Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles.

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.

Hospital care in the first 10 years of life of children with congenital anomalies in six European countries: data from the EUROlinkCAT cohort linkage study.

OBJECTIVE: To quantify the hospital care for children born with a major congenital anomaly up to 10 years of age compared with children without a congenital anomaly. DESIGN, SETTING AND PATIENTS: 79 591 children with congenital anomalies and 2 021 772 children without congenital anomalies born 1995-2014 in six European countries in seven regions covered by congenital anomaly registries were linked to inpatient electronic health records up to their 10th birthday. MAIN OUTCOME MEASURES: Number of days in hospital and number of surgeries. RESULTS: During the first year of life among the seven regions, a median of 2.4% (IQR: 2.3, 3.2) of children with a congenital anomaly accounted for 18% (14, 24) of days in hospital and 63% (62, 76) of surgeries. Over the first 10 years of life, the percentages were 17% (15, 20) of days in hospital and 20% (19, 22) of surgeries. Children with congenital anomalies spent 8.8 (7.5, 9.9) times longer in hospital during their first year of life than children without anomalies (18 days compared with 2 days) and 5 (4.1-6.1) times longer aged, 5-9 (0.5 vs 0.1 days). In the first year of life, children with gastrointestinal anomalies spent 40 times longer and those with severe heart anomalies 20 times longer in hospital reducing to over 5 times longer when aged 5-9. CONCLUSIONS: Children with a congenital anomaly consume a significant proportion of hospital care resources. Priority should be given to public health primary prevention measures to reduce the risk of congenital anomalies.

Ten-year survival of children with trisomy 13 or trisomy 18: a multi-registry European cohort study.

OBJECTIVE: To investigate the survival to 10 years of age of children with trisomy 13 (T13) and children with trisomy 18 (T18), born 1995-2014. DESIGN: Population-based cohort study that linked mortality data to data on children born with T13 or T18, including translocations and mosaicisms, from 13 member registries of EUROCAT, a European network for the surveillance of congenital anomalies. SETTING: 13 regions in nine Western European countries. PATIENTS: 252 live births with T13 and 602 with T18. MAIN OUTCOME MEASURES: Survival at 1 week, 4 weeks and 1, 5 and 10 years of age estimated by random-effects meta-analyses of registry-specific Kaplan-Meier survival estimates. RESULTS: Survival estimates of children with T13 were 34% (95% CI 26% to 46%), 17% (95% CI 11% to 29%) and 11% (95% CI 6% to 18%) at 4 weeks, 1 and 10 years, respectively. The corresponding survival estimates were 38% (95% CI 31% to 45%), 13% (95% CI 10% to 17%) and 8% (95% CI 5% to 13%) for children with T18. The 10-year survival conditional on surviving to 4 weeks was 32% (95% CI 23% to 41%) and 21% (95% CI 15% to 28%) for children with T13 and T18, respectively. CONCLUSIONS: This multi-registry European study found that despite extremely high neonatal mortality in children with T13 and T18, 32% and 21%, respectively, of those who survived to 4 weeks were likely to survive to age 10 years. These reliable survival estimates are useful to inform counselling of parents after prenatal diagnosis.

Risk factors for hospitalisation in Welsh infants with a congenital anomaly.

OBJECTIVES: To investigate risk factor associated with hospitalisation of infants with a congenital anomaly in Wales, UK. DESIGN: A population-based cohort study. SETTING: Data from the Welsh Congenital Anomaly Register and Information Service linked to the Patient Episode Database for Wales and livebirths and deaths from the Office for National Statistics. PATIENTS: All livebirths between 1999 and 2015 with a diagnosis of a congenital anomaly, which was defined as a structural, metabolic, endocrine or genetic defect, as well as rare diseases of hereditary origin. MAIN OUTCOME MEASURES: Adjusted OR (aOR) associated with 1 or 2+ hospital admissions in infancy versus no admissions were estimated for sociodemographic, maternal and infant factors using multinomial logistic regression for the subgroups of all, isolated, multiple and cardiovascular anomalies. RESULTS: 25 523 infants affected by congenital anomalies experienced a total of 50 705 admissions in infancy. Risk factors for ≥2 admissions were younger maternal age ≤24 years (aOR: 1.17; 95% CI 1.06 to 1.30), maternal smoking (aOR: 1.20; 1.10 to 1.31), preterm birth (aOR: 2.52; 2.25 to 2.83) and moderately severe congenital heart defects (aOR: 6.25; 4.47 to 8.74). Girls had an overall decreased risk of 2+ admissions (aOR: 0.84; 0.78 to 0.91). Preterm birth was a significant risk factor for admissions in all anomaly subgroups but the effect of the other characteristics varied according to anomaly subgroup. CONCLUSIONS: Over two-thirds of infants with an anomaly are admitted to hospital during infancy. Our findings identified sociodemographic and clinical characteristics contributing to an increased risk of hospitalisation of infants with congenital anomalies.

Methadone, Pierre Robin sequence and other congenital anomalies: case-control study.

OBJECTIVE: Methadone is a vital treatment for women with opioid use disorder in pregnancy. Previous reports suggested an association between methadone exposure and Pierre Robin sequence (PRS), a rare craniofacial anomaly. We assessed the association between gestational methadone exposure and PRS. DESIGN/SETTING: This case-malformed control study used European Surveillance of Congenital Anomalies population-based registries in Ireland, the Netherlands, Italy, Switzerland, Croatia, Malta, Portugal, Germany, Wales, Norway and Spain, 1995-2011. PATIENTS: Cases included PRS based on International Classification of Disease (ICD), Ninth Edition-British Paediatric Association (BPA) code 75 603 or ICD, Tenth Edition-BPA code Q8708. Malformed controls were all non-PRS anomalies, excluding genetic conditions, among live births, fetal deaths from 20 weeks' gestation and terminations of pregnancy for fetal anomalies. An exploratory analysis assessed the association between methadone exposure and other congenital anomalies (CAs) excluding PRS. Methadone exposure was ascertained from medical records and maternal interview. RESULTS: Among 87 979 CA registrations, there were 127 methadone-exposed pregnancies and 336 PRS cases. There was an association between methadone exposure and PRS (OR adjusted for registry 12.3, 95% CI 5.7 to 26.8). In absolute terms, this association reflects a risk increase from approximately 1-12 cases per 10 000 births. A raised OR was found for cleft palate (adjusted OR 5.0, 95% CI 2.7 to 9.2). CONCLUSIONS: These findings suggest that gestational methadone exposure is associated with PRS. The association may be explained by unmeasured confounding factors. The small increased risk of PRS in itself does not alter the risk-benefit balance for gestational methadone use. The association with cleft palate, a more common CA, should be assessed with independent data.

Linker region of Akt1/protein kinase Balpha mediates platelet-derived growth factor-induced translocation and cell migration.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway(s) is activated by a variety of agonists to regulate cell migration. Here, we show that the stimulation of mouse embryonic fibroblasts with platelet-derived growth factor (PDGF) induces migration in a PI3K-dependent manner. Cells lacking Akt1/PKBalpha exhibit impaired migration and peripheral ruffling in response to PDGF stimulation, whereas cells lacking Akt2/PKBbeta are normal. In addition, over-expression of Akt1/PKBalpha but not Akt2/PKBbeta is sufficient to restore PDGF-induced cell migration in an Akt1/PKBalpha and Akt2/PKBbeta deficient background. In response to PDGF stimulation, Akt1/PKBalpha selectively translocates to membrane ruffles, however, this localization is abrogated by substituting the linker region of Akt2/PKBbeta. Similarly, expression of an Akt2/PKBalpha chimera containing the linker region of Akt1/PKBalpha restored PDGF-induced migration in cells lacking both Akt1/PKBalpha and Akt2/PKBbeta. Finally, over-expression of constitutively active Rac rescues PDGF-induced migration defects in cells lacking Akt1/PKBalpha. Given these results, we suggest that Akt1/PKBalpha controls cell migration by selectively translocating to the leading edge and activating Rac.

Epidemiology of congenital cerebral anomalies in Europe: a multicentre, population-based EUROCAT study.

OBJECTIVES: To describe the epidemiology and geographical differences in prevalence of congenital cerebral anomalies in Europe. DESIGN AND SETTING: Congenital cerebral anomalies (International Classification of Diseases, 10th Revision code Q04) recorded in 29 population-based EUROCAT registries conducting surveillance of 1.7 million births per annum (29% of all European births). PARTICIPANTS: All birth outcomes (live births, fetal deaths from 20 weeks gestation and terminations of pregnancy after prenatal diagnosis of a fetal anomaly (TOPFA)) from 2005 to 2014. MAIN OUTCOME MEASURES: Prevalence, proportion of associated non-cerebral anomalies, prenatal detection rate. RESULTS: 4927 cases with congenital cerebral anomalies were identified; a prevalence (adjusted for under-reporting) of 9.8 (95% CI: 8.5 to 11.2) per 10 000 births. There was a sixfold difference in prevalence across the registries. Registries with higher proportions of prenatal diagnoses had higher prevalence. Overall, 55% of all cases were liveborn, 3% were fetal deaths and 41% resulted in TOPFA. Forty-eight per cent of all cases were an isolated cerebral anomaly, 25% had associated non-cerebral anomalies and 27% were chromosomal or part of a syndrome (genetic or teratogenic). The prevalence excluding genetic or chromosomal conditions increased by 2.4% per annum (95% CI: 1.3% to 3.5%), with the increases occurring only for congenital malformations of the corpus callosum (3.0% per annum) and 'other reduction deformities of the brain' (2.8% per annum). CONCLUSIONS: Only half of the cases were isolated cerebral anomalies. Improved prenatal and postnatal diagnosis may account for the increase in prevalence of congenital cerebral anomalies from 2005 to 2014. However, major differences in prevalence remain between regions.

Isoform-specific regulation of adipocyte differentiation by Akt/protein kinase Balpha.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway tightly regulates adipose cell differentiation. Here we show that loss of Akt1/PKBalpha in primary mouse embryo fibroblast (MEF) cells results in a defect of adipocyte differentiation. Adipocyte differentiation in vitro and ex vivo was restored in cells lacking both Akt1/PKBalpha and Akt2/PKBbeta by ectopic expression of Akt1/PKBalpha but not Akt2/PKBbeta. Akt1/PKBalpha was found to be the major regulator of phosphorylation and nuclear export of FoxO1, whose presence in the nucleus strongly attenuates adipocyte differentiation. Differentiation-induced cell division was significantly abrogated in Akt1/PKBalpha-deficient cells, but was restored after forced expression of Akt1/PKBalpha. Moreover, expression of p27(Kip1), an inhibitor of the cell cycle, was down regulated in an Akt1/PKBalpha-specific manner during adipocyte differentiation. Based on these data, we suggest that the Akt1/PKBalpha isoform plays a major role in adipocyte differentiation by regulating FoxO1 and p27(Kip1).

Phosphorylation of GATA-6 is required for vascular smooth muscle cell differentiation after mTORC1 inhibition.

Vascular smooth muscle cells (VSMCs) undergo transcriptionally regulated reversible differentiation in growing and injured blood vessels. This dedifferentiation also contributes to VSMC hyperplasia after vascular injury, including that caused by angioplasty and stenting. Stents provide mechanical support and can contain and release rapamycin, an inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1). Rapamycin suppresses VSMC hyperplasia and promotes VSMC differentiation. We report that rapamycin-induced differentiation of VSMCs required the transcription factor GATA-6. Inhibition of mTORC1 stabilized GATA-6 and promoted the nuclear accumulation of GATA-6, its binding to DNA, its transactivation of promoters encoding contractile proteins, and its inhibition of proliferation. These effects were mediated by phosphorylation of GATA-6 at Ser(290), potentially by Akt2, a kinase that is activated in VSMCs when mTORC1 is inhibited. Rapamycin induced phosphorylation of GATA-6 in wild-type mice, but not in Akt2(-/-) mice. Intimal hyperplasia after arterial injury was greater in Akt2(-/-) mice than in wild-type mice, and the exacerbated response in Akt2(-/-) mice was rescued to a greater extent by local overexpression of the wild-type or phosphomimetic (S290D) mutant GATA-6 than by that of the phosphorylation-deficient (S290A) mutant. Our data indicated that GATA-6 and Akt2 are involved in the mTORC1-mediated regulation of VSMC proliferation and differentiation. Identifying the downstream transcriptional targets of mTORC1 may provide cell type-specific drug targets to combat cardiovascular diseases associated with excessive proliferation of VSMCs.

Epidemiology of partial urorectal septum malformation sequence (or 'persistent cloaca'): a population-based study in seven regions of England and Wales, 1985-2010.

BACKGROUND: Partial urorectal septum malformation (pURSM) sequence (or 'persistent cloaca') is a rare congenital anomaly characterised by a joining of the urethral, anal, and genital openings into a single common channel. This study describes the epidemiology of pURSM sequence in England and Wales including prevalence, additional anomalies, and pregnancy outcomes. METHODS: All cases of pURSM sequence prospectively notified to seven congenital anomaly registers in England and Wales during 1985-2010, whether delivered as live births, spontaneous fetal deaths (≥20 weeks' gestation), or elective terminations of pregnancy for fetal anomaly (TOPFA, any gestation), formed this population-based cohort. The risks of spontaneous fetal and infant death were examined by Kaplan-Meier analysis. Differences in prevalence over time, and between regions, were examined by multilevel Poisson regression. RESULTS: 117 cases were recorded among 4,251,241 total births. Six (5%) pregnancies resulted in spontaneous fetal deaths, 53 (45%) in TOPFA, and 58 (50%) in live births. The prevalence was 2.8 (95% CI 2.3 to 3.4) per 100,000 total births, increasing significantly over time (p=0.002) and differing significantly between regions (p=0.005). 77 cases (66%) had at least one additional major congenital anomaly outside the perineum, including 67 (57%) renal, 29 (25%) musculoskeletal, 26 (23%) digestive system, and 24 (21%) cardiovascular anomalies. The risks of spontaneous fetal and infant death were estimated as 8.9% (95% CI 4.1 to 18.8) and 26.3% (95% CI 15.1 to 43.4) respectively. CONCLUSIONS: This is the largest study of the epidemiology of pURSM sequence. The information will be valuable for families and health professionals whenever a case of pURSM sequence is diagnosed.

A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus E2 protects from disease.

The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

Outcome of fetuses with Turner syndrome: a 10-year congenital anomaly register based study.

OBJECTIVE: To describe the characteristics and outcome of fetuses with Turner syndrome reported to a national congenital anomalies register. METHODS: All cases with a diagnosis of Turner syndrome reported to Congenital Anomaly Register and Information Service for Wales (CARIS) between 1 January 1998 and 31 December 2007 were included. The cases were grouped in five categories based on their outcomes: fetal loss (FL), termination of pregnancy (TOP), live birth (LB), and postnatal (PN) detection and comparison was undertaken between the groups. RESULTS: One hundred twenty-four cases were reported during the study period. The prevalence of Turner syndrome was 1 in 4901 live female births. Seventy-four percent had 45 X karyotype while the rest had some form of Mosaic Turner karyotype. Pregnancy was terminated in 66% of antenatally diagnosed cases. FL and TOP groups had 92% and 87%, respectively, of 45 X karyotype - far greater than in the LB and PN groups. Increased nuchal thickness was the commonest anomaly noted in antenatal ultrasound and was a predictor for 45 X karyotype, FL, and termination. CONCLUSION: Termination was the most common outcome of fetuses diagnosed antenatally with Turner syndrome. This has modified the natural history of Turner syndrome particularly in cases with Mosaic karyotype.

Insulin regulates adipocyte lipolysis via an Akt-independent signaling pathway.

After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.

Differential regulation of Akt/protein kinase B isoforms during cell cycle progression.

Phosphatidylinositol 3-kinase pathways play key regulatory roles in cell cycle progression into S phase. In this study, we demonstrated that Akt1/PKBalpha isoform plays an essential role in G(1)/S transition and proliferation. Cells lacking Akt1/PKBalpha showed an attenuated proliferation as well as G(1)/S transition, whereas cells lacking Akt2/PKBbeta showed normal proliferation and G(1)/S transition. The effect of Akt1/PKBalpha on cell proliferation and G(1)/S transition was completely abolished by swapping pleckstrin homology (PH) domain with that of Akt2/PKBbeta. Finally, full activation of Akt/PKB and cyclin D expression was achieved by the Akt1/PKBalpha or chimeric proteins containing the PH domain of Akt1/PKBalpha indicating that the PH domain of Akt1/PKBalpha provides full kinase activity and is necessary for the G(1)/S transition.

Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration.

The Akt/PKB isoforms have different roles in animals, with Akt2 primarily regulating metabolic signaling and Akt1 regulating growth and survival. Here we show distinct roles for Akt1 and Akt2 in mouse embryo fibroblast cell migration and regulation of the cytoskeleton. Akt1-deficient cells responded poorly to platelet-derived growth factor while Akt2-deficient cells had a dramatically enhanced response, resulting in a substantial increase in dorsal ruffling. Swapping domains between Akt1 and Akt2 demonstrated that the N-terminal region containing the pleckstrin homology domain and a linker region distinguishes the two isoforms, while the catalytic domains are interchangeable. Akt2 knock-out cells also migrated faster than wild-type cells, especially through extracellular matrix (ECM), while Akt1 knock-out cells migrated more slowly than wild-type cells. Consistently, Akt2 knock-out cells had elevated Pak1 and Rac activities, suggesting that Akt2 inhibits Rac and Pak1. Both Akt2 and Akt1 associated in complexes with Pak1, but only Akt2 inhibited Pak1 in kinase assays, suggesting an underlying molecular basis for the different cellular phenotypes. Together these data provide evidence for an unexpected functional link between Akt2 and Pak1 that opposes the actions of Akt1 on cell migration.