Plenty of studies have demonstrated the prognostic value of various inflammation-based indexes in cancer. This study was designed to investigate the prognostic value of the C-reactive protein/albumin (CRP/Alb) ratio in esophageal squamous cell carcinoma.A retrospective study of 423 cases with newly diagnosed esophageal squamous cell carcinoma was conducted. We analyzed the association of the CRP/Alb ratio with clinicopathologic characteristics. The prognostic value was explored by univariate and multivariate survival analysis. In addition, we compared the discriminatory ability of the CRP/Alb ratio with other inflammation-based prognostic scores by evaluating the area under the receiver operating characteristics curves (AUC), including the modified Glasgow Prognostic Score (mGPS), neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR).P < 0.001), poorer differentiation (P = 0.019), deeper tumor invasion (P = 0.003), more lymph node metastasis (P = 0.015), more distant metastasis (P < 0.001) and later TNM stage (P < 0.001). The CRP/Alb ratio was identified to be the only inflammation-based prognostic score with independent association with overall survival by multivariate analysis (P = 0.031). The AUC value of the CRP/Alb ratio was higher compared with the NLR and PLR, but not mGPS at 6, 12 and 24 months of follow-up. In addition, the CRP/Alb ratio could identify a group of patients with mGPS score of 0 who had comparable overall survival with those with mGPS score of 1.The optimal cut-off value was identified to be 0.095 for the CRP/Alb ratio. A higher level of the CRP/Alb ratio was associated with larger tumor size  contains supplementary material, which is available to authorized users. Esophageal cancer (EC) is one of the most common malignancies in the digestive system. The main pathological subtypes include adenocarcinoma and squamous cell carcinoma. Esophageal adenocarcinoma (EAC) is the major subtype in some Western countries , while tIn addition to the histopathological factors and tumor stage, some other prognostic indicators have been discovered by previous studies ,7. NutriThe C-reactive protein albumin (CRP/Alb) ratio, a novel inflammation-based prognostic score, has been demonstrated to show outstanding prognostic value in hepatocellular carcinoma compared with other established inflammation-based prognostic scores . In thisAll patients have provided written informed consent for their information to be stored and used in the hospital database. Study approval was obtained from independent ethics committees at Sun Yat-sen University Cancer Center. This study was conducted in accordance with the ethical standards of the World Medical Association Declaration of Helsinki.We retrospectively reviewed the medical records of 649 cases with newly diagnosed esophageal malignancies from October 1, 2006 to November 30, 2010 in Sun Yat-sen University Cancer Center in Guangzhou, China. Pathological diagnoses were carefully checked. We excluded patients without pathological diagnosis and patients diagnosed with other malignancies, such as EAC, esophageal small cell carcinoma, esophageal carcinosarcoma and so on. Only patients pathologically confirmed with ESCC were enrolled in this study. One patient with cervical cancer diagnosed within five years before the diagnosis of ESCC was also excluded. Moreover, we also excluded patients without pretreatment information of nutrition and/or inflammation-based prognostic indicators, patients lost to follow-up, as well as patients died of non-cancer causes. Furthermore, to eliminate the influences of non-cancer diseases on inflammation-based prognostic scores, we excluded patients with rheumatoid diseases and acute infection. Finally, there were 423 cases enrolled in our study. Clinicopathologic information and pretreatment nutrition and inflammation-based indexes were retrospectively collected.The tumor stage was classified according to the AJCC/UICC TNM staging system (the 7th edition). For the T classification, most of the cases with stage I – III ESCC were classified by post-operative pathological specimens. There were thirteen cases classified as T4b after an exploratory thoracotomy and with unresectable locally invasive tumors founded. The tumor size was defined as the long diameter measured with post-operative pathological specimens. The tumor locations were classified into upper esophagus, middle esophagus and lower esophagus. Because of the small numbers of tumors located in cervical esophagus and gastroesophageal junction, we categorized tumors located in cervical esophagus into the upper esophagus group, and tumors located in gastroesophageal junction into the lower esophagus group in this study.2. mGPS: the Glasgow Prognostic Score, it was the combination of CRP and albumin. Patients with CRP < 10 mg/L were allocated a score of 0. Patients with both CRP > 10 mg/L and albumin > 35 g/L were allocated a score of 1. Patients with both CRP > 10 mg/L and albumin < 35 g/L were allocated a score of 2. PNI: the prognostic nutritional index, it was calculated by the formula of 10 × albumin (g/dL) + 0.005× lymphocyte count/uL. NLR: the neutrophil-lymphocyte ratio . PLR: the platelet-lymphocyte ratio. CRP/Alb: the CRP (mg/L)-albumin (g/L) ratio. All the indicators involved in the calculation of the nutrition and inflammation-based prognostic scores were tested before surgery, chemotherapy and radiotherapy treatment. Patients without pretreatment information for these indicators or patients who had received surgery, chemotherapy or radiotherapy in other hospitals before they came to our center were both excluded from this research. Therefore the impact of surgery, chemotherapy and radiotherapy on these scores could be avoided.The nutrition and inflammation-based prognostic scores in this study were defined and calculated as follows. BMI: body mass index, calculated by weight (Kg)/height (m) Patients of all TNM stages were enrolled in this study. The treatment strategies were made according to the National Comprehensive Cancer Network (NCCN) Clinical Practice guidelines. Because patients involved in this study were diagnosed from October 1, 2006 to November 30, 2010, the modality of therapy combination was diversiform. To analyze the impact of treatment on survival of patients, we categorized all the patients into two groups, including curative treatment group and palliative treatment group. The treatment purpose was determined according to both the pretreatment examinations and the operation notes.Follow-up schedules were established and applied referring to the NCCN Clinical Practice Guidelines. For patients who received curative treatment, they were followed up at out-patient department every three months for the first two years, then every six months for another three years and every one year for the rest of time. Patients with incurable disease continued to attend clinics or be hospitalized. For patients who didn’t follow the advice to come back to our hospital, we had a special follow-up department to make follow up telephone interviews.U test or Kruskal-Wallis H test based on the type of the data and comparison. Overall survival (OS) was the time interval from the date of diagnosis to death from ESCC or to the last date of follow-up. OS curves were plotted with the Kaplan-Meier method, and differences were compared with log-rank test. A Cox regression was used for univariate and multivariate analysis. Hazard ratio (HR) and 95% confidence interval (95% CI) were computed with the Cox proportional-hazards model. Variables significantly prognostic in univariate analysis were selected for multivariable analysis using the forward stepwise method. The optimal cutoff values for continuous prognostic indexes were determined with the method established by Jan Budczies et al. at http://molpath.charite.de/cutoff/ [P value <0.05 was considered statistically significant.Differences of baseline and clinicopathological parameters between groups were evaluated by chi-square test, Mann–Whitney /cutoff/ . To evalThere were 423 patients pathologically confirmed with ESCC enrolled in this study. The median age was 58 years old, with an age range of 24 – 88 years old. The majority of patients were males ). The numbers of patients from staged I to IV were 54 (12.8%), 168 (39.7%), 142 (33.6%), 59 (13.9%) respectively. Thirty six (8.5%) patients were with tumors located at upper esophagus, while there were 252 (59.6%) and 135 (31.9%) patients with tumors located at middle and lower esophagus respectively. There were 363 (85.8%) patients received tumor resection. The numbers of patients receiving curative and palliative treatment were 358 (84.6%) and 65 (15.2%) respectively.P = 0.015), deeper tumor invasion (P = 0.003), more distant metastasis (P < 0.001) and more advanced TNM stage (P < 0.001). It, besides, was also associated with larger size of esophageal tumors (P < 0.001) and poorer tumor differentiation (P = 0.019). In addition, there were more patients without resection of esophageal tumors and received palliative treatment in the higher CRP/Alb ratio level group (both P < 0.001).The value of the CRP/Alb ratio ranged from 0.0 – 7.9 with a median of 0.055. The optimal cut-off value of the CRP/Alb ratio was determined to be 0.095 for the OS. We analyzed the association of the CRP/Alb ratio (≤0.095/> 0.095) with clinicopathologic characteristics of patients Table . There wP < 0.001, Figure The median follow-up time was 35.7 months, with a range of 0.6 – 95.6 months. The median OS was 60.5 months for the whole cohort of patients. Compared with a lower CRP/Alb ratio (≤0.095), a higher CRP/Alb ratio (>0.095) was associated with significant worse OS , the TNM stage (P < 0.001), treatment purpose , BMI  and the CRP/Alb ratio .These variables were selected for multivariate analysis using a forward stepwise method, and five indexes were identified to be independent prognostic factors for OS. They were age . Interestingly, in terms of the mGPS score, most of the patients  were classified into the group of score 0, and they were classified by the CRP/Alb ratio (≤0.095/> 0.095) into two groups. However, no patients with mGPS score of 1 and 2 had the CRP/Alb ratio ≤ 0.095  with other established nutrition and inflammation-based prognostic indexes, including the mGPS, PNI, NLR, PLR, CRP, WBC, albumin and BMI Table . It was nts n = 35, 81.6% P <0.001). Patients with mGPS score of 0&CRP/Alb ≤ 0.095 had the best OS, while patients with mGPS score of 0 & CRP/Alb > 0.095 and mGPS score of 1 had comparable moderate OS, and patients with mGPS score of 2 had the worst OS.We further categorized patients into four groups according to the mGPS score and CRP/Alb ratio level: mGPS score of 0 &CRP/Alb ratio ≤ 0.095, mGPS score of 0 & CRP/Alb ratio > 0.095, mGPS score of 1 and mGPS score of 2. The Kaplan-Meier curves were shown in Figure It has been recognized that inflammation is an important regulator in the genesis, progression and metastasis of malignancies ,17. Inflet al. [et al. explored its prognostic value in hepatocellular carcinoma. They found that it had comparable performance with mGPS and better performance than NLR [The CRP/Alb ratio was primarily developed to identify patients with serious illness on an acute medical ward by Fairclough, E et al. . Then anet al. . More rethan NLR . Our stuThe the CRP/Alb ratio and mGPS were both calculated with the same indexes, CRP and albumin. Since the mGPS was one of the best acknowledged and demonstrated inflammation-based prognostic scores in varieties of cancer scenarios , it drewIn this study, a higher CRP/Alb ratio was associated with lower BMI level and albumin level Table . As a maOur study indicated that the CRP/Alb level was associated with aggressive behavior. The mechanism of how inflammation regulates tumor behavior and host status was complicated. The production of CRP was independently mediated by IL-6 level . This waThe main limitation of our study was that it was conducted retrospectively in a single center, and the prognostic value of the CRP/Alb ratio was not verified in a validation cohort. Besides, there was large heterogeneity in the patient treatment, thus it was hard to analyze the impact of the CRP/Alb ratio on patients outcome in different treatment patterns. In addition, compared with the mGPS, the continuity of the CRP/Alb ratio required an optimal cut-off value in clinical practice. However, it might be feasible to find an optimal cut-off value for each tumor stage. In conclusion, our study demonstrated the CRP/Alb ratio to be a promising inflammation-based prognostic score. It was significantly associated with more invasive clinicopathologic characteristics and worse patient outcomes in ESCC. It was superior to some established inflammation-based prognostic indexes, including the NLR and PLR. Last but not least, it was a valuable coadjutant for the mGPS to predict OS of patients with ESCC. The prognostic value of the CRP/Alb ratio should be further evaluated in larger prospective studies and other malignancies.P < 0.001), tumor differentiation (P = 0.019), T stage (P = 0.003), N stage (P = 0.015), M stage (P < 0.001) and TNM stage (P < 0.001). In addition, a higher CRP/Alb ratio was associated with worse OS . Compared with the NLR and PLR, the CRP/Alb ratio showed a superior discriminatory ability. Although no statistical difference of discriminatory ability was found between the CRP/Alb ratio and the mGPS, the CRP/Alb ratio could identify a group of patients with mGPS score of 0, who had comparable overall survival with those with mGPS score of 1 (P < 0.001). In conclusion, our study demonstrated the CRP/Alb ratio to be a novel and promising prognostic inflammation-based factor in ESCC.Our study demonstrated that the CRP/Alb ratio was associated with some important clinicopathological characteristics in ESCC, including tumor size (


USP34 (Ubiquitin-Specific Protease 34) were significantly different between PCOS cases and controls. This study was aimed to replicate the previous results in another independent cohort.Polycystic ovary syndrome (PCOS) is a complex multifactor disorder and genetic factors have been implicated in its pathogenesis. Our previous genome-wide association study (GWAS) had identified allele frequencies in several single nucleotide polymorphisms (SNPs) in gene USP34 gene were performed by TaqMan-MGB probe assay and genotype-phenotype analysis was conducted subsequently.One thousand two hundred eighteen PCOS cases and 1057 controls were recruited. Genotyping of two SNPs (rs17008097 and rs17008940) in P = 0.006, OR = 1.042, 95% CI 1.012–1.073; TT vs CC P = 0.037, OR = 1.050, 95% CI 1.003–1.100).The differences of allele or genotype frequencies were not significant statistically between PCOS and controls, even after age and BMI adjustment. For clinical and metabolic features  analysis in PCOS cases, no statistical differences among three genotypes of rs17008097 and rs17008940 were found. However, rs17008940 was shown to be slightly associated with BMI in PCOS cases rather than in controls, even after age adjustment (TC vs CC USP34 gene polymorphisms (rs17008097 and rs17008940) may not be associated with PCOS in the Han Chinese women.The online version of this article (doi:10.1186/s13048-015-0158-y) contains supplementary material, which is available to authorized users. USP34, remain intriguing and might also be potential risk factors of PCOS.Polycystic ovary syndrome (PCOS) is a kind of reproductive and metabolic disorder characterized by hyper-androgen and insulin resistance, which affects 6–8 % of reproductive-aged women in Caucasian and 5.6 % in Chinese –3. CliniUSP34 gene is located on chromosome 2p15 and encodes a kind of deubiquitinating enzyme, which belongs to ubiquitin-specific protease family. Data obtained from COSMIC database [USP34 are related to ovary tumor. Moreover, USP34 positively regulates Wnt signaling pathway [USP34 may also have relationship with PCOS. As an extension of GWAS, here we conducted an independent case-control replication study to evaluate the association between USP34 and PCOS susceptibility.The database  shows th pathway , which p pathway . The exp pathway –13. TakeThis study was approved by Institutional Review Board for Reproductive Medicine of Shanghai Jiaotong University and Shandong University. A total of 1218 PCOS cases and 1057 unrelated controls were recruited consecutively from 2009 to 2013 at the Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University and the Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University. Among them, 94 PCOS cases, also born from Northern China, were collected at Renji Hospital from 2012 to 2013. Signed informed consent was obtained from each participant of this study.PCOS diagnosis was based on the 2003 Rotterdam PCOS consensus criteria and other related diseases  were excluded. In detail, PCOS can be diagnosed if at least two of the following three features are met: oligo- or anovulation, clinical and/or biochemical signs of hyperandrogenism and polycystic ovaries. Oligo- or anovulation was referred to menstrual cycles of more than 35 days in length or a history of ≤ 8 menstrual cycles in a year ; polycysSerum luteinizing hormone (LH) and testosterone (T) levels of subjects were measured by a chemiluminescent analyser . The plasma glucose was measured by AU640 automatic biochemistry analyser  and insulin was measured by chemiluminescent analyzer. Insulin resistance was calculated as fasting glucose (mmol/L)*fasting insulin (mIU/L)/22.5 using homeostasis model assessment (HOMA-IR).USP34 were selected for replication study according to the following criteria: SNPs that exist in Affymetrix Genome-Wide Human SNP Array 6.0 ; can stand for a block; minor allele frequency (MAF) > 5 % in Chinese Han population; statistically different (P < 10e-4) from our previous GWAS . Genotyping of SNPs was carried out by TaqMan-MGB probe assay , probes and primers were shown in Additional file α = 0.05), assuming higher risk allele frequency (A) of 0.05 and a genotype relative risk (Aa) of 1.5. Frequencies of genotype and allele between PCOS subjects and controls were compared by Pearson Chi-square test, and the p value was adjusted by logistic regression to eliminate the effect of age and body mass index (BMI) using SPSS v.19.0 . P < 0.05 was considered statistically significant.Numerical variables of clinical characteristics of PCOS cases and controls were expressed as mean ± SD. Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) tests were performed by Haploview software. The case-control genetic power was analyzed by Genetic Power Calculator . A samplFor genotype-phenotype analysis in PCOS patients, additive model (+/+ vs +/− vs −/−) was selected after comparison; one-way analysis of variance (ANOVA) test was used for phenotype comparison among different genotypes. Linear regression was used for age and BMI adjustment. Conservative Bonferroni test was used for multiple testing corrections.P < 0.001). In addition, PCOS group had higher BMI, LH level and T level than the control group (P < 0.001). In PCOS group, 90 patients present with hyperandrogenism and oligo-anovulation , 10 patients present with hyperandrogenism and polycystic ovaries , 620 patients present with oligo-anovulation and polycystic ovaries , and 233 patients present with full-phenotype .As shown in Table P > 0.05). Genotype and allele frequencies were summarized in Table The genotype frequencies of the two polymorphisms in PCOS cases and controls were all in Hardy-Weinberg equilibrium (P = 0.028) . But in control group, no significant difference was found in BMI among the three genotypes of rs17008940 (P = 0.256). Additionally, there were no significant differences in LH, T or HOMA-IR among the PCOS cases carrying different genotypes of the two SNPs, even after age and BMI adjustment  Table , howeverYAP1 and LPP for PCOS from these SNPs [USP34 to validate whether USP34 was associated with PCOS. No association was replicated in the present study. Therefore, SNPs with p value around 10e-5 in GWAS were controvertible and demanded for independent and large cohort of samples for verification.As a powerful technique, GWAS shed new light on genetic study for complex diseases. GWAS data is obtained from computing and statistical analyses following SNP chips detection, so the results are bioinformatics rather than biological. However, GWAS itself owns some limitations and it is necessary to be validated through further replication studies. In general, p value of 5*10e-8 was used as significant level for random variations in case-control GWAS with a power of 0.8 , 18. In ese SNPs , 19. So FTO and MC4R were associated with increased BMI in PCOS subjects rather than PCOS itself [Although rs17008940 was not shown to be related to PCOS, it might confer slight risk to the elevated BMI in PCOS. However, this slightly association possibly results from a selection bias derived from the patients and controls being recruited in an infertility clinical center and not from the general population. Hence, the results need further validation. Higher BMI was one of the important characteristics in PCOS and over 50 % PCOS women were overweight or obesity , 21. ConS itself . AbundanS itself , 24 and S itself . For exaS itself ; and ChoS itself . MoreoveS itself  and DNA S itself , 30. USPOR = 1.010; rs17008097, OR = 1.007) and this replication study maybe not sufficient to detect the potential association between USP34 gene and PCOS. Second, only 2 SNPs were chosen which may cause incomplete coverage of the gene variations. Third, the recruited subjects were all Han Chinese women and the result could not represent other population.Some limitations of the present replication study should be mentioned. First, the sample size of this replication study was relatively small (rs17008940, USP34 gene may not be associated with PCOS women among Han Chinese population. SNPs with p value around 10e-4 ~ 10e-6 in GWAS were disputable and requiring replication studies for validation. Large well-designed and population-based studies are warranted to confirm our findings.In conclusion, the present study found that polymorphisms of


Fasciola hepatica causes chronic liver disease, fasciolosis, leading to significant losses in the livestock economy and concerns for human health in many countries. The identification of F. hepatica genes involved in the parasite’s virulence through modulation of host immune system is utmost important to comprehend evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, to identify the parasite’s putative virulence genes which are associated with host immunomodulation, we explored whole transcriptome of an adult F. hepatica using current transcriptome profiling approaches integrated with detailed in silico analyses. In brief, the comparison of the parasite transcripts with the specialised public databases containing sequence data of non-parasitic organisms (Dugesiidae species and Caenorhabditis elegans) or of numerous pathogens and investigation of the sequences in terms of nucleotide evolution  and cytokine signaling relation were conducted.50 = 522 bp). A number of the parasite transcripts  were predicted to be virulence-related on the basis of the exclusive homology with the pathogen-associated data, positive selection or relationship with cytokine signaling. Of these, a group of the virulence-related genes (n = 62), not previously described, were found likely to be associated with immunomodulation based on in silico functional categorisation, showing significant sequence similarities with various immune receptors , cytokines , cluster of differentiations (e.g. CD48 and CD147) or molecules associated with other immunomodulatory mechanisms (such as regulation of macrophage activation). Some of the genes (n = 5) appeared to be under positive selection (Ka/Ks > 1), imitating proteins associated with cytokine signaling .NGS of the whole transcriptome resulted in 19,534,766 sequence reads, yielding a total of 40,260 transcripts (NF. hepatica (n = 62), which are firstly described here, could be employed for the development of better strategies (including RNAi) in the battle against both zoonotically and economically important disease, fasciolosis.With a comparative transcriptome profiling approach, we have identified a number of potential immunomodulator genes of The online version of this article (doi:10.1186/s12864-015-1539-8) contains supplementary material, which is available to authorized users. Fasciola hepatica, the liver fluke, is a digenetic trematode helminth, causing highly damaging hepatobiliary disease (fasciolosis) in mammalians including economically important ruminants (such as cattle and sheep) and humans .Workflow illustrating the experimental steps of the study is demonstrated in Figure ± 14.1%) . The ideF. hepatica transcript , in addition to the morphological identification of the isolated parasite. The amino acid sequence for this protein at the correct frame of the transcript sequence was valine (V-599) as in F. hepatica, but not leucine (L-599) as in F. gigantica  under PDR subgroup.Approximately half of the total liver fluke transcript number i.e. 51.87%) showed sequence homology with nucleotide or protein sequences of non-parasitic organisms including .87% showF. hepatica transcripts was assumed to be virulence-related transcripts (ascribed as VRs under VR group) on the basis of the degree of homology with sequences from publicly available databases; i) pathogen-associated data , ii) observation of the signs of positive selection  and iii) cytokine signaling relation , yielding a total of 1,671 transcripts   were also observed in PDR subgroup . Of the cytokine signaling related transcripts (CSRs), one transcript showed the signs of positive selection and sequence homology with the HSD database (#20661), three transcripts  showed the signs of positive selection without sequential similarity of any pathogen related databases, and the others showed sequence homology with the non-parasitic databases (n = 21). A number of CSRs (n = 10) showed non-similarity with any specialised databases used in this study.Overall, 4.15 % of all identified The InterProScan search for whole transcriptome of the parasite revealed a total of 5,089 unique accession number indicating protein families, domains or functional sites. Protein sequences inferred from a total of 20,160 transcripts  were categorised in various functional groups by considering the biological functions of the parasite in previous publications ,25,27. TSubcellular localisation signals of the parasite protein sequences were mostly related to cytosol (21.68%), extracellular space 20.57%) and nucleus (19.15%) Figure a. Simila7% and nun = 1,671), the immunomodulation categorised PDRs, PSRs, and all CSRs, a total of seventy-one transcripts, corresponding to 64 putative genes, were named virulence and immunomodulation-related transcripts (VIRs) under VIR set (Table Of VRs (et Table . Furtheret Table .Table 2n = 15) showed sequential similarities with MHC I and an important part of VIRs indicated the relationship with TGF-β signaling based on sequence homologies with TGF-β, TGF-β receptor or other proteins associated with stimulation or inhibition of TGF-β  individually were potentially located at extracellular space (n = 63) or localised within plasma membrane (n = 1)  Table , possiblGene ontology categories  for whole transcriptome of the liver fluke, in comparison with VR group, its subgroups  and VIR set are shown in Table A total of 96 different KEGG biological pathways (195 enzyme types) were determined in whole transcriptome of the parasite, in which the purine  and pyrimidine metabolisms map00240; 7.93%) were the biological pathways with higher abundant transcript numbers, where the relative abundances of the other pathways, in transcript number, were less than 5% . The observed GO categories of F. hepatica transcripts in terms of biological processes were mostly concordant with the previous classification , S. haematobium and S.mansoni databases , 4) C. elegans is a well studied free-living organism and a comprehensive sequence information for this organism is publicly available . Nucleotide sequences of Dugesia sp.  and of Schmidtea sp.  were downloaded from the DDBJ resource on 10 and 11 February 2014, respectively. In addition, a small number of nucleotide sequences (n = 125) for other organisms belonging to the Dugesiidae family, including Cura sp., Girardia sp., Neppia sp., Romankenkius sp. were downloaded from the same resource (11 February 2014). Protein coding nucleotide sequences (cds) and protein sequences of C. elegans  were obtained from the ftp site of WormBase (http://www.wormbase.org). Nucleotide sequences of all the F. hepatica transcripts were searched against nucleotide sequences of the Dugesiidae species and protein sequences of C. elegans using the standalone tblastx and blastx, respectively .To detect non-parasitism homology of the liver fluke transcripts, sequence data of F. hepatica transcript sequences were searched against protein sequences of the helminth secretome database , obtained from Gagan Garg .http://geneontology.org/), and KEGG biological pathways  were analysed for the detected protein motifs  using the blast2GO software.Gene ontology categories at parental level 2  (


To assess whether familial non-medullary thyroid cancer (FNMTC) represents an independent risk factor for increased aggressiveness of the tumor, as concern as the clinical presentation and the long-term follow-up in respect of sporadic differentiated thyroid cancer (SDTC).Retrospective study; 1976–2014.Seventy-four FNMTC families : family relationship and number of affected family members were evaluated. Clinical and histopathological features and outcome were compared to that of 643 SDTC patients followed in the same period according to the same institutional protocols. Median follow-up was 57.7 months (range 12–136) in FNMTC and 59.7 (range 15–94.6) in SDTC patients.p < 0.005). Papillary/follicular histotype distribution was similar (86%). Papillary tumors were more frequently multifocal in FNMTC (p = 0.004) and with lymph-node metastases (p = 0.016). Disease-free survival (DFS) was shorter in FNMTC vs. SDTC (p < 0.0001) with 74.8 vs. 90.8% patients free of disease at the last control (p < 0.005). Three patients died in FNMTC group vs. 1 in SDTC (p = 0.02).Three cases occurred in 3 families and 2 cases in the other 71. F:M was 3.7:1 in FNMTC and 4.3:1 in SDTC (NS). The family relationship was siblings in 62.2%. Mean age at diagnosis was lower in FNMTC than in SDTC  responsible for susceptibility for FNMTC has not yet been identified, a low frequency periodic screening of relatives DTC patients may be useful to identify FNMTC patients at early stage of disease. Familial non-medullary thyroid cancer (FNMTC) represents approximately 3–7% of all thyroid cancers that originate from thyroid follicular epithelial cells .Since the genetic alterations responsible for this disease are not known, FNMTC is defined as the condition in which two or more first-degree relatives are affected by thyroid cancer in the absence of a known familial syndrome.However, due to the high prevalence of differentiated thyroid carcinoma (DTC), it is estimated that when two members of the same family are affected, there is a 53% probability that the disease has a familial origin but also a 47% probability that it is sporadic. When three or more family members are affected, the probability rises to 99.9% .As already mentioned, to be defined familial, FNMTC should not represent the phenotypic expression of a genetic familial syndrome. There are, in fact, familial cancer syndromes regarding non-thyroid tumors where thyroid carcinoma may also be present. These include the Cowden syndrome , the familial adenomatous polyposis (FAP) characterized by multiple and diffuse adenomatous polyps in the colon, osteomas, epidermoid cysts, desmoid tumors, hamartomas of the upper digestive tract, congenital hypertrophy of retinal pigment epithelium, hepatoblastoma, and the Carney complex , the Werner syndrome , and finally syndromes in which thyroid cancer is associated with clear cell renal carcinoma , 4.Many studies suggest that FNMTC is a defined entity with early cancer onset and aggressive features relative to sporadic thyroid cancer but the topic remains controversial . The preWe retrospectively analyzed a consecutive series of 4120 thyroidectomized DTC patients in the 1976–2014 period, all followed at the Thyroid Clinic, Division of Endocrinology, Garibaldi Medical Center in Catania, Italy.Among this series, we identified 264 (6.4%) patients with a positive family history that were classified FNMTC because at least one first-degree relative was affected by confirmed thyroidal cancer of follicular origin.Only patients having all family members with FNMTC in continuous follow-up in our Center were included: 151 patients belonging to 74 families.All patients underwent total thyroidectomy, with a central and/or lateral-cervical lymph-node dissection in 53 cases (35.1%).Tumors were staged according to the VII TNM edition : T  was administered to 131/151 patients (86.7%) who met one or more of the subsequent criteria: tumor size >1.0 cm, tumor extension to soft tissues adjacent to the thyroid gland, multifocal tumor, and/or nodal metastases. Follow-up procedures included neck ultrasound and serum thyroglobulin (Tg) after either L-T4 withdrawal or recombinant human TSH stimulation.Persistent/recurrent disease was defined by one or more of the following criteria: (1) serum Tg, either under suppressive L-T4 therapy or after TSH stimulation, at detectable levels, as defined by the assay sensitivity limits at the time of measurement; (2) evidence of metastatic lymph nodes identified at neck ultrasound and confirmed by FNAB with Tg measurement in the aspirate washout; (3) positive 131I-WBS. Computed tomography, magnetic resonance imaging, bone scan, and positron emission tomography were performed when appropriate in patients with persistent/recurrent disease.Data in FNMTC patients were compared to those obtained in a control group of 643 SDTC patients matched at 1:4 ratio for the long-term follow-up. In the control group, post-operative radioiodine treatment was administrated in 390 cases (60.6%).Median follow-up was 57.7 months (range 12–136) in FNMTC and 59.7 (range 15–94.6) in SDTC patients.All procedures involving human participants were in accordance with the ethical standards of institutional research committee and with the Helsinki declaration as revised in 2013. Informed consent of the present retrospective study was waived.t-test, and qualitative data were compared by the chi-square analysis. The disease-free survival (DFS) curves were determined by Kaplan–Meier curves. p values <0.05 were considered statistically significant.Quantitative data were compared using the Seventy-one (95.9%) of the 74 families with FNMTC had two affected family members, and three families patients 4.1%) had three affected family members. The transmission of the disease was maternal in 40 cases from 20 families and paternal in 11 cases from 5 families. In 94 cases (47 families), the disease was present in brothers and sisters. In six cases (two families) in addition to the first-degree also a second-degree relative with FNMTC was identified  (Table % had thrTable p = 0.57).Of the 151 FNMTC patients studied, 119 (78.8%) were females (F) and 32 (21.2%) were males (M), with an F/M ratio of 3.7/1; in the control group (643 patients with SDTC), there were 521 F (81.0%) 122 M (19.0%), with an F/M ratio of 4.3/1 (p < 0.005).The median age at diagnosis was 45.4 ± 13.5 years for FNMTC (range 17.1–81.5) and 48.0 ± 13.7 years (range 16.8–82.6) for SDTC .Table In patients with FNMTC, the medium tumor size was 1.5 ± 1.2 cm (range 0.1–7.0). In addition, the tumor was multifocal in 69 cases (45.7%), bilateral in 48 (31.7%), and with extrathyroidal extension in 33 cases (21.8%).In the control group, tumor medium size was 1.5 ± 1.3 cm (range 0.4–6.0), multifocal in 214 cases (33.2%), bilateral in 158 (24.5%), with extrathyroidal extension in 126 (19.6%).p = 0.004).The percentage of multifocal tumors in patients with FNMTC compared to patients with SDTC was highly significant . No significant difference was found among the various groups’ pT, while there was a more significant presence of lymph-node metastases in familial tumors .In FNMTC, we found a lower proportion of microcarcinomas than in sporadic thyroid cancer  than that observed in patients from the control group [median 55.9 months (range 6–95)]. In FNMTC patients, at the last control visit, we observed a lower percentage of cases free from disease (p < 0.0001) than we observed in the control group , we did not find any significant differences with respect to the control group (316 patients), either regarding the DFS (6 recurrences in FNMTC vs. 14 in SDTC) or the percentage of disease-free patients at the last control visit (persistence of disease in 3 FNMTC patients vs. 13 in SDTC).p = 0.07) or for the percentage of disease-free patients at the last control visit (p = 0.23) compared to patients with SDTC (189 cases).Regarding familial microcarcinomas, which according to the TNM system can be classified as T1a N0/NX M0 (29 cases), there was no significant difference for DFS (p = 0.23 for DFS and p = 0.28 for the percentage of patients free of disease at the end of follow-up); on the contrary, we found that DFS was significantly longer (p = 0.038) in patients in whom the family relationship was brother–sister than in other relationships   , followed by maternal transmission (26.5%) and paternal transmission (7.3%) and in two families (4%) in addition to the first-degree also a second-degree relative with FNMTC was identified. There are few data regarding the mode of transmission in the literature, with the exception of the study by Rumianteseva et al. in which, in a series of 48 patients with FNMTC belonging to 24 families, was found a greater frequency of maternal transmission (75.0%) . The early onset of FNMTC may be due to an early diagnosis because of the familial nature of the disease or to the phenomenon of anticipation typical of familial cancers which, according Capezzone et al. , 10, derFrom a histological viewpoint, FNMTC is indistinguishable from sporadic forms of well-DTC, and these data were confirmed in our study. Regarding the histopathological features of the tumor, we found a greater number of multifocal tumors in FNMTC compared to the control group. This feature, as reported in many studies, constitutes one of the peculiarities of this form of cancer. The largest study regarding the characteristics of patients with FNMTC is from Japan. Uchino et al. examined 6,458 patients and identified 258 cases of FNMTC, with a median age at diagnosis of 49 years, and although the tumor size at diagnosis was not different compared to sporadic cancers, multifocality was observed more frequently (42 vs. 30%) . AccordiSimilar data were found by Alsanea  and Ito In our study, FNMTC more frequently exhibited nodal involvement at diagnosis compared to sporadic forms, although we did not observe a major incidence of extrathyroidal tumors.This is in agreement with other data in the literature: in 1995, Grossman documented an incidence of lymph-node metastases at diagnosis in 57% of patients with FNMTC vs. 38% of patients with sporadic thyroid carcinoma . Similarp = 0.0005) with a DFS significantly shorter (p = 0.004)  .Patients with differentiated thyroid microcarcinoma typically have an excellent prognosis. Lupoli et al., evaluating a series of tumors in patients with familial and sporadic microcarcinoma, showed that in familial forms, the tumor was multifocal in 71% of cases (vs. 19% in sporadic forms) and bilateral in 43% (vs. 19%), with high frequency of lymph-node metastases (57 vs. 28%) and vascular invasion (43 vs. 5%). The recurrence rate in these patients was significantly higher (43 vs. 5%), and one patient died of widespread metastasis . This stIt is now widely recognized that familial cancers are more aggressive than sporadic ones, even if it is difficult to separate the two forms, especially when only two family members are affected; in fact, in this case, there is a 69% probability that the cancer is familial, but it might also be a sporadic thyroid cancer .In families where three or more family members are affected and where genetic alteration is almost certain, the high aggressiveness of the tumor is clearly demonstrated by the prognosis and the final outcome, as showed in the study by Triponez et al. . He showThis suggests that early diagnosis and treatment are very important to improve the quality of life and survival of patients with FNMTC. Therefore, pre-screening is recommended in patients who have family members with FNMTC (approximately when they are 10 years younger than the affected family members)  at the time of diagnosis, central lymph-node dissection should be considered for these patients. Involvement of lateral neck cervical lymph nodes should be assessed pre-operatively.Furthermore, because FNMTC has a high recurrence rate, which is an important cause of reduced patient survival, many experts recommend that all patients should undergo I131 radioactive iodine treatment and post-surgery L-T4 suppressive therapy.At present, FNMTC follow-up guidelines are not available, but due to the points discussed above, surgical and post-operative treatment and follow-up should be planned because of the aggressiveness of these tumors. It will be useful to separate these patients, even if belonging to the same class of risk, according to the presence or absence of familial cases. Therefore, the majority of patients should undergo radioiodine ablation independently of tumor size to achieve complete ablation of residual thyroid tissue , 17.The genetic basis of FMNTC remains unknown, but it is believed that the genetic alterations are transmitted as an autosomal dominant incomplete penetrance and variable expressivity.Some of the chromosomal loci identified could play a role in the development of cancer, but none of them seem sensitive to propose a genetic screening test for high risk-families.Familial non-medullary thyroid cancer is found more frequently in siblings.Familial non-medullary thyroid cancer is diagnosed at an earlier age than sporadic thyroid carcinoma; it is less frequently a microcarcinoma but more often shows multifocal foci and lymph-node metastases at diagnosis.Familial non-medullary thyroid cancer has a worse prognosis than SDTC, with a disease-free time significantly lower than that observed in patients with sporadic thyroid cancer.Siblings of individuals with FNMTC have a better prognosis than FNMTC with other relationships ones.In conclusion, our study showed that:Further studies are still needed to define the best management for FNMTC. Because there is not a genetic test to identify at-risk patients, an early screening of patients who have family members with FNMTC is essential to identify the tumor at an early stage.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.


Poncirus trifoliata) and white clover (Trifolium repens) were planted in a two-compartmented rootbox, separated by a 37–μm nylon mesh and then inoculated with an arbuscular mycorrhizal fungus (AMF), Diversispora spurca. Inoculation with D. spurca resulted in formation of a CMN between trifoliate orange and white clover, whilst the best AM colonization occurred in the donor trifoliate orange–receptor white clover association. In the trifoliate orange–white clover association, the mycorrhizal colonization of receptor plant by extraradical hyphae originated from the donor plant significantly increased shoot and root fresh weight and chlorophyll concentration of the receptor plant. Enzymatic activity of soil β-glucoside hydrolase, protease, acid and neutral phosphatase, water-stable aggregate percentage at 2–4 and 0.5–1 mm size, and mean weight diameter in the rhizosphere of the receptor plant also increased. The hyphae of CMN released more easily-extractable glomalin-related soil protein and total glomalin-related soil protein into the receptor rhizosphere, which represented a significantly positive correlation with aggregate stability. AMF inoculation exhibited diverse changes in leaf and root sucrose concentration in the donor plant, and AM colonization by CMN conferred a significant increase of root glucose in the receptor plant. These results suggested that CMN formed in the trifoliate orange–white clover association, and root AM colonization by CMN promoted plant growth, root glucose accumulation, and rhizospheric soil properties in the receptor plant.Common mycorrhizal network (CMN) allows nutrients and signals to pass between two or more plants. In this study, trifoliate orange ( In the field, sod culture is widely used in citrus orchards to enhance organic matter and mineral contents in soil, benefit propagation of AMF, and reduce the consumption of agrichemicals and chemical fertilizers [Trifolium repens L.) as less mycorrhizal dependent plant is widely applied into citrus orchards [Trifoliate orange [tilizers . Meanwhiorchards , where iA two-compartmented rootbox was made of plexiglass. Each compartment was 6-cm length, 6-cm width, and 17-cm height. A pipe with 2×4×4 cm (length×width×height) size was fixed to a 2-cm location of the compartment bottom. A 37-μm nylon mesh was directly wrapped on the front of the pipe. The two compartments were connected by the two joint pipes and separated from the 37-μm nylon mesh, where AM hyphae, but not plant roots, are allowed from a compartment to the other for the establishment of a CMN.Diversispora spurca  C Walker & A Schüßler, were inoculated into the designed compartment at the time of transplanting. The details of the AM fungal strain in origin and propagation were described previously by Wu et al. [The soil used was Ferralsol (FAO system) collected from a citrus orchard of Yangtze University campus. The soil had a pH of 6.0 and 15.7 mg/kg Bray-P. A nine-leaf-old trifoliate orange seedling grown in autoclaved  sand without AMF was transplanted into a compartment, and then 15 seeds of white clover sterilized with 70% alcohol for 10 min were sown into the other compartment. Approx. 690 g of soil sterilized with 121°C and 0.11 Mpa for 2 h, was supplied into each chamber. The white clover was thinned to 8 plants per chamber after one week of sowing. Approx. 1,050 spores of an AM fungus, u et al. .2/s, day/night temperature 28/21°C, and relative humidity 85%. The position of rootboxes in the greenhouse was rotated weekly. During the experiment, no additional nutrition was added into each chamber of the rootbox, in order to maintain a considerably low soil P level for better AM functioning.All the rootboxes were placed in a glass greenhouse in Yangtze University campus from March 28, 2014 to August 16, 2014, where photosynthetic photon flux density was 740–960 μmol/mD. spurca (TO+/WC+); (2) the donor plant trifoliate orange was inoculated with D. spurca, but the receptor plant white clover was not inoculated with D. spurca (TO+/WC–); (3) the donor plant white clover was inoculated with D. spurca, but the receptor plant trifoliate orange was not inoculated with D. spurca (TO–/WC+); (4) both trifoliate orange and white clover were not inoculated with D. spurca (TO–/WC–). Each treatment had four replicates, creating a total of 16 rootboxes.The experiment consisted of four treatments with a completely randomized blocked arrangement: (1) both trifoliate orange and white clover plants were inoculated with After 20 weeks of the AMF inoculation, the trifoliate orange and white clover seedlings were harvested and divided into the shoot and the root, whose fresh biomass was determined. The soil adhered to the root systems was collected, air-dried, and sieved through a 4 mm mesh.Leaf chlorophyll concentration was determined by Lichtenthaler and Wellburn  with theExtraction of fructose, glucose, and sucrose in leaves and roots was done with both 8 mL of 80% ethanol and 50 mg of 1.0-mm-sieved dry samples at 80°C for 40 min, which was centrifuged at 2,500×g for 5 min. Concentrations of these carbohydrates were determined as per the colorimetrical protocol described by Wu et al. .xi is the diameter of the i sieve (mm), wi is the proportion of the i size fraction in the total sample mass, and n is the number of size fractions (n = 4).Soil β-glucoside hydrolases, proteases, and phosphatase activities were determined respectively by the method of Stot et al. , Cao et The easily-extractable glomalin-related soil protein (EE-GRSP), difficultly-extractable GRSP (DE-GRSP) and total GRSP concentration (T-GRSP) concentration was determined by the protocol of Wu et al. .P<0.05 was used to compare the significant differences between the treatments.Data were statistically analyzed with one-factor of variance (ANOVA) in SAS (v 8.1), and the Duncan’s multiple range test at D. spurca  transferred through CMN or to the receptor plant infected by CMN. Compared with the TOIn this study, AMF inoculation showed significantly higher leaf sucrose and fructose and root sucrose and glucose in the donor trifoliate orange, and higher leaf sucrose and fructose and higher root sucrose, fructose, and glucose of the receptor white clover. Wu et al. , 19 also14C or 13C labeling study revealed the C transfer by CMN from the donor plant to the receptor plant [In donor white clover–receptor trifoliate orange system, root AM colonization caused by CMN markedly decreased root sucrose but increased glucose and fructose in the receptor plant. Possibly, root colonization caused by CMN in trifoliate orange–white clover association might accelerate root sucrose cleavage into fructose and glucose in the receptor plant, resulting in an increase of root glucose in the receptor plant, which is beneficial to AM colonization and CMN development . A 14C oor plant –11, and +/WC−treatment than under the other three treatments. Higher acid and neutral phosphatase activity in the rhizosphere of the donor and receptor plants was under the TO–/WC+ treatment than under the other three treatments. Greater soil enzyme activities and soil structure by mycorrhization indicated that AMF inoculation conferred better soil structure and soil fertility in mycorrhizosphere. In the present work, significantly higher EE-GRSP and T-GRSP concentration was found in rhizosphere of trifoliate orange–white clover association, irrespective of the donor or receptor plant species. This is in agreement with the findings of Wang et al. [This study indicated that AMF inoculation significantly increased WSA percentage at the size of 2–4 and 0.5–1 mm and soil β-glucoside hydrolases, proteases and acid and neutral phosphatase activities of the donor plant and the receptor plant. Meanwhile, greater soil protease and β-glucoside hydrolases activity in the rhizosphere of donor and receptor plants was under the TOg et al.  in trifo+/WC+ treatment, as compared with TO–/WC−treatment. Possibly, under the TO+/WC+ treatment condition, both trifoliate orange and white clover dispensed more C to maintain own AM development and formed respective CMN to compete with their neighbors in soil C cycle. The present study showed that CMN presence significantly increased EE-GRSP and T-GRSP levels in receptor rhizosphere. This is a known fact that GRSP exhibited a strongly positive correlation with soil aggregate stability under various soil types and ecosystem conditions [r = 0.58, P < 0.05) and T-GRSP  in trifoliate orange chamber and positively with EE-GRSP , DE-GRSP , and T-GRSP  in white clover chamber. It suggests that CMN would induce well-developed mycelium and GRSP release  in the receptor plant rhizosphere, resulting in the enhancement of soil aggregate stability, and subsequently promoting the growth of the host plant.It is well known that mycorrhizal extraradical hyphae could release acid phosphatase . Soil β-nditions , 14, 28,D. spurca formed in trifoliate orange–white clover association, whilst the best AM colonization occurred in donor trifoliate orange–receptor white clover system. AMF inoculation significantly increased shoot and root fresh biomass, root glucose, EE-GRSP, and T-GRSP concentrations, and soil acid phosphatase, neutral phosphatase, and proteases activities in trifoliate orange or white clover as a donor plant. CMN presence originated from donor plant also holp to enhance plant growth, induce carbohydrate accumulation (mainly glucose in root), and imporve the rhizospheric soil properties in the receiver plant of the trifoliate orange–white clover system. As a result, CMN may exhibit the important functionings in sod culture of citrus orchards.The CMN with


Background: Trihalomethanes (THMs) are water disinfection by-products that have been associated with bladder cancer and adverse birth outcomes. Four THMs  were measured in blood and tap water of U.S. adults in the National Health and Nutrition Examination Survey (NHANES) 1999–2006. THMs are metabolized to potentially toxic/mutagenic intermediates by cytochrome p450 (CYP) 2D6 and CYP2E1 enzymes.Objectives: We conducted exploratory analyses of blood THMs, including factors affecting CYP2D6 and CYP2E1 activity.Methods: We used weighted multivariable regressions to evaluate associations between blood THMs and water concentrations, survey year, and other factors potentially affecting THM exposure or metabolism .n = 4,232 total THM; n = 4,080 bromoform; n = 4,582 chloroform; n = 4,374 bromodichloromethane; n = 4,464 dibromochloromethane). We detected statistically significant inverse associations with diabetes and eating cruciferous vegetables in all but the bromoform model. Medications did not consistently predict blood levels. Afternoon/evening blood samples had lower THM concentrations than morning samples. In a subsample (n = 230), air chloroform better predicted blood chloroform than water chloroform, suggesting showering/bathing was a more important source than drinking.Results: From 1999 to 2006, geometric mean blood and water THM levels dropped in parallel, with decreases of 32%–76% in blood and 38%–52% in water, likely resulting, in part, from the lowering of the total THM drinking water standard in 2002–2004. The strongest predictors of blood THM levels were survey year and water concentration (Conclusions: We identified several factors associated with blood THMs that may affect their metabolism. The potential health implications require further study.http://dx.doi.org/10.1289/ehp.1306499Citation: Riederer AM, Dhingra R, Blount BC, Steenland K. 2014. Predictors of blood trihalomethane concentrations in NHANES 1999–2006. Environ Health Perspect 122:695–702;  In vitro studies show that brominated THMs  are activated to mutagenic intermediates by glutathione S-transferase-theta-1 (GSTT1)  are formed during drinking-water disinfection as by-products of the reaction of chlorine/chloride with organic material and with bromide and iodide in source waters. The U.S. Environmental Protection Agency (EPA) regulates total THM (TTHM)—the sum of chloroform, bromoform, bromodichloromethane (BDCM), and dibromochloromethane (DBCM)—in public drinking water to 0.08 mg/L to reduce potential cancer and reproductive/developmental health risks . The U.S (GSTT1) . The U.S (GSTT1) , 1993. E (GSTT1) , 2007. T (GSTT1) .Because of their complex chemistry and other factors, THM exposure assessment is challenging and has been a key weakness in epidemiologic studies . Blood lGSTT1-null (inactive enzyme) participants had higher postshower blood chloroform than GSTT1-positive participants.A number of factors affect blood THM levels. Swimming in chlorinated pools and/or spending time at indoor pools is positively associated with blood THM concentrations . In smalChloroform is oxidized in the liver, kidney, and nasal mucosa to trichloromethanol, which degrades to phosgene, which forms cytotoxic adducts . In humaCYP2D6 polymorphisms indicating decreased activity had higher postshower blood chloroform and BDCM and higher postshower changes in BDCM and DBCM versus baseline.Gene polymorphisms may be important. CYP2E1 expression is influenced by age, obesity, diabetes and other chronic diseases, fasting, diet, and exposure to CYP2E1 inducers  and substrates  (S-transferase (GST) isozymes GST-α and GST-π  . Expressinophen) . Crucifend GST-π . The relnd GST-π .THMs were measured in blood and tap water in the 1999–2006 U.S. National Health and Nutrition Examination Survey (NHANES). We used multivariable regression and the NHANES data to explore blood THMs in relation to prescription medications (our primary research interest) and other factors potentially influencing THM metabolism, controlling for tap-water concentrations. Characterizing toxicokinetic and other environmental factors that influence blood THMs will help in understanding how exposures translate into blood concentrations and may provide further justification for using these biomarkers in epidemiologic studies. It may also potentially reduce bias in THM epidemiologic studies by identifying potential confounders and effect modifiers of exposure, even in studies without blood THM data.NHANES data collection. NHANES participants provided informed consent, thus we had a waiver from Emory University’s institutional review board for the present analyses. Detailed data collection methods are available at the NHANES website , and; c) toxicokinetic influences [BMI ]; pregnancy status ; any alcohol, caffeine, garlic, raw cruciferous vegetables, green/black tea, and/or watercress consumed in the previous 24 hr ; total grams fat consumed the previous 24 hr; smoking ; diabetes ; fasting. For race/ethnicity, we created a simple variable indicating whether or not a participant was from the major NHANES race/ethnicity category (non-Hispanic white). Participants were requested to fast overnight before the NHANES examination , which had higher mean levels in the no-tap-water-outside-the-home group. Because we did not have data for other survey years, we could not include this covariate in our models. We did have 1999–2006 data on hot drinks  consumed outside the home in the 24 hr before the examination. We assumed these were made with tap water and included a hot-drinks-consumed-outside-the-home variable (any vs. none) in our model-building efforts.We did not have complete data on tap-water consumption outside the home. Data available for 2005–2006 showed 19% of participants consuming some tap water  outside the home in the 24 hr before the NHANES examination. Unweighted Wilcoxon rank sum tests showed no statistically significant differences in blood THMs between those who did or did not drink tap water outside the home, except for chloroform , and SAS, version 9.2 , for statistical analyses. To obtain point and range  estimates, we created 8-year sample weights from the THM subsample weights, then used the NHANES survey design variables and 8-year weights to calculate descriptive statistics  variable in separate models for each THM, but neither version met our criterion for inclusion in any final models. We forced the six drug ingestion terms in at each step because estimating the effects of prescription medications on blood THMs was an original motivation for our study. We tested two-way interactions between water concentration and other variables in the final model, based on the a priori assumption that water concentrations would be the strongest influence on blood levels. Our criterion for including an interaction term in the final model was the same as that for main effects . We checked correlations between main effects variables in the final models and evaluated model assumptions  by examining plots of predicted values versus residuals and normal probability plots of residuals. We examined effects of extreme values, identified visually on box plots of the log-transformed blood data, by fitting the final models with and without these observations and comparing results.We used weighted linear multivariable regression to evaluate associations between natural log-transformed blood THM levels and the selected covariates, controlling for water concentrations . Five models were constructed—one for each individual THM and one for TTHM. Model building consisted of fitting a main effects full model with all 22 selected variables and NHANES survey year, then removing variables with the highest Wald statistic one by one until all remaining had Although the 1999–2000 air chloroform measurements were collected after the blood draw, we considered them a reasonable proxy for typical inhalation exposures. We conducted a subanalysis to evaluate the influence of air versus water chloroform on blood levels, fitting the final chloroform model to the 1999–2000 data both with and without the air measurements and comparing results.Descriptive statistics. n = 6,924); the statistics were similar when the sample was restricted to observations with non-missing blood THM measurements. The majority of participants (82.6%) had a private/public water company as their source of tap water versus 15.7% who had a private/public well (data not shown). Overall, 28.8% used home water treatment devices , with 25.9% of those on water-company water and 45.8% of those on well water using them. The most commonly eaten raw cruciferous vegetables (data not shown) were mustard/horseradish (11.7% of participants) and cabbage . Approximately half the participants took any prescription medication in the previous month (us month . Of thesp < 0.01) (data not shown). Approximately 50% of the water samples in 1999–2000, 67% in 2001–2002, and 80–90% in 2003–2006 had matching blood measurements. Detection frequencies and geometric mean (GM) water concentrations were nearly identical for the matched versus total data (data not shown), thus only the total data are presented.√–2). In general, GM blood and water THMs decreased during 1999–2006, regardless of imputation strategy used for the nondetects , with the largest between TTHM and chloroform in blood (ρ = 0.91) and TTHM and chloroform and TTHM and BDCM in water , and the smallest between chloroform and bromoform in blood (ρ = 0.04) and water (ρ = 0.02) .All correlation coefficients (ρ) were statistically significant at the α = 0.05 level, except for chloroform and bromoform in water  and substrates (p = 0.05), the effect of which would be to increase metabolism and lower parent compounds in the blood. A significant interaction (p = 0.02) was seen for water DBCM and those taking CYP2E1 substrates, indicating that the positive effect per unit of water DBCM on blood DBCM was diminished by 30% among those taking CYP2E1 substrates. This is consistent with the idea that more substrate would increase metabolism, which might diminish the parent compound, and that this in turn might diminish the positive effect of water concentrations on blood concentrations. However, this might simply be a chance finding, despite the nominally significant p-value.Taking different classes of drugs did not have marked effects on blood levels of any THM, although there were borderline significant effects in the anticipated direction for bromoform with respect to taking CYP2E1 inducers (n = 230) are shown in Supplemental Material, Table S4. Statistically significant (p < 0.01) associations were detected between log air chloroform and blood chloroform. Including the air term improved model fit (multiple R2 = 0.24 vs. 0.19). Including air chloroform reduced the water chloroform coefficient by about half, and increased its p-value from < 0.01 to 0.05, indicating that air chloroform is a stronger predictor of blood chloroform than water chloroform. Because chloroform volatilizes during hot water use, air chloroform is an intermediate on the causal pathway between water and blood when dermal or ingestion exposures  are considered and should not be included in models of water concentrations as predictors of blood levels. However, if air concentrations were available, they would be the preferred predictors of blood chloroform based on our findings and others’ . We did not include the NHANES water source variable in our models because we assumed that water concentrations would reflect source. However, we conducted subanalyses restricting the TTHM, chloroform, and BDCM models to participants on water-company water (85% of participants) . Results did not differ markedly, although inverse associations with diabetes, cruciferous vegetables, afternoon/evening examination session, and being pregnant were stronger in the subanalyses. In the restricted TTHM model, for example, the absolute value of the regression coefficients increased by 30%, 13%, and 14% for the morning examination session, diabetes, and cruciferous vegetables terms, respectively, compared with the unrestricted model.Decreasing blood and water THMs in 1999–2006. The U.S. EPA lowered the TTHM drinking-water standard from 0.10 mg/L to 0.08 mg/L for large surface-water systems on 1 January 2002 and smaller systems on 1 January 2004, anticipating that this would produce a 24% average national reduction in THM levels  was associated with lower blood BDCM. Diabetes and obesity are complex, interrelated conditions associated with increased CYP2E1 mRNA expression and/or activity in humans  and gut microflora to isothiocyanates, some which inhibit CYP2E1, others which induce GST-α and GST-π, although we are unaware of evidence that these isozymes conjugate THMs . In our p = 0.08 and p = 0.05 respectively), possibly indicating increased bromoform metabolism in people taking these drugs. Taking prescription CYP2D6 substrates was associated with higher blood bromoform (p = 0.09), possibly indicating competitive inhibition or similar toxicodynamic mechanism. The prescription CYP2E1 inducer–log water concentration interaction was borderline (p = 0.07) significantly associated with lower blood BDCM, and the prescription CYP2E1 substrate–log water concentration interaction was significant for blood DBCM (p = 0.02). The direction of these interactions was consistent with the idea that having more inducer or substrate could increase THM metabolism, which in turn may attenuate the positive effect of water THM levels on blood THM levels. Because approximately half the U.S. adult population takes prescription medications, many of which affect CYP2D6 or CYP2E1, their influence on THM toxicodynamics may be worthy of further investigation.Other potential toxicodynamic influences in our models included prescription medications and pregnancy status. Alcohol consumption, smoking, and education were also associated with blood THMs, but only in the bromoform (education) and BDCM  models. In the bromoform model, taking prescription CYP2E1 inducers or substrates was associated with lower blood levels at borderline significance , whereas nonpregnant women had significantly higher levels . Pregnancy involves a number of changes affecting xenobiotic metabolism. Increased plasma volume, changes in blood protein binding, and fat accumulation during the first two trimesters could increase the volume of distribution of many compounds , resultiLimitations. A major limitation is the cross-sectional nature of the NHANES data, which limits our ability to identify the factors driving the observed associations. In addition, our models explained only 22–44% of the variability in blood THMs, thus other factors appear to be important. We lacked detailed data on factors likely to be important to blood levels. Including personal air concentrations in our models, particularly if measurements were taken the morning of the blood draw, would have improved their explanatory power. More detailed data on frequency and duration of showering, and time since showering at the time of blood sampling, would have been useful. We had no detailed data on possible occupational THM exposures, or blood sample dates if there was seasonal variation. We also lacked data on swimming activity close to the blood draw. Such variables could have confounded the relationships we observed.Other limitations include the dichotomization of the medication terms, which did not allow us to evaluate the possible influence of taking, for example, multiple CYP2E1 inhibitors, or taking higher versus lower doses; NHANES does not include dose information. Summary scores of exposures to inducers and inhibitors may help explain blood THMs better than dichotomous classifications. Other than a toxicokinetic study of BDCM , we are A final caveat concerns the use of blood THM measurements. Although one-time blood concentrations are thought to reflect steady-state levels in humans , they maSurvey year and water THMs were the strongest predictors of blood THMs in our analyses. We also found statistically significant associations between blood THMs and potential toxicodynamic influences, controlling for water levels. Inverse associations may indicate that these factors alter THM metabolism, although additional research is needed to determine whether changes in THM absorption and distribution also play a role and to evaluate the potential health implications of these findings.(365 KB) PDFClick here for additional data file.


Candida spp. can often be identified in pulmonary samples. The impact of prompt antifungal therapy in these patients is unknown.In critically ill patients, Candida spp. colonization admitted to the intensive care unit (ICU) between 2010 and 2012 were included. The patients were analyzed according to whether or not they received antifungal therapy, which was administered at the discretion of the attending physician. Logistic regression analysis was performed to investigate the impact of antifungal therapy on hospital mortality and new onset of ventilator-associated pneumonia. In a stepwise backward elimination, the impact of age, cancer as an underlying disease, Simplified Acute Physiology Score (SAPS) II, and Sequential Organ Failure Assessment (SOFA) score were considered.In this retrospective study, 500 adult patients with pulmonary Candida spp., isolated pulmonary Candida spp. colonization was found in 322 patients (cohort 1). Pre-existing pneumonia was found in 147/322 patients. Out of the remaining 175 patients (cohort 2), 44 patients received any antifungal therapy, and 131 were defined as the control group. Patients who received antifungal therapy had higher hospital mortality  and pneumonia rates  than those who did not. In Cox regression analysis, antifungal therapy was not independently associated with favorable outcome ; new pneumonia: 1.048 (0.536–2.046)), but SAPS II and SOFA score were significantly (p < 0.05) independent covariates for worse outcome.After excluding 178 patients with multifocal Candida spp. colonization, antifungal therapy may not have an impact on the incidence of new pneumonia or in-hospital mortality after adjustment for confounders.In critically ill patients with pulmonary The online version of this article (doi:10.1186/s40560-015-0097-0) contains supplementary material, which is available to authorized users. Candida spp. can very often be identified in pulmonary samples taken from tracheal aspirates or bronchoalveolar lavage. However, the key question remains whether a simple colonization or an invasive Candida spp. infection exists. Meersseman et al. recently published that 57 % of their deceased patients had findings of Candida albicans in the pulmonary secretion, but none of these patients had a “true” Candida spp. pneumonia [Candida spp. isolation from respiratory secretions alone should never prompt treatment” [Yeasts are part of the physiological flora of the oral mucosa and the intestine tract in about 40–65 % of healthy adults . In immuCandida spp. findings increase the risk of a systemic Candida spp. infection, and thereby increase risk for morbidity and mortality [Candida colonization index as a risk score for systemic Candida spp. infection including the status of (a) multifocal colonization, (b) surgery, (c) parenteral nutrition, and (d) severe sepsis [In contrast, multifocal ortality . León ete sepsis .Candida spp. colonization was significantly associated with an increased risk of nosocomial pneumonia and prolonged length of stay at the intensive care unit (ICU) [Candida spp. colonization was found in 56 % of these patients and represents an independent risk factor for multidrug-resistant bacterial super-infection associated with an increased risk of mortality [Interestingly, Azoulay et al. found in a multicenter cohort of 800 patients that pulmonary ortality .Candida spp. colonization remains to be elucidated. In our retrospective study, we hypothesized that initiation of any antifungal therapy was associated with a reduced risk of new onset of ventilator-associated pneumonia and death compared to no antifungal therapy.In this respect, whether or not antifungal therapy should be initiated in critically ill patients with pulmonary Candida spp. The patients were treated between 2010 and 2012 at the intensive care unit of the Department of Anesthesiology, Intensive Care Medicine and Pain Therapy, University Hospital Frankfurt, Germany. This study was approved by the ethical committee of the Faculty of Medicine, University Hospital Frankfurt (379/12). The ethic committee did not have any ethical concerns and confirmed that no patient consent form is needed as this study retrospectively analyzed routine data.This is a retrospective study including 500 intensive care patients with at least one pulmonary finding of Candida spp. growth in the culture medium. The different Candida isolates were identified at species level. Serologic biomarkers for Candida spp. were not used routinely. Indication for antifungal treatment was based on an individual decision considering the underlying risk factors of the critically ill patient, respectively. These factors particularly covered neutropenia, renal replacement therapy, long-term ventilation, or multifocal colonization.As a clinical routine, pulmonary samples were taken as tracheal aspirates tracheal aspirates for microbiological analyses twice a week and additionally when a new pulmonary infection was considered . The samples were created in laboratory natively and after dilution quantitatively on nutrient agar. The results were considered positive in the presence of Primary endpoints were in-hospital mortality or any new onset of ventilator-associated pneumonia. Ventilator-associated pneumonia was defined as present (a) if the attending physician declared a new onset of a pulmonary infection and indicated broad-spectrum antibiotics or (b) if a new onset of pneumonia was defined according to the clinical and microbiological criteria of the CDC (Centers for Disease Control and Prevention) within the German Hospital Infection Surveillance System .Candida spp. was reported (baseline). The observation time period for microbiological results and therapy was limited to a maximum of 28 days of ICU therapy.Secondary endpoints were length of stay at the intensive care unit, duration of ventilation based on the documented time of ventilation within the DRG coding, length of hospital stay, SAPS II (Simplified Acute Physiology Score), SOFA  score at baseline and day 7 (without nervous system), and systemic inflammatory mediators at baseline and days 3, 7, and 14 including C-reactive protein, procalcitonin, leukocytes, thrombocytes, interleukin-6, and lipoprotein-binding protein. Also, we documented gender, primary discipline, cancer, any antibiotics and antifungal therapy, any pulmonary microbiology findings, and occurrence of sepsis and septic shock. Data collection was started as soon as the first positive microbiological finding with Candida spp. have been performed.This study has a retrospective observational design and is exploratory in nature and undertaken for description. Therefore, no direct comparisons between different antifungal strategies and Candida spp. colonization (24 % out of 214 patients vs. 19 % [Candida spp. colonization by 50 % [n = 500) with pulmonary Candida spp. colonization would have received any antifungal drug resulting in about 80 patients for the therapy group compared to 350 untreated patients (control group).For sample size calculation, we took into account previous study results. Risk of ventilator-associated pneumonia was significantly increased in patients with vs. 19 %  and 32 % by 50 % . We estiCandida spp. colonization (control group). We assumed that antifungal therapy reduces the incidence of pneumonia by 50 % with a level of significance of 0.05 and a power of 80 % by a two-sided test.Based on the above-mentioned clinical studies, incidence of ventilator-associated pneumonia was assumed to be about 30 % in untreated patients with positive p values were determined by using the two-sided Wilcoxon-Mann-Whitney test. Nominally scaled variables were presented in absolute terms and percentage, based on the type of group. The p values were determined by using the Fisher-Yates test.Metrically scaled variables were presented as mean ± standard deviation (SD) or median . The To investigate the impact of any antifungal therapy in pulmonary colonized patients on the incidence of pneumonia and on in-hospital mortality, we performed Kaplan-Meier analysis with Mantel-Cox logrank test for survival time and pneumonia-free time as the dependent variables. Additionally, we performed Cox regression analysis considering the impact of age, cancer as an underlying disease, SAPS II, and SOFA score . The regression results are displayed as odds ratio . The number of cases and these five predictor variables in Cox models stick to the rule of thumb, using around ten outcome events per predictor variable , 13. In p < 0.05.All statistics were two-tailed. Statistically significance was considered with a The data have been analyzed using IBM SPSS statistics 22 for Mac OS X and Bias 10.12.Candida spp. In the first step, we excluded patients with multifocal Candida spp. findings (n = 178), as multifocal colonization increases risk of systemic Candida spp. infection and triggers antifungal therapy. Isolated pulmonary Candida spp. colonization was found in 322 patients (cohort 1), of whom 102 patients received any antifungal therapy. Pre-existing pneumonia at baseline was found in 147/322 patients. Out of the remaining 175 patients (cohort 2), 44 patients did receive (therapy group) and 131 did not receive any antifungal therapy . The patients with antifungal therapy were more critically ill with significantly (p < 0.001) higher SAPS II and SOFA scores, higher percentage of pre-existing pneumonia (56.9 vs. 40.5 %), higher use of carbapenems, quinolones, and glycopeptides, and higher incidence of Candida non-albicans species at baseline. Additional file Candida spp. were C. albicans (69.6 vs. 81.8 %). Interestingly, significantly more therapy patients had any pulmonary bacterial findings at baseline .Baseline characteristics of patients with isolated pulmonary p < 0.005), 47.7 % of patients developed new pneumonia (vs. 16.8 %), durations of mechanical ventilation, hospital and ICU stay were longer (p < 0.001), more patients developed sepsis (p < 0.002) or septic shock (p < 0.006), and higher levels of pro-inflammatory mediators were found , SOFA score (1.094 (1.030–1.163); p = 0.004), and age (1.020 (1.004–1.037); p = 0.016) as independent covariates for mortality, but antifungal therapy had no independent impact on chance of survival (1.198 (0.810–1.771); p = 0.366; Additional file Using the Kaplan-Meier estimate, survival curves did not show any significant difference between the groups  and SOFA score were significantly higher in the therapy group (p < 0.003). Additional file p < 0.005) and ICU (p < 0.001) stays, a higher incidence of newly acquired pneumonia (p < 0.001), and died more often . During the observation period, 52.3 % had any evidence of pulmonary bacterial colonization/infection. Gram-negative bacteria were the most common co-infection , but SAPS II (1.027 (1.003–1.052), p = 0.025) and SOFA score (1.096 (1.004–1.198), p = 0.041) were independently associated with higher risk of death  (not shown).In the final step, competing risk analysis with the Aalen-Johansen estimator for pneumonia and death as the competing events was performed. Again, the time until new event was significantly shorter in the therapy group (p = 0.477). The rate of new hospital-acquired pneumonia did not differ significantly (therapy group 2/8 (25 %) vs. control group 3/12 (25 %)). Antifungal therapy had no significant impact using Cox regression analysis (0.448 (0.11–18.71), p = 0.673).In case that blood culture shows false negative candidemia, we defined a subgroup of high-risk patients of suspected candidemia who had a permanent pacemaker placement (cohort 3). The baseline characteristics are presented in Table Candida is frequently found in pulmonary secretions of patients in ICU known as Candida spp. colonization, if clinical and pathological evidence of invasive candidiasis is absent. In this respect, Candida spp. colonization may not play an important pathogenic role in the context of ventilator-associated pneumonia, at least in immunocompetent patients [Candida spp. in pulmonary secretion [patients . Consequecretion .Candida spp. colonization has been shown to be significantly associated with an increased risk of nosocomial pneumonia [Candida spp. colonization is suggested to be an independent risk factor for multidrug-resistant bacterial super-infection associated with an increased risk of mortality [On the other hand, pulmonary neumonia . Additioortality . In thisCandida spp. might be beneficial [Candida spp. in the tracheal secretions.Very recently, Albert et al. included 60 patients into a randomized trial and analyzed 29 patients within an observational study, whether antifungal therapy in critically ill patients with a clinical suspicion of ventilator-associated pneumonia with positive airway secretion specimens for neficial . MarkersCandida spp. colonization. Not surprisingly, patients that received antifungal therapy were more critically ill as evident from higher SAPS II and SOFA score and had a higher degree of systemic inflammation. In this respect, these patients showed prolonged duration of mechanical ventilation, ICU, and hospital length of stay.The main results of our retrospective study including 322 critically ill patients are in line with these recent findings. Using Cox regression analysis to account for confounders, any antifungal treatment did not had a beneficial effect on the incidence of new pneumonia or in-hospital mortality in a cohort of heterogenic intensive care patients with isolated pulmonary Consistently, our Cox regression analysis revealed SAPS II, SOFA score, and age of patient as independent variables for a higher risk of in-hospital mortality. These results are not surprising, as SAPS II “…provides an estimate of the risk of death without having to specify a primary diagnosis” , and theCandida spp. colonization.In a subgroup of patients (cohort 2), we focused on the time until new pneumonia. If any antifungal therapy is suggested to be beneficial, then free time to any new pneumonia should reasonably be longer, at least in theory. Surprisingly, our results showed contradictory effects, and time until new pneumonia was significantly shorter in patients that received antifungal therapy during ICU stay. This is in line with our findings that patients with antifungal therapy were more critically ill with significantly higher incidence of new pneumonia, higher SAPS II and SOFA score, higher incidence of sepsis, and longer duration of hospital and ICU stays. Thus, severity of critical illness may have a large bias on outcome variables. To account for these confounders, we considered a couple of known covariates for pneumonia in our Cox regression analysis. Nevertheless, antifungal therapy may not have an independent effect on time to new pneumonia or survival chance. There are three additional interesting observations: (a) most of the patients developed new pneumonia within the first 10 days after microbiological detection of yeasts, (b) preemptive antifungal therapy has been observed in 38 % of the patients, and (c) median time to initiate antifungal therapy was 4 days in non-preemptive-treated patients. Under these circumstances, it remains speculative if a faster initiation of antifungal therapy at the first day of microbiological result or even a more preemptive antifungal strategy in critically ill patients would otherwise prevent any yeasts and would result in a better outcome. Nevertheless, these factors are of minor importance regarding the key question of this analysis—whether or not to initiate antifungal therapy in critically ill patients with Candida spp. in the lung could be interpreted as harmless colonization rather than representing a true infection requiring any treatment [Candida spp. colonization had no significant impact in this study.Our results also suggest that the absence of any beneficial effect after antifungal therapy is irrespective of any pre-existing pneumonia. Thus, even in higher risk critically ill patients with pre-existing ventilator-associated pneumonia (cohort 1), the presence of reatment . Very rereatment , but simCandida spp. colonization irrespective of the severity of critical illness.Thus, our results support the recent ESCMID recommendations, not to initiate antifungal therapy in patients with isolated pulmonary Candida spp. in the blood culture. To elucidate the role of antifungal therapy in these high-risk patients, we performed a subgroup analysis of 20 patients with implanted pacemaker and isolated pulmonary Candida spp. colonization without pre-existing pneumonia. Again, antifungal therapy was not independently associated with a favorable outcome.Importantly, blood culture may show false negative results of candidemia among high-risk patients with central venous catheter or permanent pacemaker placement due to relatively slow growth of Candida spp. colonization. Secondly, we did not consider additional risk factors for ventilator-associated pneumonia as covariates, e.g., chronic obstructive pulmonary disease [Candida spp.  have different pathogenicity and sensibilities to drugs, but we did not perform subgroup analyses regarding different types of Candida spp. or kinds of antifungal drugs, as the sample size is reasonable small.Our study has the following limitations. Firstly, this study has a retrospective and explorative design, and a large prospective randomized controlled study might be needed to provide more evidence whether or not a fungal therapy may be beneficial in patients with isolated pulmonary  disease , the use disease , diabete disease , and the disease , as thesCandida spp.Our study suggests that antifungal therapy may not have an impact on the incidence of new pneumonia or in-hospital mortality after adjustment for confounders in critically ill patients with isolated pulmonary


Neurological abnormalities, such as Parkinson-like disorders (PlD), are often co-morbidities of Type 2 Diabetic (T2D) patients, although the epidemiological link between these two disorders remains controversial. The PED/PEA-15 protein represents a possible candidate linking T2D and PD, because it is increased in subjects with T2D and is highly expressed in the brain. To test this hypothesis, we have analyzed the neurological and neurochemical phenotype of transgenic mice overexpressing PED/PEA-15 (tgPED). These mice develop impaired glucose tolerance and insulin resistance, accompanied by neurological features resembling PlD: feet clasping, slow and delayed locomotor movements in different behavioral tests in absence of clear cognitive deficits, ataxia or anxiety. Morphological analysis of the brains showed selective modifications of metabolic activity in the striatal region. In the same region, we have observed 26% decrease of dopamine fibers, confirmed by immunohistochemistry and Western Blot for tyrosine hydroxylase. Moreover, they also showed 48% reduction of dopamine levels in the striatum. Thus the tgPED mice may represent a genetic animal model of neurological disease linked to T2D. PED/PEA-15 is a cytosolic protein enriched in several cells  of patients with Type 2 Diabetes (T2D) and their first degree relatives139Notwithstanding the clinical and therapeutic relevance of this relationship, the molecular mechanism underlying T2D and PD remains largely unknown.PED/PEA-15 might represent a possible molecular element subserving both T2D and PlD. In fact, several interactors of PED/PEA-15 are expressed in brain and are involved in neuronal physiology and neurodegeneration. Specifically, ERK1/2 and Phospholipase D , both binding partners of PED/PEA-15, are involved in cell death and differentiation in different neuronal systems15Transgenic mice over-expressing PED/PEA-15 (tgPED) were viable and fertile with normal litter size, as previously describedWhen tested on an inclined platform, tgPED mice exhibited normal righting response. However, tgPED mice showed two fold increased time to climb  and an iTgPED mice did not display significant differences in stride length and width compared to the wt mice , therebyTo test the motor learning, mice were trained on a Rotarod during five consecutive trials. TgPED mice did not show significant differences in time spent on rotating rod compared to wt during the first exposure of animals . Both wt3,36 = 9.1, p ≪ 0.01, trial * genotype interaction effect: F3,36 = 9.1, p = 0.5 , In a typical spatial learning task, the Barnes’ maze, tgPED mice reached the same performance level of control mice, demonstrating a preserved overall learning ability . Nevertheless, this change was significant only in the Striatal region, as shown by multiple post-hoc comparisons This behavior likely parallels the hypokinesia present in human PlD. Accordingly, the slower learning ability on Rotarod of tgPED mice can be linked to the amount of dopamine, as occurs in other hypodopaminergic animal models22In addition, TgPED mice also show later latency in negative geotaxis, which resembles the difficulty to start movements in PlD patients.Indeed, the hypoactivity of tgPED mice was paralleled by lower dopamine content and an increased metabolic activity in the CPu. This phenotype (locomotor hypoactivity + hypodopaminergic + increased CPu metabolism) has been already observed in another PlD model, the Girk2Wv (weaver) miceTgPED mice also show foot clasping, a symptom reported in several other transgenic mice bearing different neurological alterations such as mice deficient for Phospholipase A2 The kyphosis observed in tgPED mice should also be regarded as an unspecific symptom, although it has been observed in another PlD mouse model, the parkin-deficient mice.tgPED animals did not show significant differences in anxiety. Unfortunately, the variability in the two groups was high, thereby making difficult the complete exclusion of an “anxious” phenotype. The hypokinesia cannot be easily disentangled from anxiety, in animal models, since anxious tests require, in turn, locomotor activity. Indeed, the total activity of tgPED mice in the EPM was significantly lower compared to wt mice, and this may further confound the interpretation of data see .Similarly, our model did not show learning and memory deficits , nor gait modifications compared to wt.These four symptoms  are usually not simultaneously found in animal models of PlD: for instance, in genetic models of PD, the behavioral phenotype is rather variable see .At variance, tgPED mice show a hypo-dopaminergic profile and a resulting hypo-locomotion, without evident modifications of memory and gait . These observations point out that the phenotype in tgPED mice is mild, and possibly additional genetic/environmental factors are needed to give a full PD phenotype.It is already known that suppression of PED/PEA-15 brain expression is accompanied by behavioural impairment and neuroprotection36These considerations foster some important translational implication . First, on the pathogenetic side, it would be tempting to speculate that the origin of PlD in T2D is not entirely due to the hyperglycemic/vascular state. It is actually possible that the dysregulation of certain molecular targets, such as PED/PEA-15, may lead to parallel and independent damages in systemic glucose metabolism and brain metabolism. Our model, in fact, does show behavioral modifications even before true hyperglycaemia is present. Second, it is possible that PlD emerges when only glucose intolerance is present, in absence of diabetes. If this conclusion holds true, one can expect even a stronger connection between dysmetabolism and PD, because glucose intolerance is rarely tested in PD patients. Finally, it is plausible that in T2D-linked PlD the therapeutic approach should not be limited to the hyperglycemic state, because brain damage could be due to a hyperglycemia-independent mechanism. However, it could be interesting to test drugs improving insulin sensitivity in the brain in a subset of PlD patients, as a potential tool for PD prevention.Generation of mice overexpressing PED/PEA -15 (tgPED) has been described elsewhereBehavioral monitoring was performed on male tgPED (n = 11) and Wt littermates (n = 11), at the age of 6 months, during the light phase between 10:00 a.m. – 17:00 p.m. A modified SHIRPA protocol has been used as a first screening to observe gross abnormalities in posture and sensorial response. The presence of foot clasping (abduction of hindlimbs) has been evaluated suspending mice by their tails. Subsequently, mice have been tested for their negative geotaxis, stride pattern, motor abilities and memory; less stressful tests  were conducted first in the same experimental session; a minimum of one day separated the testing sessions.All animal procedures were approved by the ethical committee of the University of Naples “Federico II” and comply with the standards of the European Union.Mice were placed on the lower part of an inclined grid , facing the floor. The time to turn (negative geotaxis) and to reach the top of the grid was recorded, with a maximum time of 5 min.Evaluation of motor coordination was conducted using a standard mouse rotarod  measuring the time a mouse remained on a rotating rod. The rubber rotating rod  rotated at increasing speed from 4 to 40 rpm in 5 min. Mice were allowed to stay on the rotating rod for a maximum of 5 min for 5 successive trials. Trials were separated by at least 60 s. The latency to fall was recorded for each trial, and improvements from trial to trial was used as an index of motor learning rate.See Forelimb force has been tested with three tests : (i) hang grip force test, (ii) wire hang test and (iii) vertical pole test  and post fixed in the same fixative at 4 °C for 2 h. After cryoprotection and freezing, 20 μ thick cryosections were mounted on SuperFrost Plus slides and stored at -80. For dopamine immunohistochemistry, mice were perfused with the addition of Sodium meta-bisulphite in the perfusion buffer, to prevent oxidation of dopamine. Basal metabolic activity of different brain regions was evaluated by cytochrome oxydase (COX) staining. Sections have been incubated for 2 h at room temperature with the following staining medium: Cytochrome C 0.04% (Sigma-Aldrich), Sucrose 5%, DMSO 0.2%, DAB 0.05% in MOPS 0.1 M pH7.4. All brains have been stained at the same time in the same staining solution to minimize variability due to the staining procedure.Immunohistochemistry for tyrosine hydroxylase (TH) was performed as follows: after rinsing in PBS, slides were incubated with a mouse monoclonal anti- TH  or with anti-dopamine (1:100 with the addition of sodium metabisulphite) in PBS overnight with shaking at + 4 °C. To allow optimal homogeneous staining among silces, the HybriWell system has been used (BioScience). After three rinses in PBS, sections were then incubated with anti-mouse biotinylated antibody  for 1 hour at room temperature with shaking, and the ABC MOM system (Vector) used to visualize the reaction product (prepared according the supplier’s instructions). The reaction was visualized using DAB as chromogen. Slides have been analysed with a Zeiss Axioskop 20, equipped with a CCD camera  and motorized XYZ stage . The entire brain sections have been acquired using tiled fields from 10x objective . At least 10 slices per brain have been imaged for each staining technique. Markers expression levels have been quantified as relative optical density (ROD = log (256/grey level). ROD units, after background subtraction, are correlated to the antigen concentration.−1 according to the manufacturer’s instructions . Tissues homogenates were then separated by SDS-PAGE and analyzed by Western blot as previously described.Mice were euthanized and brain samples were collected rapidly, snap-frozen in liquid nitrogen and stored at −80 °C. Tissue samples were homogenized in a Polytron Homogenizer  in 20 ml T-PER reagent (g tissue)Data have been analyzed using Excel and SPSS software . Comparisons among groups have been analyzed using Student’s t-test for non paired data in case of continuous variables and Mann Whithey U test in case of discrete variables. Rotarod and Barnes’ maze data have been analyzed by two way ANOVA using training session and genotype as factors. Cytochrome oxidase activity levels has been analyzed with two way ANOVA using brain region and genotype as factors. Data are expressed as mean ± SEM. Rejection level of the null hypothesis has been set to p < 0.05.How to cite this article: Perruolo, G. et al. Parkinson-like phenotype in insulin-resistant PED/PEA-15 transgenic mice. Sci. Rep. 6, 29967; doi: 10.1038/srep29967 (2016).