Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding.

Overexpression of the transmembrane matrix metalloproteinase MT1-MMP/MMP14 promotes cancer cell invasion. Here we show that MT1-MMP-positive cancer cells turn MT1-MMP-negative cells invasive by transferring a soluble catalytic ectodomain of MT1-MMP. Surprisingly, this effect depends on the presence of TKS4 and TKS5 in the donor cell, adaptor proteins previously implicated in invadopodia formation. In endosomes of the donor cell, TKS4/5 promote ADAM-mediated cleavage of MT1-MMP by bridging the two proteases, and cleavage is stimulated by the low intraluminal pH of endosomes. The bridging depends on the PX domains of TKS4/5, which coincidently interact with the cytosolic tail of MT1-MMP and endosomal phosphatidylinositol 3-phosphate. MT1-MMP recruits TKS4/5 into multivesicular endosomes for their subsequent co-secretion in extracellular vesicles, together with the enzymatically active ectodomain. The shed ectodomain converts non-invasive recipient cells into an invasive phenotype. Thus, TKS4/5 promote intercellular transfer of cancer cell invasiveness by facilitating ADAM-mediated shedding of MT1-MMP in acidic endosomes.

Automated tracking of cell migration in phase contrast images with CellTraxx.

The ability of cells to move and migrate is required during development, but also in the adult in processes such as wound healing and immune responses. In addition, cancer cells exploit the cells' ability to migrate and invade to spread into nearby tissue and eventually metastasize. The majority of cancer deaths are caused by metastasis and the process of cell migration is therefore intensively studied. A common way to study cell migration is to observe cells through an optical microscope and record their movements over time. However, segmenting and tracking moving cells in phase contrast time-lapse video sequences is a challenging task. Several tools to track the velocity of migrating cells have been developed. Unfortunately, most of the automated tools are made for fluorescence images even though unlabelled cells are often preferred to avoid phototoxicity. Consequently, researchers are constrained with laborious manual tracking tools using ImageJ or similar software. We have therefore developed a freely available, user-friendly, automated tracking tool called CellTraxx. This software makes it easy to measure the velocity and directness of migrating cells in phase contrast images. Here, we demonstrate that our tool efficiently recognizes and tracks unlabelled cells of different morphologies and sizes (HeLa, RPE1, MDA-MB-231, HT1080, U2OS, PC-3) in several types of cell migration assays (random migration, wound healing and cells embedded in collagen). We also provide a detailed protocol and download instructions for CellTraxx.

Timing of Primary Surgery for Cleft Palate.

BACKGROUND: Among infants with isolated cleft palate, whether primary surgery at 6 months of age is more beneficial than surgery at 12 months of age with respect to speech outcomes, hearing outcomes, dentofacial development, and safety is unknown. METHODS: We randomly assigned infants with nonsyndromic isolated cleft palate, in a 1:1 ratio, to undergo standardized primary surgery at 6 months of age (6-month group) or at 12 months of age (12-month group) for closure of the cleft. Standardized assessments of quality-checked video and audio recordings at 1, 3, and 5 years of age were performed independently by speech and language therapists who were unaware of the trial-group assignments. The primary outcome was velopharyngeal insufficiency at 5 years of age, defined as a velopharyngeal composite summary score of at least 4 (scores range from 0 to 6, with higher scores indicating greater severity). Secondary outcomes included speech development, postoperative complications, hearing sensitivity, dentofacial development, and growth. RESULTS: We randomly assigned 558 infants at 23 centers across Europe and South America to undergo surgery at 6 months of age (281 infants) or at 12 months of age (277 infants). Speech recordings from 235 infants (83.6%) in the 6-month group and 226 (81.6%) in the 12-month group were analyzable. Insufficient velopharyngeal function at 5 years of age was observed in 21 of 235 infants (8.9%) in the 6-month group as compared with 34 of 226 (15.0%) in the 12-month group (risk ratio, 0.59; 95% confidence interval, 0.36 to 0.99; P = 0.04). Postoperative complications were infrequent and similar in the 6-month and 12-month groups. Four serious adverse events were reported (three in the 6-month group and one in the 12-month group) and had resolved at follow-up. CONCLUSIONS: Medically fit infants who underwent primary surgery for isolated cleft palate in adequately resourced settings at 6 months of age were less likely to have velopharyngeal insufficiency at the age of 5 years than those who had surgery at 12 months of age. (Funded by the National Institute of Dental and Craniofacial Research; TOPS ClinicalTrials.gov number, NCT00993551.).

Protrudin-mediated ER-endosome contact sites promote phagocytosis.

During phagocytosis, endosomes both contribute with membrane to forming phagosomes and promote phagosome maturation. However, how these vesicles are delivered to the phagocytic cup and the phagosome has been unknown. Here, we show that Protrudin-mediated endoplasmic reticulum (ER)-endosome contact sites facilitate anterograde translocation of FYCO1 and VAMP7-positive late endosomes and lysosomes (LELys) to forming phagocytic cups in a retinal pigment epithelial-derived cell line (RPE1). Protrudin-dependent phagocytic cup formation required SYT7, which promotes fusion of LELys with the plasma membrane. RPE1 cells perform phagocytosis of dead cells (efferocytosis) that expose phosphatidylserine (PS) on their surface. Exogenous addition of apoptotic bodies increased the formation of phagocytic cups, which further increased when Protrudin was overexpressed. Overexpression of Protrudin also led to elevated uptake of silica beads coated with PS. Conversely, Protrudin depletion or abrogation of ER-endosome contact sites inhibited phagocytic cup formation resulting in reduced uptake of PS-coated beads. Thus, the Protrudin pathway delivers endosomes to facilitate formation of the phagocytic cup important for PS-dependent phagocytosis.

Proteomic Changes in Methicillin-Resistant Staphylococcus aureus Exposed to Cannabinoids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to ß-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In this study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

Does group size matter during collaborative skills learning? A randomised study.

BACKGROUND: Collaborative skills learning in the form of dyad learning compared with individual learning has been shown to lead to non-inferior skills retention and transfer. However, we have limited knowledge on which learning activities improve collaborative skills training and how the number of collaborators may impact skills transfer. We explored the effects of skills training individually, in dyads, triads or tetrads on learning activities during training and on subsequent skills transfer. METHODS: In a randomised, controlled study, participants completed a pre-post-transfer-test set-up in groups of one to four. Participants completed 2 hours of obstetric ultrasound training. In the dyad, triad and tetrad group participants took turns actively handling the ultrasound probe. All performances were rated by two blinded experts using the Objective Structured Assessment of Ultrasound Skills (OSAUS) scale and a Global Rating Scale (GRS). All training was video recorded, and learning activities were analysed using the Interactive-Constructive-Active-Passive (ICAP) framework. RESULTS: One hundred one participants completed the simulation-based training, and ninety-seven completed the transfer test. Performance scores improved significantly from pre- to post-test for all groups (p < 0.001, ηp2 = 0.55). However, group size did not affect transfer test performance on OSAUS scores (p = 0.13, ηp2 = 0.06) or GRS scores (p = 0.23, ηp2 = 0.05). ICAP analyses of training activities showed that time spent on non-learning and passive learning activities increased with group size (p < 0.001, ηp2 = 0.31), whereas time spent on constructive and interactive learning activities was constant between groups compared with singles (p < 0.001, ηp2 = 0.72). CONCLUSION: Collaborative skills learning in groups of up to four did not impair skills transfer despite less hands-on time. This may be explained by a compensatory shift towards constructive and interactive learning activities that outweigh the effect of shorter hands-on time.

Selective Direct Laser Writing of Pyrolytic Carbon Microelectrodes in Absorber-Modified SU-8.

Pyrolytic carbon microelectrodes (PCMEs) are a promising alternative to their conventional metallic counterparts for various applications. Thus, methods for the simple and inexpensive patterning of PCMEs are highly sought after. Here, we demonstrate the fabrication of PCMEs through the selective pyrolysis of SU-8 photoresist by irradiation with a low-power, 806 nm, continuous wave, semiconductor-diode laser. The SU-8 was modified by adding Pro-Jet 800NP (FujiFilm) in order to ensure absorbance in the 800 nm range. The SU-8 precursor with absorber was successfully converted into pyrolytic carbon upon laser irradiation, which was not possible without an absorber. We demonstrated that the local laser pyrolysis (LLP) process in an inert nitrogen atmosphere with higher laser power and lower scan speed resulted in higher electrical conductance. The maximum conductivity achieved for a laser-pyrolyzed line was 14.2 ± 3.3 S/cm, with a line width and thickness of 28.3 ± 2.9 µm and 6.0 ± 1.0 µm, respectively, while the narrowest conductive line was just 13.5 ± 0.4 µm wide and 4.9 ± 0.5 µm thick. The LLP process seemed to be self-limiting, as multiple repetitive laser scans did not alter the properties of the carbonized lines. The direct laser writing of adjacent lines with an insulating gap down to ≤5 µm was achieved. Finally, multiple lines were seamlessly joined and intersected, enabling the writing of more complex designs with branching electrodes and the porosity of the carbon lines could be controlled by the scan speed.

Protrudin-mediated ER-endosome contact sites promote MT1-MMP exocytosis and cell invasion.

Cancer cells break tissue barriers by use of small actin-rich membrane protrusions called invadopodia. Complete invadopodia maturation depends on protrusion outgrowth and the targeted delivery of the matrix metalloproteinase MT1-MMP via endosomal transport by mechanisms that are not known. Here, we show that the ER protein Protrudin orchestrates invadopodia maturation and function. Protrudin formed contact sites with MT1-MMP-positive endosomes that contained the RAB7-binding Kinesin-1 adaptor FYCO1, and depletion of RAB7, FYCO1, or Protrudin inhibited MT1-MMP-dependent extracellular matrix degradation and cancer cell invasion by preventing anterograde translocation and exocytosis of MT1-MMP. Moreover, when endosome translocation or exocytosis was inhibited by depletion of Protrudin or Synaptotagmin VII, respectively, invadopodia were unable to expand and elongate. Conversely, when Protrudin was overexpressed, noncancerous cells developed prominent invadopodia-like protrusions and showed increased matrix degradation and invasion. Thus, Protrudin-mediated ER-endosome contact sites promote cell invasion by facilitating translocation of MT1-MMP-laden endosomes to the plasma membrane, enabling both invadopodia outgrowth and MT1-MMP exocytosis.

Scandcleft Project Trial 3: Comparison of Speech Outcomes in Relation to Sequence in 2-Stage Palatal Repair Procedures in 5-Year-Olds With Unilateral Cleft Lip and Palate.

OBJECTIVE: To compare speech outcome following different sequencing of hard and soft palate closure between arms and centers within trial 3 and compare results to peers without cleft palate. DESIGN: A prospective randomized clinical trial. SETTING: Two Norwegian and 2 British centers. PARTICIPANTS: One hundred thirty-six 5-year-olds with unilateral cleft lip and palate were randomized to either lip and soft palate closure at 3 to 4 months and hard palate closure at 12 months (arm A) or lip and hard palate closure at 3 to 4 months and soft palate closure at 12 months (arm D). MAIN OUTCOME MEASURES: A composite measure of velopharyngeal competence (VPC), overall assessment of VPC from connected speech (VPC-Rate). Percentage of consonants correct (PCC), active cleft speech characteristics (CSCs), subdivided by oral retracted and nonoral errors, and developmental speech characteristics (DSCs). RESULTS: Across the trial, 47% had VPC, with no statistically significant difference between arms within or across centers. Thirty-eight percent achieved a PCC score of >90%, with no difference between arms or centers. In one center, significantly more children in arm A produced ≥3 active CSCs (P < .05). Across centers, there was a statistically significant difference in active CSCs (arm D), oral retracted CSCs (arm D), and DSCs (arms A and D). CONCLUSIONS: Less than half of the 5-year-olds achieved VPC and around one-third achieved age-appropriate PCC scores. Cleft speech characteristics were more common in arm A, but outcomes varied within and across centers. Thus, outcome of the same surgical method can vary substantially across centers.

WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling.

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.

Plasma progesterone, estradiol, and unconjugated estriol concentrations in twin pregnancies: Relation with cervical length and preterm delivery.

INTRODUCTION: The aim of this study was to examine the association between plasma hormone concentrations, cervical length, and preterm delivery in twin pregnancies, including the effect of progesterone treatment. MATERIAL AND METHODS: This study included 191 women pregnant with twins from a randomized placebo-controlled trial. A baseline blood sample was collected at 18-24 weeks before treatment with vaginal progesterone (n = 95) or placebo pessaries (n = 96), and 167 (87.4%) women had a second sample collected after 4-8 weeks of treatment. At baseline, 155 (81.2%) women had their cervical length measured. Progesterone, estradiol, and unconjugated estriol concentration was measured, and the association between hormone concentrations, cervical length, and gestational age at delivery was examined. Hormone concentrations were compared in the placebo and progesterone group. Statistical analysis included Spearman's rho, Mann-Whitney U test, Cuzick's test for trends, and linear regression analyses. RESULTS: A short cervical length was associated with preterm delivery. Cervical length and hormone concentrations were not associated (Spearman's rho; progesterone -.05, estradiol .04, estriol .08). Decreasing gestational age at delivery was associated with higher progesterone and estradiol concentrations at baseline (P trend; progesterone 0.04, estradiol 0.02) but not in the second sample or in the weekly change between samples. Progesterone treatment did not increase the progesterone concentration. CONCLUSIONS: Plasma concentrations of progesterone, estradiol, and unconjugated estriol at 18-24 weeks are not associated with cervical length or preterm delivery in twin pregnancies. Vaginal progesterone treatment does not increase the circulating progesterone concentration in twin pregnancies. Cervical length, but not hormone concentration, is predictive of preterm delivery in twin gestations.

Concerted ESCRT and clathrin recruitment waves define the timing and morphology of intraluminal vesicle formation.

The endosomal sorting complex required for transport (ESCRT) machinery mediates cargo sorting, membrane deformation and membrane scission on the surface of endosomes, generating intraluminal vesicles (ILVs) to degrade signaling receptors. By live-cell imaging of individual endosomes in human cells, we find that ESCRT proteins are recruited in a repetitive pattern: ESCRT-0 and -I show a gradual and linear recruitment and dissociation, whereas ESCRT-III and its regulatory ATPase VPS4 display fast and transient dynamics. Electron microscopy shows that ILVs are formed consecutively, starting immediately after endocytic uptake of cargo proteins and correlating with the repeated ESCRT recruitment waves, unraveling the timing of ILV formation. Clathrin, recruited by ESCRT-0, is required for timely ESCRT-0 dissociation, efficient ILV formation, correct ILV size and cargo degradation. Thus, cargo sorting and ILV formation occur by concerted, coordinated and repetitive recruitment waves of individual ESCRT subcomplexes and are controlled by clathrin.

PtdIns3P controls mTORC1 signaling through lysosomal positioning.

The mechanistic target of rapamycin complex 1 (mTORC1) is a protein kinase complex that localizes to lysosomes to up-regulate anabolic processes and down-regulate autophagy. Although mTORC1 is known to be activated by lysosome positioning and by amino acid-stimulated production of phosphatidylinositol 3-phosphate (PtdIns3P) by the lipid kinase VPS34/PIK3C3, the mechanisms have been elusive. Here we present results that connect these seemingly unrelated pathways for mTORC1 activation. Amino acids stimulate recruitment of the PtdIns3P-binding protein FYCO1 to lysosomes and promote contacts between FYCO1 lysosomes and endoplasmic reticulum that contain the PtdIns3P effector Protrudin. Upon overexpression of Protrudin and FYCO1, mTORC1-positive lysosomes translocate to the cell periphery, thereby facilitating mTORC1 activation. This requires the ability of Protrudin to bind PtdIns3P. Conversely, upon VPS34 inhibition, or depletion of Protrudin or FYCO1, mTORC1-positive lysosomes cluster perinuclearly, accompanied by reduced mTORC1 activity under nutrient-rich conditions. Consequently, the transcription factor EB enters the nucleus, and autophagy is up-regulated. We conclude that PtdIns3P-dependent lysosome translocation to the cell periphery promotes mTORC1 activation.

Validity of auditory perceptual assessment of velopharyngeal function and dysfunction - the VPC-Sum and the VPC-Rate.

Overall weighted or composite variables for perceptual auditory estimation of velopharyngeal closure or competence have been used in several studies for evaluation of velopharyngeal function during speech. The aim of the present study was to investigate the validity of a composite score (VPC-Sum) and of auditory perceptual ratings of velopharyngeal competence (VPC-Rate). Available VPC-Sum scores and judgments of associated variables (hypernasality, audible nasal air leakage, weak pressure consonants, and non-oral articulation) from 391 5-year olds with repaired cleft palate (the Scandcleft project) were used to investigate content validity, and 339 of these were compared with an overall judgment of velopharyngeal competence (VPC-Rate) on the same patients by the same listeners. Significant positive correlations were found between the VPC-Sum and each of the associated variables (Cronbachs alpha 0.55-0.87, P < 0.001), and a moderately significant positive correlation between VPC-Sum and VPC-Rate (Rho 0.698, P < 0.01). The latter classified cases well when VPC-Sum was dichotomized with 67% predicted velopharyngeal competence and 90% velopharyngeal incompetence. The validity of the VPC-Sum was good and the VPC-Rate a good predictor, suggesting possible use of both measures depending on the objective.

Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 4. Speech outcomes in 5-year-olds - velopharyngeal competency and hypernasality.

BACKGROUND AND AIM: Adequate velopharyngeal function and speech are main goals in the treatment of cleft palate. The objective was to investigate if there were differences in velopharyngeal competency (VPC) and hypernasality at age 5 years in children with unilateral cleft lip and palate (UCLP) operated on with different surgical methods for primary palatal repair. A secondary aim was to estimate burden of care in terms of received additional secondary surgeries and speech therapy. DESIGN: Three parallel group, randomised clinical trials were undertaken as an international multicentre study by 10 cleft teams in five countries: Denmark, Finland, Sweden, Norway, and the UK. METHODS: Three different surgical protocols for primary palatal repair were tested against a common procedure in the total cohort of 448 children born with a non-syndromic UCLP. Speech audio and video recordings of 391 children (136 girls, 255 boys) were available and perceptually analysed. The main outcome measures were VPC and hypernasality from blinded assessments. RESULTS: There were no statistically significant differences between the prevalences in the arms in any of the trials. VPC: Trial 1, A: 58%, B: 61%; Trial 2, A: 57%, C: 54%; Trial 3, A: 35%, D: 51%. No hypernasality: Trial 1, A: 54%, B: 44%; Trial 2, A: 47%, C: 51%; Trial 3, A: 34%, D: 49%. CONCLUSIONS: No differences were found regarding VPC and hypernasality at age 5 years after different methods for primary palatal repair. The burden of care in terms of secondary pharyngeal surgeries, number of fistulae, and speech therapy visits differed. TRIAL REGISTRATION: ISRCTN29932826.

Scandcleft randomised trials of primary surgery for unilateral cleft lip and Palate: 9. Parental report of social and emotional experiences related to their 5-year-old child's cleft diagnosis.

BACKGROUND AND AIM: Parents of children with a cleft lip and palate may be emotionally affected by the child's diagnosis. Their experiences and perceptions are important when evaluating the complexity of satisfactory treatment outcomes. The objective was to examine parents' social and emotional experiences related to their child's cleft diagnosis, and their perceptions of the child's adjustment to living with a visible difference. DESIGN: International multicentre study by 10 cleft teams in five countries: Denmark, Finland, Sweden, Norway, and the UK. METHODS: A cohort of 448 children born with a non-syndromic UCLP were included. A total of 356 parents completed the Scandcleft Parent Questionnaire. RESULTS: The majority of parents experienced practical and emotional support from family, friends, and health professionals. Nevertheless, parents had to cope with other people's reactions to the cleft, experiences that were described as ranging from hurtful to neutral and/or positive. According to parents, 39% of the children had experienced cleft-related comments and/or teasing. More than half of the parents reported specific worries related to their child's future. CONCLUSION: While the majority of the parents experienced positive support and coped well with the child's diagnosis, some parents were at risk for psychological and emotional challenges that should be identified by the cleft team. To optimise outcomes and the child's adjustment, these parents should be offered psychological support when necessary. TRIAL REGISTRATION: ISRCTN29932826.

Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 5. Speech outcomes in 5-year-olds - consonant proficiency and errors.

BACKGROUND AND AIM: Normal articulation before school start is a main objective in cleft palate treatment. The aim was to investigate if differences exist in consonant proficiency at age 5 years between children with unilateral cleft lip and palate (UCLP) randomised to different surgical protocols for primary palatal repair. A secondary aim was to estimate burden of care in terms of received additional secondary surgeries and speech therapy. DESIGN: Three parallel group, randomised clinical trials were undertaken as an international multicentre study by 10 cleft teams in five countries: Denmark, Finland, Norway, Sweden, and the UK. METHODS: Three different surgical protocols for primary palatal repair were tested against a common procedure in the total cohort of 448 children born with non-syndromic UCLP. Speech audio- and video-recordings of 391 children (136 girls and 255 boys) were available and transcribed phonetically. The main outcome measure was Percent Consonants Correct (PCC) from blinded assessments. RESULTS: In Trial 1, arm A showed statistically significant higher PCC scores (82%) than arm B (78%) (p = .045). No significant differences were found between prevalences in Trial 2, A: 79%, C: 82%; or Trial 3, A: 80%, D: 85%. Across all trials, girls achieved better PCC scores, excluding s-errors, than boys (91.0% and 87.5%, respectively) (p = .01). CONCLUSIONS: PCC scores were higher in arm A than B in Trial 1, whereas no differences were found between arms in Trials 2 or 3. The burden of care in terms of secondary pharyngeal surgeries, number of fistulae, and speech therapy visits differed. TRIAL REGISTRATION: ISRCTN29932826.

Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 10. Parental perceptions of appearance and treatment outcomes in their 5-year-old child.

BACKGROUND AND AIM: Few studies have explored children's emotional and behavioural reactions to cleft surgery and treatment-related stress. The objective was to investigate parents' evaluations of appearance and treatment outcomes in their 5-year-old child with unilateral cleft lip and palate (UCLP), and their perceptions of how their child was coping with treatment, comparing this information with recorded postsurgical complications. DESIGN: Three parallel group randomised clinical trials were undertaken as an international multicentre study by 10 cleft teams in five countries: Denmark, Finland, Sweden, Norway, and the UK. METHODS: Three different surgical procedures for primary palatal repair were tested against a common procedure in the total cohort of 448 children born with a non-syndromic UCLP. A total of 356 parents completed the Scandcleft Parent Questionnaire, and 346 parents completed the Cleft Evaluation Profile. RESULTS: The results indicated that the majority of parents were satisfied with cleft-related features of their child's appearance. Further, most children coped well with treatment according to their parents. Nevertheless, 17.5% of the children showed minor or short-term reactions after treatment experiences, and 2% had major or lasting difficulties. There were no significant relationships between parent perceptions of treatment-related problems and the occurrence of post-surgical medical complications. CONCLUSIONS: Most parents reported satisfaction with their child's appearance. However, treatment-related problems were described in some children, urging cleft centres to be aware of potential negative emotional and behavioural reactions to treatment in some young children, with a view to preventing the development of more severe treatment-related anxiety. TRIAL REGISTRATION: ISRCTN29932826.

Differential Roles of AXIN1 and AXIN2 in Tankyrase Inhibitor-Induced Formation of Degradasomes and ß-Catenin Degradation.

Inhibition of the tankyrase enzymes (TNKS1 and TNKS2) has recently been shown to induce highly dynamic assemblies of ß-catenin destruction complex components known as degradasomes, which promote degradation of ß-catenin and reduced Wnt signaling activity in colorectal cancer cells. AXIN1 and AXIN2/Conductin, the rate-limiting factors for the stability and function of endogenous destruction complexes, are stabilized upon TNKS inhibition due to abrogated degradation of AXIN by the proteasome. Since the role of AXIN1 versus AXIN2 as scaffolding proteins in the Wnt signaling pathway still remains incompletely understood, we sought to elucidate their relative contribution in the formation of degradasomes, as these protein assemblies most likely represent the morphological and functional correlates of endogenous ß-catenin destruction complexes. In SW480 colorectal cancer cells treated with the tankyrase inhibitor (TNKSi) G007-LK we found that AXIN1 was not required for degradasome formation. In contrast, the formation of degradasomes as well as their capacity to degrade ß-catenin were considerably impaired in G007-LK-treated cells depleted of AXIN2. These findings give novel insights into differential functional roles of AXIN1 versus AXIN2 in the ß-catenin destruction complex.

Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity.

In canonical Wnt signaling, the protein levels of the key signaling mediator ß-catenin are under tight regulation by the multimeric destruction complex that mediates proteasomal degradation of ß-catenin. In colorectal cancer, destruction complex activity is often compromised due to mutations in the multifunctional scaffolding protein Adenomatous Polyposis Coli (APC), leading to a stabilization of ß-catenin. Recently, tankyrase inhibitors (TNKSi), a novel class of small molecule inhibitors, were shown to re-establish a functional destruction complex in APC-mutant cancer cell lines by stabilizing AXIN1/2, whose protein levels are usually kept low via poly(ADP-ribosyl)ation by the tankyrase enzymes (TNKS1/2). Surprisingly, we found that for the formation of the morphological correlates of destruction complexes, called degradasomes, functional proteasomes are required. In addition we found that AXIN2 is strongly upregulated after 6 h of TNKS inhibition. The proteasome inhibitor MG132 counteracted TNKSi-induced degradasome formation and AXIN2 stabilization, and this was accompanied by reduced transcription of AXIN2. Mechanistically we could implicate the transcription factor FoxM1 in this process, which was recently shown to be a transcriptional activator of AXIN2. We observed a substantial reduction in TNKSi-induced stabilization of AXIN2 after siRNA-mediated depletion of FoxM1 and found that proteasome inhibition reduced the active (phosphorylated) fraction of FoxM1. This can explain the decreased protein levels of AXIN2 after MG132 treatment. Our findings have implications for the design of in vitro studies on the destruction complex and for clinical applications of TNKSi.