The objectives of this study were to examine nutrient intakes of tuberculosis (TB) patients and to identify their associated factors.In this cross-sectional study, 300 adult TB patients were surveyed in two impoverished counties in China. Nutrient intakes were evaluated through two consecutive 24-h dietary recalls and compared with the Chinese Dietary Reference Intakes (DRIs) 2013. The potential socio-demographic and behavioral factors were analyzed using multivariate logistic model to identify strong influential factors.p < 0.05) and out-home-eating was a protective factor for low protein intake (p < 0.01).We found that mean daily energy intake was 1655.0 kcal  and 1360.3 kcal  for male and female patients, respectively. The mean daily energy intake was significantly lower than that has been recommended by DRI , with 87.4% of the male patients and 59.9% of female patients failed to consume adequate energy. The protein intakes were 44.6 g (SD: 18.2 g) and 35.9 g (SD: 12.3 g) for male and female patients, respectively, which were lower than the recommended values by DRI . Most male (90.8%) and female (58.4%) TB patients had insufficient daily protein intake. Further analyses suggested that mean daily intakes of many micronutrients, were insufficient, while for most of patients, intakes of vitamin E and sodium were sufficient. We identified that unemployment was a risk factor for low energy intake (In impoverished areas in China, intakes of macronutrients and most micronutrients in TB patients were inadequate compared with DRIs, especially for unemployed patients and patients eating at home. These findings suggested that public health actions are needed to promote education on TB patients about significance of nutritional support, and, further interventions in TB patients’ nutritional intakes are also required. Mycobacterium tuberculosis and is the leading cause of death from single infectious agents. According to World Health Organization (WHO), there were estimated 10.0 million people developed TB disease and 1.6 million people died from TB in 2017. China is in 2nd place among 30 TB high burden countries, with a reported incidence of 63 per 100,000 persons per year in 2017 [Tuberculosis (TB) is an airborne disease caused by The interactions between tuberculosis and malnutrition are well appreciated. TB can cause weight loss or cachexia and malnutrition can predispose to TB. On one hand, TB patient requires more energy to maintain body function that caused by increasing basal metabolic rate (BMR), leading to weight loss . On the Assessment of nutrient intakes is critical in nutritional management of patients with TB. Enough nutrient intakes is essential for combating TB. Insufficient protein and caloric intakes can hinder the functions of some generalized host defense mechanisms that are essential for combating TB . AdditioThis cross-sectional study was undertaken as part of the project “Investigation of nutrition and diet of patients with pulmonary tuberculosis in poor areas in China” supported by the World Health Organization Regional Office in the Western Pacific. It was conducted from November 2015 to April 2017 in two counties, Lingyun county in Guangxi province and Lin county in Shanxi Province. These two counties were national impoverished counties, and did good job in TB management. TB case notification rates in 2016 were 106.97 per 100,000 and 63.16 per 100,000 in Lingyun county and Lin county, respectively.Tuberculosis management information system from Nov 1st, 2015 through May11th, 2017. Patients with extrapulmonary tuberculosis, and those aged 18 years and below or with severe complications were not eligible for the study. Pregnant or breast-feeding women, and those who declined to sign the consent form were also excluded.We focused our study on adult patients (age ≥ 18 years) with active TB who were registered in As BMI is commonly used in nutrition assessment, we applied the prevalence of BMI < 18.5 which is also defined as malnutrition  in deterData for associated factors were taken from a questionnaire, including socio-demographic attributes  and behavioral variables .In line with Chinese Dietary Reference Intakes (DRIs) 2013 , we cateTuberculosis management information system and before anti-tuberculosis treatment.Trained staffs performed face-to-face interviews for each participant to obtain dietary intake data through a 2-day 24-h dietary recall (24hdr) questionnaire, which was adapted from the method of 3-day 24hdr . ParticiTotal energy, four macronutrients and sixteen micronutrients were evaluated. Nutrient intakes of each patient were converted to calories, weight of protein and weight of micronutrients based on Chinese Food Composition Tables (CFCT) 2004 .To evaluate whether the patients’ nutrients were sufficient, we compared mean daily nutrient intakes of TB patients to Recommended Nutrient Intakes (RNIs) and Adequate Intakes (AIs) by Chinese dietary reference intakes (DRIs) 2013. Recommended Nutrient Intakes is an estimate of the amount of a nutrient that meets the requirements of most people (97–98%) within a specific physiological group . Adequate Intakes means a recommended intake value based on observed or experimentally determined approximations or estimates of nutrient intake by a group (or groups) of healthy people .P-value of less than 0.05 was considered statistically significant. All analyses were performed using SAS 9.4 .Data of daily nutrient intakes were presented as means ± standard deviation (SD). As dietary recommendations are different for men and women, we compared TB patients’ daily nutrient intakes with DRIs by gender. Since protein-calorie malnutrition (PCM) is the most common form of undernutrition in TB patients, we only examined factors related to insufficient energy and protein intakes. Univariate logistic regression analysis was used to identify potential risk factors associated with inadequate energy and protein intakes in 300 TB patients. Age, gender, county and severity of TB were considered to be possible confounders in the multiple logistic regression model with stepwise selection. Patients were classified into two groups: below RNI/AI and above RNI/AI. A The study was carried out on the basis of China Health and Nutrition Survey (CHNS) 2015, from which the investigating method and tool were used in our study. All on-site investigations were carried out by the county-level CDC and interviewers were trained with a standard protocol. Data was checked for completeness and accuracy on the day of investigation and sampled by provincial CDC for verification later. All data was double-entered into a database specially designed for this project.All TB patients registered from Nov 1st, 2015 to May 11th, 2017 in Lin county were included in our study, while less than 40% of notified TB patients in Lingyun county have participated in this study.The majority (53.7%) of TB patients aged between 18 and 49, with the mean age being 45.5 years old (standard deviation (SD):18.7). More than half (68.7%) of them were males. Most TB patients were married, employed and didn’t attend junior middle school and above, with annual household income less than ¥20, 000. Only 39.7% of them consumed alcohol, while 46.7% of them were smokers  and 1360.3 kcal SD: 552.1 kcal) for males and females, respectively, which were significantly lower than recommended daily energy intake by DRIs . The protein intake was 44.6 g (SD: 18.2 g) and 35.9 g (SD: 12.3 g) for male and female TB patients, respectively. The protein daily intakes for our surveyed TB patients was significantly lower than daily protein intakes that were recommended by DRIs, which were 65 g and 55 g for males and females, respectively . Meanwhile, lower protein intake was associated with eating at home. The possibility of low protein intake for people eating out-of-home was much lower than those eating at home  (p < 0.05).Our univariate analyses suggested that employment status influenced energy intake, with lower energy consumption was found for the patients that were unemployed Table . FurtherOur study demonstrated that intakes of both macronutrients and selected micronutrients were inadequate for most TB patients, regardless of their gender. Protein-calorie malnutrition (PCM), characterized by inadequate intakes of both protein and total calories , was comWe found that mean daily intakes of many micronutrients were below RNI/AI, especially calcium. One potential explanation is that TB patients do not consume enough fish, shrimp and dairy products , which aFurthermore, we discerned that eating out-of-home was related to higher intake of protein. Relevant studies conducted among other populations in China –31 also Malnutrition can also have a negative impact on the treatment of TB. Malnutrition will reduce the level of protein in the patient’s body, and thus delayed the recovery of lesions. This is probably because sufficient protein not only can promote the recovery of lesions during the treatment, but also can increase the number of anti-tuberculosis drug carriers in the treatment, therefore, concentration of anti-tuberculosis drugs in the blood will rise, promoting sputum conversion . A studyOur study initiated in examining factors associated with TB patients’ nutrient intake and was also the first study in China to compare nutrient intakes of TB patients with the latest Chinese Dietary Reference Intakes (DRIs) 2013. However, there are still some limitations in our study. Firstly, the 24hdr method has its own shortcomings. Reliance on the memory of participants and recall bias could result in an underestimation of nutrient intakes . SecondlIn this study, we showed that in poor areas of China, the intakes of macronutrients and most micronutrients for TB patients were lower than DRIs, especially for those unemployed and eating at home. To reach effective treatments for TB patients, our study calls for promotion of awareness of significance of nutritional support in treatment process. In some cases, interventions in TB patients’ nutritional intakes are also required. Most importantly, the government should provide financial support for the patient so that they can afford enough food supplies.


The gut microbiota is recognized as having major impact in health and disease. Sample storage is an important aspect to obtain reliable results. Mostly recommended is immediate freezing, however, this is not always feasible. Faecal occult blood test (FOBT) papers are an appealing solution in such situations, and most studies find these to be applicable, showing no major changes within 7 days storage at room temperature (RT). As fieldwork often requires RT storage for longer periods, evaluation of this is warranted. We performed 16S rRNA gene sequencing of 19 paired faecal samples immediately frozen or kept five weeks and five months at RT on FOBT papers. Alpha-diversity evaluation revealed no effect of FOBT storage, and evaluation of beta-diversity showed that host explained 65% of community variation, while storage method explained 5%. Evaluation of community dispersion and the Firmicutes/Bacteroidetes ratio revealed a larger effect of storage time for fresh-frozen samples. Single taxa evaluation (order-to-genus level) showed significant alterations of four (of 37) genera after five weeks and five genera after five months. When comparing the two timepoints, alterations were only detectable for fresh-frozen samples. Our findings reveal that long term storage on FOBT papers is an applicable approach for microbiota research. Evidently, the gut microbiota modulates both innate and adaptive immune responses within the host14, and is crucial for host physiology and defense15.There is an increasing attention towards the human microbiota, i.e. the collection of microbes that inhabit internal and external surfaces of the human body. Over the past decade, our understanding of the microbiota and its impact on health and disease has expanded immensely. The human gut microbiota is the most widely studied, and has been linked to a wide range of both intestinal and extra-intestinal conditions, including inflammatory bowel diseases16. As microbial DNA might be degraded by oxidation, hydrolysis or enzymatic degradation17, sampling and storage methods are important factors in microbiota studies. Further, compositional changes of the microbiota can be observed due to deviating abilities of taxa to survive and replicate when exposed to oxygen. In the literature, it is claimed that storage of faecal samples at room temperature (RT) may induce alterations within 24 hours from defecation18. Thus, rapid freezing to −80 °C is generally considered best-practice in cases where immediate DNA extraction is not possible19, and is, among others, recommended by the Human Microbiome Project20. Some studies suggest that long-term storage at this temperature might alter the ratio between the two predominant bacterial phyla of the gut microbiota, Firmicutes and Bacteroidetes21.Uniform and suitable sample collection and storage procedures are crucial for clear and unbiased evaluation of the microbiota. Faecal samples reflect the luminal bacterial composition of the distal colon, and is, due to its non-invasive nature, an acceptable proxy for sampling the distal gut microbiota23. Although RNA preservation reagents have proven to be a convenient solution to stabilize the microbiota for up to two weeks at RT18, some studies find that this preservative performs poorly in microbiota-related studies25. Additionally, this and other preservatives are often costly, and by so, not feasibly for larger studies. Further, concerning field studies, the transportation of liquid biohazardous samples to central laboratory facilities can be cumbersome. Thus, the use of storage on FOBT cards without any liquid additives has gained ground. The majority of studies examining the use of FOBT-cards in microbiota research conclude that storage-induced differences are minor, compared to intra-individual differences28. However, these studies do investigate storage time at RT on FOBT-cards for up to 7 days, which might not be adequate time to reach freezing facilities in fieldwork.However, rapid freezing is not always feasible, in which case long-term storage at room or ambient temperature is inevitable. This is especially true in fieldwork in remote locations, or in regions, where electrical supply is unstable or freezing capacity is limited. Different preservation kits and buffers, including faecal occult blood test (FOBT)-cards and RNA preservation reagents have been used to extend storage time at RT prior to DNA extraction and investigation of microbiota compositionWe aimed to verify that FOBT-cards are useful as a long-term storage method in microbiota research, and further to investigate if five weeks to five months storage time on FOBT-cards at RT induce alterations in the faecal microbiota. By 16S rRNA gene sequencing, we performed pairwise comparisons of bacterial diversity and composition of faecal samples from 22 healthy individuals. One part was immediately frozen and kept at −80 °C, and one part kept on FOBT-card. Both parts were kept for five weeks (denoted timepoint 1 (TP1)) and five months (denoted timepoint 2 (TP2)), respectively, at either −80 °C or room temperature. Technical variability was examined, and sequencing data from the conventionally stored sample, which was immediately frozen, was regarded as reference.2 (range 19.4–26.6). Sample-pairs from 19/22 participants successfully underwent 16S rRNA sequencing with a minimum read count of 10,000 post quality control, whereas either DNA extraction or sequencing was unsatisfactory in 3/22. An overview of cohort characteristics is provided in Table Faecal samples were collected from 22 healthy normal-weighted individuals , free of any medication 2 months prior to inclusion. Median age (at time of sampling) was 30.9 years (range 23–64) and mean body mass index (BMI) was 22.3 kg/m2 range 9.4–26.6.The effects of RT storage on microbiota diversity was evaluated by pairwise comparisons of dissimilarity (Bray-Curtis and Jaccard), homogeneity of groups dispersions, sources of variability, alpha-diversity, the ratio between the two dominant phyla (Firmicutes-to-Bacteroidetes (F/B)-ratio) Fig. , and sin2 = 0.5%, p < 0.05), and less than the effect of storage at TP2 , while there was a small but significant difference for fresh-frozen samples .Finally, we found that dispersion for filter-paper samples was reduced compared to conventionally stored samples at TP1, while there was no significant difference at TP2  or between storage methods considering samples from TP1 (p > 0.05). However, there was a significant difference between storage methods at TP2 (p < 0.05), as well as between timepoints for fresh-frozen samples (p < 0.05). This difference was not observed for filter-paper samples (p > 0.05). F/B-ratio in each sample is available for visual inspection in Supplementary Fig. A previous study suggested that the F/B-ratio was affected by storage method30 analysing Bray-Curtis pairwise dissimilarities, including fixed effect term for storage and mixed effect term for subject, using r.squaredGLMM in R to calculate R2 values). The model found variation explained by storage to be 0.5% and 4.9% at timepoint 1 and 2, respectively. Therefore, both variables accounted for variation in the microbiota community, but the far majority of variation originated from subject. The corresponding analysis with Jaccard gave very similar results, as seen in Supplementary Table In order to defend the use of filter paper when immediate freezing is not feasible, it is essential that the individual or sampling site is the dominating source of variation, and that it dominates over the variation originating from storage type and time. To evaluate the sources of variability, we calculated Bray-Curtis dissimilarity and estimated the percentage of microbial variability that was explained by subject while correcting for storage type and timepoint. Using a linear mixed model for the data separated into the two timepoints, we found that subject explained 32% and 65% of the variation in the microbiota community at TP1 and TP2, respectively . ANCOM was included as a supportive analysis, as this method accounts for the underlying structure in microbiota data, and is superior to control for false positive inflation. Significant differential taxa were selected as those detected using both methods (Wilcoxon adjusted p < 0.05 and a true detection with cut-off 0.6 for ANCOM). We found two genera (out of 37 core genera) to significantly deviate between storage methods at TP1 and one genus deviating at TP2 . No genera or family-level taxa were found to be significantly altered by storage time in the neither fresh-frozen samples nor in the filter-paper stored samples . The genera in question are abundant in the healthy gut microbiota31, and are important in dietary fibre digestion and production of butyric acid32. Although we cannot exclude contamination of the utilized FOBT papers, we are rather confident that these alterations arise from the storage procedure, and that the genera are not a part of the FOBT papers.Importantly, we demonstrate only minor compositional differences in gut microbiota due to storage, and further validate that the microbiota of filter papers reflects that of conventionally stored samples and that any changes induced by the storage method are minor. Out of 37 identified core genera, we found that the relative abundance of only two genera, all from the Clostridiales order, increases upon five weeks storage at room temperature , of which most belonged to the Gammaproteobacteria class33. In the present study, we do not find any alterations in the Gammaproteobacteria class at TP1 or TP2. However, at TP2 we find increased levels of Sutterella (using the Wilcoxon signed rank test), a genus from the Betaproteobacteria class, and Betaproteobacteria itself increased in filter-paper samples . This finding indicates no specific selection of facultative anaerobic bacteria as only one of the four genera increased at TP2 belong to the Proteobacteria phylum. This implies that facultative anaerobic bacteria are not able to continued growth following storage at RT on FOBT paper, as would be one of the main concerns regarding this storage method.One major concern of storing faecal samples at room temperature is the fact that some bacterial taxa common to the gut, predominantly members of the Proteobacteria phylum, are able to grow in oxygen at room temperatureOur findings are in line with previous publications and supports the use of FOBT-cards for sample storage in microbiota research projects, where immediate freezing is not possible. Further, our results might suggest that samples stored at filter paper may preserve some aspects of the microbiota structure better than fresh frozen samples. Although we identify some alterations induced by storage, these are all minor. FOBT cards are suitable for storage for up to five months, without limitations to sequence data quality.Faecal samples were collected from 22 healthy individuals in January 2018. Although we aimed to limit the number of covariates known to profoundly affect the gut microbiota, we assessed intra-individual differences due to storage, which limits the importance of a homogenous cohort. None of the participants had received any antibiotics or other medication two months prior to inclusion, and none reported any gastrointestinal complaints. Female subjects were not pregnant. Faecal samples were collected in sterile faeces tubes , and handled within 15 minutes from defecation. The faecal matter was manually homogenized, and approximately 0.5 mL was subsequently applied to two faecal occult blood test (FOBT) filter card  with a clean wooden spatula, and left to dry under laminar airflow for 2–6 hours, protected from light. The remaining faecal sample was immediately frozen and kept at −80 °C. The filter paper samples were kept in air-tight zip-lock bags with desiccant , and stored at room temperature protected from light for five weeks and five months, respectively, after which the samples were moved to −80 °C, prior DNA extraction.Filter papers from FOBT-card samples were handled with ethanol-cleansed instruments , and DNA was extracted using the QIAamp DNA Stool Mini Kit  on a QIAcube platform (QIAGEN), according to manufacturer’s instructions with minor modifications. Briefly, the filter paper underwent bead-beating in PowerBead Tubes with Garnet beads (QIAGEN) on a SpeedMill PLUS instrument , prior to QIAcube extraction. DNA from conventionally stored samples were extracted using the QIAamp DNA Stool Mini Kit (QIAGEN) on a QIAcube platform (QIAGEN), according to manufacturer’s instructions. Extracted DNA was stored at −80 °C prior to PCR amplification. Blank extraction controls were included during extraction of samples. DNA quality from all samples was evaluated by standard gel analysis, to identify possible fragmentation of genomic DNA.et al.34. In brief, the V1-V2 regions of the 16S rRNA gene were amplified as described by Kozich et al.35, using the 27F-338R primer pairs, and further dual-barcoded, as described by Caporaso et al.36. Sequencing was performed on the Illumina MiSeq platform , according to manufacturer’s instructions.PCR amplification and 16S rRNA sequencing was performed as described by Wang 37 in PE (paired-end) mode with a sliding window of 0.1 readlength. Trimming was performed when average quality within the window was below 20, and reads were all >100 bp after trimming. Reads were stitched using VSEARCH38 with a length between 280 and 350 bp. Further, VSEARCH filtered reads with more than 1 expected error. Further quality filtering was performed using the FastX-Toolkit::fastq_quality_filter39 to exclude sequences with >5% nucleotides with a quality score below 30. Files were subsequently converted to FASTA format, and chimeras were removed in VSEARCH, using the gold.fa database. The remaining reads were classified using the UTAX algorithm, where reads classified as chloroplasts or not classified at domain level were removed.Sequencing was performed on the Illumina MiSeq platform, using the MiSeq Reagent Kit v3 according to manufacturer’s instructions. MiSeq FastQ files were trimmed using sickle40, implemented in VSEARCH. After removal of replicates and singletons, reads were clustered based on 97% similarity. Chimeras were once again filtered using VSEARCH in de-novo mode. To generate OTU abundance tables, all reads per sample were mapped to OTU tables using VSEARCH. Using the SINTAX classifier41 at lowest possible level with minimum 80% bootstrap confidence, one representative sequence for each OTU was annotated. OTUs with identical annotations were grouped into taxonomic bins.OTU tables were generated in UPARSESamples with fewer than 10,000 reads were removed (n = 9) as were subjects with < 4 samples post processing leaving 19 samples (76 samples) for further analysis.42 was used for statistical analysis of the microbiota data, and adjusted p-values (P.adj) were obtained using the p.adjust function in R package stats v3.5.1 with Benjamini-Hochberg (method = “BH”).The R programming environment v3.5.1Profiles for beta-diversity and single taxa analysis was transformed to adjust for deviating sequencing depth by dividing the counts by sample sum and multiply by 100 to obtain relative abundances between zero and 100.The alpha diversity measure considered was phylodiversity, as a measure of total unique phylogenetic branch length .The profiles for single taxa analysis were filtered to keep most abundant taxa as follows; minimum mean abundance across all samples of 0.05, and a minimum abundance of 0.05 in at least one sample. Further, taxa with ≥40% none-zeroes across samples were removed, which left 11 classes, 19 families and 37 genera. The relative abundance of single taxa was evaluated at phyla, class, family and genus level.43. This method accounts for the underlying structure in microbiome data and may therefore be superior in controlling for false positive inflation. ANCOM analysis was performed using abundance profiles with count data, not rarefied, and only including taxa selected for Wilcoxon analysis. ANCOM settings were adjusted = F, repeated = F, multcorr = 2, sig = 0.05 and prev.cut = 0.90. Due to the low sample size and often non-normality of the data we deemed this test appropriate.Analyses of F/B-ratio and alpha diversity were performed using the non-parametric paired Wilcoxon signed rank test in R package stats with conf.int = TRUE to obtain information of the location shift. Analyses of single taxa were performed using the described Wilcoxon signed rank test. However, as this test might result in high false positives, we supplied the analysis of differential abundances with the analysis of composition of microbiomes (ANCOM)44 (type = “centroid”). Significance of the difference in dispersion between groups was estimated using the anova function in R package stats.Analysis of dispersion was performed using the betadisper function in R package vegan30 with default settings. LMM was used to allow specification of the data structure with multiple samples from same subject. Association with storage time: Bray-Curtis pairwise dissimilarities were calculated between all samples stored with same storage method. For each distance measure, ids for the compared subjects and timepoints (same or different) were generated. Then a model was constructed including timepoint variables as fixed effect and intercepts for subject-pair variables as random effects. For association with storage method, pairwise dissimilarities were calculated between all samples stored for the same time duration. For each distance measure, ids for the compared subjects and storage method (same or different) were generated. Then data was separated according to timepoint and a model constructed including storage method variables as fixed effect and intercepts for subject-pair variables as random effects. The described analysis for storage time and method was also performed using Jaccard similarity measure. The r.squaredGLMM function in R package MuMIn45 was used to calculate conditional (R2c) and marginal (R2m) coefficient of determination. Variance explained by subject (R2) was calculated as R2c (variance explained by the entire model) minus R2m (variance explained by the fixed effects). Finally, hierarchical clustering was evaluated using the hclust function in R package stats, with Bray-Curtis, average agglomeration method, and square root transformed genera profiles.Evaluation of the association between storage method, storage time or host and microbial community structure was performed using linear mixed-effects models (LMM) in the lmer function in R package lme446, grid v3.5.1, gridExtra v2.347, gridBase v0.4.748, gridGraphics v0.3.049, plyr v1.8.450, ggplot2 v3.1.151, ggpubr v0.252, ggsignif v0.4.053, extrafont v0.1754, scales v1.0.055, vegan v2.5.444, lmerTest v3.0.156, lme4 v1.1.1930, metafor v2.0.057, genefilter v1.64.058, MuMIn v1.42.145, dendextend v1.1259.reshape2 v1.4.3Individuals were included voluntarily, and informed consent was obtained. The study was approved by the Regional Ethics Committee of Region of Southern Denmark (reference no. S-20170165) and conducted according to the principles of the Declaration of Helsinki.Supplementary information for the manuscript


In the mice with Ehrlich tumors who received Avns, there appeared to be improvement in NF-κB TNF-α, tumor markers (AFP and CEA), electrolytes, liver and kidney function enzymes, and lipid profiles; reduced MDA level; improved antioxidant parameters; normalised liver protein, P53, and PCNA; and reduced Bcl2 expression. Pathological examination of tumor lesions also indicated improvement. These results suggest that Avns exhibit antineoplastic activity and possess antioxidant properties that enhance the antioxidant defence system, thus reducing the oxidative stress caused by Ehrlich solid tumors.Interest is growing in finding natural sources of effective antitumor agents that generate fewer side effects than conventional chemotherapeutic drugs. Avenanthramides (Avns) are such compounds; these phenolic molecules naturally occur in oats and have antioxidant, anti-inflammatory, and antiproliferative effects making them worthy of further research. The aim of this study is to characterise Avns' curative ability and antineoplastic activity on solid-form Ehrlich tumors. For the study, 75 female mice were randomly and equally allocated to five groups . Mice with Ehrlich solid tumors exhibit increased tumor volume; elevated expression of AFP, ALT, AST, Bcl2, CEA, cholesterol, creatinine, urea, MDA, PCNA, potassium, triglycerides, TNF- Alongside cardiac disease, infectious disease, malnutrition, and war, cancer is one of the foremost causes of mortality worldwide. Cancer is the loss of the normal cell cycle, resulting in the unregulated cell growth and the lack of differentiation, characterised as malignant growths. Cancer can occur at any time, in any tissue or organ , 2. The Avena sativa), but also present in white cabbage. Avns are one such phenolic compound identified in oats, which, due to their antioxidant, anti-inflammatory, and antiproliferative effects, are considered beneficial to health . They were given ad lib access to a standard diet and water. All experiments were performed in accordance with guidelines for animal studies issued by the Ethical Committee of Faculty of Science, Tanta University, as approved by the Institutional Animal Care and Use Committee (IACUC-SCI-TU-0041).6 mouse cells) were subcutaneously inoculated into the left thigh of the mouse's rear limb.Mice with Ehrlich ascites carcinoma (EAC) were purchased from the Egyptian National Cancer Institute . The female Swiss albino mice were intraperitoneally injected with 2.5 million cells per mouse to maintain the tumor line. Prior to intraperitoneal injection, the EAC cells were counted using a bright line haemocytometer; dilution was performed using a physiological sterile saline solution. A volume of 0.2 mL containing the desired number of cells was injected. To assess Ehrlich solid tumors (Ehrlich), 0.2 mL EAC cells (2.5 × I0Group 1 (G1): negative control; as negative controls, these mice were untreatedGroup 2 (G2): DMSO: 50 mg/kg of DMSO were administered by oral gavage, 3 times per week, for two weeks Group 3 (G3): negative control receiving Avns; doses of 50 ng/kg/day were administered by oral gavage for two weeks Group 4 (G4): mice with Ehrlich solid tumor Group 5 (G5): cotreatment of Avns and Ehrlich solid tumor: for two weeks, starting at day zero of the Ehrlich induction, mice were treated with AvnsThe mice were given one week to acclimatise, then they were randomly divided into five equal groups (15 mice per group). After completion of the procedure, animals were euthanized with intraperitoneal injection with sodium pentobarbital and subjected to a complete necropsy. Blood samples were obtained from the inferior vena cava of each rat and collected in nonheparinised glass tubes and left for 30 min at room temperature to clot. The samples were then centrifuged at 5000 rpm for 10 min. Sera were separated and stored in aliquots at −80°C until required. Prior to assay, samples were thawed at room temperature.An automated quantitative enzyme-linked fluorescent assay (ELFA) using mini-VIDAS® AFP  was used to measure alpha-fetoprotein (AFP).Carcinoembryonic antigen (CEA) was measured by using the quantitative sandwich immunoassay, MyBioSource Mouse Carcinoembryonic Antigen Elisa Kit .α-specific monoclonal antibodies in accordance with the manufacturer's instructions.A quantitative sandwich enzyme immunoassay  was performed using TNF-Both aspartate transaminase (AST) and alanine transaminase (ALT) activities in serum were assayed by using a commercial kit that was supplied by HumanN (Germany) according to the method of Reitman and Frankel .The level of serum urea was analyzed by using a commercial kit that was purchased from Diamond, Egypt. Serum urea was determined according to the method of Patton and Crouch . Serum cCommercial assay kits, Spinreact , were used to measure the serum level of cholesterol following the method proposed by Allain et al. , whereasTumors were carefully removed from the carcasses, weighed, and their dimensions measured. To remove extraneous materials, tumor tissues were then washed with ice-cold saline three times then chilled on ice. Tumors were divided into four sections, wrapped in aluminium foil, and stored at −80°C until required for the preparation of tissue homogenates . The samw/v homogenisation. The homogenate was centrifuged at 4°C for 20 min at 10,000 g. The resultant supernatant was used for enzyme assays.After the two-week experiment, the mice were fasted, anaesthetised, and euthanized with ether; then, 10-12 hours after fasting, a full necropsy was performed. Tumor tissues were homogenised using ice-cold 1.15% KCl and 0.01 mol/L sodium potassium phosphate buffer (pH 7.4) and a Potter-Elvehjem-type homogeniser to achieve 10% 2O2 (the substrate of the enzyme) at 240 nm according to the method described by Aebi [To determine the malondialdehyde (MDA) component, a noxious product of lipid peroxidation was detected according to the following: lipid peroxidation was evaluated on the basis of MDA level, and MDA in liver was determined using the method described by Mesbah et al. . Assay o by Aebi . Assay o by Aebi . Assay o by Aebi .Immediately after dissection, Ehrlich tumors were removed and fixed in 10% neutral buffered formalin. Fresh isolated tumor from different groups was stained using routine haematoxylin and eosin counterstain methods, in accordance with Bancroft and Cook .κB [μm thick) of fixed tumor that had been mounted on gelatin chrom alum–coated glass slides were dewaxed. To reduce nonspecific background staining, sections were then washed for 5 min with distilled water, followed by a PBST rinse for 10 min before being incubated with 10% normal goat serum for 15 min. Then, the sections were incubated with anti-rabbit p53 or anti-rabbit Bc1-2 or anti-rabbit PCNA  or rabbit polyclonal anti-NF-κB/p65 (Rel A) Ab-1 antibody  at room temperature for 1–2 h. The manifestation of a dark, brown-coloured, intracytoplasmic precipitate is indicative of p53, Bcl-2, PCNA, and NF-κB being present. The primary antibody was excluded in the negative control; this was to protect against any false-positive results that could arise nonspecific reactions.The avidin-biotin complex (ABC) method was used to assess the immunoreactivities of p53, Bc1-2, PCNA, and NF-κB , 30. ParT-test was performed to compare the significant difference between groups. The criterion for statistical significance was set at P < 0.05.The analysis of results was done using the Statistical Package for the Social Sciences (SPSS software version 16). Data were presented as mean ± standard error of mean (SEM) and statistically analyzed by one-way ANOVA  followed by Dunnett test. Dunnett test comparisons were performed to assess the significance of differences between groups. Unpaired ∗∗P = 0.0037) in tumor volume in G5 mice.The effect of Avns treatment upon the growth and proliferation of subcutaneously injected Ehrlich cells is presented in P = 0.0001) and CEA (P = 0.0001) levels in the cotreated Ehrlich solid tumor group with avenanthramides (Avns) groups when compared with the Ehrlich group (P < 0.0001) ( 0.0001) .P = 0.0001) in the plasma levels of TNF-α in G4 compared to the control groups (P = 0.0005). Also, the levels of TNF-α in G5 (P = 0.0342) were significantly lower than the levels detected in G4.P < 0.0001), the enzyme levels were significantly greater than G1 (P = 0.0001), G3 , and G5 (P = 0.0001) (Comparing the serum levels of ALT and AST in G4 with the other groups shows that in G4 ( 0.0001) .P < 0.0001), urea (P < 0.0001), and potassium (P = 0.0286) (P = 0.0080) in the serum level of sodium compared to the control group (P = 0.0207)  . G4 also 0.0207) .P value = 0.0002) and triglycerides (P = 0.0010) compared to the levels observed in G4 (P < 0.0001) (As  0.0001) .P < 0.05) increase in the levels of CAT, GSH, and SOD and a significant (P < 0.05) decrease in the level of MDA in tumor tissue as compared with the Ehrlich solid tumor group  under a light microscope showed compact aggregation of the tumor tissue cells which spread within the muscular tissues Figures . SubcutaThe detection of Bcl2 expression in tumor sections in the Ehrlich solid tumor (Ehrlich) and Ehrlich cotreated with Avns is revealed in Figures The detection of P53 expression in tumor sections in the Ehrlich solid tumor (Ehrlich) and Ehrlich cotreated with Avns is revealed in Figures The detection of PCNA expression in tumor sections in Ehrlich solid tumor (Ehrlich) and Ehrlich cotreated with Avns is revealed in Figures κB expression in tumor sections in the Ehrlich solid tumor (Ehrlich) and Ehrlich cotreated with Avns is revealed in Figures κB expression, while the tumor section in Ehrlich cotreated with Avns inhibits NF-κB, where mild NF-κB-positive reactions for NF-κB expression were detected in Ehrlich cotreated with Avns , unique polyphenols found exclusively in oats and oatmeal, exhibit antioxidant and anti-inflammatory activity , 14. In Control or untreated mice that were intramuscularly inoculated with Ehrlich ascites developed a solid tumor in the right thigh of the hind limb within 13 days of inoculation. This is consistent with previous research based on the same model . Followiα and IFN-γ, which are released by macrophages [α were significantly elevated in G4 compared to G1 and G3. This increase in TNF-α levels in the tumor-afflicted mice could be due to a rise in macrophage-produced ROS, which increases lipid peroxidation. This is considered to be important by pathogenically contributing to liver damage [α levels in female mice with Ehrlich ascites carcinoma. However, the results are inconsistent with those generated by Mansour and Anis [α and IL-10 in Ehrlich solid tumor-bearing mice were significantly decreased compared to controls. Cotreatment of Ehrlich solid tumor with Avns depletes the increase in TNF-α levels in Ehrlich solid tumor. As postulated by Guo et al. [Cancer patients often suffer from the condition, cachexia, in which there is a progressive loss of fat and muscle mass leading to overwhelming weakness; it is attributed to the action of cytokines such as TNF-rophages , 33. TNFrophages . In thisr damage . The resr damage  who repoand Anis  who noteo et al. , the mecSerum AFP is one of several tumor markers that become elevated where certain cancers are present. In the G4 group of this study, the levels of serum AFP and CEA were found to be significantly greater than in G1, G3, and G5. Based on these findings, the indications are that in G4, liver and kidney functions were increased. This upregulation might be indicative of hepatic and renal damage caused by the effect of cancer cell invasions.Liver damage induced by tumor cells typically indicates disruption of normal liver cell metabolism, resulting in changes in the activity of serum enzymes. In the current study, there was a significant increase in the serum levels of ALP, ALT, and AST, together with a concomitant decline in the levels of serum albumen and total proteins in G4 mice, indicating liver toxicity. The increase in liver enzymes might arise from a generalised destruction of hepatocytes and the release of AST into the plasma following the induction of the tumor. Albumin, which is the most abundant plasma protein, and other plasma proteins are synthesised in hepatocytes in the liver; they are useful, easily monitored markers of liver function . Thus, tα) [Also, in the present study, treatment with Avns caused significant attenuation of this elevation in ALT, AST, and ALP liver enzymes and came back to be close to normal values indicating the antitumor effect of Avns agent. Equally, the level of serum albumin, which had become considerably depressed in G4 mice, was improved in G5 mice that all received treatment of Avns. Elevated serum levels of creatinine and urea are indicative of renal dysfunction. In the present study and consistent with the results observed by Abou-Zaid et al. , Husseinα) , 46.Oxidative stress is considered key in the development of numerous health conditions including cardiovascular diseases and obesity. The evidence also indicates it has a major role in tumorigenesis , 48. Stuin vivo, this can induce lipid peroxidation [α, and increase antioxidant parameters to reduce oxidative stress.This study found a positive correlation between the changes in antioxidant mechanisms, apoptosis, and the proliferation of Ehrlich solid tumor cells. There was a statistically significant increase in the level of MDA and a decrease in the levels of catalase, GSH, and SOD in the tissues received from the G4 tumors. As stated earlier in this report, Avns have the ability to modulate antioxidant enzyme activity by promoting the levels of catalase, GSH, and SOD, as well as inhibiting the level of MDA. Marklund and Marklund  also repxidation , 55. TheκB and a negative expression of P53. Evaluations of cell proliferation are one of the key indicators used to determine cancer prognoses. In clinical settings, the number of cells undergoing mitosis is counted to assist with the histopathological grading or labelling of cells in the S phase. PCNA is also an important parameter of proliferation, which has an important role in initiating the onset of the cell cycle and increasing the G1-S phases of the cell cycle [Carcinogenesis is effectively the loss of equilibrium between the process of cellular proliferation and apoptosis. Elevated rates of apoptosis have been linked with inhibiting the growth of cells that are potentially oncogenic . The apoll cycle . In thisκB) pathway, which has emerged as an important therapeutic target in cancer. Activation of the NF-κB transcription factor is associated with nuclear translocation of the p65 component of the complex. NF-κB regulates the expression of various molecules important in tumorigenesis, such as matrix metalloproteinases, cyclooxygenase-2 (COX-2), iNOS, chemokines, and inflammatory cytokines, all of which promote tumor cell invasion and angiogenesis [κB was inhibited in tumor sections treated with Avns through the inhibition of NF-κB-dependent intracellular signalling.The regulation of numerous cellular processes such as proliferation, apoptosis, and survival is done through the nuclear factor kappa B  are important phenolic compounds found in oats, which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. Here, we describe the antineoplasic activity and the curative role of avenanthramides (Avns) on Ehrlich tumor in the solid forms. Avenanthramides revealed potent antineoplasic activity and antioxidant properties by augmenting the antioxidant defence system hereby protecting the tumor against oxidative stress induced by Ehrlich solid carcinoma tumors.


A retrospective case-control study was performed to evaluate whether PCOS-specific serum markers would change in women with polycystic ovary syndrome (PCOS) during the course of two consecutive cycles of clomiphene citrate (CC)-stimulation, which did not lead to a pregnancy.n = 41) and anovulatory PCOS controls who chose an observational approach for two months (n = 24) were included in the study. The main outcome measures were levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), anti-Mullerian hormone (AMH), total testosterone, androstenedione, and sexual hormone binding globulin (SHBG).Anovulatory PCOS patients who underwent two consecutive CC-cycles (p > 0.05). In the CC-group, there were decreases in LH , the LH:FSH ratio , and AMH , as well as an increase in SHBG . A higher age and lower baseline total testosterone and AMH levels were predictive of an AMH decline (p < 0.05).In the control group, PCOS-specific serum parameters did not change during two months (Two cycles of CC-stimulation that did not lead to a pregnancy were accompanied by mean LH, AMH, and LH:FSH ratio declines and an SHBG increase. The clinical significance seems of minor relevance. Polycystic ovary syndrome (PCOS) is a commonly occurring endocrine disorder that affects 6–10% of women of reproductive age . In anovMoreover, according to the recently posited “two triangles theory,”  follicleThus, it was the aim of this retrospective study to evaluate, in anovulatory women with PCOS, whether baseline levels of luteinizing hormone (LH), FSH, AMH, and androgens would change during the course of two cycles of CC stimulation that did not lead to a pregnancy, and to compare these patients to anovulatory women with PCOS who had chosen an observational approach.There were no differences between the groups with regard to baseline patient characteristics Table . In the In a sub-analysis, we compared women who received CC 50 mg twice to those who were stimulated with CC 50 mg followed by CC 100 mg , whereas a higher baseline LH:FSH ratio and a lower baseline AMH level were associated with decreases in the LH:FSH ratio after two cycles of CC stimulation.We then analyzed possible predictive parameters for AMH and LH:FSH ratio dynamics in generalized linear models in the CC stimulation group Table . A higheThe general patient characteristics quite closely represented the average PCOS population , 18. MorThe main findings of this study were the decreases in AMH and the LH:FSH ratio after two CC cycles that did not lead to a pregnancy. Both markers are thought to somehow represent the severity of PCOS . It is dhttps://pubchem.ncbi.nlm.nih.gov/compound/dydrogesterone#section=Metabolism-Metabolites]. All follow-up serum blood samples were retrieved on the 2nd to 5th cycle day after a withdrawal bleeding. Thus, we consider a residual suppression effect on LH unlikely. We can rule out that the observed changes might be due to ovulation induction with recombinant human chorionic gonadotropin (rHCG), since a previous study showed no impact of rHCG administration in PCOS women on AMH up to 48 h thereafter [The observed AMH and LH:FSH ratio decreases reflect a close association, which has already been reported , 24. Expereafter .In the present dataset, the AMH declines were associated with lower total testosterone levels, as well as higher age and baseline AMH levels Table . The latThe decline in the LH:FSH ratio could have been predicted by the higher baseline LH:FSH ratio and a lower baseline AMH level, with an only modest effect of the latter . The following exclusion criteria were applied: (i) use of co-medications, i.e., metformin, myo-inositol, cortisone/prednisolone, or any study-specific medications (n = 28); (ii) any ovarian stimulation within the last three months (n = 3); and (iii) women who had become pregnant after the second CC cycle or had chosen not to undergo any treatment in the subsequent cycle, since no third pre-stimulation hormonal blood-testing was available in these cases (n = 18). Thus, 41 women were included in the CC group.The Clinical Division of Gynecologic Endocrinology and Reproductive Medicine of the Medical University of Vienna, Austria, serves as a referral center for gynecologic endocrinology and reproductive medicine. The diagnosis of PCOS was confirmed according to the revised Rotterdam criteria of 2004 . All womFor the control group, we identified 24 anovulatory PCOS women who did not undergo ovarian stimulation or PCOS-specific treatment for two months after initial hormonal testing, but delayed treatment by request. Selected women were diagnosed within the period mentioned above and underwent a second pre-stimulation hormonal testing after two months. None of them had undergone any previous ovarian stimulation nor were any of them included in the CC group in case of a later CC stimulation. None of the patients had undergone previous ovarian drilling.For the CC group, menstrual bleeding was induced through oral administration of 20 mg dydrogesterone for ten days. Subsequently, CC stimulation was done as published previously  and begaPre-stimulation hormonal testing was performed on the 2nd to 5th day of each stimulation cycle to obtain the most recent predictive parameters. Although this is not necessary according to established guidelines , this haIn the control group, bleeding was also induced by administration of 20 mg oral dydrogesterone, as described above. Thus, all hormonal parameters were retrieved on the 2nd to 5th cycle day.We included the following parameters: patients’ age at the first hormonal testing used in this study; body mass index (BMI); type of sterility (primary/secondary); parity; initial baseline serum levels of thyroid stimulating hormone (TSH), prolactin, and estradiol; and serum levels of FSH, LH, AMH, total testosterone, androstenedione, and sexual hormone binding globulin (SHBG), measured initially and after two CC cycles (CC group) or two months of observation (control group). All hormone measurements were performed in ISO 9001 certified and ISO 15189 accredited clinical diagnostic laboratories at the Department of Laboratory Medicine, Medical University of Vienna. IVD CE certified electrochemiluminescence immunoassays (ECLIA) on Cobas e 602 immunology analyzers  were used for all parameters, except androstenedione. Intra- and inter-assay coefficients of variation (CV) were ≤ 11.1 and 11.9% for TSH , 1.7 and 2.0% for prolactin , 6.7 and 10.6% for estradiol , 2.8 and 4.5% for FSH , 1.2 and 2.2% for LH , 1.7 and 3.5% for AMH , 14.8 and 18.1% for total testosterone , and 1.7 and 4.0% for SHBG , respectively. Androstenedione determinations were performed by an IVD CE certified chemiluminescence immunoassay (CLIA) on an Immulite 2000 XPi immunology analyzer  with intra-assay and total CV ≤ 15.1 and 17.8% , respectively. Free testosterone was calculated according to the algorithm published by Vermeulen et al. .p < 0.05.Variables are described by numbers (frequencies) for categorical parameters, mean ± standard deviation for normally distributed numerical parameters and median (interquartile range) for non-normally distributed numerical parameters. Statistical analysis was performed with SPSS 24.0 for Windows  using unrelated t-tests and paired t-tests , Wilcoxon signed rank tests  as well as the Chi-square test or the Fisher’s exact test . Possible influencing factors on hormone dynamics were tested using generalized linear models. For these, regression coefficients (ß) and their standard deviations (SD) are provided. Differences were considered statistically significant if Additional file 1: Table S1. The course of PCOS-specific serum parameters in the group of women who received CC 50 mg twice and the group of women who received CC 50 mg followed by 100 mg.


Children require anesthesia for MRI to maintain immobility and reduce discomfort; clear indications about the best anesthesiologic management are lacking and each center developed its own protocol. Moreover, children with neuropsychiatric disorders more likely require sedation and are described in literature as more prone to general and respiratory complications. Aim of this study was to analyze the applicability of a sevoflurane-based approach, to describe general and respiratory complications and to identify risk factors in a pediatric neuropsychiatric population.p ≤ 0.05) and clinical judgment.Retrospective cohort study, university Hospital (January 2007–December 2016). All the 1469 anesthesiologic records of children addressed from Neuropsychiatric Unit to undergo MRI under general anesthesia were analyzed; 12 patients equal or older than 18-year-old were excluded. We identified post-hoc nine macro-categories: static encephalopathies, metabolic/evolutive encephalopathies, epileptic encephalopathies, neuromuscular diseases, autistic spectrum disorders, migraine, psychiatric disorders, intellectual disabilities, others. A logistic regression model for events with low frequency  was carried out to identify the mutually adjusted effect among endpoints (complications) and the independent variables chosen on the basis of statistical significance . Complications’ rate was low . The risk for general complications increases with ASA score > 1 , male sex , multi-drug anesthesia . For respiratory complications, it increases with ASA score > 1 , autumn-winter , neuromuscular disorders . We had no major complications compromising patients’ outcome or requiring admission to ICU.Of 1457 anesthesiologic records (age 4.0 (IQR 2.0 to 7.0) year-old, males 891 (61.2%), weight 17.0 (IQR 12.0 to 24.9) kg), 18 were cancelled for high anesthesiologic risk, 50 were cooperative, 1389 were anesthetized. A sevoflurane-based anesthesia was feasible in 92.3%; these patients required significantly less mechanical ventilation (8.6 vs. 16.2%; Sevoflurane anesthesia is feasible and safe for children affected by neuropsychiatric disorders undergoing MRI. Specific risk factors for general and respiratory complications should be considered. Pediatric patients frequently require sedation for medical procedures to reduce pain and stress, or to maintain immobility. In the case of magnetic resonance imaging (MRI), image quality is significantly improved by complete stillness .Since 1985, scientific societies emanated guidelines to perform pediatric non-operating room anesthesia (NORA), in order to reduce complications . In the Moreover, children affected by neuropsychiatric disorders are considered at higher risk of anesthesiologic complications . A higheAmong neuropsychiatric disorders, neuromuscular diseases are considered at the highest risk of perioperative respiratory complications since they may affect respiratory muscles strength either directly by muscle fibres weakening or indirectly by degenerative nerve supply and neuromuscular junction weakness –26. A caAs in other institutions, in our center we systematically applied a standardized approach with sevoflurane-based NORA as described in infants and neonates , 17, eveAim of this study was to analyze a ten-year experience of pediatric NORA for MRI performed with a standardized sevoflurane-based approach in children affected by neuropsychiatric disorders, focusing on applicability of the approach, anesthesia management, rate of general and respiratory complications and risk factors’ identification.Ethical approval for this study  was provided by the Ethical Committee NAC of Fondazione IRCCS Policlinico S. Matteo University Hospitals, Pavia, Italy (Chairperson Prof M. Cazzola) on December 5th, 2017; the need for written consent was waived by the ethics committee.We retrospectively enrolled all patients addressed from Pediatric Neuropsychiatric Unit of Fondazione Mondino IRCCS, a university Italian neurological hospital, to undergo MRI under general anesthesia (GA) from January 2007 to December 2016. We excluded from final analysis patients equal or older than 18-year-old. Data were collected from anesthesia records. A simplified classification of neuropsychiatric disorders was performed post-hoc; nine macro-categories were identified: static encephalopathies , metabolic/evolutive encephalopathies , epileptic encephalopathies, neuromuscular diseases, autistic spectrum disorders, migraine, psychiatric disorders, intellectual disabilities, others. In particular, we focused our attention on children affected by neuromuscular diseases, being more exposed to respiratory complication –30.2 monitoring (capnography) as measured by nasal and/or oral sampling lines. If airways’ patency is judged satisfactory, the patient is maintained in spontaneous breathing, either spontaneously or with an oral cannula, with inhaled sevoflurane by bag mask. Otherwise a laryngeal mask airway (LMA) is electively placed and positive pressure ventilation in a closed circle system started by the same Drager Fabius© machine. Oro-tracheal intubation set is also available. We consider loss of airways patency as a complication in patients with altered capnography or desaturation after the induction phase. A cervical collar is frequently placed to prevent head involuntary movements, eventually impairing image quality. A peripheral vein catheter is placed under GA and glucose 5% 20 ml/h administered to prevent hunger symptoms from fasting at awakening. Italian recommendations for fastening were followed [Anesthesia team is composed by two intensivist-anesthesiologists and one MRI nurse; the hospital is neurological only and no other medical-emergency team is available. Standard practice consists in sevoflurane-based anesthesia induction and maintenance, obtained with sevoflurane inhaled by a reservoir bag mask at 8.0 and 2.5% respectively. Anesthetic gas was delivered by a Drager Fabius© machine and dispersion was prevented by a canopy and aspiration system. If a premedication is indicated, midazolam (0.1 mg/kg administered intramuscularly (IM) 30 min before anesthesia) is administered. Anesthesiologist is free to leave the standard approach, if required according to pre-anesthesia evaluation. After induction, airways’ patency is judged on the basis of respiratory thoracic excursion, peripheral oxygen saturation and expiratory COMean values and standard deviations (SD) or median and interquartile range (IQR) were used for the quantitative variables, and percentages for the categorical ones. Normal distribution was assessed by Shapiro-Wilk test.Primary end-point was the applicability of the sevoflurane-based approach, as measured by the percentage of anesthesia conducted with the standardized approach.Secondary end-points were: to identify differences between standard and non-standard anesthesia, in terms of patients’ characteristics, anesthesia characteristics and need of mechanical ventilation; to describe anesthesiologic management in this specific population in terms of anesthetic drugs for induction and maintenance, need of mechanical ventilation and associated factors, indication to additional procedures during MRI anesthesia, length of anesthesia and time for full recovery; to measure the rate of general and respiratory complications and associated risk factors.Comparisons among the categorical variables were evaluated with the Pearson chi-square test; Fisher’s exact test and Student’s T Test for independent data or Wilcoxon/Mann-Whitney U-test were used for quantitative variables.p ≤ 0.05) and of the clinical judgment; age and sex were used as adjusting variables. P-value ≤0.05 was considered significant (two-sided). All the analyses were conducted with STATA/SE for Macintosh, version 14.2.To evaluate the associations between endpoint variable (development of complications during the procedure) and the patients characteristics / anesthesia management, odds ratios (ORs) and the corresponding 95% confidence interval (CI) were calculated. Given the limited number of cases with complications , a logistic regression model for events with low frequency  was carried out to identify the mutually adjusted effect among endpoints and the independent variables chosen on the basis of the statistical significance  and required significantly less mechanical ventilation when compared to the other pharmacological approaches .Standardized sevoflurane-based anesthesia was significantly less used in children affected by neuromuscular diseases  was preferred .p = 0.016), with ASA score > 1 , undergoing a non-sevoflurane-based anesthesia  and affected by neuromuscular disease  were significantly more frequently ventilated.Among the 9.1% of ventilated patients, only one was intubated, all the others received an LMA. Patients of male sex  were associated in 16.9% of cases.p < 0.0001) and in patients with respiratory complications (20 (IQR 15 to 25) vs. 15 (IQR 10 to 20) minutes; p = 0.0236).Median anesthesia length was 50 (IQR 40 to 60) minutes. Median time for full recovery was 15 (IQR 10 to 20) minutes; it was not affected by the length of GA or patients’ characteristics but was significantly longer in mechanically ventilated patients (20 (IQR 15 to 30) vs. 15 (IQR 10 to 20) minutes; We registered 86 complications (6.2%): 43 respiratory complications (50.0%), 13 post-operative nausea and vomiting (15.1%), 10 hiccup (11.6%), 8 agitation (9.3%), 5 seizures (5.8%), 4 bradycardia (4.7%), 1 extravasation of glucose 5% (1.2%), 1 prolonged awakening (1.2%) and 1 inadequate sedation (1.2%). Among the 43 respiratory complications, we observed 23 airways obstructions (53.5%), 10 laryngospasms (23.3%), 5 depressions of the central respiratory drive (11.6%), 3 bronchospasms (7.0%) and 2 cough and hyper-salivation (4.7%).p = 0.095).We found statistically significant differences for general complications in ASA score, age, sex, the choice of a multi-pharmacological anesthesia and anesthesia length superior to 50 min, and for respiratory complications in ASA, a diagnosis of neuromuscular diseases and season autumn-winter Table . A lowerp = 0.003), in male patients  and in patients undergoing anesthesia with more than one drug . For respiratory complications, the risk increases with ASA score > 1 , in autumn-winter season  and in patients affected by neuromuscular diseases .Table In 35 cases, respiratory complications required an active intervention by the anesthesiologist; in 66.7% of cases this consisted in the placement of LMA. We observed no major complications compromising the patient’s outcome or requiring admission to ICU; 4 respiratory complications determined the definitive interruption and reschedule of MRI (0.29% of the 1389 GA).The main findings of this retrospective cohort study are: 1. A standard approach with inhaled sevoflurane-based anesthesia could be applied in a high percentage of MRI sedation in pediatric patients affected by neuropsychiatric disorders. 2. With a standardized approach, anesthesia complications’ rate in this specific population is low and similar or lower to what reported in literature on general pediatric population. 3. Risk factors for general and respiratory complications could be identified.Procedural anesthesia in children, performed outside the operating room, concerns an increasing number of patients every year, since required to reduce pain, stress and discomfort associated to health care procedures. Initially performed without specific guidelines, first recommendations appeared in 1985  to standIn the context of MRI, the goal of anesthesia is to facilitate this painless procedure providing immobility, safety and comfort; it has been shown that adequate sedation and airways management significantly improves image quality , thus rePrevious studies focused on specific pharmacological approaches for NORA in children. Chloral hydrate has been widely used in patients affected by neuropsychiatric disorders undergoing MRI –9; howevHalogenates have been successfully used in newborns and infants , 17; theWe therefore systematically applied in the last ten years a standardized approach with sevoflurane-based anesthesia in children with neuropsychiatric disorders undergoing MRI. This standard approach could be used in 92.3% of cases and was associated to a significantly lower rate of mechanical ventilation when compared to all the other pharmacological approaches combined. Interestingly, a mono-pharmacological NORA was also a protective factor for overall complications’ development and in 96.0% it consisted in a sevoflurane-based NORA. Despite the remarkable length of anesthesia, median recovery time was very short when compared to literature, possibly thanks to the speedy awakening provided by sevoflurane and the possibility to avoid mechanical ventilation in more than 90% of patients.2 waves analysis, thus confirming the need for a complete respiratory monitoring. However, advanced airways management was required in 9.1% of cases, confirming the need for adequately trained personnel.Overall, spontaneous breathing was maintained along the entire MRI exam in a high percentage; airways patency assessment was mainly based on thoracic excursion and EtCOFinally, children affected by neuropsychiatric disorders may present specific anesthesiologic issues –30. PrevThe main weakness of this study is the retrospective design: anesthesiologic records were compiled manually and parameters were reported every 10 min. However, we had no missing data on pharmacological anesthesia management. Moreover, the study was not built to compare different pharmacological approaches. Future studies should focus on the comparison of different pharmacological approaches in specific neuropsychiatric classes.Finally, IM midazolam administration could be easily replaced by less invasive approaches such as nasal administration.A standardized sevoflurane-based NORA for pediatric patients with neuropsychiatric disorders undergoing MRI is highly feasible and safe. In this population, children with ASA score > 1, of male gender, undergoing prolonged procedures in winter-autumn months and with neuromuscular diseases should be considered at higher risk of general and respiratory complications.


Perfluoroalkyl and polyfluoroalkyl substances (PFAS) have been reported to disrupt endocrine system and reproduction. However, epidemiological evidence on the association between PFAS and preeclampsia is inconsistent. We aimed to investigate the association between prenatal PFAS exposure and hypertensive disorders of pregnancy (HDP) in humans.PFAS were measured by liquid chromatography system coupled with tandem mass spectrometry in 687 umbilical cord plasma samples collected between 2011 and 2012 in Shanghai, China. Information on HDP including gestational hypertension and preeclampsia was abstracted from medical records. Multiple logistic regression was used to examine the association of each PFAS with gestational hypertension, preeclampsia, and overall HDP in separate models. Elastic net regression with logit link was used to identify independent associations between exposures and outcomes. Logistic regression was used to obtain the unpenalized estimates of the selected PFAS components for the associations with outcomes, adjusting for age, education level, pre-pregnancy BMI, parity, and mutual adjustment of selected PFAS.The risk of gestational hypertension and preeclampsia was 3.3% and 2.8% in our subjects, respectively. Perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), perfluoroundecanoic acid (PFUA) were associated with preeclampsia based on elastic net penalty regression. In the fully adjusted statistical model, women with a higher level of standardized ln-transformed PFBS had an increased odds of preeclampsia , and overall HDP .Prenatal exposure to PFBS was positively associated with the risk of preeclampsia and overall HDP.The online version of this article (10.1186/s12940-018-0445-3) contains supplementary material, which is available to authorized users. Hypertensive disorders of pregnancy (HDP) are among the most common complications of pregnancy. A national survey involving 112,386 pregnant women from 38 secondary and tertiary hospitals in China in 2011 reported that HDP occurred in 5.2% of pregnancies . HDP arePerfluoroalkyl and polyfluoroalkyl substances (PFAS) are a large group of manufactured compounds widely used in both industrial and consumer products . Humans Epidemiological studies have provided inconsistent results on the association between PFAS and preeclampsia. Two studies focusing on women who lived in areas with a high PFOA level but with background PFOS level found that PFOA and PFOS were significantly positively associated with preeclampsia with an odds ratio (OR) of 1.1–1.2 across the upper three quintiles for PFOA and OR of 1.13 in relation to a shift from 25th to 75th percentile for PFOS , 17, whiThe present analysis is a cross-sectional study. From 2011 and 2012, 687 women who had a singleton pregnancy and came for delivery at two large hospitals in Shanghai were recruited. A face-to-face interview was conducted by trained nurses to collect information on maternal age, education level, pre-pregnancy weight, and height. Information on parity and pregnancy-related complications was abstracted from medical records. Cord blood samples were collected shortly after birth. Among the 687 subjects, 13 subjects were excluded because of missing information on maternal age, education level, pre-pregnancy BMI, and parity, resulting in 674 valid subjects. A written consent was obtained from each woman. This study was approved by the Ethics Committees of all involved research institutions and hospitals.As noted above, information on chronic hypertension before pregnancy, gestational hypertension, and preeclampsia was obtained from medical records. Gestational hypertension was defined as new onset of hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg) after 20 gestational weeks. Preeclampsia was defined as new onset of hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg) after 20 gestational weeks accompanied by proteinuria (a urine dipstick of at least +). According to the International Society for the Study of Hypertension in Pregnancy, hypertensive disorders of pregnancy (HDP) included gestational hypertension, preeclampsia, chronic hypertension , or pre-eclampsia superimposed on chronic hypertension . ChronicUmbilical cord blood samples were collected at delivery and centrifuged at 4000 rpm for 10 min immediately after collection, with plasma separated and stored at − 80 °C until shipping on dry ice to the laboratory for analysis. Detailed method of measuring PFAS has been described elsewhere . A totalConsidering the multiple correlations between these PFAS, elastic net regression was used to select the exposures that are associated with outcome, while simultaneously accounting for other PFAS exposures. Elastic net regression is a penalized regularization method combined with the properties of ridge and least absolute shrinkage and selection operator (LASSO) penalty . It is wN = 10, proportion = 1.5%) and during pregnancy  in our subjects, smoking is unlikely to confound the association assessed in this study and was not included as a confounder. These covariates were included in the elastic net regression without penalization. Before regression analysis, PFAS concentrations were first ln-transformed to mitigate the effects of their right-skewed distribution and then were centered and standardized with one-standard deviation based on ln-transformed scale. Additionally, PFAS concentration was analyzed as both a continuous variable  and categorical variables in tertiles (T1/T2/T3) with the lowest tertile (T1) being the reference group. P-values for linear trend of odds ratio for hypertensive disorders of pregnancy in relation to different level of each PFAS concentration was obtained by treating the categorical PFAS variables as continuous variables in the regression model. Preeclampsia might affect kidney function and alter PFAS concentration, which may be most prominent for PFAS with a short half-life, such as PFBS. To improve the assessment of whether such as effect might have occurred, a sensitivity analysis was conducted for PFBS with further adjustment of birthweight and gestational age. Results were given in Additional file For regression models, a set of covariates that were selected based on a directed acyclic graph were set as confounders  of HDP vs Normotensive women: 30.6 (4.5) vs 29.2 (3.8), P = 0.02], and had a higher pre-pregnancy BMI than normotensive women .The risk of gestational hypertension, preeclampsia and overall HDP was 3.3, 2.8, and 6.1%, respectively, in our study. Table A total of 8 PFAS components were quantifiable in more than 70% of blood samples Table . PFOA haTable EN]~ = 0) for the overall HDP, while PFBS, PFHxS and PFUA were selected for preeclampsia, none of the PFAS components was selected for gestational hypertension.Table Table Additional file We also explored the joint effect of the PFAS on hypertensive disorders in pregnancy using the structural equation model Additional file 2:Table S1. Logistic regression models for the selected exposures and hypertensive disorders of pregnancy/preeclampsia. (DOCX 15 kb)Additional file 3:Figure S2. Structural equation model including a joint latent PFAS concentration. The latent PFAS concentration is manifested by the observed PFOA, PFOS, PFHxS, PFNA, PFDoA, PFDA, PFUA, and PFBS. “Confounders” are age, education level, parity, and pre-pregnancy BMI. (PNG 29 kb)


Severe secondary hyperparathyroidism (SHPT) is a serious problem in patients undergoing hemodialysis. The efficacy and safety of microwave ablation (MWA), a minimally invasive treatment, for severe SHPT are as yet unclear. To clarify the role of MWA, we administered it to patients with severe SHPT and assessed its efficacy and safety. This was a prospective, single-center, single-arm, clinical trial. We enrolled patients with severe SHPT attending our hemodialysis center who met the inclusion and exclusion criteria. We then assessed primary outcome measures (serum concentrations of intact parathyroid hormone) and secondary outcome measures . Twenty-six patients were enrolled in this study, 10 of whom (38.46%) were responsive to MWA and 16 (61.54%) of whom were not. The main complication was hypocalcemia , which had occurred in all cases by one week after administration of MWA. Responding patients with hypocalcemia all achieved normal serum calcium concentrations within seven months and non-responding patients within three months. There were no changes in serum phosphorus concentrations after MWA in either responders or non-responders. Microwave ablation is relatively ineffective in patients with severe SHPT undergoing maintaining hemodialysis and should not be the initial therapy in such cases. Secondary hyperparathyroidism (SHPT) is a common and serious problem in patients undergoing maintenance hemodialysis. The major associated pathophysiological changes are high serum concentrations of intact parathyroid hormone (iPTH), and disorders of calcium (Ca) and phosphorus (P) metabolism; these are associated with increased mortality.,Parathyroidectomy is the standard treatment for severe SHPT.This prospective, single-center, single-arm, open-label clinical trial was designed to test the efficacy and safety of MWA in patients with severe SHPT undergoing maintenance hemodialysis. The study was approved by the institutional review board of Beijing Friendship Hospital, Capital Medical University (No. 20120179) and written informed consent was obtained from all study patients. This study was registered with ClinicalTrials.gov (No. NCT01640184).Patients undergoing maintenance hemodialysis between 1 January 2013 and 31 December 2014 in our hospital were eligible to participate. Specific inclusion and exclusion criteria were as follows.(1) Age: 18–75 years. (2) Had been undergoing maintenance hemodialysis ≥6 months. (3) Severe SHPT: high serum iPTH concentrations (>800 pg/mL) and resistance to combination therapy with a phosphorus-restricted diet, phosphate binder, and calcitriol.(1) Previous parathyroidectomy. (2) Known history of parathyroid or other neoplasms in the neck region. (3) Treatment with cinacalcet within the previous three months. (4) Currently participating in other clinical trials.All participants consented to receiving MWA. A single ultrasound specialist administered the MWA therapy to all subjects using a KY2000 ablation system  at a frequency of 2450 MHz; the ultrasound was guided in all subjects by another single ultrasound specialist.Briefly, the procedure was as follows. First, a parathyroid ultrasound was performed to determine the blood supply of the parathyroid nodule(s) and the ablation path. Second, after the neck skin had been sterilized, local anesthesia was induced with 2% lidocaine hydrochloride, following which 5 mL 2% lidocaine hydrochloride was diluted to 20 mL and then injected into the area around the parathyroid nodule(s) to create a heat insulation layer to protect the adjacent nerves and blood vessels. Third, an ablation needle was inserted into the parathyroid tissue under ultrasound guidance. Ablation was then performed, the MWA power being between 25 and 35 W according to target nodule size. When hypoechoic signals were obtained from the whole nodule(s) and no flow signals were detected, ablation was terminated.Serum iPTH, Ca, and P concentrations were measured at baseline (within one month pre-ablation), immediately (within 1 h after MWA), one week (±3 days), and one month (±3 days) after ablation, and then bimonthly.Outcomes were classified as Response or No Response.The target value of 124–558 pg/mL for iPTH concentration was chosen according to Kidney Disease: Improving Global Outcomes (KDIGO) guidelines.Secondary outcome measures were the target serum Ca (2.0–2.5 mmol/L) and P concentrations (0.97–1.62 mmol/L) in the KDIGO guidelinesFollow-up was ended when either death or recurrence occurred.All statistical analyses were performed with SPSS 19.0 . All data with normal distribution are expressed as the mean (±standard deviation), and all data with skewed distribution as the median (interquartile range). To compare changes in serum iPTH, Ca, and P concentrations before and after MWA, analysis of variance of repeated measures was used when the data were normally distributed and Wilcoxon tests when not normally distributed.Of the 351 patients receiving hemodialysis during 2013–2014 in our hemodialysis center, 27 (7.7%) were enrolled in this study, one of whom was excluded for refusing to undergo lab tests. Thus, data for 26 subjects  of average age 55.1 (±9.6) years and duration of dialysis 87.0 (±46.0) months were assessed.All parathyroid gland nodules were ablated in patients with one, two, or three nodules. All nodules were ablated in two of the four patients with four parathyroid nodules and two in each of the other two patients, the remaining two in each patient being located too deeply to permit ablation. The number of ablated nodules averaged two per patient. The power of MWA administered was 32.9 ± 5.13 W.p<.001, p<.001) (The primary and secondary outcomes are displayed in  p<.001) . From on p<.001) .p values being <.001, .005, and .013, respectively. However, iPTH concentrations did not decline to the target range (p=.021), but increased by the one-month follow-up to concentrations that did not differ significantly from baseline values  (Sixteen patients with No Response stopped follow-up at three months (when they were identified as having No Response). Compared with baseline values (1691.42 ± 354.48 pg/mL), iPTH concentrations declined significantly immediately after MWA (1022.02 ±  427.05 pg/mL), and at the one-week (1161.82 ± 437.21 pg/mL) and one-month (1210.61 ± 505.34 pg/mL) follow-ups, et range . Further p=.099) . Serum p p=.099) . One patThe most common complication was hypocalcemia ; this was identified at the one-week follow-up. Seven patients with Response  and six with No Response  presented with Ca concentrations of 1.76 (1.58–1.96) mmol/L and 1.86 (1.78–1.93) mmol/L, respectively. These patients were injected with calcium gluconate after each hemodialysis session and/or prescribed calcium carbonate and calcitriol. The calcium concentrations increased to within the normal range at the seven-month follow-up in patients with Response and at three months in those with No Response.Two patients (one each with Response and No Response) presented with hoarseness after MWA; this resolved spontaneously at two weeks and three months, respectively.In this study, we assessed the efficacy and safety of MWA in patients with SHPT undergoing maintenance hemodialysis and found that 10/26 patients were responsive and 16/26 non-responsive to it, indicating poor efficacy.,,MWA has been widely used in interventional therapy, particularly for treating tumors. Radiofrequency ablation has proven efficacy in treating SHPT.First and most importantly, there may have been some residual parathyroid cells after ablation. As this type of ablation is performed under ultrasound guidance and not direct vision, nodules may be incompletely ablated. As has been well established, disorders of calcium and phosphorus metabolism are the most important reasons for patients undergoing hemodialysis developing SHPT, a syndrome that is common in these patients.Second, we were unable to ablate all parathyroid nodules in all patients. In two of our patients, each of whom had four nodules, we elected not to ablate two of the four nodules because they were too deep and too close to the carotid artery to ablate them without injuring it. Another consideration is that ectopic parathyroid tissue is present in about 14% of subjects with SHPTThird, efficacy is highly dependent on the technique of the operator. The parathyroid glands are close to the recurrent laryngeal nerve, which is not visualized by ultrasound. Operators who believe that a nodule is very close to this nerve may thus reduce the volume ablated to avoid injuring it, resulting in incomplete ablation and lack of response or early recurrence.This study had several other limitations. First, it was a single-center study and the sample was therefore small. Larger studies are required to clarify the efficacy of MWA in subjects with SHPT. Second, because the ablations were all performed by a single operator, our findings may not be reproducible.In conclusion, we do not recommend MWA as first-line treatment in patients with severe SHPT undergoing maintenance hemodialysis because it is not sufficiently efficacious. However, further studies are required to more accurately determine the efficacy and safety of MWA in such patients.