FFP and PCC can be associated with volume complications, undesirable thromboembolic events, and increased costs. We describe the case of a 63-year-old female with acute warfarin toxicity following a massive ingestion of warfarin (420 mg–450 mg) in an attempt to commit suicide. Upon arrival to the emergency department, serial INR checks were initiated to help guide dosing strategy and later adjusted based on INR response to treatment using only phytonadione.Treatment strategies for acute toxicity following massive ingestion of warfarin are not well described in the literature. Warfarin is the primary oral anticoagulation agent used in the treatment of thromboembolic disease, and patients with acute toxicity are at risk for life-threatening hemorrhages. Treatment options include phytonadione (vitamin K While the use of new oral anticoagulation agents is on the rise, the vitamin K antagonist (VKA) warfarin remains a primary agent for oral anticoagulation in the treatment of thromboembolic disease . Due to Acute warfarin toxicity management is complicated by its well described pharmacokinetic profile including rapid and complete absorption, delayed anticoagulation effects, and having a 29- to 45-hour half-life , 4. VariTo date, there are only a limited number of cases reports available describing massive warfarin ingestion (>300 mg) –15. HereA 63-year-old Caucasian female was brought to our emergency department (ED) after ingesting approximately 84 to 90 tablets of warfarin 5 mg (420 mg–450 mg), 6 tablets of mycophenolate mofetil 500 mg, an estimated 62 tablets of gabapentin 300 mg, and an unknown amount of over-the-counter sleeping pills. Pill counts were reported by the patient or estimated based upon the pharmacy fill date on the patient's prescription bottles and the day of intentional ingestion. All three pill bottles were found empty next to the patient after the self-reported ingestion in an attempt to commit suicide. The patient had a past medical history significant for hypertension, large vessel cerebral vascular disease, stroke, neurovasculitis, pulmonary embolism (PE), deep vein thrombosis (DVT), inferior vena cava filter placement, and depression. Her home medication regimen included lisinopril 10 mg, pantoprazole 40 mg, topiramate 25 mg, and venlafaxine 75 mg, all taken once daily, and gabapentin 600 mg, mycophenolate mofetil 500 mg, and ranitidine 150 mg, all taken twice daily, as well as clonazepam 1 mg and pramipexole 0.125 mg taken daily at bedtime. Her home warfarin regimen was 5 mg on Sunday, Tuesday, Thursday, and Saturday and 7.5 mg on Monday, Wednesday, and Friday.2) was within normal limits (WNL). Electrolytes were WNL with the exception of mild hypokalemia (3.4 mEq/L) and mild hyperchloremia (108 mmol/L). An initial computed tomography (CT) scan of the brain was performed to look for any intracranial hemorrhaging which showed no acute findings. The patient was subsequently transferred to the ICU, and, at the recommendation of the Poison Control Center, serial INR checks were initiated at 12-hour intervals.Upon her initial presentation to the ED, the patient was extremely drowsy but responded to tactile and painful stimuli. Vital signs at time of admission were as follows: blood pressure (BP) 176/120, heart rate (HR) 86 beats per minute (bpm), respiratory rate (RR) 18 breaths per minute, 100% oxygen saturation, and a temperature of 36.3°C. Other pertinent lab tests at admission included a hemoglobin of 13.8 g/dL, hematocrit of 42.5%, an INR of 2.8, and a prothrombin time (PT) of 29.1 seconds. An arterial blood gas (ABG) test performed in the ED showed the patient was acidotic with a blood pH of 7.3, bicarbonate level of 22, and a base excess of negative 5. Her partial carbon dioxide  and keeping it in a therapeutic range using repeated 300 mL infusions of FFP . One adoGiven the paucity of information available discussing intentional overdoses of warfarin and varying management strategies, we describe a case of acute warfarin toxicity after massive warfarin ingestion in an attempt to commit suicide. While similar to the aforementioned cases, her treatment strategy was more conservative than those previously mentioned. Massive warfarin ingestion can be an alarming scenario for healthcare professionals especially if clinical signs and symptoms of bleeding are present. While the emergence of bleeding should result in more aggressive treatment strategies with phytonadione and FFP or PCC administration, this case demonstrates if no signs or symptoms of bleeding are present, conservative management of acute warfarin toxicity is manageable using serial INRs to individualize and guide dosing strategies with oral and IV phytonadione. By utilizing this conservative management, it is possible to avoid not only unnecessary volume and thromboembolic complications that may be associated with FFP and PCC but additional costs as well.


The reported range of seroma formation in the literature after TEP repair is between 0.5 and 12.2% and for TAPP between 3.0 and 8.0%. Significant clinical factors associated with seroma formation include old age, a large hernia defect, an extension of the hernia sac into the scrotum, as well as the presence of a residual indirect sac. Seroma formation is a frequent complication of laparoendoscopic mesh repair of moderate to large-size direct  inguinal hernia defects. This present analysis of data from the Herniamed Hernia Registry now explores the influencing factors for seroma formation in male patients after TAPP repair of primary unilateral inguinal hernia.In total, 20,004 male patients with TAPP repair of primary unilateral inguinal hernia were included in uni- and multivariable analysis.p < 0.001). Multivariable analysis showed that glue compared to tacks  and non-fixation  led to an increased seroma rate. A large hernia defect  presented a significantly higher risk of seroma formation. Likewise, medial compared to lateral inguinal hernias had a higher seroma rate .Univariable analysis revealed the highly significant impact of the fixation technique on the seroma rate (non-fixation 0.7% vs. tacks 2.1% vs. glue 3.9%; Mesh fixation with tacks or glue, a larger hernia defect, and medial defect localization present a higher risk for seroma development in TAPP inguinal hernia repair. According to the Guidelines of the International Endohernia Society (IEHS) and the European Association of Endoscopic Surgery (EAES) –3, seromp = 0.003) [The reported range of seroma formation in literature reviews after TEP repair is between 0.5 and 12.2% . In a me= 0.003) . If no m= 0.003) . In larg= 0.003) , 9. Sign= 0.003) , 11. SerWhile certain studies had identified significantly less seroma formation on using extra lightweight meshes , a meta-This present analysis of data from the Herniamed Hernia Registry now explores the influencing factors for seroma formation after TAPP repair. Preventative measures will then be discussed.As of October 10, 2016, 577 participating hospitals and office-based surgeons mainly from Germany, Austria, and Switzerland have entered prospective data into the multicenter internet-based Herniamed Hernia Registry on their patients who had undergone routine hernia surgery and signed an informed consent agreeing to participate . As partThe most commonly used meshes ≥ 2%) are given in Table % are givp value 0.05 represents a significant result. To first discern the differences between the groups in unadjusted analyses. Fisher’s exact test was used for categorical outcome variables, and the robust t test (Satterthwaite) for continuous variables. For mesh size (cm2), a logarithmic transformation was applied and re-transformed mean and range of dispersion are given.All analyses were performed with the software SAS 9.4  and intentionally calculated to a full significance level of 5%, i.e., they were not corrected in respect of multiple tests, and each 2), mesh size (cm2), defect size (I/II/III), risk factors (yes/no), preoperative pain (yes/no/unknown), EHS classification , drainage (yes/no), and mesh fixation (non-fixation/tacks/glue). Estimates for odds ratio (OR) and the corresponding 95% confidence interval based on the Wald test were given. For influence variables with more than two categories, pairwise odds ratios were given. For age (years), the 10-year OR estimate, for BMI (kg/m2) the five-point OR estimate, and for mesh size (cm2) the 10-point OR estimate were given. Results are presented in tabular form, sorted by descending impact.To identify influence factors in multivariable analysis of seroma, a binary logistic regression model was used. Potential influence factors were: ASA score (I/II/III/IV), age (years), BMI (kg/mp < 0.001) (Table p < 0.001).Univariable analysis of the relationship between the fixation technique (non-fixation/tacks/glue) and the patient and operative characteristics revealed highly significant differences Tables , 5. For 1) Table . Seroma p < 0.001). The fixation technique and hernia defect size exerted a highly significant influence on seroma formation (in each case p < 0.001). Glue compared to tacks  and glue vs non-fixation  resulted in an increased seroma rate. Similarly, tacks compared to non-fixation  were associated with a higher risk of seroma formation. A large hernia defect  presented a significantly higher risk of seroma formation. That was also true for medial compared to lateral inguinal hernia , and for medial compared to combined . Besides, a high ASA score  led to an increased risk of seroma formation. The presence of risk factors  reduced the risk of seroma development. Only a trend towards a reduction in the risk of seroma formation was identified for the use of a drain .The results of multivariable analysis of the postoperative complications associated with seroma are summarized in Table p < 0.001). Seroma accounted for the greatest proportion of postoperative complications. Here, too, a highly significant difference was detected in relation to mesh fixation . Multivariable analysis confirmed the highly significant influence exerted by the fixation technique on the seroma rate. Glue compared to tacks and non-fixation resulted in a higher seroma rate. But also tacks compared to non-fixation led to a higher seroma rate. Accordingly, no mesh fixation clearly had the lowest seroma rate. That could be one explanation for the lower seroma rates after TEP than after TAPP [This present analysis of data from the Herniamed Hernia Registry reveals highly significant differences in the postoperative complication rate in relation to the fixation technique for male patients with TAPP repair of primary unilateral inguinal hernia. For example, the overall postoperative complication rate for non-fixation was 1.8%, for tack fixation 3.0%, and for glue fixation 4.8% , and in particular for medial hernias. Otherwise, seroma formation is commonly encountered for large inguinal hernias, and especially for large medial inguinal hernias. Therefore medial defects should either be secured with a suture to Cooper’s ligament or reduced with a Roeder loop. Furthermore, there appears to be a trend towards a positive role for drain placement after TAPP in reducing seroma formation.


CONNEXIN37 gene variations are associated with atherosclerosis diseases, such as coronary heart disease and stroke, but its association with stroke in distinct human populations remains to be determined. We report here the analysis of the association of the single nucleotide polymorphisms (SNPs) of CONNEXIN37 with ischemic stroke in Han Chinese population.Stroke has a high fatality and disability rate, and is one of the main burdens to human health. It is thus very important to identify biomarkers for the development of effective approaches for the prevention and treatment of stroke. Connexin37 is an anti-inflammatory cytokine and is involved in chronic inflammation and atherosclerosis. Recent studies have found that CONNEXIN37 gene were analyzed in 385 ischemic stroke patients and 362 hypertension control patients using ligase detection reaction (LDR) method.Two SNPs of CONNEXIN37 were associated with an increased risk of ischemic stroke, and that G allele of rs1764390 is a risk factor for ischemic stroke. Further, we found that SNP rs1764390 and SNP rs1764391 in CONNEXIN37 were associated with ischemic stroke under additive/dominant model, and recessive/dominant model, respectively.Logistic regression analysis demonstrated that, AG and GG genotypes of SNP rs1764390 and CC genotype of rs1764391 of CONNEXIN37 gene polymorphism is an ischemic stroke risk factor in Northern Han Chinese.Our results indicate that  Stroke is a complex disease with very high mortality and morbidity . Age, geACE) related gene, endothelial nitric oxide synthase (NO), methylenetetrahydrofolate reductase (MTHFR), brain natriuretic peptide (BNP), protein kinase C (PKC), PDE4D, and ALOX5AP have been found to be associated with stroke susceptibility CCCCAAGTCGTCCCAGCAGCTCTGC); rs1764390 (GGCGAGTCAGTGTGGGGTGACGAGCA[A/G]TCAGATTTCGAGTGTAACACGGCCC). The minor allele frequency (MAF) of these SNPs was greater than 0.1 and pair-wise r2 was more than 0.8. Ligase detection reaction (LDR) method was used for genetyping [http://www.generay.com.cn/). The sequences of primers and probes are listed in Tables Genomic DNA was extracted from EDTA anti-coagulated whole blood in Experimental Center and Virology Laboratory of Shengjing Hospital of China Medical University using TIANamp Blood DNA Kit  according to manufacturer’s protocols. According to the previous literature and NCBI database, genotype detection was selected at the site of secondary allele frequency (MAF) > 0.1 in Beijing Chinese Han population. Tag SNP strategies were used to select the following two potential functional SNPs of netyping –32. Prim2 test. Comparison and analysis of genotype and allele frequencies between cases and controls was performed using Pearson X2 test. The genotype frequencies were tested for Hardy-Weinberg equilibrium (HWE) using X2 test. SHEsis statistical analysis platform was used to compute haplotype frequencies and linkage disequilibrium coefficient (D’ and r2). Associations between gene polymorphisms and ischemic stroke risk were estimated by computing odds ratios (ORs) and 95% confidence intervals (95%CI) from unconditional logistic regression models. P < 0.05 was considered statistically significant. Multivariate logistic regression analysis were used after adjusting for age, systolic blood pressure, diastolic blood pressure, body mass index, smoking and drinking history, triglycerides, high density lipid cholesterol, low density lipid cholesterol and fasting plasma glucose to test the association of gene variation and ischemic stroke risk.Statistical analyses were performed using SPSS for Windows software  and SHEsis platform. Data are presented as mean ± standard deviation (SD) with normal distribution by using the t test. Count data are presented as number of cases and percentage and analyzed using chi-square XCONNEXIN37 rs1764391 and rs1764390 between cases and controls and their associations with ischemic stroke are summarized in Table CONNEXIN37 gene polymorphisms in controls and cases and their associations with ischemic stroke are shown in Table The characteristics of 385 ischemic stroke cases and 362 controls with hypertension are summarized as previously reported . The genp < 0.001; OR 0.470, 95%CI 0.201-1.094 for TT, p = 0.08, respectively). Under recessive and dominant models, rs1764391 was significantly associated with IS . After adjustment for stroke risk factors, only under recessive model was rs1764391 associated with IS with OR 0.138 . The T allele was not associated with ischemic stroke risk with OR of 1.247 . These data indicate that the T allele of rs1764391 is not a risk factor for stroke.In the logistic regression models, compared with CC genotype of rs1764391, CT but not TT genotypes were associated with an increased risk of ischemic stroke . Under additive and dominant models, rs1764390 was associated with IS with OR and 95% CI 1.317 (1.058-1.639), p = 0.014; 1.552 (1.104-2.182), p = 0.011, respectively. After adjustment for stroke risk factors, the associations did not existed with OR and 95% CI 1.149 (0.851-1.552), P = 0.364; 1.456 (0.913-2.321), P = 0.115, respectively. The G allele was associated with ischemic stroke risk with OR of 1.272 . These data indicate that the G allele of rs1764390 is a risk factor for stroke.In the logistic regression models, compared with AA genotype of rs1764390, AG and GG genotypes were associated with an increased risk of ischemic stroke , P = 0.048; 1.403 (1.080-1.822), P = 0.011, respectively.We next analyzed the distribution of haplotypes in the cases and controls. The haplotypes with frequencies greater than 0.03% are shown in Table P > 0.05 for two SNPs). For rs1764391, the χ2 and p values were 3.67 and 0.06 for control group, and 12.26 and 0.0005 for the patient group; for rs1764391, the χ2 and p values were 3.098 and 0.078 for control group, and 9.76 and 0.001 for the patient group. These results indicate a balanced genetic and Mendel population.The observed genotype frequencies of two SNPs followed Hardy-Weinberg equilibrium among the controls (2) found that genotype frequencies of the two SNPs followed linkage disequilibrium but at a low level of Linkage Disequilibrium between these SNPs . Wong et al. found that the expression of protein Cx37-pro319  was less adherent than the expressed protein Cx37-ser319  in mononuclear cell lines, suggesting that Connexin37 may inhibit mononuclear cells adhesion, the initiating factor of arteriosclerosis, to reduce atherosclerosis [Stroke and coronary heart diseases are all arteriosclerosis-related diseases. It is thus possible that the two diseases may share common susceptible genes. Gap junction proteins are a series of transmembrane proteins that form the gap between cells and allow the exchange of ions and metabolites between cells. Connexin37 is a member of the gap junction protein family, which is mainly expressed in vascular endothelial cells, monocytes and macrophages . These tclerosis .CONNEXIN37 gene were focused on rs1764391 locus (C1019T), where the changes in the T and C alleles result in the conversion of proline to tryptophan (P319S), which increases the incidence of myocardial infarction [CONNEXIN37 gene mononucleotide polymorphism is associated with ischemic stroke in the northern Chinese Han population. Our findings have provided additional evidence of the importance of CONNEXIN37 in human diseases, especially ischemic stroke.Previous studies on the farction , 37. Thefarction , 25, 37.http://Biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize) revealed a power of 0.9 based on the lowest MAF among SNPs [There are some potential limitations of the present study. First, we only examined the rural elderly patients in Northern China, which may not present on behalf of the populations of other regions, ethnic or social background. Secondly, although Power and Sample Size Calculation  (ong SNPs , the oveCONNEXIN37 SNPs rs1764390 and rs1764391 in the development and progression of ischemic stroke risk in northern Chinese Han population.Our findings have provided new insights into the roles of


There are no therapies shown to improve outcome after severe traumatic brain injury (TBI) in humans, a leading cause of morbidity and mortality. We sought to verify brain exposure of the systemically administered antioxidant N-acetylcysteine (NAC) and the synergistic adjuvant probenecid, and identify adverse effects of this drug combination after severe TBI in children.IRB-approved, randomized, double-blind, placebo controlled Phase I study in children 2 to 18 years-of-age admitted to a Pediatric Intensive Care Unit after severe TBI  requiring an externalized ventricular drain for measurement of intracranial pressure (ICP). Patients were recruited from November 2011-August 2013. Fourteen patients (n = 7/group) were randomly assigned after obtaining informed consent to receive probenecid  and NAC , or placebos via naso/orogastric tube. Serum and CSF samples were drawn pre-bolus and 1–96 h after randomization and drug concentrations were measured via UPLC-MS/MS. Glasgow Outcome Scale (GOS) score was assessed at 3 months.There were no adverse events attributable to drug treatment. One patient in the placebo group was withdrawn due to adverse effects. In the treatment group, NAC concentrations ranged from 16,977.3±2,212.3 to 16,786.1±3,285.3 in serum and from 269.3±113.0 to 467.9±262.7 ng/mL in CSF, at 24 to 72 h post-bolus, respectively; and probenecid concentrations ranged from 75.4.3±10.0 to 52.9±25.8 in serum and 5.4±1.0 to 4.6±2.1 μg/mL in CSF, at 24 to 72 h post-bolus, respectively (mean±SEM). Temperature, mean arterial pressure, ICP, use of ICP-directed therapies, surveillance serum brain injury biomarkers, and GOS at 3 months were not different between groups.Treatment resulted in detectable concentrations of NAC and probenecid in CSF and was not associated with undesirable effects after TBI in children.NCT01322009ClinicalTrials.gov  Traumatic brain injury (TBI) is a significant cause of morbidity and mortality worldwide and the primary cause of death in children in the U.S. . To-dateN-acetylcysteine (NAC) is an antioxidant and putative neuroprotective agent , 5. BiocProbenecid has been used as an antimicrobial adjunct since World War II, increasing systemic exposure of antimicrobial agents via inhibition of drug elimination through membrane transporters including organic anion transporters 1 and 3 (OAT1 and 3)  found inImportantly both probenecid and NAC are in clinical use, are approved by the U.S. Food and Drug Administration (FDA), and have favorable safety profiles. Accordingly, we hypothesized that probenecid could be used as an adjuvant in combination with NAC to increase and/or maintain NAC concentration in brain after TBI. In this randomized, double-blind Phase I study we sought to determine whether NAC and probenecid administered systemically resulted in detectable drug levels in CSF, and whether the drugs used in combination resulted in attributable adverse events in children after severe TBI.We performed a randomized, double-blind, Phase I study of the combination of probenecid and NAC versus placebos in children 2 to 18 years-of-age after severe TBI  recruited from November 2011-September 2013 at a single, tertiary Children’s Hospital. All patients were admitted to the pediatric intensive care unit (PICU) on the pediatric neurocritical care/trauma service. The study was approved by the University of Pittsburgh Institutional Review Board and written informed consent was obtained from parents and/or legal guardians of all children enrolled in the study. In addition, a Data Safety Monitoring Board from the University of Pittsburgh evaluated the safety of the study throughout the time of enrollment. The Phase I trial was stopped based on duration of funding (R01 NS069247). The study was registered in ClinicalTrials.gov (Identifier NCT01322009).The research team screened patients for eligibility for this study. Inclusion criteria included (i) age > 2 y and < 18 y, (ii) severe TBI (GCS ≤ 8) after resuscitation, (iii) placement of an EVD as a part of their standard care, and (iv) placement of a naso/orogastric feeding tube for medication administration. The trauma Attending Physician was consulted regarding the safety of administering medications into the stomach prior to approach for informed consent. Exclusion criteria included (i) examination consistent with brain death after resuscitation, (ii) documented pregnancy, (iii) contraindications to probenecid , (iv) contraindications to NAC , (v) lack of ability to obtain consent from both parents/guardians, and (vi) inability to start medications within 24 h from the time of injury.After obtaining written consent, children were randomized by the use of a blind envelope system to: (1) probenecid  and NAC  or (2) placebo of equal volume and timing administered via naso/orogastric tube. The regimen for NAC was based on dosing for treatment of acetaminophen/paracetamol overdose, and the regimen for probenecid was based on dosing for use as an antibiotic adjuvant. Placebo contents included equal volumes and dosing regimens of lactose powder (for opacity) suspended in Ora-Plus and normal saline. Study drugs or placebo were prepared by a dedicated Research Pharmacist and administered by the bedside nurse. Clinicians caring for the child and all research staff–with the exception of the Research Pharmacist and the Data Manager at the Data Coordinating Center–remained blinded to study assignment. Research staff remained blinded until all data were entered into the research database and all analyses were performed on clinical samples. Compliance with the timed drug administration was monitored by the study team and deviations from expected drug delivery times > 1 hour were considered a protocol deviation.2 ~ 35 mm Hg), hyperosmolar therapies (mannitol or 3% NaCl) and barbiturate administration. As mentioned above, continuous CSF diversion was employed in all children.All children were treated using a standardized neurocritical care protocol that has been previously described . Brieflyg for 10 min and collecting supernatant. Blood was immediately centrifuged at 3000 ×g for 10 min and serum was obtained. All processed samples were frozen at -80°C for batch analysis.Samples (CSF and serum) were collected over 4 days and were obtained prior to initial drug administration, 1 hour after the first dose, and daily prior to subsequent dosing (trough levels). An additional CSF sample was obtained at 6 h after drug administration. CSF was immediately processed by centrifuging at 3000 ×Data including patient demographics, physiological parameters, and adverse events were recorded and entered into a database. Demographic information included age, patient sex, and various injury scores  measured at the appropriate time period. Physiological parameters included hourly variables  recorded within the medical record as well as results of other tests done as part of standard practice  and gross assessment of therapies required for management of ICP . A priorm/z 289.1→158.1) and probenecid (m/z 284.1→240.1) was performed on a TSQ Quantum Ultra mass spectrometer with a heated electrospray source . NAC and probenecid assay linear ranges (r2 > 0.992) in serum and CSF were 10–10,000 ng/mL (NAC serum), 10–2,500 ng/mL (NAC CSF), 0.25–800 μg/mL (probenecid serum), and 0.25–100 μg/mL (probenecid CSF). Assays were validated in each matrix to ensure linearity, precision, and accuracy with acceptable intra-/inter-assay variability (<10.5%).Total NAC and probenecid concentrations were quantified in serum and CSF using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using validated assays . NAC samDescriptive statistics were summarized as counts  for categorical data, mean ± standard deviation (SD) or median  for normally or non-normally distributed continuous data as appropriate. Serum and CSF concentrations of NAC are presented as mean ± standard error of the mean (SEM). Fisher’s exact test was used to compare the frequency distribution of categorical variables between the groups. The Wilcoxon-Mann-Whitney test was performed to assess differences between groups for non-normally distributed continuous data. Linear mixed models were used to test the main effects of time  and group (placebo and Pro-NAC), and time by group interactions on the brain injury biomarkers NSE and GFAP. All tests were two-sided and the significance level was defined at 0.05. Analyses were conducted using SAS, version 9.3 statistical software .F = 0.60, P = 0.441; GFAP F = 0.29, P = 0.596; F = 0.08, P = 0.989; GFAP F = 1.06, P = 0.387).Patients were recruited from November 1, 2011 to August 31, 2013. Twenty patients were screened for eligibility, Seventeen patients met inclusion criteria and 14 patients were randomized after obtaining informed consent, with seven patients randomized to each group . BaselinIn addition to adverse events and drug concentrations in serum and CSF, physiologic and other clinical variables were also recorded. There were no differences between groups in rectal temperature, MAP, ICP, or CPP during the study period . Note thIt has been estimated that the average time for translation of therapies from laboratory to patient is 17 years, a gap that is being scrutinized as being unacceptably wide . Our ratNAC has been, or currently is being, evaluated in clinical trials targeting neurological diseases including TBI –13, despFor probenecid, the CSF concentration-time profile mirrors that seen in serum, but this was not the case for NAC. This apparent lag from serum to CSF observed for NAC is consistent with poor BBB permeability or transporter interactions. With co-administration of probenecid in rats, NAC levels are detectable for up to 8 h in normal brain and the area under the curve (AUC) for NAC in brain is 2.4-fold (and in serum 1.7-fold) higher versus administration of NAC alone, suggesting that NAC is a substrate for transporters inhibited by probenecid, later revealed to be OAT1 and OAT3 , found iTo our knowledge, only one other published study has reported CSF NAC concentrations in humans. Katz et al. treated 12 patients with Parkinson’s disease with oral NAC doses ranging from 7 to 70 mg/kg twice daily for two days . Peak CSProbenecid was developed as an antibiotic adjuvant during World War II to reduce OAT-mediated renal excretion of penicillin for treatment of wound infections in soldiers . In humaGiven that this study was appropriately powered to serve as a phase I pharmacokinetic study, the sample size was sufficient to define the primary goal of drug levels, but it was not designed to be able to inform on acute or chronic outcomes, and is thus only exploratory for those parameters. Another limitation to the present study includes measurement of total NAC and not reduced NAC. Thus, our measurements include oxidized NAC and all NAC conjugates. Reduced NAC levels may be more pharmacologically active. However, the pharmacokinetic portion of our study was specifically designed to determine overall exposure following dosing, and total NAC measurement is ideal in this regard. Finally, we did not include a NAC-only treatment arm in this pilot study, and given the number of studies using NAC for treatment of neurological disease –13, follTo our knowledge, this study represents: 1) the first reported use of probenecid as an adjuvant for medications unrelated to their antimicrobial activity; 2) the first CNS pharmacokinetic study of NAC and/or probenecid in children; and 3) the first CNS pharmacokinetic trial in pediatric TBI of any kind. Treatment with the combination of NAC and probenecid resulted in readily detectable CNS levels of both drugs in children with severe TBI. Given that adverse effects including undesirable physiological derangements and alterations in contemporary brain injury biomarkers related to administration of the drug combination were not observed, progression to a phase II/III trial appears warranted.S1 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


With limited sample sizes and single-institution designs, how complete response (CR) after neoadjuvant chemotherapy (NAC) influences breast conserving surgery (BCS) and its value in prognosis are not clear.Q test to determine heterogeneity.A systematic research review was conducted using electronic database. The rate of clinical complete response (cCR) in BCS after NAC and these pathological CR (PCR) and non-pCR BCS patients’ local recurrence-free survival (LRFS), distance recurrence-free survival (DRFS), overall survival (OS), and disease-free survival (DFS) rates were collected. A pooled analysis was performed using a fixed or random effects model and a Sixteen studies with a total of 4639 patients were included. The pooled data revealed that cCR patients compared with non-cCR patients had significantly higher rates of BCS, with a summary estimate odds ratios (OR) of 4.54 (95% CI 2.03–10.17). The pooled data revealed that BCS patients who achieved pCR after NAC had significantly lower rates of LRFS  and DRFS . Better DFS  and OS  were also seen, but OS was not significantly different.The rate of successful BCS is higher in the cCR group than in the non-cCR group, means cCR after NAC can encourage patients to receive BCS. The achievement of pCR after NAC in BCS patients was associated with a good prognosis in terms of LRFS and DRFS, but its value in DFS and OS requires further investigation.The online version of this article (10.1186/s12957-017-1273-6) contains supplementary material, which is available to authorized users. Breast cancer is known as the most common cancer with the second highest mortality in females . RecentlThe survival benefit for patients who achieve pCR after NAC is controversial. Some studies found that pCR patients have a better prognosis than non-pCR patients but Cortazar P put out in his meta-analysis that in trial-level studies, the association between pCR and both event-free survival (EFS) and OS was weak , 9. RecuHow CR influences BCS is unclear because of limited high-quality data. For these reasons, we collected data on diagnosis, treatment, and prognosis from patients who received BCS after NAC and pooled the results for analysis in this study. The goal was to explore the rate of BCS in cCR compared to non-cCR patients after NAC and if other factors influenced this outcome. We also investigated whether pCR is a prognostic factor in BCS patients relative to LRFS, DRFS, DFS, and OS compared with BCS patients who did not achieve pCR. The potential prognostic value of pCR status may be very useful in BCS patients who received NAC because it can help in the determination of follow-up treatments and monitoring.We carried out a systematic literature search using PubMed, Embase, and the Web of Science through June 20, 2017. The search headings were as follows: (breast conserving surgery OR breast conserving operation OR lumpectomy OR quadrantectomy) AND  AND (exp breast neoplasms OR cancer OR neoplasm OR carcinoma OR exp. neoadjuvant therapy OR neoadjuvant OR exp. preoperative care) AND . In all, we found 1587 articles, including 247 from PubMed, 865 from the Web of Science, and 475 from Embase  a focus on breast cancer patients who were treated with neoadjuvant chemotherapy and then received surgery, (2) the presence of evaluations of the associations between CR and outcomes in patients who received either breast conserving surgery or mastectomy surgery, (3) the presence of the necessary information to calculate outcomes  of interest, and (4) availability as a full-text English-language publication. When multiple publications from the same author or institution appeared that presented duplicate data, we chose the most applicable one. Systematic reviews, meta-analyses, case studies, and articles that did not meet our terms were excluded.The following information was extracted from each eligible study: the first authors’ names, the country in which the data were collected, the publication year, the applied neoadjuvant chemotherapy regimens, the total number of patients, mean age, tumor size, follow-up time, the number of patients who received BCS, the number patients who achieved CR, and the survival data (percentages and numbers of events) of BCS patients who achieved CR and those who did not. Other information was also recorded if the author found it useful. We used The Newcastle-Ottawa Scale (NOS) to assess the quality of each study by two authors. The scores included three parts: the selectivity of patients (0–4), comparability of groups (0–2) and assessment of outcome (0–3). Scores > 5 were considered high-quality studies.Q statistic and the I2 value presented significant homogeneity as I2 > 50% or Cochran Q < 0.1, a random effects model was used. If significant heterogeneity appeared, a subgroup analysis according to neoadjuvant chemotherapy type and a sensitivity analysis to exclude unsuitable studies were used to determine the source of heterogeneity.A meta-analysis was conducted for each endpoint of interest when there were two or more studies available. The data were pooled with the fixed-effects model by default, but if homogeneity across the tests was identified by the Using the data on the extent to which CR influences BCS conduct, we analyzed each study and calculated pooled odds ratios (OR) and 95% CIs. LRFS, DRFS, DFS, and OS were compared between the CR group and non-CR group of BCS patients using risk ratios (RR) and 95% CIs. A 95% CI that did not include zero was considered significantly different. A power analysis was performed for each study, and the pooled results were evaluated using Power and Precision software following the manufacturer’s instructions.P < 0.05.We investigated whether funnel plots presented symmetry to generally assess the publication bias. All the collected data were analyzed using RevMan 5.3 analysis software , and all statistical tests were two-sided with statistical significance defined as We identified 1587 records, and through careful assessments, 16 studies were identified as eligible for the data abstract , 14–28. I2 = 85% with P < 0.001 . Heterogeneity testing revealed I2 = 7% with P = 0.38 .Eleven studies reported LR rates in BCS patients (2197 patients). In BCS patients, the overall LR rate was 3.9% (range 0–10.8%) in pCR patients and 8.1% (range 2.3–17.2%) in non-pCR patients. The pooled data revealed that the BCS patients who achieved pCR after neoadjuvant chemotherapy had significantly lower rates of LR, with a summary estimate RR of 0.59 (95% CI 0.38–0.92). Heterogeneity testing revealed I2 = 2% with P = 0.38 . In BSC patients, the overall DR rate was 3.5% (varied 0–14.3%) in pCR patients and 21.5% (varied 10.8–31.0%) in non-pCR patients. The pooled data revealed that BCS patients with pCR had significantly lower DR rates, with a summary estimate RR of 0.27 (95% CI 0.13–0.55). Heterogeneity testing revealed I2 = 0% with P = 0.80, Additional file I2 = 13% with P = 0.28, Additional file Two studies that reported LR data and another two studies that reported both LR and DR data were excluded by the sensitivity analysis because of the pCR definition they used, which was the absence of an invasive component in the primary breast tumor instead of in both the breast and the axillary node. However, the advantage of pCR in BCS remained significantly different, with an RR of 0.44 (95% CI 0.22–0.86, heterogeneity testing I2 = 0% with P = 0.84, Fig. I2 = 63% P = 0.10, Fig. Only two studies provided DFS and OS rates in pCR and non-pCR patients who received BCS. The median follow-up times were 66 and 46 months. We evaluated these two studies in the meta-analysis and found that DFS rates were higher in BCS patients with pCR, with a summary estimate RR of 0.09 (95% CI 0.04–0.25, heterogeneity testing The publication bias was evaluated with funnel plots. The funnel plots showed no obvious evidence for a publication bias as the plot was basically inverted and funnel-shaped with bilateral symmetry Fig. .Fig. 4FuRecently, NAC has been widely used in breast cancer patients to downstage tumor size, increase the rate of resection, or increase eligibility for breast conserving surgery , 30. BCSBCS rates reportedly increased overall after NAC . In our Higher response and safe of BCS after tumor downstage can inspire more patients to receive BCS .LRFS and DRFS were not significantly different between the BCS and MT groups , 35 as wIn our study, the DFS rates were significantly higher in BCS patients with pCR. OS was higher in BCS patients with pCR, but no significant differences were observed. In another meta-analysis that included BCS and MT patients, pCR achievement following NAC indicated a good prognosis in terms of DFS and OS , 44. BecThere are some limitations in our study. First, this analysis relied on a slightly different definition of pCR. Although we separately analyzed pCR only in the breast, pCR definition was still somewhat different compared to some studies that included in situ contents and some that did not. Second, adjuvant therapy and BCS methods including lumpectomy or quadrantectomy with sentinel lymph node biopsy or axillary dissection were mixed and matched according to the judgment of the doctor, without a united protocol. Almost all patients who received BCS also received radiotherapy, but different doses, regions, and methods were used. Adjuvant chemotherapy was administered to some patients, especially non-pCR patients. More effective treatments were used in non-pCR patients, and this may offset the advantage of pCR values in BCS eligibility. Third, the data in this study were limited. We calculated the power of each study and the pooled power, and all values were higher than 0.8, except for the data on OS. Less data was available on OS, and the follow-up times were short, so the acceptance of these results warrants caution and critical thinking.Successful conduct rate of BCS was higher in the cCR group than in the non-cCR group; however, the relationship of cCR and pCR needs more research. BCS patients who achieved pCR after NAC demonstrated a good prognosis in terms of LRFS and DRFS; though also seem favors in DFS and OS, the value of pCR in long-time survival of BCS needs further investigation.


The mean LV ejection fractions (LVEF) were 62.7, 62.6, 60.8, and 53.8%, respectively, whereas the prevalence of AF was 10.4%, 10.5%, 14.8%, and 16.8% in patients with normal geometry, concentric remodeling, concentric hypertrophy, and eccentric hypertrophy, respectively. In conclusion, the prevalence of AF was increasing according to LV geometric remodeling patterns in association with LA size and LVEF.This study investigated the relationship between atrial fibrillation (AF) and left ventricular (LV) geometric patterns in a hospital-based population in Japan. We retrospectively analyzed 4444 patients who had undergone simultaneous scheduled transthoracic echocardiography (TTE) and electrocardiography during 2013. A total of 430 patients who had findings of previous myocardial infarctions (n = 419) and without the data on body surface area (n = 11) were excluded from the study. We calculated the LV mass index (LVMI) and relative wall (RWT) and categorized 4014 patients into four groups as follows: normal geometry (n = 3046); concentric remodeling ; concentric hypertrophy ; and eccentric remodeling . The mean left atrial volume indices (LAVI) were 22.5, 23.8, 33.3, and 37.0 mm/m However, as LVH progresses, it is associated with considerable cardiovascular (CV) morbidity and mortality3. Recent studies have focused on the prognostic impact of more subtle LV geometric abnormalities. Patterns of LVH and geometric remodeling have previously been investigated extensively in patients with hypertension and valvular heart diseases5. Concentric LVH has a high mortality risk with preserved ejection fraction (EF)7 or a high mortality risk in patients without regression of abnormal LV geometry8; other studies have reported that relative wall thickness has less impact on prognosis in patients with coronary heart disease3.Left ventricular (LV) hypertrophy (LVH) may be considered a compensatory effect since increasing LV wall thickness reduces LV wall stress9. Atrial fibrillation (AF) is more prevalent among older people with pronounced morbidity and mortality13. AF can cause heart failure, affect quality of life, lengthen the hospitalization period, and increase mortality14. Left atrial (LA) remodeling, LA enlargement, and LV remodeling are related to AF development15. However, the association between LV geometric patterns, LA enlargement, and AF remains unknown. Therefore, in this study, we investigated the association between LV geometric patterns, LA enlargement, and AF.LVH and LV dilatation cause an in increase in end-diastolic pressure, followed by enlargement of the left atriumWe retrospectively analyzed 4444 patients who had undergone simultaneous scheduled transthoracic echocardiography (TTE) and electrocardiography (ECG) at the Cardiovascular Center of Kitano Hospital during 2013. A flowchart of the study population is shown in Fig. The research protocol was approved by the institutional review board of Kitano Hospital . Informed consent was not obtained from each patient since this was a retrospective study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee. Patients’ records and information were anonymized and de-identified before analysis.From the TTE database, we extracted data for patients’ wall thickness, LV diastolic diameter (LVDd), E/e’, LA diameter (LAD), LA volume index (LAVI), and LV ejection fraction (LVEF). We also extracted the body surface area from the TTE report. From the ECG database, we extracted cardiac rhythm and recorded it as it was documented. Therefore, we could not determine whether the AF was paroxysmal or persistent.3 − (LVDd)3] + 0.6, where LVDd was the LV diastolic diameter, IVSTd was the diastolic interventricular septal wall thickness, and LVPWTd was the diastolic LV posterior wall thickness. In line with the ASE recommendations, a high LV mass index (LVMI) was defined as >115 g/m2 for male patients and >95 g/m2 for female patients. Relative wall thickness (RWT) was calculated using the following formula: (2 × LVPWTd)/(LVDd), which permits physicians to categorize an increase in the LV mass as either concentric (RWT >0.42) or eccentric (RWT ≤0.42) hypertrophy and identify concentric remodeling 17. The LAVI was calculated using the biplane area-length method and body surface area and defined high as a value >42 mL/m215. Data from two-dimensional TTE were analyzed at baseline. The LVEF was measured using the Teichholz method or the modified Simpson rule method and defined low as LVEF <50%.The LV mass was calculated using the formula recommended by the American Society of Echocardiography (ASE), and it was indexed to the body surface area as follows: LV mass = 0.8 × 1.04 [(LVDd + LVPWTd + IVSTd)We then categorized 4014 patients into four groups as follows , hyperlipidemia (ICD-10 code E78), diabetes mellitus , and chronic kidney disease (CKD) (ICD-10 code N18), from electronic medical data at our institution.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.2). The adjusted odds ratios and 95% confidence intervals were calculated. To show the association of two variables , we presented scatter plots, lines of best fit, and correlation coefficients. To visualize the effect size of each variable, the LogWorth ) scales were presented18. In LogWorth sclaes, a highly significant p-value had a large LogWorth value and a nonsignificant p-value had a low LogWorth value18. We generated the second multivariable regression model (model 2) including the LA size and LVEF to visualize the effect size of the LA size and LVEF as the LogWorth scales. In addition, we generated a multivariable logistic regression model using the same variables in AF for adjustment to analyze factors associated with a high LAVI and a low LVEF. Finally, from a logistic regression model with nominal responses (AF or not) using LVEF and LAVI, we generated a formula for linear combinations of the response levels (x = a + b x LVEF + c x LAVI) and prediction formulas for the response levels predicting the presence of AF (1/ (1 + Exp(x)) and the absence of AF (1/(1 + EXP(-x)), then we compared the two levels and predicted the presence or absence of AF according to the larger response level. We showed a receiver-operating curve with an area under the curve and provided the sensitivity and 1-specificity of this prediction formulas.Categorical variables are presented as numbers and percentages, and were compared using the chi-square test or Fisher exact test. Continuous variables are expressed as a mean ± standard deviation or median (interquartile range). Based on their distributions, continuous variables were compared using the Student t-test or Wilcoxon rank-sum test. To analyze factors associated with AF, we used a multivariable logistic regression model (model 1) involving the following potential independent, clinically relevant variables: age >70 years; sex; LV geometric remodelings that was defined as ordered variables ; and the presence of comorbidities such as ishemic heart disease, hypertension, diabetes mellitus, hyperlipidemia, CKD and overweight .Baseline characteristics of patients are provided in Table 2 in patients with a normal geometry, concentric remodeling, concentric hypertrophy, and eccentric hypertrophy, respectively.Baseline characteristics of echocardiographic findings are provided in Table Overall, 3460 patients had a normal sinus rhythm, 455 had AF, 95 had a pacemaker rhythm, and 15 had other rhythms. The prevalence rates of AF were 10.4, 10.5, 14.8, and 16.8% in patients with a normal geometry, concentric remodeling, concentric hypertrophy, and eccentric hypertrophy, respectively  in end-diastole during sinus rhythm15. Previous work has highlighted the complex mechanisms contributing to AF progression23, as the morphological, electrical, or neurohormonal remodeling along with the ventricular responses were related. Diastolic impairment and an increasing diastolic filling pressure are related to the LA size. With an increase in LA filling pressures, the atrial wall stretches and enlarges. In this study, overweight was not significantly associated with AF, which was inconsistent with the previous studies24. One of the reasons may be the differences in baseline characteristics in Japanese population and in Western population. Body weights in Japan are much lower than those in the US and Europe. Other possible reason may be due to small numbers studied in our study, considering that a marginally significance existed.Concentric remodeling and hypertrophy were often caused by pressure overload that increased the RWT. This was an adaptation to normalize the systolic wall stress. However, LV was shown to be a substrate for LV diastolic dysfunction in concentric remodeling and an incremental risk for diastolic heart failure in concentric hypertrophy. Direct underlying triggers of AF was mainly due to atrial remodeling. Atrial remodeling is caused by multiple factors23. Consequently, LA enlargement in eccentric hypertrophy is suggested to be highest among groups with pressure and volume overload. A larger LA volume is associated with a higher risk of AF15. In addition, patients with AF and patients with a larger LA showed a higher LVMI and a low LVEF in a sub-study of the AFFIRM trial25. In our study analysis of both AF and non-AF populations, LA enlargement and LVEF were significantly related to LV geometric remodeling. Therefore, the rate of AF was increasing per the LV remodeling patterns. This was true for patients with non-valvular AF. The prevalence of LVH was high in patients with non-valvular AF26. Patel et al. reported that LAVI was associated only with LVMI, not RWT21. This is consistent with our present study which showed that the relationship between LA size and RWT was different for normal and high LVMI.Eccentric hypertrophy is typically induced by volume overload, such as in valvular heart disease or systolic heart failure. The prevalence of systolic dysfunction is mostly noted in eccentric hypertrophy. Systolic dysfunction affects LV geometry through enlargement of the LV chamber. A large LV and low LVEF cause congestion of the left atrium26. Thromboembolic events may be one of the mechanisms for the higher mortality in LVH27. Although there are reportedly many factors related to AF and LA enlargement, such as age, the presence of hypertension, and obesity24, LV geometric remodeling has an impact on the presence of AF through the association with LA enlargement and a low LVEF. However, atrial fibrillation can develop without left atrial enlargement28. Atrial size increases with time in patients with atrial fibrillation, without any structural or functional abnormality of LV and the valve28. Since our study is cross-sectional, we could not identify the cause-effect relationship in each patient. Longitudinal follow-up could address this issue in each patient.In addition, LAVI and LVEF accurately predicted the presence of AF in our study; therefore, we performed multivariate analyses excluding these co-linear factors. A linear association with LVMI and LA enlargement was observed; however, the effect of RWT on a low LVEF was different between patients with a normal LVMI and those with a high LVMI. Because LV geometric remodeling was clearly associated with LA enlargement and a low LVEF, a schematic in which that LV morphology was linked to the LV function and LA size is provided Fig. . Further29 and severe AS patients30. In our study, we provided the link between LV geometric patterns and AF prevalence through the contribution of atrial enlargement and decreased LVEF. AF and LA enlargement is a risk factor for ischemic stroke and may contribute to doubling all-cause31 and CV deaths14, and, in a specific condition, non-CV deaths32. However, anticoagulants were sub-optimally prescribed in these patients. Attention should be paid to paroxysmal AF in the LVH population to improve our daily practice.In clinical practice, LVH is a risk factor in hypertensive patients24. Third, we did not consider the effect of valvular diseases such as mitral valve regurgitation. Fourth, patients with short-lasting paroxysmal AF were excluded, because we included patients with AF when they had an AF rhythm according to an ECG examination. Lastly, the data presented provide only hypothesis-generating associations between LV geometry and AF. Since this study had a cross-sectional design, a cause-effect relationship could not be assessed. Further prospective studies are needed to determine the association of LA remodeling, LV geometric changes, LV function, mortality, and the time-course for the development of AF.There are several limitations to the present study. First, the subjects are heterogeneous because of the TTE and ECG findings. The ordering criteria for ECG and TTE were not set. Second, patients’ data were extracted from electronic medical data. A lack of information regarding exercise was an important limitationThe prevalence of AF was increasing according to LV geometric remodeling patterns in association with LA size and LVEF.


Treatment of Chiari malformation can include suboccipital decompression with resection of one cerebellar tonsil. Its effects on ocular motor and cerebellar function have not yet been systematically examined.To investigate whether decompression, including resection of one cerebellar tonsil, leads to ocular motor, vestibular, or cerebellar deficits.Ten patients with Chiari malformation type 1 were systematically examined before and after (1 week and 3 months) suboccipital decompression with unilateral tonsillectomy. The work-up included a neurological and neuro-ophthalmological examination, vestibular function, posturography, and subjective scales. Cerebellar function was evaluated by ataxia rating scales.Decompression led to a major subjective improvement 3 months after surgery, especially regarding headache (5/5 patients), hyp-/dysesthesia (5/5 patients), ataxia of the upper limbs (4/5 patients), and paresis of the triceps and interosseal muscles (2/2 patients). Ocular motor disturbances before decompression were detected in 50% of the patients. These symptoms improved after surgery, but five patients had new persisting mild ocular motor deficits 3 months after decompression with unilateral tonsillectomy  without any subjective complaints. Impaired vestibular  function improved in five of seven patients with impaired function before surgery. Posturographic measurements after surgery did not change significantly.Decompression, including resection of one cerebellar tonsil, leads to an effective relief of patients’ preoperative complaints. It is a safe procedure when performed with the help of intraoperative electrophysiological monitoring, although mild ocular motor dysfunctions were seen in half of the patients, which were fortunately asymptomatic. Chiari malformation comprises a group of pathological entities characterized by the presence of anatomical deformities of the cerebellum and brainstem. Typical symptoms include occipital headaches, which are exacerbated by coughing; motor and/or sensory disturbances especially of the upper limb; and clumsiness , 2.Current treatment includes a decompression of the suboccipital region to enlarge the diameter of the foramen magnum and resection of the posterior arch of C1. But there has been much critical discussion of whether one should open the arachnoid and remove the cerebellar tonsils as well . The posExperiences with cerebellar lesions, i.e., due to ischemic infarcts, teach us that tonsil lesions can lead to significant ocular motor deficits causing disabling vertigo . HoweverFourteen consecutive patients with radiological evidence of Chiari malformation type 1 were screened for this study in the Departments of Neurology and Neurosurgery of the University Hospital Munich. Three patients had mild clinical signs and were treated conservatively. Eleven underwent surgical intervention with resection or reduction of one cerebellar tonsil and were included in the study. One patient was lost to follow-up.Patients were examined before and at two time points after surgical intervention: one directly after and one in follow-up approximately 3 months after surgery. Patients received a standardized questionnaire for typical complaints. Patients were asked about headache or neck pain (type and provoking factors), gait unsteadiness, vertigo or dizziness (type and provoking factors), visual problems, audiological problems, motor signs, sensory deficits, and lower cranial nerve deficits.®  and air-conducted cervical and bone-conducted ocular vestibular-evoked myogenic potentials (c/oVEMP) using the Interacoustics Eclipse System® , head-shaking nystagmus, and determination of the subjective visual vertical (SVV). Deviations above 2.5° were set as pathological . Cerebel System® . The asy System® . Posturo System® . Subject System® , 16. TheThis study was carried out in accordance with the recommendations of the Institutional Review Board of the ethics committee of the Ludwig-Maximilian University Munich with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board of the ethics committee of the Ludwig-Maximilian University Munich. Indication for surgical intervention was provided independent of participation in this study.3 . Finally, the region of the obex was inspected to provide free outflow of the central canal within the spinal cord whenever safely possible. To further enlarge the craniocervical space, a dural substitute  was implanted and sutured in a watertight fashion. Finally, the wound was meticulously closed in several layers, e.g., muscles, fascia, subcutis, and cutis to prevent CSF leakage.Patients underwent intraoperative electrophysiological monitoring for lower cranial nerves, motor-evoked potentials, and somatosensory-evoked potentials in order to avoid an additional effect on the brainstem as a result of exaggerated head inclination. In the prone position, the patient’s head was fixed in a Mayfield clamp. Surgical intervention consisted of a suboccipital medial dissection of the neck muscle exposing the arch of C1 and the occiput around the foramen magnum. First, the foramen magnum was enlarged by osteoclastic craniectomy of approximately 3 cm × 4 cm, the width adapted to the size of the foramen magnum. Thereafter, a laminectomy of the arch of C1 was performed to the same width of the spinal canal. Intraoperative ultrasound allowed the visualization of the tips of the tonsils in order to decompress as far down as necessary. Sometimes a laminotomy of the upper rim of the C2-arch was necessary. C2 laminectomy was never performed in order to avoid cervical instability. Then, the dura was opened in a “Y” fashion and stitched to the periosteum of the occiput. After careful dissection of the arachnoid, the longer and larger cerebellar tonsil as well as the lower brainstem and the spinal cord were identified. The identified tonsil was first shrunk by coagulation of the outer and medial surface without affecting intracranial vessels. The pia of the coagulated tonsil was incised and tonsillar tissue removed by gentle suction and bipolar coagulation. Finally the reduced tonsil laid at the level of the foramen magnum. The mean volume of tonsillar tissue resected ranged from 0.6 to 2.7 cmU-test for between-group comparisons. Differences were considered significant if p < 0.05. This study design was exploratory.Statistical analysis was performed using SPSS Statistics 20 . As data were not normally distributed, non-parametric paired testing was performed using the Wilcoxon matched pairs test to compare two related samples and the Mann–Whitney n = 10) and at two time points after surgical intervention [time point 1  and time point 2 ]. For further details on clinical data, see Table Ten patients with a Chiari malformation type 1 confirmed by MRI  were included in this study. The following values are mean values ± standard deviation. Participants were examined preoperatively , headache, and neck pain, which worsened during coughing and specific head movements (50%) and dys-/hypesthesia in upper limbs (50%).In terms of clinical–neurological characteristics, the main findings were ataxia in the upper limbs  (6/10 patients) and dys- and hypaesthesia (5/10 patients). For further details of the clinical examination, see Table n = 3), gaze-evoked-nystagmus, disturbed fixation suppression of the VOR, pathological head impulse test (each n = 2), deviation of SVV, head tilt, hypermetric saccades, rebound nystagmus, saccadic smooth pursuit, and downbeat nystagmus (DBN) (each n = 1). For further information, see Table In the neuro-ophthalmological examination, five patients had abnormal ocular motor findings including head-shaking nystagmus . Mean 8MW was 5.1 ± 0.5 s. 9-Hole peg test (9HPT) of the dominant hand was 20.1 ± 2.2 s and of the non-dominant hand 21.3 ± 2.6 s. The mean PATA rate was 27.9 ± 3.9. For further information see Table Nine out of 10 patients underwent caloric testing before surgical intervention, eight o/cVEMP. Six patients had abnormal vestibular function in these tests: three patients  had a pathological side difference in caloric testing, suggesting a deficit in horizontal canal function; three other patients had pathological o/cVEMP testing, indicating a deficit in otolith function .The total mean DHI was 17 (range 0–70), suggesting a mild handicap. For further information, see Table Patients had a tendency to poorer performance in posturography compared to healthy controls published in other studies in our center . StandinShort-term follow-up examination was performed in five patients. New documented deficits included finger-chase dysmetria  and gait ataxia (no. 4). This could be confirmed in ataxia scores .Neuro-ophthalmological examination revealed a deviation of the SVV to the right after resection of the left tonsil (patient no. five 5.2° and patient no. ten 4.5°) and new smooth pursuit deficits (no. 1). For further information, see Table Sufficient suboccipital decompression led to improvement of symptoms  in 9/10 of patients in follow-up without influence of the side of the resected tonsil. Two patients were free of complaints after the neurosurgical intervention no. 1, 2). Symptoms improved in seven patients . One patient had major complications due to CSF leakage and subsequent meningitis. He needed surgical revision for dural leak, and symptoms improved slowly over several weeks (no. 9). In follow-up, he had a residual mild right-sided hemiparesis. There were no other complications in any of the other patients. The size and length of the preexisting syringomyelia decreased in all patients. For further information, see Tables . Symptomp = 0.018; 9HPT from 20.7 ± 2.2 to 19.8 ± 2.3; p = 0.028 ]. Speech measured with the PATA test remained unchanged . The same applies to the SARA score . For further information, see Table Especially hyp-/dysesthesia (5/5 patients), ataxia of the upper limbs (4/5 patients), and paresis of triceps and interosseal muscles (2/2 patients) improved in the follow-up examination after surgical intervention. Fine motor skills and gait improved significantly [SCAFI 8MW from 5.1 ± 0.5 to 4.6 ± 0.6 s; Neuro-ophthalmologically, head-shaking nystagmus, VOR, ocular tilt reaction, and DBN improved in all affected patients. A partial improvement could be observed in gaze-evoked nystagmus and fixation suppression of the VOR.New ocular motor disturbances after surgery were smooth pursuit deficits , DBN no. 4), and gaze-evoked nystagmus and rebound nystagmus (no. 9). All of these patients were asymptomatic. Patient number 9 with major postoperative complications had further new vestibulocerebellar dysfunctions, including gaze-evoked nystagmus, rebound nystagmus, vertical smooth pursuit deficit, and vestibular deficit in the caloric testing and reported more impairment in the DHI. For further information, see Table , and gazp = 0.273), especially based on two patients , who had a significant improvement of impairment due to vertigo and dizziness. For further results, see Table The two patients with pathological cVEMP improved, as did one out of three patients with abnormal caloric testing. The mean DHI improved from 17 to 6 after surgical intervention  did not change significantly. There was no typical cerebellar 3 Hz-sway. For further information, see Table The major findings of this prospective study are as follows.First, preoperative neuro-opthalmological examination revealed abnormal findings in 50% of patients, including head-shaking nystagmus, gaze-evoked-nystagmus, disturbed fixation suppression of the VOR, deviation of the SVV, head tilt, hypermetric saccades, rebound nystagmus, saccadic smooth pursuit, and DBN. 70% of patients had abnormal vestibular function: two patients had a pathological head impulse test indicating a high-frequency deficit of the VOR, three patients had a pathological caloric test testing the low-frequency function of the VOR and three had abnormal otolith function tested with vestibular-evoked myogenic potentials.Patients had only mild to moderate cerebellar signs . The clinical symptoms of our Chiari patients were similar to those in other studies , 2, 18 eSecond, surgical intervention including tonsillectomy led to subjective and objective improvement of the symptoms in 9/10 of patients. Fine motor skills and gait measured with SCAFI improved significantly. Impaired vestibular  function improved in five of seven patients with impaired function before surgery, especially in terms of high-frequency VOR function and saccular otolith function. Ocular motor disturbances improved in all affected patients in terms of head-shaking nystagmus, VOR, ocular tilt reaction, and DBN. In some patients, further improvement could be observed in terms of gaze-evoked nystagmus and fixation suppression of the VOR.The subjective improvement in our series exceeds that of case series and reviews which generally show an improvement of around 75% . HoweverThird, neuro-ophthalmological examination after suboccipital decompression, including tonsillar reduction, revealed new, but mostly asymptomatic, ocular motor deficits in five patients, including smooth pursuit deficits, horizontally gaze-evoked nystagmus, rebound nystagmus, and DBN. Contralateral (to the side of tonsillar reduction) deviation of the SVV was temporary in 20% of patients and was compensated in follow-up. Previous retrospective studies about vestibulo-ocular manifestations in Chiari malformation or syringomyelia showed a remarkable resolution of ocular motor disorders of 75–100% after decompression without tonsillectomy  compared to retrospective studies , confirming the anatomical role of the tonsil in smooth pursuit and the gaze holding network; however, symptoms are mild and asymptomatic in our Chiari patient cohort.This study was carried out in accordance with the recommendations of the Institutional Review Board of the ethics committee of the Ludwig-Maximilian University Munich with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board of the ethics committee of the Ludwig-Maximilian University Munich. Indication for surgical intervention was provided independent of participation in this study.NG: research project , statistical analysis , manuscript preparation . KF: research project , manuscript preparation (review and critique). FI and MK: research project (execution), manuscript preparation (review). SK: research project (interpretation of the data), manuscript preparation (review). JT: research project , manuscript preparation (review and critique). MS: research project , manuscript preparation (review and critique) including medical writing for content. AP: research project , manuscript preparation (review and critique) including medical writing for content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the therapeutic effect of mycophenolate mofetil(MMF) on Chinese Takayasu’s arteritis(TAK) patients. Thirty consecutive TAK outpatients were prospectively enrolled during 2013 to 2015. MMF combined with glucocorticoid was the primary treatment regimen. If clinical stable disease could not be reached, another traditional immunosuppressive agent could be added. All patients were evaluated and followed up every 3 months and vascular image studies by Doppler ultrasonography were repeated every 6 months. The effectiveness of MMF was defined as:(1) ESR < 20 mm/hr;(2) CRP < 10 mg/L or hs-CRP<3 mg/L;(3) stable or improved in vascular image studies;(4) clinical assessment is stable, improved or in remission;(5) the dosage of glucocorticoid could be tapered to less than 15 mg/day. ESR < 40 mm/hr, CRP < 20 mg/L or hs-CRP < 6 mg/L, but meet the other three criteria is defined as partial effectiveness. MMF alone combined with corticosteroid was effective in 12(40.0%) patients. When MMF combined with methotrexate less than 15 mg/week, the effective rate was 30.0%(9/30), including partial effective in 3 patients. When MMF combined with azathioprine 100–150 mg/day, the effective rate was 10.0%(3/30), including partial effective in 1 patient. Four patients withdrew due to side effects. Two patients failed to show response. The overall effective rate of therapy including MMF in treating TAK is 80%. Takayasu’s arteritis (TAK) is a rare, chronic, granulomatosis idiopathic systemic vasculitis mainly involves aorta and its major branchesApproximately 50% of TAK patients are sensitive to the treatment of glucocorticoids (GCs)Mycophenolate mofetil (MMF) has been reported in a few studies as a useful alternative for TAK treatment1011Thirty TAK patients were treated with MMF in this study. The demographic and baseline data were summarized in Malaise (63.3%) and headache (53.3%) were the common complaints. Vascular bruit (83.3%) was the most common findings in physical examination in this group of patients, followed by asymmetric blood pressure (80.0%), pulsation (66.7%) and hypertension (56.7%). Elevated C-reaction protein (CRP) and/or high sensitive CRP (hs-CRP) (86.7%) were the most common abnormal laboratory tests, and elevated erythrocyte sediment rate (ESR) was found in 76.7% of patients. Mild anemia was found in 30.0% patients (10/30), in whom the level of hemoglobin was less than 110 g/L .The angiographic classificationSeventeen patients  received MMF as the first immunosuppressive agent, while the other thirteen patients received MMF as an alternative or additive immunosuppressive drug (ISD) due to uncontrolled disease activity. All patients received glucocorticoids (GCs) at the same time, and the dosage of steroids were tapered during the study period .The mean dosage of GCs when MMF was initiated was 40.0  mg in 24 patients who showed effectiveness during follow-up, and the mean dosage of GCs on last follow-up in these patients was 10.0  mg. The duration for dosage of GCs less than 15 mg was 12.0  months, and the duration of GCs less than 10 mg was 9.0  months. Three patients among them  stopped GCs at the last follow-up .In this study, 4 patients withdrew MMF due to adverse events. One patient developed skin rash 2 weeks after MMF administration, 2 patients complained of severe gastrointestinal discomfort, and HBV flare occurred in 1 patient. Fourteen patients received MMF and steroid combined with other immunosuppressive agent when their diseases was still active. The follow-up time after initiation of MMF in MMF combined with GCs group (median 24.5 months) was longer than that in patients with MMF combined with another immunosuppressive agent treatment group (median 11.0 months) . Excludi40.0% (12/30) patients in the MMF combined with GCs group could reach and maintain stable disease. With adding MTX less than 15 mg/week or AZA 100–150 mg/day, the dosage of steroid could be tapered to less than 15 mg/day in 12 patients (40.0%). Two patients failed to reach stable disease after being treated with combination of several ISDs including MMF . Thus thp = 0.014) and prior to initiation of MMF  were shorter in MMF combined with GCs group (n = 12) than in MMF combined with other ISD and GCs group (n = 12). And the follow-up time after initiating MMF was longer in MMF combined with GCs group .We compared the clinical features and angiographic types between patients who responded to MMF combined with GCs (n = 12), and MMF combined with other ISD and GCs (n = 12) . There wp = 0.024) and 10 mg  was longer in the former group.There was no significant difference in the dosage of GCs used when MMF treatment was initiated and on last follow-up between MMF combined with GCs group (n = 12) and MMF combined with other ISD and GCs group (n = 12). But the duration for the dosage of GCs tapered to less than 15 mg . The distribution of vessels involved between the MMF combined with GCs group (n = 12) and the combined treatment including MMF group (n = 12) was not significantly different .TAK is a chronic progressive large vessel vasculitis45781011As an inosine monophosphate dehydrogenase inhibitor, MMF could interfere DNA synthesis by inhibiting guanine nucleotide synthesis. Since lymphocytes depend primarily on de novo purine synthesis while neutrophils utilize salvage pathway, MMF inhibits proliferation of lymphocytes specifically16p = 0.005). And four patients in the combination regimen group is partial effective, demonstrating no progression of vascular lesions but with elevated level of ESR/CRP, which was lower than two times of the upper normal limit.In this study, we have demonstrated that MMF may be effective in controlling disease activity and vessel damage progression, either combined with low-dosage GCs or combined with MTX or AZA, with the total effective rate up to 80.0%. But in the combination of immunosuppressive agents (the combination regimen) and MMF group, the follow-up time after administration of MMF (median 11.0 months) is shorter than in MMF plus GC group (median 27.5 months) , the disease duration at diagnosis (median 5.0 months) and prior to commencing of MMF (median 10.0 months) were significantly shorter than in the combined regimen group (n = 12).The immunosuppressive effect of MMF in autoimmune disease was speculated by inhibiting lymphocyte-mediated immune process. Therefore, we may speculated that early administration of MMF in TAK patients may be beneficial in slowing down or preventing the disease progression.et al.et al.For the side effects of MMF, Shinjo Doppler ultrasonic imaging was repeated to measure and compare the thickness of the vessel walls and lumen diameter of vessels by one technician specialized in vascular ultrasonic examinations. As there is no widely accepted image modality to evaluate the disease activity of angiographic lesions, various image modalities have been used in different clinical trials. Magnetic resonance angiography (MRA)21The major limitation of this study is the relatively small sample size, though this is the largest cohort reported in the literature according to our knowledge. Another limitation is the relatively short follow-up time for a chronic disease as TAK. In this study, the median follow-up time was 17.0 months. As TAK is a chronic disease, so 17 months follow-up duration might not be long enough to provide solid evidence to assess disease progression. Perhaps further follow-up of these patients is needed to confirm the effectiveness of these regimens in delaying or halted the progress of the disease. Therefore, large, long-term follow up studies are needed to verify the effectiveness of MMF in TAK treatment.In summary, this study has demonstrated that, MMF, and MMF combined with MTX or AZA, when combined with daily low dose GCs, are tolerable and effective in 80.0% of TAK patients. Neither angiographic progression shown on Doppler ultrasonography nor toxicity of vital organ/system has been observed during follow-up. Therefore, we may conclude that MMF may be an effective alternative for disease control and tapering of steroid in the treatment of TAK. It offers a good choice for active TAK patients.Thirty consecutive TAK patients were treated with oral MMF, and were followed-up at the outpatient Vasculitis Clinic, Department of Rheumatology of Peking Union Medical College Hospital (PUMCH), a Chinese nationwide referral center during January 2013 to September 2015. All patients fulfilled the classification criteria of modified 1990 American College of Rheumatology (ACR) criteriaThese patients received oral MMF, 1.5 to 2 gram per day, and were followed up for a minimum period of 6 months. All patients were evaluated and followed up every 3 months and vascular image studies were repeated every 6 months. Clinical manifestations of these patients were reviewed, including symptoms, physical findings, laboratory findings, and vascular involvement. Their medication records and response to MMF were categorially summarized. The effectiveness of MMF was defined as: (1) ESR < 20 mm/hr after treatment; (2) CRP < 10 mg/L or hs-CRP < 3 mg/L after treatment; (3) stable or improved in vascular image studies; (4) clinical assessment of disease activity is stable, improved or in remission after treatment; (5) the dosage of corticosteroid could be tapered to less than 15 mg/day . ESR < 40 mm/hr, CRP < 20 mg/L or hs-CRP < 6 mg/L, but fulfilled for the other three criteria was defined as partial effective. The primary end-point of this study is the percentage of patients with full and partial effectiveness.These patients were prospectively enrolled and followed-up every 3 months. The demographic data, disease duration, clinical symptoms, physical findings, disease activity status, laboratory findings, angiographic presentations, and treatment modalities, including medication regimens and adverse drug reactions, were collected at baseline and at each follow-up visit. During follow up, we repeated the examination of involved vessels by vascular Doppler ultrasonic imaging, measuring and comparing the thickness of the vessel walls and lumen diameter of vessels.http://www.chictr.org.cn) on Jul 25, 2016. The registration number is ChiCTR-OPC-16008907.The study protocol was approved by Institutional Review Board of PUMCH, and all methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained for each participant. This study was registered in Chinese Clinical Trial Registry .Due to the small sample size of this study, the variables were not distributed in a normal pattern. We described the numerical variables as median (quartiles), and the categorical variables as number (percentage). Comparisons between groups were made using Mann-Whitney U test for numerical data, and Chi-square tests for categorical data. Fisher’s exact tests were performed when the expected frequencies were less than 5. A two-sided How to cite this article: Li, J. et al. The efficacy of Mycophenolate mofetil for the treatment of Chinese Takayasu’s arteritis. Sci. Rep.6, 38687; doi: 10.1038/srep38687 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Oribacterium and Fusobacterium could distinguish LC patients from healthy subjects. LEfSe outputs show microbial gene functions related to categories of nickel/iron_transport, amino_acid_transport, energy produced system and metabolism between LC patients and healthy subjects. These findings firstly identify microbiota dysbiosis of tongue coat in LC patients, may providing novel and non-invasive potential diagnostic biomarker of LC.Liver carcinoma (LC) is a common malignancy worldwide, associated with high morbidity and mortality. Characterizing microbiome profiles of tongue coat may provide useful insights and potential diagnostic marker for LC patients. Herein, we are the first time to investigate tongue coat microbiome of LC patients with cirrhosis based on 16S ribosomal RNA (rRNA) gene sequencing. After strict inclusion and exclusion criteria, 35 early LC patients with cirrhosis and 25 matched healthy subjects were enrolled. Microbiome diversity of tongue coat in LC patients was significantly increased shown by Shannon, Simpson and Chao 1 indexes. Microbiome on tongue coat was significantly distinguished LC patients from healthy subjects by principal component analysis. Tongue coat microbial profiles represented 38 operational taxonomic units assigned to 23 different genera, distinguishing LC patients. Linear discriminant analysis (LDA) effect size (LEfSe) reveals significant microbial dysbiosis of tongue coats in LC patients. Strikingly,  Liver carcinoma (LC) is a common malignancy worldwide, which is associated with high morbidity and mortality368The oral cavity is closely associated with the external environment, and harbours the most diverse microbiome that includes representatives of the phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes, Synergistetes and TenericutesThe tongue is a “mirror” of the body. A principal diagnostic method of Traditional Chinese Medicine (TCM) is the inspection of the tongue, which examines the shape, size, colour, and texture of the tongue body and coat and helps reveal the state of organ function and progression of diseasesOur aims were to explore the relationship between microbial diversity and LC to contribute to the prevention of LC and to improve patients’ outcomes. For this purpose, here we conducted the first analysis, to our knowledge, of 16S ribosomal RNA gene sequences of microbes present in the tongue coat of patients with LC with cirrhosis.p < 0.001). Serum concentrations of albumin and total protein were significantly decreased in the LC patients versus healthy controls . Serum levels of total bilirubin and direct bilirubin were significantly increased in the patients . The number of platelets was significantly reduced in the patients (p < 0.001). The details of the clinical characteristics of all subjects are shown in After strict inclusion and exclusion criteria, this study finally enrolled 35 patients in the early stages of LC patients with cirrhosis who were diagnosed according to the Barcelona Clinic Liver Cancer (BCLC) staging classification as well as 25 healthy subjects. There were no significant differences between the groups in age, sex distribution, and body mass index. We diagnosed 15 of 35 (42.9%) patients and 20 (57.1%) with very early and early stages of LC, respectively. Of these patients, 21 (60%) had <20 ng/ml serum alpha-fetoprotein, and the remainder (40%) had >20 ng/ml. All patients with LC were diagnosed with cirrhosis and were hepatitis B virus soluble antigen (HBsAg)-positive without complications. Serum levels of alanine aminotransferase, aspartate aminotransferase and glutamyltranspeptidase were significantly elevated in patients   to generate 466,000 filtered sequences. Rarefaction curves of numbers of observed operational taxonomic units (OTUs) per sample showed that the mean number of observed OTUs reached a plateau of approximately 2500 sequence reads , and theameters) .The unweighted (quantitative)  and weigAll qualified reads  clustered into qualified OTUs that clustered with randomly chosen qualified reads. Using 97% as the similarity cutoff, 158 qualified species-level OTUs were delineated, while 50 OTUs were discarded because of their low coverage, and 97.6% and 91.6% of all reads were assigned into families and genera, respectively . The spePrevotella, Neisseria, Fusobacterium, Porphyromonas, Veillonella, Streptococcus and Haemophilus   .Pasteurellaceae, Streptococcaceae and Pseudomonadaceae were more abundant in the HT microbiome, and 13 were more abundant in the LCT microbiome, including Fusobacteriaceae, Leptotrichiaceae, Actinomycetaceae and Lachnospiraceae  > 3), whereas the HT microbiome was characterized by a preponderance of Bacteroidetes and Gammaproteobacteria (LDA Score [log10] > 3). Two microbial biomarkers (Oribacterium and Fusobacterium) showed a significant difference in abundance between patients with LC and the HCs (p < 0.001) with ROC (receiver operating characteristic curve)-plot AUC (the area under the parasitemia curve) values of 0.814 and 0.775, respectively (We used LEfSe to compare the estimated phylotypes of the LCT and HT microbiota . The LCTectively .http://picrust.github.com)We next used Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) ; liver and renal function tests; electrolyte concentrations; liver ultrasound findings and electrocardiogram and chest X-ray results. Exclusion criteria for volunteers included hypertension, diabetes, obesity, metabolic syndrome, irritable bowel syndrome, non-alcoholic fatty liver disease, coeliac disease and liver cirrhosis. All enrolled individuals who received antibiotics, probiotics or both within 8 weeks before enrolment, or consumption of unhealthy substances  were also excluded.We enrolled 25 matched healthy volunteers who met the inclusion criteria described in our previous studyWritten informed consent and questionnaires addressing previous and current diseases, lifestyles and medication  were obt3Before sampling, the subjects were asked to rinse and gargle twice with sterile water and extend their tongue as far as possible. The tongue was divided into six functional segments according to WinkelMicrobial DNA on the tongue coat was extracted using a Qiagen Mini Kit  following the manufacturer’s instructions as described previously335The universal target V3–V4 region of the 16s rRNA gene was PCR-amplified using primers 338F 5′-barcode-ACTCCTACGGGAGGCAGCA-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′, where barcode is an eight-base sequence unique to each sample. PCR reactions contained 4 μL of 5× Fast Pfu Buffer , 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of Fast Pfu Polymerase, and 10 ng of template DNA. Four PCR reactions were run for each sample in a thermocycler (Eppendorf Mastercycler) as follows: 95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s and a final extension at 72 °C for 10 min. PCR reactions from the same sample were pooled, purified using agarose gel separation and extraction  and quantified using a fluorometric kit . Purified amplicons were pooled in equimolar amounts for library preparation. The construction of sequencing libraries and paired-end sequencing (2 × 250 bp) was performed using the MiSeq System (Illumina) at the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases  according to standard protocols. The raw reads were deposited into the European Nucleotide Archive database .PCR primers incorporated sample-specific barcodes for multiplex sequencing using the Illumina MiSeq System (paired-end 250-nt reads). Paired V4-16S rRNA sequences were trimmed to 200 bp and merged into a single sequence using FLASH v1.2.10 software. Merged sequences were filtered to exclude low-quality reads and binned according to their specific barcodes. Sequences were assembled according to the criteria as follows: (1) Ambiguous bases (N) and mismatches in barcode and primer regions were not allowed; (2) the maximum mismatch rate in the overlap region was 0.05; (3) maximum length and minimum length of reads (without barcodes and primers) were 100 nt and 500 nt, respectively. Chimeras were removed using USEARCH software. The sample size of each sample was equalized by random subtraction to 2500 reads. The remaining effective sequences were binned into OTUs using USEARCH software with a cutoff of 97% identity2. Simpson returns values of 1–D and invSimpson returns values of 1/D. PCA using weighted and unweighted UniFrac distance metrics was conducted using a random sample of 2,500 sequences per samples, and the R package (http://www.R-project.org/) was used to visualise the interactions among the bacterial communities of different samples.Representative sequences were assigned at different taxonomic levels (from phylum to genus) to the bacterial SILVA dataset following the Bayesian approach with a 97% cutoff valuehttp://enterotype.embl.de/enterotypes.html#dm). The heat map of key variables was generated using Heatmap Builder. Sequences were annotated using the Ribosomal Database Project version 2.4 classifier with a confidence level = 0.5http://huttenhower.sph.harvard.edu/galaxy/) was used to identify taxa that differed consistently between sample types according to a published studyTo reduce the possibility of PCoA calculation errors, we used three different calculation methods, including the Hellinger distance, JSD analysis and the Spearman coefficient distance to conduct distances analysis using a custom R program function provided by the EBML  curve for each crucial taxa was generated, and the area under the parametric curve (AUC) was computed by numerical integration using the R software pROC package . Validated tongue coat biomarkers were fit to logistic regression models, and the sensitivity and specificity of biomarker combinations were estimated by identifying the cutoff point of the predicted probability that yielded the highest sum of sensitivity and specificity according to published studiesGraphPad Prism V.6.0  was used for all analyses and preparation of graphs. The results are expressed as the mean ± SEM, and statistical analyses were performed using the two-tailed non-parametric Kruskal–Wallis test to evaluate the significance of differences in microbial taxa, clinical measures and diversity indexes. Differences with a How to cite this article: Lu, H. et al. Deep sequencing reveals microbiota dysbiosis of tongue coat in patients with liver carcinoma. Sci. Rep.6, 33142; doi: 10.1038/srep33142 (2016).