Type 2 Diabetes (T2D) and other chronic diseases are caused by a complex combination of many genetic and environmental factors. Few methods are available to comprehensively associate specific physical environmental factors with disease. We conducted a pilot Environmental-Wide Association Study (EWAS), in which epidemiological data are comprehensively and systematically interpreted in a manner analogous to a Genome Wide Association Study (GWAS).We performed multiple cross-sectional analyses associating 266 unique environmental factors with clinical status for T2D defined by fasting blood sugar (FBG) concentration ≥126 mg/dL. We utilized available Centers for Disease Control (CDC) National Health and Nutrition Examination Survey (NHANES) cohorts from years 1999 to 2006. Within cohort sample numbers ranged from 503 to 3,318. Logistic regression models were adjusted for age, sex, body mass index (BMI), ethnicity, and an estimate of socioeconomic status (SES). As in GWAS, multiple comparisons were controlled and significant findings were validated with other cohorts. We discovered significant associations for the pesticide-derivative heptachlor epoxide , and the vitamin γ-tocopherol . Higher concentrations of polychlorinated biphenyls (PCBs) such as PCB170  were also found. Protective factors associated with T2D included β-carotenes .Despite difficulty in ascertaining causality, the potential for novel factors of large effect associated with T2D justify the use of EWAS to create hypotheses regarding the broad contribution of the environment to disease. Even in this study based on prior collected epidemiological measures, environmental factors can be found with effect sizes comparable to the best loci yet found by GWAS. It is becoming clear that most non-communicable diseases are a result of a complex combination of genetic processes and the environment Perhaps the lack of impact of GWAS comes from not comprehensively considering environmental factors in disease. T2D provides an specific example: while genetics play a large role The current paradigm to search for the effects of multiple environmental chemicals utilizes molecular tools and model systems The EWAS consists of two methodological steps that have analogs in a GWAS. First, we consider a panel of 266 unique environmental assays, or environmental “loci”, measured across cases of diabetics and controls, yielding several environmental factors with significantly high association with T2D while controlling for multiple hypotheses. Second, we validate the associations by taking advantage of data from other cohorts in NHANES. With EWAS, we are able to hypothesize about new associations with T2D and reconfirm others. The results from EWAS can better inform about environmental factors that need to be measured in genetic studies to begin to provide us insight in regards to disease etiology.The NHANES is a publicly available dataset made available by the CDC and National Centers for Health Statistics and all participants have provided written consent.LBXVIE from 1999–2000 described “A-Tocopherol ug/dL” and variable with symbol LBXATC from 2001–2002 also described “a-tocopherol ug/dL”, we harmonized onto the single symbol for both, LBXATC.We associate 266 unique environmental factors with T2D status from the NHANES. We downloaded the all of the available NHANES data for 1999–2000, 2001–2002, 2003–2004, and 2005–2006 cohorts and collated corresponding variables across them. For example, if a variable with symbol There was a total of 543 factors in our EWAS, but not all factors were present in all cohorts: 111 factors measured in the 1999–2000 cohort, 146 from 2001–2002, 211 from 2003–2004, and 75 from 2005–2006. This comprised of 266 unique environmental factors in total, with 157 factors measured in more than one cohort. Using NHANES categorization, we binned factors into 21 “class” groupings in order to discern patterns among related groups of factors, analogous to chromosomal units in GWAS . DiffereTrichomonas vaginalis, an infectious pathogen found primarily in women.We omitted from our EWAS 73 factors that varied little across individuals in our sample. Specifically, we omitted those that had a majority (>90%) of the observations below a detection limit threshold as defined by in the NHANES codebook. We also removed factors that targeted a subset of the population, such as the test for T2D cases were individuals who had a fasting blood glucose (FBG) level greater or equal to 126 mg/dL, as advised by the American Diabetes Association (ADA) R with the survey module to conduct all survey-weighted analyses, and replicated our results with the STATA program We used survey-weighted logistic regression to associate each of the 543 environmental attributes with diabetes status while adjusting for age, sex, body mass index (BMI), ethnicity, and an estimate for SES . We acknExposures were captured either as continuous or a categorical variable. Most chemical exposure data arising from mass spectrometry or absorption measurements occurred within a very small range and had a right skew; thus, we log transformed these variables. Further, we applied a z-score transformation  in order to compare odds ratios from the many regressions. Similarly, for categorical variables, we made the definition of the referent consistent, defining them to be the “negative” results of the test.We calculated the false discovery rate (FDR), the estimated proportion of false discoveries made versus the number of real discoveries made at a given significance level, to control for type I error due to multiple hypotheses testing in associating the factors to disease status To improve our power, we used the four independent cohorts to validate significant findings . We consWe fit a final logistic regression model with data combined from multiple NHANES cohorts utilizing all measurements for a specific environmental factor, attaining an overall odds ratio. The covariates of the final model were age, sex, BMI, ethnicity, SES, and cohort. We computed new sample weights for the combined datasets by taking the average of the original sample weights as described by the NHANES analytic guidelines We conducted 3 secondary analytic tests for the validity and sensitivity of our final estimates. We first attempted to check for reverse causality, or association of exposure due to T2D diagnosis. Our second test attempted to take into account the lipophilic characteristics of the environmental factors found. Our last test attempted to take into account recent food and supplement consumption as a potential bias for exposure measures. For adequate sample size and ease of comparison to the final fit model, we utilized all available data combining multiple NHANES cohorts as the sample to conduct these three tests, described below.To attempt to account for reverse causality, we recomputed our models omitting those individuals who had been diagnosed with diabetes, defined here as those answering yes answers submitted on a NHANES health questionnaire (“Doctor told you have diabetes?”). We then refit our final models with individuals only showing biochemical evidence of T2D without actual diagnosis.Our second test attempted to account for the lipophilic chemical characteristics of our significant factors. Many of the environmental factors measured in NHANES absorb readily in fatty tissue; presence of fatty tissue is also associated with T2D and a potential confounder. Thus, we recomputed the models taking into account total triglycerides and cholesterol measured in blood specimen of participants.In our third test, we attempted to compare dietary and supplement consumption of cases or controls gathered from 24- and 48-hour recall and supplement use questionnaires reasoning that recent intake may confound exposure-disease association. The NHANES data contains amount of food components consumed based on the dietary recall available for all participants examined above. Specifically, amounts of food components are computed from the questionnaire using the United States Department of Agriculture (USDA) Food and Nutrient Database. Some of the vitamin and nutrient components included vitamin A, vitamin B-6, vitamin B-12, vitamin C, vitamin E, vitamin K, carotenes, lycopene, thiamin, riboflavin, niacin, folate, calcium, iron, magnesium, phosphorus, potassium, sodium, iron, zinc, copper, and selenium. Other components included macronutrients, such as protein, carbohydrates, fat, fiber, and cholesterol. The total amount of food components considered numbered 51 to 63 for the different cohorts. Further, the 2003–2004 and 2005–2006 cohorts contained both 24- and 48-hour recall data. Supplement use included count of consumption of vitamins, minerals, botanicals, and/or their mixture of them over the past month prior to the survey. To check for possible confounding by recent consumption, we added each food and supplement variable to the logistic regression models specified above and re-evaluated significance and effect size of the validated environmental factors. We coded food component content as the logarithm (base 10) of the amount entered. We coded supplement use as an integer count value. We acknowledge the potential of bias with the use of questionnaire data and a pre-determined database of food items but assumed it was a reliable proxy of consumption and behavioral data in lieu of other information.Using GWAS as inspiration, we systematically and comprehensively assessed the association of 266 unique environmental factors measured in the NHANES with T2D. Further, we validated these associations by observing the significance of factors in other NHANES cohorts.2 test) except for 2005–2006. Furthermore, there was a significant association between lowest SES (first tertile of poverty index) and T2D  in for the 1999–2000, 2001–2002, and 2005–2006 cohorts respectively. While we did not see a univariate association between ethnicity and T2D as diagnosed by FBG, we did confirm previously reported associations of ethnicity with T2D when stratifying by age and sex Several vitamins were found to have levels inversely associated with T2D. The first type included an antioxidant in the isoforms of β-carotene  of 0.6; 95% confidence interval (CI) 0.5–0.7; p<0.001). For the prototypical participant, high levels of trans or cis β-carotene equated to a 9% improvement in risk (15 vs. 6%) for T2D status. We were able to confirm the inverse association of β-carotenes seen in multiple epidemiological studies in Saudi Arabia We discovered a vitamin that increased risk for T2D. Surprisingly, γ-tocopherol, a form of vitamin E, was highly significantly and positively associated with T2D  in two cohorts  and nearly significant in the two others . For the prototypical participant, low levels of the γ-tocopherol equated to a 7% improvement in risk (13% vs. 6%). To our knowledge, this is a novel association between γ-tocopherol and T2D.We found organochlorinated pesticides and polychlorinated biphenyls (PCBs), both related pollutant factors, to be a highly positively associated with T2D. Among the PCBs, we specifically discovered PCB170 . The effect sizes in the individual cohorts for PCB170 were large . The models predicted up to 15% T2D risk for the prototype participant, more than double the risk of those with low concentrations of PCB170. The association between the class of PCBs with T2D has been well described within Native American Heptachlor epoxide, an oxidation product of the organochlorine pesticide heptachlor, was among the most highly associated factor  in our EWAS. The effect sizes in the individual cohorts were also large . The predicted probability for the prototypical participant with high levels of the pollutant was 7%, more than 2 times greater than those who had lower levels of this pollutant.We then attempted to test the validity of our final estimates by conducting 3 additional analytic tests. In the first test, we attempted to consider the possibility of “reverse causality” or differential exposure status due to T2D diagnosis. Second, we attempted to assess the effect of potential confounding bias due to the lipophilic characteristics on our final environmental factor effect estimates. Third, we attempted to assess the effect of recent nutrient and supplement consumption on our final effect estimates.To consider T2D diagnosis as a modulator of exposure, we removed all individuals who answered yes when questioned about a past history of diabetes in the NHANES health questionnaire (“Doctor told you have diabetes?”). We then recomputed the effect of exposure, adjusted for age, sex, SES, ethnicity, BMI, and cohort using the remaining individuals who showed biochemical evidence of T2D, but not carrying a diagnosis of T2D . For allWe next attempted to account for potential confounding bias of lipid levels. To assess the degree of possible confounding we refit the logistic regression adjusting for the logarithm (base 10) of total triglyceride and cholesterol levels in addition to age, sex, BMI, SES, ethnicity, and cohort . We did We then searched for differences in food and supplement consumption patterns between diabetics and non-diabetics for all 4 cohorts close to the time of survey derived from dietary recall and supplement use questionnaires. In comparing dietary nutrients, we did not observe a difference for any dietary nutrient except one between cases and controls. This exception included a lower total carbohydrate intake for diabetics versus controls, confirming that many diabetics may have known about their disease; specifically, the adjusted OR was 0.7  for a 10% increase in total carbohydrate consumption, adjusted for sex, age, ethnicity, SES, and cohort. We also observed an inverse association between any supplement use and T2D, with an adjusted OR of 0.6 , also consistent with our expectation of increased health awareness for those with T2D. However, we specifically could find no difference in consumption of carotenes or tocopherol (p = 0.85 and 0.2 respectively) between cases and controls, two of the validated nutrient factors found in our EWAS .Having observed some difference in consumption behavior between cases and controls, we then attempted to assess the influence of recalled dietary consumption on the environmental associations by recomputing the logistic regression models in presence of dietary and supplement use variables. Adding the new dietary or supplementary vitamin consumption variables did not attenuate the odds ratios (maximum change of 1–2%), nor did they lessen the strength of the associations for all of the 5 validated environmental factors described in We took a further step in assessing the strength of the environmental associations, adjusting for total triglycerides and cholesterol, any supplement use, and food intake simultaneously . SpecifiTo summarize of our secondary tests for validity, we concluded that reverse causality, recent food and supplement consumption, and total lipid levels did not substantially bias our effect estimates for the 5 validated factors. These tests were made possible by the extensive list of co-variates available in the NHANES.We have described a prototype Environmental-Wide Association Study (EWAS) and applied it to the study of Type 2 Diabetes (T2D), and validated many of our significant findings across independent cohorts and confirmed some of them through the literature. This pilot study is made possible by the examination of multiple cohorts present in the nationally representative NHANES dataset. We have rediscovered factors such as carotenes and PCBs with previously known association with T2D. Unexpectedly, we found higher levels of γ-tocopherol were associated with higher likelihood of T2D, independent of dietary intake. Of the components of Vitamin E, γ-tocopherol is the most abundant form in the US diet Another novel finding was in the significant association between heptachlor epoxide levels and T2D. Heptachlor is a pesticide; most uses of heptachlor were discontinued in the late 1980s While this study successfully demonstrates a prototype EWAS for T2D, this methodology can be reconfigured to measure the relationship between environmental factors and other disorders, such as obesity, lipid level abnormalities, hypertension, and/or cardiovascular disease. Methodologically, the EWAS takes inspiration from GWAS, which have been used to assess the correlation between genome-wide variability and disease.Like GWAS, the utility of EWAS lies in two types of hypothesis generation. First, the EWAS framework can be used to propose targets for further study. For example, many factors are correlated; some are similar structurally, such as the isomers of β-carotene, or co-occur in the environment, such as the PCBs and organochlorine pesticides. As we extend the GWAS analogy, these and other environmental factors could be said to be in “linkage disequilibrium” with each other. Just as is done for preliminary GWAS findings, EWAS findings can and should be used to identify further factors that may be in “disequilibrium,” for further detailed measurement and causal identification.We acknowledge that the measurement of 266 environmental factors is hardly a comprehensive study of the environment, but this is still a greater number of factors measured than the 30 microsatellite markers Another type of hypothesis we may generate is regarding the complex cause of disease. For example, we can now use an EWAS to hypothesize about “gene-environment” interactions and their relation to disease etiology. A future study addressing gene-environment interactions might be designed as a combination of both a GWAS and EWAS, where genetic variability is assessed simultaneously along with key environmental factors. While marginally more resource intensive, this type of study design could perhaps facilitate an explanation of disease causation that has eluded genomic-wide scans in addition and provide more accurate estimates of attributable risk.The EWAS allows for comprehensive and systematic analysis of the effects of the environment in association with disease on a broad scale. While many investigators have already utilized the NHANES to address the effect of a limited number of factors on disease, they do not provide a global view of these associations Limitations of this study remind us that measuring environment-wide aspects in relation to phenotypic states such as disease will be a difficult undertaking TCF7L2 gene While GWAS has allowed us to find novel variants associated with T2D of possible mechanistic importance and provided a model for a comprehensive study of the environment described here, associated variants have had only moderate effect sizes to date. Most of the risk loci identified with GWAS have small individual odds ratios, generally less than 1.3 FTO with T2D was nullified by accounting for BMI However, the correlated and dynamic nature of a multitude of environmental factors will hinder causal inference to a greater degree than GWAS It is imperative not only for epidemiologists and geneticists but also physicians and their patients to understand how multiple environmental factors may influence disease in a systematic fashion. Individuals are already demanding information regarding their “body burdens”, or the number and amount of chemicals present in their system, as evidenced by the “Human Toxome Project” In conclusion, the EWAS is a promising way to search and consider potential environmental factors as associated with disease or other clinical phenotypes. These results demand a rethinking and restructuring of studies that study disease in the genomics context. The time is ripe to usher in “enviromics” File S1The tables in this file describe the baseline demographics of the NHANES cohorts  per T2D status (Fasting Plasma Glucose >125 mg/dL). T2D cases were determined by a clinical threshold of ≥126 mg/dL fasting blood glucose. Unweighted total samples were similar across cohorts. Age and BMI were significantly different between each of groups and the proportion of sex was significantly different in 3 of the 4 cohorts . Low, medium, and high estimates of SES were computed by tertile of poverty index. Low SES (lowest tertile of poverty index) is also associated with T2D status in 3 of the 4 cohorts (3rd tertile SES referent group). Ethnicity was not seen to be associated with T2D status (“white” ethnicity referent group). * denotes unweighted number. All other statistics are weighted.(0.09 MB DOC)Click here for additional data file.Figure S1Trans-β-carotene vs. Diabetes Status for 2001–2002, 2003–2004, and 2005–2006 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (1.17 MB DOC)Click here for additional data file.Figure S2Cis-β-carotene vs. Diabetes Status for 2001–2002, 2003–2004, 2005–2006 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (1.01 MB DOC)Click here for additional data file.Figure S3γ-tocopherol vs. Diabetes Status for 1999–2000 and 2003–2004 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (0.83 MB DOC)Click here for additional data file.Figure S4Heptachlor Epoxide vs. Diabetes Status for 1999–2000 and 2003–2004 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (0.49 MB DOC)Click here for additional data file.Figure S5PCB170 vs. Diabetes Status for 1999–2000 and 2003–2004 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (0.50 MB DOC)Click here for additional data file.Table S1Percent of significant  environmental factors found by environmental class and cohort in first stage of T2D association. Number found of total per class is also shown in parentheses. * denotes that there were no factors measured for that particular environmental class as of this writing. There were a total of 21 environmental classes explored.(0.05 MB DOC)Click here for additional data file.Table S2Adjusted odds ratios for validated factors for individuals at risk for T2D diagnosis. Individuals who answered yes to having T2D in the NHANES questionnaire (“Doctor told you have diabetes?”) were omitted from the sample, leaving only those who were at risk for T2D diagnosis. Estimates were adjusted for age, sex, BMI, SES, ethnicity, and cohort. Odds ratios are for a change in 1SD of the logarithm of exposure in association with T2D diagnosis risk.(0.03 MB DOC)Click here for additional data file.Table S3Adjusted odds ratios for validated factors, adjusting for age, sex, BMI, SES, ethnicity, cohort, log10, log10. Odds ratios are for a change in 1SD of the logarithm of exposure in association with T2D.(0.03 MB DOC)Click here for additional data file.Table S4Adjusted odds ratios for validated factors, adjusting for age, sex, BMI, SES, ethnicity, cohort, log10(triglycerides), log10, log10, log10, log10, supplement use (yes/no) . Odds ratios are for a change in 1 SD of the logarithm of exposure in association with T2D. * denotes models did not include β-carotene as these data were not available for the 1999–2000 cohort.(0.03 MB DOC)Click here for additional data file.


L-arginine:glycine amidinotransferase) and GAMT (guanidinoacetate methyltransferase). Creatine is taken into cells via the creatine transporter (CrT), where it plays an essential role in energy homeostasis, particularly for tissues with high and fluctuating energy demands. Very little is known of the fetal requirement for creatine and how this may change with advancing pregnancy and into the early neonatal period. Using the spiny mouse as a model of human perinatal development, the purpose of the present study was to comprehensively examine the development of the creatine synthesis and transport systems.Creatine synthesis takes place predominately in the kidney and liver via a two-step process involving AGAT . In the fetal kidney and liver, AGAT and GAMT mRNA and protein expression were also relatively low until 34–37 days gestation. Between mid-gestation and term, neither AGAT or GAMT mRNA or protein could be detected in the placenta.Our results suggest that in the spiny mouse, a species where, like the human, considerable organogenesis occurs before birth, there appears to be a limited capacity for endogenous creatine synthesis until approximately 0.9 of pregnancy. This implies that a maternal source of creatine, transferred across the placenta, may be essential until the creatine synthesis and transport system matures in preparation for birth. If these results also apply to the human, premature birth may increase the risk of creatine deficiency. L-arginine:glycine amidinotransferase) and GAMT (guanidinoacetate methyltransferase). The first step involving AGAT occurs mostly in the kidney where arginine and glycine form guanidinoacetate, which later undergoes methylation to form creatine, occurring mostly in the liver via the actions of GAMT.The creatine/phosphocreatine (PCr) system plays an essential role in cellular energy homeostasis, serving as a spatial and temporal energy buffer in cells with high and fluctuating energy demands (for detailed reviews see -4). In aFrom the liver, creatine is carried in the blood to creatine-requiring tissues, where it is transported into cells against a large concentration gradient by a creatine-specific, high affinity, sodium- and chloride-dependent creatine transporter protein (CrT) located at the plasma membrane ,5. Once The recently discovered congenital defects in humans affecting creatine synthesis (AGAT or GAMT deficiency), or creatine uptake (CrT deficiency), are characterized by a severe depletion of cerebral creatine/PCr . In earlThe reported success of this early intervention creatine supplementation suggests that the fetus only becomes depleted of cerebral creatine after birth. It may be that the mother and/or placental unit sustain the fetal creatine requirement for all of pregnancy ,13. The Very little is known of the fetal requirement for creatine and how this may change with advancing pregnancy and into the early neonatal period, particularly for tissues known to have a high creatine requirement in the adult  . Braissade novo synthesis [In the adult mouse brain, CrT expression at the blood brain barrier has been shown to be a major pathway for supplying creatine to the brain . Howeverynthesis ,19. In tynthesis . It woulynthesis ,21.In preparation for birth it is probable that, as for AGAT and GAMT activities in the adult, the fetal kidney and liver must develop an independent capacity for creatine synthesis. In the developing rat, GAMT mRNA expression in the liver shows a steady increase in expression between E12.5–18.5, whereas AGAT mRNA in the kidney is not detectable until E18.5 . These rPrevious studies in rodents have provided insight into the temporal development of the fetal creatine synthesis and transport system ,22,23. H(Acomys cahirinus) is a precocial species that can be considered an appropriate animal model for perinatal development in the human. Unlike conventional rats and mice, the spiny mouse has a long gestation (38–40 days), small litter size , and is developmentally more advanced at birth; the body is covered with fur, eyes and ears are functional, they show active olfaction and are capable of thermoregulation and coordinated locomotion [The spiny mouse comotion . The devcomotion , liver [comotion , small icomotion , and thecomotion , indicatUsing the spiny mouse as a model of human perinatal development, the purpose of the present study was to comprehensively examine the development of the creatine synthesis and transport systems. We measured the creatine content of fetal and placental tissues, and sought to determine if the fetus had the capacity to meet its creatine requirement independently of a maternal-placental source.All experiments were approved in advance by Monash University School of Biomedical Sciences Animal Ethics Committee, and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The spiny mice used in this study were obtained from our own laboratory colony and housed, bred and time-mated as previously described .Placental and fetal tissues were collected at gestational days 20, 25, 30, 34 and 37, and neonatal tissues on postnatal days 2 and 10, from at least 4 different litters for each age (litter size range 2–4). Placentas and fetal and neonatal brain, heart, liver and kidneys were dissected, weighed and snap frozen in liquid nitrogen and stored at -80°C. Heart samples were collected only from gestational day 25 and kidney samples from day 30 due to the limited mass of tissue.The concentration of creatine and PCr were measured on gestational days 20, 30, 34, 37 and postnatal day 10, as previously described . Due to Real-time polymerase chain reaction (qPCR) was used to measure mRNA expression of CrT , AGAT , and GAMT  on gestational days 20, 25, 30, 34, 37, and postnatal days 2 and 10, from 4 animals (of different litters) at each age.Total RNA was extracted and DNase treated using the commercially available RNeasy Kits  for all samples except heart, which were extracted using PerfectPure RNA Fibrous Tissue Kit . Sample RNA (0.5–1.0 μg) was reversed transcribed to form cDNA using AMV reverse transcriptase and Random Primers according to the manufacturer's instructions , and diluted 1:2 with nuclease-free water.CrT, AGAT, GAMT and 18S primers . Each 20 μl reaction contained 1–3 μl template  and 0.5 μM of each forward and reverse primer. A 3-step PCR was used to amplify mRNA with an initial template denaturing of 95°C for 2 min, followed by 40 cycles of; 95°C for 15 sec, 64.4, 55.4 or 59.6°C for 15 sec , and 68°C for 20 sec. A fourth step of 80.5°C for 20 sec was included when amplifying AGAT and GAMT mRNA to remove primer-dimer artefact that occurred with low expression of the genes of interest. Heart samples were assayed for CrT using a Stratagene MX3000p thermal cycler system with SYBR Green PCR Mastermix . Each 20 μl reaction contained 2 μl template and 0.2 μM of each forward and reverse primer. A 3-step PCR was used to amplify mRNA; initial template denaturing of 95°C for 10 min, and 40 cycles of; 95°C for 30 sec, 60.0°C for 60 sec, and 72°C for 30 sec. Fluorescence readings were measured during the last step of cycling.For all samples except heart, qPCR was performed using an Eppendorf MastercyclerA melt curve of fluorescence versus temperature was performed after each qPCR to ensure a single product had been amplified per primer set. The DNA product of each gene of interest, housekeeping gene, and negative controls were run on a 2 percent agarose gel to confirm single product at the expected size Figure .T (cycle of threshold fluorescence) method. Briefly, relative expression in each sample were calculated by subtracting the mean CT value for 18S from the mean CT value of the gene of interest; ΔCT value. The mean ΔCT value of the calibrator sample was then subtracted from each individual sample to give ΔΔCT. This number was inserted into the formula 2-ΔΔCT and divided by the mean 2-ΔΔCTvalue of the 37 day gestation group, therefore expressed relative to the mean of the 37 day gestation group for the gene of interest within each organ. The expression stability of the housekeeping gene 18S between gestational day 20 and postnatal day 10 was verified for all organs of interest against β-actin and cyclophilin A using geNorm  [Data were analyzed and differential expression determined using the comparative ΔΔC organs) .Western blotting technique was used to measure AGAT protein expression in placenta, brain, and kidney, as well as GAMT protein expression in placenta, brain, and liver homogenates from 4 animals of different litters at select ages between 20 days gestation and postnatal day 10. The proteins were detected with affinity purified rabbit monoclonal (GAMT) and polyclonal antibodies (AGAT) made through injection of the following antigenic peptides: GAMT N-terminal aa 125–145; and AGAT N-terminal aa 62–77 and 410–423. Specific immunoglobulins against AGAT and GAMT were obtained by peptide affinity chromatography. The antibodies detected a positive band at the predicted molecular mass  and 1 mM serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF). Protein concentrations were determined using the Lowry method with a bovine serum albumin (BSA) standard curve. Samples were prepared with Laemmli sample buffer  and heated at 95°C for 5 min, denaturing the tertiary structure. Prepared samples (10 μg) and 2 μl broad range molecular mass marker were separated using 15% SDS-PAGE gel, and wet electro-transferred  to nitrocellulose blotting membrane. Transfer to membranes was verified with reversible Ponceau S, and gels stained with Coomassie Blue. Membranes were blocked for 120 min in 5% BSA in 0.1% Tween-20 TBS , and incubated in primary antibody with 5% BSA in TBST overnight at 4°C . Following incubation, membranes were washed in TBST, incubated with HRP-conjugated goat-anti-rabbit secondary antibody  and StrepTactin-HRP conjugate  with 5% BSA in TBST at room temperature for 60 min, and given a final wash in TBS. All incubations were performed with gentle agitation. Reactive protein were detected with chemiluminescence  and exposed to X-ray film . Each blot contained a positive control sample, and samples from 2 animals of each age group. Western blot data were analyzed using ImageJ 1.40 g, with values expressed relative to the positive control sample.Unless otherwise specified, all reagents were obtained from BioRad Laboratories .All data are presented as mean ± SE, and were analyzed using a one-way analysis of variance with Tukey HSD post hoc. Significance was set at p < .05. Statistical comparisons were carried out using the computer based program SPSS.Figure The developmental expression of the two key enzymes involved in creatine synthesis, AGAT and GAMT, were measured in the kidney and liver, respectively, at the mRNA and protein level from mid-gestation until the second postnatal week Figure . ExpressThere was relatively low expression of hepatic GAMT mRNA between gestational days 20–30, with a significant 50-fold increase in expression by day 37 of gestation .The expression of CrT mRNA was determined from mid-gestation until postnatal day 10 Figure . In the The provision of creatine to the tissues of a developing embryo is likely to be important for normal fetal development, particularly for energy homeostasis in the brain and heart. Despite this, very little is known about when creatine synthesis and its transporter system develops during embryonic, fetal and neonatal life. In this study we used a species that, like the human, has a relatively long gestation during which considerable fetal development and maturation occurs. We determined that the amount of TCr in the spiny mouse placenta and fetal brain increased progressively across the second half of gestation. However, expression of the two principal creatine synthesizing enzymes AGAT and GAMT in the fetal kidney, liver and brain was low until very late in gestation (days 34–37), and expression of these enzymes in the placenta was not detected at all. These results suggest that in precocial species the developing fetus is almost completely reliant on a maternal source of creatine until as late as 0.9 of pregnancy.The fetal brain showed a steady and significant increase in the estimated amount of TCr from mid-gestation until term, and increased further in the postnatal period. The high amount of creatine found in this organ is not surprising, as the adult brain is known to have a high basal creatine concentration, presumably to cope with its large and fluctuating cellular energy requirements ,4.In the adult rat brain, there is a limited capacity for creatine to cross the blood brain barrier. The widespread expression of creatine synthesizing enzymes has lead to the suggestion that the creatine requirements of the brain can be met, at least in part, independently of extra-CNS sources (i.e. that synthesized via the kidney and liver) . In the The temporal difference in the appearance of AGAT and GAMT mRNA in the fetal spiny mouse brain is an interesting phenomenon. A similar result was found for the embryonic rat, where AGAT could be detected from E12.5, yet GAMT was barely detectable even shortly before birth at E18.5 . These rWe showed that CrT mRNA expression in the spiny mouse brain increased approximately 2-fold between mid-pregnancy and term; we were unable to measure CrT protein for lack of an appropriately specific antibody (as detailed in Methods). This prenatal increase is in agreement with that described in the embryonic rat , and conAGAT and GAMT expression in the fetal kidney and liver were also relatively low until the very late stages of pregnancy; a 30 to 50-fold increase in mRNA expression was seen at 34–37 days of gestation (term is ~39 days). We expected that in preparation for birth these key organs would develop a capacity for creatine synthesis in the latter half of pregnancy, but these results suggest a limited capacity for endogenous creatine synthesis exists until very late (~0.9) in pregnancy. AGAT and GAMT expression levels in the kidney and liver remained unchanged between gestational day 37 and postnatal day 10. It would appear that shortly before birth, the spiny mouse attains the capacity to meet the postnatal requirement for endogenous creatine synthesis. It would be interesting to know how the uptake of creatine from breast milk contributes to the neonatal requirement for creatine in the spiny mouse.Similar to the brain, the amount of TCr measured in the placenta of the spiny mouse increased significantly with advancing pregnancy. Being metabolically very active, it is likely that the placenta itself has a requirement for creatine – creatine kinase expression, which is tightly coupled with cellular energy requirements, peaks in term human placenta . HoweverTo our knowledge, the transcriptional pathways controlling the regulation of the CrT, AGAT and GAMT genes have not been identified. In the fetal spiny mouse, circulating thyroid hormone increases steadily between 30 days gestation and term . AnalysiAlthough the fetal heart, liver and kidney undergo considerable growth from mid-gestation until term in the fetal spiny mouse, the estimated amount of TCr did not increase until after birth. Although heart muscle does not synthesize creatine, as with skeletal muscle it has a large requirement for creatine and therefore a considerable capacity for uptake and storage . Our finUnlike the brain and skeletal muscle, the kidney and liver have less requirement for creatine . The obsThese results suggest that, for a species where considerable maturation of the fetus occurs before birth, there appears to be a limited capacity for endogenous creatine synthesis until approximately 0.9 of pregnancy. This implies that a maternal source of creatine, transferred to the fetus via the placental unit, may be essential until the creatine/PCr synthesis and transport system matures in preparation for birth. If these results also apply to the human, it may be that infants born prematurely would be at risk of becoming creatine deficient, unless sufficient creatine can be absorbed by the immature gut from the limited amounts of creatine present in breast milk . Organs ZI carried out all animal studies, molecular work, participated in design of study and drafted the manuscript. APR participated in molecular work and drafting of manuscript. TW designed and synthesized antibodies, provided technical assistance and advice in molecular techniques, and drafting of manuscript. DWW participated in design and coordination of study, and drafted manuscript. RS participated in design and coordination of study, technical assistance, and drafting of manuscript. All authors have read and approved the final manuscript.


Nasonia vitripennis, a model for hymenopteran reproduction and development. Our work demonstrates that N. vitripennis, like other examined hymenopterans, exhibits characteristics indicative of an abortive meiosis, including slight enlargement of spermatocytes preceding meiotic initiation. However, we saw no evidence of cytoplasmic buds containing centrioles that are produced from the first abortive meiotic division, which occurs in the honeybee. In contrast to other previously studied hymenopterans, N. vitripennis males produce sperm in bundles that vary widely from 16 to over 200, thus reflecting a range of cellular divisions. Our results highlight interesting variations in spermatogenesis among the hymenopteran insects, and together with previous studies, they suggest a pattern of progression from meiosis to a more mitotic state in producing sperm.Males of hymenopteran insects, which include ants, bees and wasps, develop as haploids from unfertilized eggs. In order to accommodate their lack of homologous chromosome pairs, some hymenopterans such as the honeybee have been shown to produce haploid sperm through an abortive meiosis. We employed microscopic approaches to visualize landmark aspects of spermatogenesis in the jewel wasp  Drosophila melanogaster. Initially, a single germ cell produced from a stem cell division, termed a gonialblast, undergoes exactly four rounds of mitosis with incomplete cytokinesis, forming a cyst of 16 interconnected germ cells referred to as spermatogonia 2. Following a round of S-phase and enlargement in volume by ~25-fold3, these cells, now referred to as primary spermatocytes, pass synchronously through the two divisions of meiosis, producing 64 round, haploid spermatids4. The chromatin of these cells becomes remodeled such that the highly basic histone proteins are removed from DNA, and it is repackaged with sperm nuclear basic proteins (SNBPs), including protamines5. Each spermatid, including its nucleus, becomes highly elongated as it forms a flagellated tail and a pair of long, tubular mitochondrial derivatives2. Grouped in bundles of exactly 642, the spermatids undergo individualization through an actin-myosin-dependent process in which they become separated into mature sperm8.Spermatogenesis is one of the most elaborate cell transformational processes in animal development. Much of what is known about spermatogenesis in insects stems from numerous studies in the dipteran fruit fly, 9. Interestingly, in hymenopteran insects, which includes all bees, ants, wasps and sawflies, males arise as haploids from unfertilized eggs (reviewed in10). To be clear, males of these organisms contain only a single set of chromosomes inherited from the maternal parent. This characteristic makes homologous chromatid alignment and recombination in the male germ line impossible, thus begging the question of how haploid males produce sperm.A central question is to what degree deviations from this general paradigm of spermatogenesis occur in other insects. One outstanding example stems from studies performed in several bee species and a handful of other related insects that pertain to meiosis. In diploids, meiosis involves the side-by-side alignment of homologous chromatid pairs and the physical exchange of genetic material between them during the first meiotic divisionApis mellifera, like in D. melanogaster, the primary spermatocytes undergo cellular enlargement before entry into meiosis11. However, interestingly, in A. mellifera the nuclear envelope fails to break down in each spermatocyte during meiosis I even though the chromatin condenses into chromatids12. Each spermatocyte becomes clavate, or tear-dropped, in shape as it appears to undergo division. However, this division fails to occur, resulting in the original primary spermatocyte as the product. In the Indian honey bee Apis cerana indica, the first meiotic division does indeed occur, but the products consist of a large daughter cell containing the undivided nuclear material and a much smaller “cytoplasmic bud” containing no nuclear material12. Additionally, previous TEM studies in A. mellifera and other bees have noted the presence of small cytoplasmic buds containing extra, unused centrioles produced before or during the abortive division13. The second meiotic division in these insects is more conventional, being symmetrical and involving complete nuclear envelope breakdown and sister chromatid separation, resulting in haploid spermatids that subsequently undergo the final stages of spermatogenesis. It is presumed that the abortive division during meiosis I is an alteration of the basic cellular meiotic programming that compensates for the lack of homologous chromosomes in haploid males that is needed to properly produce haploid sperm – i.e., the reductive division step is eliminated because it is neither necessary nor desirable in haploid individuals.Previous work has shown that in the honey bee, Nasonia vitripennis, noted a less pronounced cellular increase of primary spermatocytes than occurs in A. mellifera14. Additionally, the number of sperm per bundle, and, therefore, the number of germ cell divisions producing sperm, varies among different species. For example, a predatory wasp Microstigmus arlei has 32 spermatids per bundle (5 divisions)15, some bee species have 32 spermatids per bundle while others have 64 spermatids per bundle (5 or 6 divisions)13, and the rose sawfly Arge pagana has either 256 or 512 spermatids per bundle (7 or 8 divisions)16. These patterns seem to follow a 2n rule where n is the number of germ cell divisions giving rise to sperm17. An interesting question is what cellular factor(s) determine the number of germ cell divisions for a given species.Although abortive meiosis I may be a common feature of spermatogenesis in hymenopteran insects, there appear to be some notable variations in aspects of spermatogenesis that have been found in a few examined species. For example, early TEM studies in males of the jewel wasp, N. vitripennis using a combination of several different microscopic approaches. N. vitripennis, a parasitoid of different blowfly species19, has gained prominence over the past two decades as a rising model insect for genetic and genomic studies aimed at understanding the evolution and development of axis patterning21, sex determination22, de novo centrosome formation23, host-symbiont interactions26, and venom production28. Additionally, recent work has focused on a paternally transmitted B chromosome known as PSR , which, through an unknown mechanism, causes complete elimination of the paternal (sperm-derived) half of the N. vitripennis genome during the first embryonic mitotic division30. Investigating these biological processes in N. vitripennis, particularly those involving the male germ line, would be greatly facilitated by a better understanding of spermatogenesis in this organism.These noted cellular variations in spermatogenesis, and the fact that this developmental process has been limited primarily to TEM studies in only a few hymenopteran species, led us to further investigate this process in the jewel wasp N. vitripennis, like other previously studied hymenopterans, undergoes an abortive first meiotic division to produce sperm. However, we observed several unique characteristics of spermatogenesis in N. vitripennis, including a relatively small increase in primary spermatocyte size compared to A. mellifera, and a highly variable number of spermatids per bundle. We propose that these and other characteristics may reflect an evolutionary progression from meiosis toward a fully mitotic state of sperm production in N. vitripennis.Our study here reveals certain cellular characteristics supporting the likelihood that N. vitripennis, we examined the male reproductive tract during wasp pupal development, when much of spermatogenesis occurs31. The male reproductive tract of N. vitripennis is bilaterally symmetrical, with each of the two sides beginning with an oval-shaped testis that connects at its base through a duct to a seminal vesicle, and then to an accessory gland, before joining to the ejaculatory duct . The two sides of the male reproductive tract connect at a position that is distal  to the two accessory glands  germ cells organized into cysts, and (ii) individual cells that contain very large nuclei and are interspersed among the cysts of germ cells , the nuclei appeared small, round, and hyper-condensed 11 and D. melanogaster (25-fold)2. Second, taking advantage of previously published transcriptome data for N. vitripennis germ line tissues37, we looked to see if any genes whose functions are restricted to meiosis are expressed in the wasp testis38. Five of eight such genes – DMC1, HOP2, MND1, MSH4, and SPO11 – are expressed at comparable levels in both the testis and ovary  would produce haploid gametes, regardless of how many mitotic divisions occur in any given cyst.Moreover, D. melanogaster, produce large amounts of specific RNAs and proteins that are actively transported through cytoplasmic bridges into the developing oocyte39. If trophocytes function in transporting needed factors to the germ cells, then their transport system would need to be different from that between the oocyte and its nurse cells because the trophocytes are not physically connected to the germ cells. However, although very speculative, we propose that trophocytes may express “diffusible” factors that help to regulate the overall mitotic/developmental progression of the germ cells of each cyst so that they are coordinated more-or-less together across large regions of the testis. Such an in trans means of developmental regulation would be expected to be somewhat imprecise, and is consistent with the variable number of spermatids per bundle within a given testis.Our observations show that the trophocytes interspersed among the cysts of germ cells have unique transcriptional properties. Our current work does not directly address the function(s) of these cells. However, it is intriguing to speculate on their possible roles based on knowledge of the polyploid nurse cells present in the female germ line. Specifically, nurse cells, which undergo 10–12 rounds of endoreplication by the end of oogenesis in The work presented here adds to a broader understanding of spermatogenesis in hymenopteran insects, especially regarding interesting variation in the cellular dynamics of gametogenesis present within this group. Additionally, these findings provide a number of hypotheses regarding the mechanics and evolution of spermatogenesis in a haploid individual that will be important to test in future studies.33. Wasp lines of the AsympC line were propagated by placing together equal numbers of males and females (10 of each sex) in glass vials containing ~10 Sarcophaga bullata blowfly pupae and a few drops of 50% honey in water. PSR crosses were conducted as previously described40. Wasps were maintained in an incubator at 25 °C on 12 hr light/dark cycles.The majority of our analyses were performed on reproductive tissues taken from male wasps of the AsymC genetic background; however, cytological analysis of a repetitive satellite sequence, PSR4317, was performed on the PSR + genetic background, which contains the supernumerary B chromosome PSRSarcophaga bullata host pupae at the appropriate stage based on wasp pupal color pattern  at 1:200; mouse anti-acetylated alpha-Tubulin (Santa Cruz Biotech) at 1:100; mouse anti-RNA Polymerase II phosphorylated at Serine residue 2 (Abcam) at 1:500. Secondary antibodies and their dilutions in the same buffer were: anti-rabbit Alexa488 and anti-mouse Cy5 .Fixed testes were stained with primary antibodies overnight on a platform rocker at 4 °C, washed three times at 15 minutes each with 1 × PBT. Secondary antibodies were then added to testes and incubated at room temperature on a platform rocker and in the dark  for 1 hour. The tissues were then washed three additional times with 1 × PBT (still covered) before mounting on a slide with Vectashield mounting medium with DAPI (Vector Laboratories).in situ hybridization (FISH) was performed as previously described33 using fluorescence labeled DNA oligonucleotide probes synthesized by IDT. PSR22 RNA was detected as in33, using a DIG-labeled LNA probe (5′-AATATCCAATCATAAGTCGAGACTTT-3′) generated by Exiqon. FISH was performed using the procedures and reagents provided by the manufacturer in the miRCURY kit. 28S ribosomal RNA and vasa RNA were detected using antisense DIG-labeled RNA probes synthesized in vitro from PCR-generated templates via conventional techniques41.DNA fluorescence Fluorescence confocal microscopy was conducted with a Leica TCS SPE confocal microscope. Images were collected either as a single image, or as Z-series and subsequently merged to capture for inclusion of details in different focal planes. All images were exported in JPEG format and then processed in Adobe Photoshop CS5 v. 12.The fixative used was Karnovsky’s Cacodylate Buffered Glutaraldehyde-Formaldehyde (1965), which consists of 3% glutaraldehyde and 3% formaldehyde in 0.1 M cacodylate buffer. All the testes were dissected while the pupa were immersed with fixative at room temperature. The testes within each group were placed into a 1.5 mL microfuge tube filled with fixative, left for 1 hour at room temperature, and then placed at 4 °C overnight. The next day, the samples were warmed to room temperature, the excess fixative removed, and a small amount of low melt agarose  added and mixed to disperse the testes. After letting the agarose cool, the agarose blocks were cut out, removed from the microfuge tubes, and placed into fresh fixative. While in new fixative, the agarose blocks were cut in half longitudinally, and the 2 pieces were placed into vials with new fixative. The vials were then kept at 4 °C overnight. The next day, the samples were warmed to room temperature, washed with 0.1 M cacodylate buffer and then post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for one hour. After washing in 0.1 M cacodylate buffer, the samples were dehydrated to 50% ethanol and then en bloc stained in 5% uranyl acetate and 50% ethanol for 60 minutes. The samples were then dehydrated in a graded ethanol series to 100% and then infiltrated with Epon 812 with propylene oxide as the transition fluid. After polymerization at 60 °C for 48 hours, the blocks were ultrathin sectioned, and the sections were stained in uranyl acetate and lead citrate. The sections were viewed in a Hitachi H7000 TEM at 75 kV. Images were captured using an AMT  2k × 2k CCD camera. All chemicals were supplied by EMS  unless otherwise specified.Supplementary Information


P value less than 0.05 were used to report association.The main objective of this study was to assess pesticide handling and storage practice, and its associated factors among farmers engaged in irrigation in Gondar town, Ethiopia, 2019. Community-based cross-sectional study was used to assess pesticide handling and storage practice, and its associated factors among farmers. Simple random sampling technique was used to select study subjects. Semi-structured questionnaires were used to collect data. Data were entered using Epi Info 7.0 and exported to SPSS 20. Descriptive statistics were used for some variables. Univariate and multivariable binary logistic regression was used to test the factors associated with the outcome. Adjusted odd ratio (AOR) and 95% confidence interval (CI) with About 409 study subjects completed the semi-structured questionnaires. Among the studies subjects, 261 (63.8%) of them had poor pesticide handling and storage practice. Knowledge [AOR = 3.23 ], attitude [AOR = 1.77  and educational status ], elementary education [AOR = 5.38 ], secondary education [AOR = 9.51 ] and certificate and above [AOR = 6.00 ]) were factors significantly associated with pesticide handling and storage practice. To improve the pesticide handling and storage practice, it is imperative to enhance the level of the farmer’s knowledge through training, and information dissemination in workshops. Pesticides are chemicals used for deterring, destroying or controlling any pest causing harm , 2. HumaA higher proportion of pesticide poisonings and deaths occur in developing countries where there are inadequate occupational safety standards, lack of use of personal protective equipment (PPE), inadequate hygienic facilities, illiteracy, and insufficient knowledge of pesticide hazards –9. FurthImproper use of pesticides affect the natural environment such as water, soil and air and from pesticide leaching, runoff, and spray drift wildlife, fish, plants, and other non-target organisms may be affected . EnvironPesticide exposure can cause health problems such as human carcinogens, dermatitis, kidney disorders, endocrine disruption, reproductive effects, blurred vision, neuropsychiatric disturbance and memory loss , 13–15. Developed countries have systems already in place to register pesticides and regulating their trade and use. However, in developing countries, such kinds of systems are weaker . DevelopTo our capacity, there have been scarce studies conducted in Ethiopia regarding pesticide handling and storage practice , 8. HoweTherefore, the main objective of the study was to assess pesticide storage and handling practice and its associated factors among farmers working in irrigation in Gondar town, Ethiopia, 2019.This community-based cross-sectional study was conducted from April to May 2019 among farmers working in irrigation in Gondar town, Ethiopia. The total number of population of Gondar was estimated to be 324,000 and among these 480 were farmers engaged in irrigation.All irrigation farmers in the selected area who were present during data collection and voluntary were included.Pesticide storage and handling practices The respondents who scored less than or equal to the mean value of the responses for 22 pesticide handling storage practice-related questions were considered having “poor level of practice”. Those who scored more than the mean value were considered as having a “good level of practice.”Pesticide handling, and storage knowledge level The respondent who scored less than or equal to the mean value of the responses for 10 pesticide handling, and storage knowledge-related questions were considered having “poor level of knowledge.” Those who scored more than the mean value were considered as having a “good level of knowledge.”Pesticide handling and storage attitude the respondent who scored less than or equal to the mean value of the response for 5 pesticide handling, and storage attitude related questions were considered having “poor level of attitude.” Those who scored more than the mean value were considered as having a “good level of attitude.”The sample size was determined using a single population proportion formula. The minimum sample size (n) for the study was calculated as follows. Where the critical value is (y 55.9%) .\documenAdding 10% for any unexpected events 10% of 379 equals to 37.9 ≈ 38 so, the total Samples were 379 + 38 = 417.From the total 480 farmers engaged in irrigation, 417 were selected by simple random sampling technique from three river basins namely Qeha, Shinta and Angereb. One hundred and fifty-seven out of the 180 farmers in Angereb, 131 out of 151 farmers in Shinta, and 129 out of the 147 farmers in Qeha were proportionally chosen.The data were collected by interviewing using questionnaires which comprised questions about pesticide storage and handling practice, sociodemographic characteristics, knowledge and attitude. The quality of data was assured by proper designing of the questionnaire. The questionnaires were prepared in English, translated to Amharic and translated back to English to check for consistency. The data were checked for completeness and entered using Epi info version 7.0 and exported to SPSS version 20. Univariate and multivariable logistic regression was computed to test the factors associated with pesticide handling and storage practice.Four hundred and nine farmers participated in the study. The majority of the respondents (90.7%) were male and the mean (± SD) age of study participants was 35.99 ± 9.5 years Table .Table 1SAbout 148 (36.2%) subjects had self-reported good pesticide handling and storage practice. Similarly, 161 (39.4%) and 201 (49.1%) of farmers reported a good level of knowledge and attitude regarding pesticide handling and storage, respectively  of participants reported that they used empty pesticide containers for storing food and non-food materials. About 322 (78.7%) reported that they were exposed for pesticide during the application, and 95 (23.2%) of the respondents reported that they mix pesticide in front of other people including children ] as compared with those who cannot read and write. Whereas respondents who had elementary education were 5.38 times more likely to report better pesticide handling and storage practice  compared to those who cannot read and write. The study subjects with a certificate and above educational status were 6 times more likely to have better pesticide handling and storage practice  about pesticide handling and storage as compared with those having poor knowledge. Farmers who have desirable/good attitude reported 1.77 times more likely better practice  as compared those with undesirable/poor attitude  of the respondents had good pesticide handling and storage practice. The overall good practice in the current study was higher than previous studies conducted in Kenya (15%) . This diPoor pesticide handling practice may expose the farmers to a number of pesticide-related health symptoms , 21.In this study, the level of good practice of disposing of empty pesticide containers was in line with a study conducted at Rift Valley in Ethiopia (97%) . On the Among the participants, 38.4% stored the purchased pesticide with the bulk of other household materials at their home. The absence of separate storage site may lead to a risk of contamination and thereby health risks. After the pesticide application, 16.4% the subjects said that they reuse the empty containers. Studies conducted in Nigeria found out that 74% and in Tanzania 68% of the farmers have suffered at least one health symptom due to the poor pesticide handling practice , 7, 18. A significant number of farmers (23.2%) responded that they practice mixing pesticide in front of people including children. This may lead to potential health risks for the mixer and people in a close range. In particular, children are more vulnerable as they are sensitive towards low dose of pesticide contamination , 28. TheFurthermore, 90.2% of respondents have revealed that they did not use personal protective equipment (PPE) during practising pesticide mixing and application. This result is better than a previous study  finding Cost of PPE has been reported as a significant factor behind farmers’ pesticide usage without PPE , 31. In The educational status, knowledge and attitude of respondents were predictors of their pesticide handling and storage practice. Similarly in another study pesticide handlers with knowledge about pesticide health risks had better practice during pesticide handling . A previThe pesticide storage and handling practice of farmers engaged in irrigation in Gondar town were poor. Educational status, knowledge, and attitude were identified as the factors having a strong association with pesticide handling and storage practice among farmers working in irrigation.This study might be affected by social desirability bias even though techniques to reduce bias has been employed. The inherent weakness of a cross-sectional study that fails to establish cause and effect relationship results difficulty to identify the true determinants of practice.Additional file 1: Table S1. Knowledge of farmers about pesticide storage and handling practice in North West Ethiopia in Gondar town April 2019 (n = 409).Additional file 2: Table S2. Attitude of farmers pesticide storage and handling practice in North West Ethiopia in Gondar town April 2019 (n = 409).Additional file 3: Fig. S1. Knowledge, attitude, and practice level of the study participants regarding pesticide handling and storage among farmers in Gondar, Ethiopia, 2019 (n = 409).


What is the association between radiographic measures of reduction and patient outcomes 12 months after distal radius fractures treatment for adults aged 60 years or older?This secondary analysis of a multicenter randomized clinical trial on distal radius fractures treatment options included 166 patients who completed 12-month assessments. Radiographic parameters were not associated with functional and patient-reported outcomes.Precise anatomic restoration does not guarantee good outcomes and may not have value in outcome evaluation for older adults with distal radius fractures. The value of precise anatomic restoration for distal radius fractures (DRFs) in older adults has been debated for many decades, with conflicting results in the literature. In light of the growing population of adults aged 60 years and older, both fracture incidence and associated treatment costs are expected to increase.To determine the association between radiographic measures of reduction and patient outcomes after DRF in older patients.Data were collected from the Wrist and Radius Injury Surgical Trial (WRIST), a multicenter randomized clinical trial of DRF treatments for adults aged 60 years and older . Data analysis was performed from January 3, 2019, to August 19, 2019. WRIST participants who completed 12-month assessments were included in the study. According to the biomechanical principle of alignment, 2-phase multivariable regression models were adopted to assess the association between radiographic measures of reduction and functional and patient-reported outcomes 12 months following treatment.Participants were randomized to receive volar locking plate, percutaneous pinning, or external fixation. Those who opted for nonoperative treatment received casts.Hand grip strength, wrist arc of motion, radial deviation, ulnar deviation, the Michigan Hand Outcomes Questionnaire (MHQ) total score, MHQ function score, and MHQ activities of daily living score were measured at 12 months following treatment.P = .004) and each millimeter increase toward normal (0 mm) in ulnar variance was associated with a 10.4-point improvement in MHQ ADL score . However, neither of these radiographic parameters appeared to be associated with MHQ total or function scores.Data from 166 WRIST participants  found that only 2 of the 84 correlation coefficients calculated were statistically significant. For patients aged 70 years or older, every degree increase in radial inclination away from normal (22°) grip strength in the injured hand was 1.1 kg weaker than the uninjured hand (95% CI, 0.38-1.76; The study results suggest that precise restoration of wrist anatomy is not associated with better patient outcomes for older adults with DRF 12 months following treatment. Surgeons can consider this evidence to improve quality of care by prioritizing patient preferences and efficient use of resources over achieving exact realignment.NCT01589692ClinicalTrials.gov identifier:  This secondary analysis of a randomized clinical trial assesses the association between radiographic measures of reduction and functional and patient-reported outcomes after distal radius fracture in patients aged 60 years and older. In 2005, the annual cost of treating DRF in the population aged 65 years and older was approximately $500 million.4 By 2025, it is estimated that annual fracture incidence and treatment costs will rise by 20%.4Nearly 90 000 older adults in the United States experience distal radius fractures (DRFs) annually.12 However, these conclusions cannot be applied to the current population of older adults who are much more active and functionally independent than previous generations.15 The perceived effect of disability from DRF will be more pronounced in the current population aged 60 years and older with greater demand in functional capacity. Studies have shown evidence that older adults place tremendous value on independent living, with reports of dependence making them feel as if they are a burden and delivering a toll on their emotional well-being.17 Some older adults even stated that they would accept increased mortality if it meant they would be functionally independent.18 Previous schools of thought on radiographic evaluation of reduction must be examined again with a more contemporary cohort of older patients.Previous studies of DRF outcomes in persons who are aged 60 years and older report that precise anatomic reduction is not necessary to achieve satisfactory functional results because this population requires less functional recovery than younger patients.20 A systematic review of studies that evaluated radiographic measures of reduction after DRF found no consistency in the types of fracture or severity of DRF included in study cohorts, definitions or methods of obtaining various radiographic measures, or the acceptability criteria for malalignment.21 The substantial variability in the current literature demonstrates a need for a study that strives to establish a model of measuring and using radiographic variables to assess outcomes after DRF.23What adds to the necessity of a renewed investigation is that many of the prior studies that found no clinically relevant differences in outcomes based on radiographic measures lacked rigor in sample size and study design.In this study, we apply the rich and robust data collected from a large randomized clinical trial evaluating DRF treatment options to investigate the value of radiographic assessment in outcomes evaluation after DRF in older adults. Our results may help derive a definitive answer to the long-standing question of DRF management for contemporary populations of older adults and provide quality evidence for hand surgeons to tailor treatment plans to better fit patients’ needs. We hypothesized that radiographic measures of anatomic restoration are positively associated with both functional and patient-reported outcomes 12 months after DRF treatment for older adults.24 At each participating site, 2190 patients aged 60 years or older were screened for eligibility from April 10, 2012, to December 31, 2016. Inclusion criteria were isolated DRFs (exception: concomitant ulnar styloid fracture) with displacement warranting surgical intervention ] type A2, A3, C1, or C2 and meeting one of the following radiographic criteria after reduction attempt: dorsal tilt >10°, radial inclination <15°, or radial shortening >3 mm). All fractures were amenable to treatment with all 3 surgical options. Patients with open fractures, bilateral fractures, prior ipsilateral DRF, or additional serious trauma were ineligible. Also excluded were patients living in nursing homes or other assisted living facilities and those with neurologic conditions affecting upper extremity sensation or movement, comorbid conditions prohibiting surgery, serious neurologic or psychiatric conditions precluding informed consent, or inability to complete study questionnaires and follow directions. The trial protocol is available in We used data collected as part of the Wrist and Radius Injury Surgical Trial (WRIST), an international, 24-site randomized clinical trial of DRF treatment in older adults.Enrolled patients were randomized (after written consent was obtained) to receive percutaneous pinning, external fixation with or without supplemental k-wire fixation, or internal fixation with volar plate, stratified by study site. Those who consented to study participation but did not wish to undergo surgery despite the recommendation and who met identical eligibility criteria as randomized participants were treated with casting and were considered an observation group  domain scores.28 For radiographic evaluation of reduction, alignment was measured on plain radiographs obtained at 12-month assessment. All radiographs were stored in Digital Imaging and Communications in Medicine format, and Picture Archive and Communicating System imaging software was used to view and measure the radiographic parameters. We included radial inclination, radial height, ulnar variation, and tilt  because those were the parameters with high interrater and intrarater agreement.22 These variables were measured by 2 trained clinical staff members, based on definitions by the American Academy of Orthopaedic Surgeons.29 All radiographs were measured twice, and those with discrepancies of more than 10% were resolved by a third reading.In our study, we included 12-month measurements of the clinical parameters recommended as standard components of outcomes assessment by the Distal Radius Outcomes Consortium, such as bilateral hand grip strength, arc of motion, patient-reported disability and functional outcomes as assessed with the Michigan Hand Outcomes Questionnaire (MHQ), and alignment as measured on plain radiographs.30Key clinical and demographic covariates included in our analysis model were sex, race, highest educational level, income, smoking status, preinjury level of activity, employment status 12 months following treatment, and status of dominant-hand injury. Race was self-reported by participants; this information was collected as required for all federally funded research projects. At enrollment, participants were asked to report their preinjury level of activity using the Rapid Assessment of Physical Activity, a 9-item questionnaire that evaluates a patient’s physical activity as sedentary, underactive, or active, based on guidelines from the Centers for Disease Control and Prevention.r = 0.94)  to clearly portray the association in each age cohort. In addition to the level of function and patient-reported outcomes in the injured hand, we calculated the difference in measurements and scores between the injured hand and uninjured hand  to capture within-person recovery of function and patient-reported outcomes. Higher positive values would indicate less recovery achieved. Among the 4 radiographic parameters, we excluded radial height from our model because it was highly correlated with radial inclination  for each radiographic variable were considered consistent with the hypothesized association based on the biomechanical principle. We used a conservative level of α = .01 to be mindful of the multiple associations that we are assessing in our analysis. Statistical analyses were performed with R software version 3.5.3 (R Foundation).P = .05). Likewise, there were more full-time workers in the younger group (16 [18.0%] vs 1 [1.3%]) and more retired participants in the older group (68 [88.3%] vs 52 [58.4%]) (P < .001). Among clinical characteristics, there were 10 active smokers in the younger group (11.2%) compared with 0 active smokers in the older group (P = .003) (P = .10). For ulnar variance, the mean (SD) displacement was less in the younger group (2.1 [2.0] mm) than the older group (3.1 [2.7] mm) (P = .01). The distribution of tilt measurements was also different between the younger (0° [11.6°] mm) and older (−5.2° [12.7°] mm) groups (P < .01).The final study cohort included 166 patients  from the parent WRIST study who completed all 3 components of 12-month assessment: function tests, patient-reported outcome questionnaires, and radiographs. We included 89 participants aged 60 to 69 years in the younger group and 77 participants age 70 years or older in the older group. Demographic and clinical characteristics were generally similar between the 2 subgroups for sex, race, educational level, preinjury Rapid Assessment of Physical Activity category, and status of dominant-hand injury . However = .003) . For radP = .01); (2) radial inclination vs difference in MHQ total score for the younger group ; and (3) ulnar variance vs difference in MHQ ADL score for the older group   radial inclination vs difference in grip strength for the older group  .P = .004). In other words, for every degree increase in radial inclination away from normal, the injured hand’s grip strength was 1.1 kg weaker than the uninjured hand (P = .02) (P = .003)  , B. This = .003) , C. The On finding only a few associations with statistical significance, we also checked to see if the results changed substantively when unadjusted for other radiographic parameters, that is, only 1 radiographic variable was included in the regression model instead of all 3 variables. The results were similar, with the same 3 statistically significant associations with the same direction of association  or overall hand health . For adults aged 60 to 69 years, we found that radiographic measures of reduction did not appear to be associated with 12-month treatment outcomes.In this study, we used data collected from an international, 24-site randomized clinical trial of DRF treatment options to investigate the value of precise anatomic restoration after DRF in older adults by examining the association between radiographic measures of reduction and outcomes 12 months following treatment. Our hypothesis was not confirmed; instead, radiographic measures were not associated with outcomes. In fact, only 3 of 14 models and 2 among 84 correlation coefficients were statistically significant. Furthermore, from the 3 regression models with appropriate fit, only 2 among 6 associations were congruent with the biomechanical principle of bony alignment: restoration of normal anatomic associations results in better function after DRF.38 whereas others have found that the quality of anatomic reduction influences patient outcomes.47 For example, Arora et al32 conducted a randomized study of 75 patients aged 65 years and older with DRF and found that volar locking plate fixation produced better restoration of normal anatomy on plain films compared with nonoperative treatment, but radiographic measures were not correlated with any improvement in functional outcomes. In contrast, Brogren et al46 examined the Disabilities of the Arm, Shoulder, and Hand questionnaire scores at various levels of radiographic displacement for 123 patients and found that patients with severe displacement had worse Disabilities of the Arm, Shoulder, and Hand scores at 2 years. Moreover, even among the studies that found a significant association between radiographic variables and treatment outcomes, there is no consensus on which parameters are the most important or the magnitude of influence.21 The heterogeneity of conclusions on the applicability and predictive potential of radiographic measures of reduction, as noted in several large Cochrane Database meta-analyses, is most likely a result of wide variability in study cohorts and design: the severity of fracture, outcomes reported and the way they were measured, and the way radiographic parameters were named and defined.50Evidence in the literature does not agree on the role of radiographic alignment in DRF management for older adults. Some studies conclude that there is limited utility in precise fracture reduction in older adults,22 and discrepancies between the 2 sets of measurements were resolved with a third reading. We also measured radiographic variables according to the American Academy of Orthopaedic Surgeons’ definitions.29There were some limitations to this study. Surgical technique, postoperative care, and therapy protocol care were not standardized in WRIST sites, and there may have been variations in care over the 24 participating sites and multiple surgeons. However, for our study, these differences increase the generalizability of our results by reflecting the variation in the real-world setting. The diversity in hospital settings and regional representation also adds to the robustness of the WRIST data. The parent WRIST study only included patients with substantially displaced DRF; thus, this is a limitation in our cohort selection. In addition, radiographic measurements on plain films are frequently shown to have low interrater and intrarater consistency. We minimized this bias by including only the radiographic parameters with a high rate of agreement,25 which promotes a standardized, unified approach for DRF outcomes assessment. Our use of a 2-phase multivariable regression model also adds rigor to our study as it reflects the biomechanical principle of bony alignment of the wrist more appropriately than single-phase regression models.The strengths of this study outweigh the limitations. We diminished the risk of selection bias and confounding by using the data collected from a multicenter randomized clinical trial. We included in our analysis the outcome measures recommended by the Distal Radius Outcomes Consortium,51 In the treatment decision-making process, surgeons can prioritize patient preferences over the need to achieve exact realignment. Our study results may help to improve the quality of care in DRF management for older patients.The results of our study suggest that radiographic measures of reduction are not associated with functional outcomes and patient-reported outcomes in older adults after DRF treatment. Clinically, this finding implies that precise anatomic realignment of the wrist is not necessary for satisfactory outcomes. With this evidence, surgeons may elect to decrease operative time, use of resources, and associated costs that would have been spent to achieve perfect or near-perfect reduction. Decreasing time under anesthesia benefits patients as well with reduced risk of morbidity and mortality.


A proof of concept demonstration is presented, showing the suitability of the materials for industrial pressure sensing applications.Polymer-based composites reinforced with nanocarbonaceous materials can be tailored for functional applications. Poly(vinylidene fluoride) (PVDF) reinforced with carbon nanotubes (CNT) or graphene with different filler contents have been developed as potential piezoresistive materials. The mechanical properties of the nanocomposites depend on the PVDF matrix, filler type, and filler content. PVDF 6010 is a relatively more ductile material, whereas PVDF-HFP (hexafluropropylene) shows larger maximum strain near 300% strain for composites with CNT, 10 times higher than the pristine polymer. This behavior is similar for all composites reinforced with CNT. On the other hand, reduced graphene oxide (rGO)/PVDF composites decrease the maximum strain compared to neat PVDF. It is shown that the use of different PVDF copolymers does not influence the electrical properties of the composites. On the other hand, CNT as filler leads to composites with percolation threshold around 0.5 wt.%, whereas rGO nanocomposites show percolation threshold at ≈ 2 wt.%. Both nanocomposites present excellent linearity between applied pressure and resistance variation, with pressure sensibility (PS) decreasing with applied pressure, from PS ≈ 1.1 to 0.2 MPa Polymer-based nanocomposites are attracting large attention in recent years both in the scientific and industrial areas. Through the inclusion of fillers, a wide range of polymer properties can be enhanced, such as mechanical , electriHence, their application as sensors materials are being widely studied due to their simple manufacturing and integration into devices ,7. Some The percolation threshold is the range in which the materials undergo a transition from nearly insulating to conductive, changing several orders of magnitude their electrical conductivity for small filler content variations . The perThe selection of the polymer matrix used for the development of a functional sensor depends on the stimulus needed for sensor response. Flexibility or stretchability, force, and environmental conditions influence the host polymer to use in view the overall properties to select.Soft polymer matrices such as natural rubbers or thermoplastic elastomers are reported to provide the nanocomposite high strain capability from low to large strains , and a wPVDF and its copolymers are excellent materials for functional applications, such as sensors, actuators, energy harvesting and biomaterials in the biomedical field . PVDF shPolymer nanocomposites can be manufactured using laboratory and industrial techniques. At the laboratory level, they can be processed by solvent casting , for exaIn this work, a comprehensive study of PVDF-based materials for force compression sensing is presented with excellent performance and linearity ,38. Diff3 and a tensile modulus between 1 to 1.5 GPa. PVDF 6010 is a homopolymer with medium viscosity, density between 1.75 to 1.8 g/cm3 and a tensile modulus between 1.7 to 2.5 GPa. Poly(vinylidene fluoride-co-hexafluropropylene) (PVDF-HFP), with reference Solef 21,216 and VDF/HFP mole ratio of 88/12, shows a density of 1.78 g/cm3 and a tensile modulus between 400 to 600 MPa.The selected polymers were poly(vinylidene fluorine) with reference 5130, 6010, and PVDF-HFP, all supplied by Solvay. PVDF 5130 is characterized by ultra-high viscosity with excellent adhesion, a density of 1.75 g/cmN,N′-dimethylpropyleneurea (DMPU) and was purchased from LaborSpirit.The solvent used to disperse the nanofillers and dissolve the PVDF was Multi-walled carbon nanotubes were supplied by Nanocyl with reference NC7000, showing an average length of 1.5 μm, an outer mean diameter of 9.5 nm and 90% purity. Reduced graphene oxide was obtained from The Graphene Box (Spain) with >99% of purity, 1–5 µm of length and 1–2 layers.Carbon nanofillers were dispersed in DMPU within an ultrasonic bath  for an average time of 4 h, assuring a correct de-agglomeration and homogeneous dispersion of the fillers in the solvent. Then, PVDF  was added to the filler/solvent solution and completely dissolved through magnetic stirring for approximately 3 h at 30 °C. It is to notice that this processing method has demonstrated to ensure good filler dispersion for both graphene  and CNT Films with the different polymer matrices and carbonaceous filler percentages were prepared, as indicated in −1 to 600 cm−1 with a resolution of 4 cm−1 employing a Jasco FT/IR-4100 spectrometer with a TGS detector.Fourier-transform infrared (FTIR) spectroscopy analysis was carried out in transmission mode at room temperature from 4000 cm−1 heating rate up to 200 °C under nitrogen atmosphere.Differential scanning calorimetry (DSC) tests were performed with a Netzsch DSC 200F3 Maia set up. Samples were placed into Al pan crucibles, stabilized at 30 °C and then subjected to a 20 °C.minR is the electrical resistance, A the electrode area and L the sample thickness. Electrical conductivity tests were performed in two sample points and repeated three times each.Electrical conductivity was obtained after I–V measurements. Samples were submitted to a voltage sweep from −10 to +10 V and the current was measured with a Keithley 6430 SourceMeter. Gold electrodes were previously deposited in both sides of the films by magnetron sputtering with a Polaron SC502 sputter coater. The electrical conductivity was obtained after Equation (1) considering sample thickness and electrode area:The mechanical characterization of the nanocomposites was performed by tensile tests up to failure with a Shimadzu AG-IS universal testing machine with a 50 N load cell, repeated for 5 measures for each sample.F is the tensile force, A the transversal area of the sample considering the thickness and width of the sample, ΔL the recorded displacement and L0 the initial interspace between clamps. Tensile tests were repeated for three times, ensuring that measurements were consistent within materials. The samples were considered as macroscopically homogeneous materials, without considering non-local effects [For the tensile tests, samples of 30 × 10 mm of the area and 20 to 60 μm of thickness were cut from the fabricated films, placed in the universal testing machine through a clamping system leaving an approximate interspace between clamps of 10 mm to assure a proper grip of the sample. The measurements were performed at speed of 1 mm/min and force and test time were recorded at a 100 ms rate. Engineering stress and strain (Equations (2) and (3)) were obtained according to: effects  due to tElectromechanical tests were performed under cyclic compression on samples with initial minimum pre-load of 10 N corresponding to 0.09 MPa to forces of 50, 100, 200, and 400 N, corresponding to 0.43, 0.86, 1.72, and 3.45 MPa, respectively, considering the contact area of the compression tests. The piezoresistive tests were performed in a Shimadzu AG-IS universal testing machine with a load cell of 500 N at speed of 0.5 and 1 mm/min, for 10 and 50 cycles and replicated twice. Force and displacement were recorded with a 500 ms time span.The acquisition of the electrical resistance between the compression pieces was performed with an Agilent 344401A multimeter. Piezoresistive tests were repeated twice.PS in Equation (4)) was performed according to:R represents the resistance, variation measured during the test and R0 the initial resistance of the sample under the minimum pre-load and P is the pressure on the sample.The quantitative evaluation of the pressure sensitivity  can be observed between the different PVDF matrixes, with PVDF-HFP presenting lower crystallization temperature, cT ≈ 132.3 °C, than PVDF5130 and 6010, with mT ≈ 158.2 and mT ≈ 168.9 °C, respectively. The thermograms of the neat polymers and the nanocomposites show a single peak corresponding to the melting of the α-phase of PVDF [Thermal properties were evaluated by DSC thermograms for neat PVDF 5130, 6010 and HFP A and the of PVDF .PVDF composites with CNT or rGO lead to crystallization temperatures slightly higher than the neat polymers, increasing less than 2 °C in all cases, demonstrating that the effect of the fillers in the melting behavior of the samples is quite small and independent of filler type and content, confirming the low interaction between fillers and polymers observed in the FTIR spectra , where t−1 (Ω.m)−1, as reported in literature for CNT/polymer materials [The volume of electrical conductivity for the different composites is shown in aterials ,44. It i−5 (Ω.m)−1.With respect to rGO/5130 nanocomposites, they show lower conductivity when compared to CNT at the same filler content, which is attributed to the lower aspect ratio and intrinsic conductivity of rGO when compared to CNT, leading to an increase of percolation threshold of the composite. The rGO/5130 up to 1 wt.% filler content shows similar electrical conductivity than neat PVDF5130 and the percolation threshold is ≈ 2 wt.% rGO, with an electrical conductivity of σ ≈ 1 × 10The intrinsic properties of CNT lead to low percolation thresholds in polymer-based composites. To tailor polymer-based nanomaterials with functional properties, low nanofiller content is typically required in order not to affect other properties of the polymer, such as thermal or mechanical. In terms of higher conductivity and lower threshold, CNT appears as more interesting nanocarbonaceous filler than rGO for conductive polymer nanocomposites.Mechanical measurements were performed to evaluate the stress-strain response of the several PVDF polymers and composites, as a function of the filler content and type. As it is shown in Among the different polymers, PVDF6010 shows the highest yield strength (larger than 40 MPa) and PVDF-HFP the lowest yield stress (lower than 20 MPa), being inversely for strain at rupture, where PVDF-HFP shows the larger strain, near 70%, and the 6010 rupture is near 12%. At rupture, composites with CNT embedded in PVDF-HFP and 5130 present larger maximum strain, reaching 200% of strain for 025CNT/5130 and 300% for 025CNT/HFP samples. In all the composite samples, except the ones including rGO, the inclusion of CNT fillers yields to higher stiffness and elongation at break. From the behavior observed in the rGO/PVDF samples, it can be concluded that the matrix-filler wetting is considerably weaker than the PVDF-CNT one, leading to brittle fracture of the samples. In fact, literature has reported on the decreasing of the maximum strain with low graphene content as reinforcement for PVDF 1010 [E ≈ 1 GPa being E ≈ 870 and E ≈ 350 MPa for 5310 and HFP, respectively. Reinforced with CNT, all PVDF matrices increase the initial modulus with increasing CNT content, excepting for the 1CNT/5310 sample. Similar behavior is found in rGO/5310 composites, where the initial modulus increases with filler content up to 1 wt.%, decreasing for samples with 2 wt.%.The initial modulus for neat PVDF is larger for 6010 near E ≈ 1.4 GPa. The larger initial modulus and yield stress of the PVDF 6010 composites are correlated with the percolation threshold, which depends on the filler dispersion, but also rigidity of the matrix [The 05CNT/6010 sample shows an initial modulus near e matrix , decreasThe strain at rupture for composites with PVDF 6010 is similar for the different CNT contents, increasing in the remaining PVDF composites, from 70 to 300% in HFP and 23 to 210% in 5130. PVDF5130 with rGO filler suffers a decrease of the strain at rupture from 23 to 4.5%, from neat polymer to the composite with 2 wt.% rGO, respectively. The yield stress and strain are comparable for both fillers (CNT and rGO) in PVDF 5130 materials. The load transfer efficiency and interfacial shear stress of the composites as a function of the dimensions of the fillers have been theoretically calculated . The CNTThose results reflect not just the different mechanical characteristics of the polymers but also the different wettability between polymer and fillers, depending on macromolecule characteristics.PVDF composites with CNT or rGO as filler are excellent candidates for electromechanical sensors, leading to high sensibility composites ,24. ElecThe composites present larger piezoresistive sensibility near the percolation threshold . The lin−1, as shown in The pressure sensibility was evaluated in the different nanocarbonaceous/PVDF composites as a function of polymer and filler type and applied pressure. The highest sensitivity is obtained for 05CNT/HFP composite with PS ≈ 1.1 MPaThe validation of the sensor in pressure sensing applications was performed using the 05CNT/6010 composite. The CNT nanocomposite was integrated into a developed test bench built to emulate the mechanical stiffness present in different mechanisms. With this use case, the capability of employing the developed materials to fabricate sensors which could be applied in industrial smart components is evaluated.The test bench, shown in The nanocomposite material was integrated between the beam and the threaded rod. The resistance change produced under the compression pressure was measured between the fixtures of the beam and the rod employing a Fluke 8845A multimeter and logged through PC. The PMDC motor (Maxon EC-4 pole 22 mm) was actuated by an EPOS2 controller.Different PVDF copolymers were reinforced with carbon nanotubes (CNT) and reduced graphene oxide (rGO) fillers to evaluate the performance of the materials for piezoresistive sensor applications.FTIR analysis shows that PVDF crystallizes in the α-phase, independently of polymer type and filler type and content. Similarly, the mechanical tensile modulus of the matrix and reinforcement filler (CNT or rGO) do not influence filler dispersion for low filler contents. Thermal measurements show the melting temperature around 132, 158, and 169 °C for PVDF-HFP, 5310 and 6010, respectively. This temperature is just slightly affected by the inclusion of the fillers. With respect to the mechanical response, PVDF 6010 presents the highest initial modulus. Tensile tests demonstrate that the inclusion of fillers reinforces the polymer matrices, leading to higher stiffness, yield strength or elongation at break depending on the percentage used. Their percolation threshold is lower for the CNT nanocomposites when compared with the rGO ones, being the percolation threshold independent on the polymer matrix.PS < 1.1. The linearity between the electrical resistance variation and pressure is present in all composites. Finally, a proof of concept is presented showing the suitability of the materials for applications. Hence, PVDF/CNT and rGO based piezoresistive nanocomposites present suitable characteristics to work as embeddable, highly sensitive and cost-effective sensors in industrial pressure sensing applications.Pressure sensibility is larger for PVDF-HFP with 0.5 wt.% CNT for low applied pressures. For the largest pressure, the PS is similar for all materials. The electromechanical pressure sensibility of the materials as a function of pressure varies between 0.2 <