A devastating earthquake with a magnitude of Mw 7.4 occurred on the North Anatolian Fault Zone (NAFZ) of Turkey on August 17, 1999 at 00:01:39 UTC . The aim of this study is to propose a new approach to automatically identify earthquake induced damage areas which can provide valuable information to support emergency response and recovery assessment procedures. This research was conducted in the Adapazari inner city, covering a 3 × 3 km area, where 11,373 buildings collapsed as a result of the earthquake. SPOT high resolution visible infrared (HRVIR) Pan images obtained before (25 June 1999) and after (4 October 1999) the earthquake were used in the study. Five steps were employed to conduct the research and these are: (i) geometric and radiometric correction of satellite images, (ii) Fast Fourier Transform (FFT) of pre- and post-earthquake images and filtering the images in frequency domain, (iii) generating difference image using Inverse Fast Fourier Transform (IFFT) pre- and post- earthquake images, (iv) application of level slicing to difference image to identify the earthquake-induced damages, (v) accuracy assessment of the method using ground truth obtained from a 1/5,000 scale damage map. The total accuracy obtained in the research is 80.19 %, illustrating that the proposed method can be successfully used to automatically identify earthquake-induced damage areas. Different fault systems in Anatolia and the surrounding regions were created due to the complex plate interactions among Arabia, Eurasia and Africa . The NorEarthquakes and other natural hazards can cause disasters of uncontrollable magnitude when they hit large urban areas. Emergency response and early recovery assessment in earthquakes require rapid and reliable damage assessment and loss estimation. In the case of suddenly occuring earthquakes, remote sensing data can be reliably used to create fast draft damage maps of the affected urban areas which provide valuable information to support emergency response teams and decision making during the recovery process. Remotely sensed images ranging from very high resolution to medium resolution have been widely used to derive information and estimation for damage assessment –7. Morevet al. : using the following equations:3.4.The inner city of Adapazari was divided into ninety nine blocks of 300 m x 300 m size and these blocks were used for the detailed accuracy assessment procedure. The results obtained after the level slicing of the difference image  illustrates the damaged and non-damaged areas. These areas were compared with the 1/5,000 scale damage map for each block on a parcel basis to investigate the applicability of this method to automatically identify earthquake-induced damage. The number of parcels in each block was evaluated individually and error matrix for each block was created as shown in Each block has four values corresponding to parcels identified as damaged both in the damage map and with the proposed method (cell 1), parcels identified as non-damaged both in the damage map and with the proposed method (cell 4), parcels identified as damaged in the damage map but non-damaged with the proposed method (cell 3) and parcels identified as non-damaged in the damage map but damaged with the proposed method (cell 2). Overall accuracy of each block was calculated by summing diagonal elements and dividing them to total number of parcels within that block. The equation of the overall accuracy for a block based on the values described in After calculating the overall accuracy of each block, total accuracy of the proposed method was calculated by rationing the sum of diagonals of all blocks to total number of parcels of all blocks. The performance of the proposed method was evaluated considering the total accuracy. 4.The original pre- and post-earthquake images are shown in The low frequencies are plotted near the origin (center) while the higher frequencies are plotted further out. Generally, the majority of the information in an image is in the low frequencies indicated by the bright areas at the center of the High pass filter was applied to satellite images in frequency domain to delineate the border of linear objects like roads and buildings precisely. Filters were applied to the low frequencies which are around the center for both pre- and post-earthquake data. After the filtering, IFFT was applied and edited Fourier images were converted back into the spatial domain.After the visual interpretation of IFFT images, a difference image was generated by subtracting post-IFFT-image and pre-IFFT-image. Level slicing method with 10 slices was conducted to identify the damaged and non-damaged areas. Different numbers of levels were tried to find out the optimum number of slices and the analyses shows that having a slice number higher than 10 did not contribute significant information since only a few number of pixels was assigned to a slice. The histogram of the difference image was investigated to see the general distribution of data and to determine a threshold value for damaged and non-damaged regions. Further analyses were conducted with different threshold values to find out the most appropriate value for the study to identify the changes. Standard deviation (σ) and mean (µ) values obtained from the difference image were used for the analysis. 3σ, 2.5σ, 2σ, 1.8σ and 1.6σ, 1.5σ and1.4σ were tried and 1.4σ was found as the best threshold value to determine changes. Using this threshold value, slices including data between µ-1.4σ and µ+1.4σ were assigned as non-damaged areas whereas slices outside this range were assigned as damaged areas. Damaged areas obtained from remote sensing methods were compared with the 1/5,000 scale damage map to quantify the accuracy of the proposed method. Cell 1: parcels identified as damaged both in damage map and with the proposed method, this value is 3 for block 1–2.Cell 2: parcels identified as non-damaged in damage map but damaged with the proposed method, 4 for block 1–2.Cell 3: parcels identified as damaged in damage map but non-damaged with the proposed method, 1 for block 1–2.Cell 4: parcels identified as non-damaged both in damage map and with the proposed method, 6 for block 1–2.Comparisons were conducted for each block in parcel base. Number of damaged or non-damaged parcels within a block were calculated from difference image  and damaIn most cases parcels identified as damaged in the damage map but non-damaged with the proposed method (cell 3) occurred because there were only one or two collapsed buildings within a parcel which could not be identified using SPOT images. Block 1–2, 1–3, 1–10, 4–5 are examples of this situation. On the other hand, most of the parcels having more than three collapsed buildings were easily identified using the proposed method. Higher resolution satellite images should be used to identify collapsed buildings individually.The overall accuracy of each block ranges from 50 % to 100 % . Most ofTotal accuracy of the proposed method was calculated by rationing the sum of diagonals of all blocks to total number of parcels. The total accuracy obtained from this ratio is 80.19 %. The results illustrated that the proposed method can be successfully used to identify earthquake-induced damage areas automatically. Considering the spatial resolution of the satellite image used in this study, this accuracy value is reasonable. The results derived from this research can provide important information to many decision-makers and local authorities to determine location and magnitude of destructions and conduct emergency operations. However, depending on the end-user needs, if higher accuracy value is desired, high resolution satellite image should be used.5.Remotely sensed data are crucial for disaster management, and rapid and reliable information extraction from these data is an important source for decision-making. Quick identification of heavily damaged areas in a disaster provides key information on potential damage and losses to buildings, transportation systems, industrial facilities and critical emergency facilities. These data can be used by urban planners and emergency managers to manage vulnerabilities of a region and develop risk mitigation plans.This research proposed a new approach which is the integration of FFT and level slicing to accurately identify the location of the damaged areas caused by an earthquake. A difference image obtained from IFFT-pre- and post-earthquake data can provide significant information about the location and degree of earthquake induced damage areas. Most of the parcels which include more than three collapsed buildings were easily determined using the proposed method. The total accuracy of the method is calculated for pre-defined blocks in parcel basis and found to be 80.19 %. Higher resolution satellite images should be used to identify collapsed buildings individually. However, in some cases high resolution data of the region is not available and available satellite images of the region obtained before and after the disaster have to be used. Therefore, it is important to process the available data set rapidly and accurately and this research proposed an approach to fulfill this aim.With the use of the proposed method, emergency managers, risk managers, and public policy/decision makers can understand the impact of earthquakes, identify the heavily damage areas to direct the emergency-response teams and incorporate the results into preparedness programs and early warning systems.


Overweight and obesity are public health issues in the United States, and veterans have a higher rate of overweight and obesity than the general population. Our objective was to examine whether copayment elimination increased use of a weight loss clinic by veterans.We examined clinic use by 44,411 new patients seen in a Veterans Affairs (VA) MOVE! weight management clinic before the copayment elimination and clinic use by 64,398 new patients seen in the year after copayment elimination. We examined clinic use via mixed-effects models for patients who were already exempt from copayment and patients who were newly exempt from copayment. We used 2 outcomes before and after copayment elimination: 1) the ratio of number of clinic visits by new users with the mean number of MOVE! clinic visits by all users, and 2) the number of clinic visits by each new user in the 6 months after their first visit. All models were adjusted for patient and clinic factors.Among newly exempt patients, the clinic-standardized rate of new use increased by 2.2% after the copayment was eliminated but increased 12% among already exempt veterans. This finding was confirmed in adjusted analyses. Analysis of number of clinic visits adjusted for patient and clinic factors also found that exempt and nonexempt veterans had similar numbers of repeat clinic visits.We saw an unexpected larger increase in demand among veterans who receive all VA care for free. These results suggest that VA should not assume that copayment reductions for selective preventive services will motivate patient change and achieve intended system-level outcomes. Obesity is a major health and health care problem; more than one-third of Americans are currently obese and another approximately one-third are overweight . ObesityDespite widespread dissemination of evidence-based weight management recommendations, only 20% of people trying to lose weight adhere to guidelines on dietary intake and physical activity . Patienthttp://www.move.va.gov/move23.asp) to develop tailored, behaviorally based dietary changes and physical activity self-management support that are delivered through individual or group face-to-face counseling and that are supported with interventions that use weight loss medications, intensive outpatient programs, and bariatric surgery. More than 40% of veterans who participated in at least 2 MOVE! treatment sessions lost at least 5% of their body weight in the short term; only 12.5% of nonparticipants matched for age, sex, comorbidities, and body mass index lost this amount  developed the MOVE! Weight Management Program for veterans in 2006. The program provides a systematic, evidence-based approach to weight management for the nearly 6 million veterans served by approximately 153 hospitals and 956 outpatient clinics . On June 15, 2008, VHA policy makers eliminated the copayment because patients and clinic staff expressed concern that the copayment was a barrier to initial and ongoing participation. The copayment change was communicated to VA staff at local sites but not to veterans through a coordinated national communications strategy. The policy to eliminate copayments is consistent with value-based insurance design principles that link cost sharing directly to clinical value so that cost-effective or cost-saving products and services have lower copayments than less cost-effective products and services . Value-bThe objective of this study was to evaluate the effect of the MOVE! copayment elimination on use of the MOVE! clinic by patients who had not previously received MOVE! clinic services. We examined whether the ratio of first-time MOVE! clinic visits to mean overall clinic use  increased after the copayment was eliminated. We also examined whether new users increased the number of visits they made to the MOVE! clinic in the 6 months after their first visit.The Durham VAMC Human Subjects Protection committee reviewed and approved this study. We employed a cross-sectional pre–post study design with a nonequivalent control group. We had 2 distinct and nonoverlapping cross-sectional cohorts: 1) a preperiod  cohort of veterans who were new MOVE! clinic users in the year before the copayment elimination and 2) a postperiod  cohort of veterans who were new MOVE! clinic users in the year after the copayment elimination. No patients in the preperiod cohort were represented in the postperiod cohort. Patients were identified as new users if they had no MOVE! clinic use in the 12 months before the index visit (their first MOVE! clinic visit) in the preperiod or postperiod. The cohort included incident, rather than prevalent, MOVE! users to enable us to identify immediate effects  of the cAlthough nonexempt veterans during the preperiod were required to pay a $15 copayment until the copayment was eliminated on June 15, 2008, for the purpose of this study we refer to veterans in the preperiod (and the postperiod) who did not meet requirements for exemption and for whom the MOVE! copayment was eliminated as the “newly exempt” cohort. We hypothesized that copayment elimination would most affect the behavior of new clinic users in the postperiod. We compared MOVE! clinic visits by newly exempt veterans to clinic visits by a subgroup of veterans in the preperiod and postperiod cohorts who were exempt from copayments for all outpatient clinic visits. Seventeen percent of the preperiod cohort were veterans who were required to pay copayments, and 16% of the postperiod cohort were newly exempt veterans who became newly exempt in the year after copayment elimination.The sample included 126 VAMCs with high-volume MOVE! clinics, defined as having 30 or more MOVE! participants during the preperiod. This threshold was chosen to identify VAMCs that had moved beyond the start-up phase of MOVE! implementation. For the clinic-standardized rate of new MOVE! use, we aggregated data to the clinic level by month for new users in the preperiod and the postperiod. The number of MOVE! visits by new users in the 6 months after their index visit was defined for 21,193 preperiod and 26,520 postperiod new users. We excluded 23,218 preperiod new users from the analysis of attendance precopayment and postcopayment removal because their first MOVE! clinic visit occurred within 6 months of the copayment elimination , so they had less than 6 months of preperiod time to make a repeat visit. We excluded 37,878 postperiod new users whose first visit occurred between December 16, 2008, and June 14, 2009, for parallel cohort construction. The final sample included 44,411 preperiod and 64,398 postperiod new users.To construct the 2 MOVE! clinic use outcomes and most patient characteristics , we used 2006–2009 VHA Outpatient Care File data, which contains information on every VHA outpatient visit. The first clinic use outcome was measured monthly at the clinic level separately for exempt and newly exempt users  during the preperiod. This outcome was a ratio in which the numerator was the number of MOVE! first-time visits within each clinic by patients who were new users, and the clinic-specific denominator was the overall mean monthly number of MOVE! visits to that clinic. This ratio, which we refer to as the clinic-standardized rate of new MOVE! use, represents the ratio of first-time MOVE! clinic visits to mean overall clinic use by all users (prior users and new users). The denominator was constant during the 24 months and the 2 copay groups, ranging from 0.0 to 4.3, so the ratio varied only according to the monthly change in the number of veterans who were using the MOVE! clinic for the first time. The denominator reflected the overall mean number of MOVE! clinic visits, so it was possible for the number of MOVE! clinic visits by new users in a given month to be larger than the overall monthly mean preperiod use . The second use outcome was a count variable that measured at the patient level in both the preperiod and postperiod , and consisted of the number of MOVE! visits by new users in the 6 months after their first clinic visit.To control for comorbidity, we obtained Diagnostic Cost Group (DCG) risk-adjustment scores for each patient in the preperiod and postperiod cohorts. DCG scores have been as predictive of veterans’ 1-year expenditures and mortality as other comorbidity scores ,15. PatiThe analysis for the first outcome was a linear mixed-effects model based on the framework of a split-plot design . In thisWe used a negative binomial mixed-effects model to estimate differences in the number of MOVE! clinic visits. The fixed effects in the model included the main factors of interest: an indicator for copayment eligibility, an indicator for the preperiod or postperiod cohort, and an interaction of the copay eligibility and pre–post indicators. This interaction term indicates whether use of the MOVE! clinic changed significantly in exempt vs nonexempt veterans between the preperiod and postperiod. Additional fixed effects included veterans’ age, DCG score, sex, race, and marital status. A clinic-level random effect was included in the model to account for the intraclass correlation of patients from the same clinic. All analyses were conducted using SAS version 9.2  and Stata version 10 .During the year before the copayment elimination, the mean clinic-standardized rate of new MOVE! clinic use was higher for new users who were copayment exempt (18.0%) than for those required to pay copayments (3.6%), which suggests that for every 2 nonexempt veterans newly using MOVE! in the preperiod, 10 exempt veterans newly used MOVE! The mean clinic-standardized rate by veterans exempt from copayments and new to the MOVE! program increased from 18% to 30% of all clinic visits, while the mean clinic-standardized rate by veterans formerly required to pay copayments and new to the MOVE! program increased from 3.6% to 5.8%. Compared with nonexempt new users, the relative increase in the clinic-standardized rate new MOVE! clinic visits by exempt veterans was 12% in the year before the copayment elimination and 2.2% after. In the adjusted analysis , we founP < .001, In the preperiod and postperiod, compared with nonexempt patients, exempt patients were younger , more likely to be female (12% vs 7%), less likely to be white (50% vs 54%), and less likely to be married (52% vs 70%) and had higher mean DCG scores (0.75 vs 0.61)  may have increased demand for MOVE! services by both patients and VHA clinicians. The increase in MOVE! use by exempt veterans after copayment elimination suggests that VHA does not need to be concerned about “a run on services” following copayment elimination. Exempt veterans may have used MOVE! at a higher rate than newly exempt veterans because they have greater VA use in general. To increase MOVE! use among newly exempt veterans, VA may want to consider direct financial incentives to veterans instead of a copayment reduction. A copayment reduction may be less motivating to individuals compared with a deposit contract approach , direct This study has several limitations. First, these results may not generalize to MOVE! clinics that had low volume in the year before the copayment elimination or to more recent years . Second, we do not have baseline obesity and overweight status for the exempt and nonexempt groups. Higher-income, nonexempt veterans may have had fewer candidates for participation in the MOVE! program, which could account for the lower enrollment increase in that population. Third, we did not have data on the number of veterans who wanted to participate in the MOVE! program but were unable to participate. If a disproportionate share of veterans required to pay copayments were placed on waiting lists to enroll in MOVE!, these results would underestimate the response to the copayment elimination by these veterans because they only reflect actual MOVE! clinic visits. Fourth, there may have been changes in MOVE! clinics themselves over time that created maturation biases that potentially confounded the effect of the copayment elimination. Fifth, the examination of the MOVE! clinic copayment using a cross-sectional time-series study design of new MOVE! clinic users, some of whom were newly exempt veterans and some of whom were nonequivalent “always exempt” veterans, may have limited the internal validity of this evaluation. However, examination of MOVE! clinic demand by new users and inclusion of a control group  controlled for regression-to-the-mean bias and other threats to internal validity.Eliminating the copayment reduced a financial barrier to access the MOVE! clinic, yet we did not observe increased use by veterans newly exempt from these copayments compared with veterans who were already exempt from copayments. Unexpectedly, we saw a larger increase in demand among veterans who receive all VA care for free, which may have been driven by clinic staff perception of greater potential benefit of MOVE! clinic use in this segment of the population or by a greater perceived value of this exemption. Copayment elimination may be less important for health behavior change services that require ongoing commitment from patients. These results suggest that VA should not assume that copayment reductions for selective preventive services will motivate patient change and achieve intended system-level outcomes.


Populus tremula, that are all associated with defensive responses against pests and/or pathogens, and have earlier been shown to become strongly up-regulated in poplars as a response to wounding and insect herbivory. Our results show that the majority of these defense genes show patterns of intraspecific polymorphism and site-frequency spectra that are consistent with a neutral model of evolution. However, two of the genes, both belonging to a small gene family of polyphenol oxidases, show multiple deviations from the neutral model. The gene PPO1 has a 600 bp region with a highly elevated KA/KS ratio and reduced synonymous diversity. PPO1 also shows a skew toward intermediate frequency variants in the SFS, and a pronounced fixation of non-synonymous mutations, all pointing to the fact that PPO1 has been subjected to recurrent selective sweeps. The gene PPO2 shows a marked excess of high frequency, derived variants and shows many of the same trends as PPO1 does, even though the pattern is less pronounced, suggesting that PPO2 might have been the target of a recent selective sweep. Our results supports data from both Populus and other species which have found that the the majority of defense-associated genes show few signs of selection but that a number of genes involved in mediating defense against herbivores show signs of adaptive evolution.Owing to their long life span and ecological dominance in many communities, forest trees are subject to attack from a diverse array of herbivores throughout their range, and have therefore developed a large number of both constitutive and inducible defenses. We used molecular population genetics methods to examine the evolution of eight genes in European aspen,  Plants have evolved numerous defensive mechanisms to protect themselves against biotic and abiotic stress i.e. defenses active against a small number of natural enemies) appear to be under stronger selection than generalist defenses (i.e. defenses active against a broad number of enemies) Earlier molecular population genetic surveys of plant defense genes have shown that specialist defenses  have become the de facto model species for studying the effects of pathogens and herbivores, both in greenhouse and under field conditions Populus tremula. These eight genes are all associated with defensive responses against pests and/or pathogens and have earlier been shown to be strongly up-regulated in poplars as a response to insect herbivory Owing to their long life span and ecological dominance in many communities, forest trees are subject to the attack of a diverse array of herbivorous insects and mammals throughout their range. Forest trees have developed a large number of both constitutive and inducible defenses to deal with this suite of natural enemies P. tremula were collected from the Swedish Aspen collection (SwAsp collection), which includes 116 individuals sampled from twelve different sites throughout Sweden. The SwAsp collection has been described in detail elsewhere and for more information please refer to Luquez et al. Allene oxide synthase (AOS5) and Phenylalanine-ammonia lyase (PAL1) encode proteins that are involved in the early steps of the octadecanoid- and phenylpropanoid biosynthesis pathways, respectively NFXL1 encodes a putative transcription factor that functions as a repressor of elicitor-induced defense responses and is involved in the crosstalk between different defensive responses associated with the SA and abscisic acid (ABA) pathways GHf10) that catalyzes the initial breakdown of xylan in cell walls and has been shown to be associated with cell wall degradation athL encodes a cysteine proteinase of the papain family which is induced through a hypersensitive response and takes part in the programmed cell death (PCD) of cells that surround the entry site of the pathogen infection PPO1, PPO2, and PPO3), which codes for a group of copper-containing enzymes induced by JA that catalyses the oxidation of diphenolic compounds into quinones that cross-link or alkylate dietary proteins and thereby decrease the nutritional value for herbivorous insects or pathogens Samples of P. trichocarpa v2.0, publicly available in Phytozome (http://www.phytozome.net/poplar). The gene models used were: POPTR_0009s11280.1 (AOS5), POPTR_0005s18520.1 (CathL), POPTR_0004s17900.1 (GHf10), POPTR_0012s04070.1 (NFXL1), POPTR_0006s12870.1 (PAL1), POPTR_0011s10950.1 (PPO1), POPTR_0001s39920.1 (PPO2) and POPTR_0011s04710.1 (PPO3), respectively). Five of the genes  are intronless, whereas PAL1 has two exons and CathL and GHf10 have four exons each. An effort was made to sequence the entire coding region of all genes, but this was not achieved for GHf10, PPO2, and PPO3. For GHf10 this was due to a region of 500 bp of missing data in the middle of the gene, which was amplified in two, non-overlapping pieces. For PPO2 and PPO3, low quality sequence data at the 3′ and 5′ ends prevented us from obtaining the entire coding region of these genes. Sequencing of AOS5, CathL, NFXL1, PAL1 and PPO1 yielded the entire coding region and short sequences of the 3′ and 5′ UTRs. The length of the sequenced regions ranged from 1035 bp for GHf10 to 3454 bp for NFXL1 . Alignments were annotated on the basis of the corresponding gene from the P. trichocarpa genome sequence (available at http://www.phytozome.net/poplar.php).Gene fragments were amplified from diploid genomic DNA using polymerase chain reaction (PCR) and directly sequenced on a Beckman Coulter CEQ 2000XL capillary sequencer at Umeå Plant Science Center. Sequences were verified manually and contigs were assembled using the computer program Sequencher v4.0. Regions with missing or low-quality data were trimmed from all sequences. Multiple sequence alignments were made using Clustal W P. trichocarpa genome . We then obtained all ESTs from the experiments of Sterky et al. http://www.arborea.ulaval.ca/) from GenBank or from PopulusDB (http://www.populus.db.umu.se/) (ESTs downloaded on 2010-10-05). We used these EST resources to obtain “digital northern” analysis of all genes. These digital northern analyses were performed to verify that we had selected genes from the Populus genome that do show wound induction.The full-length coding regions for all eight genes were obtained from the annotated Populus, but the low sequence divergence seen in coding regions between different species in the genus suggest that this will not influence the estimation of expression profiles from the EST libraries Publicly available EST data sets can confound such digital expression analyses in a number of ways and several measures were taken to minimize these effects. First, ESTs are usually sequenced from only one end of the cDNA clone but in some cases EST clones are sequenced from both ends. To identify such cases, all clone IDs were retrieved and duplicate IDs were excluded from the analysis. Second, a relevant comparison of EST abundance is best made if ESTs originate from non-normalized cDNA libraries. Normalization will introduce a certain level of noise in the data set and will lead to an underestimation of mRNA abundance for highly expressed genes. However, it is unlikely that it should change the rank order of genes with respect to gene expression, and data from normalized data still provide semi-quantitative data on transcript abundance. We included data from normalized libraries but these have been labeled in subsequent analyses to aid in interpreting the data. The EST data sets used were further derived from a number of different species of Full-length coding sequences were filtered with the XBLAST program to mask out repetitive elements. Expression profiles were then obtained for all genes BLASTN searches against each EST data set using stringent matching criteria. Alignments were required to show at least a 90% identity across 100 bp to be recorded as a match, to avoid over-estimating transcript abundance of genes belonging to small gene families. The number of BLASTN hits was used as a proxy for expression levels and normalized by the total number of ESTs in each data set, to enable comparisons between different treatments and/or tissues. Because wounding has been shown to affect the expression of most genes involved in both primary and secondary metabolism http://www.ub.es/dnasp/). For analyses requiring an outgroup, a single sequence of the homologous region in P. trichcarpa was used. To provide an estimate of the distribution of genome-wide levels of polymorphism we used data from 77 loci corresponding to 76 unique genes evenly distributed throughout the genome of Populus. These reference genes were chosen without prior knowledge of their function Estimates of nucleotide polymorphism (segregating sites (S), nucleotide diversity (π)), between-species divergences, for synonymous and non-synonymous sites separately, and statistical tests of neutrality , were obtained using the computer program DnaSP v.4.50.2 (http://home.uchicago.edu/~rhudson1/source/mksamples.html). For each summary statistic the null distribution under neutrality was obtained from 104 simulations, conditioned on the original sample configuration and sequence length. The demographic history of P. tremula is characterized by a moderately strong bottleneck that occurred roughly 770 kya and to account for this, coalescent simulations were performed based on the bottleneck model described in Ingvarsson In order to evaluate significance statistics of Fay and Wu's H and Tajima's D, we performed coalescent simulations using Richard Hudson's ms program  vary across genes. The sliding window analysis of diversity at synonymous sites was performed using a 75 bp window that was moved in 8 bp increments across the gene, while the rest of the analyses were performed using a 250 bp window moved in 25 bp increments. Smaller window sizes yielded too little polymorphism in many windows, while larger windows increase the risk of averaging over too many sites with independent evolutionary histories, thereby reducing the power to detect regions with enhanced genetic differentiation. We used coalescent simulation to determine whether specific window segments showed significant deviations from neutral expectations. We simulated the same number of alleles that were present in our sample and simulations were run conditional on the number of segregating sites observed in each window. We performed simulations without recombination as we are simulating only short gene-segments. Furthermore, ignoring recombination is conservative with respect to identifying outlier-values for Tajima's D and Fay and Wu's H. For the KA/KS ratio we adopted a slightly different method to assess outlier windows. We calculated the 95% percentile from the distribution of KA/KS from the 76 control loci and used this value as a cutoff limit to assess which window-regions that show elevated KA/KS ratios. We regard the sliding window analyses as exploratory and as a way to identify regions worth studying in greater detail and we therefore do not correct for multiple testing in the analysis.Since LD extends only a few hundred bps in t-test, t = 3.07, p = 0.017, AOS5 which showed very low expression across all data sets we analyzed. This confirms that the majority of the genes we studied are indeed wound induced, showing higher gene expression following wounding.We compared gene expression across different tissues using digital northerns based on available EST data sets . AlthougAOS5, CathL, GHf10, NFXL1, PAL1, PPO1, PPO2 and PPO3  while only a part of the coding regions was obtained for GHf10, PPO2 and PPO3. Seven of the genes show substantial levels of polymorphism, with the number of segregating sites ranging from 38 to 87  and PPO2 have a significantly negative Fay and Wu's H . The other six genes  do not show any deviations from neutral expectations . An excess of intermediate frequency variants in the SFS, as indicated by a positive Tajima's ctations , with PPctations .PPO1, a region at the 5′ end of the coding region harbors an excess of both intermediate- and high frequency variants  have lower or equal divergence at synonymous sites compared to the genome-wide average, whereas the other five defense genes are showing a higher degree of divergence. At non-synonymous sites only PAL1 and NFXL1 exhibit a lower or equal degree of divergence compared to the genome-wide average  test, which compares the rate of divergence to levels of standing nucleotide polymorphism within species, also identifies PPO1 as a gene whichs show a significant deviation from neutral expectations with a -log10 neutrality index (NI) of 0.721 , suggesting again preferential fixation of non-synonymous mutations at PPO1. None of the other seven genes show any deviations from expectations in the MK-tests (None of the eight defense genes are showing KMK-tests .P. tremula). These genes are representative of the two main pathways that control wound-induced defense responses, the octadecanoid and the salicylate pathway AOS5) are wound induced , patterns of nucleotide polymorphism in the eight wound induced genes conform to neutral expectations, and most genes also show low KPPO1 and PPO2, show several signs that are indicative of the action of natural selection at these two genes. Both PPO1 and PPO2 show significant skew in their site frequency spectra, at least for parts of their coding regions. PPO1 show an excess of intermediate frequency variants across the entire gene , and the sliding window analysis of the SFS shows that this patterns is most pronounced in the regions close to the 5′ and 3′ ends of the gene. In addition, the 5′ end of the gene shows a clear excess of high frequency derived variants , we found that the 600 bp region with elevated KA/KS ratio and reduced synonymous diversity is located close to the end of the tyrosinase superfamily domain, the domain that contains the active site for all PPOs. This domain consists of a di-nuclear copper center, with two copper ions, each coordinated to three histidine side chains A/KS ratio and the deepest dip of the intraspecific diversity lies within the second histidine residue of the tyrosinase domain, hinting at the possibility that the possible target of selection might affect the enzymatic catalytic properties of PPO1. Further biochemical characterizations of the different allelic variants of PPO1 are required to test this hypothesis.When searching for conserved domains in PPO1, PPO2 does not show an elevated KA/KS ratio, with the exception of a short region in the 3′ end of the gene . The sliding window analysis also identified a region in the middle of the gene that shows no deviation from neutrality, but that is flanked on both sides by regions showing an excess of high frequency derived variants (PPO2 went through a recent selective sweep PPO2 (p = 0.107), but the MK-test only detects significant deviations from neutrality when the gene has been under long-term adaptive evolution. Interestingly, judging by the location of possible flanking regions showing significant deviations in the SFS, a putative target of selection in PPO2 would also be located in the tyrosinase domain of the protein, just as in PPO1.In contrast to the gene  in a domvariants - a sign Populus genome Populus both PPO1 and PPO2 have been shown to be wound-inducibe, with PPO1 being exclusively expressed in damaged leaves while PPO2 is primarily expressed in stems, petioles and roots The two genes showing signs of either long-term or more recent natural selection are members of the polyphenol oxidase gene family, a small gene family consisting of up to 10–12 different members in the PPO, involved in generalized defense, often show few signs of natural selection compared to genes involved in specialized defenses PPO genes in this study show patterns of selection that are reminiscent of the patterns found from trypsin inhibitor (TI) genes in PopulusPPO genes due to the highly selective nature of TI genes when targeting specific protases PPO proteins are not only resistant to proteolysis in insect guts, but that limited proteolysis actually is required to activate the protein from the latent form in which it is stored in plant leaves PPO1, and to a lesser extent also in PPO2, is located close to the active site of the PPO protein and also close to a region that has been speculated to be involved in controlling conditions under which the PPO protein becomes activated in the insect gut PPO1 and PPO2 are linked to natural selection in herbivores, preventing or reducing the activation of the PPO protein, and to counter-selection in plants for maintaining this function. Such a co-evolutionary scenario is clearly a speculation at this point and more data on both the functional consequences of mutations at sites targeted by natural selection and possible counter-selection in herbivores are needed before this question can be answered conclusively.Genes like PPO1, also long-term adaptive evolution. Our results support the notion that the majority of wound-induced genes are not under strong selection in Populus. However, the results show that some biochemical herbivore defenses, that are strongly induced following mechanical damage or insect attack, show signs of adaptive evolution in PopulusTo conclude, we have shown that patterns of polymorphism and divergence at most of the eight wound-induced genes we studied showed little deviation from neutrality. The exceptions a two polyphenol oxidase genes which both appear to have been the target of recent selective sweeps, and in the case of Table S1D and Fay and Wu's H and their simulated 95% confidence intervals for the sliding window analysis.Number of segregating sites, nucleotide diversity, observed values of Tajima's (PDF)Click here for additional data file.


The metal-induced matriptase autoactivation is suppressed by N-acetylcysteine, supporting the putative role of altered cellular redox state in metal induced matriptase autoactivation. Furthermore, matriptase knockdown rendered cells more susceptible to CdCl2-induced cell death compared to control cells. This observation implies that the metal-induced matriptase autoactivation confers cells with the ability to survive exposure to toxic metals and/or oxidative stress. Our results suggest that matriptase can act as a cellular sensor of the chemical environment of the cell that allows the cell to respond to and protect itself from changes in the chemical milieu.The ability of cells to rapidly detect and react to alterations in their chemical environment, such as pH, ionic strength and redox potential, is essential for cell function and survival. We present here evidence that cells can respond to such environmental alterations by rapid induction of matriptase autoactivation. Specifically, we show that matriptase autoactivation can occur spontaneously at physiological pH, and is significantly enhanced by acidic pH, both in a cell-free system and in living cells. The acid-accelerated autoactivation can be attenuated by chloride, a property that may be part of a safety mechanism to prevent unregulated matriptase autoactivation. Additionally, the thio-redox balance of the environment also modulates matriptase autoactivation. Using the cell-free system, we show that matriptase autoactivation is suppressed by cytosolic reductive factors, with this cytosolic suppression being reverted by the addition of oxidizing agents. In living cells, we observed rapid induction of matriptase autoactivation upon exposure to toxic metal ions known to induce oxidative stress, including CoCl Both the intracellular and extracellular chemical environments of a cell play important roles in physiological and pathological processes. To maintain an optimal chemical environment, cells have developed complex mechanisms to monitor and regulate factors such as pH, the concentrations of specific ions, and redox potentials that comprise the chemical milieu of these environments. The secretory pathway maintains a gradient of decreasing pH from near neutrality in the endoplasmic reticulum (ER) (pH 7.2), to mildly acidic in the Golgi (pH 6.7–6.0), to even more acidic within the secretory granules (pH 5.7–5.2) Ion channels and G protein-coupled receptors are the two major molecular mechanisms responsible for maintenance of the chemical environment of cells There is growing evidence that the activity of the type 2 transmembrane serine protease, matriptase, is tightly regulated by the cellular chemical environment The potential role of the chemical environment in the regulation of matriptase activation was initially suggested by the observation that matriptase activation was robustly induced when cells were exposed to a mildly acidic buffer In the current study, we set out to systematically analyze the impact of the major chemical components of the extracellular environment on matriptase activation. Our results reveal that matriptase zymogen activation is an autonomous process that can proceed spontaneously at physiological pH and that is accelerated by an acidic pH both in a cell-free setting as well as in living cells. Matriptase zymogen activation is also affected by chloride ion concentration and thio-redox state. Given the rapid induction within minutes of acidification and increased thio-oxidative potential, our current study suggests that the cells could utilize matriptase to sense and respond to alterations in the cellular chemical milieu.2.The human mammary epithelial cells 184 A1N4  ® Chemiluminescence Reagent Plus .Cells or the insoluble fractions of cell homogenates were lysed in a buffer consisting of 1% Triton X-100 and 1 mM 5,5′-Dithio-bis-(2-Nitrobenzoic Acid) (DTNB) in phosphate buffered saline (PBS). The Ellman's reagent DTNB was added to the lysis buffer to prevent cleavage of the disulfide linkage that connects the serine protease and the noncatalytic domains of activated matriptase Matriptase zymogen activation assays were conducted in both the “cell-free” and whole living cell setting using a similar approach. In the cell-free setting, cell homogenates were collected by scraping the cultured cells from the culture dish in PBS and homogenized using a Dounce homogenizer. Aliquots of the homogenate were centrifuged at 12,000 rpm for 5 minutes using an Eppendorf benchtop centrifuge. The pellets of insoluble cell fragments were re-suspended with buffers of different pH and incubated at room temperature for designated times to allow activation to occur. Matriptase activation was terminated by adding 20% Triton-X 100 to a final concentration of 1%. For induction of matriptase zymogen activation in living cells, the cells were washed with PBS three times in 60 mm dishes and incubated with the activation inducers, such as buffers of different pH or modified IMEM containing metal ions at room temperature for the designated times. The cells were then harvested by scraping in PBS. After centrifugation, the cell pellets were lysed in 1% Triton X-100 and 1 mM DTNB in PBS.Cells were homogenized in ice-cooled 150 mM phosphate buffer pH 6.0 with a Dounce homogenizer. The homogenates were centrifuged to separate the insoluble cell fractions from the cytosolic fraction. The cytosolic fraction was dialyzed against 150 mM phosphate buffer pH 6.0 using dialysis tubes . The insoluble fractions were incubated with the cytosolic fractions  at room temperature for 20 min. After the incubation, 20% Triton-X 100 was added to the samples to adjust them to 1% Triton-X 100 to stop further matriptase activation. To test the impact of DTNB, oxidized glutathione (GSSG), or N-ethylmaleimide (NEM) on the cytosolic suppressor, these reagents were added to the cytosolic fractions to the indicated concentration before incubation with the insoluble fractions.184 A1N4 cells were transduced with lentiviral vectors expressing either a non-targeting control shRNA sequence (NT) or shRNAs targeting the human matriptase gene (MTP) . The MTP shRNA targeting sequence was5′-CCGGCAATGACTTCACCTTCGACTACTCGAGTAGT<@?show=[fo]?>CGAAGGTGAA GTCATTGTTTTTG-3′ and has been used and validated by us in previous studies 2 was added to the cells, with final concentrations ranging from 0 to 90 μM. The cells were incubated in CdCl2 for 24 hours. Cell survival was determined by estimating the number of cells remaining on the plate using the crystal violet assay. Briefly, the medium was removed from the wells and the cells were fixed and stained with crystal violet by incubation for 10 minutes with 0.52% crystal violet in 25% methanol. The excess stain was removed by washing the plate in deionized water three times after which the plate was dried. Staining intensity was measured by dissolving the crystal violet in 100 mM sodium citrate in 50% ethanol and measuring optical density at 570 nm which is proportional to cell number.Both matriptase knockdown (MTP KD) and control non-target (NT) 184 A1N4 cells were seeded in 96-well plates at 20,000 cells per well. Sixteen hours later, CdClMatriptase is synthesized as a zymogen that undergoes autoactivation to acquire enzymatic activity We sought to examine the impact of a lower pH environment on the activation of matriptase in the insoluble fractions by incubating the homogenates with buffers of decreasing pH. When the insoluble fractions were exposed to phosphate buffer at pH 7.0, it took approximately 10 min for the onset of matriptase activation, and about 20 min to reach the maximal matriptase activation plateau . When thin vitro occurs in intact cells. To this end, 184 A1N4 mammary epithelial cells were exposed to 150 mM phosphate buffer at pH 7.4, 7.0, 6.5 or 6.0, and the level of matriptase activation was assessed at various time points by Western blotting  is responsible for mediating the attenuation of matriptase activation, we compared the effects of various salts containing either sodium or chloride ions on matriptase activation in acidic conditions. Both sodium and magnesium chloride inhibit matriptase activation in a dose-dependent manner  to prevent premature intracellular matriptase zymogen activation. As described above , which is known to induce rapid ROS production 2 induced cellular matriptase activation in a dose-dependent manner  and/or depletion of cellular sulfhydryl reserves t manner . We next 2 hours . We founhours  was also able to induce matriptase activation and was more potent in this respect than CoCl2, producing a similar level of activation at lower concentrations  184 A1N4 cells using a matriptase targeting shRNA construct, as well as control non-targeted (NT) 184 A1N4 cells using a nonspecific shRNA construct. We verified by immunoblot assay that matriptase protein levels in the MTP KD cells were indeed significantly lower than in the control NT cells (2 (0 to 90 μM) for 24 hr. Representative results from one of our experiments are shown in 2 for the MTP KD cells was 26 μM, compared to 39 uM for the NT cells. Four independent experiments were conducted, and the average LD50 for MTP KD cells was 23±2 μM; while the average LD50 for the control NT cells 32±5 μM; t-Test, ρ = 0.011). In other words, the MTP KD cells were almost 40% more susceptible to CdCl2 than the control NT cells.CdClNT cells . We thenIn the current study, we show that pH, thio-redox state and chloride ion concentration impact the ability of matriptase to undergo autoactivation, both in a cell free system and in living cells. Interestingly, all three of these factors are considered to be major indicators of the cellular chemical environment that are altered by numerous biological processes. As changes in these cellular indicators also impact matriptase activity, we propose that matriptase may function as an interface between cells and the surrounding chemical environment. This unusual feature could confer matriptase-expressing cells with the ability to sense, respond to, and survive in unfavorable environments such as in the presence of toxic metals.While living cells resemble the insoluble fraction in the cell-free system with respect to acid-accelerated matriptase zymogen activation, there were some notable differences. At physiological pH, matriptase activation takes longer in living cells than it takes in the cell-free system, and the proportion of total matriptase that undergoes activation is less in living cells at the plateau phase compare  and 2A. The molecular basis for the rapid and robust activation of matriptase in response to pH, thio-redox state, as well as for the inhibitory effect of chloride ion concentration, remains to be elucidated. It has been previously reported that the intrinsic proteolytic activity of the matriptase zymogen is enhanced in a mildly acidic buffer The molecular mechanism underlying the effect of the thio-redox state on matriptase activation is also not completely understood. One hypothesis is that altered redox state might result in the stabilization of conformations of matriptase that favor the activated form. Matriptase contains 20 pairs of disulfide bonds The mechanism through which halogen ions inhibit matriptase activation may also be attributed to their potential impact on matriptase conformation. It should be noted that the hierarchy for matriptase activation inhibition by the halides follows the hierarchy of their sizes (iodide>bromide>chloride). These halogen ions serve as electron acceptors when associated in halogen bonds, with the hierarchy of increasing electron accepting potency also being iodide>bromide>chloride The role of matriptase in many biological processes has been revealed through the identity of its substrates, such as hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), protease activated receptor-2 (PAR2), and prostasin. HGF activation appears to contribute to the oncogenic potential of dysregulated matriptase in the development and progression of squamous cell carcinoma In addition to expression on the plasma membrane, significant amounts of matriptase are located intracellularly, likely in the synthetic pool and the secretory pathway Matriptase autoactivation is dependent on the cellular chemical environment, with changes in the environment resulting in rapid and robust activation. This unique characteristic of matriptase must be tightly regulated since inappropriate matriptase activation could be extremely damaging to cells if it occurred prior to the delivery of the enzyme to the cell surface. En route to the plasma membrane, matriptase zymogen must traffic through the secretory pathway from the ER, to the Golgi to secretory vesicles, each of which has distinct pH and redox conditions. It is possible that cells have evolved several systems to regulate matriptase so that even if conditions that favor matriptase activation are encountered prematurely, a second mechanism to counteract matriptase activation can suppress it. For instance, in the ER, redox conditions are favorable for matriptase autoactivation, however, this may be sufficiently counteracted by the unfavorable neutral pH. Similarly, in the secretory vesicles, the favorable acidic pH might be negated by the unfavorable reductive potential. On the cell surface, the physiological concentration of chloride ions may serve to moderate excessive matriptase activation potentially induced by extracellular acidosis. Furthermore, in most matriptase-expressing cells, HAI-1 is co-expressed with matriptase at considerable molar excess In summary, the results presented in this study suggest that matriptase, a widespread cell surface serine protease, can function as an interface between cells and their local chemical environment. Matriptase undergoes spontaneous autoactivation at a slow rate in a physiological pH environment, and much of this autoactivation can be suppressed by sodium chloride at physiological concentrations. Increased acidity in the cellular environment accelerates matriptase autoactivation, a mechanism that may be important for certain pathological processes such as cancer. Matriptase autoactivation is also under the tight control of the thio-redox state of the cell environment, with matriptase activation favored under oxidative conditions. The reductive milieu of the cytosol could provide a mechanism to prevent premature matriptase zymogen activation while the protease is trafficking through the gradually acidifying secretory pathway en route to plasma membrane. When the redox balance is altered, such as after exposure to transition metals, matriptase is activated, which may facilitate cell survive under such conditions. Through these positive and negative chemical modulators of matriptase zymogen activation, cells can regulate pericellular proteolysis and respond to the very dynamic cellular chemical environment in a timely and tightly-controlled manner.


Obese or overweight children have an increased risk for chronic diseases. Targeting diet and exercise in schools could help prevent childhood obesity. We have previously shown the effectiveness of a 90-minute nutrition program in improving elementary school students’ food-label literacy. The objective of this study was to investigate the effectiveness of a 45-minute version of the program.We conducted a pre–post study in a public school district, with no control group. We provided teacher training and program materials. Participants were 5th-grade students in 5 schools who had parental consent and were willing to take part. We condensed the program to a 45-minute lesson with a presentation and hands-on activity. The lesson showed students why and how to make healthful food choices based on Nutrition Facts panels and ingredient lists. The district’s physical education teachers taught the lesson. The primary outcome measure was food-label literacy .A total of 212 students completed pre–post measures. Following program delivery, we observed a significant gain of 16.2 percentage points in scores overall, ranging from 4.3 percentage points to 23.6 percentage points among schools. Results were similar to those achieved with the 90-minute program.The condensed nutrition program improved students’ food-label literacy while requiring a minimal allocation of time. Further studies in other school districts would be useful. In 2009–2010, nearly 17% of children and adolescents aged 2 through 19 years were obese, and approximately 32% were either overweight or obese . Thus, tTargeting modifiable risk factors such as diet and exercise early in life is likely to contribute to the prevention of childhood obesity. Schools can become an effective environment for obesity prevention, because no other institution has as much continuous and intensive contact and influence on children during the first 2 decades of life . SchoolsEnhancing children’s awareness of and interest in more healthful foods is a potential part of the solution –10. One This was a pre–post study conducted during March through June 2010, after approval by the Griffin Hospital Institutional Review Board (IRB) (IRB study no. 2010/03) by the Yale–Griffin Prevention Research Center (PRC) in collaboration with the New Haven Public Schools (NHPS) District Wellness Committee. The PRC provided teacher training and program materials, developed informed consent materials, and collected pre–post data. The NHPS district was responsible for teaching the program, scheduling data collection, distributing informed consent materials, collecting opt-out forms, and informing the PRC about students who had parental permission to participate in the study. To reduce burden on the district in scheduling data collection, no control schools were used for comparison.The 5 participating schools were a subset of the district’s elementary/middle schools selected by the NHPS District Wellness Committee and whose principals agreed to participate . Of the Because the program was incorporated into the health curriculum, all 5th-grade students in participating schools who were present when the Nutrition Detectives lesson was taught took part in the lesson. Study inclusion criteria were 1) being a 5th-grade student in a participating school; 2) having parental consent to complete the quizzes; 3) willingness, availability, and ability to complete the quizzes; and 4) presence at school on the day and in the room where the lesson was taught. Exclusion criteria were students who lacked parental consent, were unwilling/unavailable/unable to complete the quizzes, or were not present for the Nutrition Detectives lesson.Information sheets and consent forms approved by the Griffin Hospital IRB were sent home to parents or legal guardians of 5th-grade students in study schools. The information sheets provided instructions for parents to opt out of having their child participate by a certain deadline date. All documents and participant information were obtained for research purposes only and strictly maintained to ensure confidentiality. All student assessment data were identified by number, not name.A few months before this study, the PRC had partnered with the NHPS District Wellness Committee to adapt the 90-minute nutrition program to a 45-minute lesson, to serve as the third of 4 nutrition lessons taught by physical education (PE) teachers in all of the district’s elementary schools. The topics of lessons 1 through 4 were 1) how food affects our bodies; 2) the Food Guide Pyramid; 3) making healthful food choices by using nutrition information on food packages and 5 detective “clues”; and 4) balancing caloric intake and expenditure to maintain a healthy body weight. The nutrition lessons were part of a 5th-grade health curriculum developed by the district and community partners that also included lessons on physical activity, reproductive health, and substance abuse. PRC staff had taught the original 90-minute program to 315 5th-grade students in 8 NHPS schools as part of an unpublished feasibility study  during the 2007–2008 and 2008–2009 school years and used the same tool to evaluate food label literacy.http://www.davidkatzmd.com/nutritiondetectives.aspx), was developed in 2002 and has been periodically updated to reflect changes in food products and refined in content and design according to feedback from teachers and students to strengthen the program’s messages and reduce the potential for misconceptions. Nutrition Detectives conveys the idea of a link between food choices and health, in addition to information on how and what nutritious foods to choose. The key concepts are that “we are what we eat”; students can become “detectives” to learn about the foods they eat; and they can apply 5 “clues” to determine whether food products are healthful (“clued-in”) or less healthful (“clue-less”) choices. The objectives are to enable students to 1) explain why making informed food choices can benefit their health; 2) recognize deception on packages of food products; 3) identify 5 clues to make healthful food choices  demoP < .01) in the Independence School District in Missouri  the schools that elected to participate in the evaluation of the lesson. In May 2010, NHPS PE teachers taught the lesson in 5th-grade classrooms as part of the curriculum.The primary outcome measure was food-label literacy and nutrition knowledge on healthful food choices. Children’s ability to choose “better-for-you” foods was assessed by using a previously validated (15) standardized test instrument (Food Label Quiz) that uses Nutrition Facts panels and ingredient lists from 5 categories of food products  discussed in the Nutrition Detectives program. The quiz consists of 10 questions. Each question asks students to select the more healthful of a given pair of food products on the basis of the nutrition information for each product. The selection of each better-for-you choice is based on one or more of the program’s 5 clues.PRC research staff administered the Food Label Quiz in participating schools. Pretest and posttest quizzes were scheduled for days and times convenient to school schedules and relative to when the Nutrition Detectives lesson was taught in each school. When the PRC researchers arrived to collect data, the teachers identified the students who would be completing the quizzes. Because of the way informed consent was managed by the schools, the researchers were unable to determine the number of students who lacked parental consent, were unwilling/unavailable/unable to complete the quizzes, or were not present for the Nutrition Detectives lesson .t tests were used to evaluate the change in the percentage of correct responses of Food Label Quiz scores from pretest to posttest. Student t tests were used to assess difference in students’ performance between the 90-minute program and the 45-minute lesson. In all analyses, a 2-tailed α of less than .05 was considered statistically significant. This study was powered at 80% to detect a minimal detectable difference of 18 percentage points between pretest and posttest scores.Data were entered in an Excel  spreadsheet and analyzed by using SPSS for Windows version 15.0 . The Food Label Quiz was scored as the percentage of correct responses. Paired P < .001) on the quiz between pretest and posttest measures (P = .04).A total of 212 students — 89 boys (42.0% of participants) and 123 girls (58.0%) — completed both the pretest and posttest. There were 26 students (12.3%) from School A, 81 (38.2%) from School B, 49 (23.1%) from School C, 26 (12.3%) from School D, and 30 (14.1%) from School E. Mean baseline quiz scores ranged from 40.0% in School A to 59.0% in School C . After dmeasures . ImproveP = .92) and 67.6%  in the posttest . The students in both studies also showed similar gains in food-label literacy scores . The overall results are also similar to the 18.1 percentage-point pretest–posttest gain in scores shown in the evaluation of the 90-minute Nutrition Detectives program in the Independence School District using the same assessment tool  in the pretest  overall instructional focus  students’ socioeconomic status; 3) percentage of gifted or talented students 4); percentage of minority students; or 5) overall level of student academic achievement. For example, the 2 schools with the highest baseline food-label literacy scores and the greatest gain in scores (School B and School C) were magnet schools with the highest proportions of gifted and talented students, the lowest proportions of minority students, and the highest proportions of students meeting state goals for academic achievement. In contrast, the 2 schools with the lowest baseline scores and small nonsignificant gains in scores (School A and School E) had lower proportions of gifted and talented students, the highest proportions of minority students, and lower proportions of students meeting state academic achievement goals. An exception was School D, which had a significant gain in food-label literacy scores but had a low proportion of gifted and talented students, a high proportion of minority students, and a low proportion of students meeting state academic achievement goals.Other possible explanations for differences in scores include differences in the lesson delivery setting, PE teachers’ styles of teaching, and students’ degree of readiness to learn the lesson. For example, in one school (School A) with only a small gain in scores, the PE teacher taught the Nutrition Detectives lesson in the gymnasium rather than the classroom and combined students from all classrooms for the lesson; in addition, student disciplinary issues led to postponement of the lesson until a day when students were more cooperative.Overall, the improvement in food-label literacy scores after the lesson was greater among girls (19.1 percentage points) than among boys (12.1 percentage points). However, this is not surprising given that throughout elementary, middle, and high school, girls tend to earn higher grades than boys in all major subjects (16).Our study suggests that schools can take a lead in offering food literacy education that will enable children to identify more healthful food choices at school, at home, and in other settings. If the Nutrition Detectives program is used to achieve this goal, it appears that either the original 90-minute program or the condensed 45-minute version of the program could be used with similar success. Whereas the 90-minute version is already designed to fit well into school curricula without requiring a great deal of time, the 45-minute version could provide an additional option for schools with a crowded curricula.This study has several limitations. The extent to which the results can be generalized can be questioned because the study group was limited to 1 school district and to schools within the district that chose to participate. The self-selection of schools to participate may have been due to a higher level of motivation to offer and assess the effects of nutrition education, which could bias the results toward the null. There was no control group for comparison in evaluating the 45-minute lesson. The differences in score improvements among schools could be attributed to differences in instructional focus, demographic makeup of students, academic achievement levels of students, or inconsistencies in program delivery among schools.Despite these study limitations, the 45-minute Nutrition Detectives program appears to be an effective means of teaching elementary school students to identify healthful food choices while requiring minimal time in the school curriculum. Compared with the full-length Nutrition Detectives program, it requires less time and fewer resources to implement. Other schools can likely use this program to achieve similarly positive results, although the program may require academic readiness for students to best comprehend and apply the program’s content. Further confirmation of the condensed program’s effectiveness, either alone or in combination with other health programs, could be obtained through a randomized controlled trial with other school populations.


This present study was aimed to investigate the localizable diagnostic value of magnetoencephalography (MEG) combined with synthetic aperture magnetometry (SAM) in childhood absence epilepsy (CAE). Thirteen CAE patients underwent MEG detection at resting state and after hyperventilation, and then the epileptic foci were located by SAM. In the thirteen CAE patients, epileptic foci were found in five cases (38.5%), and they were all located in the bilateral frontal lobe, suggesting that the frontal lobe in some CAE patients may serve as the epileptic foci. Our findings indicate that MEG combined with SAM could be of diagnostic value in localizing the epileptic foci in certain CAE patients. Childhood absence epilepsy (CAE) is a common form of idiopathic generalized epilepsy (IGE), accounting for approximately 5%-15% of all childhood epilepsiesMagnetoencephalography (MEG) is a functional cerebral imaging technique that non-invasively records extracranial magnetic fields generated by the electrical activity of the brainCurrent absence epilepsy studies focus on aspects of animal models, genetics or EEG. However, MEG detection combined with SAM analysis in localization diagnosis of CAE has not been previously reported. Therefore, this study focused on MEG detection combined with SAM analysis of children with absence epilepsy in locating epileptic foci in order to investigate the localizable diagnostic value of MEG combined with SAM in CAE.Thirteen subjects were recruited from the Department of Neurology of Nanjing Children's Hospital from October 2008 to June 2009. Everyone of them met the CAE diagnostic criteria of the International League Against Epilepsy (ILAE) in 2001All children were required to stop taking anti-epileptic drugs two days before their MEG detection as well as staying up late and getting up early so as to reduce sleeping time. During the examination, to obtain children' cooperation, all subjects were well informed to act in the following ways during the detection: keeping quiet, being physically relaxed, staying still especially with the head, trying to avoid teeth clenching, frowning, swallowing and other facial movements except for hyperventilation movements during hyperventilation trials, keeping eyes slightly closed, and avoiding blinking and eye movement.CTF-275 guided by the whole head type magnetoencephalography system  was employed for MEG detection. A whole head 275-SQUID sensors MEG device simultaneously measured the corresponding induced magnetic fields. The head position was determined with localization coils at the nasion and preauricular fiducial points. Before and after each MEG recording, these points were localized by detecting the magnetic signals transmitted by the three coils. The two localizations were compared to check the head movement of each participant during the experimental session. In the direction perpendicular to the scalp, the brain magnetic field was measured and the magnetic source signal was collected. The information was stored in the computer. The coordinate system was set in each child using the digitizer. The rightward direction through the points before the two ears was set as positive in X-axis, and the forward direction from the bridge to tip of the nose was set as positive in Y-axis, and Z-axis was perpendicular to the X- and Y-axis, with the upward direction being its positive direction. While MEG signal was acquired, data were digitally filtered at a bandpass width 0.03-3.00 Hz in off-line data analysis and digitized at a sampling rate of 1,200 Hz. For the signal acquisition process, spontaneous, cerebral activities were recorded at an eye-closed resting state for approximately 2 min, for a repetitive 10 times, followed by a 3-min hyperventilation trial, and a 2-min recording.Anatomical images were obtained during a separate session with a 1.5-T Sigma magnetic resonance imaging (MRI) scanner for each patient. All patients underwent cranial MRI positioning: Head position was controlled using three coils placed at the left and right preauricular points and the nasion, serving as reference landmarks for anatomical MRI registration. In order to co-register the MRI and MEG data, three fiducial markers were placed on the MRI at the same locations used during MEG data acquisition. With sponge pad to get the head fixed, superconducting MRI instrument  was applied and quadrature head coil was adopted. T1-SPGR axial images were acquired through line 3D-SPGR sequential scanning. Scan parameters were as follows: field of view (FOV) = 24×24×16.2, matrix = 256×256×54, repetition time (TR) = 33.333 ms, echo time (TE) = 9 ms, inversion time = 0, number of averages = 1, imaging frequency = 63.849,208.The scalp data used for source reconstruction were the raw MEG data of reference and seizure periods. Raw MEG data was transferred to the MEG workstations. Software designed by Canadian CTF Company was used to superpose MEG physiological data on MRI anatomical information to produce magnetic source imaging (MSI). MSI was defined as the integration of functional data derived from MEG recordings with structural MRI. MSI showed functional and structural information in a single image. Magnetic source signals within the frequency range of 20-70 Hz were selected and automatically calculated by SAM analysis. Head model standardizing was then applied to make sure that the processed signals were compatible with each participant's MR surface. As a result, the magnetic signal source was displayed in the corresponding parts of MRI images so as to form the ultimate brain function image.A total of 13 subjects met our inclusion criteria and were recruited for the current study. They included 3 males and 10 females. The subjects aged 3 years 2 months to 12 years with an average of (8.4±3.2) years. Their course of disease was 3 months to 3 years, (1.6±0.9) years on average. Seizure onset ranged from 5 to 20 times per day. The seizure duration ranged from 6-30 s. All the diagnosed patients post-release accepted the medical treatment of sodium valproate tablets or Depakine syrup. The seizure frequency was significantly reduced, and the remaining seizures in children were generally 2 to 3 times/d, or every 2 to 3 d for 1 up to 10 attacks per day with each episode lasting 6-10 s. One of the cases was nearly 1 y with no further episodes before MEG detection.MEG and MRI information of all thirteen subjects was successfully obtained. However, the hyperventilation operation of one subject was not standardized. Thus, we did not capture the magnetic source signal of hyperventilation, which though did not affect the results. According to the SAM analysis, epileptic foci located in the bilateral frontal lobe were found in five patients, while no clear localization of epileptic foci was detected for the remaining eight children. The positive rate of MEG detection combined with SAM analysis in locating the epileptic foci of CAE was 38.5%. The five patients (two boys and three girls) aged between 6 years 8 months to 12 years with an average age of (8.4±3.2) years. Their course of disease ranged from 6 months to 20 months with an average of 14 months. Still having relatively high seizure frequency, most of them failed to be treated effectively. Three of them had an onset of 6 to 10 times per day. The other two had 3 to 5 times per day and no further attack for one year, respectively.According MEG detection combined with SAM analysis, the magnetic source waveform and possible epileptic foci location magnetic source imaging map at rest and after hyperventilation of the children with absence epilepsy are shown in MEG performs noninvasive functional imaging by recording the magnetic flux on the head surface associated with electrical currents in activated sets of neurons. MEG has rapidly evolved in the last two decades because of the introduction of whole head systems and advances in computer technology. MEG is now the imaging modality of choice where a precise and high degree of localization is required. MEG can be used to localize the primary sensory cortices , areas involved in receptive language function, and the irritative zone in epilepsy patients, and identify children with anomalous language developmentRecently developed SAM is a new MEG signal analysis introducing a high performance spatial filtering technique. SAM is not an inverse solution like dipole analysis, but rather an adaptive beamformer for estimating source activity at each selected voxel inside of the brain. SAM can estimate source changes as a function of time, or power changes subjected to statistical analysis from the non-averaged raw MEG dataSo far, possible mechanisms for childhood absence epilepsy have not been fully clarified. There were several main theorieset al.et al.et al.et al.et al.Roche-Labarba This study combined MEG detection with SAM analysis identified some part of the brain in children with absence epilepsy may be the epileptogenic zone; the epileptic foci were all found in the frontal, further indicating that the frontal lobe plays an important role in absence seizures. Our results were consistent with the “focal cortical theory” of absence epilepsy as well as findings of EEG researches on childhood absence epilepsy, which showed bilaterality.The present study indicated that the seizure frequency of patients whose epileptic foci were found by MEG detection was higher than those who tested negative, indicating that the higher the frequency of attacks was, the more positive rate of epileptogenic zone was. Many researches emphasize the important role that the hypothalamus plays in absence epilepsyIn summary, with MEG detection combined with SAM analysis, this study found that children with absence epilepsy may have epileptogenic foci, part of which in the bilateral frontal lobe. The frontal lobe plays an important role in the occurrence and development of CAE. MEG detection combined with SAM could be of a certain diagnostic value in localizing the epileptogenic foci of CAE; however, our study could not identify the origin of epileptic foci of childhood absence epilepsy. Further dynamic study is needed to ascertain the problem. This study also provided an objective basis for the assessment of severity and the effects of drug treatment of CAE. We believe that along with improvement in MEG detection technology and with advancement in analyzing software, researches can go beyond the preliminary epileptogenic foci localization and the pathophysiological mechanisms of childhood absence epilepsy, such as the origin of epileptic seizure foci, and localization and propagation path will be better understood.


The zebrafish truly seems to be coming of age. These powerful resources invoke the question of whether zebrafish can be increasingly used to model human disease, particularly common, chronic diseases of metabolism such as obesity and type 2 diabetes. In recent years, there has been considerable success, mainly from genomic approaches, in identifying genetic variants that are associated with these conditions in humans; however, mechanistic insights into the role of implicated disease loci are lacking. In this Review, we highlight some of the advantages and disadvantages of zebrafish to address the organism’s utility as a model system for human metabolic diseases.The zebrafish research community is celebrating! The zebrafish genome has recently been sequenced, the Zebrafish Mutation Project (launched by the Wellcome Trust Sanger Institute) has published the results of its first large-scale ethylnitrosourea (ENU) mutagenesis screen, and a host of new techniques, such as the genome editing technologies TALEN and CRISPR-Cas, are enabling specific mutations to be created in model organisms and investigated  Genome- disease . Can simin vivo. In combination with this, zebrafish are genetically tractable, amenable to large-scale forward genetic approaches and, as of 2013, their fully sequenced genome is available , and alleles are archived and can be requested from the Zebrafish International Resource Center (ZIRC).The Zebrafish Mutation Project (ZMP) was launched in 2011 as an initiative to create a knockout allele in every protein-coding gene in the zebrafish genome. The project involves sequencing of exon-enriched DNA from first-generation offspring (F1) produced from a mating of an ENU-mutagenised male with a wild-type female and then identification of induced mutations using a modified version of the 1000 Genomes Project variant-calling pipeline . Each alAn exciting new development is the establishment of a transcript counting (TC) pipeline to complement the morphological analysis. The TC identifies differentially expressed transcripts between mutant embryos and sibling controls using a 3’ end based RNAseq protocol. This analysis is limited to those mutant alleles that show a morphological or behavioural phenotype. In vitro studies cannot recreate the complexity that exists in vivo, so whole-animal approaches are required to study metabolism as it plays out in a multicellular context. Zebrafish are a good model in which to study metabolism because they possess all the key organs required for metabolic control in humans, from the appetite circuits that are present in the hypothalamus, through to the pancreas and insulin-sensitive tissues .Metabolic control and regulation of whole-body energy homeostasis involve a complex interplay between multiple organs and endocrine signals to carefully balance energy intake, utilisation and storage. Ucp1) knockout mouse. Ucp1 is a mitochondrial protein that is specifically expressed in BAT and is required for the dissipation of energy and production of heat by non-shivering thermogenesis. Ectopic expression of Ucp1 in skeletal muscle prevents diet-induced obesity and insulin resistance in mice , and brown adipose tissue (BAT) depots have not been identified. Therefore, the study of pathways that are activated by non-shivering thermogenesis, for example the β-adrenergic system, will be more limited in zebrafish than in mice. On the other hand, most standard animal facilities house small rodents at an ambient temperature substantially below the temperature associated with thermoneutrality in mice; thus, metabolic analysis is routinely carried out using animals that are chronically challenged by cold stress. This condition induces a range of physiological responses – such as increased food intake, metabolic rate and sympathetic activity – that could mask the metabolic phenotype under analysis . The dra in mice , and it ody mass . Howeverody mass . This hiMC4R) are the most common genetic cause of monogenic, or single-gene, obesity  in awake, freely behaving zebrafish. The authors observed a bioluminescent signal from the reporter that was associated with periods of increased locomotor activity, in line with the role of this neuronal population in regulating arousal, wakefulness and appetite  that carry out several other functions in addition to lipid storage  promoter drives expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis, and this line has been previously used in conjunction with time-lapse multiphoton laser scanning microscopy to directly observe angiogenesis  commitment to the adipogenic fate by mesenchymal stem cell progenitors and (2) induction of the adipocyte gene expression programme, which drives terminal differentiation. The pathways regulating the fate choice between adipocyte, osteoblast, myocyte and chondrocyte in step 1 remain unclear, and studies investigating this would benefit from the availability of novel fluorescent reporter lines for carrying out lineage-tracing studies elopment . It woulogenesis .FABP4 is fabp11a, and there is evidence that it is expressed in preadipocytes , CCAAT/enhancer binding protein α (C/EBPα), C/EBPβ and C/EBPδ . The zebipocytes  and adulipocytes ; thus, te depots ; in conte depots . The Glue depots . StudiesIn vivo imaging of changes in subcutaneous and visceral WAT depots in the zebrafish during development and under different metabolic challenges might help to provide insight into the intrinsic differences between these two adipose depots.The reasons behind the functional and biological differences between subcutaneous and visceral adipose depots are another unresolved issue in adipose biology. Subcutaneous fat is located beneath the epidermis, whereas visceral fat is located within the peritoneal cavity close to the endodermal organs of the abdomen. It is not clear whether it is the location of visceral fat and its exposure to particular paracrine or endocrine signals that determines its properties or if distinct developmental programmes drive subcutaneous and visceral adipocyte differentiation. It is also not understood why the health consequences of excess subcutaneous and visceral adipose tissue differ, with increased metabolic and cardiovascular risk being associated with the latter but not the former . In humaZebrafish have been used extensively to study pancreas development. The pancreas in zebrafish is comprised of exocrine and endocrine compartments connected by a ductal system to the digestive tract, as in mammals. Zebrafish pancreatic islets consist of a central core of insulin-producing β-cells surrounded by glucagon-producing α-cells, δ-cells (which produce somatostatin) and ε-cells (which produce ghrelin). The primary islet can be identified as early as 24 hours post-fertilisation (hpf) , and theZebrafish strains in which the pancreatic β-cells can be visualised with the use of a fluorescent reporter  and condQuantitative analysis of glucose homeostasis can be carried out in mature zebrafish via whole-blood analysis . Howeverpck1) promoter [Tg(pck1:Luc2)] (Tg(pck1:Luc2) line faithfully recapitulates the endogenous regulation of Pepck expression in mammals by demonstrating a robust increase in bioluminescence in Tg(pck1:Luc2) zebrafish in the fasting state and the appropriate response to pharmacological modulation. Gut et al. then went on to test 2400 bioactive compounds for their ability to modulate pck1 promoter activity. They also measured whole-larva glucose levels for 60 of these compounds. They identified two novel compounds capable of lowering blood glucose, but these compounds also paradoxically activated pck1 expression. Gut et al. reasoned that the increased pck1 expression observed was a compensatory gluconeogenic response to the low glucose levels induced by these molecules and suggested that the two molecules induced a fasting-like metabolic state. Investigation of one of these compounds found that it is able to protect against diet-induced hepatic steatosis and glucose intolerance in mice. Thus, by using this transgenic zebrafish line the authors were able to identify a novel drug that might be useful in the treatment of metabolic dysregulation. Future studies to uncover molecules that can lower Pck1 expression or that act synergistically with known modulators of pck1 will be of value.Recently, another transgenic zebrafish line was developed to facilitate the identification of new modulators of glucose homeostasis. Gut and colleagues generated a transgenic bioluminescent and fluorescent reporter line in which reporter expression is under the control of the phosphoenolpyruvate carboxykinase (1:Luc2)]  Fig. 1Bpck1 promThe zebrafish has also been used to study the process of β-cell neogenesis. In a recent study by Maddison and Chen, the authors found that continuous administration of both a glucose-rich diet and a lipid-rich diet for 8 hours can induce β-cell neogenesis in 6-dpf larvae . The gluZebrafish have also been used in a chemical screen to identify small molecules that are capable of inducing β-cell neogenesis . In thisuncoupling protein 2, pck1 and carnitine palmitoyltransferase 1A are readily detectable by quantitative reverse transcriptase PCR (qRT-PCR). The relative expression levels of these genes is similar to those seen in the adult mouse liver, suggesting that by day 6 the zebrafish liver is functionally mature, at least in terms of metabolism . Currently, apart from its association with obesity and insulin resistance, relatively little is known about what triggers and what drives the progression of NAFLD. In the zebrafish, the formation of the liver primordium and the differentiation of hepatocytes and cholangiocytes can be observed by 48 hpf . The deptabolism . A fattytabolism . Lipid utabolism . Screenitabolism . This stducttrip line, which harbours mutations in s-adenosylhomocysteine hydrolase (ahcy), a metabolic enzyme that is required to produce methyl donors for use in numerous biological processes. Mutant fish lacking ahcy develop hepatic steatosis and liver degeneration  can induce the same pathophysiology see , with thlar wall . Additiod an HCD .lyz:Ds Red line could also be used to study macrophage infiltration into adipose depots in zebrafish subjected to an overfeeding regime. This would allow in vivo monitoring of the early pathogenic processes of obesity, in particular the inflammatory response and its consequence on adipose tissue morphology and function. Many studies have implicated a role for inflammation in obesity . This line has been used to study the spatiotemporal gradients of H2O2 in wound response (Tg(phd3:EGFP), has also been generated. Experiments with this line revealed that fish with a mutation in the tumour suppressor gene pVHL display a systemic hypoxia response   . The zebs (PNDM)  (Table 1s (PNDM) .lkb1 mutant zebrafish exhibit an increased metabolic rate (The small size of the zebrafish does hamper some types of metabolic investigation; for example, intraperitoneal and intracerebroventricular injections, although possible in adult zebrafish , are teclic rate  (Table 1Another point to consider is the high genetic diversity observed between individual zebrafish genomes, even within fish of the same strain . To overCollectively, there are many exciting possibilities emerging that use zebrafish to model human metabolic disease. It is envisaged that the unique tools provided by the zebrafish system will advance our understanding of metabolic disease onset and progression, and identify currently unknown targets for disease treatment.