Enterocytozoon bieneusi (E. bieneusi) have consistently been reported worldwide, garnering public attention; however, the molecular epidemiology of E. bieneusi in the giant panda remains limited. We surveyed captive giant pandas in China for the presence of E. bieneusi by using PCR and sequence analysis of the ribosomal internal transcribed spacer (ITS) revealing a 34.5% positive rate, with seven known genotypes  and five novel genotypes  identified. We similarly analyzed water samples, and E. bieneusi was detected in two samples, with genotype SC02 identified. Phylogenetic analysis revealed that CHB1 did not cluster with any recognized group, while the remaining genotypes belonged to group 1. The predominance of zoonotic group 1 genotypes indicates a public health threat that giant pandas could spread E. bieneusi to humans. The identification of E. bieneusi in water samples suggests giant pandas could contribute to water contamination. Effective control measures are therefore needed to minimize the contamination of the water and prevent a human microsporidiosis outbreak.Human and animal infections of  Enterocytozoon bieneusi, which is responsible for ~90% of all cases of human microsporidiosis1. The first case of human E. bieneusi infection was reported in 19852, and increasing numbers have been reported worldwide ever since. In humans, E. bieneusi infection can cause persistent diarrhea, malabsorption, and wasting diathesis in immunocompromised patients, particularly those suffering from acquired immunodeficiency syndrome (AIDS), who can develop life-threatening chronic diarrhea. Immunocompetent patients can also suffer from self-limiting diarrhea for up to one month5. In addition to infecting humans, this parasite also has a wide host range, including domestic animals and wildlife. However, there remains no reliable effective treatment for the disease6. Owing to their significance and potential threat to public health, microsporidia are on the National Institute of Allergy and Infectious Diseases (NIAID) Priority Pathogens List and are considered Category B pathogens (https://www.niaid.nih.gov/research/emerging-infectious-diseases-pathogens).Microsporidiosis is an emerging infectious disease caused by microsporidial parasites, including the most common and environmentally ubiquitous species, E. bieneusi isolates cannot be discriminated morphologically, the internal transcribed spacer (ITS) region of the rRNA gene of E. bieneusi has a high degree of diversity. Thus, it is commonly used in many studies for the detection and identification of E. bieneusi genotypes. To date, over 200 genotypes have been identified and classified into eight groups (groups 1–8) based on phylogenetic analysis. Among these, genotypes belonging to group 1 are considered zoonotic and pose a major threat to humans, while those in other groups are regarded as having little or no public health significance.While Ailuropoda melanoleuca) are bears native to south central China on the International Union for Conservation of Nature (IUCN) Red List and are considered living fossils. Currently, the number of captive giant pandas is just over 370 according to the results of the fourth national giant panda survey in China. There are many factors that have led this animal to be endangered, such as its low reproductive rate, loss of habitats, and lack of food. The common occurrence of parasites in giant pandas also poses a threat to their survival7. Moreover, in China, human E. bieneusi infections have been reported in several areas, with infection rates ranging from 0.2% to 11.9%10, and the transmission route remains unknown. While the giant panda could be a source of human microsporidiosis, the role of the giant panda in the transmission of E. bieneusi has been poorly investigated. Thus, we examined the occurrence of E. bieneusi in giant pandas and their potential role in the zoonotic transmission of human microsporidiosis.Giant pandas , 13.89% among red pandas (Ailurus fulgens), 12.25–27.7% among foxes (Vulpes lagopus), 4.1–10.5% among raccoon dogs (Nyctereutes procyonoides), and 16.4% among blue foxes (Alopex lagopus)15. Our results therefore reveal the common occurrence of E. bieneusi infection in pandas. While pandas of different ages and genders were found to be infected with E. bieneusi, there were no significant differences in infection rates among yearling (20%), sub-adult (40%), and adult giant pandas (35.2%)  or between males (42.7%) and females (31.4%)   (Table E. bieneusi was found in 5/5 (100%) conservation bases and 8/15 (53.3%) zoological gardens  was observed among conservation bases, while an infection rate of 45.2% (14/31) was observed among zoological gardens. No significant difference was found in terms of the infection rates of the conservation bases and zoological gardens  (found in Sichuan province) and type I-like genotype (n = 4) (found in Shaaxi province)11. In the present study, we identified seven known genotypes  and five novel genotypes , with SC02 (n = 50) being predominant, followed by genotypes D (n = 3), SC06 (n = 2), CHB1 (n = 2), F (n = 2), EbpC (n = 2), SC01 (n = 2), and SC04 (n = 2); SC05, SC07, SC08, and Peru 6 were only detected in one specimen each , Malayan sun bear , horse, and red-bellied tree squirrels 17, and it has also been found in humans (GenBank accession number KY465443). The giant panda belongs to the family Ursidae; in studies reporting infections of E. bieneusi in this family, several genotypes have been identified, including Peru 6, CHB1, SC02, horse2, ABB1, ABB2, and J. These results indicate that cross-species transmission may be occurring. Among the genotypes observed in humans in China, in addition to SC02 (GenBank accession number KY465443), genotypes EbpC and D have been described9. The presence of SC02 and D in giant panda may pose a threat to the public.Among the seven known genotypes found in giant pandas, the most prevalent, SC02, has been previously reported in other animal hosts  have been previously found in water samples from China25. The presence of SC02 genotype in water may support that giant pandas could be a source of water contamination due to the close contact between giant pandas and water , further contributing to the spread of E. bieneusi. Notably, as the giant panda is considered one of China’s national treasures, some giant pandas are sent to foreign countries, which may broaden the geographic range of E. bieneusi and facilitate the global transmission of this pathogen.The transmission of microsporidia can include airborne, person-to-person, zoonotic, and waterborne routesusi Fig. . Microspter Fig. , human iE. bieneusi infection (34.5%) in captive giant pandas in China and reports 11 E. bieneusi genotypes identified for the first time in giant pandas. Zoonotic group 1 genotypes predominated among giant panda isolates, indicating a potential public health threat. We also detected E. bieneusi in water samples genotyped as SC02. Our study indicates that giant pandas could spread E. bieneusi to humans and be a source of water contamination. Further research on the contamination of more water samples and the potential spread of E. bieneusi to humans is needed to elucidate E. bieneusi transmission routes.Our study reveals the common occurrence of This study complied with the guidelines of the Regulations for the Administration of Affairs Concerning Experimental Animals and was approved by the Animal Ethical Committee of Sichuan Agricultural University. No animals were harmed during the sampling process. Permission was obtained from China Giant Panda Protection and Research Center for the collection of fecal specimens. All the procedures were carried out in accordance with the approved guidelines.From May 2016 to June 2017, 200 fecal samples were collected from captive giant pandas from five conservation bases (n = 169) and 15 zoological gardens (n = 31) in China. Eight water samples were collected from Chengdu research base of giant panda breeding where the fecal samples were mainly collected. Also, samples were collected from water ponds where giant pandas took bath. All samples were placed on ice in separate containers that were marked with sample information such as gender, age  and collection location (for water samples) and then were transported to the laboratory immediately. None of giant pandas had any apparent diarrhea at the time of sampling Fig. .19 and 300 μl sample concentrates were used for DNA extraction. The DNA samples were stored in 200 μl of the solution buffer from the kit at −20 °C until use.We used E.Z.N.A.® Tool DNA Kits  for DNA extraction following the manufacturer’s protocol. For fecal samples, genomic DNA was extracted directly from approximately 200 mg of fecal sample. Whereas, for each water sample, prior to DNA extraction, we concentrated the pathogen by centrifugation at 6000 × g for 10 min as described previouslyE. bieneusi using primers and amplification conditions described by Sulaiman et al.28. To amplify the ITS region of the rRNA gene, we use primers 5′-GATGGTCATAGGGATGAAGAGCTT-3′ and 5′-AATACAGGATCACTTGGATCCGT-3′ for the primary PCR, and 5′-AGGGATGAAGAGCTTCGGCTCTG-3′ and 5′-AATATCCCTAATACAGGATCACT-3′ for the secondary PCR. The annealing temperature is 55 °C for both primary and secondary PCR. Secondary PCR products were visualized by staining with Golden View following 1% agarose gel electrophoresis.The ITS gene was amplified for the identification of E. bieneusi reference sequences downloaded from the GenBank database using Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and ClustalX software . Phylogenetic analysis was performed by constructing a neighbor-joining tree using Mega 6 software (http://www.megasoftware.net/) based on the evolutionary distances calculated using a Kimura 2-parameter model. The reliability of the tree was assessed using bootstrap analysis with 1,000 replicates.Amplicons of the expected size (392 bp) were sent to Invitrogen  for sequencing. A two-directional sequencing method was applied to ensure sequence accuracy. All nucleotide sequences obtained in this study were aligned with 2) conducted with SPSS version 17.0 software . A P-value < 0.05 was considered significant.Differences between the tested conservation bases and zoological gardens in infection rates and the prevalences among different age groups and genders were compared using Chi-square tests (χRepresentative sequences identified in this study were deposited in the GenBank database (KY950533-KY950544).All data generated or analysed during this study are included in this published article and its Supplementary Information files.Supplementary Figure 1


Genetic improvement of soybean protein meal is a complex process because of negative correlation with oil, yield, and temperature. This review describes the progress in mapping and genomics, identifies knowledge gaps, and highlights the need of integrated approaches.Glycine max (L) Merr.] seed is the primary source of protein in poultry and livestock feed. Protein is a key factor that determines the nutritional and economical value of soybean. Genetic improvement of soybean seed protein content is highly desirable, and major quantitative trait loci (QTL) for soybean protein have been detected and repeatedly mapped on chromosomes (Chr.) 20 (LG-I), and 15 (LG-E). However, practical breeding progress is challenging because of seed protein content’s negative genetic correlation with seed yield, other seed components such as oil and sucrose, and interaction with environmental effects such as temperature during seed development. In this review, we discuss rate-limiting factors related to soybean protein content and nutritional quality, and potential control factors regulating seed storage protein. In addition, we describe advances in next-generation sequencing technologies for precise detection of natural variants and their integration with conventional and high-throughput genotyping technologies. A syntenic analysis of QTL on Chr. 15 and 20 was performed. Finally, we discuss comprehensive approaches for integrating protein and amino acid QTL, genome-wide association studies, whole-genome resequencing, and transcriptome data to accelerate identification of genomic hot spots for allele introgression and soybean meal protein improvement.Meal protein derived from soybean [The online version of this article (doi:10.1007/s00122-017-2955-8) contains supplementary material, which is available to authorized users. Such cultivars are inherently less valuable than their higher protein counterparts. This is a concern for the US soybean growers and breeders. Commercial soybean cultivars typically contain about 38–42% seed protein on a dry weight basis. The seed protein content needs to be at least 41.5% on a dry weight basis to produce meal with ≥47.5% protein from a soybean cultivar with 22% oil content . In addition, demand for soybean food products  has increased fivefold (from $1 billion in year 2000 to $4.5 billion in 2013) due to increasing awareness of nutritional value and health benefits of soybean in the human diet (http://www.soyfoods.org/). Corn (Z. mays), sorghum (S. bicolor), and other cereal grains are also commonly fed to swine and poultry. Yet, these meals are commonly lower in protein content, crude fiber, and lower in tryptophan, threonine, isoleucine, and valine than soybean meal. Soybean meal is not an ideal protein source because it is typically (1) lower in the amino acids, methionine, and cysteine, often leading to additional supplements to the meal; (2) high in oligosaccharides that reduce metabolizable energy, and (3) high in potassium leading to higher moisture content in litter, which indirectly affects growth in chickens   and South America (http://www.soyinfocenter.com/) did not begin cultivation of soybean until 1765 and 1882, respectively . This use increased the demand for higher protein content and convinced breeders towards breeding for higher seed protein in recent years. On the other hand, soy foods including tofu, soymilk, natto, and edamame have generated tremendous interest especially with Korean and Japanese soybean breeders because of their nutritional value and human health benefits  and meal (feed), and it benefited other crops in rotation Brown . The suchttp://www.soystats.com). In the US, a larger portion of soybean (79%) is intended for export and a small amount is used for human food (~2%), and the rest (~18%) are processed to extract seed oil, and the resulting protein meal undergoes complex processing to make defatted soybean meal . In 2015, about 31% of the crop area (82.7 M acres) was planted to soybean in the US, which was slightly less than corn planting (34%). There was an increase of 6.9 M acres in soybean planting compared to that of 2013. Soybean production in Brazil and Argentina has grown rapidly in the recent years and soybean exports have grown accordingly. The average soybean yield (from 1980 to 2014) in the US (2.5 MT/h) exceeded that of Brazil (2.2 MT/h) and Argentina (2.3 MT/h) ] followed by Brazil with 30% (94.5 MMT), and Argentina with 18% (56 MMT)  leads the world soybean meal export followed by Brazil (22%) and US (18%). This is because about 75% of soybean meal produced in the US is used domestically , Feb. 2016). Growing conditions as well as processing techniques affect the digestibility of amino acids in the soybean meal. To determine the relative feeding value of alternative protein sources, the lysine level is estimated, which in-turn determines the comparative economic value of the protein source. A comparative study of soybean meal from Argentina, Brazil and the US showed that the US soybean meal samples contain relatively higher lysine, cysteine, and threonine levels compared with South American soybean meal. Similarly, Ravindran et al.  and Kim http://www.soybase.org), only two QTL, one on Chr. 15 (cqPro-15) and another one on Chr. 20 (cqPro-20) are designated as officially confirmed QTL based on error rate (lower than 0.01) and confirmation study showing alleles at the same locus are segregating in all the test populations (http://soybase.org/). The QTL on Chr. 20 has been the focus of several studies, including the testing of the high protein allele in different genetic backgrounds and showed a large additive effect , Asian landraces, and several wild soybean lines (G. soja) using the SoySNP50K data set. The analysis result in three clades where Danbaekkong clustered with NAA as expected (data not shown). However, when the SNPs in the 27–32 Mb on Chr. 20 were analyzed, the Danbaekkong clustered separately from NAA as well as landraces and G. soja accessions  and about 75% of the genes in soybeans are present in multiple copies  family . In a recent study, major QTL for essential amino acids and crude protein were identified on Chr. 20 in the Benning × Danbaekkong population  revealed limited studies of QTL for amino acid composition (Supplementary Table 3). Panthee et al.  identifiB. napus) and soybean, which significantly improved the lysine content by twofold, however, the germination and seed quality were poor  phenotypic data for total seed protein content. The phenotypic variation showed the advantage of protein content in late maturity group (MG V—X) lines compared to early maturity group lines (MG 000—II)  Fig. . It has II) Fig. , suggestII) Fig. . In two II) Fig. . They foII) Fig. . Wolf etII) Fig.  studied II) Fig. . These aII) Fig.  observedII) Fig. . AdditioII) Fig. . The keyII) Fig. .Fig. 6CoE3 and E4. Hudson (cis-regulation) by circadian cycle.It is a well-known phenomenon that day length (photoperiod) and day/night temperature (circadian cycle) controls flowering time  is a unique resource to study regulation of protein and amino acid biosynthesis because the seed concentration of these components are higher in G. soja compared with G. max. However, these resources are still mostly untapped by breeders and others in soybean research community. The reason for underutilization of wild soybean for seed composition could be due to linkage drag on favorable agronomic characteristics . Researchers have successfully introgressed the high protein allele from cv. Danbaekkong into locally adapted lines, however, it may reduce the concentration of some essential amino acids (unpublished). Therefore, it may be necessary to utilize wild species accessions as introgression libraries as well as developing inter-specific populations. On the other hand, landraces and elite cultivars can be used to develop mapping populations, reference/core sets and training populations, and elite breeding lines can be included in training populations  have benefited some breeders and accelerated the potential rate of genetic improvement by molecular breeding . Nevertheless, it is important to take into account that induced mutations can also make undesirable changes in the genome leading to yield drag and other detrimental effects  have been useful in crop improvement  have greater applications in trait discovery, functional characterization, and crop improvement. These approaches have been utilized in the past to improve seed composition traits in soybean with varying success rates , precise and high-throughput phenotyping, and considering mutation breeding and genome engineering/editing. Integrating these aspects and comprehensive learning will extend our current genetic and genomic portfolio far beyond that of traditional breeding. Soybean meal will, however, only remain a leading source of protein if its quality  can exceed that of other competing meal sources. To accelerate this demand on a larger scale, an integration of breeding and genomics-assisted approaches is required at a greater extent than currently being implemented.GP, TV, VP, QS, PC, GS, and TC have performed the ground work and inquest for all the relevant literature and written the review. GP performed haplotype and synteny analysis. RM and HTN conceptualized and edited the review.Supplementary material 1 (DOCX 14 kb)Supplementary material 2 (DOCX 35 kb)Supplementary material 3 (DOCX 26 kb)Supplementary material 4 (DOCX 20 kb)Supplementary Figure 1: Syntenic analysis of major protein QTL on Chr. 20 (Gm20: 29803077–31462800) (JPEG 1461 kb)Below is the link to the electronic supplementary material.


BMI, that comprises both fat mass (FM) and lean mass (LM) may contribute to peak bone mass achievement in different ways. This study explored the influence of body composition in terms of total body LM and FM on hip aBMD-values in adolescence.Positive association between body weight and bone mass is well established, and the concept of body mass index (BMI) is associated with higher areal bone mineral density (aBMD) and reduced fracture riskFN) and total hip (aBMDTH) (g/cm2), total body LM and FM (g) were measured by dual energy X-ray absorptiometry (DXA). Height and weight were measured, and BMI calculated. Lifestyle variables were collected by self-administered questionnaires and interviews, including questions on time spent on leisure time physical activity. Stratified analyses of covariance and regression models included 395 girls and 363 boys. Crude results were adjusted for age, height, sexual maturation, physical activity levels, vitamin D levels, calcium intake, alcohol consumption and smoking habits.In 2010/2011, 93% of the region’s first-year upper-secondary school students (15–17 years old) in Tromsø, Norway attended the Tromsø Study, Fit Futures. Areal BMD at femoral neck (aBMDp < 0.001), but higher aBMD at higher FM-levels were found only in girls (p < 0.018). After multiple adjustments, aBMDFN-levels in girls were associated by 0.053 g/cm2 and 0.032 g/cm2 per standard deviation (SD) change in LM and FM (p < 0.001). Corresponding values in boys were 0.072 and 0.025 (p < 0.001). The high LM groups accounted for the highest aBMD-levels, while aBMD-levels at the LM/FM-combinations indicated different patterns in girls compared to boys. The adjusted odds ratio (95% CI) for low levels of aBMDFN was 6.6  in boys, compared to 2.8  in girls per SD lower LM.Unadjusted distribution indicated higher aBMD-levels at higher LM-levels in both genders (. A gender specific difference indicated that high lean mass is of crucial importance prominently in boys. In adolescents with low lean mass, especially in girls, high fat mass may partially ameliorate the effect of deficient lean mass levels.LM and FM should be regarded as strong predictors for bone mass and hence bone strength in adolescents Osteoporoses as well as osteoporotic fractures are major health problems in Western Societies, and areal bone mineral density (aBMD) is a strong predictor of future fracture risk . An indiIn a previous study of an adolescent population, association between BMI and aBMD levels at the hip were modest, but statistically significant . HoweverThe concept of BMI comprises stature and weight, while body weight is a compound of bone, fat and muscle. Lean mass (LM) is likely to be responsible for the positive association between BMI and bone mineral parameters , 13–16, In a former study we found that higher BMI-levels were related with lower levels of physical activity  . SurprisOn this basis we wanted to study the influence of body compositions measures on aBMD at the hip in a large representative sample of Norwegian adolescents, homogenous in age and ethnicity. We hypothesized that the relationship between fat mass and lean mass and areal bone mineral density would vary between girls and boys. We furthermore aimed to examine whether there are certain gender specific combinations of total body lean mass and fat mass, which are more beneficial for bone health.The Tromsø Study  is a popIn 2010/2011 all first-year upper-secondary school students in the two neighbouring municipalities Tromsø and Balsfjord were invited to participate in the first cross sectional study TFF1 [Information about the study was given in classrooms and written information was distributed through the schools web sites. Participants signed a declaration when arriving at the study site, in addition participants younger than 16 had to bring written permission from their guardians. Dedicated research technicians performed the examinations in a well-established research unit at UNN HF. The Norwegian Data Protection Authority (reference number 2009/1282) and The Regional Committee of Medical and Health Research Ethics (2011/1702/REK Nord) approved the study in July 2010 and October 2011, respectively.FN) and at total hip (aBMDTH) expressed as g/cm2, measured by dual x-ray absorptiometry (DXA) , and analysed with enCORE paediatric software [TH and aBMDFN respectively [The main outcomes in the present study were areal bone mineral density at femoral neck (aBMDsoftware . The sofectively .2). Non-fasting blood samples were collected, and analysed for serum 25 hydroxy vitamin D levels, with a reference value of 50–113 nmol/l, and a CV < 6% [Height and weight were measured in all participants to the nearest 0.1 cm and 0.1 kg on an automatic electronic scale, the Jenix DS 102 stadiometer . Measurements were performed according to standardized procedures in the Tromsø Study, with participants wearing light clothing without shoes. BMI was calculated as weight (kg) divided by the height squared  like hypothyroidism (E03), diabetes type 1(E10), eating disorders (F50.9), celiac disease (K90.0) and arthritis (M13) were separated from other diseases. Just like medication (ATC); plain corticosteroids (D07A), thyroid preparations (H03A), antiepileptic (N03A), corticosteroids (R01AD), glucocorticoids  (R03BA) and corticosteroid for systemic use (H02A), were separated from other medications.Sexual maturation was based on girls’ self reported menarche age and boys’ rating at the Puberty Development Scale (PDS) . The boyFN, aBMDTH, serum vitamin D levels and puberty score. Percentage and numbers described physical activity levels, smoking habits, and calcium intake and alcohol consumption. Correlation between body composition and anthropometric measurements were explored. As body weight was highly correlated to BMI and FM we excluded weight from further analyses (Pearson’s correlation coefficient > 0.7). Mean values of body composition at the four physical activity levels were compared, and trends estimated by linear regression.All analyses were performed sex stratified, as magnitude and tempo of bone mass acquisition differs between girls and boys . We calcWe explored the distribution of aBMD across FM and LM tertiles and estimated trends by linear regression Fig. . In girlWhen we explored the associations of BMI, LM and FM with aBMD, there was a statistical significant interaction between LM and FM. According to Kirkwood and Stern  it is noBy one-way between-group analyses of covariance (ANCOVA) we compared means and confidence intervals (CI) for aBMD across FM and LM tertiles in simple as well as in more complex models adjusted for age, height, sexual maturation, physical activity levels, calcium intake, vitamin D levels, alcohol consumption and smoking habits. The trends were estimated by linear regression, and by 2-ways ANCOVA Fig.  we compaFN-score ≥ 1 SD below mean versus not. This cut-off value was based on the hypothesis of a fracture risk reduction by 50%, when the bone mass amount is raised by one standard deviation at the end of skeletal maturation [At the end we estimated by logistic regression, the impact of lower LM and FM levels together with the above described covariates, on the likelihood of having an aBMDturation . We checp < 0.05 were considered significant.All analyses were performed by Statistical Package of Social Sciences software (SPSS v. 22) and values of Complete data sets were available for 395 girls and 363 boys aged 15–17 years old i.e. 68% of the eligible population, 73% of the participating youths  (data not shown).The waist majority (98%) of the cohort reported white ethnicity. Among girls, 123 (31%) reported use of oral contraceptives. A total of 10  and 18  participants reported diseases and medication known to affect bone, respectively (data not shown). There were no statistically significant relationships between overall BMI and physical activity levels, whereas higher levels of physical activity were significantly associated with higher levels of LM and lower levels of FM (p < 0.001), whereas across FM-tertiles such trends were seen in girls only (p ≤ 0.018)  and FM tertiles (p ≤ 0.016) at femoral neck and total hip. In boys there were positive trends across LM tertiles at both femoral sites (p < 0.001). However, across FM tertiles a weak, but significant positive trend (p = 0.010) was found at the femoral neck site, but not at total hip (data not shown).Distribution of crude aBMD-levels at the hip sites indicated trends of higher median values across LM-tertiles in both genders (18) Fig. . The ANC2 in girls and 0.083 g/cm2 in boys (p < 0.001) at femoral neck per SD higher LM. Correspondingly per SD higher FM; 0.028 g/cm2 (p < 0.001) and 0.011 g/cm2 (p = 0.15) for girls and boys respectively. In multiple regression models, LM and FM were significantly related to aBMD along with a statistically significant LM*FM interaction term, in girls minus 0.011 (p = 0.006) and in boys minus 0.016 (p = 0.005) (data not shown).Simple linear regression analyses Table  showed pFN in all FM groups, with the highest increment per SD change, in the lowest FM group . Correspondingly FM was significantly associated with aBMDFN in the adjusted models, for all LM groups (p ≤ 0.017), with the highest beta  seen in low LM group , with highest beta value in the medium tertile (0.095 g/cm2), while FM only had significant relationship with aBMD in the low LM group . For both genders analyses at the total hip site revealed similar patterns, suggesting a consistent association between LM and FM and femoral aBMD.Furthermore, in girls LM was significantly associated with aBMDHigher aBMD-values at femoral neck across LM/FM-combinations in boys were contrasted by the more complex patterns seen in girls Fig. . The higThe odd ratio (OR) for having aBMD score one SD below mean values, also suggested sexual dimorphism; for each SD lower LM the OR raised by 6.6 (95% CI: 3.4–13.0) in boys and 2.8 (95% CI: 1.6–4.9) in girls after multiple adjustments (data not shown). Similar analyses for FM turned out statistically non significant.We found a gender specific variation in LM’s and FM’s relationships with hip aBMD, after adjustments for powerful covariates known to influence bone acquisition. In girls there was a trend of higher aBMD-levels across LM tertiles and FM tertiles at femoral neck and total hip. In boys, a positive trend was observed across LM tertiles at both sites. By contrast, across FM tertiles, such a trend was only present at the femoral neck site. In addition, low LM levels suggested a doubled likelihood for lower aBMD levels in boys compared to girls. Compared to FM, LM explained more of the variation in hip aBMD, in both genders. LM/FM-combinations including high LM stood out as most beneficial for aBMD, whereas in adolescents with low LM levels, FM was of considerable importance, observed more prominently in girls.FN and whole body BMC for both genders, an association not seen in the other LM tertiles. Despite Kouda et al’s smaller study cohort (n = 235), the similarities in findings are striking, especially the association between FM and aBMD among adolescents with low LM. As summarized by Kouda et al., several studies of adolescents confirm the beneficial effect of LM on bone health, whereas studies on the associations between FM and bone provided conflicting results, some indicating a positive independent relationship [In a cross-sectional study of Japanese adolescents in a comparable age group , Kouda etionship , 14 whiltionship –37. The tionship . Also Fotionship . They foFN was significantly higher than the correlation between FM and aBMDFN. As in our study the effect of LM was greater in men than in women. Also in a Norwegian cohort consisting of middle aged and elderly men and women, LM was more strongly correlated to aBMDFN compared to FM [Our results are in agreement with previous findings in adults, like the meta-analysis on the association between LM, FM and aBMD , which ied to FM . WhereasFN, Ho-Pham et al. concluded that physical activity is an important intervention for prevention of bone loss and osteoporosis in the adult population [With the strong association between LM and aBMDpulation . Our finpulation  may explpulation , in addipulation . The propulation , indicatpulation .Several mechanisms contribute to the understanding of FM’s relationship with bone. Except greater load to the skeleton, secretions of adipocyte hormones (leptin) and pancreatic beta cell hormones (insulin etc.) are directly influenced by FM and associated with higher bone mass measures . MoreoveFN differences between the low LM/FM- and the high LM/FM-combinations were 0.129 g/cm2 and 0.149 g/cm2 in girls and boys respectively. These numbers equals one SD aBMD in this study population close to PBM level and correspond to a fracture risk difference of 50% in adulthood [An important implication of the findings from our study is that modifiable lifestyle factors seem to have a strong influence on peak bone mass attainment, and possible later fracture risk. The aBMDThe strength of this population-based study is connected to the large representative sample of Norwegian adolescents, quite homogenous according to age and ethnicity. The survey setup ensured high quality data acquisition, allowing control for important confounding factors. The results are in line with previous findings, and the large sample size allows sub-group analyses. Due to incomplete data 15% girls and 26% boys younger than 18 were excluded from analyses, mainly because of missing vitamin D-levels (girls) and puberty score (boys). The excluded girls were shorter, with higher BMI and FM levels. While the excluded boys were slightly younger, at an earlier stage of puberty, reported lower physical activity levels and more frequent smoking. It is not likely an inclusion of those participants would have altered the conclusions, but it may have strengthened the findings in girls and attenuated the boys’ results.The main limitation is the cross sectional design, which capture only a temporal window of young adolescent life. In this age-group rapid changes in height, weight and pubertal status may still occur. The inconsistency in conclusions of FM’s relationship with bone in adolescents may be a result of a delicate and changing relationship during age and hormonal state . UnfortuThe main outcome, aBMD derived by DXA-scans has limitations connected to being two-dimensional, rather than three-dimensional. By not take into account the third dimension, the technique is unable to measure the true volumetric density . HoweverTo our knowledge in addition to the study of Foley and colleagues, two longitudinal studies in corresponding age groups have been published, both on structural analyses of bone strength. Jackowski et al. investigated LM’s influence on bone during adolescence into early adulthood, and concluded that LM contributes to structural strength at the hip in both genders , while SThis cross sectional study among 15–17 year old Norwegian adolescents, supports the importance of LM as a strong independent predictor for bone mass, along with FM’s significant contribution to bone mass in girls and in adolescents with lower lean mass levels. Our observations indicate a gender specific variation; high lean mass levels were strongly associated with boys’ aBMD-levels at the hip, which highlights the negative relationship between a sedentary lifestyle and bone strength. In contrast, the results indicated a more complex balance between lean mass and fat mass in adolescent girls. The gender specific delicate balance between fat and lean mass pin points the significance of nutrition and physical activity for sound bone health in adolescence.


Our results highlight no robust evidence that the 2014 EBOV outbreak is fast-evolving and adapting to humans. Therefore, the unprecedented nature of the 2014 EBOV outbreak might be more likely related to non-virological elements, such as environmental and sociological factors.Following its immergence in December 2013, the recent Zaire Ebola virus (EBOV) outbreak in West Africa has spread and persisted for more than two years, making it the largest EBOV epidemic in both scale and geographical region to date. In this study, a total of 726 glycoprotein (GP) gene sequences of the EBOV full-length genome obtained from West Africa from the 2014 outbreak, combined with 30 from earlier outbreaks between 1976 and 2008 were used to investigate the genetic divergence, evolutionary history, population dynamics, and selection pressure of EBOV among distinct epidemic waves. Results from our dataset showed that no non-synonymous substitutions occurred on the GP gene coding sequences of EBOV that were likely to have affected protein structure or function in any way. Furthermore, the significantly different  Filoviridae family, is an enveloped, non-segmented, negative strand RNA virus approximately 19 kb in length, which has caused considerable morbidity and mortality in West African human populations236789The Zaire Ebola virus (EBOV), a member of the dN) and synonymous substitutions (dS) can provide information on the type of selection that has acted on a given set of protein-coding sequences, and has become a standard measure of selective pressuredN/dS ratio is also referred to as the ω ratio or KA/KS, with ~1, <1, and >1 indicating neutral evolution, negative or purifying selection, and positive or diversifying selection, respectively.Understanding the natural selection that has shaped genetic variation is a critical aspect for the evaluation of how certain pathogens adapt to host populations. Great efforts have been made in recent decades to understand and identify the “molecular footprint” left in nucleotide and protein-coding sequences by the past action of natural selection. Therefore, identification of genes or genomic regions that have been targeted by natural selection, then pinpointing the genotypic variation that causes a certain pathogen to adapt to a host population, is essential for the development of vaccines and therapeutics. Comparison of the relative rates of non-synonymous . In cases of multiple sequences per patient, only one sequence was randomly selected. The final dataset included 756 GP gene sequences  of EBOV from the period of 1976–2015 with known sampling dates and geographic locations were retrieved from the GenBank database of the National Center for Biotechnology Information .Maximum-likelihood (ML) phylogenetic trees were constructed using RAxML v8.0.9http://tree.bio.ed.ac.uk/software/pathogen/) and second by checking the posterior distribution of the coefficient of variation statistics. Evolutionary rates were estimated using Bayesian Markov Chain Monte Carlo (MCMC)2426http://beast.bio.ed.ac.uk/software/tracer). Maximum-clade credibility (MCC) trees summarizing all MCMC samples were generated using TreeAnnotator v1.8.2 with a burn-in rate of 10%To investigate the temporal signal and “clocklikeness” of molecular phylogenies of the dataset, linear regression was performed on the root-to-tip distances of samples versus the date of the isolate on a ML phylogeny inferred using RAxML v8.0.9dS) and non-synonymous (dN) substitutions averaged over the entire alignment can provide a crude measure of the overall strength of selection on the coding region. Global estimates of dS and dN can be obtained by fitting a codon substitution model to a given alignment and corresponding tree. Global dN/dS ratio estimation was performed using the AnalyzeCodonData.bf batch file available under the Standard Analyses menu in HyPhy v2.2.4 (http://www.hyphy.org)Comparing the global estimates of synonymous  was used to reconstruct evolutionary histories of individual sites to find evidence of co-evolution between sites in our dataset by the QuickSelectionDetection.bf batch file in HyPhy v2.2.4, which is found in the Standard Analyses menu, under the heading Positive SelectiondN/dS ratio, ω ratio, or KA/KS) for all GP codon sites and all branches of the phylogeny of EBOV, three different model classes were examined using the CODEML program in phylogenetic analysis by maximum likelihood (PAML) v4.8To assess non-synonymous/synonymous substitution rates  and 0.342 (95% confidence bounds: 0.254–0.449), respectively. The estimation for the internal branch separating the 2014 West African outbreak and earlier outbreaks was 0.194 (95% confidence bounds: 0.101–0.334), indicating a lower value compared with the above two branches. The likelihood ratio test comparing the single rate model resulted in a p-value of 0.008, indicating significantly different global dN/dS ratios between the 2014 West African lineage and earlier lineages. However, testing global dN/dS variation among branches using the TestBranchDNDS.bf batch file in HyPhy v2.2.4p = 0.249) and SharedNS1 was ~0.305. Most notably, the 12 BGM analyses showed zero edges with high (≥0.95) posterior support, although a small number of edges with weak (≥0.5) posterior support were identified.The global dN/dS ratios between the 2014 West African outbreak and earlier outbreaks , in accordance with the PAML analysesp = 0.014). Parameter estimates under Model D with k = 3 site classes indicated a large set of sites (56.338%) evolving under strong purifying selection (ω = 0.01242), a small set of sites (1.13%) evolving under selective pressure (ω = 6.28165), and a large set of sites (42.532%) evolving under divergent selective pressure, with purifying selection in earlier outbreaks (ω = 0.29709) and weak purifying selection in the 2014 West African outbreak (ω = 0.86160).The one-ratio model (M0) yielded an estimated ω of 0.379, indicating that purifying selection dominated the evolution of the EBOV GP gene. This finding was based on an average over all sites and branches. However, the one and two-ratio model comparisons of branch-specific models using the CODEML program in PAML v4.8On 29 March, 2016, the WHO stated that the EBOV situation in West Africa no longer constituted a public health emergency of international concern and the temporary recommendations adopted in response to the outbreak could be terminatedThe GP gene is the most widely studied of the EBOV genome, and its expression on the particle surface is the only transmembrane protein of EBOV responsible for receptor binding and membrane fusion495152169dN/dS ratios between the 2014 West African outbreak and earlier outbreaks were detected, suggesting significantly positive selection or purifying selection , or just strictly or relaxed selective constraints or divergent selection pressures in the 2014 West African outbreak. However, no evidence of positive selection was detected .


Pneumocystis jirovecii infection requiring intensive care in a pediatric patient with inflammatory bowel disease (IBD). The literature was reviewed and there were 92 reported cases of Pneumocystis pneumonia (PCP) in patients with IBD. Most sources were case reports and there was likely reporting bias toward patients receiving immunomodulators, anti-tumor necrosis factor (anti-TNF) therapy, and those who died. Overall, 56% of patients were males and 58% had Crohn’s disease. The median age was 45 years  and 86% of patients were lymphopenic. The case-fatality rate was 23%. Corticosteroids were used as IBD treatment in 88% of patients who subsequently developed PCP, 42% received thiopurines, 44% used anti-TNF therapy, and 15% received either cyclosporine or tacrolimus. Rates of mono, dual, triple, and quadruple immunosuppression therapy were 35, 35, 29, and 2%, respectively. This report highlights the importance of considering PCP in immunosuppressed lymphopenic pediatric IBD patients who present with unusual symptoms. Moreover, it should give gastroenterologists the impetus to limit immunosuppressive therapy to its minimal effective dose and consider options such as exclusive enteral nutrition wherever possible. Although there is no place for global PCP prophylaxis in IBD given the low incidence, in an era when there is increasing use of biologic agents with combination immunosuppressive therapy, the risk-benefit profile of PCP chemoprophylaxis should be revisited in selected cohorts such as patients on triple immunosuppression with corticosteroids, thiopurines, and a biological agent or calcineurin inhibitor, especially in lymphopenic individuals.Immunosuppressive therapy is a known risk factor for opportunistic infections. We report the first case of severe  Pneumocystis jirovecii (P. jirovecii) infection requiring intensive care in a pediatric patient with IBD.Immunosuppressive therapy is a known risk factor for opportunistic infections . We repo8 , 6-MMPN 734 pmol/8 × 108 ].A 12-year-old Caucasian girl was diagnosed with gastric and ileocolonic Crohn’s disease (CD), Paris classification L3, L4a, B1, G1, having presented with growth failure and abdominal pain and having undergone endoscopic and magnetic resonance enterography assessment. She failed exclusive enteral nutrition (EEN) and was treated with oral prednisone tapered over 3 months with maintenance azathioprine (AZA) (2 mg/kg/day). Treatment resulted in resolution of clinical symptoms and improved biochemical markers. She had recurrence of symptoms after 9 months with abdominal pain and diarrhea. Infective work-up was negative and she was commenced on oral budesonide 9 mg and her AZA dose was increased (2.5 mg/kg/day). Thiopurine metabolites at this stage were normal ; however, after AZA initiation, she had intermittent lymphopenia (0.4–1.2 × 109/l). Thiopurine methyltransferase activity testing was unfortunately unavailable at diagnosis.Prior to discharge, she was investigated for an underlying primary immunodeficiency. Human immunodeficiency (HIV) testing was negative and immunoglobulin levels were normal. B and T cell panel revealed a low absolute count of CD3, CD4, and CD8  and normal T-cell numbers. She developed an urticarial rash thought to be secondary to TMP–SMX 4 days post discharge and was changed to oral clindamycin and primaquine to complete the 21-day treatment course. She was then started on prophylactic oral dapsone, which was discontinued when she developed arthralgia. She has not subsequently taken PCP prophylaxis.She was discharged on oral TMP–SMX for 21 days, having had 3 days of IV therapy. She received 3 days of methylprednisone followed by 5 days of oral prednisone, which was tapered over 2.5 months. AZA was restarted at a reduced dose (1.5 mg/kg/day). Within 2 months, she had normal lymphocyte numbers with a normal absolute CD4 count . This resulted in resolution of symptoms, catch-up growth, and progression through puberty. She has had no further significant infections in the last 5 years and her lymphocyte count has remained stable.Pneumocystis jirovecii (formerly known as Pneumocystis carinii) is a ubiquitous opportunistic fungus, which causes pneumonia [Pneumocystis pneumonia (PCP)]. It was first described in 1940s malnourished infants  due to excellent tissue penetration, rapid response, and low cost . AtovaquPCP mortality rates are between 20 and 60% in immunocompromised non-HIV infected individuals in contrast to rates of 10–20% in HIV-infected patients , 24, 25.A retrospective cohort study determined that IBD patients were at an elevated risk of PCP compared to the general population with an increased relative risk  but low absolute risk (0.03%) . The incThere is only one published case of a child with IBD developing PCP. The 8-year-old CD patient, on infliximab monotherapy, developed PCP with concurrent disseminated histoplasmosis after 15 months therapy. His disease course was not severe. He did not require respiratory support and responded to TMP–SMX. He was maintained on budesonide with no PCP prophylaxis . PCP hasWe performed a literature review of PCP in IBD. A database search for studies on MEDLINE, EMBASE, and the Cochrane Controlled Trials Registry was performed. References of included articles were searched for further studies. Table Corticosteroids are known to reduce CD4 lymphocytes, which predisposes to PCP development . CorticoThiopurines (AZA and 6-mercaptopurine) can inhibit cell-mediated immunity, which influences PCP development. In the literature, two IBD patients on thiopurine monotherapy developed PCP, 10 were on dual therapy, as was the case with our patient, and 10 were on triple therapy. The CFR in this group was 19% (3/16), where outcome data were available.Cyclosporine works by inhibiting production of IL-2 by helper T-cells and by affecting T-cell, B-cell, neutrophil, and mast cell function. In the literature, 50% (4/8) of IBD patients with PCP died , 37, 38.Three patients in the IBD literature developed PCP while on methotrexate in combination with corticosteroid and anti-TNF therapy. Methotrexate has been implicated in the development of PCP in rheumatoid arthritis with 28 documented cases, 25% of whom died , 56.P. jirovecii colonization and actual disease can be challenging.Cytokines inhibited by anti-TNF agents are involved in the host response to PCP resulting in reduced PCP clearance. Moreover, anti-TNF therapy can lower CD4 counts making patients more susceptible to PCP . Review There are no published data on PCP associated with vedolizumab, ustekinumab, or other novel IBD treatments , 64. Mor3) has been associated with increased risk of PCP in 60–95% of cases  . MoreoveA population-based cohort study in juvenile idiopathic arthritis patients reported an incidence of 7/100,000 PY . MultiplIn a 2012 survey of PCP prophylaxis practice by gastroenterologists, 11% prescribed prophylaxis to their patients with IBD on combination therapy. Gastroenterologists were more likely to prescribe if they had previous practical experience of PCP, or practiced in an academic center .The effects of PCP can be severe, however, prevention entails using drugs with adverse effects that may counterbalance the benefits, as occurred in our patient. The guidelines for PCP prophylaxis in HIV-infected patients have been universally adopted, but there is a lack of consensus on prophylaxis with TMP–SMX in IBD patients. A recent Cochrane meta-analysis of prophylactic treatment with TMP–SMX in non-HIV infected patients included 1,412 patients, of which 520 were children. The authors were unable to find published literature addressing prophylaxis in IBD. In patients with hematological cancers and transplant recipients, they reported an 85% reduction in PCP incidence with prophylaxis and PCP-related mortality reduced by 83% with few adverse events reported (There is insufficient evidence to recommend PCP prophylaxis for all IBD patients on immunosuppressive monotherapy; however, with increasing number of immunosuppressive agents, the risk of opportunistic infection is known to increase , 27, 62.3 as a means of identifying patients at risk of infections, although not all patients who develop PCP have lymphopenia, therefore, precluding its use as an isolated risk identification guide (A study using simulation modeling to address cost and comparative effectiveness of PCP prophylaxis in CD concluded that at the present incidence, routine chemoprophylaxis was not cost effective but, based on limited data, it may be effective in triple immunosuppressive therapy . The 201on guide , 65, 75.We described a case of severe PCP in an immunosuppressed lymphopenic pediatric IBD patient. This case reiterates the importance of limiting immunosuppression to its minimal effective dose. Although the incidence of PCP in the IBD population is low, it is an aggressive condition that has a higher relative risk in IBD patients compared to the general population. Moreover, it is associated with significant morbidity and mortality. PCP should be considered in the differential diagnosis of immunosuppressed pediatric IBD patients who develop respiratory symptoms, with a low threshold for treatment. Although there is no place for global PCP prophylaxis in IBD, in an era when there is increasing use of biologic agents with combination immunosuppressive therapy, the risk-benefit profile of PCP prophylaxis should be revisited in selected cohorts such as patients on triple immunosuppression with corticosteroids, thiopurines, and a biological agent or calcineurin inhibitor especially in lymphopenic individuals. Further studies are required to guide definitive PCP prophylaxis in high risk subgroups of IBD patients.Written informed consent for publication of the case report and figures was obtained from the patient and parents.SL performed the literature review and wrote the manuscript. MS and KJ critically reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Muscle quantity at intensive care unit (ICU) admission has been independently associated with mortality. In addition to quantity, muscle quality may be important for survival. Muscle quality is influenced by fatty infiltration or myosteatosis, which can be assessed on computed tomography (CT) scans by analysing skeletal muscle density (SMD) and the amount of intermuscular adipose tissue (IMAT). We investigated whether CT-derived low skeletal muscle quality at ICU admission is independently associated with 6-month mortality and other clinical outcomes.This retrospective study included 491 mechanically ventilated critically ill adult patients with a CT scan of the abdomen made 1 day before to 4 days after ICU admission. Cox regression analysis was used to determine the association between SMD or IMAT and 6-month mortality, with adjustments for Acute Physiological, Age, and Chronic Health Evaluation (APACHE) II score, body mass index (BMI), and skeletal muscle area. Logistic and linear regression analyses were used for other clinical outcomes.p < 0.001), and more IMAT . Higher SMD was associated with a lower 6-month mortality (hazard ratio (HR) per 10 HU, 0.640; 95% confidence interval (CI), 0.552–0.742; p < 0.001), and also after correction for APACHE II score, BMI, and skeletal muscle area . Higher IMAT was not significantly associated with higher 6-month mortality after adjustment for confounders. A 10 HU increase in SMD was associated with a 14% shorter hospital length of stay.Mean APACHE II score was 24 ± 8 and 6-month mortality was 35.6%. Non-survivors had a lower SMD (25.1 vs. 31.4 Hounsfield Units (HU); Low skeletal muscle quality at ICU admission, as assessed by CT-derived skeletal muscle density, is independently associated with higher 6-month mortality in mechanically ventilated patients. Thus, muscle quality as well as muscle quantity are prognostic factors in the ICU.NCT02817646.Retrospectively registered  at ClinicalTrials.gov:  Muscle wasting is a severe complication of critical illness . PuthuchNot only the quantity, but also the quality of muscle seems important . Along wThis is a retrospective analysis of CT-derived muscle quality at a single time point at ICU admission in critically ill patients admitted to a mixed medical-surgical ICU of a university hospital from September 2003 to April 2013. Patients were included if they were aged 18 years or older, stayed in the ICU for at least 4 days, required mechanical ventilation during their ICU stay, and had an abdominal CT scan made 1 day before or up to 4 days after admission to the ICU. Patients were excluded if the CT scan was not eligible for analysis, or if data on body weight or height or the Acute Physiological, Age, and Chronic Health Evaluation (APACHE) II score was missing. By searching the hospital information system for any patients meeting inclusion criteria, we expanded our previously reported cohort of ICU patients .Patient data including age, sex, weight, height, admission diagnosis, APACHE II score, length of ventilation (LOV), ICU length of stay (ICU-LOS) and hospital length of stay , discharge destination, and ICU and hospital mortality was obtained from the ICU patient data management system  and the hospital information system . If mortality data were not registered, these were collected from the civil registry or from the general practitioner.r = 0.83–0.99; p < 0.01) and IMAT  at this level are closely related to whole body skeletal muscle and IMAT volumes as assessed by magnetic resonance imaging (MRI) [The precision of single slice CT scan analysis at the third lumbar vertebra (L3) level is high  . Both skng (MRI) –21.CT scans made 1 day before to 4 days after ICU admission for diagnostic purposes were imported from the hospital radiology system and stored on a secure computer system. Scans were analysed using Slice-O-matic versions 4.3 and 5.0  by two trained and certified investigators  who had frequent consultation with each other if there was any doubt about eligibility, landmarking, or analysis.The CT scans were analysed for eligibility and rejected if the scan quality was too low for analysis or if they contained artefacts, or if muscle was cut off due to windowing. Landmarking was performed by identifying the L3 and isolating the CT slice that depicted the whole vertebra the best. A bony landmark was used to ensure reproducibility and consistency between patients.2 [2 for male patients and below 110 cm2 for female patients [Different tissues were identified using boundaries in Hounsfield Units (HU) set to –29 to +150 for muscle, –190 to –30 for IMAT and subcutaneous adipose tissue, and –150 to –50 for visceral adipose tissue . SMD was2 . PreviouBecause muscle quality is important for dealing with recovery after ICU and hospital discharge, we chose 6-month mortality as the primary endpoint. Secondary endpoints were the odds of being discharged from the hospital to home, length of ventilation, and ICU and hospital LOS in survivors.t tests were used to compare survivors and non-survivors for normally distributed continuous variables, and Mann-Whitney U tests for non-normally distributed continuous variables. Fisher exact and Chi2 tests with post-hoc Bonferroni analysis were used to compare survivors and non-survivors for categorical variables. Kaplan-Meier plots were made to visualize the effect of SMD and IMAT (divided into two groups based on the median) on 6-month mortality, with log-rank tests to compare the survival curves of the two groups. Cox regression analysis was used to evaluate the association between SMD or IMAT (as continuous variables) and 6-month mortality. After univariable analyses, APACHE II score was added to the models to adjust for severity of illness (model 2). In the second adjusted model, body mass index (BMI), and skeletal muscle area were included as well (model 3). Age is included in the APACHE II score and was therefore not separately included in the adjusted models. Additionally, we performed analyses on the subgroup of patients with available data on visceral and subcutaneous adipose tissue in which BMI was substituted with visceral and subcutaneous adipose tissue as a measure of total body fatness (model 4).Independent sample Logistic and linear regression analyses were used to evaluate the association between SMD or IMAT and the secondary outcome measures discharge to home, LOV, ICU-LOS, and hospital-LOS in survivors. LOV, ICU-LOS, and hospital-LOS were non-normally distributed and positively skewed; therefore, the analysis was performed on the natural logarithm of the variables. By re-transforming by using the inverse, the influence of a given predictor was calculated as a percentage change in outcome.p < 0.05 was considered statistically significant.IBM SPSS Statistics 22  was used for statistical analysis. Values are reported as mean ± standard deviation (SD) or median and 25–75% interquartile range (IQR). All statistical tests were two-sided. A A total of 13,434 patients were admitted to the ICU during the study period with a mean APACHE II score of 17.4 ± 9.2. Six hundred and seventy-eight patients fulfilled inclusion criteria and had their CT scans imported from the radiology system to be analysed for eligibility. CT scans that were found not to be eligible were due to artefacts (78 scans), muscle cut-off (50 scans), or low quality (47 scans). Finally, 491 patients (72%) with complete clinical data and good quality CT scans were included for the statistical analysis. However, due to windowing or artefacts, visceral and/or subcutaneous adipose tissue could not be analysed in 154 patients. We therefore performed subgroup analyses that included visceral and subcutaneous adipose tissue in a subgroup of 337 patients (50%). Figure p < 0.001), had a lower BMI , higher APACHE II score , and were more often medical patients  than survivors.Patient characteristics are presented in Table 2, comprising 9.1% of total tissue within muscle fascia  at the L3 level. Non-survivors had a lower skeletal muscle area , lower SMD , and more IMAT (17.1 (10.5–27.1) vs. 13.3 (7.9–23.2) cm2; p = 0.004) than survivors.Mean SMD at ICU admission was 29.9 ± 11.7 HU. Median IMAT at ICU admission was 13.6 (8.4–24.3) cmMortality was significantly higher in patients with low muscle quality with SMD values below the median or IMAT values above the median Fig. .Fig. 3Kap < 0.001; Table p = 0.006).Cox regression analysis showed that higher SMD was associated with lower 6-month mortality (hazard ratio (HR) per 10 HU, 0.640; 95% confidence interval (CI), 0.552–0.742; 2, 1.153; 95% CI, 1.042–1.277; p = 0.006). However, when adjusted for APACHE II score alone or the confounders APACHE II score, BMI, and skeletal muscle area the association between IMAT and 6-month mortality was not significant .Cox regression analysis showed that higher IMAT was associated with higher 6-month mortality , more often male , and had a lower BMI . In this subgroup, we found both SMD  and IMAT  were significantly associated with 6-month mortality. In multivariable analyses both SMD  and IMAT  remained significantly associated with 6-month mortality, adjusted for APACHE II score, skeletal muscle area, and visceral and subcutaneous adipose tissue.Additional Cox regression analyses were performed in the subgroup of patients with available data on visceral and subcutaneous adipose tissue  are important prognostic markers. Unfortunately, the updated APACHE III and IV scores were not available for all patients.Of interest, IMAT was independently associated with 6-month mortality in a subgroup, but not in the entire cohort. The patients in the subgroup were younger, more often male, and had a lower BMI. Apparently, visceral tissue on CT scans can more often not be analysed in older patients with high BMI, mostly because a part of the scan is often cut-off in the windowing process.Previous studies have shown that inactivity, as seen in pre-existing illness and advancing age, can cause an increase in myosteatosis (as seen by a decrease in SMD and an increase in IMAT) and that these changes are associated with decreased muscle strength –26. DuriAdipose tissue has been noted as a major endocrine organ. To date, hundreds of adipokines, cytokines secreted by adipose tissue, have been identified . Myosteaduring critical illness has a large impact on survival, successful weaning from ventilation, and long-term functioning [Previous studies have shown that muscle wasting as occurring ctioning –8, 38. Hctioning  and Iwasctioning . In two Our study has strengths and limitations. This is the first study up to now investigating the relation between muscle quality assessed with CT scans and clinical outcomes in a large group of critically ill ventilated patients. However, we only included patients who had a CT scan made and the resulting selection bias might limit the generalizability of our findings to the overall ICU population. The APACHE II score of the study population was higher than the overall ICU population, all patients were ventilated, and had an ICU length of stay of at least 4 days, indicating that the study patients were severely ill. Low muscle quality at admission likely has greater impact in the more severely ill patients, because the effect of additional critical illness-related muscle wasting is greater in this population.Muscle quality is typically defined as muscle strength per unit of muscle mass or cross-sectional area. However, measuring muscle strength in ventilated critically ill patients is not feasible. Therefore, we used SMD and IMAT as proxy markers for muscle quality . To dateA further limitation to our study is its observational design, precluding any deduction of causality. In addition, the complexity of critical illness may obscure residual confounding. Finally, the focus of our study was the prediction of long-term mortality at ICU admission, e.g. whether muscle quality at admission is a predictor of long-term mortality independent of muscle mass and of the best validated predictive score (APACHE). Further studies are needed to determine the risk factors for poor muscle quality and to determine the additional impact of ICU-acquired weakness on long-term mortality.Low skeletal muscle quality at ICU admission, as assessed by skeletal muscle density on CT scans, is associated with higher 6-month mortality in mechanically ventilated patients, independent of muscle quantity, APACHE II score, and BMI. Low muscle quality was also associated with longer hospital length of stay in survivors. Therefore, muscle quality appears to be as important for outcome as muscle quantity. Future intervention studies, including nutrition and early exercise, should not only focus on preventing further deterioration of muscle quantity, but also of muscle quality.


Severe iodine deficiency during pregnancy has been associated with pregnancy/neonatal loss, and adverse pregnancy outcomes; however, the impact of mild–to–moderate iodine insufficiency, though prevalent in pregnancy, is not well-documented. We assessed whether mild iodine deficiency during pregnancy was associated with pregnancy/infant loss, or with other adverse pregnancy outcomes. We used samples and data from the Avon Longitudinal Study of Parents and Children (ALSPAC), from 3140 singleton pregnancies and from a further 42 women with pregnancy/infant loss. The group was classified as mildly-to-moderately iodine deficient with a median urinary iodine concentration of 95.3 µg/L  124 µg/g, IQR 82–198). The likelihood of pregnancy/infant loss was not different across four UI/Creat groups . The incidence of pre-eclampsia, non-proteinuric gestational hypertension, gestational diabetes, glycosuria, anaemia, post-partum haemorrhage, preterm delivery, mode of delivery, being small for gestational age, and large for gestational age did not differ significantly among UI/Creat groups, nor were there any significant differences in the median UI/Creat. We conclude that maternal iodine status was not associated with adverse pregnancy outcomes in a mildly-to-moderately iodine-deficient pregnant population. However, in view of the low number of women with pregnancy/infant loss in our study, further research is required. Iodine is essential for the production of thyroid hormones and is particularly important during pregnancy and early life owing to its role in brain development. While there has been enormous progress in the eradication of iodine deficiency in many countries worldwide , iodine deficiency remains a problem in pregnant women in many countries, including the UK . The WorSignificant neurodevelopmental impairment occurs with severe maternal iodine deficiency during pregnancy, including the development of cretinism . CorrectBeyond the effects on brain development, severe maternal iodine deficiency has also been associated with increased perinatal and infant mortality, recurrent miscarriage and preterm delivery, and significant improvements were observed with iodine supplementation ,12. WhetEven women living in countries classed as iodine replete are commonly iodine deficient in pregnancy  because This study used samples and data from pregnancies in the UK Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort, where we have previously documented an adverse impact of maternal iodine insufficiency on child neurocognitive development . In thisALSPAC recruited pregnant women from the county of Avon in the south-west of the UK, with expected dates of delivery 1 April 1991 to 31 December 1992 ,29. Amonn = 3524). Furthermore, we selected women who experienced pregnancy/infant loss up to the age of 1 year and who had an available antenatal urine sample for iodine analysis (n = 46). In all cases where there was a repeat urine sample, the earliest available sample was used; this was done as we postulated that the impact of iodine status on pregnancy outcome is greatest during early pregnancy.For the current study, women were selected on the basis of a singleton pregnancy and if they had at least one existing measure of urinary iodine-to-creatinine (UI/Creat) during pregnancy; these existing measures were from previous and ongoing studies examining the relationship between maternal iodine status and child IQ at age 8 years and the measures were already available in the ALSPAC resource (n = 3524); the details of this method have been described in full previously [Iodine concentration was measured in the urine samples from women with fetal/infant loss up to 1 year in the same laboratory  and using the same methodology ) as was used for all previous measures of iodine concentration in ALSPAC urine samples (eviously . The resThe median urinary iodine concentration (UIC) value of the group was compared with the WHO UIC cut-offs for iodine adequacy in pregnancy . As UIC We report iodine status in two ways, as the iodine concentration (UIC) in µg/L and as the iodine-to-creatinine ratio (UI/Creat) in µg/g. As in our previous research on iodine status in the ALSPAC cohort, we grouped women according to their UI/Creat result. The groups were defined as <50 µg/g, 50–149.9 µg/g, 150–249.9 µg/g, and ≥250 µg/g; these groups, broadly speaking relate to severely deficient, mildly-to-moderately deficient, sufficient, and more-than adequate iodine, respectively, based on WHO . We inveWe examined the following obstetric outcomes: (i) hypertensive disorders in pregnancy; (ii) glucose derangement; (iii) anaemia; (iv) post-partum haemorrhage; (v) preterm birth; (vi) mode of delivery; and (vii) birthweight. Details of obstetric outcomes were extracted from hospital records following delivery. Hypertensive disorders in pregnancy was defined by a systolic BP > 139 mmHg or a diastolic BP > 89 mmHg on 2 or more occasions beyond 20 weeks gestation, as previously defined in this cohort , and incn = 414, 11.7%; those with pregnancy/infant loss n = 5, 10.9%). If a woman had a later urine sample (with a result that was considered to be uncontaminated) that was used to replace the contaminated result . We also excluded women who were taking thyroid hormone medication during pregnancy . This resulted in a total of 3140 women with a child alive at 1 year, and 42 women with a pregnancy/infant loss.As a result of concern that the urine samples had been contaminated with iodine from urine test strips , women wUI/Creat was not normally distributed and, therefore, differences between groups were compared using a non-parametric test (Mann–Whitney U test). Additionally, UI/Creat were stratified into four iodine status groups as described above. Chi-square tests were used when comparing groups of iodine status with the presence/absence of pregnancy outcomes, and one-way analysis of variance (ANOVA) was used to compare continuous data between grouped UI/Creat.Likelihood of fetal and neonatal death events was compared to live controls. Adverse pregnancy outcomes were studied within those with live babies. Logistic regression was used to examine the association between maternal iodine status (as the four groups of UI/Creat) and likelihood of adverse pregnancy outcomes, with women in the 150–249 µg/g group as the reference category. For each pregnancy outcome, crude and adjusted odds ratios of the pregnancy outcomes (OR and AOR) were calculated. The likelihood of being small or large for gestational age were calculated against odds of being appropriately grown for gestational age. The models were adjusted for mother’s BMI (pre-pregnancy) and age, parity , cigarette smoking during early pregnancy (yes/no) and trimester of UI/Creat collection.The statistical significance was assumed a priori at 0.05 level; the 95% confidence intervals (CI) were calculated accordingly. All analyses were conducted using IBM SPSS Statistics version 23 .n = 11), neonatal deaths within the first month of life (n = 24), and child deaths between 1–12 months (n = 7). We also had uncontaminated UI/Creat results from a total of 3140 women with babies who survived beyond the first year.In total we included 42 cases of pregnancy/infant loss up to age 1 year. This included fetal deaths and stillbirths between 20–42 weeks’ gestation (n = 3182) was 95.3 µg/L (IQR 57.0–153.0), indicating mild-to-moderate iodine deficiency in this group of pregnant women [n = 161) of the cohort, 50–149.9 µg/g in 56.4% (n = 1794), 150–249.9 µg/g in 21.8% (n = 693) and greater than 250 µg/g in 16.8% (n = 534).The median UIC in the entire cohort of women included in this study (nt women . The medn = 3140) to those with pregnancy/infant loss (n = 42) (p < 0.001). The median UI/Creat of those with pregnancy/infant loss and those with babies alive at 1 year was not significantly different .We compared women with infants alive at 1 year ((n = 42) . There wAmongst those who experienced a pregnancy/child loss, the likelihood of being iodine insufficient or more-than adequate during the pregnancy were no different to those whose child was alive at one year. Adjustment for confounders  did not alter the conclusions.There were significant differences between the four UI/Creat status groups in terms of BMI, age, parity and trimester of UI/Creat collection . The <50n = 95), 50.7% (28.9) with UI/Creat 50–149.9 µg/g (n = 1132), 48.7% (28.4) with UI/Creat 150–249.9 µg/g (n = 392) and 46.9% (29.1) with UI/Creat greater than 250 µg/g (n = 335)) and the incidence and odds of being both small for gestational age and large for gestational age were not significantly different between iodine groups. We also modelled the likelihood (odds ratio) of adverse pregnancy outcomes in various UI/Creat status groups compared with sufficient UI/Creat, adjusting for the confounders of mothers’ BMI, age, parity, smoking and trimester of UI/Creat collection , which is most likely a chance finding (Type 1 error). There were no significant differences in the median UIC/Creat between the groups with and without a pregnancy complication of interest  were less likely to experience an operative delivery, both instrumental and caesarean section . This isThe reasons for these discrepancies are unclear. It may suggest that different confounding factors associated with iodine insufficiency could be at play in influencing obstetric outcomes in different settings. In the absence of good methods which can be used to assess individual iodine status we are limited to assessing iodine status of whole populations, thus the challenge has always been to determine the normal baseline rate of specific obstetric complications in an iodine-sufficient population, and whether the observed complication rate in an iodine-insufficient population could be improved with better iodine status. Even the recently reported clinical trial conducted in India and Thailand, which showed no change in the rates of preterm birth or low birth-weight infants with iodine supplementation in pregnancy, was confounded by the spontaneous improvement in urinary iodine concentration in the placebo group as gestation progressed, such that controls were no longer iodine insufficient by the end of pregnancy  and the The strength of our study is the relatively large sample size of a pregnant population that is not exposed to any universal iodination programme, and the prospective nature of the study where iodine status was evaluated in a urine sample taken before the occurrence of the adverse events of interest. Because of the sample size we were able to stratify pregnant women into four categories of iodine status for comparison. A limitation of our study is the use of a single time-point spot urinary iodine measure for categorisation of iodine status which may be a poor proxy of individual iodine status , so miscIn conclusion, our present study in a mildly-to-moderately iodine-deficient population without an iodised salt programme did not find an association between iodine status and pregnancy outcomes or child loss. Further research in this area is required, given the small number of studies that have evaluated pregnancy outcomes and iodine status, and given the low numbers of women in our study with pregnancy/infant loss.


Serious neurological adverse events (NAE) have occurred during treatment with high-dose thiotepa regimens of children with high-risk solid tumours. The objective was to assess the incidence of NAE related to high-dose thiotepa and to identify potential contributing factors that could exacerbate the occurrence of this neurotoxicity.From May 1987 to March 2011, children with solid tumours treated with high-dose thiotepa were retrospectively identified. Each NAE detected led to an independent case analysis. Potential contributing factors were pre-specified and univariate/multivariable analyses were performed.2. 81 NAE (26%) were identified. 46 NAE were related to high-dose thiotepa during the first course (18.3%) and 11 during the second course (19.6%). The symptoms appeared in a median time of 2 days after the introduction of thiotepa. Central and peripheral symptoms were headaches, tremors, confusion, seizures, cerebellar syndrome, and coma. High-dose thiotepa was reintroduced in 18 cases and symptoms reappeared in 5 children. For 3 patients who had seizures during the first course, premedication with clonazepam for the second course has prevented recurrence of NAE. As contributing factors, brain tumour and tramadol treatment increased the risk of thiotepa-related neurotoxicity by 2 to 6 times respectively.Three hundred seven courses of thiotepa (251 patients) were identified. The total dose per treatment ranged from 600 to 900 mg/mThe incidence of neurotoxicity was 18.3%. Brain tumours and tramadol treatment are risk factors to consider when using high-dose thiotepa. The outcome of patients was favourable without sequelae in all cases and rechallenge with thiotepa was possible. Little is known about the possible relationship between exposure to high-dose thiotepa and the occurrence of neurological disorders.High-dose thiotepa administered below the Maximum Tolerated Dose has never been studied in terms of neurological outcomes.This study estimates the incidence of thiotepa-related neurological complications, unlike the neurotoxicity described when the drug was used beyond the defined limiting dose or combined with another cytotoxic drug or irradiation.Our findings show that brain tumour and tramadol treatment could be independently associated with thiotepa-related neurotoxicity.High-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) has improved the survival of children with high-risk solid tumours. The rationale for HDCT is that escalated doses of HDCT may increase survival by capturing putative remnant malignant cells . The rat2H4)3, is one of the few drugs to have demonstrated clear activity at high doses in childhood tumours [2 [The HDCT regimens are generally based on the use of alkylating agents . Among t tumours . Thiotep tumours , 11. Thi tumours , 13. Fol tumours . Thiotep tumours . Thiotep tumours , 16. Due tumours . Relatio tumours –21. Toximours [2 , 21, 22.Little is known about the possible relationship between exposure to high-dose thiotepa and the occurrence of neurological disorders. To our knowledge, high-dose thiotepa administered below the MTD has never been studied in terms of neurological outcomes. One reason for the lack of knowledge is that thiotepa is rarely used alone. In our experience, serious neurological adverse events (NAE) have occurred during treatment with high-dose thiotepa regimens. Some of these NAE could lead to life-threatening complications. The objective of the present study was to assess the incidence of NAE related to high-dose thiotepa with ASCT in children with solid tumours and to identify potential contributing factors that could increase the occurrence of this neurotoxicity.The institutional review board of the Gustave Roussy Cancer Campus approved this study. The parents/guardians gave their written informed consent for the retrospective analysis of clinical data according to the institutional review board of the Gustave Roussy Cancer Campus. From the institutional pediatric transplantation register, we established a retrospective cohort of all patients treated for a solid tumour with high-dose thiotepa followed by ASCT between May 1987 and March 2011 at Gustave-Roussy, center with expertise in the treatment of complex malignancies. Our analysis focused on neurotoxicity exclusively due to thiotepa, therefore patients treated with thiotepa combined with another cytotoxic drug or radiotherapy were excluded.All medical and nursing records were reviewed. Demographics data , race aimed at assessing the influence of the type of metabolism , clinical data , and drug exposure (thiotepa and drugs administered concomitantly) from admission to hospital until the day of ASCT were collected for each patient. To assess the impact of potential organic disturbances, biological monitoring of renal and hepatic function from admission to hospital until the day of ASCT was taken into account. Biological monitoring of renal function took into account blood urea and creatinine. The glomerular filtration rate (GFR) was estimated using the Schwartz formula . MonitorCausality assessment of adverse drug reactions (imputability) was performed using the algorithm devised by Begaud et al. which is based on a three-stage process: assessment of chronological criteria, clinical and biological findings and symptom evaluation . The metPotential contributing factors were pre-specified and were: (1) sex; (2) age; (3) race; (4) primary tumour site; (5) history of neurological disorders; (6) metabolic disruption; and (7) use of concomitant therapy . Concomitant drugs were determined according to their potential ability to interact with thiotepa metabolism. They were captured the week before, during, and the week after thiotepa exposure.P value below 0.05 in the univariate analysis were selected for the multivariable analysis. The results are expressed as the Odds Ratio (OR) of NAE for each factor; an OR equal to 1 is associated with the reference category of each factor. All statistical analyses were two-sided, with P values of 0.05 or less deemed statistically significant. The software used was SAS, version 9.1.Some patients had received more than one course of high-dose thiotepa. We decided to retain only one course per patient in order to avoid skewing the statistical analysis by including patients with individual susceptibility. In principle, the first course of high-dose chemotherapy was taken into account in the analysis. The second courses were analyzed separately to study the effect of rechallenge with thiotepa when NAE occurred during the first course. The results are expressed as percentages , or medians and the interquartile range (quantitative data). Concerning the analysis of factors associated with the occurrence of NAE, univariate and multivariable analyses were performed using logistic regression. Variables with a 2 for 46 patients (18%), 720 mg/m2 for 76 patients (30%), and 600 mg/m2 for 129 patients (51%). One hundred and sixteen patients (46%) had a primary brain tumour. Twenty children (8%) had a neurological disorder due to disease when they were admitted to hospital: moderate cerebellar syndrome (n = 9), central neuropathy (n = 1), peripheral neuropathy (n = 3), hemiparesis (n = 3), and facial paralysis (n = 4). Thirty children exhibited early-stage renal failure upon admission to hospital with a median GFR of 80.5 mL/min  and a median blood urea level of 4.65 mmol/L . The 221 patients without renal insufficiency upon admission to hospital had a median GFR of 123.5 mL/min  and a median blood urea level of 3.9 mmol/L . On the day of ASCT, early-stage renal failure had persisted with a median GFR of 79 mL/min  and a median blood urea level of 2.6 mmol/L  in 12 of these 30 children. No patients had liver disruption at upon admission to hospital . Thirteen patients had reversible hepatic cytolysis at the end of the thiotepa regimen without cholestasis . During treatment with thiotepa, 92 patients had required analgesics (42%). According to the World Health Organization analgesic ladder, 76 patients (35%) had received simple analgesics such as acetaminophen or nefopam for mild pain, 53 patients (24%) had received weak opioids such as tramadol, codeine, or dextropropoxyphen for mild to moderate pain, and 9 patients (4%) had received strong opioids such as morphine or pethidine for moderate to severe pain. Sometimes, patients had received a combination of different analgesics. Regarding anti-emetic support, every child had received at least a 5-HT3 antagonist. The anti-emetic treatment added was alizapride (43%), or aprepitant (3%). None of the patients had declared an allergy to analgesics or anti-emetic drugs upon admission to hospital.Between May 1987 and March 2011, 307 courses of high-dose thiotepa with ASCT were administered to 251 patients (56 patients received 2 courses of high-dose thiotepa). Baseline characteristics of these 251 children are shown in Table After reviewing medical and nursing records, 81 NAE (26.4%) were unveiled during the 307 courses, 69 NAE had occurred during the 251 first courses (27.5%), and 12 NAE during the 56 s courses (21.4%). After assessment of intrinsic and extrinsic imputability, NAE were considered “very likely” related to thiotepa for 13 patients (16.0%), “likely” related to thiotepa for 35 patients (43.2%) and “possibly” related to thiotepa for 9 patients (11.1%). Twenty-four NAE (29.6%) were assessed as “dubious” or “unlikely” to be related to thiotepa, and were therefore excluded  after the beginning of the course of thiotepa. Table P = 0.03), a neurological disorder at baseline , alizapride , and weak opioids  were identified as risk factors for thiotepa-related NAE. The weak opioids used were tramadol (n = 46), codeine (n = 5), and dextropropoxyphen (n = 2). NAE had occurred in 21 cases when tramadol had been used , in two cases with codeine , and in one case with dextropropoxyphen . The risk of NAE was not significantly associated with gender (P = 0.41), age (P = 0.48), race (P = 0.51), a history of seizure (P = 0.27), renal failure (P = 0.98), or other concomitant drugs. Although not significantly associated with the dose of thiotepa (P = 0.18), we observed a trend towards an increasing dose-dependent rate of neurotoxicity .Table P = 0.04). The use of tramadol for analgesia was confirmed as a highly significant contributing factor for NAE .In the multivariable analysis, a brain tumour and a weak opioid (tramadol) were identified as independent risk factors for thiotepa-related NAE Table . Patient2) in all the courses, in accordance with the consensus guidelines [In this imputability analysis, the incidence of neurotoxicity due to high-dose thiotepa was estimated at 18.3%. To our knowledge, this is the first study to describe the incidence of neurotoxicity related to high-dose thiotepa, which had been administered below the defined limiting dose (1005 to 1125 mg/midelines . Neuroloidelines  or with idelines , busulfaidelines , carboplidelines , etoposiidelines . It is nidelines . CausaliVarious pathomechanisms can explain drug-induced neurological disorders. In this study, we showed that a brain tumour could be an independent risk factor, which could increase the risk for thiotepa-related neurotoxicity 2.2-fold. Drugs with the highest neurotoxicity are therefore those that readily cross the blood-brain barrier. Lipid-soluble agents with a low molecular weight, such as thiotepa and tepa enter the brain easily . Damage In our study, we found no influence of renal failure on the occurrence of high-dose thiotepa- related NAE. However, these results should be considered with caution. Only 12 children had renal disruption, classified as an early-stage disorder. Among them, two children had experienced a NAE. Even moderate renal disruption has been described as possibly capable of increasing exposure to thiotepa by 43% and exposure to tepa by 157% . TherefoIn this study, we showed that intake of a weak opioid, especially tramadol, could be an independent risk factor for thiotepa-related NAE. Drug-related iatrogenia is a major source of treatment failures, especially when multiple medications are used. CYP3A4 and CYP2B6 are the main cytochrome enzymes involved in the metabolism of thiotepa to tepa , 13. ThiOtherwise, interindividual variability in alkylating agent pharmacokinetics may lead to unpredictable toxicity and efficacy. A poor metabolizer phenotype was described as responsible for an increase in exposure to thiotepa and for an increase in the severity of mucositis . This vaIn our study, we have shown that tramadol used concomitantly with high-dose thiotepa could increase the risk of NAE 6.3-fold. Tramadol is a synthetic opiate that is chemically similar to codeine. Its antinociceptive effects are mediated via a combination of the agonist effects of μ-opioid receptors and inhibition of the reuptake of serotonin and norepinephrine . This seAn alternate hypothesis is that there is an unmeasured confounder that leads to both increased risk of NAE and increased need for pain control, hence the observed association with exposure to the weak opiates.In clinical research, the gold standard is the prospective randomized controlled trial. Retrospective cohort studies, as performed in our study, are in principle subject to multiple risks of bias . SelectiThis study estimates an incidence of 18.3% of high-dose thiotepa-related NAE. These neurological complications resolved without sequelae and rechallenge with thiotepa was possible. A brain tumour and tramadol treatment were found to be possible independent risk factors for thiotepa-related neurotoxicity. Identifying markers for individual susceptibilities is mandatory in order to develop customised treatment approaches for cancer patients treated with intensive chemotherapy.


Although chemerin, an adipokine, increases the cardiovascular (CV) risk in obese people, it is associated with a survival advantage in incident hemodialysis (HD) patients. We explored the potential effects of chemerin on CV outcomes in prevalent HD patients. This prospective study included 343 prevalent HD patients. The composite outcome was the occurrence of CV events and death during follow-up. We used multivariate Cox regression analysis to test the predictive power of different chemerin and adiponectin levels and geriatric nutritional risk index (GNRI) for the outcomes. HD patients with higher chemerin levels (≥211.4 ng/mL) had a lower risk of CV events  and composite CV outcome  than those with lower chemerin levels (<211.4 ng/mL). When evaluating CV outcomes, we identified an interaction between chemerin levels and GNRI, but not between chemerin and adiponectin levels. The findings remained robust in the sensitivity analysis. Thus, in prevalent HD patients with negligible residual renal function, higher chemerin levels predict more favourable CV outcomes. We also determined the relationship between chemerin and adiponectin and their effect on the CV outcomes of prevalent HD patients with negligible residual GFR.The baseline characteristics of the participants are summarised in In total, 97 participants experienced CV events, 21 died because of CV events, and 118 presented the composite CV outcome. Of the 97 patients with CV events, 17 had intracranial haemorrhage or ischemic stroke, 56 had coronary artery disease , 7 had peripheral arterial occlusion disease, and 17 were hospitalised for heart failure. Of the 21 patients with CV death, 8 deaths occurred because of coronary artery disease and 13 had sudden cardiac deaths. The median follow-up period was 2.5 years .P = 0.03) and with serum albumin levels . However, chemerin levels were not correlated with hs-CRP levels  in our patients. Notably, chemerin levels were negatively correlated with adiponectin levels . Adiponectin levels were negatively correlated with hs-CRP levels , GNRI , and serum albumin levels .Chemerin levels were positively correlated with GNRI  had a lower incidence of CV events  and of composite CV outcomes  compared with patients with lower chemerin levels . As shown in Given that chemerin and adiponectin levels were negatively correlated with each other, we elucidated a non-significant potential interaction between chemerin and adiponectin levels in the prediction of CV outcomes  at the Far Eastern Memorial Hospital, Taiwan, were enrolled from February 2010 (first cohort) and March 2012 (second cohort). The enrolled HD patients underwent 3.5–5 h of HD with a bicarbonate dialysate, three times a week. The median HD vintage before recruitment was 4.8 years , and 93% enrolled patients had anuria.Serum chemerin levels were determined by using a commercial enzyme-linked immunosorbent assay kit from R&D Systems, Inc. . The intra- and inter-assay coefficients of variation were 4.5% and 7.9%, respectively. The minimum detectable dose (MDD) of chemerin was 1.08–7.80 pg/mL. The mean MDD was 4.13 pg/mL . The blood samples used to measure chemerin levels were obtained after recruitment and were immediately centrifuged and stored at −70 °C until use.Serum adiponectin levels were determined using a commercial enzyme-linked immunosorbent assay kit from AssayPro . The intra- and inter-assay coefficients of variation were 3.0% and 8.3%, respectively. The MDD of adiponectin was 0.781–50 ng/mL. The blood samples used to measure adiponectin levels were obtained after recruitment and were immediately centrifuged and stored at −70 °C until use.2) = 175 × −1.154 × −0.203 × .Patient demographic data, concurrent CV disease history, waist circumference (WC), and smoking status were recorded. Venous blood was sampled after fasting for more than 8 hours before the HD patient’s mid-week HD session. Whole blood samples were obtained for haemoglobin measurement, whereas serum was used for determining creatinine, calcium, phosphorus, potassium, uric acid, albumin, and lipid profiles. Intact parathyroid hormone levels were determined using immunoassay . hs-CRP levels were determined using an immunonephelometric method with a Tina-quant CRP (Latex) ultrasensitive assay . Estimated GFRs were measured using the Modification of Diet in Renal Disease Study equation as follows: GFR ] + [41.7 × body weight/WLo], where WLo is the ideal body weight calculated in the Lorentz equation. The GNRI has been validated in HD patients, and a higher GNRI indicates high nutritional statusThe primary outcomes were CV events and composite CV outcomes . CV events were defined as the new occurrence of a CV event, including coronary events , hospitalised heart failure, hospitalised incident stroke (either ischemic or hemorrhagic stroke), and incident peripheral arterial occlusive disease requiring surgery. CV mortality was defined as death caused during the aforementioned CV events. Analyses of CV events and composite outcomes were performed while accounting for the competing risk of death as well. CV mortality was not included in the primary outcome analysis due to the low incidence of CV death during follow-up (<3% per year in this cohort). The outcome information was centrally adjudicated, in accordance with pre-specified definitions, by trained clinicians and nephrologists.Follow-up began in February 2010 (first cohort) or March 2012 (second cohort) and was censored on the date of the first CV event, at the end of the study , on the date of death or renal transplantation, or when patients were transferred to other HD facilities and were no longer followed, whichever occurred first.P < 0.001 by the Kolmogorov–Smirnov or Shapiro–Wilktest), we constructed plots of chemerin levels and HRs of the composite outcome by using the ‘lowess’ function. The results revealed a non-linear relationship; hence, we stratified the patients into two groups according to their chemerin levels in the outcome analysis. Differences in the baseline characteristics and biochemical parameters between subjects with higher and lower chemerin levels were compared using Student’s t and Mann–Whitney U tests. The chi-squared test was used for categorical variables.Continuous data are presented as the mean ± SD or median (interquartile range), and categorical data are reported as percentages. The correlations among chemerin, adiponectin, and hs-CRP levels and GNRI were tested using the Spearman’s correlation. Because chemerin levels were not normally distributed in the HD patients . Given the pathophysiological links among GNRI and chemerin and adiponectin levels, we further tested the association among them and the potential interaction of these three factors for predicting CV outcomes: we first adjusted for sex, age, HD vintage, DM status, hypertension history, patient cohort, haemoglobin level, WC, GNRI, intact parathyroid hormone (iPTH), calcium phosphate product level, and hs-CRP level; we further adjusted for adiponectin levels in the fully adjusted model. Sensitivity analyses were performed to test the robustness of our findings. Analyses of CV events or composite outcomes were performed while accounting for the competing risk of death using the method of Fine and GreyHow to cite this article: Chen, H.-Y. et al. Reappraisal of effects of serum chemerin and adiponectin levels and nutritional status on cardiovascular outcomes in prevalent hemodialysis patients. Sci. Rep.6, 34128; doi: 10.1038/srep34128 (2016).