 eyes of 27 healthy volunteers were included into the prospective observational, cross sectional, comparative study. The control group was recruited among healthy coworkers at the University of Hamburg-Eppendorf. Ocular comorbidities resolved in exclusion from the study. Patients demographics are displayed in Table To assess the retinal morphology, SD-OCT imaging  was performed and acquired images were further processed and analysed with ImageJ .referred to as quantitative score of HRF, was performed by a blinded examiner in a standardized fashion. The temporal and nasal parafoveal region (0-300 μm) were outlined in a foveal cross section with the aid of ImageJ and quantified with the counting tool at 300% magnification  evaluation was attained by analysing macular scans, which were acquired using horizontal raster pattern scans (20 × 20° (5.4 × 5.4) scan field). CRT values were extracted from the central Early Treatment Diabetic Retinopathy Study (ETDRS) subfield. The retinal nerve fiber layer (RNFL) was obtained performing an additional automatized “RNFL scan”. For statistical correlation analysis, averaged CRT and RNFL measurements of both eyes were calculated. Manual quantification of HRF, ion Fig. . Inner rcalculated vessel tortuosity score by measuring the true length (a) and the end-to-end length (b) of the retinal vessels and dividing a/b , mild (1), moderate (2), severe (3) on the basis of the acquired cSLO images similar to previous described retinal vessel tortuosity grading [Prior to the calculated analyses, a clinical grading of retinal vessel tortuosity, referred to as  grading .p < 0.05 in all statistical tests. The Wilcoxon-Mann–Whitney U test (nonparametric two-independent sample test) was used to compare all retinal parameters between FD patients and the control group, whereas the Independent T-Test was applied in the normally distributed analyses. Correlation analyses were performed using Spearman Rank Order Test.Statistical analysis was performed with SPSS . The level of significance was ampullae) in 14 eyes of 7 FD patients. One patient presented with a mild subcapsular Fabry cataract. CV was noticed in 19 of 27 patients . Of note, CV was mainly found in patients with classical Fabry mutations (15/16), while only 4 of the 11 patients with the oligosymptomatic or non-classical genetic variants p.D313Y and p.A143T or the cardiac variant p.N215S presented with CV.In the present study, we analysed the retinal phenotype of patients with genetically confirmed FD. Demographic data of the 27 patients are shown in Table r = 0.7; p < 0.001). The quantitative score of HRF showed a significant correlation with the laboratory parameter lyso-Gb3, which is used for monitoring of FD in clinical practice . Furthermore, a significantly higher number of HRF was found in male FD patients compared to females (Table p = 0.011). In addition, the subjective score of HRF was significantly higher in males  than females .In the macular SD-OCT-images intraretinal HRF were observed within the inner retinal layers  in most analysed FD patients. In SD-OCT images of the retinas of FD patients a significantly higher quantitative score of HRF  was obtained in the defined parafoveal region compared to the age-matched control group . Furthermore a significantly higher subjective score of HRF was seen in FD patients  compared to the control group ; . Also the subjective clinical score revealed a more severe vessel tortuosity score in FD patients  than in the control group  compared to the age-matched control group .Furthermore, a significant positive correlation between the subjective score of retinal vessel tortuosity and the calculated values of the vessel tortuosity was visible  also shows a moderate positive correlation with the laboratory parameter lyso-Gb3.The calculated vessel tortuosity score (p = 0.013). The subjective vessel tortuosity score presented significantly higher in males  than females .A significantly higher vessel tortuosity was noticed in male FD patients compared to females Table . The mean = 16 vs. oligosymptomatic form (n = 11)) and gender on mean quantitative and subjective score of HRF and vessel tortuosity. Classically affected patients (n = 16) presented with a significantly higher values in objective vessel tortuosity (p = 0.019), subjective vessel tortuosity (p = 0.033), quantitative score of HRF (p = 0.0096) and subjective score of HRT (p = 0.0066). In particularly male patients with the classic phenotype (n = 9) revealed significantly higher values in calculated vessel tortuosity (p = 0.0081), subjective vessel tortuosity (p = 0.0018), quantitative score of HRF (p = 0.0015) and subjective score of HRT (p = 0.00098) in comparison with females with the classic phenotype (n = 7). Patients with oligosymptomatic phenotype (n = 11) did not present gender specific score discrepancies in vessel tortuosity and HRT  to estimate the burden of intraretinal HRF. Compared to an age-correlated control group of healthy volunteers a profound accumulation of HRF was detected on SD-OCT cross-sections in patients with FD.Further longitudinal investigations are necessary to clarify whether the presented HRF are changing with disease progression or under treatment. Prospectively, an automated quantification of the HRF could be used for monitoring as such an automated segmentation of HRF in SD-OCT images of patients with diabetic retinopathy was reported recently .Along the intraretinal cross-sectional analysis, we evaluated the previously described classical vascular tortuosity  in our cLyso-Gb3 is acknowledged as an important marker in FD and correlates with disease severity as well as ERT response –49. AddiInterestingly, a strong correlation was present between the quantity of HRF and the concomitant lyso-Gb3. Quantification of vessel tortuosity also showed a moderate positive correlation with serum lyso-Gb3 levels. These findings align with the report by Sodi et al. who highlighted the correlation of vessel tortuosity and disease severity . FurtherA further subanalysis of the Fabry cohort revealed significantly higher HRF and vessel tortuosity scores in the classic phenotype of FD, in particularly in male individuals Fig. , furtherIn summary, the present study describes novel intraretinal HRF in patients with FD and further provides a simple, yet differentiated grading system to estimate the presence of HRF as well as vessel tortuosity based upon noninvasive SD-OCT imaging. As FD is a multisystemic lysosomal storage disorder and clinical decision making is based on an interdisciplinary approach, we suggest SD-OCT imaging as a further adjunctive tool to increase the ophthalmologic risk assessment. Automated quantification of the intraretinal findings detected by SD-OCT imaging could serve as an easily accessible, quick monitoring tool. A major limitation of the present study is the cross-sectional design and the limited acquisition of global disease parameters. The underlying pathology remains to be explored as no histological and/or animal retinal documentations are published at present. As the retina and in particular the macular area are highly vascularized and perfused, capillary dysfunction and concomitant endothelial glycosphingolipids deposition  are poteFurther investigations to clarify the retinal involvement have been initiated at our department. In addition, a prospective, longitudinal clinical study is currently ongoing at the university medical center Hamburg-Eppendorf that might further elucidate the impact of HRF in FD and the potential effect of systemic enzyme replacement therapy on the distribution of inner retinal HRFs.


Aphelenchoides ritzemabosi, is a migratory, plant-parasitic nematode that is widely distributed and infects the aboveground parts of many plants. The fatty acid- and retinoid-binding proteins (FAR) are nematode-specific proteins that are involved in the development, reproduction, and infection of nematodes and are secreted into the tissues to disrupt the plant defense reaction. In this study, we obtained the full-length sequence of the FAR gene (Ar-far-1) from CFN, which is 727 bp and includes a 546 bp ORF that encodes 181 amino acids. Ar-FAR-1 from CFN has the highest sequence similarity to Ab-FAR-1 from A. besseyi, and they are located within the same branch of the phylogenetic tree. Fluorescence-based ligand-binding analysis confirmed that recombinant Ar-FAR-1 was bound to fatty acids and retinol. Ar-far-1 mRNA was expressed in the muscle layer, intestine, female genital system, and egg of CFN, and more highly expressed in females than in males among the four developmental stages of CFN. We demonstrated that the reproduction number and infection capacity of CFN decreased significantly when Ar-far-1 was effectively silenced by in vitro RNAi. Ar-far-1 plays an important role in the development, reproduction, infectivity, and pathogenesis of CFN and may be used as an effective target gene for the control of CFN. The results provide meaningful data about the parasitic and pathogenic genes of CFN to study the interaction mechanism between plant-parasitic nematodes and hosts.The chrysanthemum foliar nematode (CFN),  Aphelenchoides ritzemabosi, is an obligate ecto- and endoparasite of the aboveground parts of plants [The chrysanthemum foliar nematode (CFN), f plants ,2. CFN hf plants ,4.The molecular biology of plant-parasitic nematodes has attracted much attention from scholars who have done a great deal of research; however, there are few studies on the molecular biology of CFN. To our knowledge, there has as yet been no report on the functional genes of CFN, and the only phylogenetic analyses of foliar nematodes that have been conducted have been based on the small subunit ribosomal DNA of CFN ,6 and trGp-FAR-1 from Globodera pallida has been shown to accumulate on the nematode body surface and is thought to interfere with plant lipoxygenase (LOX)-mediated defense signaling [Meloidogyne javanica may adjust expression of lipid-, cell wall-, and phenylpropanoid-related genes during nematode infection of hosts [M. incognita possibly influenced the ability of nematodes to move [Rs-far-1 from Radopholus similis was involved in the regulation of allene oxide synthase (AOS) expression in Anthurium thaliana [The fatty acid- and retinoid-binding (FAR) proteins are helix-rich, lipid-binding proteins that are unique to nematodes and may play an important role in acquiring fatty acids and retinoids from the host . After nignaling . It has of hosts , the FAR to move , and thethaliana .A. besseyi and Bursaphelenchus xylophilus, are Aphelenchida nematodes.With the development of transcriptome sequencing technology, the nematode-specific FAR proteins were being extensively discovered and studied ,25,26,27Ar-FAR-1 protein was obtained through prokaryotic expression after the FAR protein gene (Ar-far-1) of CFN was cloned using the transcriptome sequencing data of CFN, and its structure and features were analyzed, and its binding activity was detected. We also studied the localization and expression of Ar-far-1 in CFN, and the role of Ar-far-1 in reproduction and pathogenesis of CFN. This is the identification and functional study of the first functional gene in CFN.In this study, the Ar-far-1) from CFN was amplified using the specific primers (Ar-far-1 was 727 bp and included a 546 bp open reading frame (ORF) that encodes 181 amino acids (GenBank accession number KX816796)  and four introns  , and includes a conserved domain similar to that in Gp-FAR-1 . Like the six FAR proteins with known 3–D structures from other nematodes , the Ar-FAR-1 protein contain conserved potential casein kinase II phosphorylation sites and protein kinase C phosphorylation sites. Secondary structure prediction algorithms applied to the multiple alignment indicated that these proteins were rich in α-helix, with no β/extended structure (Ar-FAR-1 and the FAR-1 protein from A. besseyi were in the same branch and had the closest relationship (confidence was up to 99%), and were clustered in the same group with those from other Aphelenchina nematodes. In addition, all these 55 FAR proteins from 28 species of nematodes were divided into six groups: Filarioidea, Ascaridoidea, Tylenchina, Aphelenchina, Strongylida, and Rhabditoidea.A full-length sequence of the FAR gene  . The fuld 45 bp) . The moltructure . A phylotructure . Ar-FAR-Ar-far-1 gene in CFN, we analyzed the gDNA of CFN by Southern blot. The results showed that the gDNA digested with restriction enzyme Dra I inside the Ar-far-1 gene had two signals, and gDNA digested with restriction enzyme EcoR I outside the Ar-far-1 gene had one signal. No hybridization signal was detected in the digested carrot gDNA (Ar-far-1 gene had two copies with close positions in gDNA.To determine the gene copy number of the rot gDNA . AccordiAr-FAR-1 protein (rAr-FAR-1) was well purified and appeared as a single band of approximately 20 kDa, which was consistent with the theoretical molecular weight (20.52 kDa) of Ar-FAR-1  undecanoic acid (DAUDA) because the peak emission of DAUDA shifted from 556 nm to 478 nm upon the addition of rAr-FAR-1. After the addition of oleic acid to DAUDA, the protein complex exhibited a pronounced decrease in fluorescence, and the maximum peak emission shifted to 506 nm, indicating that oleic acid can displace DAUDA from the binding site  than that in other developmental stages and mixed-stage nematodes of CFN and was approximately 3.1, 3.6, 3.7, and 2 times higher than that in males, juveniles, eggs, and mixed-stage nematodes, respectively. The expression of Ar-far-1 was not significantly different (p > 0.05) among males, juveniles, and eggs, but Ar-far-1 expression in these groups was significantly lower (p < 0.05) than that in mixed-stage nematodes of CFN  by 46.82%, 49.71%, 69.45%, 88.98%, and 74.25%, respectively, compared with that in the corresponding control CFN treated with egfp dsRNA. The relative expression of Ar-far-1 in CFN treated with Ar-far-1 dsRNA decreased with the treatment time increased from 12 to 48 h but rebounded at 72 h. The relative expression of Ar-far-1 in CFN treated with Ar-far-1 dsRNA was lowest at 48 h, and showed a significant difference (p < 0.05) compared with that at 12 and 24 h, but no significant difference (p > 0.05) compared with that at 36 and 72 h. There was no significant difference (p > 0.05) in the expression of Ar-far-1 among CFN treated with egfp dsRNA.and 72 h A. The reAr-far-1 dsRNA exhibited significantly lower (p < 0.05) reproduction than the untreated CFN (CK) and CFN treated with egfp dsRNA at all time points  than that of CFN treated for 12 and 24 h; there was a significant difference (p < 0.05) between the reproduction of nematodes treated with Ar-far-1 dsRNA for 12 and 24 h and no significant difference (p > 0.05) between the reproduction of CFN treated with Ar-far-1 dsRNA for 24 and 36 h (p > 0.05) among the reproduction of nematodes treated with egfp dsRNA regardless of the treatment time , but no significant difference (p > 0.05) was observed between the latter two treatments  than that of nematodes treated with ddH2O and egfp dsRNA  Tzvel in Kunming City, Yunnan Province, China. They were maintained by serial subculture on excised carrot (Daucus carota) disks in Petri dishes at 25 °C in a dark incubator as previously described [CFN used in this research were collected from the leaves of escribed .Ar-far-1 gene of CFN, the full-length cDNA and gDNA of Ar-far-1 were amplified using the specific primers, FAR1-F and FAR1-R  and verified as previously described . cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit , and genomic DNA (gDNA) was extracted with a tissue DNA kit  according to the manufacturer’s instructions. To study the function of the d FAR1-R , based oAr-far-1 sequence analysis was performed with Molecular Evolutionary Genetics Analysis version 6.06 software . An ORF was identified and sequence homology comparisons were conducted using the NCBI ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and the blastx, tblastn and blastp programs from NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), respectively. The signal peptide was predicted with the SignalP 4.1 server [http://www.expasy.ch/tools/). Multiple protein sequence alignments were conducted with Clustal Omega , and phylogenetic tree analyses were performed with MEGA 6.06 by the neighbor-joining method [1 server . Proteing method ,39.w/v) agarose gel electrophoresis and transferred to a Hybond N+ membrane  [Ar-far-1 were used as a control group [Primers in England) ,40. The ol group .Ar-FAR-1 protein, full-length Ar-far-1 was amplified from the plasmid with the primers FAR1FBamHI and FAR1RXhoI (E. coli DH5a for sequence confirmation. Recombinant plasmid DNA was introduced into E. coli BL21(DE3) for expression. Expression of the recombinant protein was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining after treatment with 1 mM isopropyl β-D-Thiogalactopyranoside (IPTG). The recombinant fusion Ar-FAR-1 protein with His-tag at the N-terminus was purified by affinity chromatography using Ni Sepharose High Performanc  according to the manufacturer’s instructions. The purity of purified recombinant protein was confirmed by SDS-PAGE [To obtain purified AR1RXhoI , and cloSDS-PAGE .Ar-FAR-1 with signal peptide was detected using the fluorescent analogs DAUDA , retinol  and oleic acid  as previously described [d) values for DAUDA, retinol and oleic acid were estimated as previously described [The fatty acid and retinoid binding activity of escribed ,10,15. Tescribed .Ar-far-1. DIG RNA labeling mix  was used to synthesize DIG-labeled sense and antisense RNA probes based on the purified PCR product according to the manufacturer’s instructions. The detection of in situ hybridization was performed as described previously [Specific sense primers, FAR1-IN-T7F and FAR1-IN-R, and antisense primers, FAR1-IN-F and FAR1-IN-T7R , were deeviously ,39,42.Ar-far-1 at four developmental stages of CFN using qPCR, total RNA samples were extracted from approximately 200 CFN eggs, juveniles, females and males with the RNeasy Micro Kit  according to the manufacturer’s instructions. The RNA from each sample was used as the template for cDNA synthesis with the ReverTra Ace qPCR RT Master Mix with gDNA Remover  according to the manufacturer’s instructions. The specific primers qPCR-F1 and qPCR-R1  were transcribed using a Script MaxTM Thermo T7 Transcription Kit (Toyobo). The dsRNA was purified as described previously [egfp (enhanced green fluorescent protein) dsRNA.The specific sense primers FAR1-IN-T7F and FAR1-IN-R and the antisense primers FAR1-IN-F and FAR1-IN-T7R  were useeviously ,43. The Ar-far-1 dsRNA and egfp dsRNA solutions (2.0 mg/mL) for 12 h, 24 h, 36 h, 48 h and 72 h in a dark rotary incubator . The egfp dsRNA solution was used as a negative control. The treated nematodes were washed with DEPC-treated water, total RNA was extracted, and qPCR was used to analyze the transcript levels of Ar-far-1 mRNA in CFN as described above. Each treatment was replicated three times. In addition, 20 female nematodes soaked in Ar-far-1 and egfp dsRNA solution (2.0 mg/mL) were then inoculated onto a carrot callus and maintained at 25 °C in a dark incubator for 56 days, after which the nematodes in the carrot callus were isolated, and the total number of nematodes was calculated as previously described [Ar-far-1 and egfp dsRNA solution for 48 h were inoculated on A. thaliana leaves. The symptoms in A. thaliana caused by CFN were tested and the nematodes were extracted from A. thaliana after 21 days of inoculation. Each treatment was repeated five times, and each experiment was conducted twice to confirm the results.Approximately 500 nematodes separated from excised carrot were collected in DEPC-treated water and then soaked in escribed . Each trolutions .0 mg/mL p > 0.05); therefore, data from two experiments were combined for analyses. Multiple post hoc comparisons were conducted at the 5% level of probability using Duncan’s multiple range rest (DMRT).One-way ANOVA was used for all statistical analyses with the SPSS 13.0 statistical software package . Similarity between the repeated experiments was tested by preliminary ANOVA using experimental runs as a factor, and no significant differences were detected (


Samples added with encapsulated PPE showed the smallest variations of color a* values (red) during the considered storage period, followed by samples added with PPE, suggesting a protective effect of the extract toward the myoglobin oxidation process. In addition, textural parameters  reached the highest levels, after 8 days of storage, in burger samples added both with PPE and encapsulated PPE, supporting the potentiality of PPE, encapsulated or not into alginate beads, to be used as a natural preservative of beef burger patty formulations for maintaining quality parameters.Beef burger patties are a very perishable food product with a maximum shelf life of 3 days at 4°C, due to a fast decrease of quality parameters and microbial growth. Although some additives listed in the Regulation EU 601 (2014) are allowed in fresh minced beef and meat preparations with antioxidant functionality, no additive with antimicrobial activity is permitted. In this study, a prickly pear extract (PPE) was added to beef burger patty formulations both by direct application and encapsulation in alginate beads. Beef burger patties were evaluated during refrigerated storage (up to 8 days at 4°C) in terms of microbial quality, pH, texture, and color variation. At the end of storage, burger samples incorporating PPE and encapsulated PPE showed significantly ( Fresh minced beef is widely used all over the world as a basic ingredient of various food preparations and especially for burger patty formulations. Upon purchase, minced beef should be immediately refrigerated or frozen to avoid the bacterial spoilage. In fact, the grinding process causes the spillage of tissue fluids that represent a rich nutritional component for a wide range of microorganisms, promoting a rapid microbial growth, also if meat is immediately packaged and chilled . The minBeing a very perishable product, food additives can be added during the preparation of minced meat products as reported in the To date, no additive with antimicrobial activity is allowed on fresh minced meat and meat preparations, but several studies investigated the use of natural antioxidants for meat preservation through the reduction both of microbial growth and lipid oxidation during storage; their involvement in maintaining quality parameters and in improving health benefits has also been studied .in vitro and in vivo the great antibacterial activity of a first-crop PPE against pathogenic and spoilage bacteria, generally involved in the decay of sliced beef stored at 4°C; moreover, the extract addition preserved both the beef color and texture over the considered storage period.Prickly pear extract (PPE) has attracted scientific interest for its healthy properties, including anti-ulcer, anti-inflammatory, diuretic, cardioprotective, and anti-diabetic effects  and for With minced beef, being a much more perishable product than the sliced one, the antioxidant/antimicrobial application mode can play a decisive role in preserving the main characteristics of acceptability. Encapsulation of active food ingredients has increased in the food industry since the encapsulated materials can be protected from moisture and oxygen, thus enhancing their stability over time. The aim of the present study was to maintain the overall quality of beef burger patties through the incorporation of PPE with two different techniques: direct application or encapsulation in sodium alginate. Beef burger patties were evaluated during refrigerated storage at 4°C in terms of microbial quality, pH, texture, and color variation.First-crop red-purple fruits have been picked in July 2017, gently brushed to eliminate thorns and transported to the Di3A laboratory within 1 h into plastic trays at room temperature. Fruits were washed with tap water and manually peeled. The pulp was lyophilized and the water extract was obtained as described by ® paper Grade 1, allowed to stabilize in the air for 15 min and then weighed to exclude any PPE loss during the spherification process. Control beads were obtained replacing the PPE with SDW.PPE encapsulation in sodium alginate  was performed as described by Alginate bead size, containing SDW or PPE, was estimated using the free license software ImageJTwo independent lots of minced meat (2 kg × 1 kg) were purchased from a local supermarket in Catania , transferred (within 30 min) to the Di3A laboratory in a portable refrigerator at 4 ± 1°C and immediately used for the subsequent meat formulations.Burger patties were prepared by mixing each lot (1 kg) of the minced beef with 0.8% (w/w) of NaCl in a bowl for 3 min to obtain a uniform mixture, which was divided into four 250 g experimental units; each unit was added with SDW , encapsulated SDW , PPE , or encapsulated PPE . PPE concentration at 5% (v/w) was adopted on the basis of a previous study carried out on sliced beef , where dEnterobacteriaceae and total Pseudomonas spp., respectively. The plates were incubated for 24–48 h at 32 or 25°C (for the Pseudomonas spp. count). Bacterial colonies were counted from three replicates, and the mean was expressed as log CFU (colony forming unit)/g of hamburger ± standard deviation.The potential antimicrobial activity of PPE added to the formulation of burger patties, either encapsulated or not in sodium alginate beads, was evaluated by monitoring the microbial population growth after 0, 2, 4, and 8 days of storage at 4 ± 1°C. In brief, a portion of each sample (10 g) was aseptically transferred into a stomacher filter bag containing 90 ml of sterile Ringers solution, homogenized for 5 min and afterward serially diluted. Appropriate dilutions were then plated in Petri dishes containing Plate Count Agar  with cycloheximide 0.1% solution (Oxoid), Violet Red Bile Glucose Agar  and Pseudomonas Agar Base , supplemented with Pseudomonas CFC selective agar supplement , to monitor the growth of Total Mesophilic Bacteria (TMB), total ®, Wilmington, NC, USA). Immediately after the sample homogenization, pH was measured by using a Eutech pH 700 Meter . Each value was expressed as mean ± standard deviation of three replicates.The pH variation of either treated or untreated beef burgers  was moniThe color of all samples  was descThe different burger patties  were anap < 0.05).Statistical analyses were performed using the Statistical package software Minitab™ version 16.0. All data from experiments were expressed as mean values ± standard deviation. Data of different assays were analyzed independently and subjected to One-way analysis of variance (ANOVA). Fisher’s test was used to compare the significance of differences among means (6 CFU/g) fixed by the European regulation (p < 0.05) with 7.17 ± 0.09 and 6.66 ± 0.32 log CFU/g, respectively  and it rapidly increased after 2 days of storage, exceeding the limit of 6.7 log CFU/g  different from the SDW sample (5.45 ± 0.04 log CFU/g). After 2, 4, and 8 days of storage, the enterobacteria counts registered the lowest values (p < 0.05) in samples PPE and Encaps-PPE compared to SDW and Encaps-SDW samples. Although some differences among samples were detected, the addition of PPE strongly inhibited the growth of enterobacteria, registering at the end of the storage (8 days) values averagely 2 log units lower in samples PPE and Encaps-PPE  in comparison to SDW and Encaps-SDW samples , the amount of Pseudomonas spp. counts (p < 0.05) lower values compared to SDW and Encaps-SDW that registered the highest values of 8.94 ± 0.02 and 8.33 ± 0.09 log CFU/g, respectively. At the end of storage (8 days) the best results were obtained by the direct addition of PPE (6.76 ± 0.04 log CFU/g) followed by Encaps-PPE (7.38 ± 0.02 log CFU/g) and Encaps-SDW  higher pH value of 6.07 ± 0.06 when compared to the control (SDW). After 2 days of storage, while the SDW and Encaps-SDW control samples showed a constant pH value (about 5.9), the pH value in the PPE and Encaps-PPE samples significantly (p<0.05) decreased, reaching 5.35 ± 0.03 and 5.27 ± 0.02, respectively. After 4 days of storage, pH value strongly increased in the SDW sample (Immediately after treatment (time 0), the samples SDW, Encaps-SDW and Encaps-PPE displayed similar starting pH values of 5.92 ± 0.03, 5.94 ± 0.02, 6.07 ± 0.06, and 5.93 ± 0.01 for SDW, Encaps-SDW, PPE and Encaps-PPE, respectively; sample PPE showed a significantly  modified the initial L*, a*, and b* values of burger patties, if compared to the respective controls SDW and Encaps-SDW.As shown in p < 0.05) value of 40.3 ± 1.3 when PPE was directly added to minced meat (PPE sample), followed by Encaps-PPE (42.06 ± 0.05) and Encaps-SDW (45.08 ± 0.03) samples. Lightness values of all samples increased after 4 days of storage, recording after 8 days 52.8 ± 1.1, 51.1 ± 0.9, 45.1 ± 0.9, and 45.8 ± 0.4, for SDW, Encaps-SDW, PPE, and Encaps-PPE, respectively  higher a* values  than SDW and Encaps-SDW ones . After 8 days of storage, SDW and Encaps-SDW samples registered, respectively, a* values of 8.94 ± 0.5 and 9.49 ± 0.4, while PPE and Encaps-PPE samples showed values of 14.45 ± 0.5 and 14.05 ± 0.1. Overall, sample Encaps-PPE showed the lowest variations of a* value during the considered storage period. Sample PPE displayed an irregular trend and registered up to 8 days a decreasing a* value of almost double compared with sample Encaps-PPE (As displayed in caps-PPE .p < 0.05) higher initial values of 16.04 ± 0.8 and 15.07 ± 0.3, respectively, in comparison to SDW and Encaps-SDW ones, which recorded the values of 10.76 ± 0.4 and 13.60 ± 0.4, respectively. After 2 days of storage, b* values strongly decreased in SDW and Encaps-SDW samples ; at the same time, samples PPE and Encaps-PPE showed a lesser decrease (10.90 ± 0.4 and 9.98 ± 0.2), which can be related to previously observed antimicrobial effect  by the addition of PPE and Encaps-PPE, according to storage time and the considered parameter.Textural data of burger patties during storage , reported in p > 0.05) among treatments (p > 0.05) was observed between the SDW and Encaps-SDW samples, which registered the lowest hardness values , hardness was the same  higher values after 2, 4, and 8 days  mesophilic bacteria, Enterobacteriaceae, and Pseudomonas spp. counts, when compared to control samples added with sterile distilled water (SDW) or encapsulated SDW. However, over storage time, and in particular after 4 days, the direct addiction of PPE to patty formulations seemed to be more effective in limiting the growth of estimated microbial populations; the reasons for this could be attributed to the fact that all the added extract immediately interacted with the bacterial cells, thus reducing their viability, or with beef tissue, thus reducing its degradation with consequent formation of simpler compounds that could be utilized by the microorganisms.Microbiological parameters and quality, color, and texture of beef burger patties, prepared both by direct addition or encapsulation in alginate beads of a PPE, were evaluated during refrigerated storage (up to 8 days at 4°C). Microbiological data evidenced at the end of storage a preservative effect both of directly added or encapsulated PPE, which significantly reduced  showed an upward trend, probably due to the formation of basic microbial metabolites or metabolites derived from the deaminations of beef proteins . The addst day and during the storage time, of both packaged and fresh minced beef samples. The authors showed also a significant correlation between the a* and b* parameters, due to the formation of metmyoglobin that conduce a decrease of b* value. Control samples (SDW and Encaps-SDW) showed a more pronounced decrease over storage of b* parameter, probably due to oxygen consumption by aerobic microorganisms and to the consequent decrease in oxymyoglobin, which contributes to the formation of the yellow color  probably due to microbial spoilage and consequent increase in pH value to which the color change toward green is typically associated . Insteadow color . Also inWithin textural parameters, at the end of storage (8 days), PPE addition significantly affected hardness, which reached the highest levels in samples added with not encapsulated PPE, probably because of the presence of carbohydrates in the extract . SimilarMicrobiological control in minced beef has been identified as one of the most important factors in improving quality, extending the shelf life, ensuring product safety and reducing waste.Enterobacteriaceae and Pseudomonas spp., up to 8 days of storage, in comparison to control samples. In addition, results showed that, over the storage time, addition of PPE maintained the minced beef at almost constant pH (average 5.3), while the pH of control samples significantly increased, probably due to the production of ammonia, amines and other basic substances by bacterial activity and protein degradation. Although the antimicrobial effect of PPE was slightly more pronounced when it was directly added to burger formulation, the encapsulation of the extract determined more desirable color and texture features. In fact, in terms of color and texture parameters, over 8 days of storage, the sample containing Encaps-PPE showed a more stable trend in comparison to the other treatments.According to microbiological analysis, the addition of PPE and Encaps-PPE to burger formulation significantly affected the counts of TMB, Certain that the application of PPE extract can be considered as an effective method to contain microbial growth during storage, further studies will be addressed to assess the influence of this extract, encapsulated or not, on the technological characteristics of the cooked product and on its overall sensory acceptability.The results obtained in this study support the idea of proposing the use of PPE, encapsulated or not into alginate beads, analogously to other extracts  as a natThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.RP, CR, and BF conceived and designed the experiments. LP and DT performed the experiments. LP analyzed the data. LP, RP, and CR wrote 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.