Gene-targeted therapy for neurofibromatosis and schwannomatosis: The path to clinical trials.

Numerous successful gene-targeted therapies are arising for the treatment of a variety of rare diseases. At the same time, current treatment options for neurofibromatosis 1 and schwannomatosis are limited and do not directly address loss of gene/protein function. In addition, treatments have mostly focused on symptomatic tumors, but have failed to address multisystem involvement in these conditions. Gene-targeted therapies hold promise to address these limitations. However, despite intense interest over decades, multiple preclinical and clinical issues need to be resolved before they become a reality. The optimal approaches to gene-, mRNA-, or protein restoration and to delivery to the appropriate cell types remain elusive. Preclinical models that recapitulate manifestations of neurofibromatosis 1 and schwannomatosis need to be refined. The development of validated assays for measuring neurofibromin and merlin activity in animal and human tissues will be critical for early-stage trials, as will the selection of appropriate patients, based on their individual genotypes and risk/benefit balance. Once the safety of gene-targeted therapy for symptomatic tumors has been established, the possibility of addressing a wide range of symptoms, including non-tumor manifestations, should be explored. As preclinical efforts are underway, it will be essential to educate both clinicians and those affected by neurofibromatosis 1/schwannomatosis about the risks and benefits of gene-targeted therapy for these conditions.

Implementation of a novel thoracostomy tube trainer with real-time feedback.

Objectives: Simulation-based training leads to improved clinical performance but may be influenced by quality and frequency of training. Within simulation training, chest tube insertion remains a challenge as one of the main pitfalls of insertion is a controlled pleural entry. This study evaluates the efficacy of a novel training model with real-time pressure monitoring, the average force to pleural entry in a model and the utility of audio and visual feedback. Methods: This proprietary training model comprised a modified Kelly clamp device with three force sensors at the index finger (sensor 1) and two finger loops (sensors 2 and 3), and a manikin with a replaceable chest wall pad. Standard force values (Newtons (N)) were obtained by experts; expert data revealed that 3-5 s was an acceptable time range to complete the chest tube insertion. Participant level ranged from Post-graduate Year (PGY)-1 to PGY-6 with 13 total participants. Each individual was provided an introduction to the procedure and chest tube trainer. Force (N) and time (ms) measurements were obtained from entry through dermis to pleural space puncture. A significant pressure drop suggested puncturing through the chest wall (completion of the procedure). Results: Force data were captured during each phase of the procedure-linear, plateau, and drop. Linear phase (~3000 ms) was from start of procedure to point of maximum force (<30 N). Plateau phase was from maximum force to just before a drop in pressure. Drop phase was a drop in pressure by 5+ N in a span of 150 ms signaling completion of procedure. All participants were able to complete the task successfully. Force for pleural entry ranged from 17 N to 30 N; time to pleural entry ranged from 7500 to 15 000 ms. There was variability in use of all three sensors. All participants used the index sensor, however there was variability in the use of the loop sensors depending on the handedness of the participant. Left-handed users relied more on sensors 1 and 3 while right-handed users relied more on sensors 1 and 2. Given this variability, only force measurements from sensor 1 were used for assessment. Conclusions: This novel force-sensing chest tube trainer with continuous pressuring monitoring has a wide range of applications in simulation-based training of emergency surgical tasks. Next steps include evaluating its impact on accuracy and efficiency. Applications of real-time feedback measuring force are broad, including vascular access, trocar placement and other common procedures. Level of evidence: Level IV, prospective study.

Neurofibromin 1 regulates early developmental sleep in Drosophila.

Sleep disturbances are common in neurodevelopmental disorders, but knowledge of molecular factors that govern sleep in young animals is lacking. Evidence across species, including Drosophila, suggests that juvenile sleep has distinct functions and regulatory mechanisms in comparison to sleep in maturity. In flies, manipulation of most known adult sleep regulatory genes is not associated with sleep phenotypes during early developmental (larval) stages. Here, we examine the role of the neurodevelopmental disorder-associated gene Neurofibromin 1 (Nf1) in sleep during numerous developmental periods. Mutations in Neurofibromin 1 (Nf1) are associated with sleep and circadian disorders in humans and adult flies. We find in flies that Nf1 acts to regulate sleep across the lifespan, beginning during larval stages. Nf1 is required in neurons for this function, as is signaling via the Alk pathway. These findings identify Nf1 as one of a small number of genes positioned to regulate sleep across developmental periods.

PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing.

DICER1 syndrome is a cancer pre-disposition disorder caused by mutations that disrupt the function of DICER1 in miRNA processing. Studying the molecular, cellular and oncogenic effects of these mutations can reveal novel mechanisms that control cell homeostasis and tumor biology. Here, we conduct the first analysis of pathogenic DICER1 syndrome allele from the DICER1 3'UTR. We find that the DICER1 syndrome allele, rs1252940486, abolishes interaction with the PUMILIO RNA binding protein with the DICER1 3'UTR, resulting in the degradation of the DICER1 mRNA by AUF1. This single mutational event leads to diminished DICER1 mRNA and protein levels, and widespread reprogramming of miRNA networks. The in-depth characterization of the rs1252940486 DICER1 allele, reveals important post-transcriptional regulatory events that control DICER1 levels.

Seizures Related to Neurocysticercosis and Cocaine Use.

Neurocysticercosis (NCC) is an infection of the central nervous system with Taenia solium cysts that most commonly results in seizures. In stable patients without recent symptoms, these seizures may be provoked by seizure threshold-lowering drugs such as cocaine. This case details a 38-year-old male with a past medical history of epilepsy presenting with seizures due to comorbid NCC and cocaine use. This case was complicated by the lack of available information regarding the patient's past medical history and medication use. We highlight the importance of obtaining a full work-up, including brain imaging, to provide optimal treatment for patients with seizures despite a history of drug use.

A systematic review of pharmacologic and cell-based therapies for treatment of lymphedema (2010-2021).

BACKGROUND: Lymphedema describes the accumulation of interstitial fluid that results from lymphatic failure. Lymphedema can be of primary or secondary origin and has been estimated to affect 200 million people worldwide. Secondary lymphedema is commonly due to damage to the lymphatic vessels after surgical procedures. Treatments include compression bandaging and exercise regimens. However, at present, no pharmacologic therapy has been approved. We performed a systematic review of randomized controlled trials (RCTs) that had investigated pharmacologic and cell-based therapies for secondary lymphedema. METHODS: We searched the databases MEDLINE, Embase, and ClinicalTrials.gov from January 2010 to May 2021. Only RCTs that had investigated pharmacologic and/or cell-based therapies for secondary lymphedema were eligible for inclusion. Those studies that had examined only active filarial infection were excluded. Two of us (J.W., S.T.) independently screened the studies for eligibility. RESULTS: We identified eight RCTs that met the inclusion criteria. Overall, the studies were of poor quality with a high risk of bias. Ketoprofen demonstrated promising improvements in skin thickness and tissue histopathologic scores. Some evidence was found to suggest that doxycycline might be beneficial for nonfilarial secondary lymphedema, and a single, small RCT demonstrated that selenium might also confer some benefit. Neither synbiotics nor platelet-rich plasma resulted in reduced lymphedema volumes or symptom severity. Also, although bone marrow-derived stem cells resulted in improved symptom scores, no significant volume reduction was detected. Although positive results were demonstrated in trials investigating benzopyrones, previous meta-analyses have cast doubt on their efficacy. No two studies assessed the same intervention; thus, we could not perform a meta-analysis. CONCLUSIONS: Although the results from some studies appeared promising, the available evidence at present is insufficient for any pharmacologic or cell-based therapy for patients with secondary lymphedema. Furthermore, large, high-quality RCTs are required before treatment recommendations will be possible.

Ischemia-Reperfusion Injury After Posterior Cervical Laminectomy.

Ischemia-reperfusion injury is a rare but serious complication encountered after spinal decompression surgery. This is only the 11th case reported in the literature. There is no current mainstay of treatment; however, several therapies have been studied. This case presents a patient with myelomalacia who underwent posterior laminectomy and developed diffuse cord edema with postoperative quadriplegia. Ischemia-reperfusion injury is believed to be mediated by oxidative and nitrosative stress leading to protein degradation and lipid peroxidation. It is characterized by myelomalacia in a chronically ischemic spinal cord and hyperintensity on T2-weighted MRI after decompression. Treatment has involved steroids and rehabilitation, and outcomes have ranged from minor improvement to full recovery. Novel treatment options have shown promise in animal models.

Mutations in EPHB4 cause human venous valve aplasia.

Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex "organization" of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.

Neurofibromin regulates metabolic rate via neuronal mechanisms in Drosophila.

Neurofibromatosis type 1 is a chronic multisystemic genetic disorder that results from loss of function in the neurofibromin protein. Neurofibromin may regulate metabolism, though the underlying mechanisms remain largely unknown. Here we show that neurofibromin regulates metabolic homeostasis in Drosophila via a discrete neuronal circuit. Loss of neurofibromin increases metabolic rate via a Ras GAP-related domain-dependent mechanism, increases feeding homeostatically, and alters lipid stores and turnover kinetics. The increase in metabolic rate is independent of locomotor activity, and maps to a sparse subset of neurons. Stimulating these neurons increases metabolic rate, linking their dynamic activity state to metabolism over short time scales. Our results indicate that neurofibromin regulates metabolic rate via neuronal mechanisms, suggest that cellular and systemic metabolic alterations may represent a pathophysiological mechanism in neurofibromatosis type 1, and provide a platform for investigating the cellular role of neurofibromin in metabolic homeostasis.

Developmental loss of neurofibromin across distributed neuronal circuits drives excessive grooming in Drosophila.

Neurofibromatosis type 1 is a monogenetic disorder that predisposes individuals to tumor formation and cognitive and behavioral symptoms. The neuronal circuitry and developmental events underlying these neurological symptoms are unknown. To better understand how mutations of the underlying gene (NF1) drive behavioral alterations, we have examined grooming in the Drosophila neurofibromatosis 1 model. Mutations of the fly NF1 ortholog drive excessive grooming, and increased grooming was observed in adults when Nf1 was knocked down during development. Furthermore, intact Nf1 Ras GAP-related domain signaling was required to maintain normal grooming. The requirement for Nf1 was distributed across neuronal circuits, which were additive when targeted in parallel, rather than mapping to discrete microcircuits. Overall, these data suggest that broadly-distributed alterations in neuronal function during development, requiring intact Ras signaling, drive key Nf1-mediated behavioral alterations. Thus, global developmental alterations in brain circuits/systems function may contribute to behavioral phenotypes in neurofibromatosis type 1.

Social Behavioral Deficits with Loss of Neurofibromin Emerge from Peripheral Chemosensory Neuron Dysfunction.

Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder associated with social and communicative disabilities. The cellular and circuit mechanisms by which loss of neurofibromin 1 (Nf1) results in social deficits are unknown. Here, we identify social behavioral dysregulation with Nf1 loss in Drosophila. These deficits map to primary dysfunction of a group of peripheral sensory neurons. Nf1 regulation of Ras signaling in adult ppk23+ chemosensory cells is required for normal social behaviors in flies. Loss of Nf1 attenuates ppk23+ neuronal activity in response to pheromones, and circuit-specific manipulation of Nf1 expression or neuronal activity in ppk23+ neurons rescues social deficits. This disrupted sensory processing gives rise to persistent changes in behavior beyond the social interaction, indicating a sustained effect of an acute sensory misperception. Together our data identify a specific circuit mechanism through which Nf1 regulates social behaviors and suggest social deficits in NF1 arise from propagation of sensory misinformation.

The deleterious effects of cannabis during pregnancy on neonatal outcomes.

OBJECTIVES: To evaluate whether cannabis use during pregnancy is associated with adverse neonatal outcomes that are independent of cigarette smoking. DESIGN: Prospective cohort study. SETTING: Adelaide (Australia), Auckland (New Zealand), Cork (Ireland), and Leeds, London and Manchester (United Kingdom). PARTICIPANTS: 5610 pregnant nulliparous women with low risk pregnancies recruited for the Screening for Pregnancy Endpoints (SCOPE) study, November 2004 - February 2011. At 14-16 weeks of pregnancy, women were grouped by self-reported cannabis use. MAIN OUTCOME MEASURES: Infant birthweight, head circumference, birth length, gestational age, and severe neonatal morbidity or mortality. RESULTS: 314 women (5.6%) reported using cannabis in the 3 months before or during their pregnancy; 97 (31%) stopped using it before and 157 (50%) during the first 15 weeks of pregnancy, while 60 (19%) were still using cannabis at 15 weeks. Compared with babies of mother who had never used cannabis, infants of those who still used it at 15 weeks had lower mean values for birthweight (adjusted mean difference [aMD], -127 g; 95% CI, -238 to -17 g), head circumference (aMD, -0.5 cm; 95% CI, -0.8 to -0.1 cm), birth length (aMD, -0.8 cm; 95% CI, -1.4 to -0.2 cm), and gestational age at birth (aMD, -8.1 days; 95% CI, -12.1 to -4.0 days). The differences for all outcomes except gestational age were greater for women who used cannabis more than once a week than for those who used it less frequently. CONCLUSIONS: Continuing to use cannabis during pregnancy is an independent risk factor for poorer neonatal outcomes.

Correlation between NF1 genotype and imaging phenotype on whole-body MRI: NF1 radiogenomics.

OBJECTIVE: To investigate the genotype-phenotype correlation between neurofibromatosis 1 (NF1) germline mutations and imaging features of neurofibromas on whole-body MRI (WBMRI) by using radiomics image analysis techniques. MATERIALS AND METHODS: Twenty-nine patients with NF1 who had known germline mutations determined by targeted next-generation sequencing were selected from a previous WBMRI study using coronal short tau inversion recovery sequence. Each tumor was segmented in WBMRI and a set of 59 imaging features was calculated using our in-house volumetric image analysis platform, 3DQI. A radiomics heatmap of 59 imaging features was analyzed to investigate the per-tumor and per-patient associations between the imaging features and mutation domains and mutation types. Linear mixed-effect models and one-way analysis of variance tests were performed to assess the similarity of tumor imaging features within mutation groups, between mutation groups, and between randomly selected groups. RESULTS: A total of 218 neurofibromas (97 discrete neurofibromas and 121 plexiform neurofibromas) were identified in 19 of the 29 patients. The unsupervised hierarchical clustering in heatmap analysis revealed 6 major image feature patterns that were significantly correlated with gene mutation domains and types with strong to very strong associations of genotype-phenotype correlations in both per-tumor and per-patient studies (p < 0.05, Cramer V > 0.5), whereas tumor size and locations showed no correlations with imaging features (p = 0.79 and p = 0.42, respectively). The statistical analyses revealed that the number of significantly different features (SDFs) within mutation groups were significantly lower than those between mutation groups (mutation domains: 10.9 ± 9.5% vs 31.9 ± 23.8% and mutation types: 31.8 ± 30.7% vs 52.6 ± 29.3%). The first and second quartile p values of within-patient groups were more than 2 times higher than those between-patient groups. However, the numbers of SDFs between randomly selected groups were much lower (approximately 5.2%). CONCLUSION: This preliminary study identified the NF1 radiogenomics linkage between NF1 causative mutations and MRI radiomic features, i.e., the correlation between NF1 genotype and imaging phenotype on WBMRI.

Discovery of a Potent and Selective CCR4 Antagonist That Inhibits Treg Trafficking into the Tumor Microenvironment.

Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.

Lifestyle and pregnancy complications in polycystic ovary syndrome: The SCOPE cohort study.

OBJECTIVES: To investigate the risk of pregnancy complications in women with and without polycystic ovary syndrome after consideration of lifestyle factors. DESIGN: Prospective cohort. PATIENTS AND MEASUREMENTS: Participants (n = 5628) were apparently healthy nulliparous women with singleton pregnancies from the Screening for Pregnancy Endpoints study in New Zealand, Australia, United Kingdom and Ireland. Multivariable regression models were performed assessing the association of self-reported polycystic ovary syndrome status with pregnancy complications with consideration of lifestyle factors at the 15th week of gestation. RESULTS: Women with polycystic ovary syndrome (n = 354) were older, had a higher socio-economic index and body mass index and were less likely to consume alcohol and smoke but more likely to do vigorous exercise and take multivitamins. In univariable analysis, polycystic ovary syndrome was associated with increased risk of gestational diabetes (OR: 2.2, 95% CI: 1.2, 4.0). In multivariable models, polycystic ovary syndrome was only significantly associated with decreased risk of large for gestational age (OR: 0.62, 95% CI: 0.40, 0.98) with a population attributable risk of 0.22%. None of the other outcomes were attributable to polycystic ovary syndrome status. CONCLUSIONS: Polycystic ovary syndrome is associated with a lower risk of large for gestational age infants. In this low-risk population, the risk of pregnancy complications was not increased in women with polycystic ovary syndrome who were following a healthy lifestyle. Further studies are warranted assessing the contribution of lifestyle factors to the risk of pregnancy complications in higher risk groups of women with and without polycystic ovary syndrome.

Metabolic syndrome in pregnancy and risk for adverse pregnancy outcomes: A prospective cohort of nulliparous women.

BACKGROUND: Obesity increases the risk for developing gestational diabetes mellitus (GDM) and preeclampsia (PE), which both associate with increased risk for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) in women in later life. In the general population, metabolic syndrome (MetS) associates with T2DM and CVD. The impact of maternal MetS on pregnancy outcomes, in nulliparous pregnant women, has not been investigated. METHODS AND FINDINGS: Low-risk, nulliparous women were recruited to the multi-centre, international prospective Screening for Pregnancy Endpoints (SCOPE) cohort between 11 November 2004 and 28 February 2011. Women were assessed for a range of demographic, lifestyle, and metabolic health variables at 15 ± 1 weeks' gestation. MetS was defined according to International Diabetes Federation (IDF) criteria for adults: waist circumference ≥80 cm, along with any 2 of the following: raised trigycerides (≥1.70 mmol/l [≥150 mg/dl]), reduced high-density lipoprotein cholesterol (<1.29 mmol/l [<50 mg/dl]), raised blood pressure (BP) (i.e., systolic BP ≥130 mm Hg or diastolic BP ≥85 mm Hg), or raised plasma glucose (≥5.6 mmol/l). Log-binomial regression analyses were used to examine the risk for each pregnancy outcome (GDM, PE, large for gestational age [LGA], small for gestational age [SGA], and spontaneous preterm birth [sPTB]) with each of the 5 individual components for MetS and as a composite measure (i.e., MetS, as defined by the IDF). The relative risks, adjusted for maternal BMI, age, study centre, ethnicity, socioeconomic index, physical activity, smoking status, depression status, and fetal sex, are reported. A total of 5,530 women were included, and 12.3% (n = 684) had MetS. Women with MetS were at an increased risk for PE by a factor of 1.63 (95% CI 1.23 to 2.15) and for GDM by 3.71 (95% CI 2.42 to 5.67). In absolute terms, for PE, women with MetS had an adjusted excess risk of 2.52% (95% CI 1.51% to 4.11%) and, for GDM, had an adjusted excess risk of 8.66% (95% CI 5.38% to 13.94%). Diagnosis of MetS was not associated with increased risk for LGA, SGA, or sPTB. Increasing BMI in combination with MetS increased the estimated probability for GDM and decreased the probability of an uncomplicated pregnancy. Limitations of this study are that there are several different definitions for MetS in the adult population, and as there are none for pregnancy, we cannot be sure that the IDF criteria are the most appropriate definition for pregnancy. Furthermore, MetS was assessed in the first trimester and may not reflect pre-pregnancy metabolic health status. CONCLUSIONS: We did not compare the impact of individual metabolic components with that of MetS as a composite, and therefore cannot conclude that MetS is better at identifying women at risk. However, more than half of the women who had MetS in early pregnancy developed a pregnancy complication compared with just over a third of women who did not have MetS. Furthermore, while increasing BMI increases the probability of GDM, the addition of MetS exacerbates this probability. Further studies are required to determine if individual MetS components act synergistically or independently.

Factors Responsible for Changes in Leafroller (Lepidoptera: Tortricidae) Species Composition on Orchards and Vineyards 1974-2015, in Hawke's Bay, New Zealand.

Leafrollers are polyphagous pests whose larvae damage fruit and cause market access problems for fruit crops exported from New Zealand. Leafroller larvae and pupae were collected mainly from four fruit crops, but also from hedges, ornamental trees, and understory weeds in orchards and vineyards of Hawke's Bay, a major fruit production region. Samples were collected from 1974 to 1977 and 1993 to 2015. This timespan was divided into periods that broadly coincided with broad-spectrum insecticide management, the transition to selective insecticides, and the full implementation of integrated fruit production (IFP) programs in apples, grapes, and stone fruit. Eight tortricid species were identified, but the accidentally introduced Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) and two native species, Ctenopseustis obliquana (Walker) and Planotortrix octo Dugdale, comprised 95% of the samples. The proportions of these three species varied according to interactions between four factors: pest management regime, collection period, property location, and host plant. In the 1970s under broad-spectrum insecticide programs, the native species dominated on all hosts. By the mid-late 1990s when IFP was introduced, all the main leafroller species were in decline. However, E. postvittana declined to a lesser extent than the two native species, and consequently, it became relatively more prominent. This change in species composition was delayed in two districts where localized cases of organophosphate insecticide resistance occurred in C. obliquana and P. octo. From 2000 onwards, E. postvittana was the dominant species in all districts and on all hosts, albeit with a much-reduced pest status, except on hedges and ornamental trees where C. obliquana prevailed.

Pre-pregnancy fast food and fruit intake is associated with time to pregnancy.

STUDY QUESTION: Is preconception dietary intake associated with reduced fecundity as measured by a longer time to pregnancy (TTP)? SUMMARY ANSWER: Lower intake of fruit and higher intake of fast food in the preconception period were both associated with a longer TTP. WHAT IS KNOWN ALREADY: Several lifestyle factors, such as smoking and obesity, have consistently been associated with a longer TTP or infertility, but the role of preconception diet in women remains poorly studied. Healthier foods or dietary patterns have been associated with improved fertility, however, these studies focused on women already diagnosed with or receiving treatments for infertility, rather than in the general population. STUDY DESIGN, SIZE, DURATION: This was a multi-center pregnancy-based cohort study of 5628 nulliparous women with low-risk singleton pregnancies who participated in the Screening for Pregnancy Endpoints (SCOPE) study. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 5598 women were included. Data on retrospectively reported TTP and preconception dietary intake were collected during the first antenatal study visit (14-16 weeks' gestation). Dietary information for the 1 month prior to conception was obtained from food frequency questions for fruit, green leafy vegetables, fish and fast foods, by a research midwife. Use of any fertility treatments associated with the current pregnancy was documented (yes, n = 340, no, n = 5258). Accelerated failure time models with log normal distribution were conducted to estimate time ratios (TR) and 95% CIs. The impact of differences in dietary intake on infertility (TTP >12 months) was compared using a generalized linear model (Poisson distribution) with robust variance estimates, with resulting relative risks (RR) and 95% CIs. All analyses were controlled for a range of maternal and paternal confounders. Sensitivity analyses were conducted to explore potential biases common to TTP studies. MAIN RESULTS AND THE ROLE OF CHANCE: Lower intakes of fruit and higher intakes of fast food were both associated with modest increases in TTP and infertility. Absolute differences between the lowest and highest categories of intake for fruit and fast food were in the order of 0.6-0.9 months for TTP and 4-8% for infertility. Compared with women who consumed fruit ≥3 times/day, the adjusted effects of consuming fruit ≥1-<3 times/day (TR = 1.06, 95% CI: 0.97-1.15), 1-6 times/week (TR = 1.11, 95% CI: 1.01-1.22) or <1-3 times/month (TR = 1.19, 95% CI: 1.03-1.36), corresponded to 6, 11 and 19% increases in the median TTP (Ptrend = 0.007). Similarly, compared with women who consumed fast food ≥4 times/week, the adjusted effects of consuming fast food ≥2-<4 times/week (TR = 0.89, 95% CI: 0.81-0.98), >0-<2 times/week (TR 0.79, 95% CI 0.69-0.89) or no fast food (TR = 0.76, 95% CI: 0.61-0.95), corresponded to an 11, 21 and 24% reduction in the median TTP (Ptrend <0.001). For infertility, compared with women who consumed fruit ≥3 times/day, the adjusted effects of consuming fruit ≥1-<3 times/day, 1-6 times/week or <1-3 times/month corresponded to a 7, 18 and 29% increase in risk of infertility (Ptrend = 0.043). Similarly, compared with women who consumed fast food ≥4 times/week, the adjusted effects of consuming fast food ≥2-<4 times/week, >0-<2 times/week, or no fast food, corresponded to an 18, 34 and 41% reduced risk of infertility (Ptrend <0.001). Pre-pregnancy intake of green leafy vegetables or fish were not associated with TTP or infertility. Estimates remained stable across a range of sensitivity analyses. LIMITATIONS, REASONS FOR CAUTION: Collection of dietary data relied on retrospective recall and evaluated a limited range of foods. Paternal dietary data was not collected and the potential for residual confounding cannot be eliminated. Compared to prospective TTP studies, retrospective TTP studies are prone to a number of potential sources of bias. WIDER IMPLICATIONS OF THE FINDINGS: These findings underscore the importance of considering preconception diet for fecundity outcomes and preconception guidance. Further research is needed assessing a broader range of foods and food groups in the preconception period. STUDY FUNDING/COMPETING INTEREST(S): The SCOPE database is provided and maintained by MedSciNet AB (http://medscinet.com). The Australian SCOPE study was funded by the Premier's Science and Research Fund, South Australian Government (http://www.dfeest.sa.gov.au/science-research/premiers-research-and-industry-fund). The New Zealand SCOPE study was funded by the New Enterprise Research Fund, Foundation for Research Science and Technology; Health Research Council (04/198); Evelyn Bond Fund, Auckland District Health Board Charitable Trust. The Irish SCOPE study was funded by the Health Research Board of Ireland (CSA/2007/2; http://www.hrb.ie). The UK SCOPE study was funded by National Health Service NEAT Grant (Neat Grant FSD025), Biotechnology and Biological Sciences Research council (www.bbsrc.ac.uk/funding; GT084) and University of Manchester Proof of Concept Funding (University of Manchester); Guy's and St. Thomas' Charity (King's College London) and Tommy's charity (http://www.tommys.org/; King's College London and University of Manchester); and Cerebra UK (www.cerebra.org.uk; University of Leeds). L.E.G. is supported by an Australian National Health and Medical Research Council (NHMRC) Early Career Fellowship (ID 1070421). L.J.M. is supported by a SACVRDP Fellowship; a program collaboratively funded by the National Heart Foundation, the South Australian Department of Health and the South Australian Health and Medical Research Institute. L.C.K. is supported by a Science Foundation Ireland Program Grant for INFANT (12/RC/2272). C.T.R. was supported by a National Health and Medical Research Council (NHMRC) Senior Research Fellowship (GNT1020749). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Emerging therapeutic targets for neurofibromatosis type 1.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited tumor predisposition syndrome with an incidence of one in 3000-4000 individuals with no currently effective therapies. The NF1 gene encodes neurofibromin, which functions as a negative regulator of RAS. NF1 is a chronic multisystem disorder affecting many different tissues. Due to cell-specific complexities of RAS signaling, therapeutic approaches for NF1 will likely have to focus on a particular tissue and manifestation of the disease. Areas covered: We discuss the multisystem nature of NF1 and the signaling pathways affected due to neurofibromin deficiency. We explore the cell-/tissue-specific molecular and cellular consequences of aberrant RAS signaling in NF1 and speculate on their potential as therapeutic targets for the disease. We discuss recent genomic, transcriptomic, and proteomic studies combined with molecular, cellular, and biochemical analyses which have identified several targets for specific NF1 manifestations. We also consider the possibility of patient-specific gene therapy approaches for NF1. Expert opinion: The emergence of NF1 genotype-phenotype correlations, characterization of cell-specific signaling pathways affected in NF1, identification of novel biomarkers, and the development of sophisticated animal models accurately reflecting human pathology will continue to provide opportunities to develop therapeutic approaches to combat this multisystem disorder.