The few studies that have assessed the association between symptomatic atherosclerotic disease and risk of cancer have had conflicting results. In addition, these studies ascertained participants either from treatment settings  or by using a records linkage system  and, therefore, were prone to selection bias. Our purpose was to estimate the risk of cancer mortality in a large population-based sample of elderly people, comparing participants with symptomatic atherosclerotic disease (atherosclerotic stroke and coronary disease) to their counterparts without symptomatic atherosclerotic disease  in the same population.In this population-based, prospective study , 5262 elderly community-dwelling participants with and without symptomatic atherosclerotic disease were identified and followed for a median of 12.1 years, after which the death certificates of those who died were reviewed.P < 0.001). In an unadjusted Cox model, risk of cancer-specific mortality was decreased in participants with symptomatic atherosclerotic disease  vs. those without symptomatic atherosclerotic disease (reference group). In an adjusted Cox model, HR = 0.58; 95% CI, 0.38−0.89; P = 0.01.A total of 2701 (53.3%) of 5262 participants died, including 314 (68.6%) of 458 participants with symptomatic atherosclerotic disease and 2387 (49.7%) of 4804 controls. Cancer mortality was reported significantly less often in those with symptomatic atherosclerotic disease (15.6%) than in controls (25.6%) (This population-based, prospective study suggests that there is an inverse association between symptomatic atherosclerotic disease and risk of cancer mortality. Population-based studies are preferable for studying this relationship because they minimize potential sources of bias and confounding in comparison to those based on hospital discharge registers, and are also preferential when investigating risk factors associated with disease incidence and mortality.19 We hypothesized that the cancer-mortality risk in participants with symptomatic atherosclerotic disease would be lower than in participants without atherosclerotic disease . To address this question, we utilized data from the Neurological Disorders in Central Spain (NEDICES) study, in which a population-based sample of participants was prospectively evaluated, thus permitting the minimization of selection bias.32 Our analyses adjusted for numerous potential confounders. In addition, we conducted secondary analyses stratified by smoking status and differentiated between the main causes of stroke .There is considerable evidence from epidemiological and clinical studies that shows a consistently lower than expected occurrence of cancer in patients with Parkinson disease, Alzheimer disease, multiple sclerosis, and Huntington disease.32Data for these analyses were derived from the NEDICES study, a longitudinal, population-based survey of the prevalence, incidence, and determinants of major conditions of the elderly, including cerebrovascular disease (transient ischemic accident [TIA] and stroke), dementia, Parkinson disease, and essential tremor.32 The survey area comprised 3 communities: Margaritas , which is a working-class neighborhood in Getafe (Greater Madrid); Lista , a professional-class neighborhood in the Salamanca district ; and Arévalo (approximately 9000 inhabitants), the agricultural zone of Arévalo County (125 km northwest of Madrid). Up-to-date lists of residents were generated from population registers. In each community, survey eligibility was restricted to residents aged 65 years or older who were present on December 31, 1993, or during 6 or more months of 1993. Eligible persons who had moved away from the survey area were not traced. In Margaritas and Arévalo, every eligible subject was selected for screening. However, due to the large number of elderly residents in Lista, proportionate stratified random sampling was used to select a subsample of 2113 subjects for screening. The selected study population was 6395 people, but 481 people were ineligible , leaving 5914 eligible subjects, of whom 5278 were enrolled. All procedures were approved by the ethical standards committees on human experimentation at the University Hospitals “12 de Octubre” (Madrid) and “La Princesa” (Madrid). Written (signed) informed consent was obtained from all participants upon enrollment.Detailed accounts of the study population and sampling methods have been published.33 at baseline, subjects were asked to rate their current health on a 5-point scale using the question, “In general terms, how would you describe your health: very good, good, fair, poor, or very poor?” A small number of subjects were in several categories . Therefore, as suggested in several previous studies,33 we collapsed response options into 3 categories. These 3 categories were very good/good, fair, and poor/very poor.Face-to-face evaluations were performed at baseline (1994–1995) and then at follow-up (1997–1998). Briefly, at the time of their baseline assessment, 5278 elderly subjects were interviewed face-to-face using a 500-item screening questionnaire during which data were collected on demographics, current medical conditions, smoking (ever vs never), and drinker (ever at least once per week vs never). As in prior studies,34 atrial fibrillation, cancer, chronic obstructive pulmonary disease, dementia, diabetes, epilepsy (treated), heart failure, myocardial infarction, depression, other psychiatric disorders , renal disease, and stroke. The presence of some items resulted in the assignment of more points than others. The score ranged from 0 (no conditions) to 28 .A comorbidity index was calculated based on the presence of the following conditions, consistent with a recently published comorbidity score developed in ambulatory care settings:35 The 37-MMSE was a Spanish adaptation of the standard MMSE. It included all of the standard MMSE items as well as 3 additional items: an attention task, that is, “say 1, 3, 5, 7, 9 backwards,” a visual order, that is, a man raising his arms, and a simple construction task, that is, copying 2 overlapping circles.35A 37-item version of the Mini-Mental State Examination (37-MMSE) was also administered.36 The waist circumference was taken as the narrowest point between the inferior rib border and the iliac crest.36Waist circumference was measured using a flexible nonelastic measuring tape. Individuals stood with feet together and arms resting by their sides.24 Have you ever been diagnosed by a physician as having suffered a stroke? Have you ever had slurred speech or problems talking to somebody? Have you ever felt mouth deviation? Have you ever felt weakness in an arm or a leg?Most important for this study, the questionnaire included 4 screening questions for stroke and TIA:23 For 47 participants (6.9% of 678) who could not be examined, medical records were obtained from their general practitioners, from in-patient hospitalizations and from neurological specialists (if they had visited 1). We applied the World Health Organization clinical definition of a stroke: “rapidly developing clinical signs of focal  disturbance of cerebral function lasting more than 24 hours (unless interrupted by surgery or death) with no apparent cause other than a vascular origin.”24 TIA was defined as a focal neurological deficit of sudden onset occurring in a cerebrovascular territory distribution and recovering without sequelae in less than 24 hours, with nonvascular causes excluded.24 Stroke and TIA classification and etiology was performed on the basis of medical records, reports of brain imaging, and the judgment of the NEDICES neurologists.24 We restricted the current analyses to those TIAs or strokes caused by symptomatic intracranial or extracranial atherosclerosis , including lacunar strokes. We included lacunar strokes in this category, since emerging evidence suggests that both large artery and small vessel disease should be considered together.37Participants who screened positive for cerebrovascular disease (N = 678) were examined by 1 of 8 senior neurologists who met at the inception of the study to establish standardized methods to perform and interpret the examination .38 The hospital records from those participants who screened positive for coronary disease were examined in order to confirm the cardiovascular event.38 The diagnosis of myocardial infarction was confirmed if data in the hospital record met standardized criteria of chest pain or equivalent, there were diagnostic electrocardiographic changes, and/or there were elevated cardiac enzyme levels.38 Angina was confirmed by hospital records.38With respect to coronary disease , the screening questionnaire included queries adapted and validated in Spanish from the Multinational MONItoring of trends and determinants in CArdiovascular disease and the Rose questionnaire on chest pain and dyspnea.Participants were classified as having symptomatic atherosclerotic disease if they were diagnosed with noncardioembolic TIA or stroke, or coronary disease .Beginning in January 1994, letters explaining the survey and inviting participation were mailed to 6395 subjects. Of these, 5914 subjects were deemed eligible for screening and 5278 subjects (89.2%) were screened. The 636 subjects who were not screened either declined , could not be located due to an address change , or had died  . In Spain, a doctor completes a death certificate on all individuals at the time of death. The certificate is then sent to the local authority in the municipality where the person had been living, and the information is collected in the National Register. The doctor completing the death certificate does so in accordance with the recommendations of the World Health Organization. The assignment of cause of death is based on the basic cause of death , defined as the illness or injury which started the chain of pathological events that directly led to death . Using the International Classification of Diseases , the NEDICES investigators classified the cause of death into 6 main categories: dementia, cerebrovascular disorders, cardiovascular disorders , respiratory diseases, cancer, and other causes .P < 0.001), even after log-transformation. Therefore, we used Mann–Whitney U test to analyze these continuous variables. The Chi-square test was used to analyze categorical variables.Data analyses were performed in SPSS Version 21.0 . Age, waist circumference, 37-MMSE score, number of medications, and comorbidity index were not normally distributed  with 95% confidence intervals (CI). The time variable was the years from the date of the baseline evaluation (1994–1995) to either May 1, 2007 in living participants or the date of death in participants who had died prior to May 1, 2007.We considered several baseline variables as potential confounders. These were age in years, gender, educational level, geographical area , ever smoked (ex-smokers and current smokers), ever drank (ex-drinkers and current drinkers), waist circumference in cm, 37-MMSE total score, arterial hypertension, hypercholesterolemia, number of medications, comorbidity index, and self-rated health .In Cox proportional hazards analyses, we estimated the risks of cancer-specific mortality in participants with symptomatic atherosclerotic disease compared with the reference group (participants who did not have symptomatic atherosclerotic disease). Then, in adjusted models, we first considered variables that were associated at the p < 0.05 level with both symptomatic atherosclerotic disease and cancer-specific mortality (“Model 1” [more restrictive criteria for confounding]) and then considered baseline variables that were associated at the p < 0.05 level with either symptomatic atherosclerotic disease or cancer-specific mortality (“Model 2” [less restrictive criteria for confounding]). For completeness, we also adjusted for all the potential confounders, independent of their statistical significance  (“Model 3”). In secondary analyses, participants with symptomatic atherosclerotic disease were stratified into 2 groups based on their smoking status.Kaplan–Meier survival curves for subjects with and without symptomatic atherosclerotic disease were assessed; the log-rank test was used to compare the differences between the curves.The 5262 participants had a mean duration of follow-up of 9.5 years . Of the 5262 participants, 458 had suffered a symptomatic atherosclerotic disease event at baseline, including 201 TIA/stroke cases (43.9%), 235 (51.3%) angina/myocardial infarction cases, and 22 (4.8%) cases with both diseases.A total of 2701 (53.5%) of 5262 participants died, with deaths occurring at a median follow-up of 6.2 years (range 0.01–13.3 years), including 314 (68.6%) deaths among 458 participants with symptomatic atherosclerotic disease and 2387 (49.7%) deaths among 4804 participants without atherosclerotic disease  versus those without symptomatic atherosclerotic disease (reference group). After adjustment for baseline age, gender, ever smoker, 37-MMSE total score, arterial hypertension, number of medications, comorbidity index, and self-rated health , the risk of cancer-specific mortality remained decreased in participants with symptomatic atherosclerotic disease . In adjusted analyses, for nonsmokers, the results remained similar . In ever smokers, the risk of cancer-specific mortality was similar in participants with versus without symptomatic atherosclerotic disease .In a secondary analysis, participants with symptomatic atherosclerotic disease were stratified into 2 groups based on their smoking status. In nonsmokers, the risk of cancer-specific mortality was decreased in participants with atherosclerotic disease . In adjusted analyses, for men, the results remained similar . In women, the risk of cancer-specific mortality was lower, but not to a significant degree, in participants with versus without symptomatic atherosclerotic disease .In an additional secondary analysis, participants with symptomatic atherosclerotic disease were stratified by gender. In men, the risk of cancer-specific mortality was decreased in participants with atherosclerotic disease  versus those without symptomatic atherosclerotic disease (reference group). In adjusted analyses, the results remained similar .In a final analysis, we excluded gender-specific cancers such as breast cancer (N = 25) and prostate cancer (N = 54). In an unadjusted Cox model, risk of cancer-specific mortality was decreased in participants with symptomatic atherosclerotic disease .The Kaplan–Meier curve for overall survival Figure  showed t39The current study suggests that people with symptomatic atherosclerotic disease are at reduced risk of mortality from malignant neoplasm. When compared to participants without symptomatic atherosclerotic disease, the risk for neoplasm as the underlying cause of death was approximately 30% to 40% lower in participants who had symptomatic atherosclerotic disease. Results from our prospective long-term follow-up population-based study support earlier results from a clinico-pathological study of an inverse correlation between atherosclerosis and cancer.15 In a retrospective cohort of nearly 70,000 patients discharged with extra-coronary manifestations of atherosclerosis, from the Danish National Registry of Patients, Dreyer and Olsen13 found no association between atherosclerosis and colorectal cancers or hormone-related cancers, except for a decreased standardized incidence ratio of 0.7; 95% CI, 0.5 to 0.9 for endometrial cancer. Again, using the same cohort, these authors found that the rates of colorectal cancer in acute myocardial infarct patients were similar to those of the general population, as were the rates for hormone-related cancers, including endometrial and postmenopausal breast cancers.12 However, they found a moderate increase in the risk for tobacco-related cancers, which was strongest for patients with early onset of acute myocardial infarct and for female patients.12 In the same cohort, atherosclerotic women had a significantly elevated HR for lung cancer .14 Finally, in the Danish nationwide cohort, patients with acute myocardial infarction and stroke were at 85% and 42% increased risk of being diagnosed with colorectal cancer, respectively, within the first year of diagnosis.15 However, no increased risk in the second and subsequent years was found.15 Our results extend prior knowledge for the overall finding of lower risk of cancer-specific mortality in the setting of symptomatic atherosclerotic disease. The fact that the risk of cancer mortality was significantly lower in nonsmokers with atherosclerotic disease, but not in ever-smokers with atherosclerotic disease, suggests that a genetic or epigenetic predisposition may mediate this inverse association.A limited number of studies on the incidence of cancer among those who suffered from cerebrovascular or cardiovascular disease have been conducted, and the results are inconclusive.40 Sirtuin functions are intrinsically linked to cellular metabolism, lifespan extension induced by caloric restriction, and gene expression.42 It has been shown that SIRT1 is a critical protective gene in the vasculature and that decreased SIRT1 expression may promote atherosclerosis and medial degeneration due to DNA damage, apoptosis, and accelerated cell ageing.42 Alzheimer disease, as well as other neurodegenerative diseases, has been associated with a reduced risk of cancer mortality,17 and down regulation of the sirtuin pathway has been shown to play a crucial role in the pathogenesis of Alzheimer disease.43 Conversely, overexpression of SIRT1 is frequently observed in various types of cancers.44 Although it remains a controversial issue, it seems likely that SIRT1 overexpression confers survival advantages to cancerous or transformed cells and accelerates carcinogenesis.44Although the current findings suggest that symptomatic atherosclerotic disease is associated with decreased cancer-specific mortality, the mechanisms underlying this association remain unknown. One of the specific links between atherosclerosis and cancer could be the sirtuin family of proteins (SIRT1–7).45 Conversely, patients with atherosclerotic vascular disease had significantly higher levels of Dickkopf-1, a potent inhibitor of Wnt signaling.46 Other crucial and evolutionarily conserved signaling pathways, such as p53, p21, and p16, are significantly upregulated in atherosclerotic disease,48 while they are frequently inactivated in human tumors.49Further, elevated levels of β-catenin protein, a hallmark of an activated canonical Wnt pathway, have been demonstrated in common forms of human malignancies, creating a favorable microenvironment for tumor growth and metastasis and contributing to therapeutic resistance.50 Third, NEDICES was a study of the elderly; hence, we only included individuals aged 65 and older. This means that our findings may not necessarily be generalized to younger populations. However, the prevalence and incidence of both cancer and symptomatic atherosclerotic disease is highest in the age group we enrolled,3 with the disease burden being of most importance in older age groups. Fourth, it is possible that risk factors could have changed during the interim follow-period. Fifth, residual confounding by unmeasured variables in Cox proportional hazards analyses is possible. Finally, competing mortality is an issue to consider – indeed, there could be a potential for survival bias, in that a small proportion of individuals may have died of cancer at younger ages. However, as we stated previously, both cancer and atherosclerotic disease are ageing-related diseases. Therefore, we do not feel that competing mortality could have biased our results.Our study has limitations. First, we did not collect data on comorbidity at death or data on who signed the death certificate . It is logical to assume that the level of expertise of the physician who signed the death certificate would predict the level of accuracy of that certificate. Second, we based the diagnoses of cancer on death certificates. The quality of cancer death certification in Spain is considered high.This study also had several strengths. First, the design was population-based, allowing us to assess people with symptomatic atherosclerotic disease not based solely on care-seeking behavior and, therefore, the results may be generalizable to the community. Second, the assessments were conducted prospectively in a standardized manner, and the follow-up was of considerable length. Third, we were able to adjust for the potential confounding effects of a number of important factors. Fourth, we had the ability to consider detailed analyses of smoking status. Finally, we performed a detailed clinician and chart based-assessment of symptomatic atherosclerosis.Using a prospective, population-based design, we demonstrated that symptomatic atherosclerotic disease was associated with decreased risk of cancer-specific mortality. Of note was that the risk of cancer mortality among those who had symptomatic atherosclerosis, but had ever smoked, was similar to the general population. It is as if tobacco were a modulator of the overall risk. This study provides evidence of an inverse association between symptomatic atherosclerotic disease and cancer.


Rhizobium sp. UR51a is a Gram-negative bacterium isolated from roots of rice plants, and it presents plant growth-promoting abilities. The nutrient uptake in rice plants inoculated with UR51a was satisfactory. The genome of strain UR51a is composed of 5,233,443-bp and harbors 5,079 coding sequences. Rhizobium genus are traditionally classified as symbiotic bacteria that fix atmospheric nitrogen within nodules of leguminous plants. Rhizobium organisms can also act as nonsymbiotic bacteria associated with nonleguminous species of great agricultural importance , in the state of Rio Grande do Sul, Brazil , with an average length of 250 bp. The 54 contigs obtained were connected to form 32 scaffolds, based on the paired-end relationships of the reads. The draft genome sequence of strain UR51a consisted of 5,233,443 bp, with a G+C content of approximately 59.25%. Fifty-one tRNA genes, 5 rRNA genes, and 5,079 coding sequences (CDSs) were assigned on the basis of the annotation.fhuABCD). Siderophores are involved in the sequestration of Fe3+ in soils with low iron content, and they can stimulate bacterial and plant growth by improving Fe3+ nutrition. Genes for auxin biosynthesis, an important regulator that can influence plant development, were also encountered. The UR51a genome encodes antioxidant enzymes that mediate the protection from reactive oxygen species generated by the plant hosts, as well as genes involved in resistance to antibiotics and toxic compounds. Similarly, the sequencing of the UR51a genome will increase the understanding of interaction mechanisms between this bacterium and rice, evidencing the future application as a biotechnological product to enhance crop nutrition and production.The UR51a genome contains genes for the siderophore aerobactin uptake system (JYFU00000000. The version described in this paper is version JYFU01000000.This whole-genome shotgun project has been deposited at GenBank under the accession number


Here we aimed to explore the integrated application of ecological two-sidedness and ecological process in agricultural production. Our results are of use to decision-makers and ecologists focusing on eco-agriculture and those farmers who hope to implement standardized agricultural production practices.While the worldwide standardization of agricultural production has been advocated and recommended, relatively little research has focused on the ecological significance of such a shift. The ecological concerns stemming from the standardization of agricultural production may require new methodology. In this study, we concentrated on how ecological two-sidedness and ecological processes affect the standardization of agricultural production which was divided into three phrases , considering both the positive and negative effects of agricultural processes. We constructed evaluation indicator systems for the pre-, mid- and post-production phases and here we presented a Standardization of Green Production Index (SGPI) based on the Full Permutation Polygon Synthetic Indicator (FPPSI) method which we used to assess the superiority of three methods of standardized production for peaches. The values of SGPI for pre-, mid- and post-production were 0.121 , 0.379 , and 0.769 × 10 Agricultural standardization is based on principles such as simplification, unification, coordination, optimization, etc. on the basis of science, technology and practical experience. The aim of agricultural standardization is to guide and specify agricultural planting, processing, management and sale activities, achieve improved crop yield and quality as well as to promote economic, social and ecological profits1Since the 1970s, global agriculture standardization has progressed rapidly, especially in some developed countries, where the pre-, mid- and post-production of agriculture have almost realized standardization of methods, and have also formed a more complete production-support system. Reportedly, the standardization levels of agricultural production in the United States, Japan, Germany, and France, to name a few, have ranked among the highest in the world. Agricultural standardization production was seen as a guarantee for increasing farmers’ income, agricultural effectiveness, and rural development1At present, the standardization of agricultural production for “green”  food is in demand in China, because sustainable agriculture has three key differences compared to conventional agriculture: 1) an emphasis on ecology and the environment, 2) quality control of the whole process , and 3) traditional techniques that are combined with modern advanced technologies, with each of the production links integrated organically. Thus, the standardization of agricultural production for sustainable agriculture is one of the most important ecological issues for sustainable development of agro-ecosystems and agriculture itself. While ecology has proven to be one of the most effective tools of applied science457Certainly, agriculture is closely related to ecology9101113Agro-ecosystems consist of social, economic and natural factors with different properties that not only have their own respective structure, function and evolution laws, but also interact and restrict one other1517181920215 ha and an annual peach yield of 4.600 × 109 kg. Standardization of agricultural production in orchards was introduced into China in the 2000s, and then was gradually applied nationwide. In recent years, the standardization of agricultural production has been considered one of the best methods of promoting sustainable agriculture in orchards. This model has been gradually accepted by a large number of farmers and extensively applied in orchards in China.China has the largest area of peach cultivation and the largest output of peach yield in the world, with an annual peach cultivation area of 4.520 × 10Here we present a research framework for the standardization of agricultural production based on ecological processes and the principle of ecological two-sidedness, establish the assessment indicator systems for the sustainable production of pre-, mid- and post- production standardization, and propose an eco-index (Standardization of Green Production Index [SGPI]) combined with the Full Permutation Polygon Synthetic Indicator (FPPSI) method, so as to provide a theoretical and practical basis for worldwide agricultural standardization production.Before standardized production was implemented, safe heavy metal levels in soils were seriously exceeded, field irrigation water quality was hardly ideal, and the demands of sustainable food production could not be met. Through adopting physical and biological control techniques of pest management and using ground cover vegetation to improve soil nutrients, the input of agrochemicals was reduced, to some extent the soil environment quality was restored and the indicator levels of heavy metals in soils were all under maximum threshold values, although the content of mercury was close to the threshold value . This inIrrigation water quality was sharply improved through installing floating vegetations in the rivers and constructing sewerage filtration ponds around the peach orchards, and the observed levels were all under critical values. By adopting healthy culture techniques, seedling quality was significantly improved, and the indicator values of excellent rates of bud seedling quality and seedling growth of peach trees increased 15.0% and 8.0%, respectively.Implementation of standardized production had a definite effect on peoples’ living conditions, as the rates of people with access to sanitary drinking water, garbage disposal, and the sanitary discharge rate of industrial pollutants all reached 100.0%. The centralized sewage treatment rate reached 90.0%, and peoples’ satisfaction rate for environment conditions reached 99.9%.Analytically, the pre-production index values of negative and positive effects were 0.113 and 0.935, respectively. Thus, the pre-production standardization green index value was 0.121 which indicated that the total conditions of pre-production standardization were suitable for sustainable agriculture as it reached level IV (“Excellent” standard).−2 and 12.970 kg.hm−2, respectively , while the product weights and active ingredient weights of conventional chemical pesticides were 12.010 kg.hm−2 and 5.000 kg.hm−2, respectively, values that were reduced by 50.6% and 40.2%, respectively, through agricultural standardization. The input amount of organic fertilizers was 1.500 × 104 kg.hm−2, while the product weight and the weight of available nutrient in the chemical fertilizers employed were 2.250 × 104 kg.hm−2 and 975.000 kg.hm−2, respectively, which were reductions of 40.0% and 39.5%, respectively.Pests in peach trees were well controlled by adopting physical and biological techniques, and this provided a theoretical and practical basis for pesticide reduction. In addition to the use of ground cover vegetation to improve soil fertility, an inorganic-organic integrated fertilization mode was widely applied to prevent nutrient loss and pollution. The product amounts and the active ingredient weights of the bio-pesticides used were 29.000 kg.hmDrainage channels and ditches in peach orchards were constructed with the specified standard design, and standard fruit-thinning and bagging techniques were employed. Use of these practices was beneficial to the yield and quality of the peaches as well as cost-saving. With the substantial reduction of chemicals and the standardization of farm operation systems, certain costs were decreased, arthropod diversity was significantly increased, the numbers of neutral groups and natural enemy groups were both substantially increased, and percentage of farmers’ acceptance of standardization production reached 93.8%. However, the input of high-efficiency, environmentally friendly new techniques increased the production cost to some extent, although these techniques provided benefits to related industries.The mid-production index values of negative and positive effects were 0.341 and 0.900, respectively. Thus, the mid-production standardization green index value was 0.379 which indicated that the implementation of standardization production techniques in this phase was beneficial to peach orchard ecosystems as it reached level III (“Good” standard).The physical environment of pre-production standardization and the techniques of mid-production standardization are both important influences on post-production indicator values of consumer perception of produce quality, physiochemical characteristics of produce, and fruit sanitary requirements. After standardization production techniques were adopted, the consumer perception of fruit quality had indicator values of perfect for fruit shape, color and luster, neatness of the fruit surface and fruit flavor rate , scores that increased by 15.0, 16.0, 18.0, and 15.0% over values in non-standard production schemes. Soluble solids and titratable acids were the most important factors affecting the flavor and quality of fruit, and soluble content and malic acid content were 13.1 and 0.26%, respectively, being increases of 1.8 and 0.06%, respectively. Individual fruit weight (average 0.212 kg) increased by 7.8%, which was a key indicator of enhanced yield and increased income for the farmers. As for sanitary indicators of peach fruits, the contents of heavy metals and chemical pesticide residues were far below maximum established allowable limits, and this guaranteed the safe quality of the produce.−2 and 0.824, respectively, so the post-production standardization green index value was 0.769 × 10−2, reaching level IV (“Excellent” standard), which indicated that the standardization of pre- and mid-production promoted the healthy development of post-production standardization.The post-production index values of negative and positive effects were 0.634 × 10While previous studies have reported on the agricultural practices involved in standardized crop production, ecological methods have not, until now, been applied to this practice. In this study we proposed an integrated methodology of ecological process and ecological two-sidedness from the macro system combined with social, economic and natural factors as well as the crops and environment. To do so, we have divided the existing agricultural process into pre-, mid- and post-production, which allowed us to optimize techniques within a given part of the process.Before implementing standardized production, farmers used to bag fruits with newspapers, neglected proper thinning of fruit load, and misused agricultural chemicals. As a result, the quality of peach fruits did not meet the requirements of sustainable agriculture. In the standard production scheme studied here, use of chemical pesticides was reduced by adopting biological and physical control techniques. Fertilizer nutrient loss was prevented and fertilizer efficiency was also greatly strengthened by adopting techniques of ground cover vegetation, accurate quantitative fertilization and organic-inorganic integration fertilization modes. In addition, the yield and quality of peaches were raised by standardizing the techniques of fruit thinning and bagging.4 kg·hm−1, increasing by 9.5%, and the peaches grown there passed National Grade A Level of Green Food Certification issued by the China Green Food Development Center. After standardized production systems were implemented, new researchable questions arose: how the complex peach orchard ecosystems responded, how best to objectively evaluate disturbed agro-ecosystems, and how to construct methodological assessments.To apply ecological principles to both problem analysis and solution, we determined the key factors limiting the development of the peach industry in the study area, and then integrated these techniques with the requirements of sustainable agriculture. The peach yield in the study areas reached 1.628 × 10The application of ecological principles to agricultural production has been widely done1323171819202122Certainly, complexity is the intrinsic attribute of any ecosystems16Xinfeng” honey peach (mid-late maturing varieties), 77% cuprousoxide WP, 2% ningnanmycin AS, 72% agricultural streptomycin SP, 45% crystal lime sulphur, 1.2% matrine EC, 50% procymidone WP, 10% difenoconazole WDG, 75% chlorthalonil WP, 65% Zineb WP, 70% thiophanate-methyl WP, 10% imidacloprid WP, 25% chlorbenzuron SC, 2.5% beta-cypermethrin EC, 20% diflubenzuron SC, urea (N 40%), compound fertilizer , organic fertilizer, yellow paper bagging with single layer, solar insecticidal lamp (12v 6w), yellow peach moth sex pheromone, oriental fruit moth sex pheromone, peach leaf miner sex pheromone, plastic tube for suspending sex pheromone, sunflower seed, white clover, irrigation water, and pesticide sprayers.“Dichocrocis punctiferalis Cuenée), one oriental fruit moth (Grapholitha molesta), and one peach leaf miner (Lyonetia clerkella L.) sex pheromone traps (with the active ingredient in a capillary vessel 10.0 cm long and 1.5 mm in diameter) were hung at 20.0 m intervals in peach orchards, and the attractants were replaced monthly. To enhance the diversity and stability of arthropods and to better control pests in peach orchards, ground cover vegetation (Trifolium repens L.) was planted in the peach orchards in the aisles between tree rows.The study was conducted from 2009 to 2011 in the town of Xinchang, Pudong district of Shanghai Municipality in eastern China , where 70.0% of Shanghai peach orchards are concentrated, of which 70.0% conform to standardized agricultural production practices, a higher rate than any other district in Shanghai. For this study, farmers were trained in standardized techniques of agricultural farming under the guidance of scientific researchers in study areas with three replicates (each replicate plot was about 2 ha). Solar insecticidal lamps were installed in each 2-ha areas, and were automatically turned on at 6:00 p.m. and turned off at 5:00 a.m. All lamps were hung 2.0 m above the ground. Clusters composed of one yellow peach moth  and at three levels , thereby splitting each tree canopy into 12 resource units2728The framework of understanding the standardization of agricultural production in complex ecosystems is shown in 313233SGPI) matrixSGPI, the more beneficial it would be to the agro-ecosystems. According to typical index classification method, the SGPI was divided into four levels  method used here has been applied to optimization of other complex systems36Iij was the standardized value of the ith indicator to the jth project for positive effect evaluation, Xij was the actual value of the ith indicator to the jth project for positive effect evaluation, and Rij was the reference value of the ith indicator. Similarly, the standardization process for the value of negative indicators can be described:where Iij can be described as follows: (1) the n indicators are axes that take a normalized value of 1.0 for each indicator, (2) from these axes, an n-sided polygon can be drawn up, the values of all indicators for different projects being scaled over the n-sided axes, and (3) the new n-sided polygon will be obtained through linking the points of each projection. Then, the area of the new polygon divided by that of a regular n-sided polygon with the radius of 1.0 value, will determine the comprehensive index for positive effect indicators:The analytic method for Ij is the comprehensive index for positive effect indicators, Sj is the area of polygon for the jth project, and St is the area of regular n-sided polygon with the radius of 1.0 value. Similarly, comprehensive index I’j for negative effect indicators is presented:where WSGPI for standardization green production evaluation to x projects is presented:Thus, the matrix SGPI, in which it was supposed that the lower the value of SGPI, the more reasonable the candidate technique is.Finally, a reasonable strategy was determined according to the values of How to cite this article: Wan, N.-F. et al. An ecological method to understand agricultural standardization in peach orchard ecosystems. Sci. Rep.6, 21675; doi: 10.1038/srep21675 (2016).


In developed countries, vitamin B12  deficiency usually occurs in children, exclusively breastfed ones whose mothers are vegetarian, causing low body stores of vitamin B12. The haematologic manifestation of vitamin B12 deficiency is pernicious anaemia. It is a megaloblastic anaemia with high mean corpuscular volume and typical morphological features, such as hyperlobulation of the nuclei of the granulocytes. In advanced cases, neutropaenia and thrombocytopaenia can occur, simulating aplastic anaemia or leukaemia. In addition to haematological symptoms, infants may experience weakness, fatigue, failure to thrive, and irritability. Other common findings include pallor, glossitis, vomiting, diarrhoea, and icterus. Neurological symptoms may affect the central nervous system and, in severe cases, rarely cause brain atrophy. Here, we report an interesting case, a 12-month old infant, who was admitted with neurological symptoms and diagnosed with vitamin B12 deficiency. Vitamin B12 or cobalamin deficiency is a rare and treatable cause of failure to thrive and delayed development in infants. In developed countries, the deficiency usually occurs in infants, exclusively breastfed ones whose mothers exhibit unrecognized pernicious anaemia or are vegetarian, causing low body stores of vitamin B12 in the infant at birth and inadequate amounts of the vitamin in the breastmilk ,4. ThereA 12-month old male infant from the province of Konya, Turkey, was referred to the Pediatrics Clinic of Konya Education and Research Hospital because of developmental regression and growth retardation in April 2012. Medical history taken from his mother revealed that he was born at term, weighing 3,500 g after uncomplicated pregnancy and delivery. He was exclusively breastfed, and his mother had been following a vegetarian diet for many years. The case showed normal developmental features up to 6 months: smiling at 2 months, controlling his head at 4 months, and starting to roll at 5 months. During the first 6 months of life, his body measurements were at the 25-50th percentile for weight and length and at the 50th percentile for head-circumference. When the child became 6 months old, his parents recognized that their baby stopped gaining weight and became less active.3/mm3, and 308×103/mm3 respectively. Mean corpuscular volume, reticulocyte count, red blood cell count, and haematocrit were 97.3 fL (reference range 80-96 fL), 6×103/mm3, 2.63×106/mm3, and 21.3% respectively. The neutrophils were seen as hypersegmented. The serum cobalamin level was 117 pg/mL (reference range 211-911 pg/mL). Serum folate level was 13.85 ng/mL (reference range 3.1-20 ng/mL). Iron and ferritin levels, biochemical profile, and urine test results were normal, with normal blood and urine aminoacidography. Cortical atrophy and enlargement of subarachnoid space were evident on MRI scan  and length (71 cm) were on the 10-25th percentile, and his head-circumference (45.5 cm) was on the 25th percentile. The results of general physical examination were normal; however, the case was determined to show rhagades around the angles of both eyelids and mouth as seen in the photo  [The babMRI scan . VitaminMegaloblastic anaemia due to vitamin B12 deficiency was diagnosed through a combination of clinical and laboratory findings, including clinical presentation, increased mean corpuscular volume (macrocytosis), hypersegmentation of neutrophils, and low vitamin B12 levels.The case was given intramuscular injection of 1 mg of cobalamin and displayed a prompt neurological recovery. Three days after the first injection, he was smiling again and was neither lethargic nor hypotonic any longer. Haematologic values improved at the second week of the treatment. His parents were pleased because the case had a great improvement in his mental and motor development. Thus, the communication skills were improved with parents, the appetite became ameliorated, and he could control his head again. He started walking when he was 18 months old. Control cranial MRI performed three months after the initiation of therapy demonstrated recovery of cerebral atrophy. Subarachnoid space width was in normal range . With hiVitamin B12 deficiency is probably the second most common vitamin deficiency, causing anaemia. It is a megaloblastic anaemia, with high mean corpuscular volume and typical morphological features, such as hyperlobulation of the nuclei of the granulocytes . In deveBecause of the lack of vitamin B12 synthesization in humans and its existence in only animal products, vegetarian, vegan or macrobiotic diets may lead to vitamin B12 deficiency . Recommeet al.  and a psychomotor and linguistic delay are reported to be associated with long-term dysfunctions after prolonged vitamin B12 deficiency in infants . In our Findings from the present case study suggest that vitamin B12 deficiency should be considered in the aetiological diagnosis of neurological symptoms in infants, especially in the presence of megaloblastic anaemia and developmental delay. Healthcare providers should take into account that patients with vitamin B12 deficiency can present unexpected signs and symptoms, such as developmental delay, cerebral atrophy, and subdural effusion as seen in our patient.Vitamin B12 deficiency is a rare but treatable cause of neurological disorders, anaemia, and failure to thrive in infants exclusively breastfed by vitamin B12-deficient mothers. Recognition of the neurological symptoms of infantile vitamin B12 deficiency may allow early diagnosis and appropriate treatment. Because of the importance of vitamin B12 in the development of the foetal and neonatal brain, vegetarian and vegan mothers should be aware of the severe and not fully-reversible damages caused by insufficient nutritional intake of vitamin B12 during pregnancy and lactation. Therefore, efforts should be directed to prevent its deficiency in pregnant and breastfeeding women on vegan diets and their infants. It is also important to take the nutritional history of both infants and their mothers for the early prevention and treatment. To increase the dietary intake of vitamin B12, the diet should be rich in foods of animal origin, such as dairy products, red meat, egg and fish. Recommended dietary intake of B12 should increase from 2 to 2.2 µg/day during pregnancy. With early awareness, potentially irreversible neurologic damages can be prevented.


Cell communication is central to the integration of cell function required for the development and homeostasis of multicellular animals. Proteins are an important currency of cell communication, acting locally  or systemically (endocrine). The fibroblast growth factor (FGF) family contributes to the regulation of virtually all aspects of development and organogenesis, and after birth to tissue maintenance, as well as particular aspects of organism physiology. In the West, oncology has been the focus of translation of FGF research, whereas in China and to an extent Japan a major focus has been to use FGFs in repair and regeneration settings. These differences have their roots in research history and aims. The Chinese drive into biotechnology and the delivery of engineered clinical grade FGFs by a major Chinese research group were important enablers in this respect. The Chinese language clinical literature is not widely accessible. To put this into context, we provide the essential molecular and functional background to the FGF communication system covering FGF ligands, the heparan sulfate and Klotho co-receptors and FGF receptor (FGFR) tyrosine kinases. We then summarise a selection of clinical reports that demonstrate the efficacy of engineered recombinant FGF ligands in treating a wide range of conditions that require tissue repair/regeneration. Alongside, the functional reasons why application of exogenous FGF ligands does not lead to cancers are described. Together, this highlights that the FGF ligands represent a major opportunity for clinical translation that has been largely overlooked in the West. In unicellular organisms the unit of natural selection is the cell, whereas in multicellular animals natural selection operates on the organism. This is a very profound difference. The driver is likely to have been simple: there is a limit on the complexity of an individual cell, beyond which it is no longer robust. However, greater organism complexity allows new ecological niches and lifestyles to be exploited. Natural selection has given rise to multicellularity and cell specialisation, as a means to allow a high level of organism complexity in concert with simple and robust cells. This requires a deep functional integration of cells in the organism, achieved through cell communication, which occurs by cells delivering information through the synthesis and secretion of signalling molecules into the extracellular space; these then elicit signals in cells possessing appropriate receptor systems. The entire biochemical landscape, from ions and small molecules to proteins and polysaccharides is used to generate the repertoire of signalling molecules.versus endocrine.In multicellular animals, proteins are common currency in cell communication and are used to transmit information between cells in the organism both locally  and systemically (endocrine). Local transmission of information may be mediated by a soluble, secreted protein, or by a protein anchored in the extracellular matrix or on the plasma membrane of a neighbouring cell. The exploitation of proteins for cell communication by multicellular animals provides access to a very subtle language. This subtlety arises in part from the fact that an individual protein species may have many different isoforms (from splice variants to glycoforms), localisations and interacting partners. Each subset of molecular interactions that an individual protein species can partake in may elicit completely different, sometimes opposing, cellular responses, and, moreover, may change the distance over which communication occurs, e.g., paracrine Most therapeutics can be considered to manipulate cell communication, with varying aims, such as reducing overactive communication channels in cancer and inflammatory diseases, or increasing particular channels of communication for tissue repair and regeneration. Clearly, manipulating cell communication therapeutically is not without danger, since the opposite of the desired effect may occur. Less obvious, but an important focus of this review, is that the drivers of scientific discovery can narrow dramatically how a particular communication system is used clinically. This is exemplified by the fibroblast growth factor (FGF) family, which contributes to the regulation of virtually all aspects of development and organogenesis, and after birth in many natural processes of tissue repair and the endocrine regulation of particular facets of organism physiology. In the West, the major focus of clinical translation has been on developing inhibitors of FGF-mediated cell communication for use in cancer therapy. In the East, particularly in China and to an extent in Japan, a major focus has been to use FGFs in regenerative/repair medical settings, differences that have their roots in a combination of research history and research aims. To increase awareness of this work, we summarise a number of published clinical reports to illustrate the breadth and depth of the successful clinical applications of FGFs.in vitro enabled the discovery of some, but not all of FGFs -3 to -9 ; FGF4, FGF5 and FGF6 (FGF4 subfamily); FGF3, FGF7, FGF10 and FGF22 (FGF7 subfamily); FGF8, FGF17 and FGF18 (FGF8 subfamily); FGF9, FGF16 and FGF20 (FGF9 subfamily); FGF11, FGF12, FGF13 and FGF14 (FGF11 subfamily); FGF19, FGF21, and FGF23 (FGF19 subfamily) . The memDrosophila. All FGFs share an internal core of similarity with 28 highly conserved, and six invariant amino acid residues  are then added by the copolymerases EXT1 and EXT2  was identified by its sequence homology to αKlotho  are type 1 transmembrane proteins that define tissue specific activities of circulating endocrine FGFs  causes the FGFR to dimerise. This in return enables phosphorylation of tyrosine residues in the kinase activation loop and then of tyrosines that are docking sites for signalling proteins . The lattivities . Previoutivities . FGF signd Spred  and MAPKnd Spred . Since tnd Spred .FGFs and FGFRs interact directly with a large number of other partners, both extracellularly and, following the internalisation of ligand-receptor complexes, intracellularly. In some instances, e.g., FGF-2 binding integrins , these mC. elegans possesses one of the simplest FGF communication systems, comprising two ligands, EGL-17  is predominantly expressed in the hypodermis (egl-15(5A) isoform is expressed in the M lineage, which gives rise to the sex myoblasts. Heterologous expression of egl-17, driven by the let-756 promoter, can stimulate EGL-15(5B) and partially rescue the larval arrest phenotype of mutants lacking LET-756, and expression of let-756 driven by egl-17 promoter can partially rescue sex myoblast migration in EGL-15-deficient worms  isoform can mediate sex myoblast chemoattraction  phenotype. Excess EGL-15 activity and clear phenotype can also be achieved by mutation of N-glycosylation sites in the extracellular domain of the EGL-15 receptor  and klo-2 . Thus, a reasonable hypothesis is that the communication system used in development, LET-756 and EGL-15, is then co-opted into endocrine homeostasis. As animals grew larger, this would no longer be possible. Diversification of the FGF family and weakening of heparan sulfate binding would then allow both the growth factor/morphogen activity, which is local due to heparan sulfate binding and the systemic hormonal activity to be retained. In support of this idea is the demonstration that a human FGF-1 with its primary heparan sulfate binding site mutated is reprogrammed from a growth factor to a FGF-21 like hormone, controlling metabolism . Similarly, the healing time in the deep partial thickness burn wounds in the FGF2 treatment group was significantly shorter than the control group .FGF2 has also been shown to accelerate healing and improves scar quality in second-degree burns . Since tSurgical incisions leave scars as part of the normal healing process. These scars vary from being narrow, wide, atrophied or hypertrophic and sometimes cause medical problems, or social ones, because of their cosmetic appearance . A studyHealing of the donor site wounds, created after skin graft harvesting, involves the regeneration of epithelial cells in the residual skin appendages . Early hp < 0.01). Moreover, FGF2-treated wounds appeared flatter, smoother and firmer and were difficult to tear off, as compared to the control wounds. The use of FGF2 yielded no adverse reactions.vs. 8.1 days, p < 0.05). The FGF2-treated groups showed good healing. In addition, exudate and swelling post surgery were milder in the FGF2 groups than in the control group. There were no adverse reactions in the FGF2 groups. Quality of wound healing was superior and the healing time was shorter in the FGF2 groups as compared to the control group, indicating that FGF2 has a favourable effect on cosmetic surgical incision healing.Wound healing quality is important in the success of cosmetic surgery. Dehiscence of caesarean section incisions may occur in the form of a superficial dehiscence, in which the skin and subcutaneous fat layer break open, most often due to fat liquefaction caused by subcutaneous fat hypertrophy in pregnant women. In addition, a long trial of labour, excessive vaginal examinations, vaginitis, and intrauterine infections may potentially lead to an increase in infected incisions. Anaemia, hypoproteinemia, malnutrition, and diabetes in the perinatal period can result in poor healing capacities of local tissues. These factors can adversely affect wound healing extending hospital stay, and increasing costs.vs. 11.2 ± 1.2, p < 0.01). In contrast, in patients with dehiscence measuring 5 cm and above secondary suturing was undertaken. In this instance there was no significant difference in the FGF2 treated group compared to their respective control group ; it is likely that this control group’s shortened healing time, compared to the control group with dehiscence measuring 5 cm or less, was due to secondary suturing.FGF signalling plays an important role in skeletal development . Tissue Oral ulcers are a common disease of the oral mucosa and tend to recur. Pathologically, ulcers of oral mucosa are mainly characterized by dissolution, rupture, and shedding of local oral mucosal epithelium to form non-specific ulcers.p < 0.05 for days 3, 4, 5, and 6) as compared to the other pastes. Ulcer size was significantly reduced  in this group. No obvious adverse drug effects were observed.A study by Radiotherapy is commonly used to treat head and neck cancer. However, when the radiation dose rises to about 20 Gy–30 Gy, acute inflammation of the oral mucosa usually occurs, the symptoms of which include, among other things, oropharyngeal pain, and oral ulcers associated with oedema or pseudomembrane formation. Food intake is affected as a result. Moreover, the severity of the symptoms increases with the radiation dose. Patients who experience serious symptoms have to suspend the treatment, and the final efficacy of the treatment is thus impaired .Myeloablative allogeneic haemopoietic stem cell transplantation is an established treatment for haematologic malignancies and oral mucositis is a known complication arising from high dose chemotherapy and radiation therapy. p < 0.05). The results show that FGF2 exhibits significant efficacy for mild recurrent aphthous oral ulcers.vs. FGF2 application in the management of traumatic perforations of the tympanic membrane. Patients were divided into 3 groups: no intervention (n = 18), edge approximation (n = 20) and direct application of FGF2 (n = 20). Otoscopy was performed before and after treatment and response measurements were made, such as closure rate, closure time and rate of otorrhoea. Perforation closure was significantly higher in the FGF2 group (100%) as compared to the edge approximation (60%) and control (56%) groups (p < 0.05). Average closure time was significantly shorter in the FGF2 treatment group (12.4 ± 3.6 days), as compared to the edge approximation (46.3 ± 8.7 days) and control (48.2 ± 5.3 days) groups (p < 0.05). While most traumatic perforations of the tympanic membrane tend to heal spontaneously, large perforations may often fail to do so. The management of these is still open to debate, with a number of specialists recommending an early myringoplasty to improve outcomes . Lou & WTreatment of pressure ulcers is a major problem for clinical care. Pressure ulcers can increase patients’ suffering, extend the duration of illness, and, when serious, may even prove to be life threatening due to sepsis resulted from secondary infection. Commonly used treatments over the years have included innovative mattresses, ointments, creams, solutions, dressings, ultrasonography, ultraviolet heat lamps, and surgery.3. The results showed that, compared with placebo-treated patients, the number of FGF2-treated patients whose ulcers shrank by 70% increased significantly . Histological analysis of FGF2-treated wounds showed a significant increase in the number of fibroblasts and capillaries.Diabetic foot is a serious complication of diabetes and an important cause of diabetes-related disability. When diabetic foot develops, the patient’s feet are prone to injury, infection, ulcers and gangrene.p = 0.025). Cure rates were 46.8%, 57.4%, and 66.7% in the placebo, 0.001% (w/v) FGF and 0.01% (w/v) FGF2 groups. This trial showed that FGF2 accelerates healing of diabetic ulcers.Repair of cerebrospinal fluid leakage is difficult, which is especially so when a large fistula, with concomitant mucosal damage and infection, has developed from repeated transsphenoidal operations. The mucosa of the vocal folds atrophies with age causing glottal insufficiency, which is difficult to treat. In Europe and N America, the substantial investment by cancer sources into growth factors such as the FGFs has resulted in oncology directed clinical translation, in the form of FGFR inhibitors . In cont


It is expected that physical dose deposition properties render charged particle dose distributions sensitive to targeting uncertainties. Purpose of this work was to investigate the robustness of scanned-beam particle therapy plans against setup errors for different optimization modalities, beam setups and ion species.For 15 patients with skull base tumors, localized in regions of severe tissue density heterogeneity, scanned lateral-opposed-beam treatment plans were prepared with the treatment planning system TRiP98, employing different optimization settings (single- and multiple-field modulation) and ion species (carbon ions and protons). For 10 of the patients, additional plans were prepared with individually selected beam setups, aiming at avoiding severe tissue heterogeneities. Subsequently, multiple rigid positioning errors of magnitude 1–2 mm (i.e. within planning target expansion) were simulated by introducing a shift of the irradiation fields with respect to the computed tomography (CT) data and recomputing the plans.%95 was up to 0.7 percentage points (pp) and 3.5 pp, for shifts of magnitude 1 mm and 2 mm respectively, however, in individual cases, the reduction reached 5.1 pp and 9.7 pp. In the corresponding proton plans similar median CTV V%95 reductions of up to 0.9 pp (1 mm error) and 3.4 pp (2 mm error) were observed, with respective individual-case reductions of at most 3.2 pp and 11.7 pp. Unconstrained plans offered slightly higher coverage values, while no relevant differences were observed between different field modulation methods. Individually selected beam setups had a visible dosimetric advantage over lateral-opposed beams, for both particle species. While carbons provided more conformal plans and generally more advantageous absolute dose values, in presence of setup errors, protons showed greater dosimetric stability, in most of the investigated scenarios.In presence of shifts, in carbon ion plans using a lateral-opposed beam setup and fulfilling clinical healthy tissue dose constraints, the median reduction in CTV VResidual patient setup errors may lead to substantial dose perturbation in scanned-beam particle therapy of skull base tumors, which cannot be dealt with by planning target expansion alone. Choice of irradiation directions avoiding extreme density heterogeneities can improve plan stability against such delivery-time uncertainties. The so-called inverse depth-dose profile and the sharp lateral penumbra make charged particles, like protons and carbon ions, apt for high-precision dose-escalated radiotherapy, e.g. in the treatment of intracranial tumors, due to the direct vicinity of critical structures ,2. CarboYet, based on physical properties of dose deposition by charged particle beams, it is expected that these advantages come to the price of greater sensitivity to delivery-time targeting uncertainties as both, the geometrical miss due to sharp lateral gradients and the displacement of the density heterogeneity interfaces within the treatment field, may result in delivered dose deterioration, which may not be completely dealt with by planning target expansion . For scaet al. [et al. [The importance of the selection of an optimal plan, not only in terms of clinical dosimetric quality , but also taking into account robustness, i.e. degree of sensitivity of the plan to the uncertainties involved in the treatment process, has been recognized by the ICRU  and emphet al. . One met [et al.  reported [et al. .et al. [et al. [While the dosimetric robustness of particle therapy treatment plans has been extensively studied for other indications, e.g. prostate tumors ,9, the pet al.  investig [et al. , focusedIn this study we apply extensive plan recomputation, on a cohort of patients with skull base tumors, in order to systematically assess dosimetric robustness of scanned beam charged particle treatment plans in presence of setup errors and critical heterogeneities for (1) various plan optimization modalities, (2) different beam setups and (3) two particle species (carbon ions and protons).The data of 15 patients with skull base tumors (P1–P15) treated at our institution with intensity-modulated radiotherapy (IMRT)  or with stereotactic radiotherapy (SRT) (remaining) were chosen, with tumor localization typical for particle therapy indications , in areas of high density heterogeneity and close to dose limiting structures.3. The planning target volume (PTV) was defined, similarly to Orecchia et al. [Planning computed tomographies (CT) were acquired, without contrast medium, with in-slice resolution and slice thickness of 0.98 mm/pixel and 3 mm , 0.59 mm/pixel and 3 mm P2, P11–P12) and 0.59 mm/pixel and 2.5 mm (remaining). The original clinical target volume (CTV) and organs at risk (OAR) contours, retained for the study, were delineated in the Pinnacle3  treatment planning system (TPS) for IMRT cases and in the Virtuos  system for SRT cases, excluding unnecessary air cavities from target volumes. The median (range) CTV volume was 45.2 (15.6–90.7) cm, P11–P12Following our institutional ethics procedures, all patients were informed of potential retrospective research use of their data and given the choice to opt out. The data presented in this manuscript do not reveal, either directly or indirectly, the patients’ identities.dcm2trip, developed at our institution and distributed with TRiP98, was used to convert the DICOM data (CT images and structure sets) to the VOXELPLAN format used by TRiP98 [Carbon ion and proton treatment plans for raster-scanning delivery  were prey TRiP98 .α/β=2 Gy, threshold dose Dt=30 Gy, nuclear radius r=4.5μm), while a fixed value of 1.1 was employed for proton RBE.The local effect model LEM I was used for calculation of the RBE of carbon ions  and at least 95% of the prescription dose to 98% of the CTV volume (V%95≥98%), if possible without compromising organs at risk.The prescription dose was set to 60–63 Gy(RBE) delivered in 20–21 fractions for carbon ion plans and 64–74 Gy(RBE) in 32–37 fractions for proton plans similarly to Nikoghosyan et al. ,16 and t [et al. . The plamax) constraint at 54 Gy(RBE) was defined for optic nerves, optic chiasm and brainstem, however, the brainstem surface (1% of volume) abutting the PTV was allowed to receive a dose of up to 60 Gy(RBE) [Also OAR dose constraints were derived from the protocols adopted in the above-mentioned studies. A maximum dose (D Gy(RBE) ,16. In p Gy(RBE) .For each patient two carbon ion treatment plans and a proton plan were prepared using two lateral-opposed beams (LR), reflecting the geometry typically available at fixed-nozzle carbon ion facilities.sc) and full plan modulation . The latter modality was also used in proton planning. Additionally, for comparison, plans without OAR constraints  were prepared for both ion species.The two carbon ion plans had, respectively, OAR constraints requiring per-field modulation  were prepared using two beams with individually selected directions. By visually analyzing potential beam directions, those were selected that appeared to minimize the presence of density interfaces along the entrance channels, that could introduce range variations in case of setup errors. These beam setups, referred to as robust (ROB) in the remainder of the text, employ a range of isocentric couch rotations (yaw) towards cranial direction, combined, in the majority of cases, with beam inclination about the longitudinal axis. Such gantry-rotation-like setups are enabled, at our facility, by combined use of 45-degree-inclined beam lines, 6-degree-of-freedom robotic couches (enabling patient roll and pitch up to 15 degrees with proper immobilization) and, when required, individualized solutions (immobilization with an advantageous head rotation or treatment in prone position).Finally, for selected patients (n=10), carbon ion and proton plans using full modulation  in air at the treatment room isocenter are a function of the beam energy . For carIn the irradiation raster, the transversal distance between neighboring spots (pitch) was set to 2 mm for carbon ion plans and 3 mm for proton plans. A tolerance of 0.4–1.0 times the spot size was set, allowing the TPS to place additional raster spots outside the target volume projection, transversely to the beam, to ensure PTV coverage without the need for high fluence spots at the target border. For carbon ion plans, a ripple filter was used to broaden the narrow pristine Bragg peaks and enable an in-depth peak positioning step of 3 mm . For proRigid positioning setup errors were simulated by introducing shifts of the irradiation fields with respect to the CT data and recomputing the dose without plan reoptimization.All plans were tested against 52 shifts, divided in two groups respectively of 3D magnitude 1 mm and 2 mm. For each magnitude the group represents a complete enumeration of shifts with components, in the patient’s coordinate system , either zero or equal in absolute value. In the figures, individual shifts are expressed through their magnitude and a triplet, with the + and - signs indicating the direction of the  non-zero components. The following formula generates the set S of all 52 tested shifts, expressed in mm, in the patient’s coordinate system: %95) and the homogeneity index (HI), calculated as the difference (D%2 – D%98), were used [max) was reported for the optic structures and the near-maximum dose at 1% volume (Dnear-max) for the brainstem. Visual and quantitative dose distribution analysis was entirely performed with trip2png [The treatment plans were compared in terms of dose distribution and dose-volume histograms (DVH). For a quantitative assessment of the dose distribution in the PTV and CTV the 95% isodose coverage index max respectively.To analyze dosimetric plan robustness, the differences of the index values between the recomputed (shifted) and the initial plan were employed and denoted in the following as For selected comparisons and indices, statistical significance was assessed through a non-parametric, paired-sample sign test with a significance level of 0.05, using the R environment for statistical computing .%95, PTV and CTV HI, PTV CI and OAR D(near-)max values for all planning scenarios are shown in Table %95≥95.0% was fulfilled, except in one of the scLR plans, where it was compromised to 93.1% in order to meet OAR sparing constraints. For the same reason, for protons the planning objective (PTV V%95≥95.0%) had to be compromised twice in mcLR plans (to 94.4% and 90.4%) and once in mcROB plans (to 93.1%).The mean (range) prescribed dose was 61.8 (60–63) Gy(RBE) for carbon ion plans and 66.9 (64–74) Gy(RBE) for proton plans. The initial (no shift) plans showed comparable target coverage for all planning approaches. The median PTV and CTV VmcLR) and a plan with multiple-field optimization and individually chosen, robust beam directions (mcROB). The lateral-opposed-beam dose distributions for both particle species show cold spots reaching into the CTV volume, despite the 2 mm CTV-to-PTV margin, a typical consequence of particle beam displacement with respect to a strong tissue density interface. The underdosage is not present in dose distributions from robust beam setups.Figure The corresponding dose-volume histograms of CTV and chiasm are presented in Figure %95 and HI induced by selected shifts, across the studied patient cohort, are shown in Figure The changes of carbon ion and proton CTV VmcLR), the median reduction in CTV V%95 was up to 0.7 pp and 3.5 pp, for the worst-case shifts of magnitude 1 mm and 2 mm respectively. In individual cases, notably stronger coverage deterioration has been observed, with CTV V%95 decreasing by up to 5.1 pp and 9.7 pp respectively. The corresponding values for the proton plans of the same type were comparable, with median CTV V%95 reduction of at most 0.9 pp (1 mm error) and 3.4 pp (2 mm error) and respective individual case reduction of at most 3.2 pp and 11.7 pp.In general, setup errors caused a reduction of target coverage and homogeneity in the dose distributions. For lateral-opposed carbon ion plans making use of beam modulation max changes are presented in Figures In order to assess and compare the overall robustness of treatment plan groups, histograms of the observed CTV VmcLR) exhibited, in presence of setup errors, higher losses in target coverage, in comparison to the unconstrained counterparts. As visible in Figure ΔV%95 were only slight in median value (within 1 pp), for all simulated shifts, while in individual cases they reached 3.7 pp. This reduction of robustness, induced by the modulation of the irradiation fields, is also reflected in the histograms in Figure %95 changes in the unconstrained plans . Additionally, in about half of the simulated shifts, the reduction of CTV V%95, induced by the additional field modulation, was found to be statistically significant, although the generally low differences in median value suggest a lack of clinical relevance.Carbon ion plans introducing OAR sparing constraints through full intensity-modulation  in absolute value. For the contralateral optic nerve this difference was 6% and for optic chiasm 10%. The increase in beam modulation almost did not affect Dnear-max stability of the brainstem: the number of observations with an absolute deviation larger than 2 Gy(RBE) varied by less than 4% of the total, between the two optimization approaches. In the majority of cases under investigation, the brainstem was abutting the PTV, with its surface located in the strong dose gradient region for all planning modalities, rendering Dnear-max subject to significant changes for all plans.Increased incidence of larger Ds Figure . In partscLR treatment plans, even smaller differences between them and the corresponding mcLR were observed. Throughout all simulated shifts the variation in CTV ΔV%95 was at most 0.4 pp in median value and 1.3 pp in single cases. Also the smaller deviations in the histograms max changes for critical structures was observed, which was mostly pronounced for the ipsilateral optic nerve (8% cases more exceeding 2 Gy(RBE) in absolute value) and less marked for the other optic nerve, for the optic chiasm and for the brainstem .With the introduction of modulation, also an incidence of larger DmcROB) exhibited, in presence of setup errors, greater dosimetric stability than the corresponding lateral-opposed plans (mcLR), in terms of preservation of both target coverage and dose homogeneity. Among all simulated shifts, mcROB plans were affected by CTV V%95 loss of at most 2.5 pp in median value, reaching 6.3 pp in individual cases, which represents a clear improvement in comparison with the mcLR plans of the same patient cohort, with respective values of 4 pp and 9.7 pp. Similarly, for the CTV HI, mcROB plans presented better median value preservation (by up to 1.6 pp) and particularly strong worst case reduction, almost halving the difference.Carbon ion treatment plans with beam setups selected to avoid severe density inhomogeneities (%95 (16% of cases more) and CTV HI (18% of cases more) for mcROB plans and in the prolonged tails toward larger variations for mcLR plans.While a direct shift-by-shift comparison between the plans is not applicable, as the influence of each setup error is specific to irradiation direction, the advantage of robust over lateral-opposed beam setups is visible in the histograms in Figure max changes, in comparison with the lateral-opposed plans, was observed for most of the critical structures: the index deterioration remained below 2 Gy(RBE) in absolute value in 25% of the shifts more for the chiasm, 11% more for the contralateral optic nerve and 7% more for the ipsilateral optic nerve. Only for the brainstem, which for nearly all patients was in contact with the target dose gradient region, no considerable improvement in Dnear-max stability was observed.Also organs at risk benefitted from the greater dosimetric stability, afforded by robust beam selection, in presence of setup errors. Reduced incidence of larger DmcROB treatment plans achieved, similarly to the equivalent carbon ion ones, better preservation of target coverage and dose homogeneity in comparison to plans with a lateral-opposed beam setup. In mcROB plans, median and worst-case CTV V%95 losses were 2.8 pp and 5.8 pp respectively, while in the corresponding mcLR plan they were 3.6 pp and 11.7 pp. HI median and worst-case variations were instead 3.4 pp and 6 pp for mcROB plans and 3.9 pp and 14.2 pp for mcLR plans. The dosimetric advantage in target dose conservation through robust beam configurations is visible in the V%95 and HI deterioration histograms in Figure mcROB exhibit a larger number of cases (respectively 7% and 8%) affected by smaller absolute changes.In presence of setup errors, proton mcROB treatment plans also offered improved OAR dose stability. In comparison to the corresponding lateral-opposed plans, in a higher number of cases D(near-)max remained in absolute value within 2 Gy(RBE) for the contralateral optic nerve (16% of cases more), ipsilateral optic nerve (9%), optic chiasm (29%) and brainstem (7%).Proton mcLR and mcROB plans, which both represent clinically valid treatment planning approaches.The preceding subsections already report data on the investigated scenarios, for both carbon ions and protons. Some results of potential interest are better described through direct comparison of the two ion species, as presented in Figure %95 reproducibility to carbon ions and making the two particle species practically equal in terms of HI preservation. This result shows that applying a planning technique aiming at reducing physical effects of setup errors, like selecting less inhomogeneous beam paths, brought greater benefit to the particle species with the most conformal dose distributions. For both mcLR and mcROB plans, protons showed greater reproducibility of planned OAR dose, in presence of setup errors, with the greatest advantage for Dnear-max in the brainstem, that is the OAR closest to the target dose gradient  for the majority of investigated indices and plan types, in terms of absolute dose values carbon ion plans used for this study remained superior in terms of both median target coverage and OAR sparing Table .Systematic plan robustness tests, like those presented in this manuscript, are necessary to understand the magnitude of delivery-time dose uncertainties, in order to optimize treatment planning methodologies and also to ensure the selection of the best treatment for individual patients.et al. [et al. [In fact, despite its heavy requirements in terms of time and computing resources, a procedure based on multiple plan recomputations, similar to the one used in our study, was reported by Jäckel et al.  as part  [et al. , to asseAlternatively, accurate dose computation on real patient geometries can be achieved through Monte Carlo methods, a particularly interesting option for proton therapy, for which a dedicated high-performance implementation was recently demonstrated . For carA critical point, when investigating the dosimetric effects of setup errors, is selecting the range of errors to simulate. Two approaches have been proposed in the literature, simulating either a small number of extreme displacements, representing the worst-case scenarios, or calculating numerous dose distributions, reflecting the entire possible range (and probability) of displacements. Data obtained in our study suggests that the former may lead to false conclusions, as the worst case is not known a priori and not always strictly correlated to the displacements magnitude. More importantly, the worst-case displacement is not the same for different beam setups, rendering direct comparisons of limited value. An example of this is the comparison of a lateral-opposed beam setup against other beam configurations in presence of mediolateral shifts, by which the former remains unaffected.Therefore, the study presented in this manuscript was based on massive plan recomputation, with real patient data and a comprehensive, fine-grained set of simulated setup errors, and is, to our best knowledge, the first of this kind investigating multiple aspects, that affect carbon ion and proton dosimetric robustness, in skull base radiotherapy, on a multi-patient cohort.et al. [%90 by 0–1 pp for 1 mm displacements and 2–4 pp for 2 mm displacements in one patient. While this work, like ours, tested a broad range of errors, it focused primarily on usability of IMPT and employed plans with systematically varying weight of constraints, rather than clinically valid ones. It should also be noted that, to counterbalance the computation-time explosion of multiple optimizations, only absorbed dose computations were performed, neglecting both the additional field modulation and the potential compensation effects, introduced by variable carbon ion RBE. More recently, Hopfgartner et al. [%95 reductions of up to about 3 pp. In contrast to this study, which aimed at quantifying robustness in presence of extreme displacements for simple and more complex beam setups, selected with sole consideration of critical structure sparing, the goals of our work included extensive investigation of the robustness loss due to field modulation as well as its improvement through avoidance of critical heterogeneities in the beam channels.The only previous study addressing the issue of robustness against setup errors for carbon ion plans, a conference communication by Ellerbrock et al. , reporter et al.  systematOur results demonstrate that residual repositioning errors may, in some cases, lead to a significant perturbation of the delivered dose in scanned-beam carbon ion and proton therapy for skull base tumors , which cannot be completely dealt with by planning target expansion alone. Only rigid setup errors were simulated, as they represent the predominant source of targeting uncertainty in skull base treatments, with magnitudes (1–2 mm) consistent with the precision reported for modern non-invasive cranial immobilization devices and patient positioning systems, in terms of residual setup errors as well as intrafraction motion -30. No ret al. [The clinical plans (fulfilling OAR constraints) displayed a slightly reduced robustness, understood as the spectrum of variation of dosimetric indexes induced by presence of simulated setup errors, as compared to plans prepared without OAR constraints, illustrating the isolated influence of the dose gradients within the individual, modulated fields. For individual shifts these differences however seldom reached statistical significance, confirming the observation by Ellerbrock et al. , that thet al. .One of the most interesting applications of robustness tests against an extensive set of setup errors is the direct comparison, in terms of dosimetric stability, of different beam-setups. In our study, for a subset of patients, improved plan robustness, with respect to both CTV and OAR dose, was demonstrated when using beam setups avoiding strongly heterogeneous tissue. It should be noted that the beam directions for this set of treatment plans were selected with sole consideration of the density homogeneity in the beam channels, i.e. not considering the position of critical structures, which were however spared through plan optimization (in compliance with the planning protocol).Currently, clinical robustness testing of irradiation setups must be usually carried out by expert personnel, on a case-by-case basis, evaluating possible tradeoffs with planning constraints, like OAR sparing. This underlines the need for automatized tools to support such multi-criteria, optimal beam setup selection. Verification of the entrance channel homogeneity through pattern matching techniques, that mimic human analysis, has been demonstrated a promising approach, which can be easily combined, through an optimization process, with clinical constraints, like patient positioning limitations or OAR involvement ,34. Methet al. [et al. [et al. [Finally, our study presents the first attempt to compare the dosimetric robustness of treatment plans employing different ion species. Between protons and carbon ions, used in our study, differences have been demonstrated in the basic dose deposition properties, that may be relevant in their application as therapeutic radiation types . In our et al. , it shou [et al. . Additio [et al. .Most notably, from a strictly numerical interpretation of our results, in most of the scenarios considered, in presence of setup errors protons appear to deviate less from the planned values, both in CTV coverage and OAR involvement, with a particular stability advantage for the brainstem, the organ closest to the target and often in direct contact with it. This observation could be of importance in clinical decision-making, e.g. when defining guidelines and thresholds for image-guided radiotherapy (IGRT) protocols, and is likely related to the more pronounced scattering protons undergo, resulting in typically shallower gradients. Another important observation, related to potential clinical applications, is that carbon ion plans, with their higher initial conformity and more advantageous absolute dosimetric values, received greater advantage, in terms of target dose reproducibility, through use of robust beam setups.best particle species, but rather to demonstrate how the differences in the physical properties of ions, and in the clinical applications they enable, may affect our understanding of robustness issues. In this sense, a certainly valid general conclusion is that per-indication studies should be carried out for each ion species and some care should be used when translating results from one particle type to another. This is particularly important in the current context of combined facilities, where protons and carbon ions are used for different indications, and as newer ion species, e.g. oxygen or helium, are being considered for introduction in clinical particle therapy [Ultimately, purpose of the comparison was not to identify the  therapy . Similar therapy ,41.When assessing the impact of these results on patient treatments, one aspect, crucial for the interpretation of this study, should not be neglected. The application of the total therapeutic dose is typically fractionated, causing delivery uncertainties to be statistically smoothed, provided that they are not systematic. It is therefore advisable that, for a thorough clinical assessment, a similar study be conducted in the future, employing e.g. the results of daily positioning verification data, to assess cumulative dose. Finally, the positioning errors are only one of many potential delivery-time uncertainties that can affect the final dose distribution, like range uncertainties , anatomy


Ethanol, a commonly available agent, has been used to successfully ablate cystic and solid lesions in the pancreas. The aim of this study is to investigate the effects of an ethanol injection into the porcine pancreas and observe the time-dependent image changes in the pancreatic parenchyma.Pure ethanol was injected into the pancreatic tail using a 25-gauge EUS needle with direct ultrasound guidance under celiotomy: 1 mL and 2 mL were injected, respectively. The abdomen was closed after the injection. MRI was performed before the procedure, immediately after, and on postoperative day (POD) seven. Blood samples were taken before the procedure and on PODs one, three, five, and seven. The pigs were euthanised on POD seven.Immediately after the injection, linear high signal areas in the pancreatic tail on T2 and rounded speckled high signal areas on DWI images were detected in both animals, measuring 35 × 32 mm in the 1 mL injected pig and 42 × 38mm in the 2 mL injected pig. After POD seven, rounded high signal areas were noted on T2 images, measuring 22 × 18 mm and 36 × 28 mm respectively. On POD one, the 1 mL injected animal had a 53% elevation in serum amylase while the 2 mL injected animal had a 66% elevation. Histologically, cystic and necrotic changes in the parenchyma were observed, measuring 23 × 22 mm and 40 × 35 mm respectively.Our results, which are limited to normal pancreas, suggested that a 1 mL injection caused localised changes within the pancreas while a 2 mL injection induced more widespread changes beyond the pancreas. The effective area of ethanol was widespread immediately after injection, and then the area was reduced with cystic and necrosis changes. Endoscopic ultrasound (EUS) and EUS-guided fine-needle aspiration (EUS-FNA) are recognised as useful and safe modalities for providing high-resolution images and tissue samples for diagnosis of pancreatic neoplasms –2. RecenEthanol is a particularly attractive agent. It is inexpensive, readily available, and has the potential to rapidly ablate tissue. Its purported mechanisms of action are cell lysis and protein denaturation . EthanolMagnetic resonance imaging (MRI) is a technology that can detect the location and local chemical environment of protons in water molecules. MRI with diffusion weighted imaging (DWI) sequences and apparent diffusion coefficient (ADC) maps is widely accepted as a very sensitive way to detect acute changes. The reduced diffusion is thought to be related to cytotoxic oedema and shrinkage of the extracellular space , 17. MRIThe purpose of this study was to investigate the area of ablation in the pancreas using US-guided ethanol injection under celiotomy and time-dependent image changes of pancreatic parenchyma after the ethanol injection by MRI.Two female Yorkshire pigs (average weight 35 kg) were used for this study. This experimental protocol was approved by the Institutional Review Board of the Intervention Technical Centre  for the Use of Laboratory Animals in the Medical Device Development Centre  before the initiation of the study. The pigs were anesthetised with intramuscular ketamine and xylazine along with isoflurane that was delivered via endotracheal tube. After adequate anesthesia was achieved, the tail of the pancreas was exposed by celiotomy. We observed the pancreas directly using US  and identified the thickest portion of the pancreatic tail. A 25-gauge EUS needle  was inserted into the thickest portion of the pancreatic tail under US guidance . The neeMRI  of the abdomen was performed before the procedure, immediately after, and on POD seven. Images of T1- and T2-weighted, fat-sat T1 and T2-weighted, magnetic resonance cholangiopancreatography (MRCP), DWI, and ADC were taken after administration of rocuronium. The slice thicknesses of T1, T2, and DWI were 4.5 mm, and MRCP images were processed to create reformatted images of 0.6-mm thick. Blood samples were taken before the procedure and on PODs one, three, five, and seven. Serum concentrations of amylase (AMY) and lactate dehydrogenase (LDH) and a complete blood count (CBC) were assessed. Animals were monitored for lethargy, severe distention, and vomiting, with observation until POD seven. For the first two PODs, each animal was administered 500 mg/day of cefazolin sodium. Animals had access to regular solid food during this time.After animals were given anesthesia on POD seven, a laparotomy was performed after which the animals were euthanised while still under anesthesia. The gross appearance of the pancreas and surrounding structures was recorded. The pancreas was harvested, examined, palpated, and sectioned to identify any gross abnormalities. Tissue samples were preserved in 10% formalin for histological analysis with haematoxylin and eosin (H.E.) staining.Each animal tolerated the US-guided ethanol injection under the celiotomy without clinical manifestations of pancreatitis or distress. Activity and dietary intake in both animals were normal over the seven-day observation period.Immediately after the injection of ethanol, linear high signal areas in the pancreatic tail were noted in both animals on T2-weighted images; high signal areas were 35 × 32 mm in the animal administered 1.0 mL ethanol and 42 × 38 mm in the animal administered 2.0 mL ethanol. On the other hand, small high signal areas were noted in both animals on T1-weighted images. On the DWI, speckled high signal areas were detected in both animals , 3a-d. OOn POD one, the animal receiving 1.0 mL of ethanol had a 53% elevation (1518–2890 U/L) in serum AMY while the animal receiving 2.0 mL of ethanol had a 67% elevation (1962–2933 U/L) of AMY . ElevatiMacroscopically, tissues around the injection site were pale and swollen. Especially, in the 2.0 mL-injected animal, the injection site of the pancreatic tail was greatly swollen and had adhered to the small intestine . Cross-sA previous study of EUS-guided ethanol injection into the normal porcine pancreas showed that 50% ethanol  caused localised and self-limited changes with focal areas 2–6 mm) of inflammation, necrosis, and fibrosis at the injection site. On the other hand, the pigs injected with 98% ethanol  developed more widespread tissue changes with large areas (8–30 mm) of inflammation, fibrosis, and necrosis, and local complications of pancreatitis: acute fluid collection in one case and inflammatory colonic stricture in the other case  mm of in. In our MRI is a technology that uses nonionising radiofrequency radiation inside a strong magnetic field to detect the location and local chemical environment of protons in water molecules. MRI with DWI sequences and ADC maps is widely accepted as a very sensitive way to detect acute changes, especially in the field of cerebral infarctions . The redClinically, ethanol ablation is commonly used to ablate hepatocellular carcinomas . PremaliThere are a few limitations to this study. Firstly, the present study investigated only on a few animals, and therefore a more detailed assessments of the safety and efficacy of this procedure is necessary. Secondly, the study findings are applicable only to normal pancreas. Ethanol ablation is usually used for malignant or premalignant lesions. Therefore, the present findings might be different from actual clinical findings. Third, although the changes in the images  were observed by MRI, no information was available after a week.In conclusion, our results which are limited to normal pancreas, suggest that a 1.0 mL injection of 100% ethanol caused localised changes within the pancreas whereas a 2.0 mL injection induced more widespread changes beyond the pancreas. Image changes in the pancreatic parenchyma after the ethanol injection could be observed by using T1 and T2-weighted and DWI imaging. It was found that the effective areas of ethanol were widespread immediately after injection, and then these areas reduced with cystic and necrosis changes. MRI without contrast agents could correctly evaluate areas of ethanol ablation.The authors declare that they have no conflicts of interest associated with this study.Matsumoto K, Kato H and Fushimi S conceived and designed the research and wrote the paper; Tsutsumi K, Iwamuro M, Oda S, and Mizukawa S, analysed and interpreted the data; Akimoto Y, Uchida D, Tomoda T, Yamamoto N, and Horiguchi S did critical revision of the article for important intellectual content; and Okada H gave final approval of this article.The study was reviewed and approved by the Institutional Review Board of the Intervention Technical Centre  for the Use of Laboratory Animals in the Medical Device Development Centre .All experimental manipulations were undertaken in accordance with the Guide for the Care and Use of Laboratory Animals , and the study was approved by the Intervention Technical Centre  for the Use of Laboratory Animals in the Medical Device Development Centre .