Information on the spatial distribution of arabinogalactan proteins (AGPs) in plant organs and tissues during plant reactions to low temperature (LT) is limited. In this study, the extracellular distribution of AGPs in banana leaves and roots, and their changes under LT stress were investigated in two genotypes differing in chilling tolerance, by immuno-techniques using 17 monoclonal antibodies against different AGP epitopes. Changes in total classical AGPs in banana leaves were also tested. The results showed that AGP epitopes recognized by JIM4, JIM14, JIM16, and CCRC-M32 antibodies were primarily distributed in leaf veins, while those recognized by JIM8, JIM13, JIM15, and PN16.4B4 antibodies exhibited predominant sclerenchymal localization. Epitopes recognized by LM2, LM14, and MAC207 antibodies were distributed in both epidermal and mesophyll cells. Both genotypes accumulated classical AGPs in leaves under LT treatment, and the chilling tolerant genotype contained higher classical AGPs at each temperature treatment. The abundance of JIM4 and JIM16 epitopes in the chilling-sensitive genotype decreased slightly after LT treatment, and this trend was opposite for the tolerant one. LT induced accumulation of LM2- and LM14-immunoreactive AGPs in the tolerant genotype compared to the sensitive one, especially in phloem and mesophyll cells. These epitopes thus might play important roles in banana LT tolerance. Different AGP components also showed differential distribution patterns in banana roots. In general, banana roots started to accumulate AGPs under LT treatment earlier than leaves. The levels of AGPs recognized by MAC207 and JIM13 antibodies in the control roots of the tolerant genotype were higher than in the chilling sensitive one. Furthermore, the chilling tolerant genotype showed high immuno-reactivity against JIM13 antibody. These results indicate that several AGPs are likely involved in banana tolerance to chilling injury. Plant cell wall modification is a frequently studied phenomenon in plant-environment interactions , solanaceous lectins, and arabinogalactan proteins (AGPs) Lamport, . Among tLow temperature (LT) is one of the most common abiotic stresses, and it adversely affects the growth and development of plants growing in temperate climates, while significantly limiting their agricultural productivity and distribution  is one of the most important fruit and food crops in the tropical and subtropical regions of the world. Because the temperature can be very low during winter in the subtropical regions  ]. After 3 months of growth, the plants were transferred to an acclimation chamber. The temperature setting was decreased by 3°C from 28 to 7°C every 3 days . Samples (three plants for each genotype and temperature) were collected at the end of the third day at 25 (the control plants), 16, 10, and 7°C. All analyses were performed on laminas of the youngest fully-developed leaves. The terms “leaf” and “lamina” were used synonymously in this text.Tissue-cultured banana plants  by boiling in 0.5% (w/v) ammonium oxalate buffer at 100°C. The content of galacturonic acid  was used to represent the pectin content and was determined colorimetrically by the metahydroxydiphenyl assay adopted from Blumenkrantz and Asboe-Hansen  and from2 (w/v) at room temperature (RT) for 2–3 h. Supernatant was collected after centrifugation for 30 min at 10,000× g at RT. The AGP content in extract was estimated colorimetrically at 457 nm using 100 μl aliquots after precipitation with β-D-glucosyl-Yariv reagent  and dissolving with 20 mM NaOH. The remaining supernatant was precipitated with a slight excess β-D-glucosyl-Yariv reagent, air dried and re-suspended in ddH2O.Classical AGPs were extracted according to Lamport and Varnai (2013) with minor modification. In brief, samples were blended in liquid nitrogen and stirred in 2% CaCl−1 CaCl2 in 50 mmol L−1 piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 7.4)  and AGPs (as 5 μl drops) respectively before spotted onto nitrocellulose membrane by a micropipette. The membrane with dots was air dried at RT for 1 h. Assays with all antibodies were carried out as described by Willats et al. . After tPrimary antibodies used in this study and their antigens are listed in Table For quantification of fluorescence signal, the integrated density  was measured with Image J 1.44 software. The fluorescence signal was selected using appropriate threshold levels for individual sections. Equal threshold levels were applied for each replicate, treatment and genotype for certain epitope to ensure exact comparison of cultivars and treatments.The sections were prepared as described above. The histological examination was performed by staining the de-waxed sections with Coomassie brilliant blue G250 according to the method described by Ma et al. .t-test.Statistical analyses were performed using ANOVA by statistics program SPSS 10.0 for Windows . At least three biological and two technical replicates were used in these experiments. Data are expressed as mean ± SD. Differences between cultivars for each LT treatment were statistically evaluated using Student's As shown in Figure Musa spp. AAA) in the elongation zone, please refer to Figure A representative cross-section of a banana leaf is shown in Figure To reveal the spatial distribution of individual AGP components in banana leaves, a systematic immuno-fluorescence labeling by employing 17 monoclonal antibodies that recognize the epitopes of individual AGPs was carried out in the present study. Individual AGP components recognized by 11 out of these 17 antibodies showed different distribution patterns.Musa spp. AAA Baxijiao leaves, this epitope is primarily localized in the phloem cells around the biggest xylem vessel of the vein, forming a ring structure , while a signal of medium-strength appeared in the mesophyll and the epidermal cells. The sclerenchyma cells were not labeled -α-GalA--Rha, a plant cell wall AGP. In e Figure . JIM14 ae Figure . The JIMe Figure . The labe Figure . The LM2d Figure . Similard Figure  and MAC2d Figure .Musa spp. ABB Dajiao leaves . The PN16.4B4 antibody recognizes an epitope in the carbohydrate portion of AGP. The labeling pattern of this antibody in both genotypes was sclerenchyma and guard cell specific and resembled that of JIM8 in Musa spp. ABB Dajiao showing no signal in vascular bundles. However, the signal in guard cells was much less frequently observed (data not shown).The JIM8 antibody binds a carbohydrate  epitope of AGPs. JIM8 labeled epitopes were very abundant in the sclerenchyma cells of s Figure . In addis Figure . A diffes Figure . The labNegligible or very weak labeling was observed with 6 more antibodies that recognized different AGP components  in the leaf sections of both banana genotypes (data not shown). This finding suggested that the above epitopes are under the immuno-labeling sensitivity threshold and are thus underrepresented in the banana leaves.Musa spp. AAA Baxijiao than Musa spp. ABB Dajiao. However, the epitope abundance in Musa spp. ABB Dajiao increased by approximately 2.5 times when the temperature dropped to 16 and 10°C. When the temperature dropped further, the epitope abundance decreased a little but was still 1.8 fold higher than that of the control in this genotype. On the other hand, the JIM4 epitope abundance decreased slightly under these conditions in Musa spp. AAA Baxijiao, remaining at the same level as that in Musa spp. ABB Dajiao at 7°C. Changes in the abundance of JIM16 epitope in banana leaves under LT stress were similar to that of the JIM4 antibody  -β-D-galactan I] appeared to be much weaker and it was hardly detectable in ) Figure . The epiy Figure . When thy Figure . After sy Figure .Musa spp. ABB Dajiao   Figure . The epio Figure . Howevero Figure .Musa spp. AAA Baxijiao to LT differed from Musa spp. ABB Dajiao, the tolerant genotype. While a slight decrease (17%) was observed in the epitope level of chilling-tolerant genotype when the temperature decreased from 25 to 16°C, a clear increase (1.6 fold) in the signal intensity appeared when the temperature dropped further. The signal in Musa spp. AAA Baxijiao increased when the temperature dropped from 25 to 16°C, and this increase also occurred at 10°C  in cotton roots was markedly up-regulated by cold stress. Furthermore, the expression of the GUS gene as driven by the GhAGP31 promoter was also dramatically enhanced in the roots of transgenic Arabidopsis seedlings under cold treatment. Additionally, the overexpression of GhAGP31 in yeast and Arabidopsis significantly improved the freezing tolerance of yeast cells and the cold tolerance of Arabidopsis seedlings. During the cold hardening of winter wheat , a transient appearance of 37-and 69-kD lectins, which were identified as AGPs, might indicate their involvement in triggering plant cell defense responses to LT stresses . As shown in Table LM2, LM14, JIM8, and JIM15 AGP epitopes showed positive correlation of their abundance with the tolerance of the cultivars at lower temperatures. It indicates that they are important for banana tolerance to strong chilling stress.The abundance of JIM16 and CCRC-M32 epitopes was higher in the tolerant cultivar at mild LT stress suggesting that these epitopes determines the tolerance of banana to milder LT stress.The epitopes recognized by JIM14 and PN16.4B4 antibodies increased in both cultivars at 10 and/or 7°C, indicating that these two AGP components are chilling-responsive, but they likely do not contribute to banana tolerance to chilling injury.In the control plants, the AGPs recognized by JIM8, JIM15, LM2, and LM14 antibodies exhibited much lower abundance in the chilling tolerant genotype when compared with the chilling-sensitive one; however, the abundances of these epitopes were reversed at 7°C. These results indicate that the tolerant genotype accumulates these AGPs faster under chilling stress. This finding is consistent with the earlier accumulation of soluble proteins and free proline in the leaves of the tolerant banana genotype under LT stress when compared with the sensitive genotype as found by our previous study  were found to be up-regulated by all three stresses . However, another two fasciclin-like AGP genes (OsFLA1 and OsFLA4) were down-regulated by cold stress (Ma and Zhao, The differential expression of different HRGP types under LT stress was also reported in rice, and the transcript levels of three AGP genes (Third, the present study not only revealed the differential changes in AGP abundance, but also in the spatial distribution of AGPs in different cell types in LT treated banana leaves. For example, the accumulation of AGPs recognized by JIM4 and JIM16 was only observed in the phloem cells of the tolerant genotype Figure . LT induIn addition, the same AGP epitopes differed in their response to LT between banana leaves and roots. Furthermore, though both leaves and roots accumulated AGPs under LT treatment, the highest AGP levels were detected in roots at 16°C while in leaves it was at 10°C. This might indicate that banana roots started to accumulate AGPs earlier than leaves or eventually, that they were more sensitive to decreased temperatures. It has to be noted that differences between roots and leaves in AGP abundance and dynamics during LT stress could be also related to different developmental stages of the analyzed plant material.in planta (Seifert and Roberts, AGP glycan-specific antibodies have been broadly used to investigate AGPs in tissue culture and CX planned experiments, YY, YW, EX, LX, XY, ZS, and CX performed experiments, CX, TT, JS, XL, and HC analyzed data and prepared data presentation, CX, TT, and JS wrote the manuscript. All authors read and approved the final 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.


As a traditional anti-inflammatory Chinese herbal medicine, Alkaloid berberine has been recently reported to exhibit anti-tumour effects against a wide spectrum of cancer. However, the mechanism was largely unknown. Gene chip array reveals that with berberine treatment, c-Myc, the target gene of Wnt pathway, was down-regulated 5.3-folds, indicating that berberine might inhibit Wnt signalling. TOPflash analysis revealed that Wnt activity was significantly reduced after berberine treatment, and the mechanism of which might be that berberine disrupted β-catenin transfer to nucleus through up-regulating the expression of adenomatous polyposis coli (APC) gene and stabilized APC-β-catenin complex. Berberine administration in ApcMin/+ mice exhibited fewer and smaller polyps in intestine, along with reduction in cyclin D1 and c-Myc expression. In clinical practice, oral administration of berberine also significantly reduced the familial adenomatous polyposis patients' polyp size along with the inhibition of cyclin D1 expression in polyp samples. These observations indicate that berberine inhibits colon tumour formation through inhibition of Wnt/β-catenin signalling and berberine might be a promising drug for the prevention of colon cancer. Colorectal cancer is the second largest cause of cancer-related deaths in Western countries in vitro and in vivo anti-cancer effects of berberine through different mechanisms and it was reported that the anti-tumour effects may vary, depending on the types of cancer cells, the duration of treatment and the doses Berberine was initially isolated from the herbs Rhizoma coptidis (Chinese name Huang-Lian), which belongs to the camptothecin family of drugs, and has been used as a traditional Chinese medicine to treat various infectious disorders for more than 3000 years The tumour-suppressing effect of berberine has been implicated to be related to its anti-inflammation functions. Recently, more and more reports have shown that chronic inflammation might induce tumourigenesis Three human colon cancer cell lines, KM12C, KM12SM and KM12L4A, kindly provided by Professor I. J. Fidler , were used as models in the present study. The parental cell line KM12C was originally established from a colon carcinoma Dukes' B2. Cells from this cell line were repeatedly injected into the caecum and spleen in athymic mice, to form two new cell lines KM12L4a and KM12L4a, both with high metastatic potential. The cell lines were maintained in Eagle's MEM medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), sodium pyruvate, vitamins and a cocktail of penicillin and streptomycin at 37°C in 5% carbon dioxide . Berberine was purchased from Sigma-Aldrich  and was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.1% DMSO in the medium, and the medium with 0.1% DMSO was used as the control. Antibodies against β-actin, β-catenin, APC and horseradish peroxidase-linked anti-rabbit or mouse IgG were purchased from Santa Cruz Biotechnology . Antibodies against cyclin D1, cyclin B1, CDC-2, c-Myc, tubulin, lamin B and Phospho-β-catenin were purchased from Cell Signaling Technology .The effect of berberine on cell proliferation was tested using CHEMICON's BrdU cell proliferation assay kit according to the manufacturer's protocol Samples were collected by lysing cells in RIPA lysis buffer . Each sample (80 mg of cellular proteins) was size-fractionated using SDS-PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) transfer membranes . Blots were incubated for 1 hr at room temperature in 5% bovine serum albumin for blocking, and proteins were detected with primary antibodies overnight and then blotted with horseradish peroxidase conjugated secondary antibodies for 1 hr. The immunoblots were visualized with ECL .The TOPflash firefly luciferase and TK-RL Renilla luciferase constructs were used to measure the activation of the Wnt pathway after berberine treatment. Cells grown on 24-well plates were transfected in quadruplicates with cDNAs (20 ng/well) for TCF-luciferase reporter (TOPflash) along with a control Renilla plasmid (TK-RL) by Lipofectamine 2000 transfection Reagent . Twenty-four hours post-transfection, the cells were treated with different concentrations of berberine. Six hours later, the cells were lysed and the luciferase activity was measured and normalized to the corresponding Renilla activity with the dual-luciferase assay kit .For IP studies, cellular extracts were normalized for protein concentration and pre-cleared for 1 hr at 4°C with 0.4 ml of protein A-coated Sepharose beads. Immunoprecipitation was carried out at 4°C by incubating the fractions for 2 hrs with an anti-APC antibody or anti β-catenin antibody, and then for 1 hr with 0.4 ml of protein A/G-Sepharose beads. Control experiments were performed in the presence of the immunizing peptides, or with control IgG antisera. The IPs proteins were recovered by boiling the Sepharose beads in SDS sample buffer.The mRNA levels of APC were evaluated by quantitative real-time PCR. Total RNA was extracted with TRIzol Reagent (Invitrogen) and treated with DNase I at 37°C for 30 min. The cDNA was synthesized from 1 mg of total RNA using 15 U MMLV reverse transcriptase (Invitrogen) in 20 ml of a reaction mixture containing 0.5 mg oligo Tumour tissue specimens were fixed in neutral formalin and embedded in paraffin after collection from the killed mice. Tissue sections 5 μm thick were dewaxed and incubated with 0.01 M natrium citricum for antigen retrieval. The slides were rinsed in phosphate-buffered saline and incubated overnight at 4°C with diluted anti-cyclinD1 or anti-c-Myc antibodies. Following steps were then performed with the immunostaining kit according to the manufacturer's instructions.Animal experiments were conducted in accordance with the regulations of experimental animal administration and the animal ethical committee of the Medical College of Xiamen University. C57BL/6J-ApcMin/+ and wild-type C57BL/6J animals were purchased from Model Animal Research Center of Nanjing University. For polyp/tumour scoring and the establishment of survival curves, all animals received high nutrient food  and water, whereas mating pairs were fed on standard mouse chow. All the mice were housed in a temperature-controlled environment (20–22°C) with a 12:12 hr light:dark cycle.Seven FAP patients who were diagnosed at the Digestive Endoscopy Center of Tianjin General Hospital, Tianjin Medical University between January 2008 and December 2012 were investigated. The mean age was 28.71 ± 6.82 years old (range: 15–36). Five were males, and two were females. Because of worries about the cardiovascular side effects and the cost of long-term use, they refused to take celecoxib, which is commonly used for FAP in Western countries. Moreover, all patients did not want to have their entire colorectum removed. All patients had a clean colon and rectum at study entry. At the baseline endoscopy, the number and size of the polyps were evaluated, and then all of the polyps below 10 cm from the anal margin were removed. After 6 months, a colonoscopy was taken and the polyps recurred to almost the same level as 6 months before. The colonoscopy result at this stage was used as control group (without berberine treatment).Then the polyps were removed again and then the patients took berberine treatment  immediately following polypectomy. The patients were followed up and asked to undergo a colonoscopy after 6 months and the result was used as berberine administration group. Evaluations at base line and month six included a medicine taking, physical examinations, colonoscopy appearances and immunohistochemistry of β-catenin and cyclinD1 with biopsies of the polyps before and after berberine administration. Informed consents for colonoscopy and the present study were granted from all the patients before the procedure and ethical committee approval was obtained from Tianjin General Hospital Ethics Committee.Illumina HumanRef-8 Expression BeadChips were used for array hybridization  according to manufacturer's protocols. Briefly, total RNA (2 μg) from untreated cells and from berberine-treated cells at each time point was converted to biotinylated cDNA with the Illumina RNA Amplification Kit . Samples were purified using the RNeasy kit . Hybridization to the Sentrix HumanRef-8 Expression BeadChip, washing and scanning were performed according to the Illumina Bead Station 500× manual. Data were extracted, normalized and background subtracted using Illumina BeadStudio software. Transcript signals above 0.95 were accepted as presence of expression.P < 0.01.All values are expressed as the means ± SD. Statistical analysis was performed by Dunnett's test. Differences were considered significant when the via passive diffusion.Figure Uncontrolled cell proliferation is a hallmark of cancer. We asked whether berberine had any effect on the cell proliferation. Using the CHEMICON's BrdU Cell Proliferation Assay Kit, we quantitatively studied newly synthesized DNA of actively proliferating cells. Berberine treatment reduced the ratio of newly synthesized DNA in dosage- and time-dependent manners Fig. D.Several studies from our and other laboratories have indicated that berberine can interact with DNA and regulate gene expression To investigate the effect of berberine on Wnt/β-catenin signal pathway, TCF/LEF luciferase reporter assay was carried on. Twenty-four hours post-transfection, cells were treated with 0, 25, 50 and 100 μM beberine for 4 hrs, and then the luciferase activity was measured. The Wnt activity was suppressed significantly after treatment with berberine, compared with control in KM12C cells  mouse model with berberine treatment. Because food composition can strongly influence intestinal tumour formation in men and mice Min/+ mice (n = 12) began to receive berberine , and killed at 24 weeks. Compared with the control group, berberine administration significantly reduced the number of polyps in colon and small intestine  by berberine treatment, compared with control groups.To further clarify the anti-cancer effect and molecular mechanism, we studied tumour development and Wnt activity in ApcP < 0.05, Fig. Seven patients took berberine treatment immediately following polypectomy. The colonoscopy results between control and berberine administration  are shown in Figure in vitro and in vivo. Further study demonstrated that berberine increased the expression of APC. APC has been renowned as a negative regulator of WNT/β-catenin signalling. APC loss is an early event in tumourigenesis, and causes an increase in nuclear β-catenin and its transcriptional activity. This is thought to be the driving force for colon tumourigenesis. As a tumour suppressor, APC controls β-catenin function in transcription Controlling gene expression with small DNA-binding molecules has been a challenge at the interface of medicinal chemistry and biology. Our study demonstrated that berberine could enter the nucleus, suggesting its gene regulation function. The studies that followed showed that berberine could inhibit colorectal cancer cells proliferation by inhibition of Wnt activity. We then studied how berberine interfered with WNT/β-catenin signalling. The result shows that berberine could disrupt the nuclear translocation of β-catenin. β-catenin is a key effector of Wnt signalling, promoting cell proliferation by inducing gene transcription through the activation of TCF/LEF transcription factors in vitro and in vivoMany studies have reported that berberine exhibits its tumour-suppressing effect through inducing apoptosis in vitro experiment. Our current study was non-randomized controlled trial with a rather small sample size (seven patients). This limitation was possibly because of the rare diseases of FAP. Thus, randomized controlled trial from multiple centres with a large sample size is needed to confirm the chemoprevention efficacy of berberine.Our preliminary study showed that berberine maybe play a role in chemoprevention of intestinal adenomas in FAP patients. It may also inhibit the Wnt signalling pathway, which was in accordance with the animal and In conclusion, our study indicates that berberine might be a promising drug in prevention and treatment of colon cancer through inhibition of Wnt signalling.


We constructed a Petri net model of Wnt/β-catenin signaling including main players from the plasma membrane (WNT ligands and receptors), cytoplasmic effectors and the downstream negative feedback target gene AXIN2. We validated that our model can be used to simulate both active (WNT stimulation) and hyperactive (GSK3 inhibition) signaling by comparing our simulation and experimental data. We used this experimentally validated model to get further insights into the effect of the negative feedback regulator AXIN2 upon WNT stimulation and observed an attenuated β-catenin stabilization. We furthermore simulated the effect of APC inactivating mutations, yielding a stabilization of β-catenin levels comparable to the Wnt-pathway activities observed in colorectal and breast cancer. Our model can be used for further investigation and viable predictions of the role of Wnt/β-catenin signaling in oncogenesis and development.The Wnt/β-catenin signaling pathway is important for multiple developmental processes and tissue maintenance in adults. Consequently, deregulated signaling is involved in a range of human diseases including cancer and developmental defects. A better understanding of the intricate regulatory mechanism and effect of physiological (active) and pathophysiological (hyperactive) WNT signaling is important for predicting treatment response and developing novel therapies. The constitutively expressed CTNNB1 (commonly and hereafter referred to as β-catenin) is degraded by a destruction complex, composed of amongst others AXIN1 and GSK3. The destruction complex is inhibited during active WNT signaling, leading to β-catenin stabilization and induction of β-catenin/TCF target genes. In this study we investigated the mechanism and effect of β-catenin stabilization during active and hyperactive WNT signaling in a combined  The Wnt/β-catenin signaling pathway is crucial for regulating cell proliferation and differentiation during embryonic development, while in adults it helps control tissue homeostasis and injury repair in stem cell maintenance , 2. Extret al. [The destruction complex consists of two scaffolding proteins, AXIN1 and adenomatous polyposis coli (APC), and two kinases, casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). β-catenin is phosphorylated by CK1 and GSK3 , 7 and tet al.  showed tAXIN2 is of particular interest. First, AXIN2 is a universal β-catenin/TCF target gene and as such it is believed to faithfully report Wnt-pathway activity in multiple tissues [in vivo due to their different expression patterns [AXIN1 is constitutively expressed [AXIN2 is induced during active Wnt/β-catenin signaling [The inhibition of the destruction complex leads to β-catenin stabilization and nuclear translocation. Nuclear β-catenin interacts with TCF/LEF transcription factors  forming  tissues , 18. Sec tissues  and medipatterns : AXIN1 ixpressed , whereasignaling . The AXIignaling . HoweverDeregulated Wnt/β-catenin signaling caused by genetic alterations can have major developmental consequences, and is the leading cause of colorectal carcinogenesis . The mosIn vitro experiments in which cells are stimulated with WNT are generally assumed to represent active signaling, whereas downstream oncogenic mutations represent hyperactive signaling. Inhibition of GSK3 using small molecule inhibitors is widely used to activate WNT signaling during cellular reprogramming and in embryonic stem cell cultures [Xenopus extracts. Later models included amongst other, extensions with (i) AXIN2 feedback, explaining effects of mutations in colorectal cancer [Investigating the mechanism and effect of β-catenin stabilization during physiological (active) and pathophysiological (hyperactive) WNT signaling is crucial for developing effective treatment, both in the field of cancer research and regenerative medicine. cultures , 36. Inhcultures , 38 to fcultures  describel cancer ; (ii) AXl cancer ; (iii) Wl cancer ; (iv) efl cancer ; (v) thel cancer  and (vi)l cancer . Howeverl cancer . Petri nl cancer , 47. A cHere we have used a combined computational and experimental approach to build the first Petri net model of Wnt/β-catenin signaling. Compared to the aforementioned mathematical models, it includes a larger number of core Wnt-pathway components. Most importantly, we have included a signaling module that captures the interactions occurring at the cell membrane. As a result, our model allows both physiological (i.e. WNT ligand induced) and pathophysiological  to be captured. Furthermore, our model offers the possibility of including the negative feedback by AXIN2. We used the model to explain how active signaling upon WNT stimulation and hyperactive signaling upon GSK3 inhibition leads to different levels of β-catenin stabilization. We corroborated our observations from the model using data from TCF/LEF luciferase reporter assays and Western blot analysis. We then used the experimentally validated model to explore plausible modes of action of β-catenin stabilization as a result of negative feedback by activating expression of AXIN2 upon WNT stimulation, or due to APC inactivating mutations that are known to play a key role in oncogenesis of colorectal and breast cancer.A Petri net model can be graphically represented with two types of nodes: ‘places’, describing the biological components, and ‘transitions’, describing the activity between the biological components, which can be constructed based on known interaction data. The places are denoted with ‘tokens’, which describe the relative availability of the biological component. Tokens are assigned on the basis of existing data from the literature, typically relative protein levels. Places and transition are connected by weighted arcs that are important for the flow of the network. –50 (see β-catenin (encoding β-catenin), but also of AXIN2, a negative feedback target gene of the Wnt-pathway, are also specifically included in the model. Production and degradation of all other proteins are assumed to have similar rates and are therefore omitted, such that the token levels of these proteins remain the same throughout the simulation .We created a Petri net model of Wnt/β-catenin signaling to investigate the mechanism and effect of β-catenin stabilization under physiological (e.g. embryonic development) and pathophysiological (e.g. cancer) conditions based on the literature as described in details below . The modβ-catenin gene, and either binds the destruction complex (t6) or translocates to the nucleus, where it interacts with TCF/LEF (t10) to activate transcription of AXIN2 (t11). Since AXIN1 and AXIN2 are functional homologs [The final model consists of 18 places , 11 transitions  and 41 arcs . In the model, WNT initiates signaling extracellularly by binding to its transmembrane receptors FZD and LRP (t1), forming the WNT/FZD/LRP complex. DVL interacts with the intracellular tail of FZD when present in the WNT/FZD/LRP complex (t2), forming the WNT/FZD/LRP/DVL complex. DVL thereafter sequesters AXIN1 to the membrane (t3) forming the signalosome consisting of WNT, FZD, LRP, DVL and AXIN1. In the model we have not specifically included the contribution of GSK3 and CK1 in the formation of the signalosome, because these two multi-tasking kinases are generally assumed not to be rate-limiting in the cell , 39. Furhomologs , they arβ-catenin and AXIN2, were set to 1 and kept at these values since these genes are always presumed to be present. In our model this means that AXIN2 is induced only when the TCF/LEF-β-catenin complex is present and β-catenin is induced once every step, because there is nothing implemented to restrain this induction (β-catenin levels are instead regulated by the destruction complex).The initial token level of the protein places were set to 5 (with exceptions described below) in order to have a concise but sufficiently large pool of the protein tokens for the model to work with. Higher levels had no effect, and lower levels restricted the expressiveness of the model, i.e. too coarse-grained. The initial token level of the β-catenin protein place and all protein complexes  were set to 0, because these are produced throughout the simulation and are assumed to be initially absent. The initial token level of TCF/LEF was set to 1 which gives the lowest rate of TCF/LEF-β-catenin interaction, and hence the induction of low levels of AXIN2 . A paramMost arc weights were set to 1, which means that the model dynamics rely on its connectivity. This has been proven successful using Petri net modeling of MAPK and AKT signaling cascades , 47, 52.We simulated WNT stimulation to predict the level of β-catenin stabilization during active signaling. To this end we ran a series of simulations with different initial WNT token levels (ranging from 0 to 5) without AXIN2 feedback (i.e. the arc weight from t11 to AXIN was set to 0). As shown in WOO cells (carrying a stably integrated β-catenin/TCF luciferase reporter) with increasing concentrations of purified, commercially available, Wnt3a for 3, 8 and 24 hours. These experiments reproduce the dose- and time-dependent increase of TCF/LEF reporter gene activity predicted by our model , where 5 initial tokens represents wildtype (i.e. no Wnt-pathway activity) and 0 corresponds to complete inhibition . The simulations revealed that the response levels depend on initial GSK3 token levels see . For GSKWOO cells with increasing concentrations of CHIR99021, one of the most potent and selective GSK3 inhibitors available to date [4 fold increase in Wnt-pathway activity in the latter  and pathophysiological (i.e. GSK3 inhibition) activation of the pathway in the same cell line, using time points and analyses best suited for connecting our experimental data and the Petri net modeling predictions.Our model predicts a dose- and time dependent response for both WNT stimulation and GSK3 inhibition Figs  and 4A. The time-delay together with the continuous sequestration and dissociation of AXIN1 to the signalosome leads to prediction of higher stabilization of β-catenin for complete GSK3 inhibition compared to maximal WNT stimulation, where the difference is almost two-fold , whereas a transition indicates the activity occurring between the places (e.g. gene expression or complex formation). Places can only link to transitions and vice versa . The direction of the arcs is important for the flow of the network. An arc goes from an input place to a transition, and from a transition to an output place. Places contain ‘tokens’, indicating the availability of the corresponding entity, while arcs have a weight, denoting the amount of tokens to consume from an input place or to produce to an output place. If the token levels of all input places of a transition fulfill the requirement of  the weights of the respective arcs, the transition is enabled. Only enabled transitions can be executed, leading to transfer (consumption/production) of tokens between places. Note that if two (or more) enabled transitions share an input place, they may be in competition if available token levels do not allow simultaneous execution of both . In our model, AXIN, β-catenin and the destruction complex with β-catenin bound, are each input places for two transitions . The initial token levels and the arc weights are generally restricted to be integer values. However, to represent a lower firing rate of a transition (i.e. a rate-limiting step) a fractional arc weight is implemented.Gene expression is modeled such that one arc goes from the gene-place to the transcriptional-transition, one arc goes from the transcriptional-transition to the gene-place, and one arc goes from the transcriptional-transition to the protein-place. Gene-places are initiated with 1 token and when the transcriptional-transition of a gene is enabled the token is produced both in the protein-place and in the gene-place itself. This way the token can be reused for another round of gene expression, reflecting the fact that the gene (DNA) is needed, but is not consumed during expression.AXIN2 induction. APC mutants have decreased binding affinity to the other components of the destruction complex to different degrees. We implemented this by reducing the formation of the destruction complex i.e. the weight on the arc going from the complex-formation-transition (t5) to the destruction complex (production) was decreased to 0, 0.05, 0.1 and 0.2. In addition, we incorporated arcs going from the complex-formation-transition to the individual destruction complex components  with arc weights of 1 minus the production-weight to equally decrease the consumption. The implemented arc weight of 0 represents no production of the destruction complex (i.e. a complete null mutation). The implemented fractional arc weights of 0.05, 0.1 and 0.2 represent a production of the destruction complex once every 20, 10 and 5 steps, respectively.We modeled active and hyperactive signaling upon WNT stimulation and GSK3 inhibition, respectively, and used these conditions to validate the model with experimental data (see below). Inhibition of GSK3 inhibits formation of the destruction complex, which we interpret to be similar to oncogenic perturbations. Therefore, for modeling purposes, GSK3 inhibition was used to mimic hyperactive signaling. For GSK3 inhibition we varied the initial token level of GSK3, respectively, from 0 to 5. For WNT stimulation we varied the initial token level of WNT from 0 to 5 and removed the AXIN2 feedback (the arc weight from t11 to AXIN was set to 0). The experimentally validated model was used to predict the level of β-catenin stabilization with the AXIN2 negative feedback upon WNT stimulation and APC inactivating mutations, respectively. Upon WNT stimulation with the AXIN2 feedback we varied the initial token level of WNT  and the arc weight from t11 to AXIN (0 (no feedback) and 0.15 ). The maximal feedback of 0.15 was chosen based on the criteria that it should i) show a peak response and ii) return to basal level for all initial WNT token levels . This arc weight represents a firing rate of three times every 20 steps. Thus, the simulation for each initial WNT token level produced two β-catenin stabilization curves . The area between these two curves was used to explain the spectra of β-catenin stabilizations at intermediate levels of The model was simulated with maximally parallel execution, cf. our previous work , where thttp://www.ibi.vu.nl/downloads/WNTmodel/.For each condition we simulated the total β-catenin token levels, i.e. free β-catenin and β-catenin bound to the destruction complex or TCF/LEF, respectively, over 100 steps repeated 100 times. To account for variations in token levels due to the non-deterministic nature of the model, the mean and standard deviation of the β-catenin token levels over the 100 simulations were calculated for each step. These 100 steps represent the (early) time scale of β-catenin accumulation and the final token count represents the stabilized β-catenin level measured in experiments. The simulation steps describe the sequence of events and should not be linearly translated to time units. Similarly, the token level is a coarse-grained quantitative representation of actual protein levels and should not be linearly translated to a concentration. Instead, for analysis of the simulations we observe relative differences of β-catenin token levels over steps between simulations (i.e. different conditions and dosages). To validate the model we compared the relative β-catenin token levels predicted by our model simulations to the relative Wnt-pathway activities measured in the experiment (see below), where we distinguish between ‘low’, ‘moderate’ and ‘high’ levels. A Python script was written to run the simulations and is available together with the model in pnml format via WOO (WNT OFF/ON) cells were generated by transfecting HEK293T cells with a 7xTcf-FFluc//SV40-PuroR (7TFP) reporter plasmid . FollowWOO cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% FCS and 1% Penicillin/Streptomycin  in 5% CO2 at 37°C. These cells respond to activation of the Wnt/β-catenin signaling pathway by expressing firefly luciferase, since the firefly luciferase in the 7TFP construct is driven by the 7xTcf promoter, which contains 7 repeats of the TCF/LEF transcription response element. Cells were plated the day prior to stimulation in a 96 well-plate at a density of 20.000 cells per well. Cells were stimulated with different concentrations (10–200 ng/ml) of purified Wnt3a protein (RnD) dissolved in 0.1% BSA in PBS, or with different concentrations (750 nM-6 μM) CHIR99021 (BioVision) dissolved in DMSO, for different amounts of time (1–24 hours). At the indicated time points following stimulation, cells were lysed in 20 μl of Passive Lysis Buffer (Promega) and cell lysate from the same experiment was used for both the luciferase assay (3 wells per condition) and Western blot analysis .HEK293TTM Lite 4.0 software (LI-COR Biosciences) was used to quantify relative protein levels. Background correction was performed according to the manufacturer’s instructions .Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were run on an 8% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane (Bio-Rad) and blocked with TBS Odyssey Blocking Buffer . Primary antibodies directed against active β-catenin , total β-catenin  and α-Tubulin  were diluted in blocking buffer supplemented with 0.1% Tween-20 (TBS-T). Staining was performed overnight at 4°C. Membranes were washed in TBS-T followed by incubation with secondary antibodies (IRDye 680LT (Cat# 926–68021) or IRDye 800CW (Cat# 926–32212) (LI-COR), 1:20000 in TBS-T) for 2 hours. Membranes were washed in TBS-T and incubated in TBS prior to scanning at 700 nm and 800 nm using an Odyssey Fc (LI-COR Biosciences). Image StudioTo measure the activity of firefly luciferase (and hence Wnt-pathway activity), 10 μl of cell lysate was transferred to a black 96-well Optiplate (Perkin Elmer). The SpectraMax L Microplate Luminometer was used to inject 50 μl Luciferase Assay Reagent II (Promega) per well followed by measurement of firefly luciferase activity.S1 FigThe “location” of the readouts of Western blot analysis and luciferase reporter assay compared to their equivalent implementation in our Petri net model. Western blot analysis measures the protein levels of β-catenin (i.e. measures the β-catenin accumulation), whereas the luciferase reporter assay measures the transcriptional activation (by β-catenin/TCF complexes) of the TOPFLASH reporter, which is followed by the production of luciferase protein. Since the luciferase reporter activation requires β-catenin to be present it takes longer for the TOPFLASH reporter to be activated than for the increase in β-catenin protein levels to occur i.e. the activation always occurs downstream of β-catenin accumulation.(PDF)Click here for additional data file.


As classical chemotherapeutics only tend to highly proliferating cells and do not address at all the issue of differentiation arrest, a better understanding of these cytogenetic aberrations would facilitate the development of targeted therapies. Recently, impressive therapeutic successes have been achieved in acute promyelocytic leukemia (APL) exhibiting striking remission rates and long-term survival up to 90%.1 In this, the PML-RARα fusion protein represses the transcription of genes, which are important for myeloid differentiation. The degradation of PML-RARα can be induced by pharmacological doses of all-trans retinoic acid (ATRA), thereby enabling transcription and terminal differentiation of immature precursor cells.2 However, ATRA is only clinically successful for the small subset of APL patients. Thus, substances that may contribute to the differentiation of other AML subtypes and their modes of action are currently investigated. In a recently published issue of Cell Death Discovery, Kühn et al.3 describe the lectin LecB as inducer of the differentiation of the AML cell line THP-1 and propose a regulation by the interplay between autophagy and β-Catenin.Acute myeloid leukemia (AML) is the most common type of leukemia and characterized by a massive accumulation of immature and non-functional myeloid precursor cells in the blood and the bone marrow. A large number of these AML cases involve chromosomal translocations that generate chimeric oncoproteins like AML1-ETO or PML-RAR3 This illustrates a remarkably fast effect as in usual laboratory routine the differentiation of monocytic THP-1 cells to macrophage-like cells is performed by the exposure to phorbol-12-myristate-13-acetate for 72 h.4 The effect of LecB is mediated by its binding to glycosylated host cell receptors as blocking of the LecB-binding sites with l-fucose prevented the induction of differentiation. Of note, tumor cells often gain substantial changes in the glycosylation pattern of their surface proteins during cancer progression and metastasis.5 Furthermore, it has been shown that LecB is cytotoxic to tumor cells and provokes strong agglutination and an attenuated tumor growth rate.6 Further striking roles of lectins have been specified, for example, in bacterial invasion7 and the activation of B cells in follicular lymphoma.8 In the future, tailor-made engineered lectins9 with controlled valency and host cell receptor specificity might become powerful tools as biomarkers and for targeted therapy.The fucose-binding lectin LecB has been shown to specifically induce differentiation of the THP-1 cell line already after 4 h.et al.3 elucidated by inhibitor studies that a functional autophagy and a low β-Catenin level were essential for the LecB-induced differentiation of THP-1 cells. Interestingly, autophagy is also implicated in ATRA-based differentiation by supporting the degradation of the aberrant fusion proteins and transcription factors.2 However, autophagy is so far considered as double-edged sword and its role is not completely clarified as it may act either cytoprotective or cytotoxic to cancer cells, for example, depending on the cell type and the environment. Moreover, an aberrant Wnt/β-Catenin signaling, modulated by oncogenic fusion proteins, is frequently implicated in the pathogenesis of leukemia and contributes to stem cell self-renewal, reduced differentiation and apoptosis.10 As well, a high β-Catenin expression was found in many primary AML samples.11 Corresponding to this, Kühn et al. demonstrated that a high β-Catenin level stabilized proliferation and, remarkably, blocked the initiation of autophagy resulting in low differentiation ability. Surprisingly, the addition of the well-known autophagy-stimulating mTOR inhibitors was not sufficient to induce autophagy as long as the β-Catenin level was stable.3 Resistances of AML patients to mTOR inhibitors and their low clinical benefit have already been moaned.12 On the basis of their observations, Kühn et al. proposed that high β-Catenin expression was responsible for this resistance, which can be circumvented by the application of LecB. Initiated by the lectin, β-Catenin was degraded, autophagy became active and differentiation took place. Moreover, this reduction of the β-Catenin level even sensitized THP-1 cells to mTOR inhibitors and enabled a cumulative differentiation-inducing effect (Mechanistically, Kühn g effect . Importaβ-Catenin occurs. An important regulator of the canonical Wnt/β-Catenin signaling pathway is the cell adhesion protein E-Cadherin. In the absence of Wnt ligands, β-Catenin is sequestered at the plasma membrane in a complex with E-Cadherin, links it to the actin cytoskeleton and contributes to the regulation of cell–cell contacts. In cancer, β-Catenin is often independent on the activation by a Wnt ligand and constitutively active, thereby increasing proliferation and stem cell self-renewal.10 The stabilization of β-Catenin in a complex with E-Cadherin would therefore decrease these tumor-promoting effects. For this reason, Kühn et al. assessed the regulatory potential of E-Cadherin in AML. Indeed, the authors could introduce E-Cadherin as important player in the pathway as its down-modulation by siRNA efficiently attenuated LecB-induced differentiation. In addition, an increased colocalization of E-Cadherin and β-Catenin followed by the cellular uptake of these complexes was apparent upon LecB treatment. These findings reveal the crucial role for LecB in the formation of E-Cadherin clusters, which may stabilize β-Catenin firmly within these complexes and thereby prevent the nuclear translocation and the target gene expression. This subsequently promotes a cell fate different to that of Wnt-induced β-Catenin signaling.3However, it has not been addressed so far how the loss of et al. highlight a regulatory role of E-Cadherin for the disruption of the aberrant Wnt/β-Catenin signaling pathway. Thereupon, β-Catenin degradation facilitates the induction of autophagy and the suppression of deregulated proliferation, which both illustrate crucial aspects of differentiation in AML. Moreover, this study encourages the use of natural and tailor-made synthetic lectins as powerful tools to decipher basic cellular mechanisms and in future therapeutic approaches.In conclusion, Kühn


P = 0.004; and 10 days, P = 0.02). Hyphema occurred significantly more frequently in the trabeculectomy group (P = 0.0025). There were no significant differences of the other primary outcomes between the two groups. Additionally, duration of anterior chamber opening was significantly shorter in the Ex-PRESS group (P = 0.0002) and the eyes that had iris contact with Ex-PRESS tube had significantly shallower anterior chambers than did the eyes without the iris contact (P = 0.013). The Ex-PRESS implantation prevented early postoperative inflammation and hyphema in the anterior chamber and shortened the duration of anterior chamber opening. Iris contact with the Ex-PRESS tube occurred more frequently in eyes with open-angle glaucoma and shallow anterior chambers.We compared early postoperative complications between trabeculectomy and Ex-PRESS implantation. Enrolled patients with 39 primary open-angle or 25 exfoliative glaucoma were randomly assigned to receive trabeculectomy (trabeculectomy group) or Ex-PRESS implantation (Ex-PRESS group). Primary outcomes were early postoperative complications, including postoperative anterior chamber inflammation, frequencies of hyphema, flat anterior chamber, choroidal detachment, hypotonic maculopathy, and the change of visual acuity. The postoperative flare values in trabeculectomy group were higher than those in the Ex-PRESS group (overall,  Trabeculectomy is the gold-standard filtering surgery for intraocular pressure (IOP) reduction in glaucoma patients2The Ex-PRESS glaucoma filtration device  is a miniature, non-valved drainage tube that has been used as an alternative to trabeculectomy; this device shunts aqueous humor from the anterior chamber into the subconjunctival area near the limbus. Ex-PRESS is designed for the stable filtration of the aqueous humor although its flow (0.09 mmHg outflow resistance at physiologic flow rates) needs to be regulated by the suture tension of scleral flapThere have been several prospective and retrospective studies that have compared the safety and efficacy of Ex-PRESS implantation with those of trabeculectomy56789101113The objective of this study, called “Complications Postoperatively of Ex-PRESS versus Trabeculectomy Study (CPETS),” was to compare early postoperative complications between trabeculectomy and Ex-PRESS implantation in Asian eyes that had medically-uncontrolled primary open-angle glaucoma (primary OAG) or exfoliative glaucoma. Our primary outcome measure included postoperative anterior chamber inflammation because this surgical procedure appeared to induce less inflammation than trabeculectomy. Moreover, there have been case reports about the effects of iris contact with the tube15CPETS was approved by the institutional review board of Fukui University Hospital, Fukui, Japan. This study was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan . The protocol adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects. Patients were recruited between August 30, 2012 and February 13, 2015 at Fukui University Hospital. The enrolled patients were randomly assigned to receive either a trabeculectomy or an Ex-PRESS implantation. Allocation was concealed until intervention was assigned. Yo. T. generated the random allocation sequence, M.I. and Yu T. enrolled participants and S.M. assigned participants to interventions. The enrolled patients were determined with the following criteria: (1) Japanese patients with primary OAG or exfoliative glaucoma , (2) minimum age of 20 years, (3) no previous history of ocular surgery, and (4) an IOP ≥18 mmHg (despite maximum tolerated administration of anti-glaucoma medication). The exclusion criteria were as follows: (1) eyes that had not been treated with anti-glaucoma medication before surgery, (2) had a history of uveitis, or (3) were allergic to metals. For each patient, if both eyes satisfied the inclusion criteria, the eye with the higher IOP was selected for the study. The preoperative IOP was the average of the measurements taken within two months prior to the operation.All trabeculectomy and Ex-PRESS implantation procedures were performed using identical processes during the study period. A 5-mm conjunctival incision was created along the limbus to construct a fornix-based conjunctival flap, and a 4-mm wide half-layer scleral flap was formed. Sponges  soaked in 0.04% mitomycin-C (0.4 mg/mL) was applied on and under the scleral flap and under the conjunctiva for 4 min, followed by irrigation with 200 mL of physiological saline. In the eyes that received a trabeculectomy, a deep block of limbal tissue was excised to create a fistula into the anterior chamber, and a peripheral iridectomy was performed. In the eyes that received an Ex-PRESS implantation, the tube was inserted into the anterior chamber after penetration with a 25-gauge needle. In both procedures, the scleral flap and conjunctiva were then sutured with 10-0 nylon. All patients received similar postoperative topical medications: 0.5% levofloxacin for 3 weeks and 0.1% betamethasone three times daily for 6 months without tapering after operation respectively. Laser suture lysis and bleb needling were completed within 1 month of surgery, depending on the postoperative IOP and formation of the bleb.The primary outcomes for this study were the early postoperative complications, including postoperative anterior chamber inflammation, the frequencies of hyphema, a flat anterior chamber, choroidal detachment, hypotonic maculopathy, and visual acuity. The inflammation in the anterior chamber was quantified using a flare meter . Hyphema was defined as blood deposition in the anterior chamber as assessed during the postoperative slit lamp examination. A flat anterior chamber was identified if the anterior chamber at the pupillary border of the iris was narrower than the corneal thickness or if cornea–iris contact was observed at the peripheral anterior chamber. Choroidal detachment was determined during the postoperative fundus examination. Hypotonic maculopathy was definedThe secondary outcomes were the duration of the anterior chamber opening during surgery, postoperative IOP, postoperative anti-glaucoma medications, laser suture lysis, bleb needling, and the relationship between the iris contact of the Ex-PRESS tube and the anterior chamber depth in the Ex-PRESS group. The time for which the anterior chamber was open during the surgery was defined as the time between starting the incision for the fistula creation in the anterior chamber and suturing the scleral flap in the trabeculectomy group. The same was defined as the time between the penetration with a 25-gauge needle and suturing the scleral flap in the Ex-PRESS group. The postoperative IOPs were measured with Goldmann applanation tonometry. Because the anterior chamber depth changed with hypotony after the surgery, it was measured preoperatively using an optical biometer . We performed bleb revision with needling if the postoperative eyes had higher IOP than preoperative IOP despite of the completion of laser suture lysis. Needling of the bleb was performed by sweeping to cut episcleral scarring that was obstructing the intrascleral pathway. The contact of the iris with the Ex-PRESS tube was judged during a slit lamp examination at 1 month or later when there was no shallow or flat anterior chamber due to hypotony present. The anterior chamber angle examination was performed by gonioscopy before surgery to exclude angle closure glaucoma and again after the surgery to examine the position of the insertion of the Ex-PRESS device in the angle.The other data regarding patient characteristics were gender, age, glaucoma type, and other preoperative ophthalmic data, including preoperative IOP, number of medications, corneal thickness, anterior chamber depth, axial length, best-corrected visual acuity, and visual field. Corneal thickness, anterior chamber depth, and axial length were measured with optical coherence interferometry . A logarithm of the reciprocal of the decimal BCVA was used to approximate the logarithm of the minimal angle of resolution (LogMAR). Visual field testing was performed using a static automated white-on-white threshold 24-2 perimetry program, SITA Standard . If the eye had advanced visual field loss, a 10-2 perimetry program was used.CPETS was planned to examine postoperative complications for five years. To determine the early postoperative complications in the study, we examined the patients at days 1, 3, 7, and 10, and also at 1 and 3 months after the surgery. If the patients required additional treatment for complications, they were examined at additional follow-up visits.The sample size which would provide 80% power to prove (at a one-sided a level of 0.05) the superiority of Ex-PRESS implantation compared with trabeculectomy for postoperative anterior chamber inflammation was 28 eyes in each group for an effect size of 0.7. Therefore, we had planned to study 64 eyes to account for a 12% dropout rate.P values of <0.05 were considered statistically significant. The data were analyzed with the JMP statistical package, version 10.0 .The univariate analysis was performed with the Mann-Whitney nonparametric test (with Bonferroni correction) and the Chi-square test. In total, 65 patients were recruited to this study. One patient whose eye had a preoperative IOP of less than 18 mmHg did not meet the criteria. Thirty-two patients were assigned to each arm. All patients completed the follow-up visits for the early postoperative complication evaluations .There were no significant differences between the trabeculectomy and the Ex-PRESS groups for the baseline characteristics, such as age, gender, the side of the operated eye (right or left), type of glaucoma (primary OAG or exfoliative), the number of preoperative medications, preoperative IOP, visual acuity (logMAR), visual field defect, corneal thickness, anterior chamber depth, or axial length .P = 0.004, the Chi-square test).P = 0.02, the Chi-square test with Bonferroni correction); 6 eyes versus 1 eyes 1 month after the surgery , in the trabeculectomy group versus the Ex-PRESS group, respectively. The trabeculectomy group also showed significantly higher flare values than the Ex-PRESS group 10 days after the surgery .Next, the statistically significant difference of each flare value at each time point between the trabeculectomy and Ex-PRESS groups was evaluated. The numbers of eyes with high flare values were 15 eyes versus 5 eyes 10 days after the surgery (P = 0.0025). There were no significant differences between the two groups for flat anterior chamber (P = 0.45), choroidal detachment (P = 0.49), and hypotonic maculopathy (P = 1.00) (Hyphema occurred in 8 (25%) of 32 eyes in the trabeculectomy group, while no eyes (0%) in the Ex-PRESS group developed a hyphema ( = 1.00) . PostopeP = 0.28). The postoperative visual acuity also did not show any significant differences between the two groups .There was no significant difference in preoperative visual acuity between the two groups as represented by logMAR values .The mean ± SD duration for an open anterior chamber during surgery was 4.9 ± 1.0 min in the trabeculectomy group and 4.1 ± 1.0 min in the Ex-PRESS group. The duration was significantly shorter in the Ex-PRESS group (P = 0.28). Bleb revision with needling was performed in 3 (10%) of 32 eyes in the trabeculectomy group and in 3 (10%) of 32 eyes in the Ex-PRESS group (P = 1.00).The postoperative IOPs are shown in P = 0.013). All seven eyes with a preoperative anterior chamber depth of ≤2.61 mm developed iris contact with the Ex-PRESS tube.Fifteen (46.9%) of 32 eyes in the Ex-PRESS group had iris contact with the Ex-PRESS tube after the surgery . The eyeThe main aim of our present study was the comparison of the frequency of early postoperative complications between trabeculectomy and Ex-PRESS implantation. The inflammation in the anterior chamber was significantly lower in the Ex-PRESS group after the surgery. The frequency of postoperative hyphema was also lower in the Ex-PRESS group than in the trabeculectomy group. The duration of the open anterior chamber during surgery was also significantly shorter in the Ex-PRESS group. Additionally, in the Ex-PRESS group, 46.9% eyes had iris contact with the Ex-PRESS tube, which was significantly associated with shallower anterior chambers before surgery.No conventional sclerectomy or iridectomy is required for the Ex-PRESS implantation. The surgery, which is categorized as a minimally invasive glaucoma surgery procedure, seems to induce less inflammation than trabeculectomy. Stable filtration after Ex-PRESS implantation may be expected to show less IOP fluctuation during the intraoperative and early postoperative periods. Hypotony due to overfiltration increases the vascular permeability of the iris and the protein effusion from the choroidal detachment. Our hypothesis is that Ex-PRESS implantation presents with a lower chance of postoperative inflammation than trabeculectomy. There have been several retrospective9111267813P = 0.0002) and a lower frequency of hyphema (P = 0.0025) than did trabeculectomy. Instead of penetration into the anterior chamber with a 25-gauge needle and Ex-PRESS tube insertion, a limbal block incision and iridectomy are performed during a trabeculectomy. The 0.8 min of time difference between the two groups implies that Ex-PRESS tube insertion was simpler to perform than trabeculectomy. Intraoperative hypotony occurs during a limbal block incision in trabeculectomy and penetration into the anterior chamber with a 25-gauge needle in the Ex-PRESS implantation, leading to the increased risk for surgical complications, such as expulsive hemorrhage, corneal decompensation, and postoperative inflammation in the anterior chamber. Shorter anterior chamber opening in the Ex-PRESS implantation might be preferable to reduce these complications. The shortened duration for an open anterior chamber offered by the Ex-PRESS implantation seems small although the difference of the duration was significant (P = 0.0002). Because all the surgeries were performed by two glaucoma specialists (M.I & Yu T), the time difference between the 2 procedures might have been small. The time difference between the 2 procedures might have been larger if other surgeons carried out the study.The Ex-PRESS implantation, in addition to less postoperative inflammation, also presented with a significantly shorter open anterior chamber duration . Sci. Rep.6, 26080; doi: 10.1038/srep26080 (2016).


Recent studies showed that Wnt/β-catenin pathway could modulate the function and the differentiation of CD4+ T cells. Therefore, Wnt/β-catenin pathway may participate in the development and progress of silicosis. To investigate the role of Wnt/β-catenin pathway, we used lentivirus expressing β-catenin shRNA to block the Wnt/β-catenin pathway by intratracheal instillation to the mice model of silicosis. Treatment of lentivirus could significantly aggravate the silica-induced lung inflammation and attenuated the fibrosis at the late stage. By analyzing CD4+ T cells, we found that blockade of Wnt/β-catenin pathway suppressed regulatory T cells (Tregs). Reciprocally, enhanced Th17 response was responsible for the further accumulation of neutrophils and production of proinflammatory cytokines. In addition, blockade of Wnt/β-catenin pathway delayed the Th1/Th2 polarization by inhibiting Tregs and Th2 response. These results indicated that Wnt/β-catenin pathway could regulate Tregs to modulate Th immune response, which finally altered the pathological character of silicosis. Our study suggested that Wnt/β-catenin pathway might be a potential target to treat the silica-induced inflammation and fibrosis.CD4 Nevertheless, IL-23 is essential to promote the proliferation rather than the generation of Th17 cells [β alone can facilitate the differentiation of Tregs by inducing Foxp3 [β plus IL-6 can inhibit Tregs and induce the differentiation of Th17 cells, so there may exist a reciprocal association between Tregs and Th17 cells, and IL-6 may be the regulator of this balance [Silicosis is a damaging kind of pneumoconiosis caused by inhalation of silica mineral dust. The basic pathological changes are diffuse pulmonary fibrosis and silicotic nodule , 2. Inhaticipant . Naïve Cnd Tregs –8. Theseammation . Whereasammation . Tregs cammation . Based o disease . Subsequ disease –17. The 17 cells . TGF-β ang Foxp3 , 20. How balance , 21.β-catenin is an evolutionarily conserved signaling pathway that controls the embryonic development and cell-fate decision [β-catenin determines the activation of canonical Wnt/β-catenin pathway. β-catenin is degraded by a destruction complex consisting of axis inhibition protein, adenomatous polyposis coli, casein kinase 1, and glycogen synthase kinase 3β. In the presence of Wnt ligands, transmembrane protein Frizzled and its coreceptor LRP5/6 are activated to disrupt the destruction complex; hence β-catenin proteins accumulate and then translocate into the nucleus to generate target gene transcription [β-catenin pathway in thymic T cells development has been fully studied [β-catenin pathway still needs to be explored. It had been proved that β-catenin combined with TCF-1 activated the GATA3 transcription to initiate Th2 differentiation [+CD25+Tregs expressing stable β-catenin inhibited inflammation more effectively due to the superior ability to survive [β-catenin pathway competed with the function of Foxp3 and thus reduced the suppressive ability of Tregs [β-catenin pathway promoted Th17 differentiation [β-catenin pathway may play an important role in CD4+ T cells mediated inflammation or fibrosis.Canonical Wnt/decision , 23. Thecription . In the  studied . Howeverntiation , 27. CD4 survive . On the of Tregs . There antiation , 31. Theβ-catenin pathway was involved in the development of experimental silicosis [β-catenin pathway may participate in regulating silica-induced inflammation and fibrosis. In the current study, we used a lentivirus expressing β-catenin shRNA to decrease target mRNA, which continuously blocked the Wnt/β-catenin pathway in the lung. We found that blockade of Wnt/β-catenin pathway suppressed Tregs and elevated Th17 response. Meanwhile, silica-induced inflammation was significantly aggravated. Blocking Wnt/β-catenin pathway also mediated the delay of Th1/Th2 polarization by inhibition of Tregs. This effect impeded the resolution of lung inflammation and restrained the Th2-mediated fibrosis.Our previous study had demonstrated that activation of Wnt/ilicosis . So we sFemale C57BL/6 mice were purchased from SLAC Laboratory Animal Co., Ltd. , at 6–8 weeks of age. All mice were maintained in specific pathogen-free conditions and fed a standard mouse chow at an environmental temperature of 24 ± 1°C and a 12 h/12 h light/dark cycle with water available ad libitum. The animal study was approved by the Animal Care and Use Committee of China Medical University (CMU62043018), which complies with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.μm diameter, 80% <3 μm diameter; median diameter 1.4 μm) were obtained from the U.S. Silica Company . Silica particles were boiled in 1 N HCl to eliminate endotoxins. Prior to exposure, suspensions were autoclaved and then sonicated for 10 min.The construction of lentivirus has been described in our previous study . Natural7 transducing units (TU) of β-catenin shRNA suspended in a total volume of 0.1 mL sterile saline (silica + β-catenin shRNA group); (4) direct oral-tracheal instillation of 1 mg silica crystals and 3 × 107 transducing units (TU) of NC shRNA suspended in a total volume of 0.1 mL sterile saline (silica + NC shRNA group). The method of exposure of the mice to silica crystals (Min-U-Sil 5) has been previously described [All the mice were randomly divided into four groups, as follows: (1) direct oral-tracheal instillation of 0.1 mL sterile saline ; (2) exposure to silica by direct oral-tracheal instillation of 1 mg silica crystals suspended in a total volume of 0.1 mL sterile saline (silica group); (3) direct oral-tracheal instillation of 1 mg silica crystals and 3 × 10escribed . Mice weThe lungs of the mice were removed and washed in cold phosphate-buffered saline (PBS) after sacrifice of the mice. Bronchoalveolar lavage fluid (BALF) was obtained by cannulating the trachea and then injecting and retrieving 1 mL aliquots of sterile physiological saline two times. The BALF was centrifuged at 1,500 rpm at 4°C for 8 min. After lysis of red blood cells (RBCs), the BALF cell pellet was washed and resuspended in PBS. Total cell counts were determined using standard hematologic procedures. After cytospin preparation, BALF was stained using the Wright-Giemsa method. Macrophages, neutrophils, and lymphocytes were identified in 200 cells using standard morphologic criteria.Hilar lymph nodes (HLNs) were harvested, dissected with needles, and digested with 0.25% trypsin for 5 min at 37°C. The digestion reaction was terminated by the addition of 3% fetal bovine serum in PBS, and samples were centrifuged at 1,500 rpm and 4°C for 8 min. The HLN cell pellets were washed and resuspended in PBS.μL/mL)  for 5 h in complete RPMI-1640. Cells were then incubated with anti-mouse CD4 PerCP-Cy5.5 clone: RM4-5 for 20 min at 4°C in the dark. After cellar surface staining, cells were fixed and permeabilized using standard reagents, according to the manufacturer's protocols (eBioscience Inc.). Cells were then stained with an anti-mouse IL-17A PE clone: eBio17B7; an anti-mouse Foxp3 eFluor® 660 clone: FJK-16s; and an anti-mouse IFN-γ Alexa Fluor® 488 Clone: XMG1.2 (eBioscience Inc.). Cells were subsequently analyzed using a FACSCanto II flow cytometer . Dead cells and silica particles were gated out according to forward scattering (FSC) and side scattering (SSC). Cells were analyzed with BD FACSDiva™ Software v6.1.2 (BD Biosciences). Cytokines in BALF were measured by using BD CBA Mouse Th1/Th2/Th17 Cytokine Kit . The operations were performed according to the manufacturer's instructions. Samples were measured on the FACSCanto II flow cytometer  and analyzed by FCAP Array™ Software . Individual cytokine concentrations were indicated by their fluorescent intensities. Fluorescence intensity was proportional to the amount of a given cytokine in a vial, and the protein concentrations of the test samples were estimated according to the standard curves acquired after analysis of standard dilutions [Cells from the HLNs were resuspended in PBS and stimulated with cell stimulation cocktail (including phorbol-12-myristate-13-acetate (PMA), ionomycin, brefeldin A, and monensin) (2 ilutions .μg) samples were reverse-transcribed (RT) separately in 20-μL volumes using a program of 37°C for 15 min and 85°C for 5 sec. using ABI 2720 . Primers and TaqMan probes sequences were as follows: IL-1β, sense 5′-TGACCTGGGCTGTCCTGATG-3′, antisense 5′-GGTGCTCATGTCCTCATCCTG-3′; IL-17A, sense 5′-GCAAAAGTGAGCTCCAGAAGG-3′, antisense 5′-TCTTCATTGCGGTGGAGAGTC-3′; IL-6, sense 5′-CAATTCCAGAAACCGCTATGAAG-3′, antisense 5′-GTAGGGAAGGCCGTGGTTG-3′; IFN-γ, sense 5′-AAGCGTCATTGAATCACACCTG-3′, antisense 5′-TGACCTCAAACTTGGCAATACTC-3′; IL-10, sense 5′-GGGGCCAGTACAGCCGGGAA-3′, antisense 5′-CTGGCTGAAGGCAGTCCGCA-3′; Foxp3, sense 5′-AAGCCCCGGAGAGGCAGAGG-3′, antisense 5′-TGCAGGCTCAGGTTGTGGCG-3′; IL-4, sense 5′-AAAATCACTTGAGAGAGATCATCGG-3′; antisense 5′-GTTGCTGTGAGGACGTTTGG-3′; IL-13, sense 5′-CCCCTGTGCAACGGCAGCAT-3′; antisense 5′-GAAGGGGCCGTGGCGAAACA-3′; and GAPDH, sense 5′-CAATGTGTCCGTCGTGGATCT-3′, antisense 5′-GTCCTCAGTGTAGCCCAAGATG-3′. The probe sequences were as follows: IL-1β, 5′-(FAM) TCGCAGCAGCACATCAACAAGAGC (BHQ1)-3′; IL-17A, 5′-(FAM) CCTCAGACTACCTCAACCGTTCCAC (BHQ1)-3′; IL-6, 5′-(FAM) CACCAGCATCAGTCCCAAGAAGGCA (BHQ1)-3′; IFN-γ, 5′-(FAM) CTTCTTCAGCAACAGCAAGGCGAA (BHQ1)-3′; IL-10, 5′-(FAM) GCACCCACTTCCCAGTCGGCCAGAGCC (BHQ1)-3′; Foxp3, 5′-(FAM) ACCACCCCGCCACCTGGAAGAATGCCA (BHQ1)-3′; IL-4, 5′-(FAM) TGGCGTCCCTTCTCCTGTGACCTCG (BHQ1)-3′; IL-13, 5′-(FAM) TGGACCTGGCCGCTGGCGGGT (BHQ1)-3′; and GAPDH, 5′-(FAM) CGTGCCGCCTGGAGAAACCTGCC (BHQ1)-3′. For T-bet, IL-23, and GATA-3, reverse-transcribed cDNA was detected by SYBR Green on an ABI 7500 system , according to the manufacturer's protocol. Their primer sequences were as follows: T-bet, sense 5′-TCAACCAGCACCAGACAGAGA-3′, antisense 5′-TCCACCAAGACCACATCCAC-3′; IL-23, sense 5′-ACATGCACCAGCGGGACATA-3′, antisense 5′-CTTTGAAGATGTCAGAGTCAAGCAG-3′; GATA-3, sense 5′-GAAACCGGAAGATGTCTAGCAAA-3′, antisense 5′-TGGAGTGGCTGAAGGGAGA-3′. Real-time RT-polymerase chain reaction (PCR) was performed with a premix Ex Taq RT-PCR kit  or a SYBR Premix Ex Taq II RT-PCR kit . A total of 2 μL of cDNA was used in each 25-μL PCR volume. Each sample was assayed in duplicate. Differences in amplification efficiencies between the target and housekeeping genes were identified by comparing standard curve slopes. Real-time PCR was performed with ABI 7500 (Applied Biosystems) according to the following program: (i) TaqMan: 95°C for 30 sec., 40 cycles of 95°C for 5 sec., and 62°C for 34 sec. or (ii) SYBR Green: 95°C for 30 sec., 40 cycles of 95°C for 5 sec., and 60°C for 34 sec. PCR analyses were performed with ABI 7500 system software.Total RNA was extracted from lung homogenates using TRIzol reagent , according to the manufacturer's protocol. Total lung RNA  and Masson's trichrome stain for pathological evaluation of inflammation and fibrosis, respectively. Lung morphology was visualized using an Olympus BX51 microscope at 200x magnification.Lung tissues were fixed in 4% paraformaldehyde in PBS. Tissues were embedded in paraffin, cut into 6-p < 0.05 was considered statistically significant.SPSS 19.0 software was used to conduct statistical analyses. The differences between values were evaluated through one-way analysis of variance (ANOVA) followed by pairwise comparison with the Student-Newman-Keuls test. β-catenin pathway in silica-induced inflammation, we observed the sections of lung tissue stained by H&E. The saline control group showed normal alveolar structure and no infiltration of inflammatory cells at all time points . We then analyzed cytokines which were crucial for the development of Th17 cells. The level of IL-6 mRNA in lung tissue significantly increased in silica + β-catenin shRNA group at days 7 and 28  and day 56 in silica + β-catenin shRNA group  or indirect (modulating Th1/Th2 polarization) mechanisms [β-catenin pathway could attenuate silica-induced fibrosis. This suggested that the Tregs/Th2 axis might play profibrotic activities.The pathogenesis of experimental silicosis contained a shift from Th1 response to Th2 response and this polarization was regulated by Tregs. The Th1/Th2 balance determined the progress of pulmonary fibrosis induced by silica. Delaying the Th1/Th2 polarization might slow down the resolution of lung inflammation, and Th2 response mediated lung fibrosis would be postponed. On one hand, our study showed that Th1 response was enhanced by the treatment of pression . So bloccytokine , 53. Howcytokine . Howeverchanisms , 41. In β-catenin pathway could inhibit Tregs and enhance Th17 response reciprocally. This effect promoted the production of massive proinflammatory cytokines and led to an aggravating silica-induced inflammation. On the other hand, blocking Wnt/β-catenin pathway delayed the Th1/Th2 polarization by inhibiting Tregs and Th2 response. As a result, the inflammation was sustained whereas the fibrosis attenuated at the late stage of experimental silicosis.Our study demonstrated that blockade of Wnt/β-catenin pathway. Expression of IL-21 (A) and RORγt (B) in lung were assayed by realtime RT-PCR using the −ΔΔCt method. Values are shown as mean ± SEM (n=4-5). .Figure S1: Th17 response was enhanced by blocking the Wnt/


To evaluate the short-term reproducibility of diurnal intraocular pressure (IOP) and ocular perfusion pressure (OPP) measurements in normal volunteers, untreated normal-tension glaucoma (NTG) and primary open-angle glaucoma (POAG) patients.Fifty-four healthy volunteers (control group), 67 NTG patients and 54 POAG patients were recruited. The IOPs of both eyes were measured with a Goldmann applanation tonometer at 3-h intervals over 2 consecutive days. Blood pressure (BP) measurements were collected at the same times. The mean IOP/OPP, peak IOP/OPP, trough IOP/OPP and IOP/OPP fluctuations on each day were also calculated. The intraclass correlation coefficients (ICCs) were used to evaluate the reproducibilities.In the control group, the ICCs of mean IOP, peak IOP, trough IOP and IOP fluctuation were 0.921, 0.889, 0.888, and 0.661, respectively, and the ICCs of the mean OPP, peak OPP, trough OPP and OPP fluctuations were 0.962, 0.918, 0.953, and 0.680, respectively. In the NTG group, the ICCs of the mean IOP, peak IOP, trough IOP and IOP fluctuation were 0.862, 0.741, 0.798, and 0.290, respectively, and the ICCs of the mean OPP, peak OPP, trough OPP and OPP fluctuations were 0.947, 0.828, 0.927, and −0.008, respectively. In the POAG group, the ICCs of the mean IOP, peak IOP, trough IOP and IOP fluctuation were 0.857, 0.666, 0.808, and 0.546, respectively, and the ICCs of the mean OPP, peak OPP, trough OPP and OPP fluctuation were 0.934, 0.842, 0.910, and 0.093, respectively.The IOP measurements within a single day were not highly reproducible in the short-term. The normal volunteers exhibited better IOP and OPP reproducibilities than the glaucoma patients. The IOP and OPP fluctuations could not be accurately evaluated based on the IOP or OPP measurements within a single day. Elevated intraocular pressure (IOP) is an identified risk factor for the progression of glaucoma , 2. MorePatients with normal-tension glaucoma (NTG) exhibit significantly greater reductions in nocturnal blood pressure (BP) than healthy people , 6, and Twenty-four-hour IOP measurements are widely used in clinical and research practices. We typically measure IOPs at various time points within a single day based on convenience, time and financial cost. IOP values at the same time points are not always stable. The reproducibilities of 24-h IOP patterns are controversial –19. If 2In this study, normal volunteers, untreated NTG patients and POAG patients underwent IOP and OPP measurements over 2 consecutive days. We analysed the short-term reproducibilities of the IOP and OPP measurements in the 3 groups.The study adhered to the tenets of the Declaration of Helsinki and was approved by the institutional review board and Ethics Committee of Tianjin Eye Hospital. Informed consent was obtained from each subject. The subjects were recruited in the Tianjin Eye Hospital, Tianjin, China. All subjects were free of treatments with anti-glaucoma medications for at least 4 weeks before the IOP measurements. Subjects with systemic hypertension were excluded. And subjects with systemic administration affected BP or IOP were excluded. None of the subjects had any history of ocular surgery or trauma.None of the normal volunteers had family histories of glaucoma. The IOP values of the normal volunteers ranged from 8 mmHg to 21 mmHg. The volunteers underwent complete ophthalmic examinations and exhibited no signs of ophthalmic disease.The NTG patients were characterized by IOPs ≤ 21 mmHg at all time points, glaucomatous visual field (VF) defects, optic disc damage, an open angle of normal appearance, and the absence of secondary causes for glaucomatous optic disc damage.The POAG patients were characterized by IOPs > 21 mmHg at all time points, glaucomatous VF defects, optic disc damage, an open angle of normal appearance, and the absence of secondary causes of glaucomatous optic disc damage.All subjects underwent a complete ophthalmic examination including central corneal thickness (CCT) measurements  and VF tests using a Humphrey Field Analyzer 750i .The IOPs of both eyes was measured over 2 consecutive days. All subjects were in a sitting position, and the IOPs were measured with a Goldmann applanation tonometer  at 3-h intervals from 0600 to 2400 h. Two experienced doctors were in charge of the IOP measurements. One doctor measured the IOPs on day 1 (6:00–24:00), and the other doctor, masked to the IOP data on day 1, measured the IOPs on day 2 (6:00–24:00). The nocturnal period was from 21:00 to 06:00 . In the The mean IOP was defined as the average IOP across all measurements over 2 consecutive days (14 time points). The IOPs at each time point were defined as the average IOPs at same time point across all subjects within each group. The mean IOPs on days 1 and day 2 were defined as the average IOPs of all measurements during each day . The peak IOP, trough IOP and IOP fluctuation (the peak IOP minus the trough IOP) were also calculated from the IOP measurements during each day. For each subject, the IOPs at each time point were calculated as the average IOPs over the 2 consecutive days. Furthermore, the time points of the peak IOPs, the maximum daytime IOPs and maximum nighttime IOPs were recorded.The systolic BP (SBP), diastolic BP (DBP) and heart rate (HR) were also measured at same time points from 06:00 to 24:00 h. All subjects were in a sitting position and kept calm for at least 5 min. The BP and HR were measured on the upper left arm with an automated sphygmomanometer  Co., Ltd.). The mean SBP, DBP, and HR were defined as the averages of the data over 2 consecutive days (14 time points). The OPPs at each time point were calculated as follows: OPP = 2/3× [DBP + {1/3 × (SBP − DBP)}] − IOP . The maiP < 0.05 was considered statistically significant.The gender, eye (right/left), and MD values of the 3 groups were analysed using Kruskal-Wallis H test. The age, CCT, IOP at diagnosis, mean IOP, mean OPP, mean SBP, mean DBP and mean HR were analysed using one-way analyses of variance (ANOVA). The reproducibilities over the 2 consecutive days were elevated with the intraclass correlation coefficients (ICCs) and Bland-Altman Plots. The ICC indicates the proportion of variance in a measurement that is due to differences among subjects. The α level (type I error) was set at 0.05. An ICC ≥ 0.75 was considered indicative of excellent reproducibility, an ICC of 0.4 ≤ ICC < 0.75 was considered indicative of fair reproducibility, and an ICC of ICC < 0.4 was considered indicative of poor reproducibility . NegativP < 0.001). The mean IOPs over 2 consecutive days exhibited no significant difference between the control and NTG groups (P = 0.271). The POAG group exhibited a significantly lower mean OPP over the 2 consecutive days (29.6 ± 4.5 mmHg) than the control (44.7 ± 4.8 mmHg) and NTG groups . The mean OPP over 2 consecutive days exhibited no significant difference between the control and NTG groups (P = 0.186). The ages, eyes (right/left), genders, CCTs, mean SBPs, mean DBPs and mean HRs over the 2 consecutive days exhibited no significant differences within the 3 groups.Fifty-four normal volunteers (control group), 67 NTG patients and 54 POAG patients were recruited, and the characteristics of each group are shown in Table The ICC estimates of the IOPs at each time point across the 2 days are presented in Table The ICC estimates of the OPPs at each time point across the 2 days are presented in Table The diurnal IOP and diurnal OPP curves were plotted according to the IOP and OPP data at each time point Fig. . The meaIn the control group, the IOP reproducibility was greatest for the mean IOP (ICC = 0.921) followed by the peak IOP (ICC = 0.889) and the trough IOP (ICC = 0.888). The IOP fluctuation exhibited the lowest reproducibility (ICC = 0.661). The reproducibility of the OPP was highest for the mean OPP (ICC = 0.962) followed by the trough OPP (ICC = 0.953) and the peak OPP (ICC = 0.918). The OPP fluctuation exhibited the lowest reproducibility (ICC = 0.680).In the NTG group, the IOP reproducibility was highest for the mean IOP (ICC = 0.862) followed by the trough IOP (ICC = 0.798) and the peak IOP (ICC = 0.741). The IOP fluctuation exhibited the lowest reproducibility (ICC = 0.290). The reproducibility of the OPP was highest for the mean OPP (ICC = 0.947) followed by the trough OPP (ICC = 0.927) and the peak OPP (ICC = 0.828). The OPP fluctuation exhibited the lowest reproducibility (ICC = −0.008).In the POAG group, the IOP reproducibility was highest for the mean IOP (ICC = 0.857) followed by the trough IOP (ICC = 0.808) and the peak IOP (ICC = 0.666). The IOP fluctuation exhibited the lowest reproducibility (ICC = 0.546). The reproducibility of the OPP was highest for the mean OPP (ICC = 0.934) followed by the trough OPP (ICC = 0.910) and the peak OPP (ICC = 0.842). The OPP fluctuation exhibited the lowest reproducibility (ICC = 0.093).For each group, the mean IOPs and mean OPPs exhibited the greatest reproducibilities, whereas the IOP and OPP fluctuations elicited the poorest reproducibilities.Figure For the control group, the mean differences between 2 days were 0 mmHg for the mean IOP and trough IOP, 0.4 mmHg for the peak IOP, 0.5 mmHg for the IOP fluctuation and peak OPP, 0.7 mmHg for the mean OPP, 0.8 mmHg for the trough OPP and −0.3 mmHg for the OPP fluctuation , whereas the control and NTG group exhibited no differences between the daytime and nighttime OPPs.The POAG patients had significantly greater nighttime OPPs (30.3 ± 5.1 mmHg) than daytime OPPs  POAG group exhibited larger IOP fluctuations than the control and NTG groups; (2) the POAG group exhibited significantly lower OPPs than the control and NTG groups; (3) the control group exhibited better IOP reproducibility across the time points than the glaucoma groups; (4) the OPPs across the time points in each group exhibited a tendency towards excellent reproducibility; (5) the mean and trough IOPs of each group exhibited excellent reproducibilities, the peak IOP of the control group exhibited better excellent reproducibility than those of the glaucoma groups, and the IOP fluctuations exhibited fair or poor reproducibilities; (6) the mean OPP, peak OPP and trough OPP exhibited excellent reproducibilities, while the OPP fluctuations exhibited fair or poor reproducibilities; and (7) the nighttime readings of the majority of the subjects were within 1.0 mmHg of the maximum daytime IOP reading and 2.0 mmHg of the maximum daytime OPP.Typical patterns of diurnal IOP and OPP fluctuations were observed in the present study . The peaThe short- and long-term reproducibilities of IOP measurements have been evaluated in some studies. Realini et al. measured the IOPs of healthy individuals with a GAT at 2 h intervals from 8:00 to 20:00 during 2 visits spaced 1 week apart. The eyes of the healthy individuals did not exhibit a sustained and reproducible diurnal IOP pattern across the 2 visits . Later, The mean IOP change between the short- and long-term periods and the short-term peak IOP were associated with progression of glaucoma . Small mSong et al. also calculated the diurnal OPPs of young volunteers every 3 h once per week for 5 consecutive weeks. The maximum OPP, minimum OPP and OPP fluctuation values exhibited excellent, fair and poor reproducibilities, respectively . In thisIn this study, the POAG group exhibited a significantly lower OPP than the NTG patients and normal volunteers. A reduction of OPP is indicative of a reduction in the vascular flow to the optic nerve and could lead to glaucomatous optic nerve damage , 27. SehThere are some limitations in this study. We did not measure the IOP at 3:00 over the 2 consecutive days. This measurement would have required us to wake up the subjects, which would have disturbed their sleep-wake rhythm. This strategy might have affected the IOP and BP measurements at the later time points. Recently, a wireless contact lens sensor (CLS) was used to measure IOPs over 24 h, and this approach did not require the waking of sleeping subjects . ApplanaThe IOP measurements within a single day were not highly reproducible in the short-term. This report is the first to document the reproducibility of OPP measurements in a different study population. The OPP measurements exhibited better reproducibilities than the IOP measurements. The normal volunteers exhibited better IOP and OPP reproducibilities than the glaucoma patients. The IOP and OPP fluctuations exhibited fair or poor reproducibilities; therefore, IOP and OPP fluctuations cannot be assessed based on IOP and OPP measurements collected within a single day.