Extended Cox proportional hazards regression models with competing risk of all-cause mortality were used to evaluate risk factors on ESRD incidence. Patients were followed till 2012.1c-CV were independent predictors of ESRD with corresponding hazard ratios of 1.20 , 1.24  in HbA1c-CV from fourth to fifth quintile and 1.23  in FPG-CV from fifth quintile.After a median follow-up period of 8.23 years, 1642 patients developed ESRD, giving a crude incidence rate of 6.27/1000 person-years . After the multivariate adjustment, both FPG-CV and HbA1c-CV predicted development of ESRD in patients with type 2 diabetes, suggesting therapeutic strategies toward a goal to minimize glucose fluctuation.One-year visit-to-visit glucose variability expressed by FPG-CV and HbA However, assessing glucose control in diabetes can be challenging. Glucose was usually measured by fasting glucose, postprandial glucose, or 3-month average glucose, glycosylated hemoglobin (HbA1c) level.2 Several studies have revealed the importance of time-averaged mean levels of glycemia, measured by HbA1c, and is currently considered the “gold standard” of glycemic control in reducing diabetes-related complications.5 However, randomized controlled trials such as Action to Control Cardiovascular Risk in Diabetes (ACCORD),6 the Action in Diabetes and Vascular Disease (ADVANCE),7 and the Veterans’ Administration Diabetes Trial (VADT)8 reported that lowering blood glucose did not appreciably reduce ESRD incidence and incidence of serum creatinine doubling or 20 mL/min per 1.73 m2 decrease in estimated glomerular filtration rate (eGFR) decline in ACCORD study,6 risk of the need for renal-replacement therapy or death from renal causes in ADVANCE study,7 and mean GFR decline and incidence of severe renal changes, defined as GFR < 15 mL/min in VADT study.8 Such findings may be partly due to the fact that these studies used HbA1c as marker of glucose control, which failed to reflect glucose variability and risk associated with extreme glucose change over a long period of time.9 These findings have raised the concern that glucose variability, irrespective of the magnitude of hyperglycemia, may confer an additional risk for the development of micro- and macrovascular diabetic complications.9Diabetes mellitus (DM) ranks as a leading cause of end-stage renal disease (ESRD) in many developed and developing countries.10 With regard to nephropathy, HbA1c variability was reported a significant predictor of microalbuminuria, independent of the mean HbA1c.11 Lin et al reported that not only coefficient of variation in HbA1c (HbA1c-CV), but also coefficient of variation in fasting plasma glucose (FPG-CV) had strong association with diabetic nephropathy.12 Although fasting plasma glucose and HbA1c were widely used to monitor glucose level, only one study has explored the association between standard deviation of HbA1c (HbA1c-SD) and ESRD.13 More data are required to characterize the effect that the glucose variation exerts on ESRD. This study used the National Diabetes Care Management Program (NDCMP) registration in Taiwan to test whether glucose variation, measured by FPG-CV or HbA1c-CV, is associated with ESRD in type 2 diabetes.Among type 2 DM studies, glucose variability showed a positive association with the development of progression of diabetic retinopathy, cardiovascular events, and mortality.NDCMP is a case management program which enrolled patients with diabetes in medical center, regional hospital, and local clinic nationwide in Taiwan since 2002. DM was clinically diagnosed based on American Diabetes Association criteria , and patients were recruited without restriction for antidiabetes medication. A total of 63,084 ethnic Chinese patients diagnosed with type 2 diabetes were enrolled from 2002 to 2004. Date of entry into NDCMP was defined as index date. We excluded patients with type 1 diabetes (ICD-9-CM code 250.x1/x3), gestational diabetes (ICD-9-CM code 648.83), ESRD at baseline (ICD-9-CM code V45.1 with catastrophic illness identification), being followed up <1 year and persons <30 years of age (n = 3942). Enrollees of NDCMP program have to complete comprehensive evaluation, including demographic data, medical and drug history, blood pressure, body weight, height, waist circumference, and fundoscopy. After 12 hours of overnight fasting, blood was drawn from an antecubital vein and sent for analysis within 4 hours postcollection. Biochemical data including urea nitrogen, creatinine, total cholesterol, triglyceride, alanine transaminase, high-density lipoprotein, low-density lipoprotein, and HbA1c were examined at baseline. Patients were followed up every 3 to 6 months and received blood tests during follow-up visits. This study was approved by the China Medical University Hospital Ethical Review Board (DMR100-IRB-292).14 Patient demographics, diagnoses, and prescriptions in hospital and outpatient claims were recorded. Claims data are randomly audited by the NHI Bureau. This study used NHI data sets for inpatient care by admission and outpatient visits during 2001 to 2004 to identify baseline comorbidity. Individuals in Taiwan carry unique personal identification numbers (PIN). All NHI and NDCMP data sets can be interlinked with the PIN. For security and privacy purposes, patient identity data are scrambled cryptographically by the NHI Research Database. We followed from 1 year after index date until ESRD, death, or withdrawal from NHI.The Taiwan government launched the National Health Insurance (NHI) program in 1995. Ninety-nine percent of Taiwan populations were enrolled in the program, and the proportion of withdrawing from NHI is very low.The primary outcome measure, ESRD, was determined by catastrophic illness certification (ICD-9-CM code 585 with V45.1) from the registry for catastrophic illness database of NHI program from 2002 to 2012. The catastrophic illness certification was issued by a nephrologist and confirmed by another nephrologist. We searched all ESRD incident events by excluding those who had ESRD events before the index date. To rule out the possibility of cause-and-effect, those who had ESRD incident events within 1 year of index date were also excluded.2), albuminuria , coronary artery disease , congestive heart failure , cancer , hyperlipidemia (ICD-9-CM code 272), atrial fibrillation (ICD-9-CM code 427.31), hypertension (ICD-9-CM codes 401–405), chronic hepatitis , chronic obstructive pulmonary disease .Ten chronic conditions were tabulated for 12 months before enrollment by using NDCMP data set as well as outpatient and inpatient claims data: morbid obesity  and their 95% confidence intervals (CIs) were reported with adjustment for age, sex, and multiple variables. We considered 2 multivariate models. The first multivariate model adjusted for age (continuous) and sex . The second one further adjusted for tobacco use (yes/no), alcohol use (yes/no), duration of diabetes , type of hypoglycemic drug , antihypertensive treatment (yes/no), obesity (BMI ≥27 kg/m2), coronary artery disease (yes/no), congestive heart failure (yes/no), cancer (yes/no), hyperlipidemia (yes/no), hypertension (yes/no), atrial fibrillation (yes/no), chronic hepatitis (yes/no), chronic obstructive pulmonary disease (yes/no), mean values of the first year follow-up of fasting glucose  and HbA1c , and eGFR . We assessed interaction of FPG-CV and HbA1c by adding their product terms into the full multivariate model with the use of likelihood ratio test for statistical significance. The level of significance was set at 0.05 and all P values were 2-tailed. We performed all analyses using the SAS statistical package for Windows .For each individual patient, FPG-CV and HbA1c or FPG measurements, or being followed up <1 year. One thousand four hundred twenty-one individuals were further excluded because of lacking sociodemographic, lifestyle, drug, or comorbidity information. A total of 31,841 individuals were included for analysis, and 1642 incident ESRD cases were identified during follow-up period with cumulative incidence rates of 4.72%, 6.03%, and 15.97% for patients with normal urinary albumin-to-creatinine ratio, microalbuminuria, and macroalbuminuria and of 1.90%, 2.53%, 9.68%, and 43.15% for patients with eGFR ≥90, 60 to 89, 30 to 59, and <30 mL/min/1.73 m2. We compared baseline characteristic between patients included and those excluded by using standardized mean differences. Except for no medication slightly >0.1 SD (0.17), the standardized mean differences for the other variables were <0.1 SD, indicating a negligible difference in means or proportions between groups. In our cohort, the median numbers of FPG and HbA1C measurements within 1 year were both 3 tests and both 25th and 75th percentiles were 2 and 4 tests, respectively. The mean age is 60.94 ± 11.16 years with lowest in the highest quintile in both FPG-CV and HbA1C-CV. In the highest quintile of FPG-CV, 17.75% use tobacco, 9.15% drink alcohol, which is highest among other quintiles and is similar to 18.07% and 9.97% in the HbA1C-CV group. The highest quintile of FPG-CV and HbA1C-CV were not with highest burden of comorbidity. The highest quintile of FPG-CV had the lowest mean BMI and prevalence of hyperlipidemia and hypertension. The highest quintile of HbA1C-CV had the lowest mean BMI and prevalence of coronary artery disease, hyperlipidemia, hypertension, and hepatitis. Tables 1c levels. Patients reaching ESRD have higher mean age, longer diabetes duration, higher prevalence of hypertension drug treatment, congestive heart failure, hyperlipidemia, hypertension, chronic obstructive pulmonary disease, stroke, and hypoglycemia, and lower prevalence of chronic hepatitis. After an average of 8.23 years of follow-up, 1642 patients developed ESRD, giving a crude incidence rate of 6.27/1000 person-years ; 6491 died, with a mortality rate of 24.77/1000 person-years . Figure 1c-CV. Patients with FPG-CV > 48.6% faced higher risk , 1.24  in HbA1c-CV from fourth to fifth quintile and 1.13 , 1.23  in FPG-CV. In addition, they were both independent predictors of all-cause mortality with corresponding HR of 1.15 , 1.23  in HbA1c-CV from fourth to fifth quintile and 1.21 , 1.29  in FPG-CV.Multivariate adjusted Cox regression was performed to examine the relative contribution of FPG-CV and HbA1c-CV from fourth to fifth quintile  and with a FPG-CV from fourth to fifth quintile . The results of HbA1c-CV and FPG-CV, further stratification according to each other . With HbA1c-CV subgrouped based on deciles, multivariate-adjusted HRs for HbA1c-CV levels from sixth to tenth decile were 1.36 , 1.20 , 1.37 , 1.66 , and 1.57 ; and for FPG-CV were 1.42 , 1.26 , 1.48 , 1.82 , and 1.77 .To eliminate potential bias caused by the existence of comorbidities, sensitivity analyses were conducted, and patients with hyperglycemic hyperosmolar nonketotic coma, diabetic ketoacidosis, myocardial infarction, atrial fibrillation, and hypoglycemia were excluded n = 1812). A similarly significant HRs for ESRD were observed among patients with a HbA. A simil1c-CV independently predict ESRD in patients with type 2 diabetes >30 years. After adjusting for mean FPG, HbA1c, and other conventional risk factors, FPG-CV and HbA1c-CV still pinpoint the association between oscillating plasma glucose and ESRD. These findings are relevant to the clinical management of type 2 diabetes such that long-term CV of both FPG and HbA1c should be evaluated, in addition to only reading the raw data. Recent therapies used should be evaluated for their potential to minimize glucose fluctuation in patients with type 2 diabetes to prevent ESRD.We found that visit-to-visit glucose variations in both FPG-CV and HbA20 including with-in day blood glucose variation, hypoglycemia and hyperglycemia episodes, and visit-to-visit variation. Preferred measures of glycemic variability lack consensus. Previous studies that examine the effect of short-term intraday glucose variability on late diabetes complications in patients with type 1 diabetes have mixed results.21 However, long-term glycemic variance demonstrated a more consistent relationship with diabetes-related complications in patients with type 1 or type 2 diabetes.22 The spectra of FPG-CV, HbA1c-CV, and with-in day glucose variation might be different. Wide variation of FPG-CV and HbA1c-CV might reflect a more complicated clinical course, suboptimal medications, and self-management in addition to oxidative stress by acute variance of glucose.23 Although visit-to-visit glucose variation may not reflect actual glucose variation like self-monitored blood glucose (SMBG) or continuous monitor of glucose, it is difficult to long term measure SMBG or continuous blood glucose. The association of HbA1c variation to proteinuria,11 chronic kidney disease (CKD),13 and ESRD,13 and FPG variation to DM nephropathy,12 had been reported. We fill the gap of FPG variation to ESRD. These findings provide evidence that these longer-term indexes of glucose variation might be proper indicators for estimating diabetic complications.Glucose variability has been measured in different ways,Patients with high glucose level may be subject to change of hypoglycemic drug use or more intensive diabetes control, that would lead to decreasing glucose level and higher variation in fasting plasma glucose. In order to rule out this possibility, we have examined change in type of hypoglycemic drug use between 1 year before index date and 1 year after index date, and the concordance of each type of hypoglycemic drug use was high, ranging from 94.91% to 99.13%. Thus, the possibility that change of hypoglycemic drug use or intensification of treatment that may explain the association between glycemic variation and ESRD would be less likely. In addition, we also evaluated the concordance of change in type of antihypertensive drug use, and the concordance rate was all above 95% for each type of medication use. Thus, the probability that changes in antihypertensive and/or hypoglycaemic medications may influence the effects of HbA1c-CV and FBG-CV on risk of ESRD was low.1c-CV predict ESRD in patients with type 2 DM. Weak association of FPG-CV and HbA1c-CV is observed . Therefore, these 2 measures capture different aspects of glycemic variation. HbA1c is regarded as mean glucose and does not reflect acute fluctuation in glucose level.24 The contribution of fasting or postprandial glucose varies with different HbA1c levels while postprandial glucose contributed more at lower HbA1c levels.26 HbA1c is also affected by test time,24 elevated blood urea nitrogen level, metabolic acidosis, anemia, blood transfusion, hemoglobinopathies, erythropoietin-stimulating agents, and protein-energy wasting,27 which might occur in CKD patients. On the contrary, FPG level captures acute fluctuation in glucose level caused by irregular eating or lifestyle episode, which was not easily detected by HbA1c. Thus, FPG might be a more sensitive indicator than HbA1c for capturing variation of glucose because of overindulgence in food given that HbA1c is considered a mean. Extending FPG and HbA1c to visit-to-visit FPG-CV and HbA1c-CV as a marker for glucose variability has 2 benefits. First, FPG and HbA1c are routinely measured in almost all patients with diabetes, and estimating their CV is easy. Second, such approach seems to provide longer observation spectrum and good prediction power to further survival,28 cardiovascular events,10 stroke,29 DM nephropathy,30 and ESRD.13Our data showed both FPG-CV and HbA31 mesangial lipid accumulation,32 mesangial and tubulointerstitial cell matrix production,33 expression of fibrinogenesis markers, circulating level of inflammatory cytokines,34 endothelial dysfunction,34 and free radicals that activate the pathogenesis of diabetic complications.36Several mechanisms explained the impact of high glucose variation and renal toxicity, including increase in glomerular permeability,1c measurements in NDCMP data set. Thus, we could not evaluate the intraindividual variation of FPG-CV and HbA1c-CV during follow-up and their dynamic effects on ESRD incidence. Third, FPG cannot possibly represent the actual glycemic level because of blood drawing without fasting, overeating, or gluttony before blood drawing because of social events and these factors cannot represent the dietary behavior of a typical day or dietary restriction before regular outpatient visits. These conditions would further result in measurement error for visit-to-visit variation for FPG represented by FPG-CV. However, these conditions would result in a consistent distortion of FPG-CV measurements to higher values. If a true association exists between FPG-CV and ESRD incidence, then this type of error would result in underestimation of the effect. Fourth, the present study did not measure postprandial glucose. The effect of postprandial hyperglycemia contributing to ESRD could not be assessed.This study has limitations. First, it is an observational study, and a few residual and unrecognized confounding variables such as diet might be present. Thus, it is likely that a reduced risk of ESRD might reflect a delay of initiation of dialysis due to amelioration of uremic symptoms possibly due to diet rather than an actual slowing of the decline in renal function. Second, we only have 1-year FPG and HbA1c-CV might be used in managing patients with diabetes in predicting clinical prognosis. Future studies should assess the effects of interventions for reducing glucose variation on progression of diabetic nephropathy or ESRD incidence.In this large cohort of Chinese patients with type 2 diabetes, both FPG-CV and HbA1c-CV were independent predictors of ESRD, after adjusting mean FPG, HbA1c, and other conventional risk factors. Our results expand existing knowledge about the relationship of glycemia variation and ESRD, suggesting glucose variation using FPG-CV and HbA


Candidatus Methylacidiphilum kamchatkense” strain Kam1 is an aerobic methane-oxidizing thermoacidophilic bacterium belonging to the Verrucomicrobia phylum. It was recovered from an acidic geothermal site in Uzon Caldera, Kamchatka, Russian Federation. Its genome possesses three complete pmoCAB gene clusters encoding particulate methane monooxygenase enzymes and a complete Calvin-Benson-Bassham cycle for carbon assimilation.“ The use of methanotrophs as biocatalysts for the conversion of natural gas to biofuels, biopolymers, single-cell proteins, and high-value products is a promising approach for biotechnological use of natural gas . Large-sCandidatus Methylacidiphilum kamchatkense” strain Kam1, is a rod-shaped and Gram-negative aerobic methane-oxidizing bacterium belonging to the Verrucomicrobia phylum, isolated from an acidic hot spring in the Uzon Caldera, in Kamchatka, Russian Federation (Ca. Methylacidiphilum infernorum” (strain V4) and “Ca. Methylacidiphilum fumariolicum” (strain SolV), have also been recovered from similar geothermal environments (http://www.gatc-biotech.com). A total of 300,132 reads accounting for 72,788,864 bases were obtained. Assembly into 41 contigs was done with Newbler version 2.9 accessed through the Lifeportal, University of Oslo .“deration , and wasronments , 4, and ronments . All thrCa. Methylacidiphilum kamchatkense” Kam1 comprises 2,210,643 bp in size, with a GC content of 40.8% and 1,805 protein-coding sequences. A single rRNA operon is present, together with 46 tRNA genes. This is comparable with the genome of the other strains, V4 and SolV, whose complete genomes constitute ~2.3 and 2.5 Mbp, respectively (http://enve-omics.ce.gatech.edu/ani/index) revealed ANI values of 78 and 93%, respectively, when the Kam1 draft sequence was compared with the complete sequences of strains V4 and SolV. This indicates that Kam1 represents a separate species, as this value is lower than the threshold value of 95%, which corresponds to a genomic DNA:DNA hybridization value of 70% and is a common threshold value for distinction between species (The draft genome sequence of “ectively . An averpmoCAB1-3, as well as one truncated pmoCA cluster and a solitary pmoC gene, have been described before (Three operons, each encoding one set of the three subunits of the key enzyme, particulate methane monooxygenase, d before . The ribd before . Genes fJQNX00000000. The version described in this paper is version JQNX01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Our results showed significant positive correlations between circulating testosterone and insulin growth factor-1 (IGF-1) with knee flexion, knee extension, one-repetition maximum (1-RM), and peak oxygen consumption (VO2 peak), while no correlation was observed with estradiol. Among the adipokines, high sensitivity C-reactive protein (Hs-CRP) and leptin negatively correlated with the modified physical performance testing (PPT), knee flexion, knee extension, 1-RM, and VO2 peak. Interleukin-6 ( Il-6) negatively correlated with knee flexion and VO2 peak and soluble tumor necrosis factors receptor-1 (sTNFr1) correlated with PPT, 1-RM, and VO2 peak. Adiponectin correlated negatively with 1-RM. Multiple regression analysis revealed that, for PPT, sTNFr1 was the only independent predictor. Independent predictors included adiponectin, leptin, and testosterone for knee flexion; leptin and testosterone for knee extension; adiponectin, leptin, and testosterone for 1-RM; and IGF-1, IL-6, leptin, and testosterone for VO2 peak. In conclusion, in frail obese older adults, circulating levels of testosterone, adiponectin, and leptin appear to be important predictors of physical strength and fitness, while inflammation appears to be a major determinant of physical frailty.In this study, we evaluated the independent and combined effects of baseline circulating gonadal, anabolic hormones and adipokines on physical function in 107 frail, obese (BMI ≥ 30 kg/m These changes are associated with loss of muscle mass and decline in strength and physical function. An increase in lean body mass, strength, and physical function has been demonstrated with testosterone , 5 and gThe objective of this study was to evaluate the independent and combined effects of circulating gonadal (testosterone and estradiol) and anabolic hormones (growth hormone/IGF-1) and adipose-tissue derived factors (adipokines) on physical function, strength and physical fitness in frail obese older adults.2, had sedentary lifestyle, (did not participate in regular exercise more than twice a week), had stable body weight (±2 kg) over the past year, and were on stable medications for 6 months before enrollment. Participants who were treated with bone-acting drugs  during the previous year were excluded from participation. At enrollment, these subjects should not have cardiorespiratory or neuromuscular diseases that would limit their ability to exercise, diabetes mellitus, osteoporosis, hyperparathyroidism, chronic liver disease, uncontrolled or untreated hyperthyroidism, or significant renal impairment.This study is a cross-sectional analysis of baseline data from subjects who volunteered to participate in a previous lifestyle therapy trial of sedentary, frail, and elderly obese adults , 9. ThisPhysical function to determine frailty was assessed using the modified physical performance test (PPT) as previously described , 9. The Isokinetic knee extensor and flexor strength were evaluated by using a Biodex System 3 dynamometer  as previously described . During Total one-repetition maximum (1-RM) is the sum of the maximal weight a person can lift at one repetition for biceps curl, bench press, seated row, knee extension, knee flexion, and leg press , 10. The2  peak, a measure of aerobic fitness, was assessed during graded treadmill walking by indirect calorimetry , as previously described [max, and the speed remained constant throughout the test, while the grade was increased by 2% every 2 minutes. The test continued until the subject could no longer exercise because of exhaustion or until other conditions, such as ECG changes or development of symptoms, made it unsafe to continue [VOescribed , 11. Bricontinue , 13.2).Body weight was measured in the morning after the subjects had fasted for 12 hours . BMI wasBlood samples were obtained in the morning after subjects fasted for at least 12 hours. Serum samples were extracted and stored at −80°C until analysis. Enzyme-linked immunosorbent assay kits were used to measure interleukin-6 (IL-6) , soluble tumor necrosis factor receptor 1 (sTNFr1) , and adiponectin . Radioimmunoassay kits were used to measure serum estradiol , leptin , and insulin-like growth factor 1 (IGF-1) (Diagnostic Products Group). High-sensitive C-reactive protein (Hs-CRP) was measured by immunoturbidimetric assay , while serum total testosterone was measured by automated immunoassay (VITROS 5600). The CVs for these assays were <10%.P value of <0.05 was considered statistically significant. Normality for outcome variables was verified by Shapiro-Wilks test. Simple correlation analysis was performed to assess the individual associations between each hormone or adipokine with PPT, knee flexion, knee extension, total 1-RM, and VO2  peak followed by multiple regression analysis to determine the independent contribution of each hormone or adipokine to the above tests.Results are expressed as means ± SD. A 2) and elderly (≥65 years old) males (n = 41) and females (n = 66). The baseline characteristics of these patients have been reported previously [Our population consisted of 107 frail (PPT of ≤ 32) obese (BMI ≥ 30 kg/meviously  and summ2  peak), while no correlation was observed between estradiol and any of these measures. Significant positive correlation was observed between IGF-1 and measures of strength and aerobic fitness but not with modified PPT. Among the different adipokines, there was a negative correlation between the proinflammatory cytokine Hs-CRP with PPT, strength, and aerobic fitness (VO2  peak). The other pro-inflammatory cytokine IL-6 also correlated negatively with knee flexion and VO2  peak. The last pro-inflammatory cytokine tested sTNFr1 also correlated negatively with PPT, total 1-RM, and VO2  peak.2  peak. The adipokine adiponectin negatively correlated with total 1-RM.The adipokine leptin negatively correlated with PPT, all measures of strength, and VO2  peak) included: IGF-1, IL-6, leptin and testosterone.Multiple regression analysis  showed t2  peak), sTNFr1 was the only independent predictor of overall physical performance (PPT), while testosterone seemed to be a consistent predictor of all measures of physical strength and fitness. Leptin and adiponectin also appeared to be independent determinants of the different measures of strength, while leptin additionally predicted VO2  peak. On the other hand, IGF-1 only independently predicted physical fitness, while estradiol appeared to have no role in physical function, strength, or fitness. Thus, our results suggest that circulating hormones and adipokines alone or in combination may contribute to frailty and physical disability in obese older adults.Our results demonstrated that although there were correlations between gonadal and anabolic hormones and several adipokines with physical function, different measures of strength, and aerobic fitness  and function, known as sarcopenia . Obesityα which suppresses muscle protein synthesis and interleukin-6 (IL-6) which inhibits the anabolic effects of IGF-1 [Both obesity and aging have additive effects on chronic inflammation , 16 puttof IGF-1 . Aside fof IGF-1 . Our resOur results also demonstrated a significant contribution of testosterone to physical strength and fitness, while estradiol appeared to have no role in physical function, strength, or fitness in our study population. Although part of decline in physical function and strength with aging is attributed to abnormalities in GH/IGF-1 production and signaling, our study showed that IGF-1 had very little role in these parameters in frail obese older men and women. Aging is associated with decrease in gonadal hormones in both sexes. In women, the drastic drop in estrogen levels with menopause is associated with reduction in muscle mass . In men In men, several randomized controlled trials (RCTs) have demonstrated thattestosterone significantly increases FFM (1.1 to 3.7 kg) and decreases fat mass (1.1 to 4.5 kg) –25. In aOn the other hand, although it is well-established that testosterone administration in postmenopausal women resulted in improvement in sexual function not only in surgically menopausal women but also in women with natural menopause –31, veryLeptin and adiponectin share similarities in that both are produced by adipose tissues, have receptors in skeletal muscle, and have effects on muscle metabolism . Leptin Our study has limitations. It is cross-sectional study and therefore does not provide a direct evidence of the actual physiologic role of each factor on physical function, strength, and frailty. In addition, our sample size was relatively small which thus needs confirmation in a larger population of obese older adults.In summary, our results indicate that circulating testosterone levels and adipokines alone or in combination influence physical function, strength, and aerobic fitness in the frail obese older adults. Our group previously demonstrated that lifestyle intervention by diet and exercise was safe and improves physical function and ameliorates frailty in this population . However


Staphylococcus aureus (MRSA) causes severe catheter-related infections in haemodialysis patients ranging from local-site infections and septic thrombophlebitis to bacteraemia but the associated virulence factors and exotoxins remain unclear.Methicillin-resistant in vitro infection model using reconstituted human epithelium (RHE) to analyse the expression profiles of 4 virulence genes and 12 exotoxin-coding virulence genes in 21 MRSA strains isolated from catheter-related infections in 21 Mexican patients undergoing haemodialysis.We employed an seg, seh, sei, eta, etb, or hla genes coding staphylococcal toxins. Eleven MRSA strains (52.3%) expressed the sea gene coding staphylococcal enterotoxin A, and two strains (9.5%) expressed the v8 gene coding serine protease. The tst, chp, and arcA genes coding toxic shock syndrome toxin 1, chemotaxis inhibitory protein, and arginine deiminase, respectively, were expressed in separate single strains (4.7%). The most frequent expression profile (42.8% of the strains) comprised seg, seh, sei, eta, etb, and hla.All 21 strains (100%) expressed the It is likely that the SEG, SEH, SEI, ETA, ETB, and Hla toxins may play a role in MRSA catheter-related infections. Consideration of these toxins in the development of a vaccine or as targets for monoclonal antibody therapy could provide an improved therapeutic strategy for the treatment of catheter-related infections in haemodialysis patients. Staphylococcus aureus (MRSA) is known to produce catheter-associated bacteraemia in haemodialysis patients because of its ability to form a biofilm in the interior of endovascular catheters [S. aureus is able to produce several exotoxins with superantigen activity, including staphylococcal enterotoxins (SEA to SEE and SEG to SEJ) encoded by the genes sea, seb, sec, sed, see, seg, seh, sei, and sej; toxic shock syndrome toxin 1  and exfoliative toxins  [S. aureus are encoded by the genes hla , chp (chemotaxis inhibitory protein), arcA (arginine deiminase), and v8 (serine protease V8) [agr) system, also known as the quorum-sensing system, plays a central role in the regulation of virulence factors [Methicillin-resistant atheters . S. aureand etb) . Other vease V8) . The exp factors . The fre factors .in vitro infection model using reconstituted human epithelium (RHE) to analyse the expression profiles of genes encoding pyrogenic exotoxins in our collection of MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis.We recently analysed the expression profiles of genes encoding adhesins in MRSA strains isolated from catheter-related infections in patients undergoing haemodialysis ; howeverThe 21 MRSA strains analysed in this study were isolated from catheter-related infections in 21 Mexican patients and have been described previously . Bacteriin vitro at the air-liquid interface in serum-free conditions in a defined medium based on the MCDB-153 medium  containing 5 μg insulin/mL. In total, 2 × 106 S. aureus cells suspended in 50 μL of 0.1 M phosphate-buffered saline (PBS) were inoculated onto the surface of the RHE and incubated at 37°C for 72 h under 5% CO2 and saturated humidity. The media were changed every 24 h.Reconstituted human epithelium  consists of human epithelial cells cultured on polycarbonate filters S. aureus RNA purification and reverse transcription were performed as previously described [sea, seb, sec, sed, see, seg, seh, sei, sej, tst, eta, etb, hla, chp, arcA, and v8 [gyrB (DNA gyrase B) was used as a reference gene. S. epidermidis ATCC 35984 and Escherichia coli ATCC 11775 were used as negative controls. S. aureus ATCC 33592 was used as the positive control.escribed . The pri, and v8 . The Rotseg, seh, sei, eta, etb, and hla, of the 16 genes studied during in vitro infection of RHE. tst, chp, and arcA were expressed in separate single strains (4.7%), and v8 was expressed by 2 strains , was seen for nine (42.3%) MRSA isolates, and profile 2, composed of 7 genes , was seen for seven strains (33.3%). Profile 3, formed by 8 genes , was found in 2 (9.5%) MRSA isolates. Three distinct profiles comprising 7–8 expressed genes in only one MRSA strain each were also found.All MRSA strains n = 21) expressed 6 genes, that is,  expressespa type t895 and having identical pulsed-field gel electrophoresis (PFGE) profiles  [spa type (t895) [arcA (S-79) or chp (S-82).MRSA strains previously reported as belonging to the S. aureus virulence factors in animal models [in vitro infection model using RHE to analyse the expression profiles of genes encoding exotoxins in MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. All of the MRSA strains studied here expressed an important set of virulence factors. All of the strains expressed three staphylococcal enterotoxins , two exfoliative toxins (ETA and ETB), and the alpha toxin (Hla)  [mec type IV, 6 of 21 harboured SCCmec type II, and 3 of 21 harboured SCCmec type I [Catheter-related bacteraemia caused by MRSA in patients who have end-stage renal disease and are undergoing chronic haemodialysis is a serious health problem . Severall models -9. In tha) Table . We had  agr II) , which h agr II) . We alsoS. aureus strains related to different types of infections [The frequency of genes encoding toxins has been previously described for fections ,11; howeS. aureus isolates could further support this point. Considering these toxins in the development of a vaccine, or as targets for monoclonal antibody therapy, could provide an improved therapeutic strategy for the prevention or treatment of catheter-related infections.The results presented here show that it is likely that the SEG, SEH, SEI, ETA, ETB, and Hla toxins may play a role in the pathogenesis of MRSA catheter-related infections. Comparative quantification of the expression of these genes in these MRSA and in other The data supporting the results of this study are included within this article.


Porcine torovirus (PToV) strain PToV-NPL/2013 was identified from a pig that tested positive for porcine epidemic diarrhea virus (PEDV). The spike protein-encoding gene from PToV-NPL/2013 had 92% identity with PToV-SH1, suggesting that PToV circulating in the United States is slightly different from the isolates circulating in China. To our knowledge, this is the first report of PToV in the United States. Coronaviridae, order Niodvirales  are considered emerging viruses with a possibility of zoonotic transmission . They ardvirales , 2. ToV dvirales , 3–5. Poirales 3––10. BecaTC], 31.18), PEDV , porcine enterovirus 9, and porcine teschovirus. To further characterize RNA viruses in the sample, we conducted metagenomics using an Ion Torrent Personal Genome Machine with the methodology previously described . Samples were positive for group C rotavirus  and was 92% identical to that of porcine torovirus SH1 (PToV-SH1) (NC_022787). The 5′ untranslated region (UTR) and 3′ UTR were 792 and 196 nucleotides in length, respectively. The polyprotein-encoding open reading frame (ORF1ab) was identified from nucleotides 794 to 20910 and encoded a polyprotein of 6,706 amino acids. ORF1a was identified from 794 to 14,047 and encoded a replicase of 4,418 amino acids. Genes encoding spike (S), membrane (M), hemagglutinin-esterase (HE), and nucleocapsid (N) proteins were 4,722, 702, 1,284, and 492 nt in length, respectively, and had predicted proteins with 94, 99, 92, and 96% identity to PToV-SH1, respectively. The spike protein-encoding gene from PToV-NPL/2013 showed only 92% identity to that of PToV-SH1, suggesting that the S gene of PToV circulating in the United States is slightly different from the isolates circulating in China. Since spike is a major glycoprotein present on the viral envelope and is shown to bind host cell receptors, induce virus-mediated cell fusion, and elicit neutralizing antibodies upon infection , 13, difKM403390.The complete genome sequence of PToV-NPL/2013 has been deposited in GenBank under the accession number


The differential diagnostic and prognosis values were also evaluated. The results showed that the ratio of tumor-infiltrating MRP8 and MRP14 positive immune cells, relative to biliary epithelial cells, was significantly increased in ICC tissues compared with nonmalignant tissues, including IHBD, BilIN1, BilIN2, BilIN3, and IPNB . In addition, over-expression levels of MRP8 and MRP14 were correlated with overall survival (OS) and time to recurrence (TTR) by univariate analysis; MRP8/MRP14 combination was an independent prognostic factor for OS and TTR. MRP8 and MRP14 expression might help to identify the benign bile duct diseases from ICC, as high expression of MRP8 and MRP14 suggests a poor prognosis after surgical resection.Myeloid-related protein 8 (MRP8) and 14 (MRP14) are abundantly expressed in several kinds of benign and malignant tumors. However, little is known about their clinicopathological significance in intrahepatic cholangiocarcinoma (ICC), biliary intraepithelial neoplasia (BilIN), intraductal papillary neoplasm of bile duct (IPNB), or inflammatory hepatic biliary ducts epithelium (IHBD). This study aimed to investigate the diagnostic and prognostic values of MRP8 and MRP14 as new biomarkers for ICC. We examined MRP8 and MRP14 expression levels by immunohistochemistry in IHBD ( Intrahepatic cholangiocarcinoma (ICC) is a poorly understood biliary malignancy that accounts for an estimated 10–15% of all primary liver cancers . HoweverEarly diagnosis of ICC is possible to carry out radical surgery. Biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasm of bile duct (IPNB) are two proposed benign bile duct diseases for the development and progression of ICC. It was reported that the protein expression pattern of mucin core proteins (MUCs)/cytokeratins (CKs) and P16/EZH2/Mmi1 in neoplastic biliary epithelia differed according to the pathway of cholangiocarcinogenesis, via BilIN or IPNB lineage , 6.Partial hepatectomy remains the gold standard of curative treatment for ICC , 8. HoweMyeloid-related protein (MRP) has been implicated in multiple stages of tumorigenesis, progression and prognosis. MRP carries out a wide range of intracellular and extracellular functions, such as regulation of calcium homeostasis, cell proliferation, apoptosis, cell invasion and motility, cytoskeleton interactions, protein phosphorylation, regulation of transcriptional factors, autoimmunity, chemotaxis, inflammation and pluripotency .In this study, we analyzed the involvement of MRP8 and MRP14 in the progression of cholangiocarcinogenesis through BilIN and IPNB, and investigated its role in tumor progression of ICC.P < 0.0001), BilIN1 (P < 0.0001), BilIN2 (P = 0.0031), BilIN3 (P = 0.0453), and IPNB (P < 0.0001). The ratio of MRP14 positive cells to biliary epithelial cells in ICC was higher than in IHBD (P < 0.001), BilIN1 (P < 0.0001), BilIN2 (P = 0.0084), BilIN3 (P = 0.0261), and IPNB (P < 0.0001)  and r = 0.823 (P < 0.001), respectively  and time to recurrence (TTR) rates were statistically better in the combined low-MRP group than those in at least one high-MRP group, with the exception of OS in the testing group , -carcino embryonic antigen (CEA), -carbohydrate antigen 199 (CA199), -albumin (ALB), -gamma-glutamyl transferase (GGT), -alanine transaminase (ALT), -alkaline phosphatase (ALP), tumor size, tumor number, micro-vascular invasion, tumor-nodes-metastases (TNM), MRP8 number, MRP8 ratio, MRP14 number, MRP8 ratio, and combined MRP8/MRP14 ratio were predictors for OS in a combined primary and testing cohort. Furthermore, liver cirrhosis, serum-CEA, -CA199, -GGT, -ALP, tumor size, tumor number, micro-vascular invasion, TNM, MRP8 number, MRP8 ratio, MRP14 number, MRP14 ratio, and MRP8/MRP14 ratio combination were predictors for TTR in combined primary and testing cohorts. Multivariate Cox regression analysis indicated that serum-AFP, -CEA, -ALB, -ALP, tumor size, tumor number, TNM, and MRP8 ratio were independent prognostic factors for OS in ICC patients after the surgical resection. In addition, liver cirrhosis, serum-CEA, -ALP, tumor size, tumor number, TNM, and MRP8/MRP14 ratio combination were independent prognostic factors for TTR in ICC patients after the surgical resection in combined primary and testing cohort Table . In the ICC is a unique liver cancer that differs from hepatocellular carcinoma (HCC) , hilar aImproving the survival rate of patients with a malignant tumor after surgical resection requires clinicians to engage in the active treatment of recurrence and investigate the biological and clinicopathological characteristics that reflect tumor behavior, such as progressive or metastatic capability , 17.MRP8 and MRP14 belong to the S100 gene family, which is the largest subfamily of EF-hand calcium-binding proteins. At least 25 distinct members of this subgroup have been described . S100 prThere is increasing interest in the S100 proteins and their relationship with inflammatory bile duct diseases , BilIN aIn this study, the diagnostic and prognostic values of MRP8 and MRP14 in BilIN, IPNB, and ICC were investigated. We show, for the first time, that MRP8 and MRP14 are significantly increased in infiltrating immune cells of ICC tissues, compared with other nonmalignant tissues, such as IHBD, BilIN1, BilIN2, BilIN3, and IPNB. Moreover, high expression levels of MRP8 and MRP14 were indicative of a poor prognosis for ICC patients after surgical resection.In addition, we introduced a novel scoring method for evaluating the MRP protein expression level, which is represented by MRP positive cells/biliary epithelial cell number. Furthermore, X-Tile plots were created for assessment of a best cutoff point for continuous variables and optimized cutoff points based on outcome. Our results found novel cutoff points, such as serum-AFP of 7.5 ng/ml, -CEA of 5.2 ng/ml, -CA199 of 40 U/L, -ALB of 37.3 mg/ml, -GGT of 54 U/L, -ALT of 50 U/L, -ALP of 113 U/L, tumor size of 4 cm, MRP8 number of 8, MRP8 ratio of 0.0498, MRP14 number of 5, and MRP8 ratio of 0.0025. Thus the novel assay method and cutoff points proposed in this study may significantly enhance the inter-laboratory assay and intra-laboratory assay reproducibility.Accumulating evidence shows that stromal cells in neoplastic tissues play central roles in tumor progression and cancer metastasis . These sIn conclusion, the expressions of MRP8 and MRP14 were significantly increased in infiltrating immune cells of ICC tissues compared with IHBD, BilIN1, BilIN2, BilIN3, and IPNB. The numbers of MRP8 and MRP14 positive cells, compared with the total number of biliary epithelial cells, are useful markers for predicting the progression and prognosis of ICC. MRP8 might have a synergistic effect with MRP14 in predicting the prognosis of ICC patients after surgical resection.n = 18), BilIN1 (n = 25), BilIN2 (n = 12), BilIN3 (n = 5), and IPNB (n = 19)  with inflammation ((n = 19) . HematoxFor prognostic evaluation, the follow-up period of all 416 ICC patients lasted until September 2014 and complete follow-up data for patients in the prognostic group were available. The overall survival (OS) was defined as the length of time between surgery and death, or the last follow-up examination. The time to recurrence (TTR) was calculated from the date of tumor resection until the detection of tumor recurrence, death or last observation. Detailed follow-up procedures are described in the 2O2/phosphate-buffered saline, and nonspecific binding sites were blocked with goat serum. Primary antibodies were diluted as follows: rabbit monoclonal antibody against MRP8 ; rabbit polyclonal antibody against calgranulin B (S100A9/MRP14) . The expression levels of MRP8 and MRP 14 were represented by the ratio of number of positive cells to the biliary epithelial cell number.Tissue microarrays, immunohistochemistry and scoring of all specimens were selected randomly and tissue microarrays were constructed from two representative cores from each specimen. Immunohistochemistry was performed and samples were measured as reported previously . BrieflyThe imaging system comprised a Leica CCD camera, DFC420, connected to a Leica DM IRE2 microscope . All samples had one field captured from each core under high-power magnification (200 ×) using Leica QW in Plus V3 software. Therefore, a total of two photographs from each sample were used to count the cell number using Image-Pro Plus v6.0 software . Next, the ratio (relative positive numbers) of MRP8 and MRP 14 positive cells (absolute positive numbers) to the total number of biliary epithelial cells was calculated . FinallyP values obtained from a lookup table  was used to perform the statistical power analyses [Statistical analyses were carried out with SPSS 13.0 software  and GraphPad Prism 5.01. The significance of expression level for markers between ICC, IHBD and neoplasia was analyzed by the Mann-Whitney test. To evaluate the discriminating efficacy, receiver operating characteristic (ROC) analysis was performed. For calculating the best cut-off points for OS and TTR, the X-Tile statistical package  was used. X-tile plot illustrates the presence of substantial tumor subpopulations and shows the robustness of the relationship between a biomarker and outcome by construction of a two dimensional projection of every possible subpopulation . X-tile analyses .


CD133 is one of the most commonly used markers of cancer stem cells (CSCs), which are characterized by their ability for self-renewal and tumorigenicity. However, the clinical and prognostic significance of CD133 in gastric cancer remains controversial. To clarify a precise determinant of the clinical significance of CD133, we conducted a systematic review and meta-analysis to elucidate the correlation of CD133 overexpression with prognosis and clinicopathological features of GC patients.A search in the Cochrane Library, Pubmed, Medline, Web of Knowledge and Chinese CNKI, CBM  was performed using the following keywords gastric cancer, CD133, AC133, prominin-1, etc. Electronic searches were supplemented by hand searching reference lists, abstracts and proceedings from meetings. Outcomes included overall survival and various clinicopathological features. Two reviewers independently screened the literature according to the inclusion and exclusion criteria, extracted the data, and assessed the methodological quality of the included studies, and then RevMan 5.2.0 software was used for meta-analysis.P < 0.00001), lymph node metastasis vs. non-lymph node metastasis , distant metastasis vs. non-distant metastasis , clinical stages III~IV vs. clinical stages I~II , as well as the accumulative 5-year overall survival rates of CD133-positive vs. CD133-negative patients .A total of 603 gastric cancer patients from 8 studies were included. The results of the meta-analyses showed that, there were significant differences of CD133 expression in the following comparisons: gastric cancer tissues vs. normal esophageal tissue (OR = 3.49, 95% CI , Overexpression of CD133 is associated with lymph node metastasis, distant metastasis, poor TNM stage. Additionally, CD133-positive gastric cancer patients had worse prognosis. Our results indicate that CD133 may be involved in the carcinogenesis of gastric cancer. Evaluation of cytoplasmic CD133 overexpression in gastric cancer tissue sections may be useful in the future as a novel prognostic factor. Nevertheless, due to the poor quality and small sample size of included trials, more well-designed multi-center randomized controlled trials should be performed. Gastric cancer ranks fourth  in incidence and second (after lung cancer) in mortality among all cancers worldwide. Nearly one million people are diagnosed with gastric cancer every year worldwide, among which of 70% are in developing countries and more than 50% in East Asia, especially China and Japan . AlthougFour hundred and seventeen articles were identified initially using the search strategy above. Through reading titles and abstracts, Three hundred and eighty-seven of those were excluded due to non-gastric-related studies, non-original articles  and duplicate studies. After reading full texts, we excluded twenty-two data which couldn't be extracted due to non-CD133 related studies, non-immunohistochemical SP method, disunited positive-criteria. Eventually, there were 8 studies (6 in English and 2 in Chinese) included in the present Meta-analysis . Figure Based on Asian population, the eight studies included eventually contained 4 from China, 3 from Japan, and the rest 1 from Singapore. A total of 1195 patients were included, containing 607 from China, 476 from Japan, and the rest 112 from Singapore, most of which were male patients (60.4% from 6 studies). The median age ranged from 51.5 to 66 years old (from 5 studies). The total positive rate of CD133 overexpression by IHC is 35.1% from 8 studies (from 9.5% to 65.0%). All detected specimen were derived from gastric cancer tissues by either biopsy or surgical resection, and was proven by IHC on membrane protein level. Furthermore, those studies were divided into four groups according to the following criteria respectively: (1) overexpression of CD133 in gastric cancer tissues and pericarcinoma tissues or normal gastric tissues; (2) overexpression of CD133 in positive and negative lymph node metastasis of gastric cancer tissues; (3) overexpression of CD133 in positive and negative distant metastasis of gastric cancer tissues; (4) overexpression of CD133 in In different clinical stages of gastric cancer tissues; (5) CD133 overexpression and 5-year overall survival. Table 1P < 0.00001). Figure 2A total of four studies , 18, 20 P < 0.00001). Figure 3A scale of six studies , 19, 20 P < 0.0004). Figure 4A total of four studies , 17, 20 P < 0.00001). Figure 5A scale of seven studies , 19, 20 P < 0.00001). Figure 6A total of three studies , 19, 20 A funnel plot of every two groups in comparison above was applied with RR as the x-axis and SE(RR) as the y-axis, respectively. The plot was symmetric, suggesting that the publication bias was little. Figure 7Nowadays, CSCs are regarded to be significantly responsible for growth, invasion, metastasis and recurrence of various solid tumors. CSCs are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. Expression of cell surface markers have been used to isolate and enrich CSCs from different tumors such as CD44, CD24, CD29, CD133, aldehyde dehydrogenase1 (ALDH1) and epithelial-specific antigen (ESA) . The disP = 0.002) and lower 5-year survival rate , compared with patients with CD133(−) ) OR “ac133” [Title/Abstract]) OR “prominin 1” [Title/Abstract]) AND (((“stomach neoplasms” [MeSH Terms]) AND “carcinoma” [MeSH Terms] OR “gastric cancer” [Title/Abstract]). The reference lists of relative articles were also screened to further identify potential studies.To be eligible for inclusion in this systematic review, a study was required to meet the following criteria: (1) published in English with the full text available, (2) the use of a case control design or a cohort design, (3) the availability of data to allow the estimation of the hazard ratio (HR) for survival with a 95% CI, (4) diagnosis of gastric cancer was proven by immunohistochemistry (IHC) methods, (5) studies of CD133 overexpression based on primary gastric cancer tissue , rather than serum or any other kinds of specimen were included. All studies on the correlation of CD133 overexpression with clinicopathological markers and the association of CD133 overexpression on disease-free and overall survival of gastric cancer were included. When duplicate studies were published, only the most recent or most informative was included in the analysis, to avoid overlap between cohorts.Data tables were made to extract all relevant data from texts, tables and figures of each included studies, including author, year, country, patient number, detection method, clinicopathological features, positive rates of CD133 overexpression, as well as the overexpression-related survival. Information was carefully extracted from all the eligible studies independently. Differences in the extraction of data were assessed by a third investigator.p < 0.10, the total variation among studies was estimated by I-square. A funnel plot was used for assessing the potential publication bias. Comparisons of dichotomous measures were performed by pooled estimates of odds ratios (OR), as well as their 95% CI. Begg's rank correlation method and Egger's weighted regression method were used to assess publication bias .Meta-analysis was supplemented when applicable; otherwise, outcomes were presented in a narrative way. Statistical analysis was performed by Cochrane RevMan 5.2.0 . Dichotomous data were presented as risk ratio (RR) and continuous variables as mean difference (MD), with 95% confidence intervals (95%CI). Statistical heterogeneity was tested using a Chi-square test with significance being set at


The hemodynamic effects of laryngoscopy and endotracheal intubation are well known. A variety of drugs, like fentanyl, esmolol and lately dexmetomidine, have been used in order to attenuate the stress response to laryngoscopy and endotracheal intubation. Esmolol is an ultrashort acting beta 1 selective adrenergic antagonist, while dexmetomidine is an alpha 2 adrenergic receptor agonist. Fentanyl is a short-acting opioid.The scientific objective of this study, was to compare the efficacy of dexmetomidine, fentanyl and esmolol for attenuation of sympathetic response during laryngoscopy and endotracheal intubation.The present study included sixty (60) elective surgery patients with ASA 1 and 2, aged 20-60 years, who needed endotracheal intubation. Patients were randomly allocated into 3 groups. Each group included twenty patients (n = 20). Patients in group D (Dexmetomidine) received 1mcg/kg dexmetomidine with infusion in 10 min, patients in group F (Fentanyl) received 2 mcg/kg fentanyl, while in group E (Esmolol), patients received 2 mg/kg esmolol two min before induction. All patients had Mallampati and Cormack-Lehane Grades 1 or 2. All patients were intubated in less than 30 sec. Systolic, diastolic, mean arterial pressures and heart rates were measured before induction, before intubation and 1, 2, 4 and 10 min after intubation.When recorded values before induction and intubation were compared to the measurements of the groups, it was found that 4 and 10 min after intubation, heart rate in group D and systolic, diastolic and mean arterial pressures in group E, were lower than the other measurements. (p < 0.05).Dexmedetomidine was superior than the other drugs in preventing laryngoscopy and intubation-related tachycardia. On the other hand, esmolol was superior in preventing systolic, diastolic and mean arterial pressure increases following laryngoscopy and intubation.


AbstractTimarcha Latreille, 1829 have been analysed. Timarcha  metallica , has 18 + Xyp male meioformula and 2n = 38 chromosomes, similar to those found in the two species of subgenus Americanotimarcha Jolivet, 1948, in agreement with morphological and molecular phylogenetic grounds. Timarcha (Timarcha) carmelenae Petitpierre, 2013 displays 9 + Xyp and 2n = 20 chromosomes as in morphologically related Andalusian species, whereas Timarcha (Timarcha) parvicollisssp.seidlitzi Kraatz, 1879 shows 11 + Xyp and 2n = 24 chromosomes, clearly differing from the previous species. These results are discussed in order to get an insight into the main trends of the chromosomal evolution in Timarcha.The karyotypes of three species of  Timarcha Latreille, 1829 comprises more than three hundred described taxa, almost all from the Palaearctic  metallica , Timarcha (Timarcha) carmelenae Petitpierre, 2013 and Timarcha (Timarcha) parvicollisssp.seidlitzi Kraatz, 1879 to enlarge the cytogenetic analysis of the genus and discuss the most relevant trends of its chromosomal evolution.Herein, we report the chromosome numbers, male sex-chromosome systems, and main features of their karyotypes of The three checked species and their geographical origins are given in Table p male meioformula, with a “parachute” Xyp sex-chromosome system  of chromosomal arms is 50.Two males of this species have displayed 2n= 38 chromosomes and an 18 + XyPageBreakPageBreakp, again with a “parachute” Xyp sex-chromosome system, that is 2n = 20(Xyp) chromosomes, and showing two autosomal bivalents a bit larger than the others , where five autosomal bivalents are larger than the remaining six ones  metallica should correct a previous miscounting report of 2n = 20 chromosomes , whose range of numbers goes from 2n = 18 to 2n = 30  is assumed to be the plesiomorphic and most frequent karyotype condition for Coleoptera of the suborder Polyphaga   chromosomes, mostly composed of metacentrics or submetacentrics, would have been the plesiomorphous state for the genus, from which all the taxa of the three present subgenera, Americanotimarcha, Metallotimarcha and Timarcha s.str. may have radiated.The diploid number of 2n = 38 chromosomes shown in omosomes . The hig 2n = 30 . However 2n = 44  and Tima 2n = 38 . These htimarcha . Althougolyphaga . Besides 20(Xyp) . And thiPageBreakTimarcha (Timarcha) carmelenae with 2n = 20(Xyp), with two larger autosomal bivalents and the remaining gradually decreasing, is similar to those of Timarcha (Timarcha) intermedia Herrich-Schäffer, 1838, and Timarcha (Timarcha) lugens Rosenhauer, 1856  Küpfer, 1974 for both Timarcha (Timarcha) carmelenae and Timarcha (Timarcha) lugens (Carrichteraannua (L.) DeCandolle, 1821 for Timarcha (Timarcha) intermedia  lugens  and Carrtermedia , in conta s.str. .Timarcha (Timarcha) parvicollisssp.seidlitzi shows a karyotype of 11 + Xyp male meioformula, thus 2n = 24(Xyp) chromosomes, which separates it strikingly from the related Andalusian species with 2n = 20(Xyp) such as Timarcha (Timarcha) insparsa Rosenhauer, 1856, Timarcha (Timarcha) marginicollis Rosenhauer, 1856, Timarcha (Timarcha) intermedia, Timarcha (Timarcha) lugens Rosenhauer, 1856 and Timarcha (Timarcha) carmelenae, sharing a bifid mesosternum and elytra covered with spare and fine puncturation.Timarcha with 2n = 24 chromosomes, Timarcha (Timarcha) pratensis  (Timarcha (Timarcha) parvicollis  , from Cevicollis .


Listeria monocytogenes, a pathogenic food-borne bacterium, is the causative agent of both sporadic and outbreak cases of human listeriosis. Here, we present the genome sequence of L. monocytogenes reference strain LI0521, isolated during an outbreak involving contaminated cheese, which has been used as the model during several proteomic studies. Listeria monocytogenes, a Gram-positive pathogenic food-borne bacterium, is the causative agent of sporadic and outbreak cases of human listeriosis  has been the subject of several studies since its isolation in 1983 during an outbreak caused by contaminated cheese in Massachusetts  running Velvet v1.2.10 (http://vicbioinformatics.com); a total of 3,133 features were identified, including 2,971 open reading frames, 44 tRNA genes, 1 transfer-messenger RNA gene, approximately 100 miscellaneous non-coding RNAs, the temperate phage PSA  indicate that this strain is closely related to lineage I clonal complex 2 (CC2), sequence type 2 (ST2). In addition, the LI0521 genome includes at least 37 putative internalin coding sequences, including inlA, inlB, inlJ, and a conserved prfA virulence gene cluster  and sequencing the genome on a MiSeq Benchtop sequencer (Illumina) for 500 cycles. Quality trimming and filtering of the reads were performed with the trim-fastq.pl module in the Popoolation v1.2.2  package  v1.2.10 , resultie typing  targets  cluster . The AscJMMW00000000. The version described in this paper is the first version, JMMW01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


The immobility is long recognized as a cause of complications in critical patients, including muscle atrophy, pressure ulcers, atelectasis and ocassionally, mortality. Therefore, the role of mobilizing in critical patients is debated.To study the relationship between immobility and hospital mortality on patients requiring mechanical ventilation (MV) during Intensive Care Unit (ICU) stay.A cohort research carried out in a medical-surgical ICU at a third level hospital for 2 years (February 2009-January 2011) on patients who require MV≥ 24 hrs. Data source: clinical trial PUPPAS (1). Primary end point was hospital mortality. The main outcome measure was the implementation of posture changes during the stay in ICU . The posture changes include the next sequence of turning schedules every 2-4 hours: supine position, right side, prone position, left side. Secondary predictors of mortality were age, gender, weight, cancer, surgery, MV duration, APACHE II score, duration of anaemia (days), reintubation (%), SOFA 3 score, albuminemia level and incidence of airway obstruction. Statistical research: descriptive, t-student, chi-square and Cox regression model.329 patients were included, of 61 ± 25 years, 33% of them were women. APACHE II score was 23 ± 4 and the MV duration was 12,7 ± 18 days. The implementation of posture changes for patients was 61%. Hospital mortality was 63,5 ± 38,6 %. In this multivariable model, the hospital mortality hazard ratio (HR) in relation with the percentage of posture changes implemented was 0,97 . Other variables included on the model were APACHE II , age , surgery , SOFA score on the 3th day , duration of anaemia .Patients immobility is associated to a risk of death in patients under mechanical ventilation in ICU.


Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species  at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P+) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2′-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P+ foci by activating GABA(A)R-mediated signaling.Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression  Valerian root extract, widely used in Europe and America as a sedative, hypnotic and anxiolytic, contains a variety of constituents, including essential oils that appear to contribute to the sedating properties of the herb. Iridoid valepotriates like bornyl isovalerenate and bornyl acetate, valeric, isovaleric, formic, malic and acetoxyvalerenic acids, alkaloids and lignans are among components with possible benefit + foci, and liver tumors induced by the genotoxic hepatocarcinogen, diethylnitrosamine, was inhibited at low doses (1 and 2 ppm) in a rat liver medium-term bioassay + foci and tumor development by low doses of PB was suggested to be related to inhibitory effects on cellular proliferation within the areas of preneoplastic lesions, and a correlation was suggested with overexpression of GABA producing enzyme glutamic acid decarboxylase 65 Valerian effects on the central nervous system (CNS) are thought to be similar to those of pharmaceutical phenobarbital (PB), a sedative and anticolvulsant which also binds to GABA(A)Rs and is used widely in clinical therapy for long-term treatment sitchensis, containing high levels of valepotriates. The effects of Valerian at doses of 50, 500 and 5000 ppm applied in the drinking water were investigated in rats with reference to preneoplastic lesion development, oxidative stress, DNA damage, cellular proliferation, apoptosis and gene expression changes in the liver.To check this hypothesis, in the present study we employed a medium-term rat liver bioassay which has been shown to be a very useful tool for detection of hepatocarcinogenicity and chemopreventive potential of chemicals DEN was from Sakai Research Laboratory . All other reagents were purchased from Sigma-Aldrich or Wako Pure Chemicals Industries .sitchensis alcohol-free extract used in the present study was obtained from Eclectic Institute Inc. . It is made first using organic sugar cane alcohol, which is then removed using the Lloyd Extractor. After the alcohol has been removed, the glycerin is added. Glycerin is a solvent similar to alcohol and is listed in the US Pharmacopoeia as an agent to administer certain constituents. Present Valerian extract contained iridoid valepotriates: valtrate, valtrate isovaleroxyhydrin, acevaltrate, valechlorine, didrovaltrate, homodidrovaltrate, deoxydodidrovaltrate, isovaleroxyhydroxydidrovaltrate, isovaltrate, 7-epi-deacetyl-isovaltrate. In addition, it contains valerosidatum (an iridoid ester glycoside). Volatile oil contains a lot of components including valeric and isovaleric acids, bornyl acetate, monoterpens , sesquiterpenes β-bisabolene, caryophyllene, valeranone, valerenic acid (traces), valerianol, valerenal,β-ionone, patchouli alcohol, ledole and terpinolene among others. Furthermore, extract includes alkaloids , choline, methyl 2-pyrrolyl ketone, chlorogenic acid, caffeic acid, β-sitosterol, tannin, gam, manganese, calcium, amino acids such as GABA, glutamine, arginine, alanine and others. Highest amounts of valerenic acid were reported in V. officinalis L., trace amounts in V. sitchensis, and none in the other Valerian species analyzed Valerian root extracts usually contain more than 100 different constituents. The Valeriana A total of 120, five-week-old male Fisher 344 rats  were quarantined for 1 week before the start of the experiment. They were housed in an animal facility maintained on a 12 h (7:00–19:00) light/dark cycle, at a constant temperature of 23±1°C and relative humidity of 44±5%, and given free access to tap water and food . All experimental procedures were conducted following approval of the Animal Care and Use Committee of the Osaka City University Graduate School of Medicine. Guidelines set by the National Institute of Health and Public Health Service Policy on the Humane Use and Care of Laboratory Animals were followed at all times.sitchensis  in their drinking water at doses of 0, 50, 500 and 5000 ppm, for 6 weeks from weeks 3 to 8. All were subjected to two thirds partial hepatectomy (PH) at week 3 to maximize any interaction between proliferation and the effects of the test chemicals. At sacrifice at week 8, livers were quickly dissected out, weighed and sections from three lobes were fixed in Bouin's solution and 10% phosphate-buffered formalin for histological and immunohistochemical analyses. In addition, samples were frozen in liquid nitrogen and stored at −80°C for molecular analyses. For AST and ALT measurements by the consensus method of Japan Society of Clinical Chemistry, blood was collected from the abdominal aorta.Before the start of the experiment, 6-week-old rats were allocated to six groups . In groups 1–4, rats were given a single i.p. injection of DEN (200 mg/kg body weight (b.w.)) dissolved in saline to initiate hepatocarcinogenesis. In groups 5 and 6, rats were administered an i.p. injection of saline as vehicle controls. After 2 weeks on tap water and basal diet, animals in groups 1–4 were administered water root extract of Valeriana Valerian doses used in the present study were selected on the basis of previously published data on humans and the findings of our preliminary experiment in which no toxicity was detected even at a dose of 5000 ppm. The doses of 50 ppm (5 mg/kg b.w./day), 500 ppm (50 mg/kg b.w./day) and 5000 ppm (500 mg/kg b.w./day) consumed by a rat (b.w. 200 g) in 20 ml drinking water in the present experiment would be equal to 0.05, 0.5 and 5 mg/kg b.w./day intake by a human with a mean body weight of 50 kg (the accepted WHO safety factor in terms of accepted dietary intake (ADI) for rats is 100). Another extrapolation from human to rat involves multiplying the human dose by 6.16  et al.+ foci was accomplished using two-dimensional evaluation 2 of liver section.Immunohistochemical assessment of GST-P was performed with the ABC method as described by Kitano + foci and background liver parenchyma (5000 cells) and expressed as percentages of positive cells for PCNA or ssDNA in all GST-P+ cells or surrounding liver cells.Double stainings for GST-P and PCNA and GST-P and TUNEL were performed in formalin-fixed sections with polyclonal rabbit anti-GST-P antibody at 1∶2000 dilution, anti-PCNA mouse monoclonal  antibody and ApopTaq Peroxidase in Situ Apoptosis Detection Kit  using alkaline phosphatase  solution for the immunohistochemical detection of GST-P and DAB for the detection of PCNA or apoptosis Liver sections of selected animals were stained immunohistochemically using rabbit polyclonal GABA(A)RA1 , mouse monoclonal GABA(B)R1 , rabbit polyclonal GABA(B)R2  and anti-phospho-Nrf2 (Ser40)  antibodies by ABC method, with color development by DAB, and assessed qualitatively DNA samples were extracted from rat liver tissues to allow measurement of 8-OHdG levels by HPLC-ECD as reported previously + RNA enrichment and generation of cDNA probes using a Affymetrix GeneChip T7-Oligo(dT) Promoter Primer Kit (Affymetrix) according to the manufacturer's protocol. Biotin-labeled antisense cRNA was synthesized by in vitro transcription reaction using an RNA Transcript Labeling Kit , purified and fragmented, and hybridized to GeneChip RAT Genome 230 2.0 arrays, with 28,700 probe sets. Affymetrix GCOS software version 1.0 was employed for normalization and for monitoring specific hybridization. Microarray data were analyzed using GeneSpring software version 12 . Each array was normalized to the 50th percentile and each gene was normalized to the control (DEN initiated rats). Microarray analysis was repeated three times to check the reproducibility of the data and mean values of gene expression were calculated for spots with at least 2 fold up- or down-regulation. One-way-Anova was applied to compare replicate mean values of control and experimental groups and to find genes whose expression was consistently altered by Valerian administration. Clustering analysis was performed with the Condition Tree algorithm. The dataset was submitted to DNA Data Bank of Japan (DDBJ) (submission ID: PSUB003813).Total RNA was isolated from rat liver tissues and 8 µg pooled aliquots from 5 rats in each group were treated with DNase 1 and processed for PolyATo assign biological significance of differentially expressed genes and identify networks of interacting genes, functional groups and pathways, the Ingenuity program  was utilized. IPA was further applied for the prediction of altered up-stream regulators by Valerian. Transcriptional regulation was measured by z-scores. A z-score of above 2 was considered significant.GABA(A)RA1 (Rn00788315_m1), histone deacetylase 4 (HDAC4) (Rn01427040_m1), c-myc (Rn00561507_m1), mafb (Rn00709456_s1), jun (Rn00572991_m1), fos (Rn02396760_g1), MAPK 3 (ERK1)(Rn00820922_g1), MAPK 14 (p38) (Rn00578842), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (Rn00477784_m1), NADPH quinone oxidoreductase 1 (NQO1) (Rn01432447_m1), heme oxygenase 1 (HO-1) (Rn00561387_m1), cyp7A1 (Rn00564065_m1), superoxide dismutase (SOD) (Rn00566938_m1), catalase (CAT) (Rn00560930_m1), cyclin D1 (Rn00432359_m1), NfkB (Rn01502266_m1), p53 (RN00755717_m1), Waf1/cip1p21  (Rn01427989_s1) and Bax (Rn02532082_g1). Results are expressed relative to the number of eukaryotic 18S RNA transcripts (4319413E) used as an internal control.Real-time Q-PCR was performed as previously described The significance of differences for each parameter  was analyzed using the StatLight–2000(C) program  or the IBM SPSS Statistics 19 Software . Statistical comparisons between groups of numerical data were conducted using the Bartlett's test. If homogeneous, the data were analyzed with the Dunnett's multiple comparison test (two sided), and if not, with the Steel's test (two-sided). Statistical comparisons between vehicle control and 5000 ppm Valerian groups for numerical data were assessed using the F test. If homogeneous, the data were analyzed with the Student's t-test (two-sided), and if not, with the Welch test. In microarray analysis, GeneSpring software version 12 was utilized to perform one-way-Anova to compare replicate mean values of control and experimental groups and to find genes whose expression was consistently altered by Valerian administration.Final body and relative liver and kidney weights and Valerian intake data are shown in + foci are given in + foci were detected in vehicle control and 5000 ppm Valerian treated rats. Importantly, both numbers and to a lesser extent areas of GST-P+ foci in rats given 50, 500 and 5000 ppm Valerian after DEN initiation were significantly decreased in a dose-dependent manner as compared to the initiation control group.Data for GST-P0). In the initiation control group several hepatic foci, consisting of more than 20 cells, contained a large number of PCNA positive nuclei . Slight but significant elevation of apoptosis was also apparent in the liver of rats treated with 5000 ppm Valerian alone as compared to the respective vehicle control.Analysis by double immunohistochemistry for GST-P and TUNEL demonstrated dose-dependent increase of apoptosis in the areas of GST-P5000 ppm . This in+ foci in initiation control rats were positive for GABA(A)RA1 with a membranous and/or rarely cytoplasmic localization  form of indicator of oxidative stress, Nrf2 (Nrf2-Ser-P), have demonstrated that its expression is high mostly in the areas of central and portal veins and inflammatory cell infiltration areas in the livers of rats of DEN initiation control group, but is suppressed by Valerian treatment .WAF1/Cip1) and inositol 1,4,5-trisphosphate receptor, type 1 (Itpr1) and transcriptional regulators such as hepatocyte nuclear factor 3 beta, Kruppel-like factor 9 and early growth response 1.The results of cDNA microarray, GeneSpring and one-way-Anova analyses of differentially expressed genes after 6 weeks of 50, 500 and 5000 ppm Valerian administration followed DEN initiation are presented in In normal rat liver parenchyma of rats treated with 5000 ppm Valerian after vehicle administration, only expression of Gsta2 and adenylate kinase 3 (Ak3) was significantly increased.Clustering analysis by Gene Spring software demonstrated clear differences in gene expression pattern between vehicle and DEN treated groups . ValeriaIPA upstream regulator analysis indicated that DEN treatment resulted in activation of c-myc, Mafb, jun and HNF1 . In contWaf1/cip1 and Bax, was checked by Q-RT-PCR analysis RA1 signaling and other intracellular pathways, and to confirm the results of cDNA microarray analysis, GABA(A)RA1, HDAC4, c-myc, Mafb, jun, fos, CD1, NfkB, ERK1, p38, Nrf2, NQO1, HO-1, Gpx2, SOD, CAT and CYP7A1, p53, p21analysis . Signifianalysis . In addianalysis . On the reatment .+ foci in a medium-term rat liver bioassay indicating prevention of hepatocarcinogenesis. Importantly, clear dose dependent effects were obvious, and significant inhibition was observed even at low dose. The mechanisms are likely to be related to significant suppression of cell proliferation and induction of apoptosis in the areas of GST-P+ foci accompanied by inhibited formation of oxidative base modifications in the rat liver DNA, due to activation of GABA(A)R-mediated signaling, coordinated with induction of HDAC4 and GABA(A)RA1, CAT, p53, p21Waf1/cip1 and Bax, and inhibition of c-myc, Mafb, CD1, CYP7A1 and Nrf2.The present study demonstrated an inhibitory effect of Valerian on formation of GST-PIn this study, Valerian was also found to suppress the serum levels of AST, a pyridoxal phosphate-dependent transaminase enzyme which was induced by DEN treatment. AST is usually found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells, and it is commonly measured clinically as a marker for liver health. In line with our data, previously AST elevation in the rat blood serum and its suppression by potential chemopreventive agents was shown after DEN injection in rats and mice + foci. Interestingly, in the present study positive expression of GABA(A)RA1 (α1 subunit) with a membranous and/or cytoplasmic localization was found in cells comprising GST-P+ foci in rats initiated with DEN. Furthermore, Valerian administration caused GABA(A)RA1 elevation in GST-P+ foci. GABA(A)R is an ionotropic receptor and ligand-gated ion channel and upon activation, exhibits chloride channel activity selectively and conducts Cl− through pores resulting in hyperpolarization of the neuron. This causes an inhibitory effect on neurotransmission by diminishing the chance of a successful action potential occurring. To date, 16 human and rat GABA(A)R subunit genes have been characterized and grouped together according to their amino acid similarity and termed: α1-6, β1-3, γ1-3, δ, ε, θ, and π Here we present the first evidence for inhibition of cell proliferation with Valerian in the areas of GST-P2++ foci or (ii) indirect influence on GABAergic neurotransmission and GABA(A)R signaling in the CNS which inhibits hepatic proliferation via suppression of sympathetic regulation (+ foci overexpress GABA(A)RA1 allows us to suggest that Valerian may directly affect the cells comprising GST-P+ foci, thus, activating GABA(A)Rs, suppressing cell proliferation and finally exhibiting inhibitory effects on hepatocarcinogenesis.For comparison, GABA(B) receptors (GABA(B)R) are metabotropic transmembrane receptors for GABA that are linked via G-proteins to potassium channels gulation . Interessitchensis, a native of northwestern America, is considered to have higher levels of valepotriates and stronger medicinal activity than other Valerian species but to contain only traces of valerenic acid in vitro and in vivo on lung, pancreas, colon, breast and prostate cancers Valeriana 2 conversion) The selected Valerian doses in this study were comparable to those applied in humans if using the extrapolation with multiplication index for rats c-myc, Mafb oncogenes, Per2, Nr0b2, Igfbp1, CD1 and others. Furthermore, it inhibited N-myc and jun oncogenes as indicated by the analysis of upstream regulators by IPA R signaling.As detected by cDNA microarray analysis, Valerian treatment at all doses suppressed expression of numerous genes affecting cellular proliferation, such as s by IPA . These aGABA(A)RA1 expression was reported to be positively regulated by HDAC4 in cultured neurons + foci observed in the present study supported this concept. Therefore, the suppression of their development by Valerian might be related to an inhibitory effect on 8-OHdG formation in the DNA of hepatocytes. The observed suppression of 8-OHdG generation by Valerian after DEN initiation might be a result of suppression of oxidative stress due to up-regulation of catalase (CAT), down-regulation of Nrf2 as well as CYP7A1 in the rat liver R-mediated signaling in rats. Our data demonstrate that Valerian suppressed 8-OHdG formation, significantly inhibited cell proliferation and induced apoptosis in the areas of GST-P+ foci, and altered expression of genes related to control of cell proliferation and apoptosis, which might explain its inhibitory effects on hepatocarcinogenesis.In conclusion, Valerian, a sedative, hypnotic and anxiolytic medicine, here inhibited the formation of GST-PFigure S1Immunohistochemistry for Nrf2-Ser-P in DEN initiation control , DEN followed by 5000 ppm Valerian , and Vehicle groups . Note the intense Nrf2-Ser-P staining in the portal central vein areas in DEN control group, but absence of staining in Valerian-treated and Vehicle groups.(TIF)Click here for additional data file.Figure S2Graphically illustrated suggested mechanisms of Valerian inhibitory activity on rat hepatocarcinogenesis.(TIF)Click here for additional data file.


Chordomas are rare primary malignant bone tumors arising from embryonal notochord remnants of the axial skeleton. Chordomas commonly recur following surgery and radiotherapy, and there is no effective systemic therapy. Previous studies implicated receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF-1R), in chordoma biology. We report an adult female patient who presented in 2003 with spinal chordoma, treated with surgery and radiotherapy. She underwent further surgery for recurrent chordoma in 2008, with subsequent progression in pelvic deposits. In June 2009, she was recruited onto the Phase I OSI-906-103 trial of EGFR inhibitor erlotinib with linsitinib, a novel inhibitor of IGF-1R/insulin receptor (INSR). Treatment with 100 mg QD erlotinib and 50 mg QD linsitinib was well-tolerated, and after 18 months a partial response was achieved by RECIST criteria. From 43 months, a protocol modification allowed intra-patient linsitinib dose escalation to 50 mg BID. The patient remained stable on trial treatment for a total of 5 years, discontinuing treatment in August 2014. She subsequently experienced further disease progression for which she underwent pelvic surgery in April 2015. Analysis of DNA extracted from 2008  tissue showed that the tumor harbored wild-type EGFR, and a PIK3CA mutation was detected in plasma, but not tumor DNA. The 2015  tumor harbored a mutation of uncertain significance in ATM, with no detectable mutations in other components of a 50 gene panel, including EGFR, PIK3CA, and TP53. By immunohistochemistry, the tumor was positive for brachyury, the molecular hallmark of chordoma, and showed weak–moderate membrane and cytoplasmic EGFR. IGF-1R was detected in the plasma membrane and cytoplasm and was expressed more strongly in recurrent tumor than the primary. We also noted heterogeneous nuclear IGF-1R, which has been linked with sensitivity to IGF-1R inhibition. Similar variation in IGF-1R expression and subcellular localization was noted in 15 further cases of chordoma. In summary, this exceptionally durable response suggests that there may be merit in evaluating combined IGF-1R/INSR and EGFR inhibition in patients with chordomas that recur following failure of local treatment. Chordomas are rare primary malignant bone tumors arising from primitive notochord remnants, and account for ~17% of primary malignant bone tumors of the axial skeleton . Germ-liClinically, there is approximately equal distribution between cranial, spinal, and sacral origin, with risk of local recurrence in ~45%, metastasis in ~30%, and median survival of 6.3 years –9. The mThe patient is a 65-year-old non-smoker who presented in mid-2003 with a 9-month history of lower back pain. Magnetic resonance imaging (MRI) revealed replacement of the L4 vertebra by a lobulated tumor that invaded the spinal canal, encroaching on the cauda equina. MRI and computed tomography (CT) scans showed no evidence of primary tumor elsewhere. She underwent biopsy under fluoroscopic control followed by surgery on 16th July 2003. The procedure involved radical tumor resection with insertion of an intervertebral cage containing a biphasic calcium phosphate ceramic (Triosite). Histological examination Figures , she undFrom April 2007, the patient experienced recurrent pain, weakness, and altered sensation in the lower limbs. Initial MRI scans were equivocal for tumor recurrence but, in August 2007, showed definite evidence of recurrence to the left of the fourth lumbar vertebral body, eroding the superior end-plate and the posterosuperior border of L5 vertebral body. She experienced further clinical and radiological progression, including an acute episode of paraplegia in December 2007, and in February 2008, she underwent major revision surgery. Laparotomy revealed considerable recurrent tumor behind the body of L4 with bleeding into the spinal canal at this level, and tumor infiltrating the psoas muscles, also with hemorrhagic change. She underwent a debulking procedure with anterior vertebrectomy and anterior and posterior fusion, leaving residual tumor in the psoas muscles. Histopathological analysis confirmed recurrent brachyury-positive chordoma in the L4 vertebral body, epidural surface, psoas muscles bilaterally, and left abdominal wall. Post-operatively, there was gradual recovery of power in the lower limbs, although with persistent altered sensation in the legs and loss of knee and ankle reflexes. MRI scans in March 2009 showed further evidence of progression, prompting referral to our Early Phase Clinical Trials Unit in June 2009.The patient was recruited onto the Phase I OSI-906-103 trial, evaluating linsitinib (OSI-906), a novel inhibitor of the type 1 insulin-like growth factor receptor (IGF-1R) and insulin receptor (INSR), in combination with the epidermal growth factor receptor (EGFR) inhibitor erlotinib . InitialSequential MRI scans revealed initial disease stabilization, with gradual improvement to partial response by response evaluation criteria in solid tumors (RECIST) in December 2010, after 18 months of study treatment . This analysis showed no evidence of mutation in EGFR, TP53, PTEN, PIK3CA, PDGFRA, KRAS, NRAS, BRAF, KIT, ERBB4, FGFR1, ERBB2, MET, FGFR3, FLT3, GNAS, SMARCB1, CTNNB1, CDKN2A, ABL1, NOTCH1, PTPN11, SMO, SMAD4, VHL, NPM1, MPL, CSF1R, HRAS, JAK3, AKT1, IDH1, CDH1, FGFR2, SRC, KDR, ALK, JAK2, MLH1, RB1, HNF1A, APC, RET, STK11, FBXW7, EZH2, GNA11, GNAQ, and IDH2. A mutation of uncertain clinical significance was detected in the ATM gene .Analysis of tumor tissue was conducted under study 07/H0606/120, approved by National Research Ethics Service (NRES) Committee South Central-Oxford C. Formalin-fixed, paraffin-embedded (FFPE) tumor blocks from the 2008 surgery yielded a small amount of genomic DNA for mutation analysis. No variants were detected in EGFR codons 18, 19, and 21 , KRAS, BRAF, or PIK3CA, whereas analysis of plasma DNA revealed E542K mutation in exon 9 of PIK3CA, as reported in Ref. . DNA wasImmunohistochemical (IHC) analysis was conducted on FFPE tumor to investigate IGF-1R and EGFR expression, using an IGF-1R IHC protocol recently optimized for sensitivity and specificity –18. InitTo determine the extent to which these appearances are representative, we performed IGF-1R IHC on 15 additional chordoma cases Figure , includivia AKT to enhance tumor cell growth and survival (in vitro chordoma cell growth by a tyrphostin EGFR inhibitor (Previous reports have implicated receptor tyrosine kinase (RTK) signaling in the biology of chordomas . A recensurvival , and thesurvival . In sevesurvival , 22–24, survival . EGFR exnhibitor . As in onhibitor , 27. A rnhibitor , but thenhibitor –32. Our nhibitor .Recent studies have investigated the contribution of the IGF axis to chordoma biology. IGF-binding protein-2 (IGFBP-2) was among the genes found to be upregulated in chordoma compared with benign intervertebral disk . In otheGenetic analysis of human chordomas has identified several additional changes that are implicated in tumorigenesis and could be relevant to response to therapy. First, PIK3CA E545K) was detected in 1 of 45 patients  and was K was detIn conclusion, we report a durable objective response to linsitinib and erlotinib in a patient with spinal chordoma that had recurred following failure of local treatment. Factors that may be relevant to the response include TP53, EGFR and PIK3CA status and the presence of nuclear IGF-1R, which may signify dependence on IGF-1R and sensitivity to IGF axis inhibition. We suggest that trials incorporating assessment of these parameters should evaluate the effects of combined IGF-1R/INSR and EGFR inhibition in patients with recurrent chordomas.VM, MW, ABH, SP, DW, and MM were involved in care of the patient; TA, LB, RP, SP, SH, NA, and OA contributed to data acquisition and analysis; VM drafted the manuscript, and the content was reviewed, edited, and approved by all authors.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.