Tp53, is mutated or silenced in most human cancers and is recognized as one of the most important cancer drivers. Homozygotic Tp53 knockout mice, which develop lethal cancers early in their lives, are already used in cancer prevention studies, and now Tp53 knockout rats have also been generated. This study assessed feasibility of using homozygous Tp53 knockout rats to evaluate the possible outcome of cancer chemoprevention.The gene that encodes tumor protein p53, Tp53 knockout rats with a Wistar strain genetic background was initiated and maintained in the animal house at our institution. Tp53 heterozygotic females were bred with Tp53 homozygous knockout males to obtain a surplus of knockout homozygotes. To evaluate the reproducibility of their lifespan, 4 groups of Tp53 homozygous knockout male rats born during consecutive quarters of the year were kept behind a sanitary barrier in a controlled environment until they reached a moribund state. Their individual lifespan data were used to construct quarterly survival curves.A small colony of Tp53 homozygous knockout rats was normally distributed, with a median of 133 days. Sample size vs. effect calculations revealed that confirming a 20% and 30% increase in the lifespan would respectively require a sample size of 18 and 9 animals . As an example, the Tp53 homozygous knockout rat model was used to test the chemopreventive properties of carnosine, a dipeptide with suspected anticancer properties possibly involving modulation of the mTOR pathway. The result was negative.The four consecutive quarterly survival curves were highly reproducible. They were combined into a single “master” curve for use as a reference in intervention studies. The average lifespan of untreated male Tp53 homozygous knockout male rat colony is required before it can be confirmed as a viable tool for assessing new methods of cancer prevention or treatment.Further evaluation of the  Potentially the best method to fight cancer could be chemoprevention, defined as the long-term intake of natural, synthetic or biological agents to reverse, suppress or prevent carcinogenesis in its initial phases or the progression of premalignant cells to invasive disease , 2. A plTp53, which encodes transcription factor p53, has become one of the most extensively studied factors involved in carcinogenesis. Mutations in Tp53 are present in more than 50% of human malignancies [Tp53 knockout mice were used in experimental research on Tp53. These animals mature normally, but soon afterwards, all spontaneously develop lethal tumors. In null homozygotes (p53–/–), the average time to tumor development is 4.5 months, whereas half of the heterozygotes (p53+/–) develop tumors by 18 months [Since its discovery in 1979, gnancies . Almost Tp53 null mice provided numerous opportunities to study cellular processes in the absence of wild-type Tp53. Using Tp53 heterozygotes as a sensitive rodent model for testing known or suspected carcinogens has been advocated as it requires fewer animals and less time than conventional rodent carcinogenicity tests [The viability of ty tests .Tp53 knockout homozygotic mice can be used to assess efficacy of cancer chemoprevention. In the first study of this type, it was found that imposing a caloric restriction on them to 60% of ad libitum consumption results in significant prolongation of survival, from a median of 16 weeks to 25 weeks [d-limonene, or all-trans retinoic acid. An average prolongation of survival by 58% was found only in the DHEA-treated group [It has also been suggested that 25 weeks . In the ed group .Tp53 knockout mice as a model for testing cancer chemoprevention strategies, only a few studies of this type have later been reported. In one [Tp53 knockout mice survival upon pharmacological blockade of anti-apoptotic proteins belonging to the Bcl family or intake of a complex nutraceutical supplement containing ca. 35 ingredients.However, despite the authors’ conclusion that their results provided validation of . In one , oral tr. In one , a proto. In one , 16 showTp53 knockout rats have been generated more recently. Tong et al. [Tp53 gene via homologous recombination in embryonic stem cells and showed germline transmission of the Tp53 gene-targeted mutation. Van Boxtel et al. [Tp53 knockout rats and reported that both null Tp53 homozygotes and Tp53 heterozygotes start to develop lethal tumors, mainly sarcomas, at 4 and 8 months of age, respectively. McCoy et al. [Tp53 knockout rat that contains an 11-bp deletion in exon 3, resulting in a frameshift and premature terminations in the open reading frame. The most recent report is that of Hansen et al. [Tp53 knockout rat based on the inbred rat strain Fisher-344. All these authors remarked that Tp53 knockout rats could be highly suitable for oncology research, with van Boxtel et al. [Tp53 knockout rats are highly complementary to existing mouse models for studying human cancer biology, and that due to early onset of spontaneous cancers, this model is unique and may be especially useful tool for testing therapeutic interventions.g et al.  disruptel et al.  used N-ey et al.  created n et al. , who crel et al.  concludiTp53) can be better predictors of cancer drug discovery than experiments with cell cultures and models that involve non-genetically engineered animals. More recently, Prasad et al. [Tp53 knockout rats have not yet been used in such experiments.Aggarwal et al.  noted thd et al.  argued td et al. , and eved et al. . HoweverTp53 knockout homozygotes, and on their possible suitability as a tool for preclinical assessment of secondary cancer prevention.In the animal house of our institution, we initiated a small colony of p53 knockout rats and maintained it over 1 year. Hereby we report on the reproducibility of the survival curves of male rat Tp53 knockout rats (p53 TGEM Rat) with a Wistar strain genetic background was initiated and maintained in the animal house of our institution. Two pairs of male and female heterozygotic rats were purchased from Charles River under the exclusive license of Transposagen. They bear a single T to A point mutation in the Tp53 DNA-binding domain that introduces a premature C to X stop codon in position 273aa [A colony of on 273aa . These aTp53 knockout rats is determined using simple allele-discriminating PCR (SAP), as described by Bui and Liu [Rattus norvegicus p53 mRNA sequence: NM_030989. The primer set is specific for the detection of a single nucleotide mutation in Tp53: Tp53 mutant reverse primer (5′- GTCTCTCCCAGGACAGGTT -3′), Tp53 wild-type reverse primer (5′-GTCTCTCCCAGGACAGGTA-3′), and Tp53 common forward primer (5′-GAAGACTCCAGGTAGGAAGC-3′).The genotype of the  and Liu , with a DNA is extracted from a tail snip using a GeneMATRIX EURx kit (EURx Ltd.) according to the manufacturer’s instructions. The tail biopsy is taken between 3 and 4 weeks of age. PCR is performed with an initial denaturation step at 94 °C for 6 min, followed by 35 cycles of 94 °C for 20 s, 52 °C for 20 s, 72 °C for 20 s, and final elongation step at 72 °C for 7 min.Tp53 appeared to be lethal for most of the female offspring, we restricted our experiment to homozygotic knockout male rats. To evaluate reproducibility of their survival, 4 groups of Tp53 knockout homozygotic male rats born during consecutive quarters of the year were kept behind a sanitary barrier in the controlled environment of a pathogen-free animal facility. They had a 12-h light and dark cycle with the temperature maintained at 22 °C ± 1 °C and a relative humidity of 40–70%. Food and water were supplied ad libitum. When the animals reached moribund state, they were killed.Because homozygotic knockout of To avoid unnecessary pain and suffering, we applied predetermined criteria for evaluating the physiological and behavioral signs of disease. This approach enabled conscious interruption of the experiment (humane endpoint), while providing accurate and reliable data acquisition. Rats with observed changes in their physical appearance  or behavior were examined with increasing intensity, namely by palpation of the whole body. Symptoms that required immediate discontinuation of the experiment  included: 1) weight loss from 15 to 20% within 7 days; 2) paralysis of limbs or neurological symptoms; 3) loss of ability to ambulate (inability to access food or water); 4) labored breathing; 5) tumor size exceeding 3 cm; 6) appearance of an open wounds, ulcers or tumor necrosis . All theR Statistical Software. The statistical significance of disturbances in sex and genotype rates were assessed with chi-squared goodness-of-fit test . Individual lifespan data were used to calculate the Kaplan-Meier estimator and its estimated standard error (square root of the Greenwood formula), and the 95% confidence interval using the log transform. The normality of data distribution was checked with the Shapiro-Wilk normality test.All the calculations were performed using t-test was conducted to assess the number of animals necessary to obtain statistically significant data  for 3 preselected powers – 0.7, 0.8 and 0.9 – and expected effect size. The effect size describes the magnitude of a treatment effect, defined as the difference between the means, divided by the standard deviation of either group.Power analysis for the two-group independent sample Tp53 homozygous knockout rats as a tool for testing chemoprevention used L-carnosine, a naturally occurring dipeptide consisting of β-alanine and L-histidine. This compound has been suggested to influence cell proliferation, cell cycle arrest, apoptosis, and the glycolytic energy metabolism of certain tumor cells [The “field test” of the or cells , 28. It or cells . This sup values of 0.002 and less than 0.001, respectively, for heterozygotic crossing and heterozygotic females and knockout male crossing litters. The same test  was used to evaluate the statistical significance of observed disturbances in the genotype ratios. In all the groups – females and males in heterozygotic crossing litters and females and males in heterozygotic female and knock out male crossing litters – the detected disturbance was statistically significant with the respective p values as follows: less than 0.001, 0.03, less than 0.001 and 0.05. We could observe a significantly reduced number of knockout pups, most probably due to a perturbation in embryo development, affecting mostly females . Moreover, the female knockout pups showed a reduced vitality.We observed disturbances in the male-to-female ratio and the expected genotype ratio in both types of litter . However, the litter size seemed not to be affected, with average size of 9 ± 3 rats. Statistical analysis using the chi-squared goodness-of-fit test  showed that the observed reduction in the number of female pups in the litter (up to 40%) is significant, with Knockout rats developed grossly visible tumors and/or suffered marked conditional decline at 4 months of age at the latest. In our in-house population, we observed solid tumors and lymphomas. In the majority of the animals, between day 80 and 128 of life, clearly visible tumors (diameter over 0.3 cm) were detected under the skin in various locations  and the rats were euthanized (according to above described humane endpoint) from 9 to 36 days afterwards. We did not find any significant correlation between the day of tumor onset and the lifespan.Tp53 knockout rats for chemopreventive studies, so we did not histologically assess the tumors. Our general impression was that the most frequent tumor type  appeared to be hemangiosarcomas present in different locations under the skin. Importantly, these tumors displayed high intratumoral hydrostatic pressure, resulting upon incision in an aggressive expulsion of the bloody fluid which filled the cyst. Also, approx. 10% of the animals had a noticeably low hematocrit.Our study aimed to assess the feasibility of using p value = 0.7939), and the average lifespan was 133 days with a standard deviation of 27 days. The reference survival curve was constructed data for all 85 male p53 knockout rats   vs. effect size (d) calculations this model can, for example, detect a 20% effect with a sample size of 18 animals and a 30% effect with only 9 animals  or Fisc126 days ) backgroApc gene [Although mice are the most popular experimental animal in many research areas , some investigators favor rats because of their larger size, which facilitates experimental interventions, e.g., repetitive blood sampling or in vivo imaging studies with magnetic resonance or positron emission techniques . The othApc gene .Tp53 gene leading to the Li-Fraumeni syndrome, sarcomas comprise approx. 95% of all tumors that occur before the age of 50 years [Tp53 homozygous knockout rats is similar to those in humans suffering from Li-Fraumeni syndrome, whereas Tp53 knockout mice predominantly develop lethal lymphomas  [It is also worth mentioning that in humans with germline mutations of the 50 years . Thus, tevented) .a priori assumed to be prohibitively expensive. However, the stability and reproducibility of survival curves of Tp53 male knockout rats may allow researchers to omit a synchronous untreated control, which will be helpful in curbing expenses. No large number of animals is required to become convinced that a compound does not display cancer-preventing properties in this testing system, as shown by the results of a field test of our approach.Both species (mouse and rat) have similar food intake expressed relative to body weight . Since rTp53 homozygous knockout rats, may develop in rodents without involvement of mutations and DNA damage. Indeed, such tumors are well known to toxicologists, because they are induced by various industrial chemicals and pharmaceuticals. Interestingly, several of the hemangiosarcoma-inducing substances are non-DNA reactive, and the mode of action of some of them  has been proposed to include hemolysis leading to iron overload [Tp53 homozygotic knockouts, a very low hematocrit was evident. This serendipitous observation may reflect a hitherto unknown link between the Tp53 gene and iron metabolism. A few years ago, it was suggested [Tp53 homozygous knockout rats. Some other explanation should be sought for the frequent development of anemia in these animals.We shall also note that hemangiosarcomas, which appear to be the most frequent type of tumor in overload . As mentuggested  that hemTp53 knockout mice [Tp53 homozygous knockout rats.Carnosine, a naturally occurring dipeptide consisting of β-alanine and L-histidine and known primarily for its antioxidative properties, is nontoxic and readily available as a food supplement. We chose this compound because its inhibitory effects on neoplastic cell lines has been described, stimulating speculations on its possible usefulness in cancer treatment . Moreoveout mice , we reasTp53 homozygous knockout rats is that, in fact, the arguments for expecting anticancer effects of carnosine were poor. Although carnosine inhibited ATP production in primary cultures of human malignant gliomas and in established cultures of human gastric cancer, this effect occurred when the cancer cells were respectivel exposed to 50 mM and 20 mM concentrations of the peptide [As we have found, this is not the case. The most probable reason for carnosine failing to protect against cancer development in the  peptide , 42. The peptide , and higTp53 knockout rats possess all or only some of these features. In any case, these animals may prove useful for evaluating a new type of anticancer drugs, proposed recently by Wang et al. [Tp53 knockout rats should also prove helpful for preclinical evaluation of gene therapy aimed at restoring the active Tp53 gene in cancer cells in vivo. Preclinical tests of Tp53 gene therapies have thus far been performed only in mice [Ma et al.  argued tg et al. . Such ne in mice , and at  in mice .


Yellow rust is one of the most destructive diseases for winter wheat and has led to a significant decrease in winter wheat quality and yield. Identifying and monitoring yellow rust is of great importance for guiding agricultural production over large areas. Compared with traditional crop disease discrimination methods, remote sensing technology has proven to be a useful tool for accomplishing such a task at large scale. This study explores the potential of the Sentinel-2 Multispectral Instrument (MSI), a newly launched satellite with refined spatial resolution and three red-edge bands, for discriminating between yellow rust infection severities  in winter wheat. The corresponding simulative multispectral bands for the Sentinel-2 sensor were calculated by the sensor’s relative spectral response (RSR) function based on the in situ hyperspectral data acquired at the canopy level. Three Sentinel-2 spectral bands, including B4 (Red), B5 (Re1), and B7 (Re3), were found to be sensitive bands using the random forest (RF) method. A new multispectral index, the Red Edge Disease Stress Index (REDSI), which consists of these sensitive bands, was proposed to detect yellow rust infection at different severity levels. The overall identification accuracy for REDSI was 84.1% and the kappa coefficient was 0.76. Moreover, REDSI performed better than other commonly used disease spectral indexes for yellow rust discrimination at the canopy scale. The optimal threshold method was adopted for mapping yellow rust infection at regional scales based on realistic Sentinel-2 multispectral image data to further assess REDSI’s ability for yellow rust detection. The overall accuracy was 85.2% and kappa coefficient was 0.67, which was found through validation against a set of field survey data. This study suggests that the Sentinel-2 MSI has the potential for yellow rust discrimination, and the newly proposed REDSI has great robustness and generalized ability for yellow rust detection at canopy and regional scales. Furthermore, our results suggest that the above remote sensing technology can be used to provide scientific guidance for monitoring and precise management of crop diseases and pests. Puccinia striiformis f. sp. tritici (Pst), and is one of the most destructive diseases for wheat [Wheat is an important grain crop in China and has extraordinary significance for ensuring national food security. Various wheat diseases have become major factors for decreased wheat yields in China . Yellow or wheat ,2. It isor wheat ,4. Yelloor wheat . Traditior wheat . In prac2 = 0.91), and proposed a vegetation index, YRI (yellow rust index), to distinguish yellow rust from healthy wheat as well as from powdery mildew and aphids, respectively. Zhang et al. [Remote sensing technology has proved to be an efficient tool for monitoring crop growth and identifying crop diseases over the last several decades. Such diseases include: rust infection ,6,7,8,9,g et al.  found thIn recent years, many methods or models have been used to select the sensitive features for crop disease detecting and discriminating between different crop diseases ,28. RandT. peregrinus damage in plantation forests [Most vegetation indexes/models are established to detect yellow rust diseases based on hyperspectral data collected by hyperspectral sensors , hence, these VIs and models can hardly be applied directly for monitoring crop diseases at large scales due to the narrow coverage, high cost, and low availability of hyperspectral data ,35. Mult forests . Yuan et forests . HoweverThe Sentinel-2 MSI sensor was launched in 2015 by the European Space Agency (ESA) and was designed to meet the needs of “Global Monitoring for Environment and Security” (GMES) . The newThis study used the relative spectral response (RSR) function of the Sentinel-2 MSI sensor and canopy hyperspectral data to simulate the reflectance of Sentinel-2 sensor channels, and explored the potential of the Sentinel-2 MSI sensor for identifying yellow rust infection in winter wheat. The objectives were to: (1) select the most sensitive bands of multispectral data (Sentinel-2) for identifying healthy wheat and both slight and severe yellow rust infection in winter wheat; (2) propose a new red-edge multispectral vegetation index for discriminating yellow-rust-infected winter wheat from healthy wheat; and (3) map yellow rust infection using realistic Sentinel-2 satellite imagery at regional scales.−1 of available phosphorus, and 116.6–128.1 mg·kg−1 of rapidly available potassium. The winter wheat cultivar known as ‘Mingxian 169’ was selected because of its moderate susceptibility to yellow rust. The yellow rust pathogens infected the winter wheat through an inoculation process (spore solution concentration of 9 mg 100−1 mL−1) according to the National Plant Protection Standard (NPPS) on 13 April 2017. For the yellow rust experiment group, 39 replicates were planted in a plot with a size of 3 m × 13 m. For the control group, 12 replicates were planted in a plot with a size of 3 m × 6 m. A gradient of disease severity was gradually generated in the field after an inoculation period of 5–7 days. To avoid cross-infection of disease, the distance between each group was no less than 5 m, the control plots had use of pesticides to prevent infections. In this study, each plot was placed under the same cultivation and management conditions (200 kg·ha−1 nitrogen and 450 m3·ha−1 water) to avoid the influence of extraneous factors such as: cultivar, water, fertilizer, and field management. The canopy scale experiment was carried out at the experimental field of the Chinese Academy of Agricultural Sciences at the winter wheat grain filling stage in 2017. The field is in Langfang , Hebei Province, China, where the average annual precipitation is 554.9 mm and average annual temperature is 11.9 °C. The soil had a nutrient content in the topsoil layer (0–30 cm depth) of about 1.41–1.47% of organic matter, 20.5–55.8 mg·kgField surveys of wheat yellow rust infection were conducted in Chuzhou and Hefei, Anhui Province, China   on 9 May4 calibration panel was performed every 10 measurements to correct the changes in the illumination condition. All experiments were measured under cloudless conditions between 10:00 a.m. and 14:00 p.m.  when minimum variations in solar view angle occurred. The reflectance spectrum of each sample was measured ten times, and their average was considered as the reflectance spectrum of the samples. The spectral reflectance of 113 winter wheat samples was selected at the canopy scale by eliminating the hyperspectral data affected by the noise, including 29 healthy samples and 84 diseased samples with different severity levels.The hyperspectral data at the canopy scale were measured using an ASD FieldSpec Pro spectrometer  at grain filling stage on 15, 18, and 25 May 2017. The spectral range of the spectrometer was between 350 nm and 2500 nm, with 3 nm and 10 nm spectral resolution in the 350–1000 nm and 1000–2500 nm regions, respectively. All canopy spectral measurements were taken with a 25° field of view at a height of 1.3 m above the ground. A 40 cm × 40 cm BaSOhttps://scihub.copernicus.eu/, and the full coverage image was mosaicked by two images acquired simultaneously. More information about the image is shown in Two simultaneous Sentinel-2 multispectral images were acquired on 12 May 2017, from n is the value of highest disease severity gradient (n = 8), and f is the number of leaves for each degree of disease severity.The disease index (DI) was used to describe the severity of crop diseases. An assessment of the DI was performed simultaneously with a spectrum acquisition process for wheat yellow rust . A totaltegories : 0%, 1%,In addition to the continuous variable DI for evaluating the diseases status, the disease severity of the canopy was also quantitatively classified into three classes for subsequent research and analysis. These include: 29 healthy wheat samples (DI = 0), 39 slight yellow rust infection samples (0 < DI ≤ 20%) and 45 severe yellow rust infection samples (DI > 20%). The criterion for these classifications was suggested by the national plant protection department.We integrated the field canopy hyperspectral data based on the sensor’s RSR function to simulate the multispectral reflectance of Sentinel-2 (Formula 2) to assess its potential for winter wheat yellow rust monitoring and detection. The formula is given as:p-value < 0.001). We found that seven Sentinel-2 spectral bands  manifested excellent potential for discriminating healthy and infected (slight and severe) samples  as shown in p-value < 0.001) SVIs were selected to identify yellow rust. These SVIs included conventional and red-edge vegetation indexes  instead of the red band (B4). NREDI is the index of normalized red-edge bands [Following the integration processing of hyperspectral data, an independent t-test was applied to examine the statistical significance of all bands based on healthy, slightly, and severely infected samples at the highest significance level  It samples a new training set with replacement at each iteration, and the new training set is the same size as the original set; (2) Rather than choosing the best split among all attributes, m attributes are randomly chosen from M at each node and then these m attributes are used to split the node according to the principle of the decision tree algorithm, where m << M, and it is held constant during the forest construction process. Because the algorithm does not use all samples for model training at one time, this makes it possible to use the remaining samples (out of bag data) to evaluate the out of bag error (OOB error). Moreover, the principle of the feature importance ranking is to compare the difference in OOB error of each feature before and after adding noise to determine the importance of each feature. Thus, the importance of each feature is directly proportional to the difference [Random forest (RF) is an ensemble of learning algorithms proposed by Breiman . It consfference . We usedFLDA is a widely used analysis method for classification ,55. The To map disease damage with a single variable, the optimal threshold segmentation method was adopted. Threshold segmentation is a method that separates an image into a number of meaningful regions according to the selected threshold values . For thiAs shown in The ratio curve of reflectance, dividing the reflectance of yellow-rust-infected wheat by healthy wheat is shown in The RF method was used to evaluate the importance of each band in the process of discriminating between healthy wheat and slightly and severely yellow-rust-infected winter wheat, and the ranking of each result is illustrated in According to the analysis in i band , and the values of 665, 705, 783 represent the central wavelength of the Red, Re1, and Re3 band, respectively.In practice, the symptoms of slight yellow rust infection  in the spectrum are not significantly different from the physiology of healthy wheat, so it is difficult to distinguish slight yellow rust infection from healthy winter wheat when using the triangular area feature. As shown in green, and NREDI2 with overall classification accuracies of 81.4%, 79.6%, and 79.6%, respectively  for the healthy and yellow-rust-infected winter wheat, followed by NREDI1, VARIectively . MoreoveWhen winter wheat is infected by yellow rust, the leaves begin to wilt and become discolored. Winter wheat presents different physiological symptoms for healthy and diseased canopies depending on the biology of the pathogens and the characteristic host–pathogen interaction . As showOur result demonstrates the potential of the red-edge disease stress index (RDESI) for discriminating yellow rust infection in winter wheat based on Sentinel-2 satellite data. The common way to detect significant bands or vegetation index for plant diseases discrimination is by connecting to biochemical or biophysical traits . For exaThe developed REDSIs are based on the triangle geometry area and the most relevant wavelength ratio regarding wheat yellow rust disease. Triangle geometry area was determined by the reflectance of B4, B5, and B7. The reflectance of B4 and B7 showed the opposite trend under the condition of disease stress. The greater the severity of yellow rust infection, the greater the reflectance of B4, and the smaller the reflectance of B7, which leads to the smaller triangle geometry area. The value the triangle geometry area divided by the reflectance of B4 would increase the separability among healthy, slightly, and severely yellow-rust-infected winter wheat. The REDSI produced good results to detect winter wheat yellow rust disease at the canopy and regional scales. At the canopy scale, REDSI’s ability to identify serious yellow rust infection is greater compared with its ability to identify wheat that is healthy and has slight yellow rust infection. It is consistent with the view of Sanakran et al.  that theIn the study, REDSI can be used for yellow rust monitoring using independent datasets from different measuring devices and spatial scales, which demonstrates the robustness and potential of REDSI for yellow rust discrimination in practice. However, it should be noted that not only yellow rust disease stress but other diseases  may occur simultaneously in the field during winter wheat growth stages. So, future work will be needed to explore REDSI’s ability to identify and monitor other winter wheat diseases. In addition, the environmental conditions are much more complicated in practice, and the occurrence of disease is also closely related to biotic conditions and the ecological environment, including: temperature, humidity, meteorological conditions, plant density, and field management strategies. For example, Danial and Parlevliet suggest that the density of vegetation can affect the severity levels of yellow rust . TherefoIn this study, we used canopy hyperspectral data to simulate the corresponding multispectral bands of Sentinel-2, based on the RSR function of the Sentinel-2 multispectral sensor, and developed a new index, REDSI , for detecting and monitoring yellow rust infection of winter wheat at the canopy and regional scale. Compared with other common spectral vegetation indexes, REDSI has excellent performance in detecting and monitoring yellow rust in winter wheat at the canopy and regional scale, with the overall accuracy of 84.1% and 85.2%, respectively. Furthermore, the index had to be continually validated with other diseases and other cultivars to guide agriculture precision management.


Their findings have prompted considerable public health debate. There is ongoing discussion as to whether cutoff points for body-mass index (BMI) categories should differ across regions or racial or ethnic groups.2 Additionally, studies differ in their assessment of the relation between BMI and mortality. In particular, BMI in the overweight category (BMI 25–<30 kg/m2) is not consistently associated with increased mortality.4 To improve precision, observational studies have been combined in pooled analyses and meta-analyses. Pooled analyses benefit from the use of harmonised methods but, unlike meta-analyses, are not generally based on systematic review.Concurrent with the global increase in obesity,The Lancet, The Global BMI Mortality Collaboration5 presents results from the largest ever pooled dataset about the relation between BMI and mortality. Their data began with 239 studies, in 32 countries, with more than 10 million participants and about 1·6 million deaths. Prespecified analyses of never smokers without pre-existing chronic disease, excluding the first 5 years of follow-up, are presented for about 3·9 million participants and about 386 000 deaths occurring in 189 studies. This study is notable for the use of standardised methods to extract hazard ratios (HRs) for mortality across the studies and for extensive and valuable appendices that examine diverse subsets of the pooled data. The authors made efforts to address reverse causation and residual confounding. Results are broadly similar to other recent studies.7 For example, HRs were J-shaped, with increased risk of mortality for both low BMI and obesity (BMI ≥ 30 kg/m2). This study documents heterogeneity in the association between BMI and mortality across different continents and shows weaker associations in older populations, especially in those aged 70 years and older. For overweight adults (BMI 25–<30 kg/m2), HRs—adjusting for age and sex and excluding individuals with baseline chronic disease—ranged from 0·99 (95% CI 0·98–1·00) after adjustment for smoking to 1·11 (1·10–1·11) after exclusion of ever smokers and the first 5 years of follow-up. The elevated HR of 1·11 in overweight adults after exclusion of about 60% of the sample and about 75% of deaths is a key result of this pooled analysis.In 8 For example, Monte Carlo simulation and analytical analyses of the association between BMI and mortality indicate that exclusion of 2 years or 5 years of early mortality does not necessarily reduce bias and can even increase bias of estimated HRs.9Two major issues are raised by this important paper. The first is whether conclusions about the relation between BMI and mortality from analyses with extensive exclusions can be generalisable and unbiased. The second is what sort of public health guidance can be obtained from analyses that pool global data. Substantial research and conceptual questions remain for each of these issues. Samples of different distributions of environmental and person-specific factors  are difficult to address consistently in large pooling studies. Exclusion of ever smokers, deaths in the first 5 years of follow-up, and pre-existing chronic disease (where available) might further increase these differences. Extensive exclusions might also limit the generalisability of resulting health recommendations. Selection bias and early mortality exclusion, to control for potential bias from weight change due to occult disease leading to reverse causation, pose additional challenges for inference from pooling studies.10 new data elements such as weight histories,11 and increased attention to BMI over the life course,12 might improve our understanding of the links between excess bodyweight and mortality. The present study compares data from four continents, pooling data across diverse racial and ethnic groups, and across countries with very different patterns of chronic disease management.2 Large studies of specific race and ethnic groups, such as that of Yi and colleagues of 12·8 million Koreans,4 can help to clarify recommendations for specific countries or demographic groups even if these studies cannot conclusively address the limits of observational studies.Challenges in deriving global public health recommendations are unlikely to be resolved by ever-larger datasets without further developments in study data and design. New study designs such as mendelian randomisation,13 many crucial questions about BMI will continue to rely on observational data. To date, few sufficiently sized randomised trials have been done to address whether weight-loss interventions reduce mortality or morbidity. One trial14 was ended after about 10 years of follow-up because no association between weight loss and cardiovascular events was found. Weight-loss interventions have only modest long-term effectiveness15 and generally target behaviours, such as diet and physical activity,13 that can lead to change in BMI rather than directly targeting BMI itself. Therefore, clinical trials are limited in their capacity to address causal relations between BMI and mortality.Despite the limitations of observational studies for causal inference of obesity and mortality,The Lancet, via the World Obesity Federation, and other coalitions such as the US National Collaborative on Childhood Obesity Research, are championing diverse approaches to this challenge including support for better measurement, systems models, and increased attention to the evaluation of obesity-related policies.Important challenges remain in the effort to translate epidemiological evidence of excess bodyweight and mortality into effective guidelines and public health interventions.


The zooarchaeological research presented here investigates Neolithic and Chalcolithic  animal exploitation strategies at Uğurlu Höyük on the Turkish island of Gökçeada in the northeastern Aegean Sea. Toward this end, we first discuss the results of our analysis of the zooarchaeological assemblages from Uğurlu Höyük and then consider the data within a wider regional explanatory framework using a diachronic approach, comparing them with those from western and northwestern Anatolian sites. The first settlers of Gökçeada were farmers who introduced domestic sheep, goats, cattle and pigs to the island as early as 6500 years BC. Our results align well with recently published zooarchaeological data on the westward spread of domestic animals across Turkey and the Neolithization of southeast Europe. Using an island site as a case study, we independently confirm that the dispersal of early farming was a polynucleated and multidirectional phenomenon that did not sweep across the land, replace everything on its way, and deliver the same “Neolithic package” everywhere. Instead, this complex process generated a diversity of human-animal interactions. Thus, studying the dispersal of early farmers from southwest Asia into southeast Europe via Anatolia requires a rigorous methodological approach to develop a fine-resolution picture of the variability seen in human adaptations and dispersals within complex and rapidly changing environmental and cultural settings. For this, the whole spectrum of human-animal interactions must be fully documented for each sub-region of southwest Asia and the circum-Mediterranean. Neolithization from 10,000–7000 BC have been the subjects of extensive scholarly debate since the 1970s  and the subsequent spread of new genes, languages, ideologies, and domesticated cereals and livestock into Europe via a process called s . Various e.g., , 4–14).–14.NeoliUğurlu Höyük is a Neolithic settlement on Gökçeada (Imbros in Greek), the largest Turkish island situated between Anatolia and the European continent in the Aegean Sea, and currently the only site with an early Neolithic component in the eastern Aegean. Thus, with its key geographical location between Southeast Europe and Southwest Asia and its early Neolithic strata, the results of zooarchaeological research presented here, more broadly, may have implications reaching beyond Anatolia and contribute to our understanding of the spread and development of agricultural societies in southeast Europe in general and the eastern Aegean in particular.More specifically, this paper focuses on animal exploitation strategies at Uğurlu and adds new zooarchaeological data to the existing body of research on the spread of domesticated animals across Neolithic western Anatolia. We address the following specific questions:Did the islanders have a diverse subsistence strategy, including foraging and marine resource exploitation, or did they heavily rely on livestock management? How did the animal economy change through time?How did island habitation affect animal management decisions compared to the mainland Anatolia? Did the islanders manage cattle, sheep, goats, and pigs differently?In investigating animal exploitation at the site, we first characterize faunal assemblages and examine their formation processes and taphonomic histories, which assist us in identifying the role of humans in the accumulation, modification, and destruction of these assemblages. Second, we examine taxonomic composition and species trends, body part distributions, age structures, and body size to probe the foregoing questions.In studying the dispersal of agricultural economies from southwest Asia to southeast Europe, archaeologists have used a dichotomized framework. The colonization or demic diffusion model entails replacement of foragers by advancing waves of farmers –17, wherThere has been a recent movement, however, toward a consensus acknowledging the complexity of the processes and mechanisms that spread the Neolithic across Europe. Toward this end, it is now recognized that farming spread into Europe by a mixture of expansion, diffusion, and adoption as the predominant mechanisms –25. NeolBenjamin Arbuckle and Levent Atici’s  broad suIn order to expand the corpus of demographic and osteometric data from Anatolia and to document the movement of sheep, goat, cattle, and pig from southwest Asia into Europe through western Anatolia, eighteen researchers working in Turkey merged their primary datasets into a single database representing seventeen sites, 42 chronological phases, and more than 200,000 faunal specimens .In central Anatolia, domestic sheep and goats first appeared by the mid-eighth millennium BC, then domestic cattle a millennium later; domestic pigs were never incorporated into Neolithic economies in this region . OutsideFurthermore, recent Neolithic investigations in western Turkey have dramatically changed our understanding of the emergence and development of animal husbandry in western Turkey. Excavations at Ulucak Höyük have revealed Aceramic Neolithic layers and pushed the appearance of herding back to the early seventh millennium BC , 35. SedThe results of biomarker and zooarchaeological research in western Turkey synergize to offer new insights into the relative importance of animal products such as meat and dairy during the Neolithic. The analysis of lipid residues in archaeological ceramics identified dairy use in the Marmara and Izmir regions beginning around 6500 BC . In the This paper employs an analytical approach similar to that of Arbuckle and colleagues  in an atThe island of Gökçeada lies about 17 km from the Gelibolu  Peninsula of the Anatolian mainland, and covers an area of 289.5 square km. During the Last Glacial Maximum (ca. 20.000–18.000 BC), sea levels were about 120 m lower than the present sea level , 44. GökThe site of Uğurlu Höyük is a low mound covering an area of approximately 250 x 200 m on a gentle slope at the eastern foot of Mount Isa (Doğanlı) on the western part of the island. The main Uğurlu-Dereköy road cuts through the site and it has also been damaged by a long trench dug for the opening of an irrigation system. The Pilon stream lies at the eastern part of the site, and there is a nearby spring. The island is mountainous, and the bedrock geology is mainly composed of volcanic rocks. The site was first discovered in 1998 and a long-term project was started in the summer of 2009 by Burçin Erdoğu . During Thanks to a rigorous dating program, we have a well-dated and established chronology for the cultural sequence . The earThe earliest occupation at the site, Phase VI, is represented in sounding trenches, yielding scattered stones, a hearth, several disc-shaped shell or stone beads and bone awls . This stDuring Phase IV the settlement seems to have enlarged, covering an area of 6 hectares. Phase IV is represented by a 2.5-meter thick deposit with at least four occupational layers. No complete buildings have been exposed. So far, two layers of Phase IV have been excavated. The first layer has yielded a compact floor, a circular hearth, extremely dense concentrations of animal bones, and bone tools such as awls, chisels, and needles. A building with damaged stone walls, a thick compact floor with multiple layers of plaster and post-holes, a large storage vessel, and an antler hammer were found in the second layer. In addition, mats preserved as phytoliths were sitting on some parts of the floor.Triticum monococcum), six-rowed barley (Hordeum vulgare), naked barley (Hordeum vulgare var. nudum), and pea (Pisum sativum L.). Large quantities of shells and some fish bones indicate the potential role of marine resources in the Neolithic diet of Uğurlu. Among shells, Patella and Mytilidae are numerous. Neolithic figurines make up a small assemblage that includes acrolithic figurines, a marble figurine head, and a pregnant-like anthropomorphic figurine.In the eastern part of the site, a building with a long exterior buttress and two architectural phases has been partly excavated . From thSpondylus gaederopys, pendants made from Cerastoderma, and bone and antler tools  were sampled randomly, generating 6061 bone fragments. Of the three strata, Phase V has generated the largest sample (N = 3967), as the faunal remains were densely packed in a small area of 2 x 4 m, enabling effective hand-picking. This area lies at the edge of the residential areas of the settlement, and might have served as a disposal area for the Neolithic inhabitants. The sediment in this area is almost totally built of bones, making up 90 to 95% of the matrix, with sporadic lithic and pottery elements. The size distribution of shaft fragments smaller than 5 cm in each of the three strata, with 85, 86, and 73%, respectively, attests to the overall efficiency of recovery at the site.The recording protocol employed in this work entailed general documentation of the entire assemblage for the purpose of characterization and included every element, element portion, and nonidentified splinter recovered (N = 6061). No pre-sorting was practiced and all of the bones were packed and stored together in the storage area of the Uğurlu Höyük dig house. Every fragment was examined first by naked eye and then with a 10–15 x hand lens under strong light, if necessary, for bone surface modifications, while sub-samples were randomly chosen for recording variables such as fracture platform angle and percussion and notches. All the fragments were identified to the maximum degree possible, refitted and mended when possible, weighed, counted, labeled, assigned unique individual specimen numbers, measured when appropriate, and entered into an automated FileMaker database . When inTaxonomic and skeletal element identifications were carried out partly using a modern comparative reference collection assembled by the authors and partly using published manuals and articles describing identification criteria. When the degree of certainty of identification was high, specimens were identified to the highest taxonomic category possible, i.e., species. When identification to a higher taxonomic category such as species, genus, or family was not possible, methodological categories, such as “medium artiodactyl” were used. For the purpose of statistical viability, the bones of sheep and goats were combined into an “O/C” (“caprine”) category and treated as a single analytical unit. According to Shipman (: 106), tNumber of Fragments (NF), Number of Identified Specimens (NISP), Minimum Number of Elements (MNE), and Bone Weight (BW) were quantitative measures employed in this paper . NF was Ovis orientalis and Capra aegagrus) specimens from Iran. For dental remains, age classes were recorded for caprines following procedures described by Deniz and Payne ).The results of zooarchaeological research presented here align well with the findings of Arbuckle and Atici  and ArbuS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 TableAuthor information and links to online databases are also included.(XLSX)Click here for additional data file.S7 TableLinks to online databases are also included.(XLSX)Click here for additional data file.S8 TableAuthor information are also included.(XLSX)Click here for additional data file.


Obesity is associated not only with an array of metabolic disorders  but also skeletal and joint abnormalities. Recently, a pleiotropic role of vitamin D has been emphasized. Obese children frequently present with vitamin D deficiency, and greater fat mass is associated with lower serum concentration of this vitamin. Although some evidence suggests that weight loss may affect vitamin D status, this issue has not been studied extensively thus far. The aim of a double-blind placebo-controlled study is to assess long-term health effects of vitamin D supplementation in vitamin D deficient obese children participating in an integrated weight-loss programme.A randomized double-blind, placebo-controlled trial analysing the effects of vitamin D3 supplementation in overweight or obese vitamin D deficient (<30 ng/ml) children participating in an integrated weight-loss programme. Children are randomized to receive either vitamin D (1200 IU) or placebo for 26 weeks. Primary endpoints include changes in BMI (body mass index), body composition and bone mineral density at the end of the study period, and secondary endpoints – the changes in laboratory parameter reflecting liver and kidney function  and glucose homeostasis .The effects of vitamin D supplementation in obese individuals, especially children, subjected to a weight-loss program are still poorly understood. Considering physiological processes associated with puberty and adolescent growth, we speculate that supplementation may enhance weight reduction and prevent bone loss in obese children deficient in this vitamin.NCT 02828228; Trial registration date: 8 Jun 2016; Registered in: ClinicalTrials.gov. The trial was registered retrospectively. Obesity is associated with an array of metabolic disorders, such as insulin resistance, hiperinsulinemia, impaired glucose tolerance, abnormal fasting glycaemia, symptomatic diabetes mellitus, dyslipidaemia and cardiovascular disorders, namely arterial hypertension. Moreover, overweight or obese subjects are at increased risk of joint and skeletal disorders, respiratory problems, kidney diseases and alimentary dysfunction, especially non-alcoholic fatty liver disease. High risk of complications associated with childhood obesity justifies early implementation of intervention programmes. Many previous studies demonstrated that the most effective form of intervention are integrated multidisciplinary weight-loss programmes, involving not only children but also their family members –3. ReducRole of vitamin D in energetic metabolism has been emphasized quite recently. Many obese children present with low blood concentrations of vitamin D –9, probaAnother important issue is contribution of vitamin D to the etiopathogenesis of metabolic syndrome. Previous studies documented inverse relationships between blood concentration of vitamin D, waist circumference, systolic blood pressure, insulin resistance, fasting glycaemia, total cholesterol, triglyceride and LDL cholesterol levels in paediatric subjects, as well as positive associations between concentrations of vitamin D and HDL cholesterol , 14, 15.Importantly, a positive association has been found between concentration of vitamin D and sensitivity to insulin in obese children; furthermore, the level of this vitamin correlated inversely with concentration of glycated haemoglobin (HbA1c) . MoreoveThese findings imply that vitamin D deficient obese children may be at increased risk of many metabolic disorders, such as insulin resistance, hiperinsulinemia, impaired glucose tolerance, abnormal fasting glycaemia, symptomatic diabetes mellitus, dyslipidaemia and arterial hypertension. Indeed, a number of observational studies documented a substantial role of vitamin D deficiency in etiopathogenesis of metabolic syndrome and other obesity-related complications. However, we still lack evidence from interventional studies confirming causal character of these relationships.Metabolic effects of obesity on bone growth and maturation are still not fully understood. Moreover, the results of previous studies analysing bone mass and bone density in obese individuals are highly inconclusive. While according to some authors, bone mass decreases with body weight , others Vitamin D plays an important biological role in the process of bone maturation and mineralization. Previous studies documented an inverse relationship between blood concentration of vitamin D and bone mineral density , 28. In Surprisingly, the results of previous studies analysing a relationship between obesity, vitamin D and bone metabolism are sparse and highly inconclusive. An analysis of 58 morbidly obese teenagers demonstrated that individuals with physiological blood concentrations of parathyroid hormone (PTH) presented with normal bone mineral density, irrespectively of their vitamin D levels .n = 24) or normal body weight (n = 25) showed that the former presented with higher bone mineral density than their normal-weight peers; this relationship turned out to be independent of blood concentration of vitamin D, physical activity level and fat-free mass content. However, in the same study, bone mineral density correlated with blood concentrations of leptin and insulin [In contrast, a recently published study including a small group of adolescents with obesity  or placebo for 26 weeks.We hypothesized that supplementation of vitamin D in obese children presenting with low serum levels of vitamin D (25(OH)D3) may exert a beneficial effect on the outcomes of an integrated weight-loss intervention, namely on changes in BMI, muscle mass, bone mass, bone mineral density and biochemical markers of metabolic complications related to obesity.The study is conducted as a continuation and extension of the “6–10-14 for Health” integrated weight-loss intervention programme for obese children from Gdansk municipality. Both participants of the programme and their family members are offered a 12-month integrated intervention, including medical, dietetic and psychological counselling, as well as educational workshops for parents. The same intervention will be offered to all subjects of our study as well.The first phase of the programme, financed by the municipality of Gdansk, has been conducted in 2011–2013. The programme comprised of a screening and survey conducted among all 6-, 10- and 14-year-old children attending primary and grammar schools. A total of 18,162 children  were examined. The aim of the screening was to identify a group of children with risk factors of civilization-related disorders, i.e. overweight or obesity defined according to BMI percentile charts developed within the framework of “OLAF” project ). This gThe intervention included four appointments with participating children and their guardians: at enrolment, as well as 3, 6 and 12 months thereafter. The appointments had form of individual consultations with various specialists. The first visit included complex medical examination, discussion on health status of a child, interpretation of laboratory findings, and consultation with a dietician, psychologist and physical education specialist. Then, individual protocol of health intervention was developed for each subject. The aim of the intervention was to implement dietary modifications, to enhance health activity of the child, and to reinforce health-oriented behaviours among his/her family members. Compliance with the protocol and the results of the intervention were verified during follow-up meetings with the specialists, and used as a basis for further tailor-made management.p = 0.0001) and fat mass content determined by means of bioimpedance .Interim evaluation of the programme’s effectiveness included a group of 300 children. Mean BMI percentiles at enrolment and following 12 months of the intervention were 92.96 and 88.83, respectively. The tailor-made intervention contributed to a significant decrease in both BMI percentile  in obese children presenting with low serum concentrations of 25OHD3 at enrolment to the integrated weight-loss programme.This is a double-blind randomized study. Upon enrolment, the subjects were randomized to one of the two groups.to analyse the effect of vitamin D supplementation on BMI, body composition and bone mineral density of obese children participating in the weight-loss programme;to analyse the effect of vitamin D supplementation on the risk profile of obesity-related complications, namely impaired glucose tolerance, insulin resistance, dyslipidaemia and arterial hypertension in obese children participating in the weight-loss programme;to analyse the prevalence of vitamin D deficiency in obese children;to verify if selected biological characteristics of obese children  may constitute a risk factor for vitamin D deficiency.To our best knowledge, this is the first randomized placebo-controlled study analysing the effects of vitamin D supplementation in obese children subjected to a weight-loss intervention. The aims of the study were:Relative changes in BMI, body composition and bone mineral density in the group of vitamin D-supplemented children, determined after 26 weeks of the intervention.blood level of vitamin Darterial blood pressurebiochemical parameters Inflammatory markers .Relative changes in the following parameters in the group of vitamin D-supplemented children and in the placebo group, compared after 26 weeks of the intervention:n = 100 + 100) was estimated for bi-factorial analysis of variance (2) group x (2) time with main effect and interaction analysis, with target statistical power of 0.90 and significance of differences at p < 0.05. Assuming probability of the event (at least 10% reduction in baseline BMI over the follow-up period) at 0.85 and 0.6 for the experimental and control group, respectively, minimum sample size providing 0.9 statistical power for alpha equal 0.05 or lower and beta equal 0.1 or lower was estimated at 130 (65 per group). These values were rounded up to 100 per group assuming up to 30% drop-out rate.The size of the sample  for 26 weeksGROUP II: medical intervention, intervention of a dietician, psychologist and specialist of physical education, parental education + daily oral administration of placebo for 26 weeks.Two hundred participants have been randomly assigned to one of the two groups:Children aged 6, 10 and 14 years, attending primary and grammar schools in Gdansk are subjected to a screening and intervention programme subsidized by the municipality of Gdansk . Participants whose anthropometric parameters correspond to overweight (BMI between the 85th and 95th percentile) or obesity (BMI > =95th percentile), according to the percentile charts developed within the framework of the “OLAF” project , are quaAll enrolled children whose parents gave consent for their participation in the “6–10-14 for Health” programme are subjected to laboratory testing  and paediatric consultation. Blood concentration of 25(OH)D3 is determined as well. Finally, an additional 4-ml blood sample is collected and stored for the purpose of further laboratory testing.Upon obtaining a written consent from their parents or guardians, all children with obesity or overweight and decreased blood concentration of 25(OH)D3 are enrolled.overweight (BMI between the 85th and 95th percentile) or obesity (BMI > =95th percentile), identified on the basis of anthropometric parametersblood concentration of 25(OH)D3 < 30 ng/mlwritten informed consent from legal guardiansInclusion criteriaChronic conditions Contraindications to administration of vitamin DAdministration of any preparation containing vitamin D, calcium, or steroid hormones during 3 months preceding the studyExclusion criteriaComputer generated random numbers were given to vitamin D or placebo packages at the time of their appearance at the University Clinical Center Clinical Trial Office Figs.  and 2.FiBoth of the study treatments: vitamin D (1200 IU) and placebo were provided from the vendor (Sequoia) as identical capsules placed in the same looking packages . The sets of 7 boxes (corresponding to 6-month treatment) are prepared and blinded at the University Clinical Centre Clinical Trial Office by an independent worker, who does not take part in the study. After assigning the numbers packages are handed out to the subjects by the investigator  during the enrolment visit, the investigator enrols the patients to the study without knowing the group assignment. The investigator and the patient is blinded for the intervention. During the last visit, the investigator collects all blisters and boxes, to assess treatment compliance on the basis of the remaining capsule number.Weight- loss intervention programmeDietetic consultation – analysis of nutritional behaviours and dietary habits, selection of appropriate diet. Dietetic consultation takes place at enrolment and 3, 6 and 12 months thereafter.Psychological consultation – reinforcement of changes in health behaviours of the child and his/her parents.Consultation with a physical education specialist – defining optimal level of physical activity, adjusted to the subject’s body weight, abilities and preferences, development of a training programme with increasing intensity and volume of physical activity.Body weight and body height are determined with a digital scale , with the child wearing an underwear and standing barefoot. Body height is measured to the nearest 0.001 m, and body weight to the nearest 0.1 kg. The scale is calibrated every day. Waist and hip circumferences are measured on a horizontal plane by an Ergonomic Circumference Measuring Tape .Arterial blood pressure is determined oscillometrically (Omron) on the left arm, with a cuff of an adequate size placed at the level of the heart, in the child seated with uncrossed legs, following at least a 5-min rest in the seated position. The width of the inflatable cuff corresponds to at least 40% of arm circumference. Three separate measurements of blood pressure are taken and averaged .The participants are subjected to a 3-min Kasch pulse recovery (KPR) step test , 44. TheDietary records from three consecutive days (2 weekdays +1 day of a weekend), collected prior to enrolment and at the end of the intervention are analysed, and dietary intakes of calcium and vitamin D are calculated with Dieta 5.0 software .Pubertal status is determined on based on the results of physical examination and expressed using the Tanner stage.Total body bone mineral content (TBBMC), total body bone mineral density (TBBMD), lean body mass (LBM), fat mass (FT) (Hologic Discovery Wi).Laboratory parameters:Complete blood count.Lipid profile determined with an enzymatic method.Oral glucose tolerance test (OGTT) with glucose concentration determined with hexokinase method.Concentration of insulin determined by means of an immunochemiluminescence assay.Concentration of creatinine.ALT, TSH, fT4, PTH, Ca, P,hs-CRP level determined by means of an immunoturbidimetric assay.Concentration of 25(OH)D3 determined by means of an immunochemiluminescence assay.medical history, physical examinationinterpretation of laboratory findings (tests conducted during screening + concentration of vitamin D),anthropometric evaluation and analysis of body composition (bioimpedance method)consultation with a dietician, psychologist and specialist in physical activity, and defining detailed protocol of the interventionKasch Pulse Recovery Testdetermination of hs-CRP, IL-6, chemerin and adiponectin concentrations in blood samples obtained during screeningfamiliarizing child’s parents with the objectives and protocol of the studyobtaining written informed consent for participation in the studyrandomization to GROUP I or IIdensitometry (DXA) – within one week after enrolmentVisit 1 (enrolment)medical history, physical examinationanthropometric evaluation and analysis of body composition (bioimpedance method)consultation with a dietician, psychologist and specialist in physical activity, and defining detailed protocol of the intervention. Within a week prior to Visit III – obtaining blood (ca. 5 ml) for laboratory testing .Visit 2 (3 months)medical history, physical examinationinterpretation of laboratory findingsanthropometric evaluation and analysis of body composition (bioimpedance method)consultation with a dietician, psychologist and specialist in physical activity, and defining detailed protocol of the interventionobtaining blood for laboratory testingtermination of active compound/placebo administrationVisit 3 (6 months)medical history, physical examinationanthropometric evaluation and analysis of body composition (bioimpedance method)consultation with a dietician, psychologist and specialist in physical activity, and defining detailed protocol of the interventiondensitometry (DXA) – within one week after the study termination Table Table 1InVisit 4 (12 months)Statistical analysis will include:- verification of normal distribution of continuous variables with the Shapiro-Wilk test.- determination of basic statistical characteristics for continuous and discrete variables.- intergroup comparisons with parametric and non-parametric tests.- intragroup comparisons with parametric and non-parametric tests.- analysis of intervention efficacy based on bi-factorial analysis of variance (2) group x (2) time with main effect and interaction analysis.The study is supported by Nutricia Foundation RG-1/2015 .The study is currently ongoing and the data are still collected. No partial results have been published thus far. Obesity, defined as an excessive accumulation of adipose tissue with resultant impairment of body functioning, is associated with an increase in morbidity and mortality risk. A dramatic increase in the prevalence of overweight and obesity among children has been documented during recent 30 years. A study conducted at the Institute of Food and Nutrition within the framework of the National Programme for Obesity Prevention showed that about 12–14% of Polish children are obese , and in Many authors emphasized the importance of a complex approach to children with obesity. Also involvement of family members, friends and acquaintances is considered to be an important determinant of a successful weight-loss intervention –3.p = 0.0001).Our preliminary findings imply that 12-month intervention is sufficient for a significant decrease in absolute BMI value, BMI percentile and fat mass content  D3 will be determined, and only the subjects with vitamin D3 levels below 30 ng/ml will be enrolled, providing consent from their parents/legal guardians. Then, all the subjects will be managed in line with the “6–10-14 for Health” programme protocol.The flow chart depicts the procedure and scheme of qualification, randomization, duration of the study and included interventions.The table provides detailed information about the procedures  included at consecutive stages of the study.


There is conflicting evidence of benefit versus harm for warfarin anticoagulation in hemodialysis patients with atrial fibrillation. This equipoise may be explained by suboptimal Time in Therapeutic Range (TTR), which correlates well with thromboembolic and bleeding complications. This study aimed to compare nephrologist-led management of warfarin therapy versus that led by specialized anticoagulation clinic.In a retrospective cohort of chronic hemodialysis patients from two institutions , we identified patients with atrial fibrillation who were receiving warfarin for thromboembolic prophylaxis. Mean TTRs, proportion of patients achieving TTR ≥ 60%, and frequency of INR testing were compared using a logistic regression model.p-value 0.95). However, the proportion of patients achieving TTR ≥ 60% was 65% versus 43.3%  and mean frequency of INR testing was every 6 days versus every 13.9 days in Institutions A and B respectively.In Institution A, 16.7% of hemodialysis patients had atrial fibrillation, of whom 36.8% were on warfarin. In Institution B, 18% of hemodialysis patients had atrial fibrillation, and 55.5% were on warfarin. The mean TTR was 61.8% (SD 14.5) in Institution A, and 60.5% (SD 15.8) in Institution B (There was no statistical difference in mean TTR between nephrologist-led management of warfarin and that of clinic-led management. However, the former achieved a trend toward a higher proportion of patients with optimal TTR. This improved therapeutic results was associated with more frequent INR monitoring. Warfarin is a mainstay therapy for reducing stroke risk in atrial fibrillation –4. The uTime in therapeutic range (TTR), as measured by the Rosendaal method , was fouAnticoagulation management directed by specialized anticoagulation clinics has been found to reduce thromboembolic and bleeding complications, as well as optimize TTR in atrial fibrillation patients . FurtherThis is a retrospective cohort study of chronic hemodialysis patients with atrial fibrillation receiving warfarin for the prevention of thromboembolic complications of atrial fibrillation in two university teaching hospitals; the McGill University Health Centre (MUHC) and the Jewish General Hospital (JGH), both located in Montreal, Canada. Between January 2015 and November 2016, patients receiving hemodialysis at the above institutions were screened for eligibility. At the MUHC, warfarin-based anticoagulation is managed by nephrologists, whereas at the JGH it is managed through an anticoagulation clinic led by hematologists. The study was approved by the Medical/Biomedical Research Ethics Committee of the West-Central Montreal Island Health Board.Patients were identified using the following inclusion criteria: a minimum age of 18 years, on maintenance hemodialysis for end-stage renal disease, a documented history of atrial fibrillation or atrial flutter, and on warfarin for a minimum of 3 months. Patients who were on hemodialysis during part of the study period but either died, received a renal transplant, or were transferred to another centre for hemodialysis were included in the study. Patients were included if their total duration of non-hospitalized INR testing while on warfarin was at least 140 days, to ensure sufficient INR data for TTR analysis.INR values on warfarin were collected retrospectively for up to 1 year starting from the last encounter with the patient during the study period. INR values during hospital admissions lasting more than 2 days were excluded as were any INRs obtained within the first week post discharge. The following additional data were collected from dialysis charts and/or electronic medical records: demographics (age and gender), CHADS2 score, total duration on hemodialysis, relevant comorbidities , medication use , and total number of days of hospitalization during the study period.The end points of the study were; 1) the mean TTR, based on the Rosendaal method, 2) the proportion of patients achieving TTR ≥ 60%, based on the Rosendaal method, 3) mean frequency of INR testing.p-values and Odds Ratio (OR): hemodialysis vintage, total duration of hospitalization, and total number of days of outpatient antibiotic use. All statistical analyses were performed using SAS version 9.4.The study cohort was stratified into two groups, Nephrologist-led management (Institution A) and Anticoagulation clinic-led management (Institution B). Continuous variables were expressed as mean ± standard deviation (SD) using paired t-test analysis, whereas, the Chi-square and Fisher exact tests were used to compare binary variables. TTR results from both institutions were fitted in a regression model using PROC GLM, and a logistic model using PROC LOGISTIC was used in the analysis of proportion of patients achieving TTR ≥ 60%. Adjustments for the following variables were performed when calculating To evaluate cost effectiveness of the two strategies, we utilized the laboratory cost of INR assay (0.50 Canadian dollars per test) and the per test professional fee allocated for warfarin management to anti-coagulation clinic physician (12.50 Canadian dollars) applicable for the Province of Quebec, Canada. The nephrologist is not eligible for the professional fee.For the period starting from January 1st, 2015 and ending on November 26th, 2016, we identified 341 patients in Institution A and 300 patients in Institution B who were undergoing hemodialysis for at least 3 months. In Institution A, 57 patients (16.7%) had documented history of atrial fibrillation, of whom 21 (36.8%) were on warfarin at the end of the study period. In Institution B, 54 patients (18%) had history of atrial fibrillation, and 30 (55.5%) were on warfarin. Following the application of the inclusion criteria, the INR data of 20 patients in Institution A and 30 patients in Institution B were included in the final TTR analysis. A flow diagram of the study is illustrated in   at Institution A, and 60.5% (SD 15.8) at Institution B (unadjusted 5) Table . The meaAnalysis of patients achieving the target TTR ≥ 60%, based on Rosendaal method, is illustrated in Table  and Fig. At InstThe average annual number of INR tests was 60 in Institution A (nephrologist-led management) and 26 in Institution B (anti-coagulation clinic-led management). Therefore, at a total per test cost of 0.50$CAN, the average annual cost of INR testing per patient in Institution A is approximately 30.00 $CAD, whereas, at a total per test cost of 12.50$CAN, the average annual cost of INR testing per patient in Institution B is 338.00 $CAN. Therefore, there is significant overall cost savings associated with nephrologist-led warfarin management despite more frequent INR testing. It is important to note that this analysis is based on laboratory and professional fees specific to the Province of Quebec, Canada, which vary somewhat among Canadian provinces and more so across other countries.Our study demonstrates a nearly equal mean TTR among chronic hemodialysis patients receiving warfarin, whether managed by specialized anticoagulation clinic or by nephrologists. In addition, both management modalities achieved mean TTRs that were superior to previously published data in hemodialysis patients, and comparable to those reported in general population. Interestingly, nephrologist-led warfarin management achieved more patients at the target TTR ≥60% than that achieved by anticoagulation clinic, with an odds ratio greater than 2. This outcome correlated with the higher frequency of INR testing that was observed with nephrologist-led management.Time in Therapeutic Range (TTR), calculated by Rosendaal method, has been used as the optimal method to assess the effectiveness and safety of warfarin anticoagulation. It was found to correlate well with bleeding and thromboembolic complications. The data on TTR in the hemodialysis population is limited to a few studies that reported TTRs ranges between 49 and 54% , 18. TheHemodialysis patients with atrial fibrillation have a higher risk of thromboembolic events when compared to the general population with atrial fibrillation , 8. The There is considerable evidence in the literature of general population with atrial fibrillation that achieved TTR can vary depending on the specialty of healthcare providers responsible for managing the warfarin therapy , 20. HowOur study has some noteworthy limitations. Firstly, the relatively small patient sample size and the inclusion of only two university-afflicted dialysis centers limits both the statistical power of the outcome measures and the broader inference to all hemodialysis patients. Secondly, the observational nature of this study may introduce the possibility of unmeasured bias. However, we did obtain data on many key factors that could influence TTR values in patients on warfarin, such as HD vintage, outpatient antibiotic use, and total duration of hospital admissions, and we adjusted for them in our TTR analysis.Our study can be considered as a hypothesis- generating for a subsequent, larger, prospective study addressing whether improved TTRs can be widely achieved in hemodialysis patients, possibly through nephrologist-directed management of warfarin dosing, and whether improved therapy will translate into stroke reductions and less bleeding complications as seen in other patient populations with atrial fibrillation.There was no statistical difference of mean TTR between nephrologist-led management of warfarin to that of anticoagulation clinic-led management. However, the former achieved a trend of higher proportion of patients with optimal TTR which was associated with more frequent monitoring and greater cost effectiveness.


Infectious gangrene of the foot is a serious complication of diabetes that usually leads to a certain level of lower-extremity amputation (LEA). Nevertheless, the long-term survival and factors associated with mortality in such patients have yet to be elucidated.A total of 157 patients with type 2 diabetes who received treatment for infectious foot gangrene at a major diabetic foot center in Taiwan from 2002 to 2009 were enrolled, of whom 90 had major LEAs (above the ankle) and 67 had minor LEAs (below the ankle). Clinical data during treatment were used for the analysis of survival and LEA, and survival was tracked after treatment until December 2012.P < 0.01). An abnormal ankle-brachial index was an independent risk factor [odds ratio 3.12 (95% CI 1.18–8.24)] for a poor outcome (major LEA) after adjusting for age, smoking status, hypertension, major adverse cardiac events, and renal function.Of the 157 patients, 109 died, with a median survival time of 3.12 years and 5-year survival rate of 40%. Age [hazard ratio 1.04 ], and major LEA [1.80 (1.05–3.09)] were independent factors associated with mortality. Patients with minor LEAs had a better median survival than those with major LEAs (5.5 and 1.9 years, respectively, Efforts to limit amputations below the ankle resulted in better survival of patients with infectious foot gangrene. An abnormal ankle-brachial index may guide physicians to make appropriate decisions with regards to the amputation level. Foot gangrene, defined as dead tissue in the foot resulting from inadequate blood flow supply, is one of the manifestations of critical limb ischemia . It can Previous reports have shown that patients with diabetes-related amputations have a high risk of mortality, with a 5-year survival rate of 40–48% regardless of the etiology of the amputation . The risA total of 157 patients with type 2 diabetes and infectious foot gangrene who were admitted to the diabetic foot care center of Chang Gung Memorial Hospital in Taiwan from 2002 to 2009 were enrolled. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (no. 104-1401B). All of the patients received multidisciplinary care including glycemic control, antibiotic treatment and wound dressing. Surgical interventions to remove necrotic tissue or limbs were determined according to the consensus of a team of physicians taking into account the risk of mortality due to co-morbidities and clinical status or an obviously poor likelihood of preserving the foot  , 12.All of the enrolled patients received some degree of gangrene tissue removal, including 90 with major lower-extremity amputation (LEAs) (above the ankle) and 67 with minor LEAs .Survival time was calculated from the discharge date to December 31, 2012, or death. Information of death was obtained from the National Health Insurance database.Clinical data were collected during the admission for foot gangrene treatment. Age, duration of diabetes and HbA1c were recorded as continuous variables. Gender, smoking status, underlying co-morbidities , and renal status), ankle-brachial index (ABI) and amputation level were recorded as categorical variables. Smokers were defined as currently smoking with at least one cigarette per day, and renal status was defined according to the National Kidney Disease Outcomes Quality Initiative (NKDOQI) guidelines as follows: normal renal function with an estimated glomerular filtration rate (eGFR) calculated according to the modification of diet in renal disease equation of ≥60 ml/min; moderate chronic kidney disease (CKD) with an eGFR <60 ml/min but not requiring dialysis treatment; and dialysis status as the continuous need for renal replacement therapy. ABI was calculated by measuring the ratio of the blood pressure of the lower leg to the blood pressure in the arm according to the 2011 American Heart Association guidelines for peripheral arterial disease , and an t test for continuous variables, and Pearson’s chi-square test for categorical variables. In addition, multivariate logistic regression analysis was performed including age, smoking status, hypertension, MACEs, renal function and PAD to identify the independent risk factors predicting the outcomes of the patients with major LEAs.Kaplan-Meier method was used to estimate the survival probability for differing survival times (time to event). Each variable was entered into a multivariate Cox regression model to identify the independent risk factors associated with mortality. Differences in survival and clinical data were calculated between the minor and major LEA groups using the Kaplan-Meier method and log rank test for survival, the Student’s P value <0.05.All statistical analyses were performed using the Statistical Package for the Social Sciences . Statistical significance was defined as a The mean age of the enrolled patients was 66.8 years, and 63.1% of the patients were male . The 5-year survival rate was 40% with a median survival time of 3.12 years and a crude mortality rate of 187.64 per 1000 patient-years.P < 0.01)  Table .Fig. 1AdP < 0.01), and also relatively higher rates of MACEs and renal dysfunction although without reaching statistical significance. A longer hospital stay has been observed in patients with minor LEAs than those with major LEAs .The patients with major LEAs had obviously abnormal ABI values at treatment (80% vs. 47.8%, P = 0.02].Since LEA status had an impact on survival, we further analyzed the factors predicting the level of LEA Table . After aIn this study, the patients with diabetes and infectious foot gangrene related amputees had a 5-year survival rate of 40% after treatment. This survival rate is not better than other studies on diabetic amputees with various etiologies, ranging from a 5-year survival rate of around 40% reported by Tseng et al. , 47.7% bIn addition to the age factor, another independent factor associated survival is the outcome of major LEA. Major LEAs resulted in a worse survival prospect than minor LEAs, which is consistent with other survival studies on diabetic amputees , 7, 14. Prompt major LEA for infectious foot gangrene is sometimes suggested by physicians to control foot infection and also to allow for early rehabilitation such as wearing prostheses and ambulation to avoid restricted activity-related disabilities during hospitalization . HoweverThe results of this study suggest that even though factors such as age and dialysis status beyond our control, avoiding major LEAs could prolong the survival of patients with diabetes and infectious foot gangrene. However, limb preservation is challenging for those patients because of difficulty in wound healing, severe comorbidities, and the high risk of progressive sepsis . FurtherIn this and our previous study, we showed that the ABI value could be used as a predictor for the outcome of minor or major LEAs . HoweverAnother interesting factor associated survival with borderline significance was the renal function status. A reduced eGFR has been reported to be associated with increased risks of death, cardiovascular events, and hospitalization , 29. LavThis study is limited by the single center and retrospective design. In addition, although we evaluated lower limb circulation, the rate of revascularization was relatively low because of clinical concerns over the risk of kidney injury, intravascular procedure complications in the patients with sepsis, and difficulty of the procedure. Therefore, the revascularization data in this study had little power in survival analysis. Further studies are needed to elucidate the importance of revascularization in patients with infectious foot gangrene.The patients with diabetes and amputations caused by infectious foot gangrene in this study had a limited 5-years survival rate of around 40%. Age and LEA level were risk factors associated with mortality. The ABI may assist with appropriate decision making with regards to limb amputation level.


Objective: To identify and compare the bacterial community profile of mucosal tissues from a gastric antrum ulcer and a duodenal ulcer in Helicobacter pylori (Hp) positive dyspeptic patients.Methods: Genomic DNA was extracted from the mucosal tissues obtained from 18 patients diagnosed with gastric antrum or duodenal ulcers. A library was constructed using 16S rRNA gene amplification, and Miseq high-throughput sequencing was used to analyse the amplified products. Bioinformatics methods, including operational taxonomic units (OTUs), hierarchical clustering, and a diversity analysis, were performed to investigate and characterize the community composition.Results: The proportion of Helicobacter in the mucosa of patients with a gastric antrum ulcer was significantly higher than that of patients with a duodenal ulcer. However, the diversity of the bacterial community in the gastric antrum ulcer mucosa was significantly lower compared with the mucosa of the duodenal ulcer. There were significant differences in microbial community structure between the gastric antrum ulcer and the duodenal ulcer. Notably, Helicobacter, Prevotella, Neisseria, and Streptococcus were also predominant genera in the bacterial community of the duodenal ulcer mucosa, and they outnumbered those species in gastric antrum ulcer mucosa.Conclusion: The bacterial community composition and the corresponding abundance differ between the mucosal tissues of Hp positive gastric antrum ulcer and duodenal ulcer patients. Additionally, the bacterial community diversity in the mucosal tissues from gastric duodenal ulcer patients is higher than that from gastric antrum ulcer patients, and Helicobacter is not the absolutely predominant genus. Helicobacter pylori (H. pylori) infection is an important causal factor , and these mechanisms are secondary to those in Crohn's disease and tuberculosis  before endoscopy, and the test results were positive. All patients never had Hp eradication before gastroscopy. And we surveyed the patients' information, such as histopathological data, diet habits, economic status, and family history.All patients were outpatients admitted in the First People's Hospital of Wenling diagnosed with gastric and duodenal ulcers. Each patient received a Hp isolation culture tube containing a brain-heart infusion and the other was in a sample tube containing DNA preservation solution.Each patient underwent endoscopic mucosal tissue sampling, and two mucosal tissue specimens were collected in parallel at the same biopsy site. Specifically, 3–5 cm of tissue around the gastric antrum ulcer surface was collected from gastric antrum ulcer patients, and tissue around the duodenal ulcer surface was collected for duodenal ulcer patients. For each patient, one of the collected mucosal tissue samples was placed in an Hp, and strains that were urease, oxidase, and catalase positive were classified as Hp positive, otherwise they were classified as Hp negative. Gastric antrum ulcer H. pylori positive patients were designated group A (antrum), and duodenal ulcer Hp positive patients were designated group B .After the sample was received, the tissue was first placed at room temperature, and the gastric mucosal tissue was sucked into a sander with a Pasteur pipette and then sufficiently ground to homogenize it; it was then inoculated on a 5% defibrinated sheep blood agar plate and evenly spread. The inoculated plate was placed in a 37°C three-gas incubator , cultured for 3–11 days, and the growth was observed. Suspected colonies were smeared for microscopic observation of bacterial morphology consistent with Genomic DNA from all the mucosal flora samples was extracted using an Invitrogen Purelink Genomic DNA kit . The operation was carried out strictly according to the kit instructions. The qualitative and quantitative detection of nucleic acids was accomplished by 1% agarose gel electrophoresis and a Qubit2.0 concentration analyser.2O to the total volume of 20 μL. PCR reaction conditions: 95°C, 3 min, 25 cycles including 95°C 30 s, 60°C 30 s, 72°C 30 s; 72°C 5 min.A specific primer with a “sequencing linker” was synthesized for the 16S rRNA V3 and V4 regions of mucosal flora samples. The first step PCR primer sequence was 356F: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′; 806R: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3′. The PCR reaction system contained 2 × HiFi buffer 10 μL, forward primer 5 μmol/L, reverse primer 5 μmol/L, DNA 10 ng, plus ddH2O to obtain the purified PCR product. Then, the second step PCR reaction system contained 2 × HiFi buffer 10 μL, forward primer 10 μmol/L, reverse primer 10 μmol/L, first PCR Purify the product template 2 μL, plus ddH2O to a total volume of 20 μL. The PCR reaction conditions were 95°C, 3 min, 12 cycles including 95°C 30 s, 60°C 30 s, 72°C 30 s; 72°C 5 min. After completion of the PCR reaction, purification was accomplished according to the previously mentioned magnetic beads purification protocol, and finally, 30 μL of ddH2O was eluted to obtain a DNA library of mucosal samples.After PCR amplification, the PCR products were purified with Backman magnetic beads. Briefly, 20 μL of the PCR reaction product was mixed with 16 μL of magnetic beads to adsorb the DNA, and then washed with 80% ethanol twice and eluted with 40 μL of ddHQuality control of all the libraries was conducted using an Agilent 2100 Bioanalyzer analyser and a Qubit 2.0 concentration analyser. The library that passed the qualification was sequenced using an Illumina MiSeq sequencer.The original image data of the sequencing results were analyzed by the CASAVA software (v1.8.2), and the sequencing data were obtained after a preliminary quality analysis. Pandaseq (v2.7) was used to compare every two sequences and assemble them according to the end of the overlapping area. Using Trimmomatic (v0.30), the primers and linker sequences, and bases with a mass <20 at both ends as well as sequences with lengths <400 bp were also removed. Using usearch (v8.0), the remaining sequences were compared with those in the database, and the chimeric sequences were removed to obtain the final validated data. Operational Taxonomic Units (OTUs) are clustered sequences into bins, clustering was performed by the UCLUST method with the parameter similarity set at 97%, and an OTU list and OTU representative sequences were obtained. The sequences were randomized using the Qiime platform (v1.7), and dilution curves were constructed using the number of sample sequences and the number of OTU they could represent. According to the results of the OTU clustering, ACE, Chao1, Shannon, and Simpson were used to analyse the abundance and diversity of the mucosal samples. Using the Qiime platform (v1.7), a principal component analysis was performed based on the UniFrac distance.t-test, and the classification of the data was analyzed using the chi-square test or Fisher's exact test. A P value ≤ 0.05 was considered as statistically significant.Statistical analyses were performed using SPSS19.0 software. The comparison of the continuity variables was analyzed using a Hp isolation and culture, one case of gastric antrum ulcer and one case of duodenal ulcer were determined to be Hp negative and removed. Thus, a total of 9 Hp positive cases remained for the gastric antrum ulcer group. The average age of the patients was 48.4 years, and the male to female ratio was 1.25: 1. For the duodenal ulcer Hp positive group of nine patients, the average age was 41 years and the male to female ratio was 2: 1. There was no significant difference between the two groups for age  or sex ratio . There was also no significant difference in histopathological data, diet habits, economic status, and so on between two groups  and 24226 in the Hp positive duodenal ulcer patients (group B) .As shown in Figure Hp positive duodenal antrum ulcer than that for the Hp positive gastric ulcer. Helicobacter accounted for the major proportion in the Hp positive gastric antrum ulcer, with an over 40% ratio. However, Helicobacter was not the predominant bacterial genus in the Hp positive duodenal ulcer, only present in less than a 10% proportion in some samples. Notably, bacterial genera including Prevotella (9.83%), Neisseria (5.03%), Streptococcus (3.96%), Veillonella (1.92%), Streptococcus (3.96%), and Porphyromonas (2.62%) were also present in the Hp positive duodenal ulcer. Furthermore, the genera Psychromonas, Frankia, Prevotella, and Porphyromonas were identified and significantly distinguished the Hp positive duodenal ulcer samples .The bacterial community composition at the genus level in the two ulcer types is shown in Figure Hp in the Hp positive gastric antrum and the duodenal ulcers. As shown in Figure Hp in the gastric antrum ulcer samples was 76.44%, and it was 24% in the duodenal ulcer samples. The difference was statistically significant .We then determined the proportion of colonization of Hp positive ulcer types by first calculating the four α-diversity indexes, including the Chao1, Shannon, Simpson, and ACE indexes. For the Hp positive gastric antrum ulcers, the four indexes were 9518.19, 3.81, 0.51, and 10224.67 , indicating a lower bacterial community diversity.We investigated the bacterial community diversity of the two Hp positive gastric ulcer types, which compared the bacterial community difference between every two samples. As shown in Figure Hp positive gastric antrum ulcer were mainly clustered in the positive-value quadrant, and their distribution was relatively concentrated. The samples of the Hp positive gastric duodenal ulcer were mainly clustered in the negative-value quadrant, and their distribution was relatively dispersed. This indicated that the intra-sample similarity is higher for the Hp positive gastric duodenal ulcer than for the Hp positive gastric antrum ulcer.We then performed clustering based on an unweighted distance matrix and a principal component analysis to characterize the β-diversity for both Hp was first discovered in the stomach, and then other microorganisms were also identified (Hp infection and peptic ulcers has long been known; for example, in Japan, 94% of patients with gastric ulcer and 98% of patients with duodenal ulcer have an associated Hp infection (Alam et al., Hp is the leading cause of ulcers, few studies found differences in the microorganism populations between the two ulcer types. In this study, we compared the mucosal flora structure in 18 cases of Hp positive gastric antral ulcer and duodenal ulcer.Previously, it has been well acknowledged that an acidic environment in the stomach and other antibacterial factors are not suitable for bacterial colonization; thus, the stomach should be in a relatively sterile state . With thentified . The cloFirmicutes were predominant in both ulcer types, which was consistent with the results reported by Bik et al. (Bik et al., Prevotella, Neisseria, and Streptococcus in the Hp positive mucosal tissues in both ulcer types, despite the predominance of the bacterium Helicobacter. These bacteria are mainly found in the mouth and in food, and their presence has been confirmed in the stomach of the Hp negative patients in earlier studies (Jiang et al., Streptococcus and Neisseria were predominant bacterial genera in Hp positive gastric mucosal tissues. Using a 16S rDNA high-throughput sequencing analysis (Li et al., Streptococcus and Neisseria were predominant bacterial genera inout Hp negative gastric antral ulcer patients, and their colonization clearly affected that of Neisseria and Streptococcus as predominantly observed in the stomach of Hp negative patients.Comparing bacteria at the phylum level, we found that Mycobacterium, Bacteroides, and Hp positive duodenal ulcer patients, the proportion of Helicobacter is significantly lower than that of Hp positive gastric antrum ulcer patients (Figure Pseudomonas, Neisseria, and Streptococcus accounted for a relatively larger proportion, so Helicobacter was not the only predominant genus. In addition, the proportion of Bacteroides and Streptomyces in patients with duodenal ulcers was significantly higher than that in patients with gastric antrum ulcers; this outcome was also the case for the bacterial genera Halophilus, Frankesia, Prevobacteria, and Porphyromonas. An explanation might be that the duodenum is part of the small intestine, which is closer to the jejunum and therefore will be affected by the diversity of the intestinal flora. It might also result from the fact that mucosal cells in the duodenum do not secrete gastric acid, and the relatively high pH value is more suitable for the growth of other flora.However, few literature reports about the duodenal mucosal floral structure exist. Our study shows that in s Figure , whereasHp positive duodenal ulcer was significantly higher than that in patients with an Hp positive gastric antrum ulcer (Figures Hp to a certain extent Song (Hp infection together with probiotic adjuvant therapy to rebuild the diversity of gastric flora and maintain the stability of the stomach micro-ecological environment.With respect to biodiversity, the bacterial diversity in the mucosal tissues of patients with an  Figures , 4. The  Figures . Anotherent Song . Thus, tHp positive duodenal ulcer and gastric antrum ulcer patients using a 16S rRNA sequencing analysis and identified substantial differences. We believe that similar studies involving more patients that use other bacterial detection methods will further elucidate the relationship between the floral structure and different types of peptic ulcers, which will finally benefit clinical therapies.In conclusion, we compared the bacterial community composition and diversity in the mucosal tissues of XC and ZW conceived and designed the study; CX, QL, and LJ contributed to collected samples; LZ performed the laboratory tests; XC and ZW participated in analyzing data and writing the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.