Fusarium oxysporum f. sp. lycopersici (Fol), a fungal pathogen of tomato. FolVam7 contains SNARE and PX (Phox homology) domains that are indispensable for normal localization and function of FolVam7. Targeted gene deletion showed that FolVam7‐mediated vesicle trafficking is important for vegetative growth, asexual development, conidial morphology and plant infection. Further cytological examinations revealed that FolVam7 is localized to vesicles and vacuole membranes in the hyphae stage. Moreover, the ΔFolvam7 mutant is insensitive to salt and osmotic stresses and hypersensitive to cell wall stressors. Taken together, our results suggested that FolVam7‐mediated vesicle trafficking promotes vegetative growth, conidiogenesis and pathogenicity of Fol.Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) are conserved in fungi, plants and animals. The Vam7 gene encodes a v‐SNARE protein that involved in vesicle trafficking in fungi. Here, we identified and characterized the function of FolVam7, a homologue of the yeast SNARE protein Vam7p in  The infection process of Fol has been well studied in tomato, where it displays apparent gene‐for‐gene relationships  by a BLAST_P search using the Vam7 protein sequence from S. cerevisiae. FOXG_02319 encodes a polypeptide of 362 amino acids, which we named FolVam7. Domain prediction tools revealed that FolVam7 contains a PHOX homology motif (PX) (6–116 residues) at the N‐terminus and a SNARE domain (302–360 residues) at the C‐terminus (http://smartemblheidelberg.de/) , minimal medium (MM) and potato dextrose agar (PDA). The results showed that the vegetative growth of ΔFolvam7 was severely attenuated on all media tested , a fluorescent dye that stains the interior of vacuoles. We found that the majority of the GFP‐FolVam7 signal partially co‐localized with CMAC at the apical region of the hyphae  in the ely Fig. A and B. ΔPX and FolVam7ΔSNARE deletion domain constructs with a GFP‐tag at the N‐terminus and transformed each construct into the ΔFolvam7 mutant  of FolVam7 for cellular localization and biological function, we generated FolVam7ant Fig. A. Both Gasm Fig. B. These am7 Fig. C. Finallvar Fig. D. TherefF. graminearum , or the cell wall stressors Congo red (CR), Calcofluor white (CFW) and sodium dodecylsulfate (SDS). After 5 days incubation, osmotic stress partially relieved the ΔFolvam7 mutant growth defect on plates containing 1 M NaCl, 1 M KCl or 1 M sorbitol  . The PCR products were purified and then transformed into protoplasts of the wild‐type strain as previously described  plasmid into yeast, using the yeast gap repair approach to obtain plasmid GFP‐FolVAM7 ‐PCR and used in PCRs. The transformants were then screened by phenotypic characterization and detection of a GFP signal during fluorescence microscopy. All primers used in this section are listed in the Supporting Information Table In order to analyse the biological function of the Solanum lycopersicum cv. C32    and water (mock) for 30 min respectively. Treated seedlings were then planted in a container with potting soil. Inoculated plants were grown at 28°C, 55%–65% relative humidity and 16‐h light. The plants were examined for typical wilting symptoms after 3 weeks.et al., et al., et al., Fungal recovery assays were performed as previously described  according to the manufacturer's recommendations, and subsequently further purified using RNeasy Mini spin columns (Qiagen). Samples containing 1 μg of total RNA were used for the first strand cDNA synthesis using HiS cript II Reverse Transcriptase  following the manufacturer's instructions. The RT2 PCR Real‐Time SYBR Green/ROX PCR master mix  was used for qRT‐PCR analysis. Relative quantification of each transcript was calculated using the 2et al., et al., et al., To examine endocytosis, strains were cultured in liquid CM medium. After growing at 28°C for 24 h, the hyphae were stained with N‐(3‐triethylammoniumpropyl)‐4‐(p‐diethylamino‐phenyl‐hexatrienyl) pyridinium dibromide (FM4‐64) . This dye is used for staining the Spitzenkorper, plasma membrane, septum, early endosomes, late endosomes and vacuolar membranes, as well as for the examination of endocytosis, as described previously (Fischer‐Parton http://eupathdb.org/eupathdb/): FolVAM7 (FOXG_02319).The gene sequences can be found using the following accession number at website  of at least three replicate measurements. Significant differences between treatments were statistically evaluated by SD and one‐way analysis of variance (ANOVA) using SPSS 2.0 . The data for two specific different treatments were compared statistically by ANOVA, followed by Student's T‐test if the ANOVA result was significant at Fig. S1. Phylogenetic and structural analysis of FolVam7 and its homologues. A. Phylogenetic analysis of Vam7 homologues from different organisms. A neighbour‐joining tree was constructed using MEGA 5 with 1000 bootstraps. GenBank accession numbers and the corresponding species names are as follows: XP_018235815.1 ; XP_957713.1 ; XP_002379361 ; XP_957713.1 ; XP_761553.1 ; NP_011303.1 . B. Identification of conserved protein domains in FolVam7 and its homologues. PX, PhoX homology domain; SNARE, Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor; aa, amino acid.Click here for additional data file.Fig. S2. Targeted gene replacement of FolVAM7 in Fol. A. Schematic diagram of the split‐marker gene deletion strategy for FolVAM7. B. Results from Southern blot analysis of genomic DNA using gene‐specific or HPH probes. DNA from the mutant hybridize only to the HPH probe, while that from wild type hybridizes only to the gene‐specific probe.Click here for additional data file.Fig. S3. Schematic diagram showing the GFP‐FolVAM7 construct.Click here for additional data file.Fig. S4 qPCR to determine relative levels of Fol in stem sections of inoculated plants. Genomic DNA was isolated from tomato stems of the Moneymaker cultivar infected with various Fol strains as described in the Materials and Methods. Quantitative PCRs were performed to evaluate the fungal biomass using primers that amplify the intergenic spacer region of ribosomal 28S. The mean values of three determinations with standard deviations are shown. Asterisks indicate statistically significant differences relative to wild type Fol (p < 0.01).Click here for additional data file.Table S1 Primers used in this study.Click here for additional data file.


We found that LPS + LF− significantly increased livebirths, pup survival, and litter size compared to LPS alone. Moreover, offspring from the LPS + LF− group exhibited significantly improved neuromotor performance and more neurons compared to those from the LPS group. Additionally, LF− treatment on human umbilical vein endothelial cells (HUVECs) increased cell viability and decreased oxidative stress after treatment with LPS. Collectively, our data demonstrates that maternal LF− supplementation during pregnancy postpones the onset of PTB, acts to increase the liveborn rate and survival time of newborns, and reduces perinatal brain injury in cases of intrauterine inflammation.Maternal periodontal disease has been linked to adverse pregnancy sequelae, including preterm birth (PTB); yet, root planing and scaling in pregnancy has not been associated with improved perinatal outcomes. Fluoride, a cariostatic agent, has been added to drinking water and dental products to prevent caries and improve dental health. The objective of this study was to explore the effects of fluoride supplementation using a mouse model of preterm birth and perinatal sequalae. Pregnant mice were fed low dose fluoride (LF Although this connection is known, interventions designed to improve periodontal disease, such as scaling and root planing, have not been useful in improving adverse pregnancy outcomes5. Additionally, studies including oral hygiene education, such as encouraging the use of mouthwash, tooth brushing, and flossing after meals, did not find a difference in the lengths of pregnancy gestation between women with and without periodontal disease7. Finally, treatment of periodontal disease that included antibiotics (metronidazole) did not have an effect on the PTB rate8. The mechanism behind the connection between PTB and periodontal disease remains unclear.Periodontal disease is a common problem worldwide and has been associated with an increased risk for many comorbidities, including hypertension9. PTB remains the leading cause of neonatal morbidity, constituting up to 70% of perinatal mortality worldwide11. Intrauterine inflammation (IUI) is strongly associated with PTB. Up to 40% of premature births exhibit clinical evidence of inflammation13, and exposure to IUI during prenatal development places offspring at risk for adverse neurodevelopmental outcomes, such as cerebral palsy, autism spectrum disorder, learning disabilities, and schizophrenia16.PTB, defined as delivery before 37 weeks gestation, represents a major public health challenge. Each year, approximately 15 million children are born prematurely around the globe17. Periodontal disease is a gram-negative anaerobic infection of the mouth that affects up to 90% of the population18 and has been shown to be even more prevalent in the pregnant population19. Periodontal disease and PTB share many of the same common risk factors, including age, smoking habits, low socioeconomic status, and systemic health status. Also, previous research has suggested that maternal infections altering normal cytokine- and hormone-regulated gestation may result in preterm labor20. It has also been shown that the placental microbiome is similar to that of the oral cavity21. Periodontitis could act as a distant reservoir of both microbes and inflammatory mediators, which may influence pregnancy and contribute to the induction of PTB25. While intervention studies  have sought to determine the effect of periodontal treatment on reducing the PTB risk, the results remain controversial17.The results of epidemiological, molecular, microbiological and animal-model studies also support a positive association between maternal periodontal disease (periodontitis) and PTB−) plays an important role in upholding good oral hygiene. It is classified as a cariostatic agent, which is a substance that blocks the formation of dental caries. Fluoridation of drinking water and dental products help to prevent dental cavities, indicating that the use of an appropriate amount of fluoride may improve oral health28. The National Academy of Medicine (NAM), previously known as the Institute of Medicine, recommends a daily intake of 3 mg of fluoride for women aged 14 to 50. The NAM also notes that most research indicates that a daily intake of 10 mg for 10 or more years is necessary to produce clinical signs of mild skeletal fluorosis; thus, the upper intake limit is regarded as 10 mg/day29. The Food and Drug Administration has also stated that 10 mg/day is the upper intake limit29. While fluoride has been linked to certain negative health outcomes, this has occurred only at extremely high doses30. The role of low dose fluoride (LF−) on pregnancy outcomes, although recommended by the NAM, has not been very well explored.Fluoride  due to its beneficial oral health properties. Specifically, we aimed to explore the effects of fluoride supplementation at levels recommended by the NAM on obstetrical outcomes using a mouse model of IUI. Eventually, we propose that fluoride could be used as a prenatal supplement for pregnant women suffering from periodontal disease to decrease the risks of PTB.− and 113 mg/L high dose fluoride (HF−) from embryonic day (E) 9 to postnatal day (PND) 19. On E17, an established mouse model of IUI was utilized as previously described35. Mice were monitored for PTB for 36 h after surgery. There were no preterm births among pregnant dams that received intrauterine phosphate-buffered saline (PBS) (n = 20) or intrauterine PBS with either LF− (n = 17) or HF− (n = 7) supplementation (Table − supplementation (n = 51) significantly reduced the PTB rate to 23.5% (Table − significantly increased the PTB rate to 91.7% (n = 12) within 24 h of LPS-induced IUI (Table − group delivered prior to 24 h in 100% of the cases with no surviving pups. Most preterm deliveries in the LPS + LF− group (43.1%) occurred at late preterm gestation between 24 and 36 h after injection compared to LPS group (23.1%) (Table − (66.7%), and LPS + HF− (91.7%) groups 36 h after intrauterine LPS injection (Table Timed-pregnant CD1 mice consumed water with 6 mg/L LF− supplementation (55.9%) significantly increased the liveborn ratio by litter compared to LPS alone (23.7%) in cases of PTB (Table − group (17.6%) were able to survive until the end of the observation period (PND19) than with no fluoride supplementation (2.6%) (Table − (n = 19) significantly increased litter size compared to LPS alone (n = 9) . With maternal LF− supplementation, more pups (0.9 ± 0.4) born preterm were able to survive without other medical treatment until PND19 than LPS alone (0.1 ± 0.1). Data are reported as mean ± SEM.Maternal LFPS alone 3.7% in cPS alone 3.7% in c− supplementation resulted in greater pup survival and litter size compared to LPS alone from PND4 to PND19 . Term liveborn pups exposed to maternal LPS continued to die after birth, resulting in decreased litter size from 6.5 ± 1.2 at PND1 to 5.6 ± 1.1 at PND19   and maternal LPS-exposed groups with and without F− supplementation   and the LF− group was 0.072 ± 0.005 , 5.327 ± 0.662  and 0.037 ± 0.004  mg/L, and in control group was 0.056 ± 0.003 , 2.792 ± 0.547  and 0.020 ± 0.001  mg/L. Maternal LF− supplementation significantly increased the fluoride content in maternal serum, urine and amniotic fluid compared to control group   Fig. .Figure 4− (0.001 mM) promoted HUVECs proliferation, while HF−  inhibited HUVECs proliferation after treatment with LPS for 18 h  were significantly increased in HUVECs in the LPS group (LPS 1 µg/mL and F− 0 mM) compared to those in the control group (LPS 0 µg/mL and F− 0 mM)  . In contrast, HUVECs in the HF− group, which were treated with 0.1 mM F− and LPS, had significantly more carbonyl proteins than those in the LPS group .The effects of different fluoride concentrations on HUVECs viability were examined. LF8 h Fig. . Proteinst) Fig. . Carbony37. Furthermore, low dose fluoride acts as an anti-inflammatory agent, to increase the liveborn rate and survival time of newborns following exposure to an intrauterine inflammatory environment. Moreover, maternal low dose fluoride supplementation prevents fetal cortical neuronal injury and results in neuromotor improvement following in utero inflammation exposure. The timing of administration and the dose of fluoride are very important, as the fluoride content increases in a dose- and time-dependent manner in all tissues and organs. High fluoride dosing has adverse effects, including increasing the PTB rate following LPS administration.For the first time, our study demonstrates that maternal low dose fluoride supplementation during IUI-affected pregnancy postpones the onset of PTB. It is clinically significant that the early PTB (Preterm delivery prior to 24 h) rate was lower in the LPS + LF group than in the LPS group before 24 hours. The gestation period in a mouse is about 20 days. Therefore, the 12-hour gestation period in a mouse represents more than one week in the gestation period in humans, when compared to a total gestation period of about 40 weeks. A one-week difference in the age of a premature baby is extremely important. As the age of preterm infants increases from week 22 to week 28 during the 28-week period, severe fetal morbidity and mortality decrease significantly38 – playing a key role39. Basha, Rai and Begum found that the ingestion of high dose fluoride may lead to oxidative stress by increasing lipid peroxidation (which can greatly damage a cell) and modifying antioxidant enzymes40. We used a well-established mouse model of IUI and PTB41. In order to evaluate the translational potential of our study, we examined the direct effect of various doses of fluoride on HUVECs.IUI is associated with PTB, with oxidative stress – an imbalance between the production of reactive oxygen species and defensive antioxidant mechanisms44. HUVECs are the most widespread in vitro model for the study of function and pathology of vasculature. The placenta represents the interface between maternal and fetal circulation, and features an extensive vascular network to facilitate nutrient provision from maternal blood, including fluoride. In our study, supplementation of low dose fluoride on HUVECs increased cell viability, and decreased oxidative stress.Many studies have used HUVECs to investigate the mechanism of preterm labor because dysfunctional endothelial cell activation and cytokines are implicated in preterm labor45. Biofilms help evade the host immune response and are known to resist antimicrobial substances. Furthermore, some bacteria have been shown to resist antibacterial substances more in biofilms than under typical conditions, perhaps through affecting oxidative stress46. Biofilms are also important in PTB, as they can be present in amniotic fluid sludge, which is the presence of minute particles aggregated in amniotic fluid near the cervix. In human studies, the presence of amniotic fluid sludge has been identified as an independent predictor of imminent preterm delivery48.More than 80% of human microbial infections – including gingivitis – have been associated with biofilms, which are clusters of microorganisms that exist in a liquid extracellular matrix49 is known to be created during dental procedures, and can even be caused by routine toothbrushing50. We propose this as a possible mechanism for oral bacteria to access the placental membranes. Oral bacteria, including Fusobacterium nucleatum and Porphyromonas gingivalis, is known to be found in the placenta, and overall, the microbiome profile of the placenta is most similar to that of the oral cavity51. The use of intrauterine injection of LPS is used as an established model of PTB because it is an endotoxin present in the cell walls of gram-negative bacteria53. We propose that endotoxins of bacteremia from the oral cavity that are released at the placental membranes contribute to adverse pregnancy outcomes, including PTB. We used LPS from Escherichia coli, which is another gram-negative species, to serve as the oral bacterial endotoxin in our study. Thus, LPS is an effective model to imitate the effects of periodontal disease on pregnancy.Transient bacteremia (bacteria in the bloodstream)et al. conducted a randomized trial, and found that more than 90% of the resultant children from both the treatment and control groups possessed no cavities. The researchers concluded that prenatal fluoride supplementation does not significantly reduce the risk of cavities in children54. Our study is the first to examine the effect of prenatal fluoride supplementation on PTB.Prenatal supplementation of fluoride has been previously examined, yet solely in the context of caries prevention. Leverett 56. Ge et al. showed that the offspring had a cognitive dysfunction in the group maternally exposed to high dose fluoride, and it was thought to most likely be caused by the disruption of the expression of synapse-associated proteins57. Similarly, our study demonstrates detrimental effects of high dose fluoride in PTB. Moreover, our research shows that low dose fluoride can potentially improve neurodevelopmental outcomes, through having an anti-inflammatory role, and a protective effect against cortical neuronal injury.Prior research in rodents has shown that high doses of fluoride have detrimental effects when ingested during pregnancy, including accumulation in the fetal brain and a negative effect on cognitive function, such as learning, attention, and recollectionOur study is limited in its use of a mouse model, as more research must be performed in order to assess the effect of fluoride in the human pregnant population. Future clinical research studies have the potential to translate our findings in mice, possibly demonstrating fluoride’s ability to alleviate adverse pregnancy outcomes, such as PTB, that are associated with periodontal disease.All animal care and treatment procedures were approved by the Animal Care and Use Committee of Johns Hopkins University (Hopkins-IACUC Protocol No. MO14M326) and were carried out in accordance with institutional standards. Timed-pregnant CD-1® mice  were used in the study.35. Briefly, dams received 25 μg LPS  in 100 μL PBS or 100 μL PBS vehicle via intrauterine injection between the first and second gestational sacs of the right uterine horn. Dams were randomly assigned to 6 groups: PBS (n = 20), PBS + LF− (n = 17), PBS + HF− (n = 7), LPS (n = 52), LPS + LF− (n = 51) and LPS + HF− (n = 12). Mouse gestation lasts 19–20 days, and we consider all deliveries prior to E19 as preterm. PTB rates were evaluated at 24 h and 36 h after LPS injection. Pup survival data and delivery outcomes were assessed. Birth time was measured in hours relative to the time of LPS injection. Live pups’ birth weights were measured.On E17, an established mouse model of IUI was utilized as previously described59 and brush their teeth twice (~0.25 mg fluoride/pea size amount of toothpaste)60 each day, they may consume up to 2.6 additional mg of fluoride daily. Thus, the maximum daily fluoride intake is approximately 5.6 mg, which is within the NAM and FDA limits. Considering a pregnant woman with an estimated weight of 80 kg, 5.6 mg/80 kg = 0.07 mg/kg/day. According to the FDA’s Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, the conversion equation for human and animal doses is as follows: Human mg/kg = Animal mg/kg dose × ; Human Km is 37, and Mouse Km is 3. The equivalent mouse fluoride dose equals 0.863 mg/kg/day. Assuming 0.04 kg/mouse, the total fluoride intake is 0.035 mg/mouse/day. According to guidance from the Johns Hopkins University Animal Care and Use Committee , average daily water intake for CD1 mice is 6 mL (1.5 mL/10 g body weight/day). Thus, mice received a low dose fluoride concentration of about 6 mg/L. Sodium fluoride (NaF) was purchased from Sigma-Aldrich . Timed-pregnant CD1 mice consumed water with 6 mg/L LF− and 113 mg/L HF− from E9 to postnatal day (PND) 19. Maternal water and food intake were recorded daily until delivery.Based on NAM recommendations, pregnant women may receive 3 mg of supplemental fluoride each day to ensure adequate intake. Considering that women each day should drink a little more than 2 L of water 61. Samples were directly analyzed for fluoride using a combination fluoride ion selective electrode  and SevenExcellence pH/Ion meter S500 . Samples were mixed 1:1 with Total Ionic Strength Adjustment Buffer II  and placed under the electrode. The fluoride content of each sample (mg/L) was determined from a standard curve prepared by analyses of a series of fluoride standard solutions conducted at the same time.After intake of low dose fluoridated water from E9 to E18, dams of each group were sacrificed on E18, and samples of serum and amniotic fluid were collected. Maternal urine was collected on E18 according to the previously described protocol− treatment on neuromotor and cognitive development in offspring. Cliff aversion and surface righting tests were performed according to the previously described protocol62. The amount of time needed to complete each test was recorded and analyzed. A total of 31 litters  were analyzed.Tests were performed at PND 5 and 9 to assess the impact of LPS exposure and LFhttp://imagej.nih.gov/ij/, National Institute of Health, Bethesda, MD, USA) on five randomly chosen fields in the frontal cortex.On E18, dams were sacrificed 24 h after surgery. Fetal brains were isolated and fixed in 4% paraformaldehyde overnight at 4 °C. Fixed tissues were immersed in 30% sucrose until saturated; they were then cryosectioned (20 μm thickness), followed by histological staining. Routine Nissl staining was performed to evaluate the cortical neuronal morphology in fetal brains. All photographs used for quantification were taken with Zeiss AxioPlan 2 Microscope System  attached to a Canon EOS Rebel Camera . Neurons were counted (field of view) based on Nissl staining using Image J  to make cells trypsinized, and Trypsin Neutralizing Solution  was used to inhibit further tryptic activity. We used the HUVECs from passage 3 to passage 6. In this experiment, the HUVECs were treated with varying concentrations of NaF . After incubation with NaF for 6 h, the LPS solution (1 µg/mL) was added to the HUVECs culture wells for 18 h.Human umbilical vein endothelial cells  purchased in proliferating flasks were grown in Endothelial Cell Growth Medium  in a 37 °C 5% COHUVECs viability was determined with Cell Counting Kit-8 Assay Kit (CKK-8) . The absorbance was measured with a microplate reader  at a wavelength of 450 nm. Protein peroxidation was quantified as a marker of oxidative stress using the OxiSelect Protein Carbonyl ELISA Kit .Statistical analyses were performed using Prism 6 . Categorical data were analyzed using either the Chi-squared test or Fisher’s exact test. Continuous data were tested for normality , and outliers were identified using Grubb’s test. Student’s t-test was used for parametric data. One-way analysis of variance (ANOVA) with Bonferonni post hoc testing was utilized for multiple comparisons of normally distributed data. Pups’ survival data and maternal oral intake data were analyzed by two-way ANOVA (group × time).


The predictive validity of postgraduate examinations, such as MRCGP and MRCP(UK) in the UK, is hard to assess, particularly for clinically relevant outcomes. The sanctions imposed on doctors by the UK’s General Medical Council (GMC), including erasure from the Medical Register, are indicators of serious problems with fitness to practise (FtP) that threaten patient safety or wellbeing. This data linkage study combined data on GMC sanctions with data on postgraduate examination performance.n = 27,561) or Clinical Skills Assessment  at first attempt between 2010 and 2016 or taking MRCP(UK) Part 1 , Part 2  or Practical Assessment of Clinical Examination Skills  at first attempt between 2001 and 2016. Exam data were linked with GMC actions on a doctor’s registration from September 2008 to January 2017, sanctions including Erasure, Suspension, Conditions on Practice, Undertakings or Warnings (ESCUW). Examination results were only considered at first attempts. Multiple logistic regression assessed the odds ratio for ESCUW in relation to examination results. Multiple imputation was used for structurally missing values.Examination results were obtained for UK registered doctors taking the MRCGP Applied Knowledge Test , with a mean Cohen’s MRCGP and MRCP(UK) performance are valid predictors of professionally important outcomes that transcend simple knowledge or skills and the GMC puts under the headings of conduct and trust. Postgraduate examinations may predict FtP sanctions because the psychological processes involved in successfully studying, understanding and practising medicine at a high level share similar mechanisms to those underlying conduct and trust. Perhaps the most serious event in a UK doctor’s professional life is to be investigated by the General Medical Council (GMC) because of concerns about their fitness to practise (FtP). FtP concerns are investigated in a quasi-legal fashion; the proceedings can be very stressful  and can Revalidation requires all doctors to demonstrate on a regular basis that they are up to date and fit to practise in their chosen field and able to provide a good level of care  for a 1 SD change in an examination score. ORs for a particular exam were expressed as the increased odds of ESCUW for a doctor on the 2.5th percentile of exam marks in relation to a doctor on the 97.5th percentile, other examinations being at mean performance.Logistic regressions were used to model the binary outcome of ESCUW in relation to predictor variables expressed as standardised (e(OR) of ESCUW in relation to MRCGP AKT and CSA, and to MRCP(UK) Parts 1, 2 and PACES, to assess the relative prediction from different examination types. Missing values were present for structural reasons, doctors who failed one part of MRCP(UK) typically not taking later parts, but results in both exams are also missing because of truncation within the time window used, some exams being taken outside of the time window, i.e. before data collection began or in the future. Missing exam results for the multiple regression were imputed 100 times using the Multiple Imputation package in IBM SPSS under the assumption that data are missing at random (MAR).Multiple logistic regression assessed the logprobability, p, which is the proportion of cases having a sanction, and hence, the probability of not having a sanction is (1 − p). The odds of having a sanction is p/(1 − p). Logistic regression models logit(p) where logit(p) = loge(odds) = loge(p/(1 − p)), loge indicates natural logarithms to base e . Logistic regression is used because probabilities are bounded by the values 0 and 1, and simple regression models with probabilities as a dependent variable can predict nonsensical probabilities of greater than 1 or less than 0. In contrast logit(p) has a range from minus infinity to plus infinity. For similar reasons, it mostly makes little theoretical sense to plot p against a predictor as the relationship is necessarily curvilinear, p being constrained to the range 0 to 1. Likewise, it rarely makes sense to plot odds against a predictor variable, particularly when p values are low  since as p approaches 0 and odds are calculated as p/(1 − p), so 1 − p approaches 1 and therefore odds ≈ p/1 = p, also making curvilinearity inevitable and difficult to interpret. Interpreting loge(odds) is not always intuitive but is simpler when p values are small. Logistic regression equations say that logit(p) = a + b.x, where a is a constant and b a multiplier of a predictor variable, x. For small p, the equation becomes log(p) = a + b.x, and b indicates the multiplicative change in p for a one-step change in b. As a simple example, consider probabilities of 0.1, 0.01, 0.001 and 0.0001, predictors of values 1, 2, 3 and 4, and let logs be to base 10, so that log10(p) is − 1, − 2, − 3 and − 4. A one-step increase in x, e.g. from 1 to 2, results in a decrease in log(p) of − 1, and hence, p is reduced by a factor of 10. Because a + b.x is a linear model, then were an intervention to reduce x by 1 then all probabilities would be reduced by a factor of 10, whether they started at 0.1 or 0.0001, indicating a common mechanism or process.Raw data can be expressed in terms of the e(OR) of a doctor having ESCUW was analysed using a multiple logistic regression on the entire LRMP in terms of a doctor’s sex, place of qualification (UK vs others), years since qualification and years since qualification not spent on the GMC Register . Doctors who had been on the LRMP for longer were more likely to have ESCUW, rising from 0.5% of doctors in the first decade after graduation to 1.3% in the second decade, 2.0% in the third decade, 2.8% in the fourth decade and 3.1% in the fifth decade, presumably due to increasing opportunity for problems to arise. The majority of doctors taking MRCGP or MRCP(UK) during the time period of this study were in the first decade or two after qualifying .Considering all the 380,583 doctors in the LRMP records at 1 Jan 2017, there were 6158 doctors who had ESCUW during the study period (1.62%), 680 (0.23%) erased, 2250 (0.59%) suspended, 2871 (0.75%) with conditions, 1263 (0.33%) with undertakings and 1735 (0.46%) with warnings. Three thousand eight hundred seventy-eight (63.0%) of the 6158 doctors with ESCUW had only one FtP sanction, the remainder having two , three , four  or five  sanctions. The loge(OR) = 1.004, SE = 0.034, OR = 2.730×), to have been qualified longer (loge(OR) = 0.065 per decade, SE = .008, OR = 1.067× per decade), not to have qualified in the UK (loge(OR) = 0.304, SE = 0.033, OR = 1.355×) and to have spent more time not on the GMC Register (loge(OR) = 0.206 per decade, SE = 0.023, OR = 1.228× per decade). Note that the vast majority of doctors who had spent longer not on the GMC Register were those who had qualified outside of the UK. It should also be emphasised that ethnicity is not available in the LRMP.Multiple logistic regression showed that those with ESCUW were 2.73× more likely to be male  having ESCUW scoring significantly lower on the exam than those without ESCUW, the Cohen’s Multiple logistic regression of ESCUW on both AKT and CSA used 100 multiple imputations for the 27,651 candidates. EM (expectation maximisation) estimation of means and SDs showed no difference in overall performance of imputed and non-imputed cases, missing CSA results being due to candidates not yet having taken the exam. The multiple logistic regression showed that AKT and CSA both had independent predictive effects after taking the other into account, with the effect of CSA being greater than that for AKT and the 95% confidence intervals for the ORs not overlapping , with AUC estimates in the paired data of 0.666 and 0.713 for AKT and CSA, respectively, showing that the CSA better predicts ESCUW than AKT.Areas under the ROC curve were 68.6% SE 1.3%) and 71.2% (SE 1.7%) for the AKT and CSA assessments, respectively , Part 2  or PACES  at a first attempt and were on the LRMP and so had data on ESCUW. Twenty thousand two hundred ninety-nine doctors had taken all three MRCP(UK) parts, 7771 had taken two parts and 20,299 had taken only one part.After merging, the database had 44,314 doctors who had taken Part 1 (d being − 0.617 (Table e(OR) = − 0.597, SE = 0.048, OR = 0.550× per SD). Multiple logistic regression taking into account sex, ethnicity, UK qualification, decades since qualification and decades not on the UK Register showed that Part 1 scores were still significant predictors of ESCUW (loge(OR) = − 0.376, SE = 0.052, OR = 0.687× per SD).Of 37,358 doctors who were on their first attempt at Part 1, 423 (1.13%) had ESCUW and had significantly lower scores on the exam, Cohen’s e(OR) = − 0.589 per SD, SE = 0.066, OR = 0.555× per SD). After taking sex, UK qualification, decades since qualification and decades not on the UK Register into account, Part 2 scores were still significant predictors of ESCUW (loge(OR) = − 0.379 per SD, SE = 0.074, OR = 0.685× per SD).For first attempt at Part 2, there were 28,285 doctors of whom 274 had ESCUW (0.97%), and they had significantly lower marks with an effect size of − 0.536. On its own, Part 2 score predicted ESCUW (logd = − 0.696 (Table e(OR) = − 0.588 per SD, SE = 0.051, OR = 0.555× per SD), and the effect remained highly significant after taking into account sex, UK qualification, decades since qualification and decades not on the UK Register (loge(OR) = − 0.356 per SD, SE = 0.063, OR = 0.700× per SD).Of 27,040 doctors taking PACES for the first time, 289 1.07%) had ESCUW and had scored significantly lower on the exam with Cohen’s .07% had z-score-transformed marks for Part 1 was − 0.1045 (.993), for Part 2 was − 0.168 (1.040) and for PACES was − 0.085 (1.016), compared with means of 0 (SD 1) in the raw z-score-transformed data. Missing data therefore were biased, not least as candidates who fail a part at a first attempt are less likely to take further parts, but if they had, they would have performed less well than those passing parts at a first attempt  a common threshold for safe practice’ , the impThe linearity of the relationships in Fig. all candidates in the database, with imputation of missing values where needed. A pre-requisite of taking PACES (and Part 2) is having passed Part 1, and most candidates taking CSA will have passed AKT. There is therefore restriction of range in candidates taking later examinations, only better candidates taking them. However, what is required is a comparative assessment of construct validity on the basis of all candidates having taken all of the assessments, and for that, missing data need to be imputed. PACES predicts at a similar level as, say, Part 1, in the simple analyses, but the variance of the candidates is less for PACES, and if the likely ability level of all candidates is taken into account, then PACES will predict better across the entire range of ability. An alternative way of viewing it is that if just one test were to be applied to all candidates with the intention of predicting FtP issues, then PACES would predict better than Part 1. A separate approach to comparing knowledge-based and clinical assessments is our comparison of ROC curves, where the paired analysis is restricted to candidates taking both types of assessment and where the area under the curve is significantly greater for clinical as opposed to knowledge-based assessments, for both MRCGP and MRCP(UK).An important finding from the multiple regressions is that clinical assessments (CSA or PACES) predict FtP significantly better than do the knowledge assessments of AKT and Part 1. That result may seem surprising given the seemingly similar slopes of the graphs in Fig. Good medical practice’ [‘Doctors have a respected position in society and their work gives them privileged access to patients … A doctor whose conduct has shown that [they] cannot justify the trust placed in [them] should not continue in unrestricted practice while that remains the case. In short, the public is entitled to expect that their doctor is fit to practise, and follows our principles of good practice described in ractice’  , for which knowledge, understanding and competency in practical skills are the essential components, most notably in ‘knowledge assessments’ such as MRCGP AKT and MRCP(UK) Parts 1 and 2 and ‘clinical assessments’ such as MRCGP CSA and MRCP(UK) PACES. Examination has neither conduct nor trust at its core, although most examiners would probably feel that those traits are implicit, but not explicit, in the assessments. It is therefore remarkable in many ways that poor performance in MRCGP and MRCP(UK) relates so strongly to FtP issues in our very large sample of doctors. A weakness of our study is that the LRMP does not specify the precise reasons for sanction . The gr


Studies on nasopharyngeal carcinoma (NPC) in five electronic databases were systematically searched online from the inception to June 5, 2018. Quality of the included studies was assessed using the updated Quality Assessment of Diagnostic Accuracy Studies 2. Data of sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and the 95% confidence intervals were pooled using a bivariate random‐effect model. Forty‐four studies with 61 groups of data and totally 3369 patients were included in the qualitative and quantitative synthesis analysis. The overall estimated sensitivity and specificity of positron emission tomography/computed tomography/magnetic resonance imaging (PET‐CT/MRI) for local recurrent/residual NPC were 0.90 and 0.85, respectively. The pooled area under the curve of (AUC) of PET‐CT/MRI in the summary receiver operator characteristic curve was 0.94. Subgroup analysis showed MRI vs PET‐CT had lower sensitivity (0.83 vs 0.92) and specificity (0.78 vs 0.89). The AUCs of MRI and PET‐CT were 0.87 and 0.96, respectively. No‐cross of 95% CI was found in MRI vs PET/CT (0.87‐0.90 vs 0.94‐0.98). Meta‐regression showed PET/CT vs MRI was a potential source of heterogeneity. PET/CT and MRI both showed quite high overall ability in diagnosing local recurrent/residual NPC, but the subgroup analysis indicated PET‐CT was superior over MRI in diagnosis of local recurrence and residue of NPC after radiotherapy. The examination methods affected the heterogeneity within studies. South China and Southeast Asia are among the pandemic areas of NPC.However, some side effects would appear after radiotherapy, such as edema, inflammation, fibrosis, and scar.2This study follows the Cochrane Handbook for Systematic Reviews and the Preferred Reporting Items for Systematic Reviews and Meta‐analysis  AND (“positron emission tomography” OR “PET” OR “PET/CT” OR “PET‐CT” OR “18‐fluoro‐2‐deoxyglucose positron emission tomography” OR “18F‐FDG PET/CT” OR “MRI” OR “magnetic resonance imaging” OR “nuclear magnetic resonance scanner” OR “magnetic resonance angiography”) in combination with some keywords: recurrent or recurrence, residue, diagnosis or diagnostic (sensitivity and specificity), receiver operating curve or ROC. The references of some reviews and articles were also reviewed in order to obtain the potentially eligible trials. Languages were restricted to Chinese and English.2.2Two authors independently scanned and screened the titles, abstracts, and full texts of the initially retrieved studies. Disagreements were resolved by explicit consensus. The inclusion criteria were as follows: (a) Recurrence and residues of NPC were confirmed by golden standard (biopsy or follow‐up); (b) the aim was to assess the diagnostic ability of PET/CT or MRI or both for recurrence or residues or both of NPC; (c) enough data were provided for further pooling analysis, including true positive (TP), false positive (FP), false negative (FN), and true negative (TN). For duplicates, the latest publication was used. Studies with duplicated or unqualified data, or focused on animal or experimental design were excluded. Reviews, comments, letter, and case reports were also excluded.2.3Two authors independently extracted data and resolved discrepancies by mutual discussion. From each included study, the following information was extracted: surname of first author, year of publication, country, examination method (PET/CT vs MRI), study design (prospective vs retrospective), age , time of examination, golden standard (biopsy vs follow‐up), sample size, four folds data , sensitivity, and specificity. The extracted data were put into a standardized Excel sheet.2.4Quality of the included studies was assessed using the updated Quality Assessment of Diagnostic Accuracy Studies 2, which consists of two parts: risk of bias and applicability concerns. The risk of bias includes four items: patient selection, index test, reference standard flow, and timing. Each item has three options: high, unclear, and low. A study with ≥1 item scored “high” is considered as high risk of bias, whereas a study with all items scored “low” is treated as low or unclear risk of bias. The applicability concerns consist of three options: high, low, and unclear.2.5I2 statistic, with the significance level at P < 0.05 or I2 > 50%.P < 0.05.Statistical analyses were performed on Stata 13 , and the quality was assessed on Review Manager 5 . Firstly, the threshold effect was evaluated by Spearman correlation coefficient, significant value of which means the existence of the threshold effect.33.1Figure 3.2The general characteristics of the included studies were presented in Table 3.3Data 3.4I2 > 50%). The other overall estimated results were as follows: sensitivity = 0.90 [95% CI: 0.86‐0.93, Figure The estimated results about the diagnostic ability of PET‐CT/MRI for local recurrent and residual NPC were shown in Table Table 3.5P < 0.001; Table Considering the high heterogeneity within studies, we conducted the meta‐regression to explore the potential influencing factors. The meta‐regression results indicated the examination method (PET/CT vs MRI) was a potential source of heterogeneity , indicating the publication bias of the current study was limited, but Egger's test showed some publication bias .The publication bias was assessed by Deek's line regression plot. The X‐ and Y‐directions were effective sample size and diagnostic odds ratio, respectively. The angle between the regression line and the X‐direction was close to zero, which means no publication bias  in diagnosing local residual/recurrent NPC,Whether there is local residue or recurrence is extremely important for NPC staging and treatment plan. As reported, NPC patients with local residue had poorer prognosis and higher risk of recurrence.The differences of overall accuracy between PET/CT and MRI may be attributed to the imaging principle. It is generally agreed that MRI outperforms CT in detecting residual and recurrent NPC.PET/CT has higher overall diagnostic accuracy for recurrent NPC and generally consists of a PET scanner, a high‐resolution spiral CT scanner, and an operating system that will combine two types of scan images. PET and CT can be obtained simultaneously with one scan. PET/CT images combine the metabolic imaging characteristics of PET scanners with the anatomical imaging characteristics of CT scanners, which make up for the unclear positioning of PET and solve the low accuracy of CT. Given the biological characteristics of specific tumor tissues and the imaging characteristics of PET/CT, PET/CT has significant advantages in differentiating post‐radiotherapy NPC from fibrosis and tumor local recurrence or lymph node metastasis. Currently, the most commonly used nuclide tracer is 18F‐FDG, imaging of which distinguishes benign and malignancy mainly according to the difference in glucose metabolism between normal tissues and tumor tissues in the human body. The principle is that SPECT 18F‐FDG after entering human malignant tumor cells is decomposed by hexokinase into an undecomposed 6‐phosphoric acid deoxidizing glucose, which largely accumulates in the tumor cells and significantly increases the metabolism activity of tumor tissues and uptake of 18F‐FDG. However, as a tumor‐nonspecific imaging agent, the uptake of 18F‐FDG in the irradiation area can also be increased by inflammatory changes.The present study has several limitations. First, the heterogeneity within studies is quite high, which was addressed here by two ways. The subgroup analysis only by the golden standard found the source of heterogeneity, but not population, study design, examination methods, or sample size. Only significant difference of overall accuracy was found. Then, multivariate meta‐regression including the above factors indicated the examination method may be associated with heterogeneity. Second, some factors and unmeasured or unreported study characteristics such age gender and stage cannot be obtained for further subgroup, which may overestimate or underestimate the overall pooled results. The reason is that the sample size was too small to further subgroup analysis in each study. Third, the golden standard was mixed , but biopsy would be better. However, the subgroup analysis did not indicate significant difference among three types. Moreover, MRI or PET ∓CT had enormously evolved during the long search period from 1991 to 2018. However, the meta‐regression indicated publication year seemingly had no effect on the estimated covariate effect.In conclusion, PET/CT and MRI both show quite high overall diagnostic ability for local recurrence/residue of NPC. But the subgroup analyses indicate PET‐CT is superior over MRI in diagnosis of local recurrent and residual NPC after radiotherapy. The examination methods affect the heterogeneity within studies. The present study provides stronger evidence for clinical practice.The authors declare that they have no conflict of interest. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Peak bone mass accrual occurs during adolescence, a time when dieting and related eating behaviors are common. Impaired bone mineral accrual is a known consequence of eating disorders in adolescents, but the effects of subclinical dieting behaviors on bone mineral content (BMC) have not been described in this age group. The goal of this analysis was to determine whether dieting behavior in preadolescence and adolescence is associated with bone mineral accrual in adolescent girls.n = 139) were followed in a longitudinal cohort study. BMC was assessed at ages 9 and 15y. Dieting to lose weight was reported every 2 years, and dietary restraint and disinhibition, eating attitudes, weight concerns, and body esteem were assessed at age 11y. Girls were classified as “early dieters” if they first dieted by age 11y (31.7%), “adolescent dieters” if they first dieted after 11y (46.8%), or non-dieters if they did not report dieting by 15 y (21.6%). The effect of dieting related variables on BMC at 15y and change in BMC from 9 to 15y was assessed using linear regression, controlling for height, weight, BMI, physical activity, and pubertal status.Non-Hispanic white girls (p = 0.02) and 3.1% lower BMC at age 15y (p = 0.005) than girls who first reported dieting after 11y or not at all. Number of weight control behaviors used, dietary restraint, and weight concerns were also negatively associated with BMC (p < 0.05).Girls who first reported dieting to lose weight by age 11y had a 4.2% lower bone mineral accrual across adolescence  contains supplementary material, which is available to authorized users. Adolescence is a critical period for bone development, with nearly 40% of adult skeletal calcium accumulated in the 4 years surrounding peak height velocity . In healWeight concerns, dietary restraint, and self-initiated dieting/weight control behaviors also emerge during preadolescent and adolescent years . Young gn = 167 girls remained in the study, and attrition was primarily due to relocation outside the area. For this analysis, girls who had bone mineral content data at ages 9 and 15 as well as complete anthropometric, pubertal status, and physical activity data were included, resulting in a sample of 139 girls. This sample size yields 80% power to detect a medium size effect of dieting status based on Cohen’s f (0.24). [n = 58), those that were included had slightly older mothers (mean (SD) 35.8 (4.7) vs. 34.2 (4.8) years at enrollment, p = 0.03), but there were no differences in parent income, education, and BMI, paternal age, or girl’s BMI percentile at enrollment. Additional details on this cohort have been published elsewhere [Participants were part of a longitudinal cohort study of the health and development of young girls, consisting of 197 non-Hispanic white 5-year-old girls living in central Pennsylvania. Families were recruited for participation in the study in 1996–97 through flyers and newspaper advertisements. Additionally, families with age-eligible daughters within a five-county radius received mailings and follow-up phone calls. Eligibility criteria for girls’ participation at the time of recruitment included living with both biological parents, absence of severe food allergies or chronic medical conditions affecting food intake, and the absence of dietary restrictions involving animal products. Girls were assessed every 2 years from age 5 to age 15 years, though bone mineral content was first assessed at age 9. At age 15, lsewhere , 15. TheHeight and weight were measured in triplicate by trained staff using calibrated scales at each assessment and were used to calculate BMI. BMI percentiles were calculated using the 2000 CDC growth charts . PubertaHabitual physical activity was assessed at age 9 using a checklist of 22 different activities . Girls wDietary intake of calcium and other nutrients was assessed using 24-h dietary recalls completed at each biennial assessment . Three 2Self-reported dieting to lose weight was assessed using a dichotomous yes/no question, “Have you ever dieted?”, at each visit. Girls were instructed that they should think of a diet as “whenever you eat less or exercise more in order to lose weight.” Girls who first reported dieting by age 11 were classified as “early dieters,” and girls who first reported dieting between 11 and 15 years were classified as “adolescent dieters.” Girls who did not report dieting by age 15 were classified as “non-dieters” . Girls wDietary restraint and disinhibition were measured using the Dutch Eating Behavior Questionnaire (DEBQ) . Disordep < 0.05.Analyses were performed in SAS 9.4. Descriptive statistics were generated for all variables of interest. Effects of dieting, behavioral measures, and dietary intake on change in BMC from 9 to 15 years (ΔBMC) and absolute BMC at 15 years were assessed using a general linear model (PROC GLM). Established predictors were first entered as a block into the model. When ΔBMC from age 9 to 15 years was used as the outcome, predictors included BMC at 9 years, change in height from age 9 to 15 years, change in weight from age 9 to 15 years, breast Tanner score at age 9 years, BMI percentile at age 15 years, and frequency of participation in physical activity at 9 years. For BMC at 15 years, the predictors were BMC at age 9, height at age 15, breast Tanner score at age 9, BMI percentile at age 15 years (weight at 15 did not predict BMC at 15 independently of BMI at 15), and frequency of physical activity at 9 years. Next, dieting variables were then added to the regression individually. Variables that were statistically significant in the individual models were then examined together. A final model for each outcome was selected using a stepwise selection process (PROC GLMSELECT). Assumptions of linearity and homoscedasticity were confirmed by examinations of residuals. To further examine potential contributors to the effect of early dieting on bone mineral accrual, differences in weight control behaviors and nutrient intake between early, adolescent, and non-dieters were assessed using Fisher’s exact test or ANOVA and Tukey’s post-hoc test. All tests were parametric and two-tailed. Significance was accepted at n = 40) at age 9 to 21.6% (n = 30) at age 15. The majority of girls reported dieting at some point during the study, with 31.7% (n = 44) of girls reporting dieting by age 11 (‘early dieters’), and 46.8% (n = 65) of girls first reporting dieting between 11 and 15 (‘adolescent dieters’). Only 21.6% (n = 30) did not reported dieting at any time point.Participant characteristics are described in Table . Girls who were classified as early dieters engaged in more weight control strategies, and had higher restraint, disinhibition, chEAT dieting and total scores, and weight concerns, and lower weight-related body esteem, than girls classified as adolescent dieters or non-dieters. Though both the adolescent dieters and non-dieter groups had not dieted at age 11, there were some characteristics that distinguished these groups at this age. Adolescent dieters had significantly higher restraint and weight concerns at age 11 years than non-dieters, suggesting elevated levels of these characteristics at 11 years preceded onset of dieting between 11 and 15 years.Differences in weight control behaviors at age 11y by dieting onset category are presented in Table 2Change in height and weight from 9 to 15 years, BMC at age 9 years, BMI percentile at age 15 years, and breast Tanner score at age 9 years were all significant predictors of ΔBMC from age 9 to 15 years Table . A modelp = 0.02). The total number of weight control strategies girls had reported ever using at age 11 was also negatively associated with ΔBMC from age 9 to 15 years . Greater restraint , chEAT dieting score , and weight concerns  at age 11 years were all significantly associated with lower ΔBMC. Disinhibition, food preoccupation, social pressure to eat, and weight related body esteem were not associated with bone outcomes.Overall, girls who reported ever dieting by age 15 did not have significantly different ΔBMC than those that never dieted. However, there was a significant negative relationship between early dieting and ΔBMC Table . After ap = 0.004, Fig. Similar patterns emerged using BMC at age 15 years as the outcome. Girls reported dieting at any age by 15 did not have significantly different BMC from those that never dieted, but early dieters had significantly lower BMC  than those that were not early dieters  average kcal, magnesium, and iron intakes than girls who never dieted, and there were also trends (p < 0.10) for lower intakes of calcium, potassium, zinc, fiber, and dairy among girls who dieted compared to non-dieters , but not healthy weight control strategies , was associated with lower intakes of protein, calcium, iron, and zinc . In anotgreater bone mineral accrual [Though we are unable to evaluate this hypothesis in the current data set, hormones may also provide a link between dieting and bone acquisition. In adult women, levels of the stress hormone cortisol have been positively associated with restraint , 42 and  accrual . Future There are limitations to this study. The sample was relatively small and primarily non-Hispanic white, two-parent families with middle to high incomes, which limits the generalizability of these findings to other groups. Though the data used for this analysis were collected from 2000 to 2007, the conclusions remain relevant today given the increasing prevalence of weight loss attempts  and contIn summary, dieting in preadolescence is associated smaller gains in BMC across adolescence and lower BMC at age 15 years. This study builds on previous findings that measures of disordered eating attitudes in healthy children are associated with poorer bone health. Further research is needed to elucidate mechanisms behind this observation. Interventions to prevent dieting in preadolescents and adolescents may also improve bone health. In light of the ongoing childhood obesity crisis, additional research is needed to identify weight management strategies that do not interfere with bone health in children.Additional file 1:Table S1. Dietary intake predictors of change in bone mineral content from 9 to 15 years. Results of regression analyses predicting change in bone mineral content from 9 to 15 years from dietary intake of bone-related nutrients/food groups, adjusting for anthropometric, pubertal development, and physical activity variables, in girls. (DOCX 13 kb)Additional file 2:Table S2. Dietary intake predictors of bone mineral content at age 15 years. Results of regression analyses predicting bone mineral content at age 15 years from dietary intake of bone-related nutrients/food groups, adjusting for anthropometric, pubertal development, and physical activity variables, in girls. (DOCX 13 kb)


Resonant peaks are clearly observed in the differential conductance of the MTJs due to the formation of QWs. Furthermore, enhanced tunnel magnetoresistance (TMR) peaks at the resonant bias voltages are realized for the MTJs at room temperature, i.e., it is observed that TMR ratios at specific and even high bias‐voltages (Vbias) are larger than zero‐bias TMR ratios for the MTJs with odd Fe atomic layers, in contrast to the earlier experimental studies. In addition, a new finding in this study is unique sign changes in the temperature coefficient of resistance (TCR) depending on the Fe thickness and Vbias, which is interpreted as a signature of the QW formation of Δ1 symmetry electronic states. The present study suggests that the spin‐dependent resonant tunneling via the QWs formed in Cr/ultrathin‐Fe/MgAl2O4/Fe structures should open a new pathway to achieve a large TMR at practically high Vbias.The quantum well (QW) realizes new functionalities due to the discrete electronic energy levels formed in the well‐shaped potential. Magnetic tunnel junctions (MTJs) combined with a quasi‐QW structure of Cr/ultrathin‐Fe/MgAl 2O4/Fe‐based magnetic tunnel junctions, quantum well (QW) statesInside Cr/ultrathin‐Fe/MgAl are established within a precisely thickness‐controlled ultrathin Fe layer. Via the QW states, the tunnel magnetoresistance (TMR) is strongly modulated at room temperature. For odd Fe atomic layer cases, the resonance appears at a high bias voltage, where the TMR is even higher than the value at zero bias. Owing to the perfect lattice matching between Fe and MgAl2O4,2O4 barrier–based double‐barrier MTJ has been reported to exhibit better QW states comparing with a MgO barrier.1‐state electrons was clearly observed with a strong modification of TMR even at room temperature (RT). The TMR ratio at resonant Vbias, at which the Δ1↑ channels are localized, is definitely larger than that at zero bias. In addition, it was found that due to the Δ1↑ states localized at specific energy levels, i.e., the formation of well‐defined QWs, positive and negative T dependence of tunneling conductance were distinguishably observed, depending on whether the conductance channels involve the Δ1↑–QW state.In this study, Cr/ultrathin‐Fe/MgAlVoutput ≡ (RAP − RP)/RAP × Vbias, particularly for magnetic sensing applications. In fact, conventional MTJs are likely to face a serious problem of the TMR degradation at high Vbias. For example, while a large RT TMR ratio around 250% near zero bias in CoFeB/MgO‐based perpendicularly magnetized MTJs (p‐MTJs) was reported, it drastically drops to ≈70% at 0.5 V bias voltage, and could not survive for over 1 V.Besides the fundamental understanding of the enhanced TMR, it is also important to achieve improved high‐bias performance of MTJs, i.e., large voltage output, 2FiguretFe)/MgAl2O4 (2)/Fe (10) stacks  were prepared without pinning and magnetically coupled layers. Further, no postannealing was performed for the top Fe layer to keep it a general free layer and to limit the QW occurring only in the bottom ultrathin‐Fe layer. As well known, the dominant states of the electron transport in epitaxial MTJs with crystalline barrier consisting of MgO or MgAl2O4 possess the Δ1 (spd‐like) symmetry, while tunneling probabilities of other states, i.e., Δ5 (pd‐like), Δ2 (d‐like), and Δ2′ (d‐like) states, rapidly decay with increasing the barrier thickness.1 symmetry, resulting in the symmetry filtering. Thus, only Δ1 symmetry electrons need to be considered for these MTJs. In addition, the TMR effect is a resistance change depending on the relative angle of magnetization of the electrodes, and therefore the TMR ratio is given by the effective spin polarization of Δ1 electrons in the Fe electrodes. Since there is no Δ1 states near the Fermi level (EF) in Cr, even the metallic Cr layer can be regarded as a potential barrier in the present heterostructure of Cr/ultrathin‐Fe/MgAl2O4/Fe, leading to a quasi double‐barrier structure for the formation of QW states of Δ1 symmetry electrons /Fe 0 stacks 4 (2)/Fe 0 stacks 4 (2)/Fe 0 stacks 4 (2)/Fe  stacks  magnetization configuration) and zero magnetic fields, respectively. Meanwhile, due to the orthogonal configuration at zero fields, the observed TMR is smaller than the conventional TMR that is calculated asRAP is the tunnel resistance of antiparallel (AP) magnetization configurations. Since RAP can be calculated from the relationGP(AP) = 1/RP(AP). The conventional TMR ratios are also calculated for comparison with the related results reported so far. We also investigated the differential conductance (dI/dV) spectra of the MTJs, since the resonant states show up as peak structures in the dI/dV spectra, as discussed in previous reports.Owing to the interface effect of Fe/MgAltFe ensured that MTJs with an integer number of Fe atomic layers existed in a certain part of the sample as FiguretFe, and then the dI/dV spectra were summarized as a conductance map onto Vbias and tFe in Figure tFe. Here, it is considered that tFe at the peak positions correspond to integer numbers of Fe atomic layers. Due to shadow effect of a linear shutter, possible intermixing at the Cr/Fe interface, and/or the formation of FeO layer at the Fe/oxide interface,tFe. Based on theoretical calculations of QWs in the Cr/Fe/MgO case,nFe. Note that tFe ≠ nFedFe due to discussion above, where dFe ≈ 0.143 nm is the thickness of a single Fe atomic layer, i.e., monolayer (ML). We attribute the peaks of tFe = 0.81, 1.02, and 1.22 nm to nFe = 5, 6, and 7 ML in this study by conductance spectrum estimation.To prepare the MTJs with an exact integer number of Fe atomic layers, a wedge‐shaped Fe layer was deposited with the designed thickness from 0.45 to 1.45 nm using a linear motion shutter. This gradually increasing I/dV, resistance (R), and current for nFe = 5, 6, and 7 ML are compared in FigureI/dV spectra, the even and odd nFe samples show clear difference, i.e., two resonant peaks arise away from zero bias for odd nFe, while a single resonant peak arises around zero bias for the even nFe. For nFe = 5 ML, the resonant peak positions are around Vbias = −0.58 and +0.55 V, as indicated with the dot lines in Figure Vbias, a little larger than those for dI/dV. The shift is presumably due to the fact that TMR at a given Vbias is calculated from the R values. The most important finding in Figure Vbias of −0.58 V is even higher than that of ≈22% at zero bias. This TMR‐enhanced behavior is in contrast to MgO‐based QW‐MTJs: TMR at resonant Vbias is 0.8 (≈0.6) times as large as that at zero bias at RT in Cr/Fe/MgO/Fe MTJVbias for odd nFe, the TMR becomes less bias sensitive: TMR varies from ≈0.9 to ≈1.1 times of its zero bias value within , which is hardly found in conventional MTJs. Benefited from the enhanced TMR ratio at high bias voltage, i.e., a large output voltage is also obtained around 0.35 V at −1 V .The TMR ratios are also summarized in the same manner as shown in Figure nFe, showing a single TMR peak as normal MTJs. This is because the resonant Vbias is just located near zero bias, where the intrinsic TMR peak is normally located. For nFe = 5 ML, the TMR at Vbias = 0 is around 32%.On the other hand, completely different behavior was observed in the MTJs with an even I/dV and R in the AP state are also deduced from those in the parallel (P) and 90° states to investigate the conventional TMR ratio. As shown in Figure I/dV in the AP states is much weakened, compared with in P states, which agrees well with the theoretical prediction mentioned above. To explore the possible performance in practical p‐MTJs, the conventional TMR calculated from R in the P and AP states is also plotted in Figure nFe = 5 ML, the TMR ratios at resonant and zero bias are 65% and 55%, respectively. For nFe = 6 ML, the TMR ratio at zero bias reaches 92%, which is comparable to the highest value in pure Fe‐based p‐MTJ.nFe, e.g., 5.5 and 6.5 ML, the peak structures in dI/dV and TMR behave as a simple sum of those with neighboring integer numbers of nFe . This suggests that it is possible to modulate the TMR behavior between single‐peak (for even nFe cases) type and multipeak (odd nFe cases) type simply by adjusting the Fe coverage.As mentioned above, the measured TMR ratios are smaller than the true values in their conventional definition due to the orthogonal configuration. Hence, dVbias, compared to the previous studies, is attributed to two major factors: improved structural coherency owing to MgAl2O4 and enhanced QW effect owing to the atomically flat ultrathin Fe layer. The former supplies a much‐improved high bias performance as previous works suggested.The significant enhancement of TMR at resonant 1 symmetry electrons can presumably be characterized by temperature coefficient of resistance (TCR). In fact, tunneling transport normally shows negative TCR, whereas positive TCR in the P state has been observed for high‐quality Fe/MgO (MgAl2O4)‐based MTJs in which the Δ1 electron transport should be dominant.1 preferential transport through Fe electrodes. FigureGP = dI/dV at the P state) normalized by the 5 K values of the MTJ with nFe = 5 ML at selected bias voltages. A clear difference was observed among the temperature dependences of GP at the bias voltages selected as around a resonant point (Vbias = −0.585 V), out of resonance (Vbias = −0.915 V) and in between the two (Vbias = −0.791 V). For the measurement around the resonant Vbias, a clear negative correlation is observed, where GP decreases as temperature increases, showing an elastic tunneling feature.Vbias, a positive correlation, where GP increases with temperature, is observed, showing an inelastic tunneling feature.I/dV and R as a function of Vbias for nFe = 5 and 6 ML at different temperature are collected as Figure1 electron transport is observed for nFe = 6 ML in the entire range of Vbias. On the other hand, it is found that the sign of TCR changes when |Vbias| exceeds the resonant positions shown by red arrows . This phenomenon of TCR can be interpreted as follows: in the well‐defined QW‐MTJs, the Δ1 transport channels are highly localized around the resonant Vbias, as suggested by the enhanced TMR appearing at the resonant Vbias. As mentioned above, R at a given Vbias is given as the integration of the inverse dI/dV from 0 bias to the Vbias, so that R at Vbias reflects dI/dV ranging from 0 bias to Vbias. Therefore, for nFe = 5 ML, R hardly includes the Δ1 electron character below the resonant Vbias, whereas above the resonant Vbias, R turns to the resistance dominated by the Δ1 states, changing the sign of TCR. Positive TCR is always observed for nFe = 6 ML, since the resonant Vbias is around 0 V. As mentioned above, the observed behavior of TCR can be well interpreted as a fact that well‐defined QW states consisting of the Δ1 symmetry electrons are formed in the present Cr/Fe/MgAl2O4 structure, in short, as a signature of the well‐defined QWs.It is well known experimentally that the tunneling transport of Δ31 electron tunneling transport via QWs that are definitely created in the Cr/ultrathin‐Fe/MgAl2O4/Fe MTJs. TMR at resonant Vbias is even higher than that at 0 bias at RT, while the behavior of dI/dV and TMR strongly depends on whether nFe is an even or odd number, suggesting that the SDRT through QWs is useful to achieve a large TMR, exclusively at high bias voltages. In addition, the sign change in TCR was discovered for the present MTJs, as evidence for the formation of well‐defined QWs.In summary, we demonstrate the enhanced TMR by modifying the ΔtFe: 0.45−1.25 nm)/MgAl2O4 (2 nm)/Fe (10 nm)/Ru (15 nm) was prepared on a single crystalline MgO(001) substrate. All the layers, except sputtered Ru capping layer, were deposited by electron beam evaporation under a base pressure of less than 1 × 10−8 Pa. The substrate was first annealed at 800 °C for degassing and cleaning surface. Then the 5 nm MgO was deposited on substrate at 450 °C as a seeding layer. Then the Cr, QW‐Fe, and MgAl2O4 layers were deposited at 150 °C wherein the postannealing was performed at 800, 250, and 400 °C, respectively. The wedge‐shaped Fe layer was deposited with a step of 0.02 nm using a linear motion shutter equipped between the Fe source and substrate. The top Fe reference layer was deposited at RT without postannealing to prevent the formation of perpendicular anisotropy at the MgAl2O4/Fe interface. Finally, the stacks were capped with Ru layer via radio frequency magnetron sputtering with the process pressure of 0.4 Pa at RT without postannealing. The prepared film was patterned into ellipse junctions of 5 × 10 µm2 in size by a conventional microfabrication method using Ar‐ion etching and photolithography. Then, the junctions were measured for magnetoresistance and the current–voltage (I–V) curves using a dc four‐probe method, with a bias voltage up to ±1 V. The magnetic field up to 2 T was applied perpendicular to the film surface for RT measurement. A low‐temperature measurement was also performed using a physical property measurement system. The differential conductance (dI/dV) spectra were mathematically calculated from the I–V curves.A fully epitaxial MgO (5 nm)/Cr (30 nm)/Fe‐QW (The authors declare no conflict of interest.SupplementaryClick here for additional data file.


This 5‐year prospective cohort study aimed to quantify possible associations between liraglutide and risk of AP and PC as compared to other antidiabetic drugs (ADs).Both acute pancreatitis (AP) and pancreatic cancer (PC) have been areas of focus for studies of incretin drugsPatients initiating liraglutide or other ADs who were enrolled in a US health plan (2010‐2014) were included. Comparisons of AP and PC incidence rates were made between matched cohorts of liraglutide initiators and initiators of other ADs. Adjudicated AP cases and algorithm‐based PC cases were identified. Propensity score‐matched intention‐to‐treat (ITT) and time‐on‐drug (TOD) analyses were completed using Poisson regression. A latency analysis was performed for PC.). In the primary AP analysis, “current” use of liraglutide was not significantly associated with elevated risk across comparators . ITT results were similar where, in the primary analysis, no RRs were significantly associated with PC ; latency and TOD analyses did not alter findings. There was no evidence of a dose‐response effect.Median follow‐up was 405 days for AP cohorts  and 503 days for PC cohorts (35 163 liraglutide, 1:1 matched to all comparatorsLiraglutide was not associated with an increased risk of AP or PC, although risk estimates were more variable for AP, and numbers of cases for both outcomes were limited because of the rarity of outcomes. The US Food and Drug Administration (FDA) and European Medicines Agency reviewed the totality of existing data but, to date, a final conclusion has not been rendered regarding a causal association between GLP‐1 RA therapies and either AP or PC; therefore, they remain safety risks for these drugs. Both agencies called for more research in this area.i]), with mixed findings.Several studies using randomized clinical trial (RCT) methods or observational methods have already been published concerning this topic. Glycaemic control trials of liraglutide,22.1This prospective cohort study was conducted using the Optum Research Database (ORD), which includes a large, geographically diverse population of commercial health insurance enrollees. The ORD contains enrollment data, medical claims and pharmacy claims; enrollment in 2014 included approximately 13.9 million individuals with both medical and pharmacy benefits and comprises approximately 4% of the US population. This study was approved by the New England Institutional Review Board and Privacy Board.2.2i  and for three summary comparators: all comparators combined, all comparators excluding exenatide, to address potential GLP‐1 RA effect, and all comparators excluding exenatide and DPP‐4i, to address potential incretin effect.A new‐user, active‐comparator design was used for this study.Follow‐up began on the day after first observed dispensing of a study drug (index date) and continued until disenrollment, censoring or end of study period , whichever took place first. The AP cohorts were restricted to the sub‐population for whom medical records could be requested to confirm AP outcome and patients with a diagnosis of AP or chronic pancreatitis (ICD‐9577.1) at baseline were excluded. Patients with a PC diagnosis during baseline were excluded from both cohorts.2.3Logistic regression modeled the PS as the predicted probability of initiating liraglutide vs comparison treatment, conditional on covariates.Many patients initiated more than one study drug. Subsequent initiators who did not match during one recruitment time block, but who initiated any study drug during a later block, were again eligible for matching with PS, re‐estimated using the baseline period prior to that initiation. Patients could enter into multiple matched cohorts over the study period but were allowed to match into a drug cohort pair only once.2.4and;Report of abdominal pain or amylase levels ≥5× ULN or 1000 U/L  and/or;Lipase levels ≥3× upper limit normal (ULN) or 300 units per liter (U/L)  Imaging diagnostic for, or suggestive of, AP.Potential AP cases were initially identified by ICD‐9 code 577.0, which does not include chronic pancreatitis, cysts or pseudocysts of the pancreas, or other diseases of the pancreas. Because the positive predictive value (PPV) for AP is typically low,Definite cases (three of the three criteria) and probable cases (criterion 1 and criterion 2 or 3) were considered confirmed cases. Final analyses included cases of AP that were confirmed through this medical chart review/adjudication.Claim with an inpatient or outpatient diagnosis of PC (ICD‐9157.xx)No diagnosis of benign pancreatic neoplasm within 60 days after PC diagnosisProcedures indicating pancreatic surgery, chemotherapy or radiation therapy within 180 days after PC diagnosisNo diagnosis of other cancers  within 60 days before or after PC diagnosisPC cases were identified, employing a validated algorithm (PPV of 0.88 [95% CI 0.62‐0.98])2.5Baseline covariates and distribution of time‐to‐event for AP and PC were summarized for liraglutide initiators and all comparators. Analytic methods separately quantified risks associated with drug initiation, recency of use and cumulative exposure. ITT analyses quantified the risk associated with initiating treatment, attributing follow‐up time to drug used at matched cohort entry. Incidence rates (IR per 100 000 person‐years [PYs]) were estimated as the number of cases divided by PYs at‐risk. Poisson regression was used to compute estimated incidence rate ratios (RRs) and 95% CIs. Generalized estimating equations (GEE) were used to account for the paired nature of the matched cohorts.For each outcome, TOD analyses evaluated the effect of treatment within ‘current’, ‘recent’, and ‘past’ categories. “Current” use included first day of follow‐up through end of days supplied, with an additional 31 days to account for medication non‐adherence. Refill dispensings that were observed during these 31 days resulted in continuous “current” use time. “Recent” use began at the end of “current” time and continued for another 31 days. Subsequent person‐time was categorized as “past” use, which persisted unless the patient re‐initiated the same treatment, thereby re‐entering “current” use. Poisson regression was used to estimate the RRs and 95% CIs for “current,” “recent” and “past” use of liraglutide vs the corresponding comparator category, adjusted by the logit of the PS to address confounding.Another TOD analysis addressed potential for a dose‐response relationship through calculation of cumulative time on liraglutide. Cumulative time exposed and unexposed to liraglutide during follow‐up was quantified for PC, and IR was determined within each exposure type. Estimates of RR were made using Poisson regression within categories of cumulative time  relative to all “liraglutide unexposed” time, adjusted for the logit of the PS. The TOD analysis was the primary analysis for AP, with focus on “current” time, given the acute nature of AP, while the ITT analysis was the primary analysis for PC.33.1Among first observed initiations for each patient, 8499 initiated liraglutide and 100 161 initiated comparators. A total of 9995 liraglutide initiations, including first and subsequent initiations, were 1:1 matched to all comparator initiations. Selected baseline covariate distributions for the liraglutide:all comparators pair before and after matching for the AP analysis are displayed in Table Overall median follow‐up time was 405 days (interquartile range [IQR] 181‐747 days), ranging from 335 days (SUs) to 469 days (pioglitazone). Within each matched set, median lengths of follow‐up were similar.Charts were requested for 350 patients with an AP diagnosis. Overall, 180 AP cases were confirmed through adjudication; 15 were dropped for administrative reasons or baseline exclusions, leaving 165. More details on the adjudication process are available in Appendix Median time from matched cohort entry until AP diagnosis was 421 days  for liraglutide compared with 286 days  for all comparators. Cases in the liraglutide cohorts experienced consistently longer times to diagnosis. Distributions of time to AP diagnosis are available for all pairs in Appendix The results of AP analyses are presented in Figure In the ITT analyses, RRs ranged from 1.1  to 4.0 , with one statistically significant elevated risk .3.2Among first observed initiations for each patient, 27 283 were liraglutide initiations and 362 539 were initiations of any comparator. A total of 35 163 liraglutide initiations, including first and subsequent, were matched to all comparators initiations. Distributions of select baseline covariates pre‐ and post‐matching for the liraglutide:all comparators pair are displayed in Table A total of 240 claims‐based cases were identified, with 10 among liraglutide initiators. Median days to PC diagnosis were 369  and 318  . RRs ranged from 0.5  to 1.6 . Most RRs were similar or lower when a one‐year latency period was taken into account (data not shown).In the primary analysis (ITT), IRs were 21.4/100 000 PYs (liraglutide) vs 31.5/100 000 PYs  to 0.7 . RRs were higher for “recent” use, but these estimates were based on small numbers of cases and limited PYs. The two highest RRs were in “past” use .In the cumulative TOD analysis, adjusted RRs were 1.0 , 1.0  and 0.4 .4This five‐year prospective cohort study included several drug cohorts sourced from administrative claims, supplemented with medical record review. Across therapies with different mechanisms of action, the data do not suggest an association between liraglutide use and increased risk of AP or PC.In the AP TOD analysis, RRs often exceeded 1.0; however, no findings excluded the null. The estimated RR was not greater than 1.0 for contrasts within “current” exposure. The question of whether patients with early symptoms of AP may have switched medications and discontinued use of liraglutide more rapidly than comparators, which would result in more cases being attributed to the “start of past use” liraglutide category relative to comparators, was investigated via sensitivity analysis. Mean time between the beginning of “start of past use” and date of AP cases was longer for liraglutide  than for all comparators , a finding that does not indicate that liraglutide patients were more likely to have discontinued or switched medications prior to diagnosis of AP.i patients. However, no consistent patterns of patient differences were observed overall, suggesting balanced characteristics in the cohorts over time, regardless of timing in relation to discontinuation.In the ITT analyses, only the CI for all comparators except exenatide and DPP‐4i excluded the null. Another sensitivity analysis that evaluated the potential selection bias that could result from preferential discontinuation following AP symptoms was completed for each of the eight matched AP cohorts. Baseline characteristics of patients who discontinued the study drug early during follow‐up (≤median) and those of patients who discontinued use later during follow‐up were compared. Occasional differences existed; for example, among patients who discontinued early, liraglutide patients were slightly older and more likely to be black than were DPP‐4i therapies .i therapies,Several observational studies, including early results from this study, found no significant association between GLP‐1 RAs  and AP.i RCT that found significant increases in AP risk in the treatment group vs controls. These studies were based on the same three RCTs with large patient populations, adjudicated cases of AP and median follow‐up periods ranging from 1.6 to 3.0 years.i use .is  independently or combined .In PC analyses, the majority of ITT RRs were below 1.0; the only statistically significant finding was a lower risk with liraglutide use, relative to SUs. No dose‐response effect was observed in the cumulative use analyses, consistent with other observational studies of the GLP‐1 RA class, with the exception of that by Elashoff et al, with the limitations already noted.The strengths of this study include the use of a large administrative claims database, supplemented with a medical record review. Case ascertainment included medical record adjudication (AP) and a validated algorithm (PC).Each cohort, including the all comparator cohorts, was created separately and was PS‐matched with the liraglutide cohort, independently, within refined calendar periods to capture changes in US market share. Three all comparator cohorts were used, so that, in addition to including a matched comparison with all study comparators, one comparison involves non‐GLP1‐RA drug classes and another involves non‐incretins.Multiple analytic approaches were used to balance the strengths and weaknesses. Because AP is an acute event, TOD was the primary analysis, with a focus on “current” use. In contrast, longer‐term exposure was important to assess the risk of PC; thus, ITT was the primary analysis, including latency analysis to reduce potential protopathic bias.Medical record confirmation was required for AP cases, restricting the sample size to patients in plans that allow access to medical records (~30% of eligible patients). Despite this restriction, the number of AP cases is comparable to that of many other individual RCTs and observational studies with chart‐confirmed outcomes.Observational studies assess associations under “real‐world” conditions; however, for a disease such as diabetes with numerous treatment options, these conditions complicate AD studies. Many patients have a history of AD use, and even restricted definitions of initiation may not fully capture earlier drug use. Concurrent or add‐on drugs in other classes are difficult to quantify. Patients switch/add therapies if side effects occur or if diabetes is not well‐managed. Baseline drug use was included in the PS; however, changes during follow‐up are not accounted for with these analytic methods. As a result, it is difficult to attribute causality among patients using multiple drugs or undergoing multiple therapies.Results should be interpreted with the understanding that comparators may not be reflective of initiators of those individual drugs in general, particularly for first‐line therapies, e.g., the comparison is made between liraglutide initiators and metformin initiators with baseline characteristics similar to liraglutide initiators, and not a comparison to all types of metformin initiators.Many measures and comparators were used to assess the association between liraglutide and pancreatic outcomes. AP analyses demonstrate no consistent pattern of increased risk with liraglutide during any period of use, including the primary analysis of “current” use. There was no support for an increased risk of PC with liraglutide use, a finding that is consistent with many other studies.In conclusion, based on observational data in a commercially insured population, liraglutide use was not associated with increased risk of AP or PC. Although high specificity of identified cases was achieved, concerns remain regarding the limited numbers because of the rarity of outcomes, as well as confounding by unmeasured factors and the question of whether the findings are generalizable to other populations.The study was supported through a research contract between Optum and Novo Nordisk A/S, who is the Marketing Authorization Holder of liraglutide. N.J.Z., J.D.S., H.N. and D.D.D. are employed by Optum and hold stock/stock options in the parent company of Optum . D.F., K.M. and L.L. were employed by Optum at the time of study conduct. A.M.‐P., H.B.‐T. and H.G. are employees and stockholders of Novo Nordisk A/S.D.D.D. had full access to all data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Because this work was undertaken to meet a regulatory request, both the FDA and Novo Nordisk reviewed and commented on the protocol and statistical analysis plan underlying this work. The final study design and analytic plan required FDA approval. D.D.D., D.F. and K.M. were responsible for study concept and design. L.L. and H.N. were responsible for acquisition of data. D.D.D., D.F., K.M., L.L. and H.N. were responsible for analysis and interpretation of data. D.F. and N.J.Z. were responsible for drafting the manuscript. D.D.D., K.M., H.G., A.M.‐P., H.B.‐T., N.J.Z. and J.D.S. were responsible for critical revision of the manuscript for important intellectual content.File S1. Supplementary Appendices.Click here for additional data file.


P=0.003), by −0.52 D at 6 months (P=0.012), and by −1.31 D at 12 months (P < 0.001). In the pseudophakic eyes, we found no significant refraction progression at any of the postsurgery follow-up visits. Our multivariable analyses showed that lens nuclear color grade change was a significant prognostic factor for refractive myopic progression (P < 0.001). Trabeculectomy causes refractive myopic progression in phakic eyes. Nuclear sclerotic cataract progression is associated with refractive myopic shift after trabeculectomy. This trail is registered with UMIN000007813.We prospectively evaluated refractive changes in the eyes of 97 patients who underwent trabeculectomy at Fukui University Hospital, Fukui, Japan. The primary outcome measure was the refractive change after trabeculectomy. Secondary outcome measures included postoperative complications and prognostic factors for refractive change. We observed a progressive and significant mean refractive myopic shift of −0.80 D at 12 months after surgery. In phakic eyes, the mean myopic refractive shifts progressed significantly by −0.46 D at 3 months after surgery ( Trabeculectomy is a common filtering surgery for patients with glaucoma and medically uncontrollable intraocular pressure (IOP). Patients treated with trabeculectomy often encounter postoperative visual disturbances. Hypotonic maculopathy, postoperative hyphema, anterior chamber inflammation, and fixation loss or wipeout diminish visual acuity after trabeculectomy .In terms of refraction, trabeculectomy causes corneal astigmatism, which also deteriorates visual acuity –7. Our sUMIN000007813; date of access and registration: April 24, 2012). Our study protocol adhered to the tenets of the Declaration of Helsinki. We obtained written informed consent from all patients.This was a prospective clinical cohort study that was approved by the institutional review board of the Fukui University Hospital . We registered this study with the University Hospital Medical Information Network Clinical Trials Registry of Japan This prospective study evaluated refractive changes after trabeculectomy. We recruited patients between April 2012 and March 2016 at the Fukui University Hospital. The inclusion criteria were being aged ≥20 years and having open angle glaucoma  without a history of intraocular surgery other than phacoemulsification. The exclusion criteria were patients with aphakic eyes, eyes with a history of glaucoma surgery before trabeculectomy, eyes with previous vitrectomy, or pseudophakic eyes previously treated with cataract extraction other than phacoemulsification.All surgeries were performed by one surgeon (MI). The surgeon made either a 5 mm conjunctival incision along the limbus to create a fornix-based conjunctival flap, or an 8 mm conjunctival incision parallel to the limbus at 7–9 mm posterior to the limbus to create a limbus-based conjunctival flap. A 4 mm wide half layer scleral flap was also created. Mitomycin C (0.4 mg/ml) was applied on and under the scleral flap and under Tenon's capsule for 4 min, and the eye was irrigated with physiological saline (100 ml). The surgeon then excised a deep limbal block to create a fistula in the anterior chamber to proceed with peripheral iridectomy. The surgeon closed the scleral flap by using three sutures of 10-0 monofilament nylon. The conjunctival flap was also sutured with 10-0 monofilament nylon. All patients received similar postoperative topical medications with 0.5% levofloxacin 3 times a day for 1 month and 0.1% betamethasone sodium phosphate 3 times a day for 6 months.We collected patient data including gender, age, glaucoma type, preoperative IOP, postoperative IOP, refractive errors, best corrected visual acuity (BCVA), number of glaucoma medications, anterior chamber depth, axial length, anterior chamber opening duration, and presence of postoperative complications. We used the logarithm of the reciprocal of the decimal BCVA to approximate the logarithm of the minimal angle of resolution (LogMAR). We scheduled the first study-related visit 2 weeks after surgery; thereafter, follow-up visits occurred 1 month, 3 months, 6 months, and 12 months after surgery. We assessed IOP, BCVA, refractive errors, and number of glaucoma medications before surgery and at all follow-up visits. We also looked for complications at all follow-up visits. We measured preoperative and postoperative refractive errors in all eyes by using an automatic refractometer . Refractive errors were measured three times at each visit, and the average of three refractive errors was used as the refraction value for analysis. We defined the anterior chamber open-time during the surgery as the time from the initial fistula creation incision in the anterior chamber to the scleral flap suturing. We quantified the nuclear cataract progression by using the Lens Opacification Classification System III (LOCS-III) nuclear color grade . The defThe primary outcome measure was the refractive change after trabeculectomy. Secondary outcome measures included postoperative complications and prognostic factors for refractive changes.t test with Bonferroni correction, unpaired t, and chi-squared tests. The longitudinal repeated measures were analyzed using one-way repeated measures analysis of variance (ANOVA). We performed multivariate analysis to determine the prognostic factors for myopic progression using a logistic regression model. We considered P values <0.05 as statistically significant.We performed univariate comparisons between groups using paired We enrolled a total of 106 patients (106 eyes) in this study. Five patients were not available for analysis within the 1-year follow-up. We excluded four patients from the analysis because of glaucoma reoperations within the 1-year follow-up. In total, we evaluated 97 patients (97 eyes) for the study. P=0.016), to −2.22 ± 3.89 D at 3 months after surgery , to −2.28 ± 3.82 D at 6 months after surgery , and to −2.73 ± 3.90 D at 12 months after surgery .P=0.008), to −2.83 ± 4.50 D at 6 months after surgery , and to −3.62 ± 4.51 D at 12 months after surgery . In the pseudophakic group, we found no significant progression of refraction at any of the postsurgery follow-up visits  into those with myopic progression (n = 22) and those without it (n = 39) and conducted subgroup analyses. We defined myopic progression as >−1 D progression at 12 months after surgery from the preoperative refraction value. We compared various factors between the two groups (P=0.003). The preoperative and postoperative BCVAs in the patients with myopic progression were significantly worse than those in patients without it . The exfoliation glaucoma and fornix-based incisions in the patients with myopic progression were significantly more frequent than those in the patients without myopic progression . The axial length in the patients with myopic progression was significantly shorter than that in the patients without it (P=0.018). The preoperative and postoperative nuclear color grades in the patients with myopic progression were significantly higher than those in the patients without it . Finally, the nuclear color grade changes (the difference between the preoperative and the postoperative nuclear color grades) in the patients with myopic progression were significantly higher than those in the patients without myopic progression (P < 0.001).We divided phakic patients , by −0.52 D at 6 months (P=0.012), and by −1.31 D at 12 months (P < 0.001), whereas there were no significant refraction changes in the pseudophakic eyes at any postsurgery follow-up visits. Our multivariable analyses confirmed that the nuclear sclerotic cataract progression was significantly associated with refractive myopic progression (P < 0.001).The aims of our study were to evaluate refractive changes after trabeculectomy and to identify prognostic factors for postoperative myopia. A myopic shift in refraction progressed significantly by −0.80 D 12 months after trabeculectomy. In the phakic eyes, the myopic shift had progressed significantly by −0.46 D at 3 months after surgery , the toxicity of mitomycin C for lens epithelium , and theExfoliation glaucoma and fornix-based incision have been associated with cataract progression after trabeculectomy –28. ConsMyopic progression occurs in eyes after lens-sparing vitrectomy due to nuclear sclerotic cataract progression –14, a reWe are aware of the limitations of our study. First, we used LOCS-III to evaluate cataract grade and, thus, were unable to objectively evaluate the nuclear cataract grade. An anterior eye segment analysis system to evaluate the lens light scattering would have provided a more objective approach. Second, refraction after trabeculectomy is influenced by many factors, such as changes in the corneal topography and axial length. In the current study, we could not evaluate the astigmatism and axial length. The correlation between myopic shift and the change of astigmatism and axial length should be evaluated in future studies. Third, we measured all refractions without mydriasis. Although, the effect of accommodation was small because of the advanced ages of our patients, future studies with mydriatic refractions should be performed. Fourth, our data still do not offer long-term results for postoperative changes after 3 years or longer. The present study will be monitored for 5 years after surgery. Further follow-up periods might provide more information about refractive changes after trabeculectomy.In all, trabeculectomy causes refractive myopic progression postoperatively in phakic eyes. Nuclear sclerotic cataract progression after trabeculectomy is the cause of refractive myopic progression.