A method of selecting a high-risk group for adjuvant therapy is necessary. The aim of this study was to assess the predictive value of One hundred forty-five patients with pathological stage IA NSCLC who underwent pretreatment with FDG positron emission tomography and CT evaluations were retrospectively enrolled. The associations among tumor recurrence and patient characteristics, maximal standard uptake value (SUVmax) of primary tumors, and CT imaging features were investigated using univariate and multivariate analyses. A receiver operating characteristic (ROC) curve analysis was performed to quantify the predictive value of these factors.P < 0.05). A higher SUVmax (≥2.5) (P = 0.021), a lower ground-glass opacity ratio (≤17%) (P = 0.014), and the presence of bronchovascular bundle thickening (P = 0.003) were independent predictive factors of tumor recurrence in the multivariate analysis. The use of this predictive model yielded a greater area under the ROC curve (0.877), which suggests good discrimination.Tumor recurrence developed in 21 (14.5%) of the 145 patients, and the 5-year recurrence-free survival rate was 77%. The univariate analysis demonstrated that SUVmax, the grade of histological differentiation, tumor size, and the presence of bronchovascular bundle thickening were significant predictive factors (The combined evaluation of FDG uptake and CT morphologic features may be helpful in the prediction of recurrence in patients with pathological stage IA NSCLC and in the stratification of a high-risk group for postoperative adjuvant therapy or prospective clinical trials. However, the rate of recurrence remains unsatisfactory and ranges between 15% and 30%, even after complete surgical resection.4 Many recent studies have recognized that lymphovascular invasion (LVI) is an important and poor prognostic factor for pathological stage I tumors, and adjuvant chemotherapy might be helpful to improve survival in this high-risk group.9 However, in clinical practice, many cases without LVI still encounter recurrence despite complete surgical resection. This observation encouraged us to investigate whether other risk factors in addition to LVI were correlated with recurrence in pathological stage IA tumors, which will help identify high-risk patients who may benefit from adjuvant chemotherapy and will help in the prediction of outcomes.Currently, tumor stage remains the most important factor correlated with survival in non-small cell lung cancer (NSCLC), and the 5-year survival rates range from 83% in pathological stage IA to 23% in stage IIIA tumors.18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) has been widely used as a noninvasive diagnostic modality for diagnosing, staging, detecting recurrence, and monitoring the therapeutic response in patients with NSCLC.10 Furthermore, the degree of FDG uptake of tumor lesions, expressed as the maximal standardized uptake value (SUVmax), is proportional to the glucose metabolic rate of viable tumor cells and is a significant prognostic factor in NSCLC.14 Thin-slice CT imaging characteristics are also important in the evaluation of malignant potential or pathological aggressiveness of NSCLC. A well-known indicator of invasiveness and a prognostic factor on CT images is the ground-glass opacity (GGO) ratio of early lung cancer.16 However, it remains unclear whether a relationship exists among FDG uptake, CT imaging features, and recurrence in pathological stage IA NSCLC. Thus, we retrospectively analyzed the clinical and radiological features of pathological stage IA NSCLC patients and developed a prediction model to facilitate the risk assessment of recurrence.17 All pathological evaluation, including histological subtype, histological grade, and LVI, was performed by a pathologist with 15 years of experience who was blind to the recurrence status of the patients.The study was approved by the Institutional Review Board for Human Investigation, which waived the requirement of informed consent for this retrospective study. Between January 2005 and December 2011, 472 consecutive patients with NSCLC underwent pulmonary surgery at our institute. The inclusion criteria of the study were as follows: pathological stage IA disease, preoperative CT and FDG PET examinations, and no preoperative or postoperative adjuvant chemotherapy or radiotherapy. Patients with multiple lung cancers or a history of malignancy were excluded. One hundred forty-five patients  who met inclusion criteria were enrolled in this study. Clinical parameters, including age, sex, smoking history, pretreatment serum carcinoembryonic antigen (CEA) level , surgical procedure type, pathological findings, and tumor stage, were recorded. Tumor–node metastasis (TNM) staging was performed in all patients based on the 7th edition of the American Joint Committee on Cancer (AJCC) staging manual.After surgical resection, all patients were followed and assessed by chest CT imaging every 3 to 6 months in the first 2 years and annually thereafter. Recurrence was confirmed by either radiological or pathological exams in all cases. When ambiguous cases occurred, the diagnosis of recurrence was made by the consensus of a multidisciplinary discussion. The recurrence-free survival (RFS) period was calculated in months from the date of surgery until recurrence or the last follow-up.18F-FDG, imaging was performed using a spiral CT scan from the head to the upper thigh with a 5 mm thickness per slice. Subsequently, PET data were acquired in a supine position using the conventional 3-dimensional mode  at 3 minutes per bed position. The CT images were reconstructed using conventional filtered back projection at 2.4 mm axial intervals to match the slice separation of the PET data. The CT attenuation-corrected PET images were reconstructed using the method of ordered subset expectation maximization (2 iterations and 8 subsets). Coregistered images were displayed using SYNGO software (Siemens Inc).PET/CT was performed using a Biograph PET/CT scanner . All patients fasted except for water at least 6 hours prior to the examination. The serum glucose level was measured prior to the administration of the radiotracer to ascertain that blood sugar was less than 150 mg/dL. Sixty minutes after the intravenous injection of 10 mCi A nuclear medicine physician with 26 years of experience who was blinded to the recurrent status of the patients interpreted the FDG–PET images. The SUVmax for each patient was measured by placing regions of interest (ROIs) on the axial image of the tumor. The SUVmax was calculated as the maximum FDG uptake within the ROI divided by the injected dose over the patient's body weight (SUVmax = maximum pixel activity/[injected dose/body weight]).Chest CT scans  from the lung apices through the adrenal glands were available for all patients and had been conducted within 1 month prior to PET scan. The routine scanning parameters were as follows: a slice thickness of 5 mm, a reconstruction interval of 5 mm, a rotation speed of 0.75 seconds, a pitch of 1.05 to 1.25 and 120 kVp, and an effective tube current × time product that ranged between 150 and 200 mA. High-resolution images of the tumor were acquired with a thin slice thickness of 1 mm and reconstructed with a high-spatial-frequency algorithm.The CT findings were analyzed in the lung window setting  using a picture archiving and communication system (PACS) workstation . All CT images were evaluated in consensus by 2 chest radiologists  who were aware that all patients had proven lung cancers, but were blinded to the final pathological result and recurrence status. The imaging features that were analyzed for each lesion included the tumor size, tumor location, border (lobulated or nonlobulated), margin (spiculated or nonspiculated), presence of air bronchogram, presence of bronchovascular bundle thickening, presence of pleural retraction, and GGO ratio. GGO was defined as hazy opacity without obscuring the underlying vascular or bronchial structures. Two reviewers also performed quantitative measurements (1 measurement by each reviewer separately) to determine the tumor size and the GGO ratio. The maximal diameter of the entire tumor and internal solid portion on axial images was recorded. The GGO ratio was calculated using the following formula: . We used the mean of the 2 measurements (1 measurement by each reviewer separately) as the final result. If the interobserver difference was larger than 5%, a third measurement was conducted by consensus. In these cases (n = 9), the third measurement was used as the final result.2 or Fisher exact test. A receiver operating characteristic (ROC) curve of the SUVmax and GGO ratio was generated to obtain the cutoff value that yielded optimal sensitivity and specificity for the prediction of recurrence. A multivariate logistic regression analysis was used to identify independent risk factors for the patients with recurrence. The predictive value of the established model was assessed by calculating the area under the ROC curve (AUC). According to Hanley and McNeil,18 an AUC value of 0.51 to 0.60 indicates the absence of discrimination, 0.61 to 0.70 indicates poor discrimination, 0.71 to 0.80 indicates acceptable discrimination, 0.81 to 0.90 indicates good discrimination, and 0.91 to 1.0 indicates perfect discrimination. The RFS curves were assessed using the Kaplan–Meier method, and the differences between the variables were determined using the log-rank test. A multivariate Cox proportional hazards model was used to determine independent variables for predicting RFS. Two-sided P values <0.05 were considered statistically significant. All statistical analyses were performed using SPSS software  and MedCalc .Descriptive data are presented as the means ± standard deviation. A categorical comparison was performed using the χThe characteristics of the 145 enrolled patients are shown in Table P < 0.001). Patients with poorly differentiated tumors were more likely to experience recurrence compared with patients with well or moderately differentiated tumors . With respect to the LVI, there was no significant difference.The ROC curve revealed that the cutoff point of SUVmax was 2.5, and the calculated AUC was 0.725  (acceptable discrimination). We dichotomized the tumors according to this threshold (SUVmax = 2.5), and the sensitivity and specificity for predicting recurrence were 90.5 and 56.5%, respectively. Among the clinical factors, SUVmax and the grade of histological differentiation exhibited a significant correlation with recurrence . Tumors associated with the presence of bronchovascular bundle thickening were more frequently observed in the recurrent group compared with in the nonrecurrent group (P = 0.002)  (acceptable discrimination). The optimal cutoff value for the GGO ratio for predicting recurrence was ≤17% . A comparison between the recurrence and no recurrence groups identified significant differences in the tumor size, the GGO ratio, and the presence of bronchovascular bundle thickening. The patients with recurrence tended to present a larger tumor size (≥2 cm) and lower tumor GGO ratio (≤17%) (P < 0.001)  Figure .P < 0.001, =0.002 and 0.007, respectively) . The 5-year RFS was analyzed according to the predictive model . The cumulative rate of tumor recurrence was also significantly higher for patients with tumors who presented a high SUVmax (≥2.5), a low GGO ratio (≤17%), and the presence of bronchovascular bundle thickening  with pathological stage IA, and the SUVmax cutoff value of 2.5 is close to that reported by Uehara et al and Kishimoto et al.24 Therefore, we suggest that this value might be an appropriate threshold of early and small adenocarcinoma.Prior series studies have reported that the preoperative SUVmax has prognostic value in early stage NSCLC and that a higher SUVmax is frequently correlated with recurrence.27 However, although the prognostic factors demonstrated in our study were similar to previous studies, most of our patients with recurrence rarely exhibited evidence of LVI in pathological evaluation. In fact, the assessment of LVI may be affected by different staining methods. It has been reported that immunohistochemical staining, such as podoplanin, D2-40, or CD34, is more accurate for the detection of LVI compared with hematoxylin and eosin staining.28 Moreover, LVI also might be missed if the vascular lumen has been obliterated by tumor cells. Therefore, based on our results, CT morphologic features still have a role in the prediction of prognosis despite the fact that no invasive finding was identified in the pathological assessment. Furthermore, we suggest that tumors with CT imaging features indicating a lower GGO ratio and the presence of bronchovascular bundle thickening may be stratified as a high-risk group for recurrence.In the evaluation of pulmonary nodules on thin-sliced CT images, previous studies have demonstrated that lower tumor GGO ratios and the presence of bronchovascular bundle thickening are unfavorable prognostic factors and that most tumors with these imaging features usually correlate with pathological invasiveness, such as LVI, a well-established prognostic factor.Although few published studies have evaluated the presence of thickened bronchovascular bundles as a prognostic factor of tumor recurrence, we demonstrated that a morphologic lesion analysis that included this feature enhanced our performance in the prediction of tumor recurrence compared with an analysis that included SUVmax or the GGO ratio alone. When SUVmax or the GGO ratio was applied alone, the AUC was 0.725 and 0.726, respectively. Considering the higher AUC of 0.877 in the multivariate analysis, we believe that including SUVmax, the GGO ratio, and the presence of thickened bronchovascular bundles can increase the predictive ability of the model, especially in comparison with the use of a single parameter alone, to identify patients at high risk of tumor recurrence. Furthermore, this model could be easily applied in clinical practice.33 Therefore, further prospective studies including these parameters will be designed.There were some limitations to our study. First, this study was a retrospective design conducted in a single institution and included a relatively small number of patients. A larger, multi-institutional prospective study is needed for further validation of the current results. Second, because our cohort mainly consisted of adenocarcinoma (90.3%), the results of our study may not represent typical stage IA NSCLC. Thus, research to further evaluate the prognostic factors of nonadenocarcinoma groups is warranted. Third, our criteria are based on subjective assessments of CT morphologic features. A CT attenuation-based approach or assessment by means of computerized schemes to improve measurement and interpretation should be made in future studies. Finally, although simplicity is a great advantage of SUVmax measurement, it may not reflect metabolic activity of the entire neoplastic lesion because of the heterogeneity of NSCLC. Recently, several published studies have reported that volume-based PET parameters such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were better than SUVmax for predicting prognosis in early stage NSCLC.In conclusion, our results highlight the predictive value of radiological factors for recurrence in pathological IA NSCLC, especially in the cases of tumors without LVI in pathological evaluation, and we suggest that a higher SUVmax, a lower GGO ratio, and the presence of bronchovascular bundle thickening on CT imaging are correlated with recurrence. The use of this predictive model might be helpful in the selection of patients for postoperative adjuvant chemotherapy or for inclusion in further prospective clinical trials.


Puberty is a stage of sexual development determined by the interaction of many loci and environmental factors. Identification of genes contributing to genetic variation in this character can assist with selection for early pubertal bulls, improving genetic progress in livestock breeding. Thyroid hormones play an important role in sexual development and spermatogenic function. The objective of this study was to evaluate the association between single nucleotide polymorphisms (SNPs) located in thyroglobulin(TG) gene with age of puberty in Angus bulls. Four SNPs were genotyped in 273 animals using SEQUENOM technology and the association between markers and puberty age was analyzed. Results showed a significant association (P < 0.05) between these markers and puberty age estimated at a sperm concentration of 50 million and a progressive motility of 10%. This is the first report of an association of TG polymorphisms with age of puberty in bulls, and results suggest the importance of thyroidal regulation in bovine sexual development and arrival to puberty. Puberty is a stage of sexual development determined by the interaction of many loci hierarchically arranged in networks, and environmental factors . Puberttrα and trβ genes) are present within gonadal tissues, suggesting that THs must have an action on these organs , which is primarily expressed in Leydig cells and plays an important role in sexual differentiation  exert a broad range of effects on metabolism, growth, homeostasis control, and other biological processes . The pr et al., ). In add et al., ). Furthe et al., , Duarte- et al., ). On the et al., ). Sox9 s et al., ). Dax1 get. al., ).Beyond the fact that THs have considerable effects on the hypothalamic-pituitary-gonadal axis (HPG) and sexual development, some studies examined the involvement of androgens in TH synthesis and metabolism. For example, gonadotropin-releasing hormone (GnRH) interferes with the hypothalamic-pituitary-thyroid axis (HPT), increasing thyroid stimulating hormone (TSH) secretion. Moreover, ARs were identified in the thyroid gland of different vertebrate species, suggesting that the androgen axis directly regulates TH synthesis and metabolism . Other Considering the existing evidence on the biological connection between metabolic status, regulated to a large extent by thyroid hormones, and sexual development, and taking into account the fact that THs play a role in the regulation of sexual function, we decided to evaluate the possible associations between SNPs located in the 3’ flanking region of TG gene with the age of puberty in bull calves.http://www.sequenom.com/). Detailed information of the studied SNPs is presented in Table In order to reach this objective, DNA was extracted from blood samples belonging to 273 Angus bulls using Wizard Genomic kit, following manufacturer instructions . Four SNPs  in TG previously reported by Hou et al.,  were genThe association between haplotype markers of TG gene and the estimated puberty ages was analyzed utilizing MIXED procedure implemented in SAS 9.0 software (SAS Inst. Inc.). The linear mixed model used to analyze the association between puberty age and genotypes was the following:ijkl = phenotypic observation of the I bull, μ = the overall mean, Si = the fixed effect of ith year, Gj = the fixed effect of jth genotype, Bk = the fixed effect of kth herd, Ol = random effect of lth sire, and eijkI = random error.Where Y2 = 1). Only two (TGT and CAG) of the eight possible haplotypes were found, with gene frequencies of 0.81and 0.19 for TGT and CAG haplotypes, respectively. Genotype frequencies values were 0.64 for homozygote TGT, 0.03 for homozygote CAG and 0.33 for heterozygote bulls. The obtained haplotypes for the 273 bulls were tested for association with phenotypic data for the two estimated puberty ages mentioned above. The association analysis showed a significant association (P < 0.05) between the haplotype markers and puberty age estimated at C50 and M10. Homozygote TGT exhibited a mean +/- S.E. age at C50 and M10 of 289.74 ± 8.13 days, while homozygote CAG showed 347.67 ± 22.51 days, resulting in a difference of 57.93 days of age. Heterozygote animals showed a mean +/- S.E. age of 299.65 ± 8.72 days of age. No significant association was found between both haplotypes and age at puberty estimated at SC28 .After analyzing the genotyping results, one SNP (rs109662686) was removed given that it exhibited a call rate lower than 57%. The LD analysis indicated that the three remaining SNPs were completely linked (rThe results obtained here constitute the first report of an association of TG gene polymorphisms with age of puberty in bulls and could be explained by the vast amount of works in which the modulatory influence of THs on male reproduction is demonstrated (Flood et al., , Duarte-Despite the broad range of effects in vertebrates and the connections in nearly every biological endocrine system, a lot of studies provide enough evidence to confirm the role of THs in sexual differentiation and gonadal development in mammalian and non-mammalian species. We can affirm that THs influence steroidogenesis and spermatogenesis and as described above there is extensive evidence that links TG gene to testicular development, existing a direct crosstalk between HPG and thyroid hormones axis (Wagner et al., , NonnemaIn conclusion, we detected an association between TG polymorphisms and age at puberty at C50 and M10 in male Angus cattle. Our results could contribute to the investigation on regulation of bovine puberty and fertility. THs are essential for normal growth, sexual development and reproductive function, and normal thyroid activity seems to be a requisite for an adequate male reproductive function. However, the knowledge about the interaction between both endocrine axis, HTP and HTG, is still rudimentary and needs further investigation.


Urine output is closely associated with renal function and has been used as a diagnostic criterion for acute kidney injury (AKI). However, urine output during cardiopulmonary bypass (CPB) has never been identified as a predictor of postoperative AKI. Considering altered renal homeostasis during CPB, we made a comprehensible approach to CPB urine output and evaluated its predictability for AKI.Patients undergoing cardiovascular surgery with the use of CPB, between January 2009 and December 2011, were retrospectively reviewed. AKI was defined as an increase in serum creatinine ≥0.3 mg/dL in the first postoperative 48 hours. We extrapolated a possible optimal amount of urine output from the plot of probability of AKI development according to CPB urine output. After separating patients by the predicted optimal value, we performed stepwise logistic regression analyses to find potential predictors of AKI in both subgroups.A total of 696 patients were analyzed. The amount of CPB urine output had a biphasic association with the incidence of AKI using 4 mL/kg/h as a boundary value. In a multivariate logistic regression to find predictors for AKI in entire patients, CPB urine output did not show statistical significance. After separating patients into subgroups with CPB urine output below and over 4 mL/kg/h, it was identified as an independent predictor for AKI with the odds ratio of 0.43  and 1.11 , respectively.The amount of urine output during CPB with careful analysis may serve as a simple and feasible method to predict the development of AKI after cardiac surgery at an early time point. Oliguria is a major diagnostic criterion of AKI15 the maintenance of urine flow may not guarantee a normally functioning kidney. In the same context, a large amount of urine output during CPB should not be interpreted as a favorable sign, because the tubular damage triggered by inflammatory and thrombotic response during CPB may increase the urine flow.16As impairment of tubular reabsorption and heterogeneity of nephron function could paradoxically increase amount of urine output,We hypothesized that a relationship between the amount of urine output during CPB and the development of postoperative AKI may not be linear, but it is rather U-shaped. Moreover, we conducted a hypothesis-generating analysis to investigate the possible independent association between them.After approval of Institutional Review Board, we retrospectively reviewed prospectively entered, protocol-based electronic medical records of all adult patients who underwent cardiovascular surgery with CPB at the Cardiovascular Hospital of Yonsei University Health System between January 2009 and December 2011 (n = 727). The need to obtain written consent from patients was waived. Patients who had had preoperative renal failure requiring dialysis (n = 21) and those without known preoperative serum creatinine levels (n = 10) were excluded. After careful examination of data, 696 patients were enrolled in the current study.3 (40 mEq), and acetated Ringer solution. Pump flows of 2.2 to 2.5 L/min/m2 and mean arterial pressure ≥60 mm Hg were targeted during CPB. Hemofiltration was performed during CPB in all patients having sufficient intravascular volume. After surgery, patients were transferred to the intensive care unit (ICU) and provided standardized postoperative care.Standardized general anesthesia was provided to all patients. CPB was facilitated by a roller pump using a circuit primed with 1600 mL of solution comprising 6% hydroxyethyl starch 130/0.4, 20% mannitol (5 mL/kg), NaHCO17Preoperative data used in our analysis were age, sex, height, weight, New York Heart Association class, history of diabetes, hypertension, and cerebral vascular accident, additive EuroSCORE, and estimated glomerular filtration rate (eGFR) derived from serum creatinine 1 to 2 days before the surgery, which was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) Eq.Operative features including type of surgery, CPB and aortic cross clamp time, use of total circulatory arrest, fluid input and transfusion of blood products, urine output during CPB and total operation time, volume of hemofiltration, inotropes and vasoconstrictors administered, and use of diuretics were included in the current analysis.18As for the postoperative data, fluid input, transfusion of blood products, urine output, inotropes and vasoconstrictors administered during 48 hours, requirement for renal replacement therapy during 48 hours and hospital stay, time to extubation, and 30-day major morbidity endpoints including myocardial infarction, stroke, pneumonia, other infections, reoperation due to any reasons, length of ICU and hospital stay, and mortality were collected. Postoperative AKI which was diagnosed by the Acute Kidney Injury Network (AKIN) criteria (absolute increase in the serum creatinine concentration ≥0.3 mg/dL) within 48 hours of operation.We investigated a possible optimal amount of urine output according to the association of urine output during CPB and the incidence of AKI. After separating cohort by the predicted optimal value, we performed logistic regression analyses in both sides to find potential predictors of AKI. And nomograms for estimating individual probability of AKI development were drawn and validated.t test. Data that did not show normal distribution were expressed as median and interquartile range (IQR) and were compared using Mann–Whitney U test. Categorical variables were expressed as percentages and compared using Fisher exact test or chi-square test.After Kolmogorov–Smirnov test, numerical data with normal distribution were presented as mean and standard deviation (SD). Patients who did and did not develop AKI were compared using the P = 0.05) factors were observed. The nomogram model was developed using the finally selected multivariate logistic model. The accuracy of the nomogram was internally and externally validated by discrimination and calibration, respectively. The discrimination of the model was measured using 1000 bootstrap samples to estimate bias-corrected concordance index, a measure of predictive accuracy of the model. The calibration of nomogram, which measures how far prediction is from observed outcome, was assessed with the calibration plot. Area under the curve (AUC) was constructed of the probability of the final model.After calculation of the probability of the incidence of AKI according to CPB urine output, we constructed a cubic spline curve. Associations between risk factors and AKI incidence were evaluated using univariate logistic regression analysis repeatedly on entire patient group and subgroups with CPB urine output below and over the detected change point. For the multivariate model, a stepwise selection method was used to select significant variables when univariately significant  and R software version 2.15.3 using for creating the nomogram and receiver-operating characteristic (ROC) curve, and 2-side Of 696 patients analyzed in the current study, AKI occurred in 257 patients 37%). Characteristics of the patients are listed in Table 7%. CharaIn a multivariate logistic regression analyses on all patients, male sex, lower preoperative eGFR, preoperative digoxin or diuretics use, double or triple valve surgery, intraoperative diuretics use, transfusion during the surgery, and number of transfused packed red blood cells (pRBCs) during surgery, number of transfused pRBCs during postoperative 48 hours were identified as statistically significant variables for predicting AKI .29 Oxidative stress and inflammatory flare-up during CPB and cardiac surgery could have damaged tubular system and consequently unmasked the impaired glomerular filtration postoperatively. Preoperative digoxin use was closely associated with postoperative AKI regardless of the group. Most of the patients who had been taking digoxin preoperatively had suffered from atrial fibrillation in our study population, and the result is consistent with the previous study reporting that preoperative atrial fibrillation as a strong risk factor for postoperative renal dysfunction.30We divided patients into 2 subgroups at 4 mL/kg/h of the CPB urine output. We could construct predictive and validated risk stratification models in both subgroups, and the amount of CPB urine output was identified as a predictor in both models. In the subgroup with CPB urine output below 4 mL/kg/h, the OR for AKI increased by 233% with every 1 mL/kg/h decrease in urine output. In patients with CPB urine output over 4 mL/kg/h, the OR for AKI increased by 11% with every 1 mL/kg/h increase in urine output. This finding underscores the need to carefully evaluate the decreased concentrating capacity resulting from the tubular damage, when faced with excessive diuresis.Although there have been numbers of risk stratification models previously, those that do not incorporate weights of the risk factors may confront patients with excessive or insufficient intervention. In the current study, we additionally constructed nomograms to predict the probability of AKI development according to the individual risk based on the logistic regression models in each subgroup. The calibration curves for the nomograms Figure  suggest Our study has some limitations. Firstly, as an inherent weakness of a retrospective study, we excluded patients for whom we could not obtain complete information from this analysis, which could possibly create selection bias. Secondly, our results do not guarantee the usefulness of CPB urine output to diagnose postoperative AKI in all cardiac surgical patients until it is validated with the database of diverse cardiac surgical cohorts.In summary, the current study demonstrates primary evidence regarding the predictive relevance of CPB urine output to the risk of postoperative AKI. Assessment of the amount of urine output during CPB may be the most simple and fastest diagnostic method, but a comprehensive approach is required. External validation and further prospective studies relating to the biomarker assay are warranted to determine its clinical utility.


Bacillus thuringiensis strains ATCC10792 and CT43 were grown in two Luria broth medium lots on four dates and transcriptomics data were generated using one lane of sequence output from an Illumina HiSeq2000 instrument for each of the 32 samples, which were then analyzed using DESeq2. Genome coverages across samples ranged from 87 to 465X with medium lots and culture dates identified as major variation sources. Significantly differentially expressed genes  were detected for cultures grown using different medium lots and between different dates. The highly differentially expressed iron acquisition and metabolism genes, were a likely consequence of differing amounts of iron in the two media lots. Indeed, in this study RNA-seq was a tool for predictive biology since we hypothesized and confirmed the two LB medium lots had different iron contents (~two-fold difference). This study shows that the noise in data can be controlled and minimized with appropriate experimental design and by having the appropriate number of replicates and reads for the system being studied. We outline parameters for an efficient and cost effective microbial transcriptomics study.RNA-seq is being used increasingly for gene expression studies and it is revolutionizing the fields of genomics and transcriptomics. However, the field of RNA-seq analysis is still evolving. Therefore, we specifically designed this study to contain large numbers of reads and four biological replicates per condition so we could alter these parameters and assess their impact on differential expression results.  B. thuringiensis is a Gram-positive, spore and Cry toxin producing bacterium  is 6,260,142 bp, was recently reannotated with 6330 genes and 13 copies for the 5S, 16S, and 23S rRNA genes predicted. The data from this well-replicated study with 32 samples, each from one Illumina HiSeq 2000 lane, generated a large number of reads per sample, and significantly differentially expressed genes were detected using DESeq2  and have Average Nucleotide Identity (ANI)-values of ≥99.63% in reciprocal genome analyses based on BLAST (ANib). Each strain was plated on Luria Bertani (LB) medium and cultured at 30°C. Single colonies were used to inoculate 5 mL LB starter cultures, which were grown at 30°C with shaking at 200 rpm  overnight. For RNA-seq experiments, 1 mL aliquots of overnight cultures were used to inoculate 500 mL baffled flasks containing 200 mL of LB medium. Cultures were grown for 3 h at 30°C with shaking at 200 rpm and harvested at approximately mid-log phase . To harvest cells for RNA extraction, 40 mL culture aliquots were collected by rapid centrifugation  at 7649 × g at 4°C for 5 min. Cell pellets were frozen in liquid nitrogen for 10 min and then stored at −80°C. All cultures were grown and harvested under similar conditions. A total of 16 samples were collected per strain, with four biological replicates for each strain, collected on four different dates and using media from two different LB broth lots (lot #1091744 and 7220443) using water from two different buildings to generate 32 samples for RNA-seq analysis  >8.5) was isolated from strain CT43 and strain ATCC 10792 using the TRIzol reagent  combined with a bead beating step, essentially as described previously  and were filtered and trimmed based on quality assessments. Sequence reads < 20 nucleotides were discarded. A modified-Mott trimming algorithm, which incorporates quality scores on a Phred scale, was used for quality trimming in CLCBio with the quality trimming parameter set to 0.02. Trimmed and filtered reads were then aligned to their respective reference genomes, using the default “prokaryote genomes” and unique reads settings. Raw data variance was observed by Principle Component Analysis (PCA) and by cluster analysis using JMPGenomics 6.0 .NZ_CM000753 and NC_017208 for strains ATCC10792 and CT43, respectively. RNA-seq data have been deposited in NCBI Gene Expression Omnibus (GEO) database under accession number GSE71189 and raw sequence data deposited at the NCBI Sequence Read Archive (SRA) under accession number SRP041628.The genomes used in this study have been described  as previously described , as previously described  were grouped in sets with replicates for another condition Figure . The numnm600, was 0.422 ± 0.04 (range 0.384–0.504) for strain ATCC10792 and 0.415 ± 0.05 for CT43 at the time of sample collection  and two-fold change) were observed to be differentially expressed between medium lots (#1091744 vs. #7220443) for strains ATCC10792 and CT43, respectively. A complete list of altered gene expression based on medium lot difference is provided in Supplementary File When analyzing the data based on different culture dates within a particular medium lot (the date effect) for ATCC10792, 403 genes were identified as differentially expressed for cultures in medium lot #1091744 when culture from the date 2/23/12 was compared with 3/6/12. Similarly, for cultures grown in medium lot #7220443 when comparing cultures from dates 3/13/12 and 3/15/12, 458 genes were identified as differentially expressed in ATCC10792 (Supplementary File R2)-values of 0.90 and 0.92 for genes chosen for medium lots and culture dates, respectively  was analyzed Table . A total8 M was aSelection of an appropriate sequencing depth or genome coverage is a concern in the field, which impacts sensitivity, detection of weakly expressed genes as well as considerations such as cost and replicate numbers  did not show up in the differentially expressed genes as their p-adjusted values were set to “NA” by automatic independent filtering based on low mean normalized counts in DESeq2. Similarly, for datasets of four and two replicates  may still be required for studies that are more discovery-based than hypothesis-driven  difference in measured iron contents was surprising and may be of broader interest to the research communities that use this media. Our results are in agreement with earlier studies showing the importance of culture medium choices in transcriptomics , Bacillus subtilis , Bacillus anthracis , and others . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This work is sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory and used laboratories supported by the BioEnergy Science Center (BESC). BESC is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This manuscript has been authored by UT-Battelle, LLC under Contract no. DE-AC05-00OR22725 with the US Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (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.


This study aimed to analyse the injuries sustained by rescue workers in earthquake relief efforts in high altitude areas for improving the ways of how to effectively prevent the injuries.The clinical data of 207 relief workers from four military hospitals in Tibet, who were injured in the Tibetan disaster areas of China during ‘4.25’ Nepal earthquake rescue period, was retrospectively analyzed. The demographic features, sites of injury and causes of injury were investigated.n = 61, 26.5 %), followed by injuries in leg-knee-calf , head-neck (4.87 %), thoracic and abdominal region (2.6 %) and lower back (3.9 %). The specific high-altitude environment increased the challenges associated with earthquake relief.The most frequently injured sites were the ankle-foot and hand-wrist (The specific plateau environment and climate increased the burden and challenge in earthquake relief. The injury distribution data shown in this study demonstrated that effective organization and personnel protection can reduce the injury occurrences.Relief workers were prone to suffering various injuries and diseases under specific high-altitude environment. The Tibetan government has implemented a rescue force of 843,096 shifts, 39,073 vehicles and all types of equipments”.On 25 April 2015 at 14:11, local time, a 7.8-magnitude (Mw) earthquake occurred in Nepal . The impact of the earthquake, which was followed by hundreds of aftershocks, has led to at least 8,000 deaths and more injuries. Additional casualties were reported in Chinese Tibet, India, Bangladesh and Bhutan. On 15The city of Shigatse, 43 km from the epicentre in Chinese Tibet, was damaged by the earthquake with serious loss of life and property. Shigatse is located in the southwestern Qinghai-Tibet plateau of China at an average elevation of more than 4000 m  with a large diurnal temperature variation, less oxygen levels and strong ultraviolet radiation in the daytime , 2. Its Most of the published studies were focused on the injuries of all the earthquake victims rather than relief personnel themselves –6. In spWritten informed consent was waived for secondary use of health data. Out-patient and in-patient records were collected from General Hospital of Tibetan Military Region, No. 8 Hospital of the People’s Liberation Army (PLA), No. 115 Hospital of PLA and No. 41 Hospital of PLA during the 20 days period after the earthquake. Patients’ records were collected when they met the criteria as follows: (1) the causes of injury or illness were related to participation in Shigatse disaster relief following the ‘4.25’ Nepal earthquake; (2) injuries caused by aftershocks during the process of disaster relief; (3) diagnosis based on clinical features, laboratory examination and/or digital radiography (DR) or multi-layer spiral computed tomography (CT) findings and (4) no history of surgery or treatment prior to visiting the clinic. To avoid bias, we eliminated patients whose conditions were related to motor vehicle accidents during disaster relief efforts. This study used descriptive statistical methods to analyse the population structure, types of injury, injury positions and relevant pathogenic factors. Initial screening generated 239 cases, from which 32 cases were excluded due to failure to meet aforementioned criteria. The remaining 207 cases were included for further analyses. This study was approved by the Medical Ethics Committee of Chengdu Military General Hospital.Original patients’ records included 23 variables, after reviewing clinical and laboratory data by two clinical experts and imaging data by two imaging experts; ten variables were included for further analyses. The ten included variables are sex, age, occupation, injury type, injury position, cause of injury, medical treatment, laboratory findings, physical examinations, and DR or CT. Based on the International Classification of Diseases (ICD)-10 (version 2014) , standarP < 0.05 was used to determine the significance.Double data entry was used to minimize data entry errors. SPSS16.0  statistical package was used to conduct statistical analysis. Continuous data was expressed as mean ± standard deviation and Pearson chi-square test was used for categorical data. One-way ANOVA was used to determine the significance of the difference among the group means. An alpha level of All of the 207 patients were disaster relief rescuers, most of them were young males. The mean age of these patients was 36.15 ± 13.72 years. The age distribution heavily skewed to 20-40 years old, with a total of 133 patients (approximately 64.3 %) aged 20–39 years Fig. , Table 1The evaluation of injury sites Fig.  showed 1A clustering analysis of the anatomic traumatic injury positions revealed that of the 138 traumatic injuries in this study, 61 44.2 %) had foot-ankle and hand–wrist injuries and 22 (15.9 %) had lower thigh-kneeleg injuries  in injury incidence in different working groups according to their PPE  as described in material and methods. The “military” relief working group was equipped with camouflage uniform and cap, combat boots and thin thread woven gloves. The “professional” relief working group was equipped with professional uniform and helmet, long boots, protective gloves with thick layers and mask. The “other” working group was not equipped with any particular equipment. A Pearson chi-square test revealed significant difference ; in addition, many relief workers had no living experience in high altitudes (elevation >3500 m). Accordingly, the environmental conditions led to different types of altitude sickness and various other types of illness , 23, parIn summary, the present study showed the distribution of injury sites for professional rescue workers during the earthquake rescue work, which might be useful for prevention of injuries in the future rescue work.The specific plateau environment and climate increased the burden and challenge in earthquake relief. Relief workers are more prone to limb and joint damage, single site injuries. The specific weather and dietary situation increased the incidence of respiratory and digestive diseases. The injury distribution data shown in this study demonstrated that effective organization and personnel protection can reduce the injury occurrences. The key is to strengthen the protection of vulnerable parts of the body. For personnel who are often involved in rescue work, such as soldiers and professional rescue team should receive the training and education of protection for reducing injuries. In the case of the poor environment in the plateau, more attention should be paid to the changes of the climate and food. This study could facilitate the organization and implementation of earthquake relief efforts in plateau areas, whilst providing counsel to related issues of injury and plateau disease prevention and clinical management in earthquake rescue.


Populus trichocarpa. Heritabilities were low to moderate (0.13–0.32) and population differentiation for many traits exceeded the 99th percentile of the genome-wide distribution of FST, suggesting local adaptation. When climate variables were taken as predictors and the 12 traits as response variables in a multivariate regression tree analysis, evapotranspiration (Eref) explained the most variation, with subsequent splits related to mean temperature of the warmest month, frost-free period (FFP), and mean annual precipitation (MAP). These grouping matched relatively well the splits using geographic variables as predictors: the northernmost groups (short FFP and low Eref) had the lowest growth, and lowest cold injury index; the southern British Columbia group (low Eref and intermediate temperatures) had average growth and cold injury index; the group from the coast of California and Oregon (high Eref and FFP) had the highest growth performance and the highest cold injury index; and the southernmost, high-altitude group (with high Eref and low FFP) performed poorly, had high cold injury index, and lower water use efficiency. Taken together, these results suggest variation in both temperature and water availability across the range shape multivariate adaptive traits in poplar.Local adaptation to climate in temperate forest trees involves the integration of multiple physiological, morphological, and phenological traits. Latitudinal clines are frequently observed for these traits, but environmental constraints also track longitude and altitude. We combined extensive phenotyping of 12 candidate adaptive traits, multivariate regression trees, quantitative genetics, and a genome-wide panel of SNP markers to better understand the interplay among geography, climate, and adaptation to abiotic factors in  Tree species and populations may respond to climate change through migration, phenotypic plasticity, evolutionary adaptation, or local extinction  and neutral molecular markers (FST), which can indicate the role of divergent selection in shaping adaptive genetic differences  is a deciduous broadleaf tree species native to western North America that occupies riparian corridors with diverse topography and climate from California to southern Alaska , though it extends farther east in the Sierra Nevada Mountains of California and in northern British Columbia. The rapid growth of P. trichocarpa, ease of propagation, intraspecific variation, and substantial hybrid vigor for many important commercial and adaptive traits have contributed to its use for pulp, paper, and bioenergy   with a null distribution of genome-wide neutral differentiation (FST) to illustrate the potential role of divergent selection in shaping patterns of trait differentiation.In 2010, branch cuttings of P. trichocarpa genotypes that span much of the species' range were collected and used to produce plantlets. The plantlets were grown for 6 months in a mist house and subsequently transplanted in May 2011 to an outdoor common garden located at the Reynolds Homestead Forest Resource Research Center located in Patrick County, Virginia (36°37′N and 80°09′W). Ramets of each genotype were planted in a randomized complete block design with 4 blocks. Annual climate variables (means of years 1981–2009) for the closest weather station  showed a mean annual precipitation (MAP) of 1275 mm, a mean annual temperature (MAT) of 14.1°C, a mean warmest month temperature (MWMT) of 24.4°C, and a mean coldest month temperature (MCMT) of 4.2°C (obtained from the weatherbase.com). For this study, we selected 124 genotypes from the 789 available genotypes that covered a wide latitudinal and altitudinal gradient (37–58°N latitude and sea level through ~2300 m) , was calculated relative to the international Pee Dee Belemnite standard  located at the Virginia Tech Forest Soils and Biogeochemistry Laboratory. The carbon isotope ratio, y Craig, .h), and insertion height of the lowest branch (Il)  was calculated according to Causton (h–Il)/H.In March 2013, while the trees were in a dormant state, growth and branch characteristics  was assessed by measuring electrolytic leakage of branch sections collected on November 5, 2012  was calculated according to Flint et al. (Bud stage was scored on a weekly basis during April 2012 (BF = bud flush) and September 2012 (BS = bud set), and the date of BF or BS was converted to the Julian day (the number of days from December 31). The bud was considered flushed when a leaf had emerged 1 cm from the bud, and set when dark red/brown scales covered the apex. Buds that set or flushed before the first week or after the last week of scoring were recorded as having occurred 1 week before or 1 week later, respectively. Fall cold injury , tR and R0 are the relative conductivities for –20°C and 4°C respectively, tL is the specific conductivity of leachate from the sample frozen at –20°C, kL is the specific conductivity of leachate from the sample frozen at –20°C and then heat-killed, L0 is the specific conductivity of leachate from control samples (4°C), and dL is the specific conductivity of leachate from the heat-killed control samples . MRT is an extension of Classification and Regression Trees (CART) that can be used to explore, describe, and predict relationships between multivariate response data and multiple predictor variables  were obtained using the ClimateWNA program . Descriptions of each variable can be found in Supplemental Table Data were visually inspected for normality and homoscedasticity, and NSyll was linearized by log transformation before analysis. To reduce the number of variables, and to deal with the high correlation between growth and branching variables, principal component analysis (PCA) was run on the growth and branch traits using the  De'ath, . A set oST) and broad-sense heritability (H2). For each trait, the clonal best linear unbiased predictors (BLUPs) and variance components were estimated using restricted maximum likelihood (REML) with block as a fixed factor and genotype and population as random factors according to the following model:Yijk is the value for the kth genotype from the jth population in the ith block, μ is the population mean, Bi is the fixed effect of the ith block, Pj is the random effect of the jth population, Gk(Pj) is the random effect of the kth genotype in the jth population, and εijk is the experimental error. QST was computed as:Samples were grouped into 20 populations according to their geographic origins  for computation of population differentiation in quantitative traits (Q2P is the among population component of variance and σ2G(P) is the genotype-within-population component of variance. These variance component estimates are based on clonal replication of trees, and have been shown by Lynch and Walsh  and within-population (H2G(P)):Where σnd Walsh  to give l et al.  showed tFinally, genetic correlations between traits were obtained by calculating correlations among the respective BLUPs, and environmental correlations were estimated according to Searle :H2G1 and H2G2 are the across-population heritabilities (H2G) of the relevant traits. Parametric bootstrapping was used to compute the 95% confidence intervals for the genetic parameters  (Table p < 0.05) relationship with latitude of origin (Table r = –0.79), while δ13C had correlations of 0.20 and –0.46 with latitude and altitude, respectively. The relationship with altitude was mostly driven by the southernmost group of samples, which had a low mean δ13C (–24.77) compared with the other populations. Among the traits, the highest correlations with climate were with temperature related variables , frost-free period (FF), EMT, EXT; Supplemental Table 13C had a weak negative but significant correlations with moisture variables (Hargreaves climatic moisture deficit (CMD): r = –0.32; Annual heat:moisture index (AHM): r = –0.22; Summer heat:moisture index (SHM): r = –0.27; Hargreaves reference evaporation (Eref): r = –0.28). Relatively strong positive genetic correlations (r > 0.50) were found between growth traits  and branch traits  (Table –20 was correlated with BS (r = 0.53) but there was no correlation between BF and either I–20 or BS. However, growth and branching traits had significant negative relationships with BF, and a positive relationship with BS and I–20. Finally, δ13C had had no significant correlations with the other traits.All traits except RNB had a significant , set buds late (high BS), and had a high I–20. The 32 genotypes from Group 2, originating between 49.34° and 40.30°N Lat. , had above average growth and branching, small BF, high BS, and the highest I–20. Trees originating between latitudes 51.05° and 49.28°N Lat. (southern BC) were further differentiated into Groups 3 and 4 according to their longitude of origin. Group 4 (interior group) originated from areas east of 123°W and consisted of 29 genotypes characterized by growth  slightly below the mean and low I–20, whereas the 24 genotypes from Group 3  originated from areas west of 123°W Long. and had growth and I–20 slightly above the sample mean. Group 5 consisted of 31 genotypes originating above 51.05°N. This group had the lowest growth, fewest branches, latest BF, earliest BS, and lowest I–20.Prior to MRT analysis, growth and branching traits were subjected to PCA to remove their colinearity. The first two principal components were retained because they explained most of the variation in growth and branching traits (88%). The loadings of the traits  exhibited early BF, had the latest BS, the highest I–20, the highest performance in growth and branch characteristics, and the highest δ13C. The eight genotypes of Group 3 (MAP < 1004 mm) had growth and branch traits close to the average, the earliest BF, late BS, and high I–20. Group 4 had 29 genotypes originating from an area characterized by Eref < 744 and MWMT > 16.85°C, respectively. Genotypes from this population were characterized by growth and branching traits, δ13C, BS, BF, and I–20 close to the average. Finally, Group 5 was comprised of 31 genotypes that originate from localities with Eref lower than 744 and MWMT higher than 16.85°C. Genotypes from this group had the lowest growth and branch traits, early BS, late BF, and the lowest I–20.MRTs using climate variables as predictors also split the 124 genotypes in five groups, which explained 39% of variation in the measured traits  and δ13C had moderate values (0.32 ≤ QST ≤ 0.44), while differentiation for branch traits  and BF was low (QST < 0.25) ) were low to moderate  ≥ 0.29), while VI had the lowest (H2 = 0.13).BS and IST often less than 0.10 , but most studies in woody species have reported low QST for BF, in agreement with our results  in their native environment. For many tree species, common garden experiments have revealed that high latitude populations achieve less height growth even when they display higher assimilation rates than low latitude populations Burtt, . InteresThe center of the range  was partitioned into three populations. Genotypes from southern BC, which is characterized by moderate temperatures, high precipitation, and low evaporative demand, were differentiated into two groups according to continentality: the coastal group, which originates from a mild climate (MWMT > 16.85) with a long growing season, displayed better growth , and was more susceptible to fall cold damage than the interior group. The interior population in southern BC was merged with the northern groups in the MRT using climate predictors, as the temperature variables of these two population locations were similar. Genotypes spanning the coast of Oregon and Washington were the tallest, and had the highest number of branches in the common garden. This area is below 400 m altitude and has a mild climate  that is similar to the common garden in terms of temperature variables. The climate variable-based MRT showed that these genotypes could be further differentiated according to MAP in spite of closely overlapping geography. This local spatial heterogeneity in climate could help these populations respond to climate change, facilitating migration by reducing dispersal distance required to find an appropriate climatic niche , than plants adapted to wet environments  compared with the other populations , for which δ13C values were higher. This may seem counterintuitive as one may expect genotypes adapted to dry locations would exhibit higher WUE . However, when grown in the common garden, genotypes of Group 1 likely encountered a moister summer than in their native environment, and consequently may have adjusted one of the physiological parameters noted above, which may have resulted in lower δ13C. On the other hand, accessions from populations originating from areas with greater summer precipitation may have experienced mild water stress and responded by reducing their water loss, which led to higher δ13C. Aitken et al. (Pseudotsuga menziesii) seedlings grown at a location with intermediate precipitation compared with source populations . This hypothesis is also supported by δ13C lower than the average for Group 3 in the climate-based MRT, for which summer moisture values  were similar to Group 1.Plants adapted to arid environments generally have higher δn et al.  found a 2 assimilation (Halle et al., 13C and growth/branch characteristics combined with moderate H2 for these traits suggests that, for P. trichocarpa, there is a possibility of improving WUE without compromising productivity. The continued development of poplars as bioenergy feedstocks depends partially on their suitability for cultivation on marginal lands that may be water limited (Berndes, P. nigra (Chamaillard et al., P. trichocarpa (McKown et al., 13C and growth means that stomatal conductance drives WUE variation, while a positive correlation between the two traits generally supports that differences in photosynthetic capacity is the main driver for WUE. Gornall and Guy (P. trichocarpa genotypes in WUE were related to variability in stomatal conductance rather than assimilation rate.Branch characteristics are closely related to productivity as they determine the quantity of light interception and COBerndes, . Such a Berndes, , P. nigr and Guy  showed tP. trichocarpa has considerable standing adaptive variation within and among populations, which represent an adaptive potential. We found strong population differentiation in growth, phenology, cold hardiness, and carbon isotope composition, but weak genome-wide differentiation at SNP loci, which suggests that local adaptation better explains patterns of variation in these traits than drift alone. Multivariate regression trees using geography or climate variables as predictors showed that population differentiation in adaptive traits could be primarily explained by either latitude or evapotranspiration, respectively. The MRT analysis also suggested that the geographically intermingled accessions from coastal California and Oregon could be separated for the traits we measured on the basis of heterogeneity in mean annual precipitation. Genotypes from these areas were the best adapted to the climatic conditions of our Virginia common garden, suggesting that they can be used for breeding purposes in areas with similar environmental conditions. Indeed, with the growing interest in poplar species for bioenergy, knowledge of suitable genotypes for specific environments constitutes valuable information for successful plantations. One limitation of this study is the location of the common garden, which was located well outside the natural longitudinal range of P. trichocarpa, and at a latitude similar to the southern species range limit. In addition to the impacts of daylength regimes the common garden location likely had on growth and phenology, this environment may also impact the rankings of genotypes due to differences in soil conditions and temperature. In addition, fungal pathogens present in the southeastern US may impact performance of this species, particularly Septoria musiva, though we did not observe cankers during the course of this study. To address these issues, we have established an additional common garden site near the center of the species range, which will help to tease apart the relationship between genetics and environment.Our data suggest that 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.


Antrodia camphorata is a parasitic fungus from Taiwan, it has been documented to possess a variety of pharmacological and biological activities. The present study was undertaken to evaluate the potential of Antrodia camphorata ethanol extract to accelerate the rate of wound healing closure and histology of wound area in experimental rats. The safety of Antrodia camphorata was determined in vivo by the acute toxicity test and in vitro by fibroblast cell proliferation assay. The scratch assay was used to evaluate the in vitro wound healing in fibroblast cells and the excision model of wound healing was tested in vivo using four groups of adult Sprague Dawley rats. Our results showed that wound treated with Antrodia camphorata extract and intrasite gel significantly accelerates the rate of wound healing closure than those treated with the vehicle. Wounds dressed with Antrodia camphorata extract showed remarkably less scar width at wound closure and granulation tissue contained less inflammatory cell and more fibroblast compared to wounds treated with the vehicle. Masson's trichrom stain showed granulation tissue containing more collagen and less inflammatory cell in Antrodia camphorata treated wounds. In conclusion, Antrodia camphorata extract significantly enhanced the rate of the wound enclosure in rats and promotes the in vitro healing through fibroblast cell proliferation. A wound can be described as the disruption of the typical anatomical shape and physiological function of a tissue. Wounds may arise due to physical, chemical, or microbial agents. Wound healing process is a dynamic procedure which consists of three main stages including inflammation, proliferation, and tissue remodeling. In the inflammation stage platelets aggregates, blood clots, and subsequently phagocytosis and vasodilatation occur. The proliferation stage (active growth stage) includes tissue granulation and epithelialization, while the remodeling stage (reconstruction stage) involves maturation and remodeling of the scar tissue .Natural products have been proven to enclose wound healing property and their primary or secondary metabolites have been shown to contribute potential repair mechanisms including activation of immune epithelial cells, cytokines, extracellular matrix (ECM), reactive oxygen species (ROS), growth factors, and different inflammatory mediators . In the Antrodia camphorata, a Basidiomycete fungus from Taiwan [ Cinnamomum kanehirai tree and it has failed to be cultivated artificially [ Antrodia camphorata possesses a variety of pharmacological functions like anti-inflammatory, antioxidant [ Antrodia camphorata have been analyzed including steroid, sesquiterpene lactone, triterpenoids, and polysaccharide and their biological activities were documented [m Taiwan , from fam Taiwan . It is rficially . Prior rioxidant , 11, antioxidant , antimetioxidant , anti-heioxidant , anti-heioxidant , and antioxidant  effects.cumented , 18. Antrodia camphorata as a wound healing agent. Therefore, this study aimed to evaluate the ability of topical application Antrodia camphorata extract in accelerating wound healing in vivo in Sprague Dawley rats and to study whether Antrodia camphorata facilitates the in vitro wound healing process by fibroblast cell line proliferation.No literature was found concerning the activity of‚5-diphenyltetrazolium bromide) was purchased from Merck, Germany. Green bean extracted powder of Antrodia camphorata was purchased from Bio-Herb Biotech. Co., Ltd., Taiwan.Intrasite gel , an amorphous gel, containing 2.3% of a modified carboxymethyl cellulose (CMC) polymer with propylene glycol (20%), was used as the reference control. Acacia Arabic gum , a complex mixture of polysaccharides and glycoproteins, was used as the vehicle control after dissolving in normal saline as described by Zahra et al. . MTT reaAntrodia camphorata extract was dissolved by using the vehicle, gum acacia, in normal saline as described by Zahra et al. [ Antrodia camphorata extract each. So one mL of each solution contains 100 and 200 mg of Antrodia camphorata extract, respectively.a et al. . Two gra Antrodia camphorata extract, and Group 3 rats were dressed twice daily with 0.2 mL of 200 mg/mL Antrodia camphorata extract. Group 4 rats were dressed twice daily with 0.2 mL of 200 mg/kg reference drug (Intrasite gel) Antrodia camphorata extract. The contraction of the wound area was measured. Wound areas were traced manually and calculated in square millimeters. The wound closure area of each animal was assessed by tracing the wound at days 0, 7, and 14 after wounding surgery and the wound closure rate was expressed as the percentage of wound area compared with that on postoperative day by using transparency paper and a permanent marker under general anesthesia (a mixture of Ketamine and Xylazil) as described by Abdulla et al. [Sprague Dawley adult male rats were randomly divided into 4 groups of 6 rats each. Each rat that weighed between 180 and 200 gm was housed separately (one rat per cage). The animals were maintained on standard pellet diet and tap water. The animals were anesthetized by light dosage of ketamin and xylazine anesthesia. The skin shaved by electrical shaver, disinfected with 70% alcohol. An area of uniform wound 2.00 cm in diameter was excised from the nape of the dorsal neck of all rats with the aid of round seal as described by [2 graph sheet and traced out. The squares were counted and the area recorded.The wound areas recorded were measured using a graph paper. The percent wounds healing on these days are determined. Number of days required for falling of scar without any residual raw wound gave the period of epithelization. The wound area was measured immediately by placing a transparent tracing paper over the wound and tracing it out. The tracing paper was placed on 1 mmA0 is wound area at day zero and Ad is wound area on corresponding day [Then the percentage of wound closure was calculated following the formula:ding day .μm section followed by staining with hematoxylin and eosin and Masson's trichrome stains and examined using an Olympus light microscope. The evaluated parameters were epithelialization, inflammatory cell infiltration, fibroblast proliferation, neovascularization, and collagen deposition, which were assessed generally whether absent, present, or abundant.For assessing the histological observations of the healed wounds, the skin specimens from wounds healed areas were fixed in 10% buffered formalin and processed by paraffin tissue processing machine. The healed skin was assessed by taking a 5 The wound tissue homogenate from each rat was prepared at 4°C by using a Teflon homogenizer . After centrifugation at 4,500 rpm for 15 min at 4°C, the supernatant was used for nitric oxide (NO) and hydroxyproline (HXP) determinations. The NO levels of the wound tissues were measured using the NO assay kit . The HXP assay kit from  was used to test the wound tissues HXP levels. Antrodia camphorata on human fibroblast cell lines, the MTT assay was performed to study its cytotoxicity. Cells were grown in DMEM medium ), supplemented with 10% fetal bovine serum  at 37°C under 5% CO2 in a humidified IR Jacketed incubator . Cells were counted (0.5 × 105 cells/mL) and transmitted into a 96-well plate and incubated for 48 hours before adding the Antrodia camphorata extracts. Serial dilutions of extracts were prepared by using distilled water and 0.25% DMSO as solvents to give final concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL and then 10 μL was injected to each well and incubated for 48 hours. 10 μL of MTT  solution was added and incubated at 37°C for four hours. Thus the solution was took out by suction and 100 μL of DMSO was added to each well and then the absorbance was recorded by the ELISA plate reader  at 595 nm. The percentage of cell growth inhibition was calculated as in vitro wound healing assay was performed using the CytoSelect wound healing assay kit  [6 cells/mL) in media containing 10% fetal bovine serum (FBS) and incubated for 48 hrs until a monolayer was formed around the inserts. The inserts were removed and cells were treated with the experimental samples (200 μg/mL) and incubated for 48 hrs. The migration and proliferation of the cells into the wound field were monitored and then the wound percent closure was measured asBefore evaluating the wound healing activity ofc., USA)  in which Antrodia camphorata was sent to the Consolidated Laboratory (M) Sdn. Bhd., for the analysis of the minerals, fats, and elemental compositions.500 gm of the fine powder of Antrodia camphorata extract when administered by oral gavage to rats at single doses of 2 gm/kg following OECD-423 guidelines [2O), low dose 2 gm/kg, and high dose 5 gm/kg of Antrodia camphorata extract, respectively. Each rat was made to fast (no food but water) overnight prior to dosing. Food was withheld for another 3 to 4 hours after the dosing. The rats were closely observed for 30 minutes and at 2, 4, 24, and 48 hours after the dosing to detect if there were any acute signs of clinical toxicity, morbidity, and mortality twice daily. Comparisons were verified between the individual animals and their respective vehicle treated animals. Detailed behavioral observations include respiration (dyspenea), salivation, skin piloerection, exophthalmos, convulsion, and locomotion changes. After keeping them alive for 14 days, at day 15, the rats were sacrificed to measure serum biochemical and (liver and kidney) histological parameters following the standard methods [The acute toxicity test was used to determine the safety ofidelines . In brieP < 0.05 was considered significant.All values are reported as mean ± SEM. and the statistical significance of differences among groups was assessed using one-way ANOVA. A value of  Antrodia camphorata or Intrasite gel showed considerable signs of dermal healing and healed faster than the vehicle group (gum acacia).  Antrodia camphorata extract on the percentage of wound healed at days after surgery. Throughout the experiment, the percentage of healing in the vehicle control group wounds was significantly lower than those of Antrodia camphorata extract-treated groups and Intrasite gel wounds  and nitric oxide (NO) proves that the effect of Antrodia camphorata extract on an excision wound model was significantly greater than that of the vehicle itself. These results were with the consistence of the previous studies published elsewhere [Moreover, this study showed thatlsewhere , 5. A prlsewhere . It has lsewhere . Numeroulsewhere , 27, 28.lsewhere , 6. Antrodia camphorata extract has remarkable activity on fibroblast cell proliferation. The cell viability increased after treatment; it is clear that the extract is not potentially toxic to the skin cells and it is proliferating their growth as shown in  Antrodia camphorata on in vitro wound closure was assessed by scratch assay in which the level of cellular fill within the wound area in response to Antrodia camphorata was recoded  signaling pathway and direct collagen synthesis and angiogenesis in wound healing [The chemical analysis of healing . Also po healing . Moreove healing . Antrodia camphorata. The animals were carefully observed for 14 days. During the observation period, animals remain living with active healthy condition and there were no obvious toxicity signs. There were no macroscopic abnormalities at any times of observation and no cases of death were recorded. The animals were sacrificed, and the blood biochemical tests and histopathology tests were made. The results obtained from blood biochemical tests and histopathology tests did not demonstrate any difference between the treated groups and the control group as shown in  Antrodia camphorata was safe in oral acute toxicity test even at these high doses.The experimental rats were pretreated with a dose of 2 gm/kg and 5 gm/kg of Antrodia camphorata is safe when administered orally in doses 2 gm/kg and 5 gm/kg because none of the animals produced toxic signs and symptoms and none died during the study period. During topical application of Antrodia camphorata, the rats did not show any signs of irritation, pain, restlessness, and biting of wound area. Moreover there were no significant differences in biochemical parameters of liver and kidney and the values are within normal range. Histology revealed no nephrotoxic or hepatotoxic effect. Similarly, large numbers of medicinal mushrooms and plant extracts with potential wound healing activity which have no hepatotoxic or nephrotoxic effects on laboratory animals have been reported by several authors [The results of the present study revealed that authors , 5, 6.Antrodia camphorata showed high potential in wound healing activity, especially at higher concentrations. The results showed that the wound area of wounds dressed with Antrodia camphorata extract had relatively less scar width at wound closure. They also showed a proliferating effect on fibroblast cells and promoted the in vitro wound healing. Such natural sources may lead to better forms of therapy for patients with acute, chronic, and surgical skin wounds.


Bacteroides fragilis isolate BOB25. It is an enterotoxigenic isolate that was obtained from a stool sample of a patient with dysbiosis.Here we present the complete genome sequence of  Bacteroides fragilis is a Gram-negative, obligate anaerobic, rod-shaped bacterium that comprises about 1 to 2% of the normal human colonic microflora and is critical to systematic and mucosal immunity and host nutrition (B. fragilis (ETBF) are implicated in inflammatory diarrhea and have been associated with colorectal cancer in humans (B. fragilis toxin (BFT), a 20-kDa, secreted, zinc-dependent metalloprotease, has been identified as a specific virulence factor for ETBF, although the mechanisms of the intracellular processing, secretion, and those accounting for the variation of expression have not yet been elucidated  with 13-fold coverage and median length of 812 bp. Another library was sequenced with the PGM sequencer system  with 16-fold coverage and median length of 206 bp. The raw reads from both runs were then combined and filtered using the spectral alignment error correction tool SAET 3  and identified a total of 4,112 coding sequences (CDSs), 18 rRNA genes, and 70 tRNA genes. The most important difference between the ETBF strain and published genome sequences of nontoxigenic (NTBF) strains  located within the conjugative transposon CTn86 of 63,282 bp. Interestingly, two copies of this transposon are present in the genome of BOB25, separated by 934,125 bp and oriented in opposite directions. Similar to those previously characterized , and it is flanked by putative mobilization genes . In addition, there are several regions that show no similarity to NTBF genomic sequences, most of which are mobile elements like prophages and conjugative transposons. However, some of these regions appear to be involved in capsular polysaccharide synthesis, making them attractive targets for further research. Finally, unlike most of the nontoxigenic strains, the complete genome sequence of the ETBF isolate BOB25 does not contain any plasmids.The complete genome of nd 638R 79), whichcterized , the patB. fragilis isolate BOB25 was deposited at GenBank under the accession number CP011073. The version described in this paper is the first version, CP011073.1.The genome sequence of


Following the Chernobyl nuclear disaster of 1986, vast areas of Ukraine became contaminated with radionuclides. We examined health effects of school-based food intervention for children in a rural region Narodichi, Ukraine, exposed to low-level radiation through diet of locally produced foods. Until 1995, children received three daily meals with low content of artificial radionuclides which were subsequently reduced to two.n = 947 children; 3,573 repeated measurements). Generalized Estimating Equation models evaluated effect of the food supplementation reduction on hematologic measures and prevalence of anemia, acute respiratory illnesses and diseases of immune system.Annual health screening data (1993–1998) were examined using a quasi-experimental regression discontinuity analysis  of previously declining anemia increased from 0.57 to 1.31 per year (pinteraction < .0001). The relation between food supplementation and hemoglobin levels was modified by residential 137Cs soil levels. After food supply reduction, PR of common cold and bronchitis increased from 1.27 to 2.32 per year (pinteraction = 0.01) and from 1.09 to 1.24 per year (pinteraction = 0.43), respectively.Prior improvement of several hematologic parameters diminished after food supplementation was reduced. From 1995 to 1996, levels of hemoglobin and erythrocytes decreased from 12.63 (95 % CI: 12.56-12.71) to 12.46 g/dL (% CI: 12.39-12.52) and from 4.10 (95 % CI: 4.07-4.12) to 4.02 (95 % CI: 4.00-4.04) × 10Food supplementation provided by the Ukrainian government likely prevented development of anemia in many of the children residing in the contaminated district. Food supplementation after the community exposure to radioactivity through a diet of locally grown foods should be considered as an effective approach to reduce adverse health effects of radiation.The online version of this article (doi:10.1186/s12940-015-0084-x) contains supplementary material, which is available to authorized users. After the Chernobyl disaster, the Ukrainian government conducted environmental monitoring of 137Cs soil contamination, and the results were published in several reports by the Ministry of Health and the Ministry of Emergency Situations of Ukraine [137Cs in locally grown foods and forest products such as mushrooms, berries and game which may cause adverse health effects in their consumers [137Cs into vegetation is strongly influenced by the type of soil and is the highest in Ukrainian and Belarusian Polyssia, a large agricultural area in northern Ukraine and southern Belarus located in close proximity to Chernobyl, where the predominant soil type is marsh and peat [137Cs intake is considered to come from milk and dairy products, 5-10 % from meat, 10-15 % from mushrooms and berries, and 10-15 % from potatoes and vegetables [137Cs, their consumption is limited to the summer and fall, while milk is available throughout the entire year in all areas and widely consumed by both children and adults [137Cs in milk vary between different areas [137Cs in organs of children is higher than in adults [Immediately after the Chernobyl nuclear disaster that occurred on April 26, 1986, the environment within the region of the nuclear power plant was contaminated with different gamma-emitting radionuclides including  Ukraine . The greonsumers . The traand peat . Furthergetables . Levels getables , 13. Altd adults , 14. Levnt areas . Throughnt areas . It is cnt areas . In chilnt areas . In addin adults . TherefoIn this study we assessed the beneficial health effects of a school-based public health intervention on a pediatric population chronically exposed to radiation through foods grown within a region of Ukraine contaminated by radiation after the Chernobyl disaster. Herein we compared different measures of health status, including blood indices and the prevalence of different health conditions in these children both before and after the public health intervention was reduced using a quasi-experimental regression discontinuity design.137Cs soil contamination levels in Ukraine [The Narodichi region is a peasant farming region located about 80 km from the Chernobyl nuclear site to the west of the evacuation zone and is one of the areas with the highest  Ukraine . As part Ukraine , 23 who 137Cs exposed population an adverse effect of the level of soil 137Cs contamination was found on hematologic parameters [137Cs soil contamination in the villages as a proxy for individual exposures. Children resided in 34 different settlements of the Narodichi region. All villages and towns had 137Cs soil measurements taken in 1992 with concentrations ranging from 51 to 356 kBq/m2. For all consecutive years (1993–1998), the average levels of 137Cs soil contamination were calculated based on the 1992 measurement and a decay function and are publically available in reports by the Ministry of Health and the Ministry of the Emergency Situations of Ukraine [137Cs  used in this study represents the interquartile range (199 kBq/m2) normalized average 137Cs soil concentration at each child’s village of residence. The interquartile range was determined for 137Cs soil concentrations in all villages and for all years of observations combined. The individual whole body content of 137Cs were measured in Bq using a gamma-spectrometer ) as was described elsewhere [137Cs used calibration coefficients to account for child's weight. To further reduce a chance of confounding by weight, we additionally adjusted the individual whole body content of 137Cs for child's weight . For the hematologic parameters, blood sampling data collected during the mandatory clinical examinations were used. Blood was collected in EDTA tubes and blood count analysis conducted using Sysmex model F-800. Blood smears were stained according to the standardized GIEMSA procedure. A novel sensitive biomarkers of chronic inflammation, the platelet/lymphocyte and neutrophil/lymphocyte ratios [In previous research within this rameters . This ine ratios , were ca137Cs, every child was provided three meals with low content of artificial radionuclides each day at school from 1986 until the program funding was decreased in 1995 and then only 2 meals each day were provided. The meals were prepared to meet nutritional standards determined by the Ukrainian Government for the appropriate age groups [To reduce the dietary exposure to e groups . The eff137Cs in the soil decreased overtime, these two variables were not balanced near the threshold (mid-year 1995); therefore, we included them as covariates in the statistical models. Since the objective of the study was to compare changes in blood indices and disease prevalence before and after the food supplementation reduction, the study period was divided into two time periods: 1993–1995 and 1996–1998. The temporal changes in different blood cell counts and the hemoglobin level during two different periods of food intervention were estimated using linear models with repeated measures (proc genmod in SAS) (time trends 1993–1995 vs. 1996–1998). Generalized Estimating Equations (GEE) with an exchangeable correlation structure were used to estimate the parameters [137Cs levels, and an interaction term between food intervention status and time (food × time). Since yearly physical examinations were always conducted in February-April, the season of the year was not a confounder and, therefore, was not included in the statistical models. For the statistical analyses, basophils, eosinophils, leukocytes, lymphocytes, monocytes, neutrophils, platelets, platelet/lymphocyte ratio and neutrophil/lymphocyte ratio were log-transformed, and the results were back-transformed to yield geometric means.In principal, the Chernobyl accident resulted in a natural experiment, since radiation was distributed independently from potential confounding variables. There were six annual measurements of the hematologic parameters between 1993 and 1998. To assess the effectiveness of the school-based food intervention which was reduced after the 1995 school year, we used a regression discontinuity analysis . This tyrameters . This strameters . The ind137Cs soil contamination in the relationships between the food intervention and blood hemoglobin levels using stratification by quintiles of soil 137Cs . The statistical models included time , food intervention , age, gender, and food × time interaction.We examined the effect modifying role of residential 137Cs levels, and food × time interaction. All data analyses were performed using SAS software  with significance level α = 0.05.To calculate prevalence ratios of anemia, diseases of respiratory and immune systems we used log-binomial models with repeated measures to infer serial correlation . The cov137Cs adjusted for body weight (Bq/kg) increased between 1993 and 1998 (Table 137Cs soil levels (Bq/m2) . Except for erythrocyte count and hemoglobin concentration, none of the hematologic markers were correlated with one another. The interclass-correlation coefficients over the total time period of 6 years averaged around 0.50, an indication of reasonable stability of the measurements.Although the pediatric health screening was mandatory, it was not enforced. Approximately 75 % of the children in the region participated in the screenings annually. In total 1,459 children were screened and included in the NCC. Of these, 1,247 provided blood samples (86 %). Of all, 947 children were 7–16 years old and included in this analysis. A total of 3,573 repeated blood measurements were available over the 6 years of observation. The characteristics of the study population are described in Table 12/L (95 % CI: 4.07-4.12) to 4.02 × 1012/L (95 % CI: 4.00-4.04) and then they started to increase again .In adjusted analyses, the mean levels of hemoglobin, erythrocytes and several other blood indices changed after the food intervention was reduced Fig. , Table 3ain Fig. , Table 3137Cs soil contamination (pinteraction < 0.0001)  Fig. . At the interaction < .0001, Table boys vs. girls = 0.80, 95 % CI: 0.64-1.01, p = 0.06). In stratified analyses (data not shown), prevalence ratio of anemia in girls was 40 % lower in the years between 1993 and 1995  and between 1995 and 1998 it was increasing by 38 % per year . In boys, the prevalence ratio of anemia in 1993–1995 was 49 % lower , and in the years 1995–1998 it increased to 22 % . Also, the prevalence ratio of common cold was higher after the reduction of food supplementation as compared to the prior period  per year.Temporary changes in prevalence of various health conditions were plotted with trend lines to demonstrate discontinuity in the regressions Figs. . Previou137Cs soil contamination. Mean increase in hemoglobin levels before and after the reduction of food supplementation was lower at the higher 137Cs soil levels.After the reduction of the school-based food supplementation the rates of improvement of several hematological markers, including hemoglobin, erythrocytes, leukocytes, monocytes, neutrophils and neutrophil/lymphocyte ratio observed in previous years decreased. Between 1995 and 1996, concentrations of hemoglobin and erythrocytes significantly decreased and blood counts of neutrophils, platelets, neutrophil/lymphocyte ratio, and serum levels of immunoglobulins A, G and M, on the contrary, increased. In agreement, after the food supplementation was reduced, in 1996–1998, prevalence of anemia, common cold and bronchitis increased. We also found that the relationship between reduction of the number of meals with low content of artificial radionuclides and levels of hemoglobin was modified by levels of residential 137Cs soil levels) makes this study similar in rigor to a randomized controlled trial; thus, near the threshold all temporally invariant confounding is adjusted for by design.One of potential limitations of this study is its quasi-experimental nature. The number of meals with low content of artificial radionuclides (3 vs. 2) per day was not randomly assigned with the purpose to investigate its effects, and analyses were performed retrospectively. However, quasi-experimental evaluations of public health interventions are essential when randomized controlled trials are not possible or ethical. The main concern with such designs is potential confounding from contemporaneous influential events , 33. Wha137Cs contamination levels as a proxy of individual  exposure which could have introduced its misclassification. This proxy measure doesn't take into account individual food intake and adherence to protective recommendations. The data on 137Cs levels in food consumed at home were not collected in this study. The magnitude of the associated error, however, would not be strong since the individual 137Cs activity in a body was positively correlated with residential soil contamination levels in previous studies [Another limitation of this analysis is that in the statistical models we adjusted for residential  studies , 13, 34 137Cs. When reducing this food supplementation, the internal radiation exposure potentially increased due to the consumption of the contaminated food at home. A prior study has shown that the individual radiation doses in Narodichi region increased up to 2–3 times from May through October due to consumption of locally grown produce and forest goods [137Cs exposure after 1995. Our finding that the mean hemoglobin levels during both periods of food supplementation (1993–1995 and 1996–1998) were lower at the higher levels of residential 137Cs soil contamination also suggests that ingestion of 137Cs from contaminated food was likely the main driving force of changes in hematologic parameters. It is likely that changes in blood indices were underestimated since all health examinations and blood tests were conducted in the spring, when the main sources of internal exposure are milk, meat and potatoes; thus, they did not capture the changes due to higher exposures from locally grown foods and forest goods consumed during summer and fall [137Cs and measured hematologic parameters was investigated [137Cs exposure. The blood cell counts were reduced with increasing soil contamination. In the observation period all parameters did improve, partly attributed to the natural weathering and decay of 137Cs in the environment and the regenerative capacity of the hematopoietic system. However, the cessation of food supplementation was not considered at that time within the study design. In this study, after the food supplementation was reduced in midyear 1995, the hemoglobin concentrations and erythrocyte counts in 1996 immediately dropped and the temporal improvement in hemoglobin levels in the following years decreased, which was corroborated by an increase in diagnoses of anemia. Three factors could potentially cause anemia in this population: increased consumption of radionuclides from locally produced food, poor nutrition , and increasing proportion of girls who reached menarche. According to previous studies, even before the Chernobyl nuclear incident the food consumed at home lacked proteins, sufficient vitamins and minerals, and provided excessive amounts of refined carbohydrates and fat [The improvement of blood indices seen from 1993 to 1995 was most likely due to the natural weathering and decay of cesium in the environment and the countermeasures introduced by the Ukrainian government aiming to reduce the internal exposures to radionuclides. These countermeasures included prohibition of farming in highly contaminated areas, warning against consumption of forest goods and game, decontamination of locally grown produce, adding cesium binders to cattle foods and importation of food with low content of artificial radionuclides from other areas , 15, 35.stigated . The res and fat . In this137Cs ingested with food affected the immune system. Altered immune reactivity may lead to increased susceptibility to infections and respiratory pathology in this population, as was previously described [Various observed changes in levels of white blood cells and concentrations of serum IgA, IgG and IgM after the food supplementation was reduced indicate that increased exposure to escribed . We foun131I, 134Cs and 137Cs. The distribution and consumption of foodstuffs with levels of contamination exceeding provisional regulation values were restricted, and infants in Tokyo were provided clean bottled water without cost [No studies are available to date that document the effect of a school-based food intervention aimed at the reduction of internal radiation exposure due to contaminated food. Another more recent large-scale public health intervention occurred that targeted all age groups. After the large nuclear incident that occurred on March 11, 2011 in Fukushima, Japan, the Japanese government introduced monitoring of contamination of tap water and foodstuffs for various radionuclides, including out cost . Howeverout cost . The effThe food with low content of artificial radionuclides provided for children by the Ukrainian government following the Chernobyl disaster likely prevented the development of anemia in many of the children residing in the contaminated districts. Food supplementation reduction was associated with increased prevalence of anemia and acute respiratory illnesses. Further research is needed over a longer period of time to forecast the concentrations of hemoglobin and other hematological measurements to better estimate the impact of this public health intervention and see if increasing food supplementation would be beneficial at this point in time after the accident. Also, measurement of whole-body radiation burden would improve future study designs.