Evaluating Gabapentin Dosing, Efficacy and Safety in Infants.

OBJECTIVE: Gabapentin for management of neuropathic pain, irritability, neonatal abstinence syndrome, rescue sedation, feeding intolerance and visceral hyperalgesia in infants has grown over the past decade. There remains little guidance for indications, initiation, titration and maintenance dosing trends and assessment of outcomes. The primary objective was to describe gabapentin dosing, and the secondary objectives were to identify outcomes to assess efficacy and describe weaning practices. METHODS: A retrospective single-center study was performed in infants younger than 1 year who received gabapentin at Boston Children's Hospital between 2015 and 2021. The primary outcome was indication, initiation and maximum gabapentin dose. Secondary outcomes included mortality, adverse reactions and impact on feeding volumes, weight-for-age Z-scores and face, legs, activity, cry, consolability (FLACC) scores. Descriptive statistics were utilized. RESULTS: Sixty-six infants received gabapentin at a mean ± SD age of 5.5 ± 2.7 months (range of 0-11 months). The mean ± SD initiation dose of gabapentin was 8.6 ± 5.4 mg/kg/day with a median interval of 24 hours (8-24 hours). The maximum mean dose was 23.2 ± 14.4 mg/kg/day at a median interval of every 8 hours (8 hours). The most common indications for initiation were irritability, rescue sedation, and visceral hyperalgesia. There was a statistical improvement in weight-for-age Z scores from 24 hours prior to gabapentin initiation to 2 weeks after the maximum dose of gabapentin (-2.23 ± 1.78 to -1.66 ± 1.91, p < 0.001) and a reduction in FLACC scores (2.29 ± 1.64 to 1.52 ± 1.76, p = 0.007) from 24 hours prior to gabapentin initiation to 3 days after the maximum dose of gabapentin. Three patients experienced minor adverse events. CONCLUSIONS: Gabapentin was well tolerated in infants. Initial gabapentin dosing of 5 mg/kg/dose every 24 hours appears safe and consistent with other published studies in infants. The improvement in outcomes with few adverse events suggests a beneficial role for gabapentin.

Automatic monitoring of neural activity with single-cell resolution in behaving Hydra.

The ability to record every spike from every neuron in a behaving animal is one of the holy grails of neuroscience. Here, we report coming one step closer towards this goal with the development of an end-to-end pipeline that automatically tracks and extracts calcium signals from individual neurons in the cnidarian Hydra vulgaris. We imaged dually labeled (nuclear tdTomato and cytoplasmic GCaMP7s) transgenic Hydra and developed an open-source Python platform (TraSE-IN) for the Tracking and Spike Estimation of Individual Neurons in the animal during behavior. The TraSE-IN platform comprises a series of modules that segments and tracks each nucleus over time and extracts the corresponding calcium activity in the GCaMP channel. Another series of signal processing modules allows robust prediction of individual spikes from each neuron's calcium signal. This complete pipeline will facilitate the automatic generation and analysis of large-scale datasets of single-cell resolution neural activity in Hydra, and potentially other model organisms, paving the way towards deciphering the neural code of an entire animal.

Efficacy and safety of antithrombin supplementation in neonates and infants on a continuous heparin infusion.

Background: Antithrombin (AT) is a natural anticoagulant and potent inhibitor of several coagulation proteins, including activated factor X (FXa) and FIIa. The therapeutic activity of heparin depends on the presence of AT. Levels of plasma AT are low in neonates and young infants compared to those in adults. Exogenous AT supplementation is postulated to enhance the activity of heparin and facilitate attainment of therapeutic anticoagulation in infants. Objectives: To describe the efficacy and safety of AT administration in infants on a continuous heparin infusion. Methods: Retrospective cohort study of 50 infants who received AT while on a heparin infusion. The primary efficacy outcome was attainment of therapeutic anticoagulation within 48 hours after AT administration. Secondary outcomes included the percent of partial thromboplastin time (PTT) levels and/or antifactor Xa (anti-FXa) activity within the therapeutic window, attainment of the target AT activity level, the incidence and severity of bleeding, and all-cause in-hospital mortality. A secondary analysis investigated the relationship between simultaneously measured PTT levels and anti-FXa activity used for heparin monitoring. Results: AT supplementation resulted in achievement of at least one therapeutic PTT level or anti-FXa activity in 90% of AT courses, though not sustained. PTT was within the therapeutic window more often than anti-FXa activity. When measured simultaneously, therapeutic anti-FXa levels were associated with supratherapeutic PTT levels. Conclusion: AT supplementation in infants on a continuous heparin infusion may transiently improve the therapeutic effect of heparin, but this is largely dependent on the laboratory parameters used for monitoring.

Update on the reliability of gait analysis interpretation in cerebral palsy: Inter-institution agreement.

BACKGROUND: Studies have shown good reliability for gait analysis interpretation among surgeons from the same institution. However, reliability among surgeons from different institutions remains to be determined. RESEARCH QUESTION: Is gait analysis interpretation by surgeons from different institutions as reliable as it is for surgeons from the same institution? METHODS: Gait analysis data for 67 patients with cerebral palsy (CP) were reviewed prospectively by two orthopedic surgeons from different institutions in the same state, each with > 10 years' experience interpreting gait analysis data. The surgeons identified gait problems and made treatment recommendations for each patient using a rating form. Percent agreement between raters was calculated for each problem and treatment, and compared to expected agreement based on chance using Cohen's kappa. RESULTS: For problem identification, the greatest agreement was seen for equinus (85% agreement), calcaneus (88%), in-toeing (89%), and out-toeing (90%). Agreement for the remaining problems ranged between 66-78%. Percent agreement was significantly higher than expected due to chance for all issues (p ≤ 0.01) with modest kappa values ranging from 0.12 to 0.51. Agreement between surgeons for treatment recommendations was highest for triceps surae lengthening (89% agreement), tibial derotation osteotomy (90%), and foot osteotomy (87%). Agreement for the remaining treatments ranged between 72-78%. Percent agreement for all treatments was significantly higher than the expected values (p ≤ 0.002) with modest kappa values ranging from 0.22 to 0.52. SIGNIFICANCE: Previous research established that computerized gait analysis data interpretation is reliable for surgeons within a single institution. The current study demonstrates that gait analysis interpretation can also be reliable among surgeons from different institutions. Future research should examine reliability among physicians from more institutions to confirm these results.

Anterior Distal Femoral Hemiepiphysiodesis Does Not Change Pelvic Tilt in Children With Cerebral Palsy.

BACKGROUND: Anterior distal femoral hemiepiphysiodesis (ADFH) is a surgical treatment choice to correct flexed knee gait and fixed knee flexion deformities in children with cerebral palsy who are skeletally immature. Increased anterior pelvic tilt has been reported after surgeries that correct knee flexion deformities, including hamstring lengthening (HSL) and distal femoral extension osteotomies, but anterior pelvic tilt has not been studied after ADFH. We hypothesized that anterior pelvic tilt would increase after ADFH, especially when combined with HSL, and it would correlate with the change in minimum knee flexion in stance and dynamic hamstring lengths. METHODS: Thirty-four eligible participants (age: 13.0, SD: 2.0) were included. Change in mean pelvic tilt across the gait cycle was compared as a function of clinical and gait parameters using linear mixed models. The relationship of change in pelvic tilt to change in other variables was examined using Pearson correlation. RESULTS: Overall, anterior pelvic tilt increased significantly after ADFH by 4.4 degrees ( P = 0.02). Further, the analysis revealed anterior pelvic tilt only increased significantly in the group that had concurrent HSL (11.1 degrees, P < 0.001). Overall, minimum knee flexion significantly decreased (increase in knee extension) in stance (-19.1 degrees, P < 0.001) and there was an increase in maximum normalized dynamic hamstring lengths (0.03, P < 0.001). The anterior pelvic tilt increased significantly in Gross Motor Function Classification System levels III to IV (5.9 degrees, P = 0.02) but did not change significantly in Gross Motor Function Classification System I to II (2.5 degrees, P = 0.37). Change in pelvic tilt was correlated with change in maximum dynamic hamstring lengths ( r = 0.87, P < 0.0001) and change in minimum knee flexion in stance ( r = -0.71, P < 0.0001). CONCLUSIONS: Anterior distal hemiepiphysiodesis without concurrent HSL for flexion knee deformities does not result in increased anterior pelvic tilt. Surgeons should consider anterior distal hemiepiphysiodesis in patients with cerebral palsy and flexed knee gait, who preoperatively have long dynamically modeled hamstrings, are skeletally immature, and when maintenance of pelvic tilt is desired. LEVEL OF EVIDENCE: Level III-retrospective comparative study.

Automatic monitoring of whole-body neural activity in behaving Hydra.

The ability to record every spike from every neuron in a behaving animal is one of the holy grails of neuroscience. Here, we report coming one step closer towards this goal with the development of an end-to-end pipeline that automatically tracks and extracts calcium signals from individual neurons in the cnidarian Hydra vulgaris. We imaged dually labeled (nuclear tdTomato and cytoplasmic GCaMP7s) transgenic Hydra and developed an open-source Python platform (TraSE-IN) for the Tracking and Spike Estimation of Individual Neurons in the animal during behavior. The TraSE-IN platform comprises a series of modules that segments and tracks each nucleus over time and extracts the corresponding calcium activity in the GCaMP channel. Another series of signal processing modules allows robust prediction of individual spikes from each neuron's calcium signal. This complete pipeline will facilitate the automatic generation and analysis of large-scale datasets of single-cell resolution neural activity in Hydra, and potentially other model organisms, paving the way towards deciphering the neural code of an entire animal.

On being a Hydra with, and without, a nervous system: what do neurons add?

The small freshwater cnidarian Hydra has been the subject of scientific inquiry for over 300 years due to its remarkable regenerative capacities and apparent immortality. More recently, Hydra has been recognized as an excellent model system within neuroscience because of its small size, transparency, and simple nervous system, which allow high-resolution imaging of its entire nerve net while behaving. In less than a decade, studies of Hydra's nervous system have yielded insights into the activity of neural circuits in vivo unobtainable in most other animals. In addition to these unique attributes, there is yet another lesser-known feature of Hydra that makes it even more intriguing: it does not require its neural hardware to live. The extraordinary ability to survive the removal and replacement of its entire nervous system makes Hydra uniquely suited to address the question of what neurons add to an extant organism. Here, I will review what early work on nerve-free Hydra reveals about the potential role of the nervous system in these animals and point towards future directions for this work.

Persistent increase in anterior pelvic tilt after hamstring lengthening in children with cerebral palsy.

BACKGROUND: Hamstring lengthening has traditionally been the surgical treatment of choice to correct flexed knee gait in children with cerebral palsy (CP). Improved passive knee extension and knee extension during gait are reported post hamstring lengthening, but concurrent increased anterior pelvic tilt also occurs. RESEARCH QUESTION: Does anterior pelvic tilt increase after hamstring lengthening in children with CP both in the short-term and mid-term, and what predicts increased post-operative anterior pelvic tilt? METHODS: 44 participants were included (age 7.2, SD 2.0 years; 5 GMFCS I, 17 GMFCS II, 21 GMFCS III, 1 GMFCS IV). Mean pelvic tilt was compared between visits, and the effect of potential predictors of change in pelvic tilt was examined using linear mixed models. The relationship of change in pelvic tilt to change in other variables was examined using Pearson correlation. RESULTS: Anterior pelvic tilt increased significantly post-operatively by 4.8° (p < 0.001). It remained significantly higher by 3.8° at 2-15 years follow-up (p < 0.001). Change in pelvic tilt was not affected by sex, age at surgery, GMFCS level, assistance during walking, time since surgery, or baseline values of hip extensor strength, knee extensor strength, knee flexor strength, popliteal angle, hip flexion contracture, step length, walking speed, maximum hip power in stance, or minimum knee flexion in stance. Pre-operative dynamic hamstring length was associated with greater anterior pelvic tilt at all visits but did not affect amount of change in pelvic tilt. Patients in GMFCS I-II showed a similar pattern of change in pelvic tilt to GMFCS III-IV. SIGNFICANCE: When considering hamstring lengthening for ambulatory children with CP, surgeons should weigh increased mid-term anterior pelvic tilt post-operatively with the desired outcome of improved knee extension in stance. Patients with neutral or posterior pelvic tilt and short dynamic hamstring lengths pre-operatively have lowest risk of excessive post-operative anterior pelvic tilt.

USP47 deubiquitylates Groucho/TLE to promote Wnt-ß-catenin signaling.

The Wnt-ß-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator ß-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the ß-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of ß-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.

The role of furosemide and fluid management for a hemodynamically significant patent ductus arteriosus in premature infants.

A patent ductus arteriosus (PDA) in infants born premature can present significant management challenges for neonatal providers. Quantifying a hemodynamically significant PDA (hsPDA) represents the first hurdle, however, identifying the best evidence-based approach amongst conservative, pharmacologic, and/or interventional management options has proven to be even more complicated. Within the conservative arm, furosemide to reduce pulmonary edema and improve lung function has spawned several discussions given the concerns for its upregulation of prostaglandin E2 in the kidneys and conflicting outcomes data. There remains no consensus regarding furosemide use in hsPDAs. In this perspective article, we summarize the approach to defining a hsPDA, review the current practice of furosemide use in the management of hsPDA, and suggest an approach to fluid management and diuresis to address the question: is the routine use of furosemide in hsPDA merited in current practice?

Variation in Functional Mobility Within Gross Motor Function Classification System Levels.

BACKGROUND AND OBJECTIVE: Variation in walking performance within Gross Motor Function Classification System (GMFCS) levels for patients with cerebral palsy (CP) is often unrecognized. The Functional Mobility Scale (FMS) rates mobility at household, school, and community distances. This study evaluated the variability of walking performance within GMFCS levels as measured by the FMS. METHODS: Retrospective review of gait analysis records for ambulatory patients with CP. FMS rating distribution at each distance was examined for GMFCS levels I-IV within age groups (below 12 or above 12 y) and compared among levels using χ2 tests. RESULTS: A total of 788 patients (499 male; age 11.2, SD 3.9 y) were included. FMS score distribution differed significantly among GMFCS levels for all distances (P<0.001). GMFCS LEVEL: I-Children walked independently on all surfaces at home and school distances at all ages. In all, 5% to 7% used wheeled mobility in the community. II-Most walked at home and school distances. Some younger children crawled at home, and 5% to 8% of all subjects used walls and furniture. Approximately 50% of subjects in both age groups used some form of walking aids or a stroller/wheelchair in the community. III-Twenty-five percent to 30% walked unaided at home, requiring walking aids or wheeled mobility at school or in the community. Forty-five percent of younger and 18% of older subjects crawled at home. Eight percent of younger and 28% of older subjects used wheelchairs at school. Seventy-three percent to 75% of all subjects used strollers/wheelchairs in the community. IV-Sixty-two percent of younger and 43% of older subjects crawled at home. Approximately 15% of all subjects did some aided walking at home. Twenty-seven percent of younger children did some aided walking at school, while only 1 older subject did so. All used strollers/wheelchairs in the community. CONCLUSION: Mobility function varies within each GMFCS level with the most variability in GMFCS II at school and community distances and GMFCS III at household distances. These findings highlight the importance of using both the GMFCS and FMS when assessing functional mobility in children with CP. LEVEL OF EVIDENCE: Level III-retrospective study.

Tracking calcium dynamics from individual neurons in behaving animals.

Measuring the activity of neuronal populations with calcium imaging can capture emergent functional properties of neuronal circuits with single cell resolution. However, the motion of freely behaving animals, together with the intermittent detectability of calcium sensors, can hinder automatic monitoring of neuronal activity and their subsequent functional characterization. We report the development and open-source implementation of a multi-step cellular tracking algorithm (Elastic Motion Correction and Concatenation or EMC2) that compensates for the intermittent disappearance of moving neurons by integrating local deformation information from detectable neurons. We demonstrate the accuracy and versatility of our algorithm using calcium imaging data from two-photon volumetric microscopy in visual cortex of awake mice, and from confocal microscopy in behaving Hydra, which experiences major body deformation during its contractions. We quantify the performance of our algorithm using ground truth manual tracking of neurons, along with synthetic time-lapse sequences, covering a wide range of particle motions and detectability parameters. As a demonstration of the utility of the algorithm, we monitor for several days calcium activity of the same neurons in layer 2/3 of mouse visual cortex in vivo, finding significant turnover within the active neurons across days, with only few neurons that remained active across days. Also, combining automatic tracking of single neuron activity with statistical clustering, we characterize and map neuronal ensembles in behaving Hydra, finding three major non-overlapping ensembles of neurons (CB, RP1 and RP2) whose activity correlates with contractions and elongations. Our results show that the EMC2 algorithm can be used as a robust and versatile platform for neuronal tracking in behaving animals.

Calcaneal Sliding Osteotomy Versus Calcaneal Lengthening Osteotomy for Valgus Foot Deformity Correction in Children With Cerebral Palsy.

BACKGROUND: Medial calcaneal sliding (CS) osteotomy and lateral column lengthening (LCL) are often performed to relieve pain and improve transverse plane alignment and gait stability for children with cerebral palsy (CP) and valgus foot deformities. The purpose of this study was to examine the effectiveness of these procedures in this population. METHODS: Retrospective medical record review (including 3D gait analysis data) of patients with CP who underwent LCL (26 subjects, 46 limbs) or CS (46 subjects, 73 limbs). Data extraction included complications (modified Clavien-Dindo system), change in standing foot position (modified Yoo system), and change in gait kinematics and kinetics preoperatively to postoperatively. Groups were compared using paired t tests, Fisher exact test, and survivorship analysis using Cox proportional hazard models. RESULTS: Subjects were 57% male, average age at surgery 11.1 (SD 2.5) years. Average length of follow-up was 3.2 (SD 2.8) years, and was longer in the LCL group (P=0.0004). Complications were minor with similar rates between groups (P=0.14). Prolonged pain and plantar hypersensitivity occurred only in the CS group. Successful maintenance of deformity correction was achieved in 52/73 limbs (71%) in the CS group and 16/44 limbs (36%) in the LCL group (P<0.001). Recurrent pes valgus and need for repeat foot surgery were more common after LCL (P=0.003 and 0.001, respectively). Recurrent pes valgus never occurred when talonavicular fusion was done concomitantly with CS. After accounting for the between group difference in length of follow-up, there was no difference in the rates of recurrent valgus or repeat foot surgery between LCL and CS. None of the variables predicted development of pes varus (P>0.20). Ankle kinematics and kinetics during gait were unchanged in both groups. CONCLUSIONS: CS and LCL have similar effectiveness in providing long-lasting correction of valgus foot deformities. Concomitant talonavicular fusion is key to success of CS for lower functioning patients with severe deformities, and obligate brace wearers. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Spontaneous electrical low-frequency oscillations: a possible role in Hydra and all living systems.

As one of the first model systems in biology, the basal metazoan Hydra has been revealing fundamental features of living systems since it was first discovered by Antonie van Leeuwenhoek in the early eighteenth century. While it has become well-established within cell and developmental biology, this tiny freshwater polyp is only now being re-introduced to modern neuroscience where it has already produced a curious finding: the presence of low-frequency spontaneous neural oscillations at the same frequency as those found in the default mode network in the human brain. Surprisingly, increasing evidence suggests such spontaneous electrical low-frequency oscillations (SELFOs) are found across the wide diversity of life on Earth, from bacteria to humans. This paper reviews the evidence for SELFOs in diverse phyla, beginning with the importance of their discovery in Hydra, and hypothesizes a potential role as electrical organism organizers, which supports a growing literature on the role of bioelectricity as a 'template' for developmental memory in organism regeneration. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

Anterior distal femoral hemiepiphysiodesis with and without patellar tendon shortening for fixed knee flexion contractures in children with cerebral palsy.

PURPOSE: Surgery is often required for fixed knee flexion contractures in patients with neuromuscular conditions. Anterior distal femoral hemiepiphysiodesis (ADFH) is an alternative to distal femoral extension osteotomy (DFEO) in skeletally immature patients. ADFH is typically not accompanied by patellar tendon shortening surgery (PTS). Our purpose was to compare ADFH alone versus ADFH with PTS for treatment of fixed knee flexion contractures and crouched gait in children with cerebral palsy (CP). METHODS: Retrospective review of pre- and postoperative gait analysis data for children with CP who underwent ADFH alone, or ADFH with PTS. Data were analysed using linear mixed models to control for covariates. RESULTS: In total, 25 participants (42 limbs) were included, 17 male and eight female, mean age at surgery 12.9 (sd 1.9) years. Both groups experienced significant improvement in popliteal angle, knee extension range of motion (ROM) and knee extension in stance phase. Greater improvement was seen for all variables in the ADFH/PTS group, mainly due to greater popliteal angle and knee flexion during gait preoperatively in that group (p ≤ 0.02) rather than the procedure performed (p ≥ 0.19). There was no difference between groups postoperatively. Rate of contracture resolution was 0.5° to 1.0° per month, faster in larger contractures (p = 0.02). CONCLUSIONS: ADFH with and without PTS is effective in improving knee extension in skeletally immature patients with CP, correcting contractures at a rate of 0.5° to 1.0° per month. Combined ADFH and PTS surgery may be preferable in patients with larger contractures of up to 30° to 35°. LEVEL OF EVIDENCE: III.

Prevalence of avian haemosporidian parasites is positively related to the abundance of host species at multiple sites within a region.

Parasite prevalence is thought to be positively related to host population density owing to enhanced contagion. However, the relationship between prevalence and local abundance of multiple host species is underexplored. We surveyed birds and their haemosporidian parasites (genera Plasmodium and Haemoproteus) at multiple sites across eastern North America to test whether the prevalence of these parasites in a host species at a particular site is related to that host's local abundance. Prevalence was positively related to host abundance within most sites, although the effect was stronger and more consistent for Plasmodium than for Haemoproteus. In contrast, prevalence was not related to variation in the abundance of most individual host species among sites across the region. These results suggest that parasite prevalence partly reflects the relative abundances of host species in local assemblages. However, three nonnative host species had low prevalence despite being relatively abundant at one site, as predicted by the enemy release hypothesis.

Local host specialization, host-switching, and dispersal shape the regional distributions of avian haemosporidian parasites.

The drivers of regional parasite distributions are poorly understood, especially in comparison with those of free-living species. For vector-transmitted parasites, in particular, distributions might be influenced by host-switching and by parasite dispersal with primary hosts and vectors. We surveyed haemosporidian blood parasites (Plasmodium and Haemoproteus) of small land birds in eastern North America to characterize a regional parasite community. Distributions of parasite populations generally reflected distributions of their hosts across the region. However, when the interdependence between hosts and parasites was controlled statistically, local host assemblages were related to regional climatic gradients, but parasite assemblages were not. Moreover, because parasite assemblage similarity does not decrease with distance when controlling for host assemblages and climate, parasites evidently disperse readily within the distributions of their hosts. The degree of specialization on hosts varied in some parasite lineages over short periods and small geographic distances independently of the diversity of available hosts and potentially competing parasite lineages. Nonrandom spatial turnover was apparent in parasite lineages infecting one host species that was well-sampled within a single year across its range, plausibly reflecting localized adaptations of hosts and parasites. Overall, populations of avian hosts generally determine the geographic distributions of haemosporidian parasites. However, parasites are not dispersal-limited within their host distributions, and they may switch hosts readily.

XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling.

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing ß-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.

A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts.

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (ß-catenin and Axin) in opposing fashion. We have now fused ß-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α.

Wnt/ß-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote ß-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and ß-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or ß-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.