The most pronounced difference in CSF sRAGE levels between good and poor outcome patients was found on days 0–3 post-SAH but did not reach the significance threshold (p = 0.234). CSF sRAGE levels did not change significantly during hospitalisation (p = 0.868) and correlated poorly with treatment outcome, systemic inflammatory markers, and other monitored parameters. Our study revealed an early and constant increase of sRAGE level in the CSF of SAH patients.Receptors for advanced glycation end-products (RAGE) mediate the inflammatory reaction that follows aneurysmal subarachnoid haemorrhage. Soluble RAGE (sRAGE) may function as a decoy receptor. The significance of this endogenous anti-inflammatory mechanism in subarachnoid haemorrhage (SAH) remains unknown. The present study aims to analyse sRAGE levels in the cerebrospinal fluid (CSF) of SAH patients. sRAGE levels were assayed by ELISA kit in 47 CSF samples collected on post-SAH days 0–3, 5–7, and 10–14 from 27 SAH patients with acute hydrocephalus. CSF levels of sRAGE were compared with a control group and correlated with other monitored parameters. In the control group, the CSF contained only a trace amount of sRAGE. By contrast, the CSF of 20 SAH patients collected on post-SAH days 0–3 was found to contain statistically significant higher levels of sRAGE (mean concentration 3.91 pg/mL,  The management of aneurysmal subarachnoid haemorrhage (SAH) has seen no significant advance since the introduction of nimodipine and the use of Guglielmi detachable coils , 2. FavoThis single-centre, observational, prospective study was conducted in accordance with the Declaration of Helsinki and its protocol was approved by the local bioethics committee. Between January 2015 and September 2016, twenty-seven patients met the enrollment criteria which are as follows: (1) SAH confirmed by computed tomography (CT), (2) early (<24 h) endovascular treatment, (3) acute hydrocephalus diagnosed on CT and managed with external ventricular drainage (EVD) < 48 h, and (4) informed consent (by patient or family). Patients below the age of 18 were excluded due to physiological differences in CSF content as well as distinct aSAH presentation, aneurysm morphology, and outcome , 22. AlsSubjects were followed until death or the completion of 3 months following SAH. The primary outcome was the functional state after 3 months, and the secondary outcome was in-hospital mortality. The functional outcome was defined using the Glasgow Outcome Scale (GOS); these were dichotomized as good (GOS 4-5) or poor (GOS 1–3) outcomes.CSF samples were collected from the EVD at three time points, on post-SAH days 0–3, 5–7, and 10–14. The final sRAGE assays comprised twenty samples from days 0–3, sixteen from days 5–7, and eleven from days 10–14. A complete set of three samples was obtained from only five patients on account of suspected EVD infection, EVD obstruction, or early EVD removal. Each sample was centrifuged and stored at −80°C until assayed. The sRAGE assays were carried out using ELISA commercial kit RAB0007-1KT . The minimum detectable level of sRAGE was 2.06 pg/mL, and linearity was conserved between 2.06 pg/mL and 1500 pg/mL. Haemoglobin (Hgb) level, C-reactive protein (CRP) level, and white blood cell (WBC) count and fibrinogen level were assessed daily by automatic analysers XT 2000i , Cobas 6000 , and ACL TOP 500 .p < 0.05 was considered statistically significant when comparing. Data were analysed using Statistica 10 .In the tables, values for numerical data have been expressed as mean and standard deviations; for ordinal numerical data, they are expressed as median and interquartile range and for categorical data as counts and percentages. In the figures, all data are presented as mean and standard deviations. The normality of data distribution was assessed using the Shapiro-Wilk test. The correlations were assessed by Spearman's test, and correlation coefficient (cc) > 0.6  (cc < −0.6) was considered significant. A value of p < 0.001) (p = 0.234)  (The control group consisted of twenty-five patients free of CNS disease. Detectable levels of sRAGE were found in only two members of this group (CSF sRAGE of 2.1 and 1.87 pg/mL). The details of the study group are presented in < 0.001) . No sRAG= 0.234) . The p v= 0.868) . Spearma= 0.868) . The sRAOur study demonstrated elevation of CSF sRAGE in patients with poor grade SAH requiring EVD insertion. Clinical studies of sRAGE in patients with neurological disorders have thus far revealed the following: (1) serum sRAGE elevation in ischaemic stroke patients , (2) serRAGE is a transmembrane protein that belongs to the immunoglobulin superfamily , 23–25. As our understanding of the SAH complications has improved, identifying mediators of its critical pathways and designing new targeted therapies becomes of primary importance in neuroproteomics research . The infConclusions are limited by the small number of patients involved in the study and lack of consecutive sampling in most cases. The enrolled patients do not represent the full spectrum of SAH as hydrocephalus was an inclusion criterion and could have contributed to brain injury before EVD insertion. Despite careful monitoring, EVD infection remains a potential bias. In addition, only a few elements of the inflammatory pathway were investigated, and further investigation will be required to elucidate the larger picture of post-SAH inflammation.CSF levels of sRAGE increase early in patients with SAH who require acute treatment of hydrocephalus and remain elevated but do not correlate with treatment outcome. The significance of sRAGE as an endogenous anti-inflammatory mechanism requires further investigation.


GNAS mutation. McCune–Albright syndrome (MAS) refers to FD combined with skin (café-au-lait) and endocrine manifestations. This study describes the clinical childhood manifestations of polyostotic FD and MAS in a Nordic cohort.Fibrous dysplasia (FD) presents as skeletal lesions in which normal bone is replaced by abnormal fibrous tissue due to mosaic n = 16) with polyostotic FD with or without MAS diagnosed and followed in two Nordic Pediatric tertiary clinics between 1996 and 2017.We retrospectively reviewed a cohort of pediatric patients (n = 8) had café-au-lait spots, and either gonadotropin-independent precocious puberty (PP)  or abnormal testicle structure . None manifested hyperthyroidism or growth hormone excess. Mild hypophosphatemia was common (11/16), but none had signs of hypophosphatemic rickets. Craniofacial bone involvement was found in 12 patients (75%); in 5 of these, skeletal lesions were limited to craniofacial area. One child with craniofacial disease had lost vision due to optic nerve damage. Eleven (69%) patients had sustained a fracture at FD lesion, over half of them requiring surgical fixation of the fracture, most commonly in the proximal femur. The first symptoms leading to FD/MAS diagnosis included skull/facial asymmetry (n = 4), PP (n = 3), abnormal gait (n = 3), pathologic fracture (n = 3), wide-spread café-au-lait spots (n = 1), headache (n = 1), and vision loss (n = 1).Half of the 16 patients with polyostotic FD presented with MAS. All patients with MAS (Polyostotic FD and MAS remain diagnostic and therapeutic challenges because of the broad clinical spectrum. Recurrent fractures, pain, and even vision loss may impair the quality of life in children with FD. GNAS gene, coding for the α-subunit of the stimulatory G protein (Gs)  is a genetic, non-inheritable skeletal disorder in which normal bone is replaced by abnormal bone structure in the dysplastic FD lesions . FD is cein (Gs) , 3. Thesed cells , 3. Gsα The clinical spectrum of these conditions is broad, depending on the distribution of genetic mosaicism. Skeletal lesions with abnormal bone structure may present only in one bone  or in multiple sites  and can lead to deformities, fractures, functional impairment, and pain . FD can Polyostotic FD, and particularly MAS is a rare condition. The prevalence of MAS ranges between 1/100,000 and 1/1,000,000 with no ethnic differences in the disease frequency . The exaAlthough each separate manifestation of MAS is well known, there are only few studies reporting the typical clinical presentation and outcome in pediatric patients with polyostotic FD. In this study, we describe clinical features of polyostotic FD and MAS in a Nordic cohort including all pediatric patients with polyostotic FD in two tertiary referral hospitals in Finland and Sweden.n = 7) and Astrid Lindgren’s Children Hospital, Karolinska University Hospital, Stockholm, Sweden (n = 9). Patients had been referred to hospital care based on clinical reasons. For this study, we reviewed retrospectively the clinical data for all patients who were diagnosed and followed with polyostotic FD between 1996 and 2017 in Stockholm and between 2004 and 2017 in Helsinki. Due to centralized care, the cohort includes the majority of pediatric patients with polyostotic FD in these two countries. In Finland, Children’s hospital has been a referral center for the whole country for pediatric patients with rare bone diseases since 2004. In Sweden, most pediatric patients with polyostotic FD and MAS are diagnosed or followed at Karolinska University Hospital. We therefore estimate that our cohort includes close to 100% of the Finnish and over half of the Swedish pediatric patients with polyostotic FD and MAS during the study period. Diagnosis of polyostotic FD was based on clinical, radiological, and histological findings; no genetic analyses were performed upon diagnosis. The diagnosis of MAS was defined as polyostotic FD together with any extra-skeletal manifestation (café-au-lait spots and/or any endocrine manifestation).Our retrospective cohort study included all pediatric and adolescent patients with polyostotic FD and MAS who were followed in two tertiary hospitals, both largest hospitals in the respective countries: Children’s Hospital, Helsinki University Hospital, Helsinki, Finland .Our cohort included eight pediatric patients with MAS and eight patients with polyostotic FD only. Based on the background population of 15 million, and approximately 20% (3 million) of them in pediatric age group, this corresponds an estimated prevalence of 1 per 375,000 for MAS. The study cohort included eight (50%) females and eight males. Median age at last follow-up visit was 13.5 years (range 5.3–18.9). Median follow-up time from the diagnosis was 8.6 years (range 0–15.9). The clinical characteristics are presented in Table The median age at diagnosis of polyostotic FD was 5.0 years (range 0.74–11 years), whereas the median age at first symptom of the disease was 3.5 years (range 0–8.5). Among the MAS patients, the median age at first symptom was 2.7 (0.25–7.3) and that at diagnosis 3.6 (range 1.8–8.1) years. In the patients with polyostotic FD only, median age at first symptom and at diagnosis were 6.0 (0.0–8.5) and 7.3 (0.7–11) years, respectively.n = 8) was facial or skull asymmetry (n = 4), abnormal gait (n = 1), pathological fracture (n = 1), headache (n = 1), and impaired vision (n = 1). In patients with MAS (n = 8), the first manifestation was PP in three girls, café-au-lait lesions in two girls, abnormal gait in two and fracture in one. In most children, the diagnosis of polyostotic FD or MAS was set within a few months of symptom onset. In one female subject, MAS first presented with vaginal bleeding in infancy, but diagnosis of MAS was made only several years later, at 8.1 years of age, when she manifested with skeletal symptoms. In addition, one girl presented with facial asymmetry already at birth but was diagnosed with craniofacial FD only at 10.8 years, and another girl with multiple café-au-lait spots in infancy was diagnosed with MAS at the age of 2 years, when she developed precocious gonadotropin-independent puberty.The first manifestation of the disease in polyostotic FD without MAS (n = 9) and orbital (n = 8) bones.To evaluate the affected skeletal areas, bone scintigraphy was performed in altogether nine subjects, in seven of seven Finnish and in two of nine Swedish patients. In most of the Swedish patients, plain radiographs were used to evaluate the distribution of FD lesions. In addition, craniofacial engagement was evaluated with magnetic resonance imaging (MRI) or CT in 11 patients. The extent of skeletal involvement is described in Table n = 9) and tibia (n = 6). Three of the 16 subjects (19%) presented with scoliosis. It was associated with extensive FD lesions at the lumbar spine, pelvis, and hip in one patient, and by FD lesions at ileum in another patient. The third affected patient had pronounced neuromuscular scoliosis. Altogether 11 (69%) patients (7/8 of those with MAS) had sustained at least one fracture by the end of follow-up; most of them had had several fractures in the same affected bone. All but one of those without fracture history had FD lesions only in multiple bones at cranial or facial area. In turn, only one of the five patients with solely craniofacial involvement had sustained a (traumatic) fracture . Of those with a fracture history, 8/11 had hypophosphatemia as compared with 2/5 of those without a fracture. In six patients (55% of those with a fracture), the fracture(s) were surgically treated. In total, nine patients  had needed at least one orthopedic surgery because of the FD lesions  patients. The most severe skeletal manifestations of polyostotic FD were recurrent fractures in the weight-bearing long bones, mostly in the proximal femur.Altogether 8  of the 16 patients with polyostotic FD (50%) were defined as having MAS  of 51 cm (range 48–54) and weight 3.5 kg (3.2–4.6). The median height Z-score at FD diagnosis (n = 15) was +0.50 (range −1.70 to +3.50) and at the end of follow-up, +0.05 (range −1.8 to +1.6); none of the subjects presented with short stature (height Z-score < −2.0). Among the five girls with PP, height Z-score was on average +1.5 at FD diagnosis (range −1.0 to +3.5) and −0.20 at the end of study follow-up (range −1.8 to +1.6)  and GnRH agonist (leuprorelin). The remaining three MAS girls with PP were followed without hormonal treatment for PP. In one of these, fluctuation of circulating estrogen level could be demonstrated biochemically. In the two girls treated with clomifen and leuprorelin, vaginal bleeding could be prevented, and further bone age advancement could be blocked. Both of them also reached normal post-pubertal height Figure . PP girlBrain MRI or CT was performed as part of diagnostic work-up or in the follow-up of 11 of 12 subjects with craniofacial lesions. None had pituitary adenomas or other neoplasms on imaging.All eight subjects with MAS presented with café-au-lait spots. Altogether 11 of our 16 patients had presented with mild hypophosphatemia (plasma phosphate below the reference range for age) and hyperphosphaturia. Serum FGF-23 was measured in four of seven Finnish patients, and it exceeded the upper reference range for age in all but one of them. None of the patients had received treatment for mild hypophosphatemia as none presented with muscle weakness or other symptoms of hypophosphatemia, or radiographic features of hypophosphatemic rickets.One female patient had congenital hepatopathy with neonatal, transient hepatomegaly, and elevated liver enzymes, and she later developed inflammatory bowel disease at adolescence. No other subjects had a history of any hepatic, biliary, or pancreatic disorders or biochemical abnormalities in liver function. Abdominal ultrasound or MRI was performed in additional three of the seven Finnish patients for other reasons, for example, due to unspecific abdominal pain, and they all showed normal findings. Abdominal ultrasound or other imaging studies were not routinely performed in our cohort patients.Three patients required psychological assessment and support. All these patients reported also recurrent headache or other pain.This study reports the clinical spectrum of polyostotic FD in a non-selected pediatric cohort. Our cohort included all pediatric patients with polyostotic FD in two Nordic tertiary referral centers between 1996 and 2017.As expected, the spectrum of skeletal manifestations was wide, from few lesions with mild or no symptoms to wide-spread disabling disease with pain and joint involvement.Polyostotic FD was part of MAS in half 8/16) of the patients. All of them presented with café-au-lait spots. In addition, approximately two-thirds of girls had PP, and one-third of boys had testicular involvement, with no other endocrinopathies. None of the patients manifested with hyperthyroidism or overt GH excess. Mild hypophosphatemia as a result of FGF-23-mediated hyperphosphaturia was evident in two-thirds of the patients. In the largest reported FD cohort (NIH cohort) by Collins and coworkers, PP was found in 50% of the female patients and gonadal abnormalities in 70% of the male patients. In the same cohort, café-au-lait spots were found in 66%, renal phosphate wasting in 43%, thyroid abnormalities in 66% (28% with hyperthyroidism), and GH excess in 21% of the patients , even without any other clinical signs of the disease as these may develop much later. MAS should also be kept in mind as an alternative diagnosis in patients evaluated for neurofibromatosis. As for those with polyostotic FD without MAS, craniofacial asymmetry and plain radiographs in case of fractures could usually lead to the diagnosis.Majority of the patients in our cohort had sustained a fracture 11/16). This is in agreement with a previous study on fracture risk in FD . As assu. This isZ-scores were within normal reference range at diagnosis and at the last follow-up visit. Prepubertal and pubertal growth pattern was normal in all subjects without MAS, suggesting that their gonads are not affected by the activating GNAS mutation. On the other hand, over half of our MAS patients with peripherally driven PP were followed without treatment, and they also had normal growth and seemed to reach normal adult height. In the two PP girls with more severe PP, the outcome of treatment with estrogen blocker and GnRH agonist was satisfactory, although one of the girls ended up with slightly shorter final height than expected by parental height Z-scores.To the best of our knowledge, this is the first study to report growth characteristics in pediatric patients with polyostotic FD or MAS. In our cohort, all subjects were born with weight and height appropriate for gestational age. On average, height The best treatment modality for peripheral, gonadotropin-independent PP in MAS patients has remained unclear , 18. A rIn general, the prevalence of MAS is estimated to be 1 per 100,000–1,000,000 . With thIn conclusion, MAS still remains a diagnostic challenge, and delayed diagnosis may have significant consequences. The prevalence of thyroid and GH excess seems to be low in pediatric MAS patients but follow-up until adulthood with careful endocrine assessments is warranted. Skeletal outcome of polyostotic FD can be severe and disabling, and craniofacial lesions can even lead to permanent vision loss. Therefore, craniofacial FD should be closely monitored with skull MRI in childhood to enable early detection of optic nerve compression. Chronic pain is a significant complication and needs to be assessed during follow-up visits in children with polyostotic FD. Pediatric patients with polyostotic FD and particularly with MAS should be followed in tertiary hospitals with experience on these rare patients with strikingly broad spectrum of disease manifestations.This study was carried out in accordance with the Declaration of Helsinki. This retrospective registry-based study protocol was approved by the Research Ethics Committees of Helsinki University Hospital and Karolinska University Hospital, and patient consents were obtained as necessitated by these permissions.PU collected and analyzed the data and wrote the manuscript text with the help of other authors; HV provided the Finnish patients, collected patient data, and participated in writing the manuscript; SB provided the Swedish patients and participated in writing the manuscript; OM planned the study and the manuscript structure and participated in writing the manuscript; and EH provided the Swedish patients, collected patient data, and participated in writing 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.


Finally, 46 genes encoding transcription factors (TFs) maintained a differential expression pattern between the two varieties regardless of the growth site; correlation analyses among TFs, GAS, GAO gene expressions and STLs contents suggest that one MYB and one bHLH may act in the pathway.Stem-chicory of the “Catalogna” group is a vegetable consumed for bitter-flavored stems. Type and levels of bitter sesquiterpene lactones (STLs) participate in conferring bitterness in vegetables. The content of lactucin—and lactucopocrin-like STLs was higher in “Molfettese” than “Galatina” landrace stalks, regardless of the cultivation sites, consistently with bitterness scores and gustative differences. The “Galatina” transcriptome assembly resulted in 58,872 unigenes, 77% of which were annotated, paving the way to molecular investigation of the STL pathway. Comparative transcriptome analysis allowed the identification of 69,352 SNPs and of 1640 differentially expressed genes that maintained the pattern independently of the site. Enrichment analyses revealed that 4 out of 29 unigenes were up-regulated in “Molfettese” vs “Galatina” within the sesquiterpenoid pathway. The expression of two  Cichorium intybus L.) is cultivated worldwide to produce food, coffee surrogates, forages pharmaceuticals, and healthcare compounds  and complex genome  were characterized in several Asteraceae species  are secondary metabolites typical of C. intybus, a reference Gal transcriptome for the “Catalogna” group was created. Digital gene expression (DGE) targeted to stems at the commercial maturity revealed those differentially expressed genes (DEGs), which maintained the patterns in Mol and Gal landraces irrespective of the cultivation area. Focusing on transcriptomic differences of the STL pathway, the Mol genotype was enriched of upregulated genes—two germacrene A-synthase and one oxidase (GAS and GAO)—acting upstream the route. The GAS and GAO transcription levels correlated positively with the contents of 11(S), 13-dihydrolactucin and 11(s), 13-dihydro-8-deoxylactucin, supporting that STL biosynthesis regulation occurs at the transcriptional level. Consequently, the GAS and GAO higher expression levels of Mol vs. Gal may account for higher contents of STLs, supporting that these genes may be good expression markers for bitterness selection. Correlation analyses among the expression levels of GAS, GAO, and transcription factor (TF) genes and STLs contents pointed at MYB and bHLH as the best TF candidates in the STL biosynthesis regulation.A major aim of this work was to characterize the factors that contribute to bitterness at the metabolic and genetic levels by comparing “Galatina”  and “Molfettese” (Mol) landraces. The STL quantification revealed a higher content of lactucins and lactupicrins in the latter compared to the former, independently of the growing sites. Due to the lack of comprehensive genomic information on Cichorium intybus L. “Catalogna” group) were previously described, including botanical classification, phenotypical traits, site coordinates, and cultivation parameters . As for Apulia, harvesting occurred on the 14/01/2013 and 25/01/2013 for Mol and Gal, respectively; the average temperatures 1 week before harvesting were 9.4 ± 0.1°C and 9.5 ± 0.3°C, and the average temperature of the 1–25/1/2013 lapse was 9.7 ± 0.2°C (www.agrometeopuglia.it). Harvested plants were brought to laboratories, selected for comparable weights  and processed. In the experiments of STL lactones quantification, transcriptional and allelic variation analyses, marketable stems were removed from rosettes (n = 15) of each landrace. A replicate batch consisted of 10 homogeneous stems . These were immediately frozen in liquid nitrogen and stored at −80°C for RNA isolation or they were lyophilized at −50°C for 72 h  and stored at −20°C for HPLC analyses. Three biological replicates were used in all the experiments.The “Galatina”  and “Molfettese” (Mol) landraces of stem chicory , the samples were prepared by adopting both cellulase hydrolytic treatment  and commercial maturation (n = 5) stages were used; distinct tissues  were sampled and ground in liquid nitrogen. 500 mg of each tissues was used to isolate total RNA by TRIzol reagent (Invitrogen) followed by an additional purification step using RNAeasy separation columns ; yields were estimated by electrophoretic and spectrophotometric analyses , and RNA integrity number (RIN > 7) was verified using BioAnalyzer 2100 (Agilent Technologies Inc). cDNA libraries were prepared using TruSeq RNA-seq sample preparation kit (Illumina) and sequenced in 100 bp paired-end mode using an Illumina HiSeq2000 .For transcriptome reference assembly, Gal plants at the transplant (n = 10) at harvesting time were sampled from Gal (n = 5) and Mol (n = 5) grown in each location. Total RNA was isolated, quantified and controlled for yield and integrity as described above. Illumina Truseq cDNA libraries were prepared and sequenced in 50 bp single-end on Illumina HiSeq2000 platform. For a given genotype, three biological replicates for each growing area were generated. RNAseq data sets have been stored in the National Center for Biotechnology Information database  under the BioProject accession number PRJNA328202.As for NGS transcriptional analyses and SNP mining, size-comparable stems (de novo) and two-steps (EST-based backbone construction followed by a de novo assembly) approaches. In the one-step approach the cleaned reads were assembled using Trinity  and assembled using the EGassembler pipeline  mining the following databases: Nr, NCBI non-redundant database ; TAIR, The Arabidopsis Information Resource (TAIR10); SwissProt and TrEMBL, the manually annotated and reviewed and the automatically annotated and not reviewed sections of the UniProt Knowledgebase (UniProtKB), respectively (release 2014_02); KOG, euKaryotic Ortholog Groups , SAMtools  was used for identification of potential simple sequence repeats (SSRs). Units with one to six nucleotides and a minimum repetition of twelve units for mono-nucleotides, six for di-nucleotides, five for tri-, tetra-, penta- and hexa-nucelotides were considered in the analysis.BWA  values.The raw single-end reads were trimmed as described above. For a given genotype, the cleaned reads were mapped on the reference assembly using BWA  of stems was isolated by the RNeasy Plant Mini Kit (Qiagen), DNase treated , and 1 μg was reverse-transcribed at 55°C by SuperscriptIII (Life Technologies). The cDNA (100 ng) was amplified by Eco Real-Time PCR System (Illumina) using 1x Quantimix easy master mix (Biotools) and 0.3 μM of each primer in a 10 μl final volume. The triplicate reaction conditions were as follows: 95°C for 10 min, 45 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 40 s. Primer specificity was checked by melting curve analysis and by agarose gel electrophoresis. Three technical replicates and three independent biological experiments were performed for each sample. The expression levels of the target unigenes were normalized with the reference genes ACT  ≥ 1 were set as the thresholds for the significance of the gene expression difference. The hypergeometric test  was applied to STL content variation in landraces grown in Apulia and Lazio cultivation sites, followed by Duncan Multiple Range Test. All procedures, General Linear Model and means separation, were carried out by Statistical Analysis System program . The principal component analysis (PCA) allowed a visual overview about spatial distribution and grouping among genotypes and growing sites; it was based on mean centered and standardized data  and results were shown as bi-plots of scores (treatments) and loadings (variables) plots . Differential expression analysis was performed using the Bioconductor edgeR package  as well as that of total lactucin-like forms  independently of the growth site , 8-deoxylactucin (dLc), lactucopicrin (Lp) and the respective dihydro-derivatives, 1,3-dihydrolactucin (DHLc), 11(s), 13-dihydro-8-deoxylactucin (DHdLc), 11(s), 13-dihydrolactucopicrin (DHdLp) were quantified in stems of Mol and Gal landraces. Overall, the total STL content was significantly higher in Mol than Gal stems  which could raise the unigene mean length from ca. 760 to 1248 bp. Finally, the unmapped reads were retrieved by Bowtie2  matches in the Nr database, 38,978 (66.2%) in the TrEMBL, 36,281 (61.6%) in the Tair, and 26,233 (44.6%) in the SwissProt databases. GO, KEGG and KOG databases allowed functional classification of unigenes. An overview of the unigene annotations are in Table C. intybus Gal transcriptome with at least one annotation signature were 45,572 ; subsequently, we selected the genes that maintained the relative expression pattern (independently of the area) for further characterization. 12,274 DEGs were identified , germacrene A oxidase  and β-amyrin synthase . GAS and GAO are two key enzymes in the synthesis of germacrene-type STLs and act consecutively in the upstream steps that generate the costunolide  mRNAs. The GAS, GAO, and COS proteins shared significant identities with several Asteraceae orthologues .Sesquiterpene lactones (STL) precursors belong to the sesquiterpenoid and triterpenoid pathway (STP); therefore, the latter was further characterized by identifying 29 unigenes putatively encoding 16 distinct enzymes Table . Four unts Table , 1 GAO aes Table ; the seqes Table . The Aste Figure  placed Cy Figure . The Aste Figure  assignedTFs) involved in the STL pathway. Globally, 2680 gene deduced products could be ascribed to 57 TF families according to PlantTFDB rules , GAO (Ci_contig7113) and the 46 TFs genes. A correlogram was built  and GAO (Ci_contig7113) showed very strong (r > 0.8) positive correlation between them and strong (0.7 ≤ r ≤ 0.8) correlation with total STL amounts  and MYB (Ci_contig49541) genes and both GAS and GAO. Conversely, strong (−0.8 ≤ r ≤ −0.7) and very strong (r < −0.8) negative correlations characterized the transcription of bHLH (Ci_contig48487) vs. that of GAS and GAO, while strong negative correlation of MIKC (Ci_contig17846) and ERF (Ci_contig18404) occurred just vs. GAO.We approached the search for transcription factor genes  showed 7 SNPs, while GAS (Ci_contig62598) contained one heterozygous SNP, which produced a synonym substitution (Tyrosin).Polymorphism identification from transcriptome sequences is useful to score gene functional variations in species without a sequenced genome. Consequently, SSR and SNP were mined and a specific focus was on the STL pathway genes. Overall, 11,672 putative SSRs were found in 9826 unigenes and 1525 of them contained more than one microsatellite Table . Excludede novo transcriptome assembly, transcript and metabolite profiling was used to achieve insights in the genetic pathway of sesquiterpenes and, more generally, valuable genetic tools using two stem-chicory “Catalogna” landraces.In this work, the combination of a 2 = 56.89; p < 0.001; Table C. intybus sugarloaf, witloof and radicchio  and endive (C. endive) leaves  and triterpenoid (β-amyrin synthase) biosynthesis  are 2-fold more expressed in Mol than Gal. Similarly, the GAO gene transcription is twice higher in Mol than Gal and the deduced protein corresponds to that of C. intybus available in public databases. Significant positive correlation occurred between GAS and GAO transcriptions and the contents of Lc-like . These results corroborate the finding that terpene accumulation goes in parallel with the expressions of respective synthase genes , it is worth noting that the expression was more abundant in Mol than Gal in Apulia; the trend was maintained in Lazio, but not at significant level  maintained differential expression pattern between the two genotypes in both Apulia and Lazio sites. This suggested that environment minimally or equally affected this gene pool, which may represent a source of transcriptional markers. Within this pool, four up-regulated DEGs  have been unknown as well as the routes of catabolism and transport. Moreover, the knowledge on the TFs involved in sesqui- and triterpenoids biosynthesis is still fragmentary  and negative  correlations with both the biosynthesis genes (GAS and GAO) and total STLs (three-way relationship). Given that MYB factors and bHLH members control sesquiterpenes synthesis in plants acting on terpene synthase genes  showed a very strong negative correlation (r < −0.8) with GAO and total STLs, but not with GAS. Similarly, the Ci_contig45269 (WRKY) had a strong positive correlation with GAO but not with GAS or total STL. Consequently, these TFs might take part in STL metabolism of C. intybus through specific routes or indirectly, consistently with their role in regulating STL biosynthesis in other species  differentiating Gal and Mol unigenes. This implies that no protein mutation occurs that might explain the STL content differences. These latter are more likely due to a diversified gene expression regulation . Nonetheless, the heterozygous SNP provides a tool for genetic mapping of this key gene in the stem-chicory populations.Finally, the landrace comparative approach and data mining of transcriptome and metabolic variations were efficient to discover genes involved in STL pathway as a precious source to comprehend regulation of bitter taste in this vegetables and support plant breeding for product quality.DG was responsible for research costs and guided the work design and manuscript writing. GT carried out transcriptome assembly, differential gene expression analysis, polymorphism mining, qPCR validation, statistics and writing. GM contributed to transcriptome assembly and software usage. MG and MR performed statistical analysis on sesquiterpenes contents and gustative test. MG, GA and AS produced plant materials. GCT carried out the sesquiterpenes quantification. CN, GF, ED, MI performed sampling, phenotyping and nucleic acid extractions. All the authors reviewed, edited and approved the manuscript.www.mezzogiorno.cnr.it). CNR solutions n. 73 and n.74 of 29/3/2011 and pp. 1–26 of the annexed executive project.This work was supported by a dedicated grant from the Italian Ministry of Economy and Finance to the National Research Council for the project “Innovazione e Sviluppo del Mezzogiorno–Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare” Law 191/2009 (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.


Concerning vulvar and cervical cancers, the sentinel lymph node procedure has been incorporated to the current guidelines in Europe and North America, whereas for endometrial cancer it is considered investigative.This review discusses the state-of-the-art of sentinel lymph node mapping in gynaecological malignancies, including cervical cancer, endometrial cancer, and vulvar cancer, with an emphasis on new technological advances. For this objective, PubMed/MEDLINE was searched for relevant studies about the sentinel lymph node procedure in gynaecology. In particular, the use of preoperative lymphatic mapping with lymphoscintigraphy and single photon emission tomography/computed tomography (SPECT/CT) was identified in 18 studies. Other recent advances as hybrid tracers (e.g. ICG- All these particular aspects of SLN procedure in gynaecological malignancies will be discussed in this review. A research of the literature was performed on PubMed/MEDLINE using the following keywords (MeSH terms) to encounter the most relevant studies about the SLN procedure in gynaecology: “SLN biopsy”, “lymphatic mapping”, “lymphoscintigraphy”, “SPECT/CT”, “intraoperative SLN detection”, “hybrid tracer”, “vulvar cancer”, “cervical cancer”, and “endometrial cancer”. The search has been restricted to the English language. The references of the retrieved articles were examined to identify additional articles. This review also includes meta-analyses published in the last five years.In gynaecological tumours, the sentinel lymph node (SLN) procedure is principally performed in vulvar cancer (VC), cervical cancer (CC), and endometrial cancer (EC). Although both preoperative lymphatic mapping and intraoperative SLN detection are common parts of SLN procedure in gynaecological tumours, the type of injection and lymphatic drainage is different for each one of these malignancies Fig. . In vulv99mTc-nanocolloid) in the four quadrants of the cervix using a 20 or 22-gauge spinal needle. In the case of previous conisation, the peri-cicatricial injection at the four quadrants is recommended [Cervical cancer (CC) is the third most common gynaecological cancer with an estimated of 12,990 new cases and 4120 deaths in the US, in 2016 . Accordiommended . The mosommended . The injommended . The earommended . Therefoommended , providi99mTc SPECT/MRI-fused images have been used for SLN mapping in preoperative assessment of SLN metastases in the early-stage cervical cancer in women. The authors found a 99mTc SPECT-MRI accuracy of 74.9 % (95 % CI 0.569–0.930) to non-invasively assess SLN metastases, including 136 SLNs of which 13 (9.6 %) in 8/79 patients (10.7 %) contained metastases [SPECT/CT has higher SLN detection rate compared to the conventional planar images (98.6 vs. 85.3 %), as reported in a recent meta-analysis, including eight studies . In genetastases .Table 1DEndometrial cancer (EC) is the most common malignancy of gynaecological cancer with an estimated incidence of 60,050 new cases and 10,470 deaths in the US, in 2016 . RadicalThe deep lymphatic drainage of the corpus uteri is a probable reason for the low correlation found between planar lymphoscintigraphy and surgical mapping . This liVulvar cancer (VC) is a rare gynaecological malignancy with an estimated number of 5950 new cases and 1110 deaths in the US, in 2016 . The cur99mTc-nanocolloid) in three or four intradermal/intramucous around the primary lesion or excision scar a few minutes after the application of an anaesthetic lidocaine spray or crème. Simultaneous anterior and lateral dynamic lymphoscintigraphy is performed immediately after injection followed by early (15 min) and delayed (2 h) static planar imaging. Subsequently, SPECT/CT is recommended as complementary modality, providing anatomical and functional information facilitating more accurate virtual surgical planning [The SLN mapping is performed through injection of radiocolloid  and to detect additional lymph nodes in the same region or in other regions with poor or even without visualization at planar lymphoscintigraphy Fig. , as well99mTc-nanocolloid in one signature represents a new hybrid tracer for the detection of SLN. Recently, Mathéron et al. reported the use of a new hybrid tracer, in the SLN identification procedure in vulvar cancer. They showed that 98 % of the SNs were radioactive at the time of excision, 96 % were fluorescent, and only 65 % were blue in 15 patients. The additional value of ICG is related to better intraoperative visualization of SN to optimize the intraoperative SLN visualization using the fluorescence component [99mTc-nanocolloid may be used in the SLN(s) identification procedure during the robot-assisted laparoscopy [The indocyanine green (ICG) added to omponent . Neverthomponent . In the aroscopy , 52. In aroscopy . Furtheraroscopy .In conclusion, the SLN procedure has widely been validated in vulvar cancer and cervical cancer. Its application in these malignancies is well standardized and has been incorporated to the current guidelines in Europe and North America. By contrast, in endometrial cancer, there are various controversial aspects (e.g. injection route) to be clarified and the use of the SLN procedure needs to be validated in larger clinical series. Beside these aspects, the present review showed that recent technological advances, such as preoperative and intraoperative use of SPECT/CT, the contribution of the hybrid tracer ICG-99mTc-nanocolloid, and technological advances like SLN robotic-guided procedure might play an increasing role to guide gynaecological cancer surgery in the future.


P < 0.001, P < 0.001, and P = 0.014, resp.). Plasma levels of pentosidine were significantly decreased in patients who received metformin compared to untreated patients (P = 0.01). However, there was no significant difference between patients treated with metformin and untreated patients in plasma CML levels. Plasma levels of sRAGE were significantly increased in patients who received metformin and ACE inhibitors . However, in a multiple stepwise regression analysis, pentosidine, sRAGE, and drugs treatments were not independently associated. Patients with metabolic syndrome showed increased levels of AGEs such as pentosidine and CML. Metformin treatment showed a decreased level of pentosidine but not of CML. Therapeutic pathways of AGEs development should be taken into account and further experimental and in vitro studies merit for advanced research.Metabolic syndrome (MetS) is considered one of the most important public health problems. Several and controversial studies showed that the role of advanced glycation end products (AGEs) and their receptor in the development of metabolic syndrome and therapeutic pathways is still unsolved. We have investigated whether plasma pentosidine, carboxymethyl-lysine (CML), and soluble receptor for advanced glycation end products (sRAGE) levels were increased in patients with MetS and the effect of metformin in plasma levels of pentosidine, CML, and sRAGE. 80 control subjects and 86 patients were included in this study. Pentosidine, CML, and sRAGE were measured in plasma by enzyme-linked immunosorbent assay (ELISA). Plasma pentosidine, CML, and sRAGE levels were significantly increased in patients compared to control subjects ( Cardiovascular diseases are one of the main causes of mortality in industrialized countries, due to an increase in prevalence of different cardiovascular risk such as abdominal obesity, insulin resistance, dyslipidemia, hypertension, and hyperglycemia. These metabolic disorders presented within the same individual give rise to metabolic syndrome. Recent estimates are that between 20 and 30% of the adult population worldwide have a metabolic syndrome . The cauε-N-carboxymethyl-l-lysine (CML) have been well characterized as biomarkers for the formation and accumulation of AGEs [Due to complex metabolic disorder in metabolic syndrome, reactive derivatives are formed via nonenzymatic reaction named Maillard reaction between the free amino groups in lysine and arginine residues in proteins and carbohydrates that undergo a series of complex reactions to an irreversible complex group of compound termed advanced glycation end products (AGEs) , 3. Thes of AGEs  and are  of AGEs –9. AGEs  of AGEs –12. RAGEPrevious studies have confirmed that interaction between AGEs and RAGE activates the generation of oxidative stress and production of proinflammatory cytokines in various types of cells that have been implicated in metabolic and many other disease statuses –16. FurtThe present study was designed to elucidate, in the first part, the level of serum pentosidine, CML, and soluble form of RAGE (sRAGE) in patients with metabolic syndrome and, in the second part, to investigate the effect of several drugs, in particular, metformin which is known by its antiglycation effect, on pentosidine, CML, and sRAGE levels in these patients.n = 55) and patients who are untreated with this drug (n = 31).This study included 80 control subjects and 86 patients who developed a metabolic syndrome recruited from Department of Endocrinology of Taher Sfar Hospital in Mahdia (Tunisia). The criteria of the American Heart Association Scientific Statements of 2009 were used to define metabolic syndrome . PatientIn all subjects, venous blood was collected in the morning after an overnight fast. After centrifuging the samples at 3000 rpm for 10 min at 4°C, plasma and serum were collected and stored at −80°C until analysis. Fasting plasma glucose, triglyceride, total cholesterol, HDL cholesterol, and LDL cholesterol were measured using enzymatic methods by CX9 autochemical analysis instrument . Hemoglobin A1C (HbA1c) was measured using G7 HPLC Analyser (Tosoh Europe NV). Plasma levels of pentosidine, CML, and sRAGE were determined by enzyme-linked immunosorbent assay (ELISA) provided by Cusabio Biotech Co., Ltd., according to the manufacturer's instructions. Detection range of pentosidine CML and sRAGE kit was, respectively, 31–2000 pmol/mL, 62–4000 pg/mL, and 78–5000 pg/mL. The intra-assay and interassay coefficients of variation were <8% and <10%, respectively. t-test or Mann–Whitney U test. A P value less than 0.05 was considered statistically significant. Correlation was determined by a linear regression analysis and a multiple regression analysis was used to further explore the linear relationships between the variables. Regression variables were estimated as well as the correlation coefficient r. ANOVA was used to assess the significance of the regression with significance accepted at P < 0.05.The study data was expressed as mean ± SD or median and interquartile ranges (IQR) using the SPSS program (version 18). Differences between groups were analyzed by the independent sample Student'sP < 0.001), and total and HDL cholesterol (P < 0.01).The clinical, anthropometric characteristics and treatment drugs of controls and patients with MetS are shown in P < 0.001, P < 0.001, and P = 0.014, resp.).P = 0.01), respectively. However, no significant difference between metformin effects on plasma CML levels had been shown. Plasma levels of sRAGE were significantly increased between patients who received metformin and patients without this treatment: 180.9 (142.33–273.85) versus 131.84 (111.75–226.98) pg/mL (P < 0.001), respectively. We cannot conclude that this increase is due to the effect of metformin because there is an increase in sRAGE plasma levels in patients who are treated with ACE inhibitors: 194.9 (138.59–468.59) versus 142.33 (116.69–194.32) pg/mL in untreated patients (P = 0.002). This finding was illustrated in Figures As shown in A linear regression analysis in patients with MetS showed negative and significant correlation between pentosidine levels and metformin treatment. sRAGE levels correlated positively with metformin treatment and/or ACE inhibitors treatment. However, no significant correlation between drugs treatments and CML levels had been shown . To explPredictors and identification of factors involved in the modulation of metabolic disorders of metabolic syndrome may be important in the prevention and the management of their complications. For this context, in the first part, the present study demonstrated new biomarkers such as plasma pentosidine, CML, and sRAGE levels, which are increased in patients with MetS. In the second part, our study evaluated the levels of these biomarkers in patients treated with drugs recognized by their antiglycation effect as metformin. Our results showed decreased pentosidine and increased sRAGE plasma levels in treated patients compared with untreated patients.A number of clinical studies have reported that AGEs accumulate at a much higher rate in diabetic than in normal population –25. Otheβ who modulates genes transcription for various factors: endothelin-1, vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), proinflammatory cytokines such as interleukins 1 and 6, and tumor necrosis factor-α (TNF-α) [The reason why AGEs are involved in the genesis of metabolic syndrome needs to be explained. At the molecular levels, AGEs including pentosidine and CML cause damage by two principal mechanisms. First, structural modification of proteins perturbs their normal function. Second, AGEs are ligands for the receptor for AGEs (RAGE) causes intracellular oxidative stress and activation of nuclear factor NF-K (TNF-α) , 39. As  (TNF-α) .A several metabolic components well established as risk factors for cardiovascular disease have also been shown to be associated with altered plasma sRAGE or esRAGE. Plasma esRAGE levels are decreased in patients with MetS and are inversely correlated with several components of MetS including body mass index, blood pressures, insulin resistance index, fasting plasma glucose, serum triglyceride, and lower HDL cholesterol levels . Hudson In addition to its action on glycemic control through the decrease in hepatic glucose production and increasing the sensitivity of peripheral cells to insulin, a number of studies have shown that metformin is beneficial in reducing diabetes associated vascular risk by inhibition of glycation process and the intermediate of AGEs such as MG , 50. The in vitro studies merit for advanced research.Patients with metabolic syndrome showed increased levels of AGEs products such as pentosidine and CML. Metformin treatment effect showed a decreased level of pentosidine but not of CML. Therapeutic ways of AGEs development should be taken into account and further experimental and


Crude oil is a complex mixture of hydrocarbons with different structures; its components vary in bioavailability and toxicity. It is important to understand how bacterial communities response to different hydrocarbons and their co-acclimation in the process of degradation. In this study, microcosms with the addition of structurally different hydrocarbons were setup to investigate the successions of bacterial communities and the interactions between different bacterial taxa. Hydrocarbons were effectively degraded in all microcosms after 40 days. High-throughput sequencing offered a great quantity of data for analyzing successions of bacterial communities. The results indicated that the bacterial communities responded dramatically different to various hydrocarbons. KEGG database and PICRUSt were applied to predict functions of individual bacterial taxa and networks were constructed to analyze co-acclimations between functional bacterial groups. Almost all functional genes catalyzing degradation of different hydrocarbons were predicted in bacterial communities. Most of bacterial taxa were believed to conduct biodegradation processes via interactions with each other. This study addressed a few investigated area of bacterial community responses to structurally different organic pollutants and their co-acclimation and interactions in the process of biodegradation. The study could provide useful information to guide the bioremediation of crude oil pollution. Over one million tons of petroleum enters marine environments, originating from both natural  and anthropogenic sources every year34Alcanivorax, Cycloclasticus, Oleiphilus, Oleispira and many other phylogenetic groups are well-acknowledged as ‘professional hydrocarbonoclastic bacteria’, since they always use hydrocarbons as priority carbon source15Alcanivorax was regarded as the typical functional group specifically degrading strait-chain or branched alkanes9Cycloclasticus showed priority in degrading PAHs18Crude oil is regarded as one of the most complex mixture of organic compounds, which is mainly composed of the saturated hydrocarbons, the aromatic hydrocarbons, and non-hydrocarbons 669101112et al.Thalassolituns and Roseobacter dominated the process of n-alkane degradation; bacteria in the genus Alcanivorax were responsible for the degradation of branched alkane, and Cycloclasticus was the dominant genus in PAHs degradation. This study provided preliminary data about partition of different petroleum hydrocarbons by microbial population. Further investigations could be conducted on revealing individual functions of different bacterial taxa and interactions between different microbes in the process of biodegradation. Here, a series of microcosms were setup with addition of petroleum hydrocarbons with different structures, to investigate 1, successions of bacterial communities under stresses of different hydrocarbons; 2, functions and contributions of different bacterial taxa in each steps of hydrocarbon-degrading; 3, co-acclimations between different bacterial phylotypes or between different functional groups. This study could provide a better understanding of co-acclimation of functional bacteria in the process of biodegrading hydrocarbons.Although preferences of different substrates by different hydrocarbon-degrading bacteria have been well-studied in laboratory by using pure cultures, little is known about functions of individual bacterial taxa and co-acclimations between different bacteria with degrading ability in the degrading process. McKew After the four times domestication with 10 d time’s interval, bacterial communities in sediments showed great degrading efficiency to different hydrocarbons . BiodegrMiseq High-throughput sequencing of 16S rRNA gene amplicons yielded 1,319,248 raw reads. After quality filtering and normalizing, 1,237,359 sequences were selected for further analysis. The numbers of sequences used for duplicate samples of original sediment were 3348 and 3387, while numbers for all other samples ranged from 4964 to 6423. This generated totally 4750 OTUs. Each duplicate sample had a mean of 1532 OTUs (AVEDEV = 101). Rarefaction curve analysis  indicateSignificant temporal variations of bacterial communities were found during the domestication period, as well as dramatically different responses of bacterial communities to structurally different hydrocarbons were also observed in this study. By comparing bacterial phylotypes which showed dramatic changes at the third domestication phase and those at the fourth domestication phase, we found that bacterial communities reached a relatively static status after 30 d incubation . For exaAlthough Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were dominant in all Miseq sequencing data, obvious variations of bacterial communities at phyla-level (including classes in proteobacteria) were detected under stresses of different hydrocarbons . For inse. g. unclassified groups in families of Desulfobulbaceae, and Desulfuromonadaceae), and Epsilonproteobacteria (e. g. Sulfurimonas and an unclassified group in Helicobacteraceae) were abundant in sediment samples, but could hardly be found after co-incubation with five hydrocarbons. Contrarily, some other groups (e. g. Pseudomonas in Gammaproteobacteria and an unclassified group in the family of Erythrobacteraceae in Alphaproteobacteria) showed significant increase after domestication in all five treatments. When different treatments were considered, obvious variations of dominant bacterial phylotypes were discovered. Three unclassified groups in families Erythrobacteraceae, Xanthomonadaceae, and Rhodobacteraceae, dominated bacterial communities in the treatment with addition of tetradecane. Sequences affiliated with the genus Pseudomonas increased in the first 20 d, but decreased after 20 d and kept steady at the two last domestication phases. When pristane was added, sequences in genera Marinobacter, Hyphomonas, Muricauda, Devosia, and the two unclassified groups in families Erythrobacteraceae and Xanthomonadaceae, became dominant groups. For the treatment of hexacosane, two Gammaproteobacterial groups, Pseudomonas and an unclassified group in the family of Pseudomonadaceae showed great superiority compared with all other phylotypes. The two dominant groups changed to Aequorivita and the unclassified groups in the family Erythrobacteraceae when phenanthrene was added as a stress. Bacterial sequences in the two genera Pseudomonas and Aequorivita and other two unclassified groups in families Alcaligenaceae and Pseudomonadaceae dominated bacterial communities after domestication with addition of pyrene.To analyze successions of bacterial communities in depth, a heatmap profile was generated based on abundance of sequences at genera level . In accoNocardia increased dramatically (81.6 times) after 40 d co-incubation with hexacosane, while sequencing numbers of the other genus Marinomonas decreased 93 times compared to the numbers in sediment samples. Meanwhile, some bacterial groups responded to two or more different hydrocarbons. For example, bacteria in genera Parvibaculum and Devosia showed greatly increasing rate with stresses of two n-alkanes, tetradecane and pristane, while bacteria affiliated into the genus Oceanimonas increased dramatically when the two PAHs were applied. Bacteria affiliated into the genus Pseudomonas showed significant increase in treatments with addition of tetradecan, hexacosane, phenethrene, and pyrene. The unclassified bacterial group in the family Syntrophobacteraceae responded negatively to pristane, tetradecane, phenathrene and pyrene, except for the C26 alkane. Bacterial phylotypes in genera Sulfurimonas, Nitrosopumilus, and Desulfococcus, and some unclassified groups in families Desulfobulbaceae, Helicobacteraceae, and Piscirickettsiaceae, and an unclassified group in the class of Gammaproteobacteria, which were aligned at the bottom of the network  and different hydrocarbons indicated varied responses of bacteria to different stresses . Some ba network , showed α/ω-hydrocy fatty acid which could be easier degraded via the fatty acid degradation pathway  for the gene encoded with the unspecific monooxygenase in the fourth step, which was 6% less than the proportion of Nocardia.Functions of different OTUs and proportions of functional bacterial groups participated in each step of biodegradation were predicted by using KEGG and PICRUSt. There were four steps for biodegrading alkane to  pathway . Four di pathway . The unchttp://www.kegg.jp/kegg/) suggested pathways for pyrene and phenanthrene degradation. Pyrene and phenanthrene could be biodegraded to 3,4-dihydroxy-phenanthrene (pathway name as rn00624) which could be further metabolized into tricarboxylic acid cycle (TCA cycles) via “Naphthalene degradation (rn00626)”, and “Benzoate degradation (rn00362)” or “Tyrosine metabolism (rn00350)”. Most of functional genes encoded with enzymes in these four pathways were successfully predicted in bacterial communities treated by pyrene and phenathrene . Bacteria in the genus Mycobacterium were found to contribute to reactions catalyzed by 39 different enzymes, including the four enzymes mentioned above. This group of bacteria were abundant (>10%) in almost of these reactions, except for the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase (EC: 1.13.11.27), which was mainly contributed by bacteria in the genus Pseudomonas. Bacteria in the genus Oceanimonas, and three unclassified groups in families of Alcaligenaceae, Comamonadaceae, and Erythrobacteraceae were also found to be abundant in different catalyzing reactions. Contributions of different bacterial phylotypes to reactions in the process of biodegrading the other PAH, phenanthrene were exhibited in Mycobacterium, Kaistobacter, Methanosaeta, Pseudonocardia and unclassified phylotypes in families Rhizobiales, Rhodobacteraceae, Rhodocyclaceae, Pseudomonadaceae, Clostridiales and Solirubrobacterales. Besides these exclusive groups, some bacterial phylotypes, e.g. Pseudomonas, Oceanimonas, and the unclassified group in the family of Erythrobacteraceae, showed great priority in catalyzing different reactions.The “Xenobiotics biodegradation and metabolism” list on KEGG website . The “functional group” 3 involved in all the four steps of biodegradation of pyrene, which mainly attributed to the key member of the group, Mycobacterium. The other four groups included different bacterial phylotypes contributed to different steps of biodegradation. For example, the “functional group” 4, which included several different phylotypes carrying degradation related genes, contributed mainly in the three steps of naphthalene, tyrosine and benzoate degradation/metabolism, but not the step of PAHs biodegradation.Bacterial communities with significant correlations during the domestication period were applied to construct networks which could demonstrate the co-acclimation of functional bacteria. Five complex networks were generated for five different treatments using different hydrocarbons respectively . All fivn-alkanes, branched alkane and PAHs. The known lists of these generalists included Roseobacter, Acinetobacter, Marinobacter, Pseudomonas and Rhodococcus27282930Sulfurimonas and three unclassified groups in families of Desulfobulbaceae, Desulfuromonadeceae and Helicobacteraceae could not be detected after 40 d co-incubation with five different hydrocarbons. The first three groups were well-acknowledged to participate in the geochemical cycle of sulfurNocardia and Devosia were observed to be mainly positively responded to n-alkanes and branched alkanes. The genus Nocardia was previously implicated in long-chain n-alkanes and functional genes  were successfully detected33Nocardia increased more than 80 times. Also, bacteria in this genus could degrade pristane which was regarded to be more recalcitrant to degradation and was always used as a slowly biodegradable biomarkerNocardia , a branched alkane , and two PAHs . Minimal salt medium (MSM) consisted of 1000 mg/L (NH4)2SO4, 800 mg/L Na2HPO4, 200 mg/L KH2PO4, 200 mg/L MgSO4•7H2O, 5 mg/L FeCl3•3H2O, 1 mg/L (NH4)6Mo7O24•4H2O, 100 mg/L CaCl2•2H2O, and 20 g/L NaCl. Different hydrocarbons at a final concentration of 400 mg/L were supplemented into MSM medium for further experiments. Sediments used in this study were collected from Bohai sea , north of China. Subsurface sediments were collected by using bottom sediment grab sampler and transferred to the laboratory immediately for further analysis.Five hydrocarbons with different structures were applied in this study , includiAliquots (10 g) of sediments were inoculated into 100 ml MSM solutions and co-incubated with different hydrocarbons at 170 rpm, 25 °C. Ten milliliters of mixed liquid culture (including sediments) were transferred into new MSM solutions with corresponding hydrocarbons every ten days for 40 d. Residuals were kept at −80 °C for bacterial community analysis. 40 dAll the treatments at the first two transformations were performed in duplicate. For accurate chemical analysis, quadruplicate treatments were setup for each hydrocarbon at the last transformation. Two of the quadruplicating samples were specially used for chemical analysis, in case of any loss of hydrocarbons in solutions or on flasks. And the other two were applied for analysis of bacterial communities at the day 40. Controls were performed by adding 400 mg/L of different hydrocarbons in MSM medium without inoculation of sediment.4). Dehydrated solutions were then concentrated by using rotary evaporator to a final volume of 10 ml. Extracted hydrocarbons were analyzed by GC-MS (Agilent 7890-5975c) equipped with a DB-5 capillary column . High purity helium (99.999%) was applied as the carrier gas with the speed of 1 ml/min. The temperature program was conducted as follows, 50–200 °C at 25 °C/min, 200–260 °C at 15 °C/min, and 260 °C for 10 min. Injector and transfer line temperatures were both set at 260 °C. Degradation efficiency was calculated by comparing peak areas of hydrocarbons.Hydrocarbons in liquid solutions were dissolved in dichloromethane, and all solutions were transferred into separatory funnel. Flasks were then washed twice by 20 ml of dichloromethane and then mixed with precious solutions. Hydrocarbons were then extracted using dichloromethane three times and all organic phase was collected. Dehydration of collected solution was performed by addition of excessive magnesium sulfate . DNA concentration and quality were determined by using a NanoDrop 2000 UV-Vis Spectrophotometer . High quality DNA was then applied for Miseq sequencing for amplifying V4 hypervariable region of 16S rRNA gene. The primer set used in this study was universal primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with 12 nt unique barcode. The sequence data were analyzed by using QIIME Pipeline–Version 1.9.0 (http://qiime.org/). After being trimmed and assigned to each sample based on barcodes, sequences with high quality  were chimera checked by using usearch quality filter (usearch_qf)50et al.Total genomic DNA was extracted by using PowerSoilNetwork profiles were constructed to verify the stationary of bacterial communities as follows. Bacterial phylotypes showing significant differences (F-test < 0.1) were used to construct networks by applying the open source platform, Cytoscapehttp://hemi.biocuckoo.org/faq.php), while network was constructed by using cytoscape. Bacterial phylotypes showing dramatic changes (F-test < 0.1) where selected as source interaction elements, and hydrocarbons were regarded as target interaction elements when network was constructed. Interactions between source and target elements were conducted based on different types of responses of bacterial communities to stresses of varied hydrocarbons .Successions of bacterial communities of the whole domesticating period were analyzed based on 16S rRNA gene sequencing data and assigned taxonomic information. First, all sequences were classified to different bacterial phyla and different classes in the phylum proteobacteria. Relative abundances of different bacterial phylotypes at phylum-level (class level in proteobacteria) were then calculated and visualized by using ggplot2 packages in RPICRUSt . Networks were also generated by Cytoscape as mentioned above and split into different functional groups (subnetworks) by using “Cytocluster” in the module. Functions were endowed to different sub-networks based on predicted, functions of dominant bacterial phylotypes in the group.To investigate the co-acclimation of bacterial communities and define functions of different bacterial groups, networks were constructed for bacterial communities with bacterial phylotypes, showing significant correlation evaluated by Pearson’s correlation analysis .How to cite this article: Wang, H. et al. Co-acclimation of bacterial communities under stresses of hydrocarbons with different structures. Sci. Rep.6, 34588; doi: 10.1038/srep34588 (2016).


After piglets were vaccinated against classical swine fever virus and foot-and-mouth disease virus, the antibody titers were significantly increased. RDS treatment showed an excellent resistance to enhance T-SOD activity. Values of blood routine and blood biochemistry showed no toxicity. These results suggested that RDS could be considered as an adjuvant to enhance immune responses to vaccines, as well as dietary additives for animals to enhance humoral and cellular immunity.Resveratrol, a polyphenolic plant antitoxin, has a wide range of pharmacological activities. In this study, we systematically evaluated the effects of resveratrol dry suspension (RDS) on immune function in piglets that were treated with different doses of RDS for 2 weeks. The results showed that the RDS has significant effects on the development, maturation, proliferation, and transformation of T lymphocytes. RDS could regulate humoral immune responses by upregulating the release of IFN- The immune system is a vital barrier against the invasion of microorganisms, and it assumes enormous importance in fight against diseases and malignant abnormal cells . Modern  Polygonum cuspidatum, was first found in red wine because of the beneficial effect on the heart [Resveratrol , a natural polyphenolic compound extracted fromhe heart .Resveratrol supplementation in rat diets showed an increase in IgM concentration and splenocyte proliferation and a decrease in the triglyceride level . In chicAlthough the function of resveratrol to regulate the immune response has been demonstrated in various animal models, it has been rarely reported in piglets. Pigs can be used as animal models for human diseases because of the great similarity between pigs and humans in lipid metabolism, cardiovascular physiology , and dig Echinacea purpurea powder was purchased from Qilu Animal Health products Co., Ltd. .The resveratrol dry suspension (RDS) was prepared in Natural Medicine Research Center of Sichuan Agricultural University , and the content of resveratrol was 3%. Resveratrol was purchased from Sigma Co., Ltd. (USA). Echinacea purpurea-treated group , respectively. The RDS and Echinacea purpurea (positive control) were suspended in water and fed to animals at 9 a.m. every morning for 14 days. The standard diet of animals was formulated based on the NRC (2012) recommendation for the nutrient requirements of 7–11 kg pigs [Animal experiments were conducted under the principles of proper laboratory animal care and were approved by the ethical committee of the Laboratory Animals Care and Use of Sichuan Agriculture University  2014-187). 40 crossbred weaned piglets (Duroc × Landrace × Big White) at 28 days of age were randomly divided into five groups of 8 animals each group . The 5 groups were as follows: saline control group (Group I), low dose of RDS-treated group , middle dose of RDS-treated group , high dose of RDS-treated group , andhe NRC 20 recommenDuring treatment period, piglets were weighed under limosis. The states of the animals were observed and recorded every day. The average daily feed intake (ADFI), average daily gain (ADG), and ratio of feed to gain (F : G) were measured.Within 24 hours of the last administration, piglets were sacrificed and the organs were weighed, including heart, lung, liver, kidney, spleen, and inguinal lymph nodes. The indexes were calculated according to the following formula: index (mg/g) = (the weight of organ)/the body weight.Each piglet was inoculated with classical swine fever vaccine (CSFV) in the first day of the trial reference to the recommended immunization program . A week Blood samples from anterior vein were collected to determine the serum antibody level at 0 d, 7 d, and 14 d during the trial, respectively. The antibody levels of CSFV and FMDV in serum were analyzed by ELISA kits  in accordance with the manufacturer's instructions.α-PRE, and CD8α-SPRD monoclonal antibodies  at temperature 37°C for 0.5 h in the darkness, followed by centrifugation and resuspending in PBS. T lymphocyte subsets were analyzed by flow cytometry .Within 24 hours of the last administration, 2 ml of blood sample of each piglet from anterior vein was collected and dealt with EDTA. The lymphocytes were separated by lymphocyte separation medium . Then, the cells were incubated with CD3e-FITC, CD46 cells/L. Blastogenic response of lymphocytes to the mitogen of ConA  was assessed by CCK-8 . Lymphocyte suspension was incubated with ConA (10 μg/mL) in 150 μL RPMI 1640 medium containing 10% fetal bovine serum  at 37°C with 5% CO2. After incubation for 48 h, 10 μL CCK-8 was added to each well. After incubation for 2 h, the absorbance at 450 nm was measured by a microplate reader .Within 24 hours of the last administration, blood sample of each piglet from anterior vein was collected with anticoagulation. Then 3 ml of blood sample was slowly injected into 6 ml of porcine peripheral blood lymphocyte separation solution  and centrifuged to obtain the intermediate white cell layer. The cells were washed and centrifuged by PBS three times and then suspended in RPMI-1640 medium  at the concentration of 2 × 10Within 24 hours of the last administration, 3 piglets from each group were sacrificed and the spleen was isolated in a sterile environment. Spleen tissue with the weight of 5 g was disrupted, and spleen cell suspensions were passed through sterile nylon mesh. Red blood cells were lysed by Erythrocyte Lysate . The spleen cells were suspended in RPMI-1640 medium and the methods of culture and detection were identical to those described above.The blood of piglets was collected from the anterior vein at the end of the trial. The serum was isolated by centrifugation. The serum concentrations of IgG, IgA, and IgM were measured by ELISA kits .The serum total antioxidant capacity (T-AOC), malondialdehyde level (MDA), and superoxide dismutase (T-SOD) in serum were determined by ELISA kits .The serum cytokine levels of interleukin, interferon, and tumor necrosis factor were determined by ELISA kits .The blood samples obtained at the end of the trial were collected into a precalibrated tube containing sodium citrate. The hematological parameters included white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), platelet count (PLT), and leukocyte differential count  .Serum biochemical indicators were detected, including albumin (ALB), total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN), creatinine (CRE), glucose (GLU), calcium (Ca), phosphorus (P), total bilirubin (TBIL), and total cholesterol (CHO).p > 0.05). The RDS and Echinacea purpurea treatment had no effect on coefficients of organs when compared to the saline control group (p > 0.05).The growth performance and visceral index of piglets were shown in p > 0.05) among all the groups. In RDS treatment, these T lymphocyte subsets were slightly higher than positive control (p > 0.05).The percentage of T lymphocytes in the peripheral blood of piglets was shown in p < 0.01) stimulated the proliferation of peripheral blood lymphocytes, while the other treatment groups did not show any differences. In splenic lymphocytes, all RDS treatments significantly increased (p < 0.05) lymphocyte proliferation, which showed RDS possessed potent effect on lymphocyte activity.The proliferation of peripheral blood lymphocytes and splenic lymphocytes under the stimulation of ConA was shown in p < 0.01) in RDS treatment (0.33 g/kg and 1.0 g/kg) compared to the saline control group, while the antibody level in Echinacea purpurea powder-treatment was also remarkably higher (p < 0.05) than that of saline control group. After 14 days of inoculation CSFV, only RDS treatment (0.33 g/kg and 1.0 g/kg) differed significantly in the saline control group (p < 0.01 or p < 0.05). Detection results after a week of vaccination with FMDV showed that all drug treatments significantly (p < 0.05) improved the antibody levels in piglets. These data demonstrated the positive effects of RDS on the secretion of antibodies.The detection of antibody levels in piglets was shown in p < 0.05 or p < 0.01) the levels of IgG and IgM in the serum, while the RDS treatment (0.33 g/kg) and Echinacea purpurea powder treatment significantly increased (p < 0.01) the content of IgA. At 14 d, the RDS treatment (0.33 g/kg) significantly promoted (p < 0.01) the secretion of IgA in serum, yet the other drug-treatment groups had no effect on the changes of immunoglobulin content compared with the saline control group.The immunoglobulins levels of serum in piglets were measured in the first and second weeks of the trial, respectively, and the results were shown in  Echinacea purpurea powder treatment significantly improved (p < 0.01) the total antioxidant capacity of serum. Similarly, the RDS treatment (0.33 g/kg) and the Echinacea purpurea treatment significantly increased the total antioxidant capacity at 14 d, while the other groups were not significantly different compared with the saline control group. All the drug treatments had no effect on MDA production. RDS-treatment groups (0.33 g/kg and 1.0 g/kg) and positive control group significantly improved the activity of serum T-SOD after 7 d (p < 0.01 or p < 0.05), and only the RDS-treatment (0.33 g/kg) and positive control group significantly improved the activity of serum T-SOD after 14 d. The results confirmed that RDS had a good antioxidant capacity at the dose of 0.33 g/kg.The result  showed t Echinacea purpurea treatment reduced the release of TNF-α (p < 0.01 or p < 0.05) at 7 d, while the RDS treatment (0.1 g/kg and 0.33 g/kg) also reduced the release of IL-12 (p < 0.05). In the second week, all RDS-treatment and Echinacea purpurea-treatment groups increased the release of IFN-γ (p < 0.05) and the RDS treatment (1.0 g/kg) increased the release of IL-2 (p < 0.01).The result  showed tp < 0.05). The urea nitrogen (BUN) and triglyceride (TG) levels were increased in the RDS-treatment (0.1 g/kg) group (p < 0.01). Alanine aminotransferase (ALT) levels were increased in the median dose group; meanwhile blood sugar (GLU) levels were lower in the RDS-treatment (0.33 g/kg) group (p < 0.01).Tables  Echinacea purpurea was shown to elicit an immune response by increasing the phagocytosis of granulocytes and the number of lymphocytes in fattening pigs as a feed additive [Our study systematically evaluated the effect of RDS on the immune function of piglets through various parameters. We found that RDS was the effective preparation of resveratrol and could significantly enhance immune function of piglets.additive . TherefoIn this study, RDS had no effect on the growth performance and organ coefficient of the piglets, which was similar to the previous study . It was γ and kill the target cells carrying the antigen when it was activated [CD3+CD4+ cell as a T helper/inducing cell secretes a variety of lymphokines which can regulate other cells involved in the immune response, while CD3+CD8+ cell as a cytotoxic T cell can secrete IFN-ctivated . The effctivated . The redctivated , and thectivated . In our ctivated , the ratT lymphocytes can be transformed into lymphoblasts for cell division and proliferation in vitro culture under the stimulation of mitogen, such as concanavalin (ConA). Antigen stimulation changed from steady state of small lymphocytes into large lymphocytes, accompanied by increased cell volume and lighter nuclear staining, nucleolus, and cytoplasmic ribosome. Then, lymphocyte division and proliferation of effector cells took place . Lymphoc Echinacea purpurea extract (0.1–1 g/kg) was significantly higher than that in control group [ Echinacea into diet had an enhancing effect on response of influenza vaccine [ Echinacea treatment significantly improved the antibody titers against CSFV and FMDV, and the activity of RDS treatment was superior to Echinacea treatment. A recent study evaluated the effects of resveratrol on inflammatory response and antibody production against Philasterides dicentrarchi induced in turbot; the results showed a good regulatory effect of resveratrol on the inflammatory response the vaccine induced [Natural products have been shown to serve as adjuvants that can enhance animal antibody levels under the stimulation of vaccines. Astragalus polysaccharide and oxymatrine have been reported to possess synergistical immunoenhancement in enhancing the immune efficacy of Newcastle disease vaccine . The antol group . Adding  vaccine . Swine f vaccine . In the  induced . These rImmunoglobulins are formed in spleen and lymph nodes and secreted by mature plasma cells. They exist in the serum, body fluids, and tissues and can be directly involved in humoral immunity. Resveratrol supplementation remarkably promoted the production of immunoglobulin G in rats . SimilarRecently, the antioxidant activity of resveratrol has been fully confirmed by various experiments. It has been shown that resveratrol can exhibit prooxidant properties, leading to oxidative breakage of cellular DNA in the presence of transition metal ions, such as copper, which hinted the anticancer and chemopreventive properties of resveratrol . Resveraα levels in diabetic rats treated with resveratrol (5 g/kg) have decreased significantly [γ and downregulating the release of TNF-α.Resveratrol can regulate the secretion of cytokines by mediating and activating immune cells. It was reported that TNG-ficantly , and thificantly . Besidesficantly . In our  Echinacea purpurea-treatment group. Resveratrol suppressed oxidative and inflammatory stress response to a high-fat, high-carbohydrate meal [Blood routine and biochemical tests are often used to assist in the diagnosis of diseases and to observe the toxicity of drugs. In our study, the increase in WBC, NEUT, LY, and MONO suggested that a slight inflammation may have taken place in the RDS-treatment groups (0.33 g/kg and 1.0 g/kg) andate meal . In the ate meal . These tγ and downregulating the release of TNF-α. It significantly increased the antibody titers of the piglets under the stimulation of CSFV and FMDV when immunized against the vaccine. It showed an excellent resistance to oxidation and enhanced the T-SOD activity, and it has low toxicity. These positive effects hint that RDS could be considered as an adjuvant to enhance the body's immune response to vaccines, as well as dietary additives for animals to enhance humoral and cellular immunity and to play antioxidant and antiaging effects.In summary, RDS significantly affects the development, maturation, proliferation, and transformation of T lymphocytes and is involved in the regulation of humoral immune responses by upregulating the release of IFN-


Changes in lipid levels/profiles can reflect health status and diseases. Urinary lipidomics, thus, has a great potential in clinical diagnostics/prognostics. Previously, only chloroform and methanol were used for extracting lipids from the urine. The present study aimed to optimize lipid extraction and examine differential lipid classes obtained by various extraction protocols. Urine samples were collected from eight healthy individuals and then pooled. Lipids were extracted by six solvent protocols, including (i) chloroform/methanol , (ii) chloroform/methanol , (iii) hexane/isopropanol , (iv) chloroform, (v) diethyl ether, and (vi) hexane. Lipid profiles of the six extracts were acquired by MALDI-TOF mass spectrometry (MS) and some lipid classes were verified by LIFT-TOF/TOF MS/MS. The data revealed that phosphatidylglycerol (PG) and phosphatidylinositol (PI) could be detected by all six protocols. However, phosphatidylcholine (PC) and sphingomyelin (SM) were detectable only by protocols (i)–(iv), whereas phosphatidylserine (PS) was detectable only by protocols (iii)–(vi), and phosphatidylethanolamine (PE) was detectable only by protocols (v)–(vi). In summary, we have demonstrated differential lipidome profiles yielded by different extraction protocols. These data can serve as an important source for selection of an appropriate extraction method for further highly focused studies on particular lipid classes in the human urine. Urine is a versatile sample widely used for clinical diagnostics as its collection from patients or healthy subjects is very simple and noninvasive5Unlike proteins or genes, which are made up of a limited number of monomeric units, lipids exhibit a much greater degree of molecular structural diversity, resulting to technical challenges for their analysis. Traditional lipid analysis, i.e. thin layer chromatography (TLC), is limited by technical sensitivity, selectivity and resolution99999Sample preparation and processing prior to analysis is a crucial step for lipid profiling. Previously, urinary lipid extraction was mostly done by using the solvent mixture of chloroform and methanol 1:1 or 2:1, v/v):1, v/v2335714The present study examined normal human urinary lipidome profile by MALDI-TOF MS and LIFT-TOF/TOF MS/MS analyses using various lipid extraction protocols . The mos2O]+ (m/z = 137.1), [M + Na]+ (m/z = 177.1), and [2M + H-2H2O]+ (m/z = 273.1). These DHB adduct ions were consistent with the previous studies using 0.5 M DHB in methanol with 0.1% TFA as the matrix solutionm/z range of 700–910, which cover the intact masses of almost all of phospholipids (PLs) and the SM class of SPs.Due to the chemical diversity of lipids, their analysis in this study was performed in both positive and negative ionization modes. Fortunately, DHB matrix could be used for lipid analysis in positive and negative ionization modes as previously described]+ m/z = 7.1, and It was obvious that the positive ionization mode covered a greater number of the lipid species as compared to that acquired by negative ionization mode, consistent with the data reported previouslym/z = 700–910) of lipid extracts from individual solvent protocols acquired by positive ionization mode. A comparison of six extraction protocols revealed markedly different lipid profiles. The assigned lipid classes, including SM, PC, PE, PG, PI and PS, and their detectability (in triplicate set of experiments) by each extraction protocol are summarized in From m/z of 855.6 corresponded to [M + Na]+ion of PI34:3. It was enriched in extracts from protocols (v) and (vi), while could be also detected in protocol (iii)  to (vi)  . Additio to (vi) . PI is aSimilar to PI, PG36:5, PG38:8, PG38:3, PG40:6 and PG40:3 were detected by all protocols, but with a gradual increase in their intensities from protocols (i) to (vi) . For PS,m/z 137.0 to m/z 273.1 (intensity of m/z 137.0 divided by intensity of m/z 273.1) had the lowest coefficient of variation (CV) at 5%. We therefore used this ratio to normalize the intensity of all the lipid species acquired throughout the present study.In addition to lipid identification, we quantitated the relative abundance of each lipid class obtained from individual extraction protocols. We observed that profiling of the 2,5-dihydroxybenzoic acid (DHB) adduct ions acquired in positive ion mode was reproducible in all extraction protocols . Among these DHB adducts, ratio of the peak intensity of m/z of 184 head group fragment ion, whereas PIs could be identified by the characteristic m/z of 284 fragment ion.Finally, tandem mass spectrometry (MS/MS) was performed on some major abundant lipid species using LIFT-TOF/TOF mode. The product ions characteristic head-group of the lipid was used to identify the lipid class. Fragment ions were generated by LIFT technology as described previouslyInvestigations of SM and PC are quite important as they are relevant to many diseases. In our present study, detection of these two lipid classes by MALDI-TOF MS and LIFT-TOF/TOF MS/MS analysis was relatively simpler as compared to other lipid classes due to the high abundant levels of SM and PC in the urine. In concordance, there are previous reports indicating the high abundance of PC and SM in human plasmaIn summary, we have successfully established methods for selective extraction of differential lipid components in the human urine. Our results showed that PG and PI could be detected by all six protocols. However, PC and SM were detectable only by protocols (i)–(iv), whereas PS was detectable only by protocols (iii)–(vi), and PE was detectable only by protocols (v) and (vi). These data can serve as an important source for selection of an appropriate extraction method for further highly focused studies on particular lipid classes in the human urine.Methanol, hexane, and isopropanol were HPLC-grade from Fisher Scientific . Chloroform was HPLC-grade from RCI Labscan . Diethyl ether was from Merck . Trifluoroacetic acid (TFA) was MS-grade from Fisher Scientific . 2,5-dihydroxybenzoic acid (DHB) was from Bruker Daltonik . Deionized water (18.2 MΩ·cm) was derived from Milli-Q water purification system .g and room temperature  for 5 min to remove cellular debris and particles. The clear supernatant was collected and processed for lipid extraction.This study was approved by the institutional ethical committee  . All the experiments involved human subjects and clinical samples were conducted according to the international guidelines, i.e. the Declaration of Helsinki, the Belmont Report, and ICH Good Clinical Practice, and informed consent was obtained from all subjects. Mid-stream urine samples were collected from 8 healthy individuals . An equal volume from each urine sample was pooled and then centrifuged at 1,000 g and 20 °C for 5 min. The organic phase layer (containing lipids) was then collected using a pipette and transferred into a new tube. The extract was then dried in a SpeedVac concentrator .Extraction of lipids from the pooled urine was performed by using 6 different solvent protocols . Equal volume (1–2 μl) of lipid extract and MALDI matrix solution  was mixed in a 0.6-ml microcentrifuge tube. Then, 1 μl of the mixture was spotted onto a MALDI plate . The mass spectra were acquired over the m/z range of 0–1,000 in both positive and negative ion Reflectron TOF modes. The laser power was adjusted to a point just above the ionization threshold of the sample, and the laser rate was set at 10 Hz with 1,000 laser shots per acquisition. Ten acquisitions were averaged for each individual sample.MALDI-TOF MS was performed using an UltrafleXtreme MALDI-TOF/TOF mass spectrometer . The TOF was calibrated using a peptide mixture  before acquisition of the sample spectra. The dried lipid extracts were reconstituted in 200 μl of CHClTandem mass spectrometry (MS/MS) analysis was performed on molecular species of interest (some major abundant lipid classes) using LIFT-TOF/TOF mode. The product ions characteristic head-group of the lipid was used to identify the lipid class. Fragment ions were generated by LIFT (laser ionization fragmentation technology) approach as described previouslym/z, and Tophat base line subtraction were used as the default parameters. The m/z values of lipid ions were searched against LIPID MAPS Structure Database (LMSD)www.lipidmaps.org/tools)+ and [M + Na]+ were selected in the positive ionization mode, whereas [M−H]− was selected in the negative ionization mode. The mass tolerance was set at ±0.2 Da. Lipid categories were limited to phospholipids (PLs) and sphingolipids (SPs). The relative intensity of each lipid class identified was normalized by intensity ratio of the two matrix ions, m/z of 137.0 and 273.1 (intensity of m/z 137.0 divided by intensity of m/z 273.1), which gave the lowest coefficient of variation (CV) among all the DHB adduct ions.A mass list was generated by peak detection algorithm within FlexAnalysis software . Signal to noise threshold of 4, peak width of 0.1 How to cite this article: Tipthara, P. and Thongboonkerd, V. Differential human urinary lipid profiles using various lipid-extraction protocols: MALDI-TOF and LIFT-TOF/TOF analyses. Sci. Rep. 6, 33756; doi: 10.1038/srep33756 (2016).