TNFAIP2 3'-UTR rs8126 T>C genetic variant which has been proved to be associated with head and neck cancer susceptibility. This SNP could disturb binding of miR-184 with TNFAIP2 mRNA and influence TNFAIP2 regulation. However, it is still unclear how this polymorphism is involved in development of esophageal squamous cell carcinoma (ESCC). Therefore, we hypothesized that the functional TNFAIP2 rs8126 SNP may affect TNFAIP2 expression and, thus, ESCC risk.Accumulated evidences demonstrated that single nucleotide polymorphisms (SNPs) in mRNA 3'-untranslated region (3'-UTR) may impact microRNAs (miRNAs)-mediated expression regulation of oncogenes and tumor suppressors. There is a TNFAIP2 rs8126 variant and ESCC risk as well as the functional relevance on TNFAIP2 expression in vivo. Genotypes were determined in a case-control set consisted of 588 ESCC patients and 600 controls. The allele-specific regulation on TNFAIP2 expression by the rs8126 SNP was examined in normal and cancerous tissue specimens of esophagus.We investigated the association between the P = 0.003) or 1.38  for developing ESCC in Chinese compared with individual carrying the TT genotype. Carriers of the rs8126 CC and CT genotypes had significantly lower TNFAIP2 mRNA levels than those with the TT genotypes in normal esophagus tissues (P<0.05).We found that individuals carrying the rs8126 CC or CT genotype had an OR of 1.89 (95%CI  = 1.23–2.85, TNFAIP2 rs8126 genetic variant is a ESCC susceptibility SNP. These results support the hypothesis that genetic variants interrupting miRNA-mediated gene regulation might be important genetic modifiers of cancer risk.Our data demonstrate that functional  As one of the most common and fatal malignancies worldwide, esophageal squamous cell carcinoma (ESCC) shows a relatively high morbidity in Eastern Asian and Eastern African compared to Western countries TNFAIP2 mRNA. In detail, the rs8126 variant C allele led to significantly lower luciferase activity and expression of TNFAIP2 mRNA, compared with the T allele Ptrend  = 0.009). Similarly, the rs8126 CC genotype was associated with a significantly elevated risk of gastric cancer , compared with the combined rs8126 TT and TC genotypes, particularly in current drinkers.TNFAIP2  is also known as the primary response gene B94 protein, which is one of the SEC6 members. Originally, TNFAIP2 was identified as a TNF-alpha-induced protein in human endothelial cells TNFAIP2 rs8126 genetic polymorphism may contribute to ESCC susceptibility via influencing TNFAIP2 expression in esophagus cells. To test this hypothesis, we conducted a large case-control study of ESCC. To reveal the biological function of this SNP, we detected the impact of its different genotypes on TNFAIP2 mRNA expression in both normal and malignant esophagus tissues.However, it is still unclear whether this functional polymorphism is involved in ESCC development. Based on the aforementioned findings, we hypothesized that the A total of 588 ESCC cases from Huaian No. 2 Hospital  and sex- and age-matched 600 controls were included in this study. Patients were consecutively recruited between January 2009 and February 2012 at Huaian No. 2 Hospital. All cases are incident ones during enrollment of the current case-control study. Cancer-free controls were chosen from 3600 individuals of a community cancer-screening program for early cancer detection. The program was conducted in Huaian city during the same time period as the patients were recruited. The diagnosis of all patients was histologically confirmed. Subjects who smoked one cigarette per day for more than one year were considered as smokers. Individuals were considered as alcohol drinkers, if they drank at least once every week. Eighteen pairs of ESCC tissues and esophagus normal tissues adjacent to the tumors were obtained from surgically removed specimens of patients in Huaian No. 2 Hospital. The normal tissues sampled at least 2cm away from the margin of the tumor. All subjects were ethnic Han Chinese. At recruitment, the informed consent was obtained from each subject. All participants have provided their written informed consents to participate in this study. This study was approved by the institutional Review Board of Huaian No. 2 Hospital.CC-3′5′- GGGGCCGGCTCTCTTGGG and 5′-CACACGTACAAAGACCTTGGGCATCC-3′. The 25 µL PCR reaction mixture consisted of 100 ng of DNA, 0.1 mmol/L of each primer, 0.2 mmol/L of deoxynucleoside triphosphate, 1.0 U of Taq DNA polymerase , 1× reaction buffer and 1.5 mmol/L MgCl2. The PCR profile contains an initial melting step of 2 minutes at 94°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 55°C, 30 seconds at 72°C, and a final elongation step of 10 minutes at 72°C. Restriction enzyme ApaI (NEB) was utilized to distinguish the rs8126 genotypes. A 10% random sample was reciprocally examined by different persons, and the reproducibility was 100%.The rs8126 T>C polymorphism was genotyped through the PCR-restriction fragment length polymorphism (RFLP) method as described previously TNFAIP2 mRNA or β-actin mRNA distributed normally. Relative gene expression quantization for TNFAIP2  was examined with the ABI 7500 real-time PCR system in triplicates using β-actin as the endogenous control . The expression of individual TNFAIP2 mRNA measurements was calculated relative to expression of β-actinTotal RNA was extracted from cancerous and normal esophagus tissue samples using TRIzol Reagent following RNase-Free DNase treatment (Invitrogen). RNA was examined in 1% agrose gel which was stained with EB to examine the integrity of each tissue RNA sample before qRT-PCR. The quantity was detected by Nanodrop and 100 ng RNA was used in the RT reaction with TaqMan Reverse Transcription Reagents (N8080234). TaqMan real-time qPCR method with the master mix reagent  was used to examine mRNA levels in cancerous and normal esophagus tissues according to the manufacturer's instructions. The expression values of TNFAIP2 rs8126 T>C polymorphism between ESCC patients and controls were calculated by Pearson's χ2 test. Associations between TNFAIP2 rs8126 genotypes and ESCC risk were estimated by OR and their 95% CIs computed using unconditional logistic regression model. All ORs and 95% CIs were adjusted for age, sex, drinking and smoking status, where it was appropriate. All statistical tests were two-sided. A P value of less than 0.05 was used as the criterion of statistical significance. All calculations were performed with SPSS software package .The differences in demographic variables and genotype distributions of P>0.05), indicating that the frequency matching was adequate , with the frequency of CC homozygote being significantly higher among patients than among controls (9.7% vs. 6.2%).Allele frequencies and genotype distributions of TNFAIP2 rs8126 genetic polymorphism and ESCC risk were estimated using unconditional logistic regression analyses (P = 0.003) for developing ESCC in this population compared with individual having the TT genotype. Additionally, the rs8126 CT carriers also showed a 1.38-fold increased ESCC risk compared with those carrying the rs8126 TT genotype   . Both ORTNFAIP2 mRNA expression in both ESCC and normal esophagus tissues in the same population. As shown in TNFAIP2 mRNA levels (mean ±SD) than those with the TT genotypes in normal esophagus tissues . However, no statistically significant differences of TNFAIP2 expression were observed between CC, CT and TT genotypes in ESCC tissue specimens .We investigated whether the ESCC susceptibility SNP rs8126 has an allele-specific impact on the TNFAIP2 3'-UTR rs8126 genetic variant and ESCC risk via a case-control approach. To the best of our knowledge, this is the first study to examine how the TNFAIP2 rs8126 SNP is involved in ESCC tumorigenesis. We found that the rs8126 genetic variant was significantly associated with increased ESCC risk in a Chinese population. Intriguingly, this polymorphism has an allele-specific impact on TNFAIP2 expression in vivo. Our data support the hypothesis that functional genetic variants at gene 3'-UTR region influence miRNA-mediated expression regulation of mRNA.In the current study, we investigated the association between the functional Genome-wide association studies (GWAS) highlighted the importance of SNPs in ESCC development TNFAIP2 mRNA could significantly decrease expression of TNFAIP2 mRNA in vitro and in vivoTNFAIP2 expression in normal esophagus specimens among C allele carriers than those in T allele carriers. After genotyping the TNFAIP2 rs8126 polymorphism in 1077 HNSCC patients and 1073 cancer-free controls in a non-Hispanic White population, Liu et al found that, compared with the rs8126 TT genotype, the variant C allele were associated with increased HNSCC risk in an allele dose-response manner  P = 0.024), compared with the combined rs8126 TT and TC genotypes, particularly in current drinkers TNFAIP2 rs8126 polymorphism might be a common risk factor among different gastrointestinal malignancies.In a previous study, Liu et al firstly identified a functional rs8126 T>C SNP in HNSCC There might be several limitations in the current case-control study. For instance, inherent selection bias may exist since it was a hospital-based study and the cases were from the hospital. Therefore, the findings of this study warrant to be validated in the future through a population-based prospective study. Liu et al presented the rs8126 as a functional variant that affects binding of miR-184 and presumably regulates expression of TNFAIP2 in head and neck cancer. However, functionality of this variant could not be generalized to other tissues without testing, even though the current analysis showed nominal association of the polymorphism with mRNA expression.TNFAIP2 rs8126 polymorphism was associated with a significantly increased risk of ESCC in a Chinese population. These data support the hypothesis that genetic variants interrupting miRNA-mediated gene regulation might be important genetic modifiers of cancer risk.In summary, our results demonstrated that the functional


N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune disorder of the central nervous system (CNS). Its immunopathogenesis has been proposed to include early cerebrospinal fluid (CSF) lymphocytosis, subsequent CNS disease restriction and B cell mechanism predominance. There are limited data regarding T cell involvement in the disease. To contribute to the current knowledge, we investigated the complex system of chemokines and cytokines related to B and T cell functions in CSF and sera samples from anti-NMDAR encephalitis patients at different time-points of the disease. One patient in our study group had a long-persisting coma and underwent extraordinary immunosuppressive therapy.Anti-P value <0.05 was considered to be significant.Twenty-seven paired CSF/serum samples were collected from nine patients during the follow-up period . The patient samples were stratified into three periods after the onset of the first disease symptom and compared with the controls. Modified Rankin score (mRS) defined the clinical status. The concentrations of the chemokines (C-X-C motif ligand (CXCL)10, CXCL8 and C-C motif ligand 2 (CCL2)) and the cytokines (interferon (IFN)γ, interleukin (IL)4, IL7, IL15, IL17A and tumour necrosis factor (TNF)α) were measured with Luminex multiple bead technology. The B cell-activating factor (BAFF) and CXCL13 concentrations were determined via enzyme-linked immunosorbent assay. We correlated the disease period with the mRS, pleocytosis and the levels of all of the investigated chemokines and cytokines. Non-parametric tests were used, a The increased CXCL10 and CXCL13 CSF levels accompanied early-stage disease progression and pleocytosis. The CSF CXCL10 and CXCL13 levels were the highest in the most complicated patient. The CSF BAFF levels remained unchanged through the periods. In contrast, the CSF levels of T cell-related cytokines  and IL15 were slightly increased at all of the periods examined. No dynamic changes in chemokine and cytokine levels were observed in the peripheral blood.Our data support the hypothesis that anti-NMDAR encephalitis is restricted to the CNS and that chemoattraction of immune cells dominates at its early stage. Furthermore, our findings raise the question of whether T cells are involved in this disease.The online version of this article (doi:10.1186/s12974-016-0507-9) contains supplementary material, which is available to authorized users. N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune disorder of the central nervous system (CNS) that predominantly affects young females [Anti- females . The pro females . This cl females . Subsequ females .The immunopathogenesis of this disease has yet to be fully elucidated, although B cells, but not T cells, have been proposed to be involved . Based oTo contribute to the current knowledge regarding the immune mechanisms that participate in anti-NMDAR encephalitis, we studied the complex system of chemokines and cytokines in CSF and sera from anti-NMDAR encephalitis patients at different time-points of the disease. Chemokines and cytokines are produced by various cell types and reflect complex immune processes. Chemokines attract immune cells to sites of action and, thus, might be responsible for the initial pleocytosis. We selected the following chemokines: C-C motif ligand 2 (CCL2) for monocytes, C-X-C motif ligand 8 (CXCL8) for neutrophils, CXCL10 for T cells and CXCL13 for B cells. B cell-activating factor (BAFF) was measured as a critical marker of B cell activation and survival. Based on the understanding that T helper (Th) cells might support B cell functions , we measNine Caucasian patients  with non-paraneoplastic anti-NMDAR encephalitis were included in this study. The patients were diagnosed and/or treated at the Motol University Hospital, Prague, Czech Republic.The patients’ CSF and sera were positive for anti-NMDAR antibodies at the time of diagnosis. Their clinical status was defined using the modified Rankin score (mRS). Magnetic resonance imaging of the brain, electrophysiological studies and oncological assessments were performed upon disease diagnosis, and the oncological assessments were repeated every 6 months. Seven of the nine patients underwent more than one CSF and serum withdrawal during the follow-up period  Fig. .Fig. 1Stn = 5), visual impairment (n = 2), anxiety (n = 2) or tics (n = 1). The findings in CSF and sera from the controls were within the normal range, and no anti-NMDAR antibodies were detected.The control group consisted of ten Caucasian patients  who underwent a single CSF and serum withdrawal due to the following symptoms: headache ; period 2  and period 3 . Stratification of the samples according to the mRS and the laboratory results is shown in Fig. In total, 27 paired CSF/serum samples from nine patients and ten paired CSF/serum samples from ten controls were tested. The patient samples were stratified into three periods after the appearance of the first disease symptom: Anti-NMDAR antibodies in CSF and sera were detected using commercial kits . This cell-based assay enabled the qualitative detection of anti-NMDAR antibodies in the samples; quantitative analysis was not performed.The routine analysis of CSF included determinations of the following: cell count, protein levels, CSF/serum albumin ratio, immunoglobulin (Ig)G and IgM levels and oligoclonal band levels.Aliquots of centrifuged CSF and serum samples were immediately stored at −20 °C and thawed once before use for chemokine and cytokine analysis.The concentrations of the chemokines CCL2, CXCL8 and CXCL10 and the cytokines IFNγ, IL2, IL4, IL7, IL15, IL17A and TNFα were measured with Luminex multiple bead technology according to the manufacturer’s instructions . The data were collected using the Luminex-100 system . The BAFF and CXCL13 concentrations were determined via enzyme-linked immunosorbent assay according to the manufacturer’s instructions using software from R&D Systems .P value <0.05 was considered to be significant.Statistical analyses were performed using GraphPad PRISM, version 6.0 . Non-parametric tests were used. The Wilcoxon signed-rank test was used for pairwise comparisons of the CSF and sera samples. The Kruskal-Wallis test was performed to compare multiple groups of samples, and Dunn’s multiple comparisons test was employed for post hoc analysis. Patient no. 1 provided multiple samples at different time-points in each period, and we used the average values in each period for multiple-group comparisons. The correlations between the parameters were calculated using the Spearman correlation. A The clinical peak of the disease manifested within 30 days of the first disease symptom onset in all of the patients . Nevertheless, the disease severity varied among the patients. Notably, patient nos. 1 and 6 differed markedly from the others Fig. . Data frP = 0.0006, Mann-Whitney U test). Clinical improvement continued into period 3 (median mRS = 1), although this result was not significant. Generally, the patients reached mRS ≤2 in a median time of 3 months (range 1–25 months).The recovery time lasted for months. The mRS significantly decreased in period 2 (median mRS = 2) compared to period 1 . The patients were treated as recommended . None ofWe analysed the complex system of chemokines and cytokines in patient CSF and serum samples at three previously defined periods compared to corresponding control samples. Samples from period 1 were obtained from 6/8 treatment naïve patients. The remaining samples from period 1 were obtained from patients who responded insufficiently to the first-line immunotherapy, and the second-line treatment was planned (nos. 8 and 9). Samples in periods 2 and 3 were from treated patients. Only one sample in period 3 was from treatment naïve patient (no. 6). Marked changes in the levels of chemokines and cytokines were observed in the CNS compartment but not in the peripheral blood Fig. –g. ImporThe CSF CXCL10 and CXCL13 concentrations were significantly increased during period 1 and the CXCL10 level remained significantly increased during period 2 in the patients compared with the controls Fig. , b. ThisINFγ, TNFα and IL17A: The CSF levels of these Th cell-related cytokines were significantly increased at all periods in the patients compared with the controls .ols Fig. –e. IL7 aols Fig. . The CSFols Fig. . BAFF, Ir = −0.41, P = 0.04) and the CSF leukocyte numbers  and chemokine levels  were observed. Additionally, CSF pleocytosis correlated with the chemokine levels . Time did not correlate with the levels of B or T cell-related cytokines .We correlated the disease period with the clinical status (mRS), pleocytosis and the levels of all of the investigated chemokines and cytokines. Correlations between time and the mRS . In particular, pleocytosis progression  and markedly increased levels of all of the investigated chemokines and cytokines were observed in the first two samples. Interestingly, the increase in the CXCL10 levels preceded the increase in the CXCL13 and BAFF levels, and these concentrations were the highest among all patients. Pleocytosis was not observed, and the chemokine and BAFF levels were not increased in the five subsequent samples that were collected during her coma Fig. , c. In cImmune mechanisms in anti-NMDAR encephalitis are currently being intensively studied. Analysis of chemokine and cytokine levels in a clinical context can contribute to a better understanding of the immune processes this disease. Our study was focused on the complex spectrum of chemokines and cytokines related to B and T cell functions and their changes over the different clinical courses of anti-NMDAR encephalitis. To our knowledge, no other cytokine study in this disease has been published except the one by Leypoldt and his colleagues .The role of B cells in anti-NMDAR encephalitis has already been demonstrated , 12, 13;We showed marked changes in the CSF levels of T and B cell-related chemokines (CXCL10 and CXCL13). The significantly increased levels of these chemokines were observed at the early stage of the disease when the clinical symptoms were the most prominent (period 1, Fig. CXCL13 was recently proposed as a biomarker of the treatment response in anti-NMDAR encephalitis . Our finB cells are expected to thrive in the CNS compartment due to the BAFF levels , which wFurther, we hypothesized that T cells could be involved in the disease immunopathogenesis. It was recently reported that Th17 cells appear to be more efficient in supporting B cell responses outside the germinal centres and in promoting antibody production than their Th1 counterparts based on animal models . IL17A wOur investigation of chemokines and cytokines in the CSF and sera of anti-NMDAR encephalitis patients supported the hypothesis that the disease immunopathology is restricted to the CNS compartment. The dynamics of CXCL10 and CXCL13 CSF levels further supported the hypothesis that chemoattraction of the immune cells dominates at the early stage of the disease. Our findings regarding CXCL10, IFNγ, TNFα, IL17A and IL15 raise the question of possible involvement of T cells in this disease. The role of these cytokines in anti-NMDAR encephalitis remains to be elucidated.


There is a lack of evidence on the relationship between prematurity and palatal abnormalities. The aim of this study was to evaluate the incidence of palatal groove, the associated factors and the persistence time in preterm infants from birth to 24 months of age.The children’s data, medical history and eating habits were collected using a questionnaire answered by the legal guardian and updated every dental visit. Natal and neonatal data were obtained from the medical records. During the orofacial examination, the presence or absence of a palatal groove was observed. In order to evaluate for associations between independent variables and the palatal groove, descriptive analyses and bivariate analyses were conducted using the Mann-Whitney, Pearson’s chi-squared and Fisher’s exact tests, when appropriate. The Poisson regression analysis was used to determine risk and protective factors for the occurrence of palatal groove. The significance level was 0.05. For the persistence of palatal groove, a survival analysis was used (Kaplan Meier method).n = 19 (25.7 %) and persisted for an average time of 12 months. Bivariate analysis showed a significantly higher occurrence of palatal groove in girls  as well as in infants that stayed longer in the neonatal intensive care unit (NICU) (median 37 days vs 20 days), that did not have exclusive breastfeeding (94.7 % vs 69.1 %), were intubated (median five days vs one day) or used an orogastric tube (median 33 days vs 15 days). The quantitative data for ‘NICU’, ‘intubation’ and ‘orogastric tube’ were correlated and estimated as risk factors for palatal groove formation in the unadjusted Poisson regression analysis.Seventy-four preterm infants were monitored. Palatal groove occurred in Palatal groove occur transiently in approximately one quarter of preterm infants, especially in infants that stay longer in the NICU, are intubated or use an orogastric tube. Preterm birth of children is a global problem that afflicts approximately 5 to 18 % of the population  in the oEtude Epidémiologique sur les Petits Ages Gestationnels (EPIPAGE) preterm infant cohort study showed that, at five years of age, 3.7 % of children  had some alteration to their palate morphology [A secondary analysis of the There is no accurate definition of a ‘normal’ palate in an infant, but a groove or sulci system printed on the alveolar ridge is the distinctive feature of the infant palate . The litNevertheless, the aforementioned studies –11 are oThis cohort study was approved by the Institutional Research Ethics Board (protocol #144/2011), and one of the parents of each child signed the informed and free consent form agreeing to participate in the study.The monitoring of the children was performed in a university hospital in the mid-west region of Brazil, which has a Neonatal Intensive Care Unit (NICU) that receives newborns both internally and from other hospitals. The population attended to this hospital is generally of low socioeconomic level. After discharge from the NICU, the children are monitored on an outpatient basis by the medical team up to two years of age. The intervals between follow-up visits are established as needed by medical recommendations. The clinic operates twice a week.For this study, all outpatient infants from that hospital service, born between February 2012 and December 2013 without facial deformities or congenital syndromes, were included, provided they were edentulous at their first dental care visit and their parents consented to participate. Children who could not be examined in the first dental care visit or did not attend any follow-up visits were excluded. The children were monitored between February 2012 and July 2014. The sample was non-probabilistic and consecutive. General data were obtained from birth, but the first dental assessment started when they began their follow-up consultations in the medical outpatient clinic.Consultations with the dental team were held on the same day as consults scheduled with the medical team. At the first dental visit, the guardian filled out a questionnaire addressing the data, medical history and feeding habits of the child. At the follow-up visits, the medical, dental and nutrition histories were updated. Additionally, data about the gestational age and the infant’s condition at birth  were obtained from the infant’s medical records. These data were obtained immediately after childbirth. Categorisation of the gestational ages was based on the criteria of the World Health Organization (WHO) : ExtremeAfterwards, orofacial examination of the infant was performed. Three trained dentists alternated for the exam in pairs, so they reached a consensus on the diagnosis of palatal groove. The dentists were not blinded to the children’s medical history. Training of examiners to evaluate the persistence of the palatal groove consisted of a theoretical study on the diagnostic criteria based on slides with examples of different shaped grooves. Two out of the three examiners performed the oral assessment simultaneously. The dentist placed the child in a knee-to-knee position with the parent and examined its oral cavity with the aid of artificial light (headlamp) and a mouth mirror. When examining the palate, the presence or absence of a palatal groove was visually observed, using the description of Erenberg and Nowak . The obsThe sample size was estimated based on a 25.0 % incidence of palatal groove in intubated preterm infants, compared with zero cases in non-intubated term infants . Thus, fThe data were analysed using IBM SPSS version 19. The quantitative variables showed non-normal distribution (except for birth weight). After descriptive analysis, a bivariate association between the independent variables and the incidence of palatal groove was sought, using the Mann-Whitney, Pearson’s chi-squared and Fisher’s exact tests, as appropriate. The correlations among quantitative variables were estimated with Spearman’s correlation test. The variables that were significantly associated with the occurrence of palatal groove in the bivariate analysis were included in the Poisson regression to identify risk and protective factors for the incidence of palatal groove in children born prematurely. The significance level was 0.05.To evaluate the persistence of the palatal groove, survival analysis was used (Kaplan Meier method) in the software Prisma GraphPad. Based on the follow-up sessions, the time in months needed for disappearance of the palatal groove was recorded. The excluded cases were children who did not have the palatal groove as of the first consultation or who were not monitored long enough to observe the disappearance of this alteration.A total of 177 children were evaluated for eligibility. Among them, 89 children were included. Of these children, fifteen were excluded due to the following criteria: five because it was not possible to complete the dental examination at the first visit and observe the presence or absence of the palatal groove, and ten because they did not come to follow-up visits. The results of this study thus refer to a cohort of 74 preterm infants.The infants attended a median of six consultations (interquartile range = 4), with a median follow-up period of 11 months (interquartile range = 4). Other characteristics of the participants are described in Table At the first examination, the occurrence of a palatal groove was observed in 19 infants 25.7 %). In the bivariate analysis, the incidence of a palatal groove was significantly associated with the female sex, a longer stay in the NICU, intubation (higher frequency and duration), a longer use of an orogastric tube and the absence of exclusive breastfeeding  and days using an orogastric tube . The number of days intubated also correlated with days using an orogastric tube . So, an adjusted model was not estimated because of the significant correlations among these variables.In the Poisson regression analysis, the odds ratio values were individually calculated for the variables that showed ve Table . As onlyP > 0.05). Disappearance of the groove was not observed in two of the 19 children. One was monitored until six months of age and the other until eight months of age.The palatal groove persisted for a median of 12 months (Fig. The most significant finding of this longitudinal study was that palatal groove occurs transiently in approximately one quarter of preterm infants. The palatal groove especially affects infants that stay longer in the NICU, are intubated or use an orogastric tube. Thus, the clinical implication of this finding is that the palatal groove cannot be held responsible for morphological deficiencies of the palate that could result in malocclusions in deciduous, mixed or permanent dentition.Surprisingly, in the bivariate analysis female children were more likely to have a palatal groove. In the EPIPAGE study, boys had more palatal alterations , but ourEach additional day in the NICU increased the chance of a child having a palatal groove by 1 %. In fact, it is understood that admission to the NICU is an indicator of the severity of the preterm condition, as are intubation and use of an orogastric tube. Therefore, our results showed that intubation was associated with the occurrence of palatal groove: each additional day of intubation increased the risk of children having a palatal groove by 4 %. Intubation has been reported as a cause of oral deformities , 11, as In this study, each additional day of orogastric tube use increased the risk of palatal groove development by 2 %. It is speculated that orotracheal and orogastric tubes can put pressure on the alveolar crest and the median region of the palate, negatively interfering with palatal growth and the “descent” of the jaw . HoweverIn this study, feeding was inversely associated with the occurrence of palatal groove in the bivariate analysis, but in the unadjusted regression it was not confirmed as a significant protection factor. Consequently, this association should be interpreted carefully. It is likely that more severely affected children did not breastfeed. So, along with a more consistent inclusion of the mode of feeding in future studies on palatal morphology of preterm infants , this reAlthough infants with a median age of 14 months were monitored, our findings show that the palatal groove does not persist for long, although it was previously reported that palatal groove persist in children for two to 10 years of age , 8. In aOne of the limitations of this study is that we did not have access to the oral conditions immediately after extubation and/or discharge, which could help in identifying the occurrence of an eventual palatal groove that may have disappeared by the first dental consultation. However, as the median age at the beginning of monitoring was three months, and most of the infants stayed in the NICU for a median time of approximately one month, it is believed that this factor did not negatively affect the results.Another point to be discussed is the palatal groove diagnosis, which in this study was strictly visual, while other studies used models and other measuring devices . HoweverThus, the results of this study suggest that the occurrence of the palatal groove in infants is transient and associated with local traumatic factors, which most likely lead to growth of the oral soft tissues and “printing” of the tube on the palate. In any event, additional studies in infants are needed to investigate whether other changes in palate morphology involve environmental or genetic factors.Approximately one quarter of preterm infants had a palatal groove, which persisted for a median time of 12 months. The factors associated with the incidence of palatal groove were longer periods in NICU, intubation or use of orogastric tubes.


Clostridium difficile infection (CDI) that often fails standard antibiotic therapy. Despite its widespread recent use, however, little is known about the stability of the fecal microbiota following FMT.Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Here we report on short- and long-term changes and provide kinetic visualization of fecal microbiota composition in patients with multiply recurrent CDI that were refractory to antibiotic therapy and treated using FMT. Fecal samples were collected from four patients before and up to 151 days after FMT, with daily collections until 28 days and weekly collections until 84 days post-FMT. The composition of fecal bacteria was characterized using high throughput 16S rRNA gene sequence analysis, compared to microbiota across body sites in the Human Microbiome Project (HMP) database, and visualized in a movie-like, kinetic format. FMT resulted in rapid normalization of bacterial fecal sample composition from a markedly dysbiotic state to one representative of normal fecal microbiota. While the microbiome appeared most similar to the donor implant material 1 day post-FMT, the composition diverged variably at later time points. The donor microbiota composition also varied over time. However, both post-FMT and donor samples remained within the larger cloud of fecal microbiota characterized as healthy by the HMP.Dynamic behavior is an intrinsic property of normal fecal microbiota and should be accounted for in comparing microbial communities among normal individuals and those with disease states. This also suggests that more frequent sample analyses are needed in order to properly assess success of FMT procedures.The online version of this article (doi:10.1186/s40168-015-0070-0) contains supplementary material, which is available to authorized users. Clostridium difficile infection (CDI) that cannot be cured with antibiotics alone [Fecal microbiota transplantation (FMT) has emerged in recent years as a highly effective treatment for refractory cs alone . The procs alone -8. Howevcs alone . This tycs alone -12. In acs alone -17. TherHere we describe both short- and long-term dynamic changes of fecal bacterial composition in four patients following FMT. All patients received microbiota from the same pre-qualified donor according to the standardized FMT protocol we described previously . Three pFour patients (CD1 to CD4) with recurrent CDI were treated with FMT using material obtained from a single donor but from different time points, and fecal samples were collected from these patients before and after the procedure as well as from the donor at times of donation. Bacterial communities from these fecal samples were characterized by sequencing the V4 region of the 16S rRNA gene. Following trimming and quality filtering from a total of 12,536,492 sequences, we randomly subsampled to 5,000 sequences/sample in order to normalize read depth across all samples. All further analyses were performed using this rarefied read depth.To better understand changes in bacterial communities following FMT, we compared the bacterial composition of patient fecal samples to those of microbial communities from various body sites from the 252 healthy individuals characterized in the Human Microbiota Project (HMP)  Figure  using unTo more closely examine temporal changes in recipient fecal samples following FMT, we analyzed fecal microbial communities from patients CD1 to CD4 and donor, as well as from 10 additional donor samples, using weighted and unweighted UniFrac  followedvia weighted UniFrac and PCoA  prior to FMT, and rapidly fell below the upper control limit within 1 to 2 days after the procedure .We focused on these changes by examining the relative abundance of e Figure . In addiNext, we compared fecal microbial communities within each patient over time to that of the initial donor sample. We generated heat maps based on Pearson correlations between every sample within a given patient set, including respective donor samples and samples from 10 additional pre-FMT patients Figure A. This avia both Pearson and Spearman analyses in all patients. After the initial time point after FMT, the Pearson correlation values of patient to donor samples were highly variable within and across patients, although Spearman correlations remained high for three patients. To examine whether this variation is similar in healthy individuals, we determined Pearson and Spearman correlations within the four donor samples used in FMT, as well as eight additional donor samples from the same individual as a control. Results of this analysis revealed that donor microbiota also changed over time . Normalization refers to the mean between-sample distance for each set of patient samples versus the set of donor samples, while dynamic range is the mean distance between each sample within a single patient set. Effectively, the normality of a post-FMT patient sample set is a measure of how similar it is to the donor  sample set, while dynamic range is a measure of variability within a given patient sample set. We found that neither the normalization nor the dynamic range of any post-FMT patient sample set was significantly different than the donor set following analysis using unweighted UniFrac , and FMT is associated with reemergence of dominance by members of the Bacteroidetes and Firmicutes phyla.It is now well understood that the fecal microbiota change substantially following FMT, typically shifting to fecal microbial communities more similar to those of the donor after transplant -8. Here Analysis of multiple donor and post-FMT samples demonstrates the dynamic behavior of fecal microbial communities over time. Both donor and recipient samples are characterized by highly dynamic shifts that nonetheless remain within the compositional range of normal fecal microbiota. This observation is consistent with known rapid responsiveness of the fecal microbiome to environmental inputs, such as dietary variations , and driThe dynamic nature of intestinal microbiota is an intrinsic property, which should be taken into account when considering how therapeutic interventions, including FMT, impact its composition over time. In long-term post-FMT follow-up, Song and colleagues also noted dynamic changes in the fecal microbiome of R-CDI patients up to 16 weeks post-FMT . These iThree of the recipients in this study received freshly prepared microbiota, while one received frozen/thawed preparation. Use of frozen microbiota preparations is increasing in clinical practice , and itsThe patients in this study did not have any significant gastrointestinal comorbidities. However, a significant proportion of patients with recurrent CDI have underlying inflammatory bowel disease, take potent immunosuppressive medications, or have multiple other medical problems ,24. The The fecal microbiota of patients with R-CDI continues to undergo change after FMT is performed, though these changes appear to fall within the range of normal variation of healthy individuals over time. Dynamic behavior is an intrinsic property of normal fecal microbiota and should be accounted for in comparing microbial communities among normal individuals and those with disease states.All patients suffered from multiply recurrent CDI refractory to standard antibiotic therapies. A single standard donor was used in the preparation of all fecal microbiota material as described previously . The Insvia colonoscopy as previously described [via the biopsy channel of the colonoscope. A total of 17 donor samples from the same individual were used in these studies. The CD1 to CD4 donor samples were given to patients CD1 to CD4, respectively. Patients CD1, CD3, and CD4 received freshly prepared fecal microbiota, while patient CD2 received a previously frozen preparation of fecal microbiota, all from the same standardized, anonymous donor.FMT was done using a standardized preparation of concentrated fresh or frozen fecal bacteria escribed . All patescribed . The dayFecal samples were collected at home by the patients using swabs to sample feces deposited into a toilet hat immediately after production and stored frozen at approximately −20°C. Samples were subsequently transferred to the laboratory and stored at −80°C until used. Donor samples for DNA extraction were collected during processing of material for FMT and stored frozen at −80°C until used. Samples from patients CD1 to CD4 were obtained prior to FMT and between 1 to 151 days post-FMT, with daily collection until day 28, and weekly collection until day 84. Fecal material prior to FMT was obtained from patients CD5 to CD14.DNA was extracted from donor and recipients’ pre- and post-FMT fecal samples using MOBIO PowerSoil DNA extraction kits , according to the manufacturer’s instructions. Fecal DNA concentrations were measured using a QuBit DNA quantification system .http://www.earthmicrobiome.org/ following the recommendations of Caporaso et al. [Extracted DNA was amplified using the EMP standard protocols at o et al. . Brieflyo et al. .DNA sequencing was performed as previously described  on an Ilet al., manuscript in preparation, https://github.com/PitchInteractiveInc/Phinch).Sequence data was processed and analyzed using QIIME  accordinn principal coordinates axes required to explain at least 80% of the variation in the distance matrix. An 80% cutoff was chosen to balance bias and overfitting. Distances were recalculated using Euclidean distances between points in PCoA space in order to convert PCoA coordinates to a distance matrix. The empirical P values for the ‘normality’ were obtained by comparing the mean distance between patient and donor samples to the histogram of within-donor distances . The empirical P values for the ‘dynamic range’ (stability) were obtained by comparing the mean distance within patient samples to the histogram of within-donor distances. These analyses were also performed using alternative parameters including, weighted UniFrac, Jensen-Shannon, root Jensen-Shannon, and Bray-Curtis.For each set of post-transplant patient samples, we assessed the similarity of that set to the set of reference samples from the donor . To reduce noise and compare patient samples along only relevant dimensions in UniFrac distance space, we applied PCoA to the unweighted UniFrac distance matrix containing only the post-transplant and donor samples for that donor-patient pair, then recalculated the distances using only the first Approval for this study was given by the University of Minnesota Institutional Review Board (Protocol Number: 0901M56962). All human subjects provided informed consent for participation in the study and collection and analysis of data. All human subjects gave their permission for their information to be published.


Streptococcus pneumoniae, is often associated with HIV co-infection and mortality rates are double those seen in better resourced settings.Bacterial meningitis in sub-Saharan Africa is predominantly caused by To investigate the cause of this excessive mortality we quantified the pneumococcal DNA load and six common pro-inflammatory cytokines in the cerebrospinal fluid (CSF) of Malawian adults with culture proven pneumococcal meningitis and correlated the results to clinical parameters and outcome. There are currently no published data relating bacterial load to outcome in adults with pneumococcal meningitis.5 copies/ml CSF) and there was no significant variation in bacterial load between survivors and non-survivors. All pro-inflammatory CSF cytokines were elevated in the CSF, with no clinically important differences between survivors and non-survivors. HIV status did not affect the CSF bacterial load or cytokine response.The mean age of patients was 32 years, 82% were HIV infected and 49% had died by day 40. CSF bacterial loads were high (median 6.5 × 10Mortality from pneumococcal meningitis in adults in sub-Saharan Africa is not related to pneumococcal bacterial load. More research is needed to understand the very high mortality from meningitis in this region. •Pneumococcal meningitis in Malawian adults has a very high mortality rate.•Bacterial load and cytokine response may predict outcome in children.•Pneumococcal load and cytokine response were not associated with outcome in adults.•Low CSF white cell count and Glasgow coma score were associated with mortality.•Anti-cytokine therapies are unlikely to be effective in bacterial meningitis. Streptococcus pneumoniae bacterial load in the cerebrospinal fluid (CSF) has been associated with increased mortality in children with meningitis in Malawi and Finland, higher bacterial loads in the blood of adults with pneumococcal sepsis in Europe are also associated with poor outcome.The incidence of pneumococcal meningitis in adults is estimated to be 0.1–1/100,000 in well-resourced countries3 with >50% neutrophils),www.mlw.medcol.mw. Diagnostic CSF was cultured on blood agar at 37 °C in 5% CO2. S. pneumoniae was identified by standard methods including optochin sensitivity and alpha haemolysis. Only culture positive samples for S. pneumoniae were included in this study, molecular diagnostics using PCR were not available. Treatment of pneumococcal meningitis was ceftriaxone 2 g twice daily for 10 days. A second CSF sample was taken 48 h post antibiotic therapy in a sub-set of patients, some of whom were treated with intramuscular as opposed to intravenous ceftriaxone as per protocol of the dexamethasone clinical trial. Poor outcome was defined as death by 6 weeks of follow up. Morbidity data were not available.Patients aged >16 years with bacterial meningitis were recruited into one of two sequential randomised placebo controlled clinical trials of adjuvant therapy between 2001 and 2004  or 2006–2008 LytA) as the amplification target.S. pneumoniae serotype 23F (P833), and quantified using the NanoDrop ND-1000.DNA was extracted from 200 μl of pneumococcal culture positive CSF supernatant and Real-Time PCR was performed as described previously using autolysin . Six bead populations with differing 650 nm fluorescence intensities were coated with cytokine specific capture antibodies and incubated with flourochrome (phycoerythrin – 585 nm) according to the manufacturer's instructions. The resulting sandwich complexes were resolved in the FACScan flow cytometer and the output analysed using manufacturer's software.Participants or accompanying legal guardians gave written informed consent for CSF samples to be stored and used for research studies. Both constituent studies were granted ethical approval for sample collection and analysis by the Liverpool School of Tropical Medicine Research Ethics Committee and the College of Medicine Research Ethics Committee, University of Malawi.t-test was used to analyse normally distributed variables. Categorical variables were analysed using Fisher's exact test. A multivariate model was generated using binary logistic regression, enter mode, and cross checked using backwards LR mode. Clinical variables entered into the multivariate model were chosen based on previous association with poor outcome.Statistical analysis was done using IBM SPSS statistics version 20. Graphs were generated using GraphPad PRISM version 5. Median (inter-quartile range) was used to summarise non-normally distributed variables, while mean (SD) was used to summarise normally distributed variables. Statistical tests were 2 tailed, non-parametric tests were used to analyse data that were not normally distributed. A Data were collected from 151 patients with stored CSF samples and paired clinical data. Data were available for all patients to day 10; day 40 outcome data were missing for 3 patients who were lost to clinical follow up.3 (IQR 181–2600) with significantly higher WCCs in survivors compared to non-survivors p = 0.02 on univariate analysis (5 copies/ml (IQR 1.08 × 105–2.96 × 106) in the admission samples and 2.96 × 104 copies/ml (IQR 3.8 × 103–2.12 × 105) in the CSF samples taken 48 h post antibiotics  was 0.49 .Baseline characteristics of the included patients are shown in analysis . The medibiotics a. There n = 18) to combine into a single model. No statistically significant mortality association was demonstrated for the CSF bacterial load or CSF white cell count, HIV status, age or gender on model 1 (n = 102); seizures at any time in the illness, GCS or altered mental status and anaemia were associated with mortality (n = 62) IL8 and IL10 were marginal predictors of non-survival; IL8 p = 0.036, OR 1.00 (95% CI 1.00: 1.00) and IL10 p = 0.029, OR 1.00 (95% CI 1.00 : 1.00); of the clinical parameters, only altered mental status or GCS retained significance in this model  were significantly higher only in HIV co-infected non-survivors as compared to HIV co-infected survivors.S. pneumoniae than excessive virulence of the pathogen.13We have previously reported results of a major proteomic analysis of Malawian adults with bacterial meningitis.Limitations exist within our data. Firstly, the small numbers of patients with both CSF genomic load and cytokine data available precluded a definitive analysis of bacterial load and cytokine levels in the same statistical model. Secondly, data for sequelae in survivors were not available, the different scoring systems used in the included studies meant significant heterogeneity in the outcome data precluded analysis of the impact of bacterial load or cytokine response on morbidity from meningitis.In conclusion, poor outcome from pneumococcal meningitis in Malawi is likely to be multifactorial and our data suggest that anti-cytokine adjunctive treatments in sub-Saharan Africa are unlikely to be effective. Alternative strategies such as pneumococcal vaccination in HIV infected adults, reducing pre-hospital delays to treatment, optimising in-hospital care, investigating alternative adjunctive treatments targeting pneumococcal toxins and optimising macrophage phagocytosis10.13039/100004440Wellcome Trust (CDF 061231 and 089671/B/09/Z)  and NIHR Biomedical Research funding to SG. The cytokine analysis was funded by the Wellcome Trust (Research fellowship to SBG). The steroid and glycerol adjunctive therapy studies were funded by the 10.13039/501100000403Meningitis Research Foundation. Neither the funding bodies nor the trial sponsors had any role in the laboratory work, data analysis, manuscript preparation or decision to publish.The bacterial load work was funded by the The authors declare no conflicts of interest.


Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased . Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders. GBA1 gene, which codes for the lysosomal glycohydrolase β-glucocerebrosidase (EC = 3.2.1.45), cause Gaucher's disease (6). Several reports have demonstrated that GBA1 mutations are an important risk factor for PD . Either the inhibition or down-regulation of β-glucocerebrosidase causes α-synuclein accumulation in cultured cells along with mitochondrial dysfunction (11). A self-propagating loop in which mutated β-glucocerebrosidase induces α-synuclein accumulation within the lysosomes that subsequently compromises lysosomal protein degradation has been postulated (12). As a further demonstration of a link between β-glucocerebrosidase deficiency and PD, decreased β-glucocerebrosidase activity has been described in the cerebellum and substantia nigra of PD patients, even in those without GBA1 mutations (13). Alterations in lysosomal chaperone-mediated autophagy have been demonstrated to cause the reduction of β-glucocerebrosidase protein levels and activity in the brain of early stage PD patients, especially in brain regions showing increased α-synuclein levels (14). β-glucocerebrosidase activity has been found to be decreased in the CSF of PD patients compared to individuals affected by other neurological disorders (OND) (15). In another study that assessed the CSF β-glucocerebrosidase activity in patients with different dementia disorders, including synucleinopathy (dementia with Lewy bodies) and tauopathies , a selective decrease was observed in patients diagnosed with dementia with Lewy bodies, while α-mannosidase (EC = 3.2.1.24) showed a marked decrease in all of the groups studied (16). Recently, increased β-galactosidase (EC = 3.2.1.23) and cathepsin E (EC = 3.4.23.34) activities and a reduced α-fucosidase (EC = 3.2.1.51) activity have been found in the CSF of PD patients compared to healthy controls, and β-glucocerebrosidase showed a trend toward reduction in the PD group (17). Most recently, significantly reduced β-glucocerebrosidase activity was found in a cohort of 71 PD patients compared to 45 neurological control patients. Interestingly, the β-glucocerebrosidase activity reduction was more pronounced in patients with an early stage of the disease (18).Lysosomes are involved in the degradation of macromolecules, and the impairment of their functionality due to a genetic lack of acidic hydrolases is the cause of lysosomal storage diseases (1). In recent years, lysosomal dysfunction has also been shown in patients with neurodegenerative disorders, i.e., Parkinson's disease (PD)  and Alzheimer's disease (AD) , where the accumulation of undegraded and misfolded proteins occurs. Mutations in the de novo vs treated PD patients and healthy volunteers vs neurological patients as control groups) and in pre-analytical confounding factors, i.e., different conditions for the collection and storage of the CSF samples. Differences in the CSF handling and analytical procedures should play a negligible role in the total variability of these assays because all of the previously cited CSF studies were performed in a single laboratory.The reasons for these different outcomes may be found both in the heterogeneity of the diagnostic groups studied , α-fucosidase, β-mannosidase (EC = 3.2.1.25), α-mannosidase, β-galactosidase and β-glucocerebrosidase, and two proteases, cathepsin D (EC = 3.4.23.5) and cathepsin E.Twenty-eight subjects were included in this study. Twelve of the subjects were diagnosed as having PD according to the United Kingdom Parkinson's Disease Society Brain Bank criteria (19), whereas the remaining 16 patients were affected by other minor neurological disorders (OND) requiring lumbar puncture (LP) for diagnostic purposes . The demographic and clinical data are reported in The study was approved by the ethical committee of the Umbria region. None of the subjects included in the study presented with neurological deterioration. LP was performed on subjects in the OND group for diagnostic purposes. Patients were included only if they were able to understand the study aim and procedures. Written informed consent was obtained from all of the subjects included in this study.The LPs were performed between 8:00 and 10:00 AM. The CSF samples (10 ml) were collected in sterile polypropylene tubes, centrifuged for 10 minutes at 2,000×g, and stored in aliquots under the various conditions described below.The activities of the CSF lysosomal enzymes were determined with the corresponding fluorigenic substrate and reaction buffer . To meas2 as a substrate dissolved in 50 mM sodium acetate buffer with a pH = 4.0. The samples were incubated with the substrate at a concentration of 30 µM for 1 h at 40°C in the presence and absence of the cathepsin D inhibitor pepstatin A (15 µM). The reactions were stopped by adding 5% (w/v) trichloroacetic acid to a final volume of 3 ml and were measured with a Jasco FP-750 spectrofluorometer at an excitation wavelength of 328 nm and an emission wavelength of 393 nm. The value obtained in the presence of pepstatin A corresponded to the cathepsin E activity. The cathepsin D activity was calculated by subtracting the value obtained in the presence of pepstatin A from that measured in the absence of the inhibitor.The cathepsin D and cathepsin E activities were determined using MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NHThe standard curves have been created using serial dilutions of the fluorochromes 4-methylumbelliferone and Mca-Pro-Leu-OH peptide (for cathepsins). In order to minimize the standard curves variability, they have been created by the same operator using the same stock reagents solutions. Moreover, substrates were prepared and frozen into aliquots, so that in every assay the substrate was used after one freeze/thaw cycle.One unit (U) of enzyme activity was defined as the amount of enzyme that hydrolyses 1 µmol of substrate/min at 37°C. All of the activities were measured in triplicate. All of the samples included in this study were assayed for the activities of the lysosomal enzymes within 30 min from the collection (fresh CSF). The remaining volume of each sample was used to assess the confounding factors.The statistical analysis was carried out with R v.2.15 (20). The descriptive statistics are presented as the mean ± SD for the continuous variables and as percentages for the categorical variables. To analyze the variability of the enzymes' activities, mixed-effects regression models were used for taking into account the repeated measurements of samples between storage techniques, freeze-thaw cycles and time points (21). The statistically significant level is considered as p<0.05.Analysis of the effect of the diagnosis on pre-analytical factors has been performed, showing no significant differences between OND and PD groups for the measured enzyme activities (data not shown). Therefore, the results are presented considering the whole group of samples independently from the diagnosis.Eight CSF samples were flash-frozen in liquid nitrogen in ten separate aliquots. One aliquot was thawed and used for enzymatic assays each day for ten consecutive days. Each enzyme was tested in duplicate while each sample was measured in triplicate. The within- and between-run assay coefficients of variation (CVs) were calculated according to guideline EP5-A2 from the National Committee for Clinical Laboratory Standards (NCCLS) (22). As an internal control, a pooled CSF sample was run in each experiment to ensure that the measured samples were within an acceptable range, which was defined as ±2 times the standard deviation (SD) from five consecutive measurements of the same pool.The values of the within- and between-run CV for each enzymatic assay are reported in A previous study found an increased stability in the activities of lysosomal enzymes when the CSF was frozen in liquid nitrogen (23). To test which freezing conditions should be recommended for the measurement of the activities of lysosomal enzymes, three conditions were compared: flash freezing in liquid nitrogen, direct freezing at −80°C and direct freezing at −20°C. Twelve CSF samples were aliquoted, placed on ice and frozen within 30 min after the lumbar puncture by placing them at −20°C, −80°C or in liquid nitrogen and then stored at −80°C. Within 3 days, the aliquots were thawed on ice and assayed.Analysis of the variance among the three conditions tested showed that only the α-mannosidase activity was significantly reduced when the sample was frozen at −20°C compared to the fresh sample (p<0.001), flash freezing the sample in liquid nitrogen and directly freezing the sample at −80°C (p<0.01). The activities of all of the other enzymes showed no significant differences among the three freezing conditions .Several pre-storage conditions, resembling the most common procedures for CSF storage before freezing, were tested and compared to the respective fresh CSF sample. Twelve CSF samples were divided into aliquots, which were kept at 4°C or RT (22°C). One aliquot of each group was flash-frozen after 1, 4, 24, and 48 hours and stored at −80°C. After 1 week from the last freezing, all the aliquots were thawed on ice and used to determine the activities of the lysosomal enzymes. The activity of each enzyme in each sample was compared to that of the respective fresh sample.Five CSF samples were flash frozen in liquid nitrogen and stored at −80°C. The samples were thawed in ice altogether, assayed for the enzyme activities and frozen again at −80°C. Five cycles were performed using this procedure.The mean percentage activity changes of the lysosomal enzymes for up to 5 freeze and thaw (F/T) cycles with respect to fresh CSF are reported in To test if a traumatic LP had a direct influence on the activities of lysosomal enzymes in CSF, three CSF samples were spiked with the blood of the same patient to obtain the following scalar erythrocyte concentrations: 50,000, 5,000, 1,000, 500 and 250 erythrocytes/µl. The spiked CSF samples were assayed for the different enzyme activities, and the results were compared with the respective neat sample.Blood contamination of the CSF samples with up to 50,000 erythrocytes/µl did not significantly modify the activity levels of any of the lysosomal enzymes in a dosage-dependent way .Aliquots of 8 CSF samples were flash frozen and then stored at −20°C or −80°C. Four samples were used to assess the longitudinal stability of the enzymes at each temperature. One aliquot of each sample was thawed in ice and used for enzymatic assays after 1, 2, 3, 4, 8, 16, 32 and 40 weeks of storage. The differences between the enzyme activities of fresh CSF and the sample stored for 1 week were also calculated. To estimate the longitudinal stability of the activities of the enzymes, the samples stored for 1 week either at −20°C or −80°C were chosen as reference samples, and the percentage difference between each time point and the respective reference sample was calculated.Initially, the change in the activity percentage between fresh CSF and the same sample stored for 1 week at -80°C was calculated for each enzyme. The results are reported in The tested activities had different degrees of variability when assayed longitudinally. The potential diagnostic value of the activities of CSF lysosomal enzymes for different neurological diseases has been investigated since the end of the seventies . Nevertheless, due to the thermal instability of these enzymes in biological fluids, this measurement has never been considered for clinical utilization .A renewed interest in these enzymes has emerged as a consequence of recent evidence highlighting the link between the impairment of the lysosomal system and PD pathology . Interestingly, some recent studies have shown that the activities of some lysosomal enzymes in the CSF of patients with PD and other synucleinopathies is altered compared to control patients or patients with other neurodegenerative diseases . Accordingly, the opportunity to use CSF lysosomal enzymes as biomarkers for PD deserves attention .Here, we systematically evaluated a series of pre-analytical factors that may affect the measurement of the activities of some lysosomal enzymes in CSF. Our results show that the total variability of these assays is minimally influenced by the within- and between-run variability, which ensures day-to-day repeatability. Other analytical conditions affected each enzyme in a different fashion.The activities of some enzymes seem to be weakly influenced by pre-analytical factors. In particular, the activity levels of cathepsins D and E showed elevated resilience to F/T cycles, high longitudinal stability up to 40 weeks and no significant change when kept at 4°C for at least 24 hours before freezing. Accordingly, the measurement of their activities in CSF may represent a promising biomarker when also considering their possible involvement in the pathogenesis of PD and AD . To our knowledge, this is the first study assessing the longitudinal stability of the cathepsin D and E activities in CSF. The β-mannosidase activity was found to be stable when the CSF samples were kept at 4°C for up to 24 hours and then stored at −80°C for up to 40 weeks similarly to the cathepsins, but its activity has a marked fluctuation after F/T cycles. Among the other lysosomal enzymes, the α-mannosidase activity was altered by almost all of the pre-analytical factors tested. For instance, after only 1 week of storage the α-mannosidase activity showed a large decrease (∼20% at −80°C and ∼60% at −20°C), which confirmed the findings of a previous study (23). The β-hexosaminidase activity also showed marked fluctuations after F/T cycles. The β-galactosidase and α-fucosidase activities had similar inconsistencies when evaluated longitudinally. These changes could be explained by the presence of unstable enzymatic modulators in the CSF (36). Moreover, some enzymes are part of multi-enzyme complexes that could be disjointed during freezing steps, which could increase the affinity of each enzyme to the synthetic substrate (37).Our results also showed that the CSF β-glucocerebrosidase activity is influenced by pre-analytical factors. A 40% decrease of the CSF β-glucocerebrosidase activity was reported when the samples were directly frozen at −80°C with respect to samples snap-frozen in liquid nitrogen (23). In this study, we were not able to find significant difference in the CSF β-glucocerebrosidase activity between the two conditions after 1 week of storage (same time frame as in the previous study). Furthermore, at −80°C, β-glucocerebrosidase was stable for up to 32 weeks. These differences may be due to the time-delay and the storage temperature before freezing, which were not specified in the previous study and significantly affect the CSF β-glucocerebrosidase activity. Moreover, the two studies are not directly comparable because in Goi et al., the β-glucocerebrosidase enzymatic assay was performed in absence of TDC. Taurodeoxicolic acid enhances the specificity of the assay inactivating specific isoenzymes, as it has been shown in leucocytes (38).The final recommendations for the proper assessment of the activities of CSF lysosomal enzymes emerging from this work are: i) the CSF samples should be centrifuged and kept at 4°C immediately after the LP and stored at −80°C within 1 h of the collection; ii) the β-mannosidase, cathepsin D and cathepsin E activities should be tested within 40 weeks of the LP, and the GCase activity should be tested within 32 weeks; and iii) the number of F/T cycles do not significantly affect cathepsins D and E for up to four cycles, but they should be avoided for β-glucocerebrosidase and the other enzymes.In conclusion, we have shown that after applying the correct pre-analytical operating procedures, the measurement of β-mannosidase, β-glucocerebrosidase, cathepsin D and cathepsin E in CSF can be reliable and reproducible. These conditions are mandatory to correctly compare the results from different cohorts aimed at confirming the role of the activities of CSF lysosomal enzymes as potential biomarkers for neurodegenerative disorders, with a special interest on Parkinson's disease.


The porion (Po) is used to construct the Frankfort horizontal (FH) plane for cephalometrics, and the external auditory meatus (EAM) is to transfer and mount the dental model with facebow. The classical assumption is that EAM represents Po by the parallel positioning. However, we are sometimes questioning about the possible positional disparity between Po and EAM, when the occlusal cant or facial midline is different from our clinical understandings. The purpose of this study was to evaluate the positional parallelism of Po and EAM in facial asymmetries, and also to investigate their relationship with the maxillary occlusal cant.The 67 subjects were classified into three groups. Group I had normal subjects with facial symmetry (1.05 ± 0.52 mm of average chin deviation) with minimal occlusal cant (<1.5 mm). Asymmetry group II-A had no maxillary occlusal cant (average 0.60 ± 0.36), while asymmetry group II-B had occlusal cant (average 3.72 ± 1.47). The distances of bilateral Po, EAM, and mesiobuccal cusp tips of the maxillary first molars (Mx) from the horizontal orbital plane (Orb) and the coronal plane were measured on the three-dimensional computed tomographic images. Their right and left side distance discrepancies were calculated and statistically compared.p = 0.001), while other groups were not. Interside discrepancy of the vertical distances from EAM to Mx in group II-B also showed the significant differences, as compared with those from Po to Mx and from Orb to Mx.EAM was located 10.3 mm below and 2.3 mm anterior to Po in group I.  The vertical distances from Po to EAM of both sides were significantly different in group II-B (The subjects with facial asymmetry and prominent maxillary occlusal cant tend to have the symmetric position of Po but asymmetric EAM. Some caution or other measures will be helpful for them to be used during the clinical procedures. Porion (Po) is an anatomical landmark point for the craniofacial evaluation. It is defined as the most superior and outer bony surface point of the external auditory meatus and can be called as the anatomical Po . It has Meanwhile, Po is frequently overlapped with adjacent anatomical structures on two-dimensional (2D) cephalometric radiographs that it is sometimes difficult to identify this anatomical Po due to its lack of visibility . The heaThe maxillary skeletal and dental position in relation to the cranium is traditionally reproduced by the facebow registration and the mounting onto the articulator. They have been conventionally used for the variety of dental procedures, including the prosthodontics treatment procedures or the pre-operative diagnosis and wafer fabrication for orthognathic surgery. The facebow device is typically used to reproduce the three-dimensional (3D) position of the maxillary and mandibular dental arch in relation to the cranium . This deSo, we can consider that the ear rods of the cephalostats in cephalometric machine and those of facebow are technically the same in that they rely on the anatomical position of EAM. In addition, they rely on the same classical assumption that EAM can represent Po with the parallel positioning. Thus, EAM can be meaningful only when Po can be reproduced by EAM. However, we sometimes suffered from the doubt of their parallelism when the occlusal cant or facial midline is different from our clinical understandings. This condition is worse when the patients have the facial asymmetries. We would not be confident to the procedures of putting the ear rods to EAM and/or the positional disparity between Po and EAM.The procedures of precise diagnosis and proper surgical treatment planning are critically important for patients with facial asymmetry. Those procedures should include the dedication to the underlying cause of the facial asymmetry. However, 2D cephalometric analysis with FH plane and/or the dental model mounting with the facebow transfer frequently fails to provide enough information for the accurate diagnosis and treatment planning. Again, it certainly has to do with the following: (1) the poor manipulation of the ear rods for cephalometrics, facebow, and articulator; (2) the designation of the reference points; or (3) the asymmetric locations of Po and/or EAM.It is our hypothesis that Po on the temporal part of the cranial bone, and/or EAM by the elastic cartilage part of the outer third ear canal may have an asymmetric position in facial asymmetries. This reasoning is based on the simple fact that the facial asymmetry can be accompanied by varying degrees of cranial base asymmetry . Most fa3D computed tomography (CT) is now a major imaging tool for craniofacial evaluation and treatment planning. The complex craniofacial structures can be radiographically investigated in various aspects and can be measured more precisely using the 3D CT images . Thus, tConsidering all these facts, the right and left symmetrical position of Po and EAM and their parallelism are the basic but essential premise for anthropologic or cephalometric analysis as well as the dental procedures with the facebow-transferred dental model mounting , 11. To The 3D CT reconstruction images from 67 subjects  were included in the present study Table . The 22 Their CT scans were acquired with the High-speed Advantage CT scanner  used with high resolution bone algorithm  at 1 s, 1 mm slice thickness, and 512 × 512 pixel reconstruction matrix. Reformatted 3D images were created from the CT scan data and were analyzed using SimPlant software .Foramen cecum (FC): the most anterior and superior point of a pit on the cribriform plate between the crista galli and endocranial wall of frontal boneCenter of foramen magnum (CFM): the midpoint of foramen magnum at the level of basionFalx cerebri (FxCe): the point of falx cerebri near bregma on the coronal sectionOptic canal (Oc): the most superior point of optic canal, both sidesEye ball center (EC): the center point of eye ball in sagittal, axial, and coronal plane, both sidesOpisthion (Op): the midpoint on the posterior margin of the foramen magnumFor the 3D reference planes, anatomical landmark points of cranium, which were already used previously , 16, werThree craniofacial reference planes were constructed on the 3D skull images for measurements. The midsagittal plane (MSP) was first determined as a plane passing through FxCe, FC, and CFM, which were selected as stable and reliable points in our previous reports . The horPo: the most superior and outer point on the ovoid bony contour of the external auditory meatus, both sides Fig. Fig. 1DetEAM: the center point of the most outer round or ovoid shadow of the external auditory meatus, both sides Fig. Fig. 2DetMx: the mesiobuccal cusp tip of maxillary first molar, both sidesAnd the reference point for Po, EAM, and maxillary first molars (Mx) point was defined and marked on 3D reconstruction image as follows:The vertical distances from Po to Orb (Po-Orb) and from EAM to Orb (EAM-Orb) were measured on both sides to obtain the vertical distances of Po and EAM Fig. . The horVertical discrepancy of Orb-Po or horizontal discrepancy of Fro-Po: the interside discrepancy of the vertical/horizontal distances from Orb/Fro to bilateral Po (right–left)Vertical discrepancy of Orb-EAM or horizontal discrepancy of Fro-EAM: the interside discrepancy of the vertical/horizontal distances from Orb/Fro to bilateral EAM (right–left)Vertical discrepancy of Orb-Mx or horizontal discrepancy of Fro-Mx: the interside discrepancy of the vertical/horizontal distances from Orb/Fro to Mx (right–left)Vertical or horizontal discrepancy of Po-Mx: the interside discrepancy of the vertical/horizontal distances from Po to Mx (right–left)Vertical or horizontal discrepancy of EAM-Mx: the interside discrepancy of the vertical/horizontal distances from EAM to Mx (right–left)The chin top deviation was evaluated by the location of menton from MSP. The maxillary occlusal cant was evaluated by measuring the vertical or horizontal distances from Orb/Fro, Po, and EAM to Mx, and the discrepancies between right and left side were calculated as follows:After the 3D image analysis, the subjects were divided into three groups. Group I was defined as the control group to include the normal control subjects without any chin deviation or occlusal cant. Group II  were subdivided into two groups, group II-A and group II-B, on the basis of the vertical maxillary occlusal cant  Table . Group It tests were also used to compare the distances between right and left side, and the maxillary occlusal cants to other measurements.To eliminate statistical confounder by the negative values related to the vertical maxillary cant direction, the 3D images and measurement data were subject to be adjusted to let all subjects have the right downward maxillary cant. The skull images with the left downward maxillary vertical cant were flipped by MSP, to have the changed right and left side. Statistical comparisons of variance and mean values for all three groups were made using one-way ANOVA and post hoc Tukey test by Statistical Package for the Social Sciences . Paired t test (p < 0.001). Meanwhile, the chin top deviation was not significantly different between that of group II-A and II-B (p = 0.056).We marked and measured the distances for Po and EAM to understand the symmetry of them and their relationship with the maxillary occlusal cant on 3D CT images. Table p = 0.554 for vertical distances and 0.611 for horizontal distances). Though the detailed data were not presented here, 48 % subjects had vertical Po discrepancies of less than 1.0 mm . In addition, only 12 % subjects showed more than 3.0 mm of the vertical distance discrepancies for Po . And 36 % subjects had horizontal discrepancies of Po being less than 1.0 mm, while 21 % subject showed 3 mm or more horizontal discrepancies of Po.Table p = 0.099 for vertical distances and 0.755 for horizontal distances). Though the detailed data were not shown here, 46 % subjects had vertical difference of EAM within the range of −1.0 to 1.0 mm . And the 12 % subjects showed remarkable vertical EAM discrepancies greater than 3 mm . And 33 % subjects had horizontal discrepancies of EAM in range of −1.0~1.0 mm, while 19 % subject showed 3 mm or more horizontal discrepancies of EAM.Again, Table p = 0.001).When we compared the vertical and horizontal distance discrepancies of the right and left side for Orb-Po and Orb-EAM in Table p = 0.001), with 10.89 mm on the right side and 9.96 mm on the left side. The vertical distance between Po and EAM of the right side was 0.93 ± 1.45 mm greater than that of the left side in group II-B. And the horizontal distances between Po and EAM did not show any significant interside differences for all groups, though that distance interside difference of group II-B reached −0.67 mm (p = 0.110).The results of Table p = 0.633~0.985). However, group II-B had the significantly different vertical measurements for Orb-Mx vs. EAM-Mx and Po-Mx vs. EAM-Mx (p = 0.026 and 0.001 each). In addition, all horizontal discrepancies of Fro-Mx, Po-Mx, and EAM-Mx were not significantly different from each other in all groups, and they did not have significant intergroup differences.On Table Po is a simple anatomical landmark that can be used in anthropometric and cephalometric analysis, and is frequently represented by EAM. If the position of EAM is not parallel with Po, it can make the distorted or inaccurate baseline to construct the reference plane or model mounting. And it can lead to the deep errors for the analysis of facial asymmetry. So, we want to evaluate the positional parallelism or similarity of Po and EAM in facial asymmetries and also to investigate their relationship with the maxillary occlusal cant.The authors first wanted to construct the reliable horizontal reference planes except FH plane for the verification of FH plane-related reference points in this study. We first established the MSP as a balanced and stable midsagittal plane, based on our previous investigation with 3D CT images of normal subjects and dry skull . The OrbOur first result showed that the vertical discrepancies of Orb-Po and Orb-EAM were significantly different in group II-B Table . But othWhen we compared the vertical distance difference for three groups for Orb-Mx, Orb-EAM, and Orb-Po, they were close to zero and greater in the order of amount of chin top deviation. It is consistent with the previous researches that the degree of facial asymmetry is increased with greater distance from the cranium , 19. HowIn our investigation, EAM was located about 10.3 mm below Po and approximately 2.3 mm anterior to Po in the normal subjects of group I. Since Po and EAM are located in such a distance, some authors stated that machine Po is unsuitable as a representation of anatomical Po , 20. OurOrthognathic patients are likely to have more significant dentofacial morphologic variations than the normal population group had . In our t test). The interside discrepancies of Po-Mx and EAM-Mx in group II-B have the average difference of 0.92 mm. The direction of EAM’s cant was same with that of maxillary occlusal cant in group II-B. Though it is not the big value, it can bring about the bad effects for the diagnosis and treatment planning for orthognathic surgery. If it is the case to be measured from EAM, this would decrease the amount of maxillary occlusal cant. Thus, the maxillary occlusal cant may be underestimated during the facebow registration or the model measurement, and in turn, it will cause the insufficient correction of maxillary occlusal cant and the undesirable chin top position during the orthognathic surgery. It also implies that the single measurement of maxillary cant based only on one reference point is likely to be inaccurate especially for the facial asymmetry with the remarkable maxillary cant.As Table Meanwhile, our investigation showed that the direction of Po’s vertical cant was not significantly related to the direction of maxillary vertical cant for all groups. And the 51.4 % of subjects had the opposite direction of maxillary occlusal and Po’s cant. So, we now believe we can rely on Po with assurance for the horizontal reference point.Additionally, group II-A had greater horizontal difference of Fro-Mx than group II-B had. We can guess that the maxillary horizontal transposition of group II-A might be greater than group II-B had, and it would also devote to the acceleration of chin deviation. However, our sample size of group II-A was too small to make a statistically significant conclusion. Further studies with more samples will be helpful in the future.This study has some other limitations. We used a classification on basis of facial asymmetry and vertical maxillary cant, but it did not address the anteroposterior facial deformities such as prognathism or retrognathism and horizontal maxillary occlusal deviation. Further study with more detailed classifications may give rise to more information about the pattern of Po and EAM locations. The evaluation of mediolateral deviations of Po and EAM were also absent in present investigation, that it was insufficient to understand overall 3D asymmetry.The Po is generally used to locate the FH plane, which is one of the gold standards for the horizontal reference plane for a long time. In spite of this tradition, many researchers have reported that the FH plane displays large individual variation (with the standard deviation ranged 4.02 °–9.1 °) . CurrentIn summary, Po tends to have symmetrical vertical locations in symmetrical as well as the facial asymmetry subjects. However, EAM can be located significantly asymmetrically in facial asymmetries with prominent maxillary occlusal cant. The possible asymmetry of EAM should be considered for the diagnosis and measurement of maxillary cant for facial asymmetry.The vertical cants of EAM and Po were significantly different in facial asymmetries with prominent maxillary occlusal cant.The EAM was located 10.3 mm below and 2.3 mm anterior to Po in normal subjects.The vertical distance of Po-EAM was significantly greater at downward-canted side in facial asymmetries with prominent maxillary occlusal cant.For facial asymmetry subjects with remarkable maxillary occlusal cant, the vertical maxillary cant measured from EAM was significantly smaller than those measured from Po or Orb.These results indicate that the subjects with facial asymmetry and prominent maxillary occlusal cant tend to have the symmetric positions of Po but asymmetric location of EAM.


On the 3rd and 4th visits, the participants performed a 1000-m TT at either 8 am or 6 pm, in randomized, repeated-measures, crossover design. The time to complete the time trial was lower in the evening than in the morning , but there was no significant different in pacing. However, oxygen uptake and aerobic mechanical power output at 600 and 1000 m tended to be higher in the evening . There was also a main effect of time of day for insulin, cortisol, and total and free testosterone concentration, which were all higher in the morning . The growth hormone, was twofold higher in the evening (p<0.05). The plasma glucose was ∼11% lower in the morning (p<0.05). Glucagon, norepinephrine, epinephrine and lactate were similar for the morning and evening trials (p>0.05), but the norepinephrine response to the exercise was increased in the morning , and it was accompanied by a 5-fold increase in the response of glucose. Muscle recruitment, as measured by electromyography, was similar between morning and evening trials (p>0.05). Our findings suggest that performance was improved in the evening, and it was accompanied by an improved hormonal and metabolic milieu.The aim of this study was to determine the effect of time of day on performance, pacing, and hormonal and metabolic responses during a 1000-m cycling time-trial. Nine male, recreational cyclists visited the laboratory four times. During the 1 Performance during a short-distance time trial (TT), such as a 1000-m cycling TT, may be optimized when an “all-out” pacing is used −1 for women and 6.0 W·kg−1 for men) in the afternoon compared with the morning, and that this greater work was associated with a larger anaerobic and aerobic contribution. The underlying mechanism explaining the greater energy expenditure and consequently improved performance in the evening is not fully understood, but a slightly increased core temperature in the evening could increase nerve conduction velocity and vasodilatation, which in turn would increase muscular supply and substrate elimination, thereby improving glycogenolysis and glycolysis To date, studies investigating the influence of time of day on performance have concentrated on intermittent, Wingate or constant-load exercise milieu and could influence performance during different times of day, is unknown. Given that performance during a short-distance cycling TT is dependent on the ability of athletes to produce and maintain a high level of power output throughout the test There is also limited research connecting circadian variations in hormone levels with pacing milieu at rest and/or during the exercise, and a greater aerobic and anaerobic contribution.The present study therefore aimed to investigate performance, pacing, energy system and muscle recruitment distributions, and hormonal responses, during a 1000-m cycling TT performed in the morning (8:00 am) or in the evening (6:00 pm). The 1000-m cycling time trial was chosen as it is energetically supported by both the aerobic and anaerobic energy systems 2peak) 49±7 ml·kg−1·min−1, peak power output (PPO) 253±52 W] participated in this study. The participants had been training more than 3 times weekly (approximately 60 km [3–4 hours] per session) during the 4 years preceding the study, and all had good competition experience (∼14 competitions per year). The participants were classified as intermediate (n = 5) or moderate (n = 4) morning types using the chronotype questionnaire of Horne and Östberg Nine recreational cyclists [Mean ± standard deviation (SD): age 31±7.3 years, height 175±7.8 cm, body mass 73.5±11.6 kg, body fat 11.6±4.7%, peak oxygen uptake , the participants performed a familiarization of the 1000-m cycling TT. The 1st and 2nd visits were performed between 10 am and 2 pm to prevent adaptation to a particular experimental time. During the 3rd and 4th visits (∼96 h after the 2nd visit and separated by 7 days for recovery), the participants performed a 1000-m cycling TT either in the morning or in the evening  in a randomized, repeated-measures, crossover design. The participants were asked to refrain from vigorous physical activity, caffeinated substances or alcohol 24 h before each test Each participant visited the laboratory four times on different days. During the 12 and carbon dioxide production (2) were measured breath-by-breath throughout the test using a gas analyzer . The volume of expired air was measured by a bidirectional flow sensor, calibrated before starting the test with a syringe containing 3 liters of air . The fraction of expired O2 was analyzed with a zirconium sensor and end tidal CO2 by infrared absorption. Both sensors were calibrated automatically before starting the test with a cylinder containing known concentrations of O2 (16%) and CO2 (5%). The HRmax and 2peak were taken as the highest value reached in the last stage and as the mean value obtained during the last 30 s of the test, respectively. The PPO was determined as the highest PO attained during the incremental test. When the participants could not maintain the power output during the entire stage (<3 minutes), the PPO was calculated using fractional time supported in the last stage multiplied by the increment rate (i.e. 30 W) The incremental test was performed on a cycle ergometer , and consisted of a 5-min warm-up at 100 W, followed by increments of 30 W every 3 min until voluntary exhaustion or when the participants were not able to maintain their pedal frequency between 80–90 revolutions per minute (rpm). The ventilation  was drained and discarded, and 20 ml of blood was then collected (baseline). After each blood sample collection, 1 ml of sterile sodium chloride (NaCl 0.9%) was injected to prevent clotting and the obstruction of blood through the stent.The experimental procedures are illustrated in Following the resting blood collection, in order to normalize the electromyographic activity (EMG) data during the cycling TT, three 5-s, two-legged maximum voluntary contractions (MVC) of the knee extensors (trunk-thigh angle at 90 and knee at 60° from full leg extension 0°), separated by a 60-s interval, were performed. A standardized knee extensor warm up, consisting of four 5-s contractions at intensities corresponding 50, 60, 70 and 80% of the maximum subjective force (30-s rest periods between the repetitions), was performed immediately before the MVC. The participants were verbally encouraged during the MVC to achieve their maximal force. Force was recorded using a load cell . Electromyographic activity signals from the vastus lateralis (VL) of the right leg were recorded via bipolar Ag-AgCl surface electrode at an interelectrode distance of 20 mm. The VL was chosen because it has been reported as the most appropriate to monitor EMG activity in the lower limb during a cycling TT 2, respiratory exchange ratio (RER), HR, EMG (5-s recordings), and aerobic and anaerobic mechanical power output  were also determined and statistically analyzed every 200-m. The Pan and Paer were calculated from the RER, 2, and the efficiency estimated during the warm up, in accordance with Hettinga et al. After completion of the MVC procedures, the participants were transferred to the cycle ergotrainer. The participants remained at rest for 5 minutes before performing a 5-min warm-up at 100 W followed by a 5-min recovery . ThereafAdditional blood samples (20 mL venous blood) were collected immediately and 60 minutes after the TT . Blood samples were transferred into tubes with EDTA (10 ml) or without non-EDTA (10 ml) and were immediately centrifuged at 3000 revolutions per minute (RPM) at 4°C for 10 minutes to separate plasma or serum, and stored at −80°C until subsequent analysis. The following hormones and metabolites were measured in the plasma: Glucagon , and lactate and glucose . Plasma norepinephrine and epinephrine concentrations were measured by HPLC using the ion-pair reverse phase chromatography coupled with electrochemical detection (0.5 V), as described previously 2/d2t was used to compare the time to complete the task between morning and afternoon and to access any possible test order effect (test 1 versus test 2). Confidence intervals (95% CI) and effect sizes for the t and F ratio, expressed as partial eta-squared (ηp2), were also calculated when appropriate to evaluate the magnitude of differences. A ηp2 of 0.1, 0.3 and 0.5 were considered as small, moderate and large effects, respectively The sample size required was estimated from the equation n = 8e2 and HR during the 1000-m cycling TT in the morning and evening are displayed in t (8)  = 2.331, p = 0.048, ηp2 = 0.63, 95% CI  = 0.7 to 12.8 s). The mean values for PO, Paer, Pan, iEMG, 2 and HR were not different between evening and morning, although a moderate effect size was found for all parameters (except iEMG) with higher values observed in the evening . There was a main effect of distance for Paer, Pan, iEMG, 2, and HR , but not for PO (p>0.10).The PO, Paer, Pan, iEMG, The hormone, glucose and lactate response to the 1000-m cycling TT in the morning and evening are displayed in Insulin, cortisol, and total and free testosterone were higher in the morning than in the evening , while GH was higher in the evening than in the morning (p = 0.02). In addition, the GH values 60′ post-TT were significantly lower in the morning than in the evening (p = 0.023), while total testosterone values at the same time point were higher in the morning than in the evening (p = 0.005). Baseline GH values were lower than immediately post-TT (p<0.05) for both morning and evening trials, but without a significant difference between post-TT and 60′ post-TT (p>0.10). Total testosterone immediately after the TT was higher than the 60′ post-TT (p<0.05) in both trials, but without a significant difference between post-TT and baseline or between 60′ post-TT and baseline (p>0.10). Similar results were obtained for free testosterone, except that values post-TT were also higher than baseline for both trials .The plasma glucose was lower in the morning than in the evening (p = 0.04). However, there was no significant effect of time or an interaction effect (p>0.10). The plasma lactate increased with the exercise but returned to baseline values after 60 min of recovery for both conditions .The normalized norepinephrine response to the exercise (post-TT less baseline) was significantly higher in the morning than in the evening . The norThere was no test order effects for any of the variables investigated .The main purpose of this study was to determine the effect of time of day on performance, pacing, energy system contribution, and metabolic and hormonal responses during a 1000-m cycling TT. The main findings of the present study were: 1) a better performance (shorter TT times) in the evening than in the morning, which was not accompanied by a significant alteration in the aerobic or anaerobic contribution; 2) Insulin, cortisol, and free and total testosterone were lower in the evening, while GH and plasma glucose concentration were higher in the evening. However, the norepinephrine and glucose responses to the exercise were higher in the morning.ηp2 = 0.63), suggesting that time of day can affect performance during a short-distance TT. This improved performance in the evening is in agreement with previous reports of better performance in the evening during a 30-s Wingate test 2max), constant-load exercise In the present study, the time for amateur, recreational cyclists to complete the 1000-m cycling TT was ∼6.5 s (∼6.9%) quicker in the evening than in the morning. This represented a large effect size  and the lowest in the evening (08:00 pm), but that the acute response to exercise is similar at different times of the day It is important to acknowledge that we recruited recreational cyclists and these results should not be extrapolated to other groups such as highly-trained athletes or sedentary/clinical populations. Further studies should be performed investigating if the effects found in the present study are reproducible in these populations. It is also important to underline that competitions do not always occur in the evening. However, training can be performed in both the morning and evening. It is necessary to recognize that training may have to be performed under unfavorable conditions, but as close as possible to the competition environment and conditions (e.g. if the competition is in the morning). Corroborating with this last statement, Edwards et al. milieu , combined with an exacerbated norepinephrine and plasma glucose response to the exercise.In conclusion, performance was impaired in the morning compared with the evening, but it was not associated with a clear alteration in pacing, an aerobic and anaerobic energy supply distribuition. Morning exercise was performed in a less favorable metabolic