Pathologic myocardial hypertrophy develops when the heart is chronically pressure-overloaded. Elevated intracellular cGMP-levels have been reported to prevent the development of pathologic myocardial hypertrophy, therefore we investigated the effects of chronic activation of the cGMP producing enzyme, soluble guanylate cyclase by Cinaciguat in a rat model of pressure overload-induced cardiac hypertrophy. Abdominal aortic banding (AAB) was used to evoke pressure overload-induced cardiac hypertrophy in male Wistar rats. Sham operated animals served as controls. Experimental and control groups were treated with 10 mg/kg/day Cinaciguat (Cin) or placebo (Co) p.o. for six weeks, respectively. Pathologic myocardial hypertrophy was present in the AABCo group following 6 weeks of pressure overload of the heart, evidenced by increased relative heart weight, average cardiomyocyte diameter, collagen content and apoptosis. Cinaciguat did not significantly alter blood pressure, but effectively attenuated all features of pathologic myocardial hypertrophy, and normalized functional changes, such as the increase in contractility following AAB. Our results demonstrate that chronic enhancement of cGMP signalling by pharmacological activation of sGC might be a novel therapeutic approach in the prevention of pathologic myocardial hypertrophy. Long term presence of pathologic myocardial hypertrophy is a major underlying cause of heart failure (HF). One of its main inducing factors is pressure overload of the left ventricle (LV), which causes concentric LV hypertrophy (LVH) with collagen accumulation and subsequent impairment of diastolic function. This adverse remodelling of the LV can result in HF with preserved ejection fraction (HFpEF), a condition that is increasingly investigated, as it equals HF with reduced ejection fraction (HFrEF) both in outcomes and numbers1Cyclic GMP (cGMP) is an important regulator of many physiological and pathophysiological processes in the cardiovascular system, including cardiac remodelling610A new group of drugs named sGC activators has been developedIn our present study, we aimed at characterizing the cardiac effects of Cinaciguat in a rat model of pressure overload-induced LVH. We used abdominal aortic banding (AAB) to induce pressure overload in our animals, which is a well-established and widely used procedure to evoke hypertension and pathologic LVH in rodents1516rd postoperative week verified significantly elevated LV wall thickness values, relative wall thickness (RWT) and estimated LV mass (LVM) in the AABCo group compared to ShamCo without significant changes in chamber dimensions  and fractional shortening (FS) remained unchanged during the whole study , EF, stroke volume (SV), cardiac output (CO), or LV end-systolic volume (LVESV), and also parameters of preload, such as LV end-diastolic volume (LVEDV) and -pressure (LVEDP) were not significantly different among the groups .LV systolic (LVESP) and mean arterial blood pressure (MAP) proximal to the site of stenosis were significantly higher in both AAB groups than in the Sham groups, and neither of these parameters were affected by Cinaciguat .max) was significantly higher in AABCo animals than in ShamCo rats (es) and preload recruitable stroke work (PRSW) (max-end-diastolic volume relationship (dP/dtmax-EDV) had a similar trend (Maximum rate of LV pressure increment (dP/dtmCo rats . Load ink (PRSW) , also shar trend .Active relaxation was impaired in the AABCo rats compared to ShamCo, as evidenced by the time constant of LV pressure decay (τ), while it was similar to ShamCo in the AABCin animals .Morphological changes were present on the microscopic level as well. Average cardiomyocyte width was significantly increased in the AABCo group compared to ShamCo , which wAnalysing immunohistochemical staining on myocardial sections for cGMP resulted in significantly higher score in AABCin rats than either in ShamCo or AABCo animals . Plasma 2+-ATPase 2a (SERCA2a) and phospholamban (Pln) was significantly elevated in the AABCin rats  and endothelial NO synthase (NOS3) , and decCin rats .Anti-apoptotic signalling was reinforced by Cinaciguat, as evidenced by the significant increase in B-cell lymphoma 2 (Bcl-2) expression and the strong tendency towards higher expression of 70 kDa heat shock protein (HSP70) in the AABCin animals .Protein density of protein kinase G (PKG) was significantly elevated in myocardial homogenates of AABCo rats, while it was comparable to ShamCo in the AABCin group . Phosphoin vivo despite the unchanged loading of the LV. In parallel with the significant morphological changes, functional alterations were normalised by the Cinaciguat treatment following AAB.In our current work we demonstrate for the first time that the chronic activation of sGC by Cinaciguat and the subsequent rise in cGMP levels efficiently reduce pressure overload-induced pathologic myocardial hypertrophy In vivo, the major drive in the background of the hypertrophic response of cardiomyocytes to chronically increased afterload is the stretching of the cell membrane18in vitro studies have shown that Cinaciguat has anti-hypertrophic effects in cultured neonatal rat cardiomyocytes2+-related signalling pathways, troponin Iin vivo myocardial anti-hypertrophic effect of Cinaciguat in previously published works was suggested to be secondary to the amelioration of the primary disease  levels in response to Cinaciguat treatment.rd and 6th week  was proposed as a fairly load-insensitive index of ventricular contractility. PRSW (the slope of the linear relation between SW and EDV) has been described as a parameter independent of chamber size and mass, and it is sensitive to contractile function of the ventricle432+-channels and activation of late rectifier K+-channels might both decrease intracellular Ca2+ concentration2+-sensitivity of cardiomyocytesAnalysis of P-V data acquired during invasive hemodynamic measurements provides more precise assessment of cardiac performance. E2+-clearance in the early phase of diastole, resulting in maintained active relaxation47A hallmark of HHD is the impairment of LV diastolic function long before systolic dysfunction occurs, resulting clinically in the HFpEF phenotype. Both the decrease of passive compliance and impaired active relaxation of the LV can be in the background of diastolic dysfunctionDiastolic dysfunction causes backward failure initially in the pulmonary circulationin vivo. Therefore, the observed increase in contractility in the AABCo group and conversely, normalization of contractility in AABCin animals might partially be caused by the structural differences of the LV wall between these groups.LV geometry and the amount of fibrotic components in the myocardial wall might both influence LV contractility parameters measureable by PV-analysis. Despite this method being the current gold standard for assessing different aspects of cardiac function, it is not possible to separate these confounding factors from the true contractility of the myocardial sarcomere in vivo irrespective of hemodynamic load. We observed the beneficial effect of sGC activation on morphological, functional and molecular levels as well. sGC activators therefore might prove to be an efficient new therapeutic approach in the treatment of pathologic myocardial hypertrophy.Our research group shows here for the first time that chronic activation of sGC by Cinaciguat prevents the development of pathologic myocardial hypertrophy For more details, see the online After acclimation, banding of the abdominal aorta  between the renal arteries and the superior mesenteric artery, or sham operation n = 16) was performed in pentobarbital sodium (60 mg/kg i.p.) anaesthesia as previously described was perf5 days after the operations, sham and AAB animals were randomized into control or treatment groups . Treated animals received Cinaciguat (10 mg/kg p.o.) suspended in 0.5% methylcellulose solution via oral gavage, while control rats were given only the vehicle every day for 6 weeks. The dosage was adjusted to body weight, which was measured three times a week during the whole study period.rd and 6th week after the operations as previously describedWe performed echocardiographic measurements at the 3max, τ (according to the Glantz method), EF and SW were computed and calculated using a special P-V analysis program . SV and CO were calculated and corrected according to in vitro and in vivo volume calibrations using PVAN software. In addition to the above parameters, the slope (Ees) of the LV end-systolic P-V relationship  staining to detect DNA strand breaks in LV myocardium. Light microscopic examination was performed with a Zeiss microscope , and digital images were captured using an imaging software .The mean value of transverse transnuclear widths of 100 randomly selected, longitudinally oriented LV cardiomyocytes represents each sample. The amount of myocardial collagen was determined by measuring the area fraction of the Picrosirius red-stained areas of five randomly selected visual fields (magnification: 200x) of subendocardial LV myocardium of each section with ImageJ software. Immunohistochemical reactivity for cGMP was examined with light microscopy at a magnification of 400x. Semi-quantitative scoring  was performed by two people blinded to the groups as described elsewhereAfter hemodynamic measurements were completed, blood samples from the inferior caval vein were collected in tubes rinsed with EDTA. The blood samples were centrifuged at 3,000 RPM for 15 min at 4 °C, then separated plasma was stored in aliquots at -80°C. Plasma level of cGMP was determined using an enzyme immunoassay kit as per manufacturer’s protocol .2+-ATPase isoform 2a (SERCA2a) and phospholamban (Pln), all purchased from Applied Biosystems. Gene expression data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expression levels were calculated using the CT comparative method (2−ΔCT). All results are expressed as values normalized to a positive calibrator .LV myocardial tissue samples were harvested immediately after euthanasia, snap frozen in liquid nitrogen, and stored at −80 °C. LV tissue was homogenized in RLT buffer, and RNA was isolated from the ventricular samples using the RNeasy Fibrous Tissue Mini Kit  according to the manufacturer’s instructions. Quantitative real-time PCR was performed with the StepOne-Plus Real-Time PCR System  in triplicates of each sample for the following targets: α- and β-isoform of myosin heavy chain , endothelial nitric oxide synthase (NOS3), atrial natriuretic peptide (ANP), B cell lymphoma 2 (Bcl-2), 70 kDa heat shock protein (HSP70), sarcoplasmic and endoplasmic reticulum Ca® Novex® Bis-Tris Mini Gel, Invitrogen, Carlsbad, CA, USA). Afterwards, proteins were transferred to nitrocellulose membrane by using a semi-dry electroblotting system . Membranes were incubated overnight at 4 °C with primary antibodies  against various target proteins as follows: members of NO signalling such as protein kinase G , vasodilator-stimulated phosphoprotein (VASP) and phospho-VASP, phospholamban (Pln) and phospho-Pln as markers of PKG activity. After washing, membranes were incubated in horseradish peroxidase (HRP) – conjugated secondary antibody dilutions at room temperature (RT) for 1 h . Immunoblots were developed using Pierce® ECL Western Blotting Substrate Kit . Protein band densities were quantified using GeneTools software . GAPDH  was used to assess equal protein loading. Values of protein band densities (after adjusting to GAPDH band densities) were normalized to the average value of the ShamCo group and were used to perform statistical analysis. Representative original immunoblots are shown in Immunoblot analysis was performed as previously describedAll drugs listed were purchased from Sigma-Aldrich  except for Cinaciguat, which is a kind gift of Bayer AG .Statistical analysis was performed on a personal computer with a commercially available software .band, ptreat) and significant banding × treatment interactions (pint). Tukey’s post hoc testing was performed to evaluate differences between the groups. Data that did not show normal distribution were transformed logarithmically before performing two-factorial ANOVA.All data are expressed as mean ± standard error of the mean (SEM). After testing normal distribution of the data using the Shapiro-Wilk test, two-factorial analysis of variance (ANOVA) (with ‘aortic banding’ and ‘Cinaciguat treatment’ as factors) was carried out to detect independent effects of the factors .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The STICH(-ES) trial showed that coronary artery bypass grafting was superior to medical therapy alone in treating ischemic heart failure. However, dosages of disease modifying drugs were not reported. We included 128  consecutive patients with ischemic heart failure and an ejection fraction ≤35% undergoing isolated elective coronary artery bypass grafting. We defined optimal medical therapy (OMT) as prescription of ≥50% dosages of guideline recommended medications (i.e. beta-blocker (BB) and renin angiotensin system (RAS) antagonist) plus prescription of a mineralocorticoid receptor antagonist (MRA). The mean logistic EuroSCORE was 12.3 ± 13.8%. The five year survival was 74%. At discharge, 111 patients (87%) were on a BB and 106 (83%) were on a RAS antagonist. Forty-nine patients (38%) received an MRA. Only 8 patients (6%) received OMT. A Cox regression analysis revealed EuroSCORE (p < 0.001) and the use of MRA (p = 0.003) and BB (p = 0.037) at discharge as significant predictors of 5 year survival. Prescription rates of heart failure medication are comparable to those reported in the STICH trial, but rates of OMT are very low at admission and discharge. Prescription of BB and MRA was associated with improved survival, highlighting the need for disease management programs and rigorous discharge management. It compared a conservative strategy of medical therapy alone versus CABG and medical therapy, and found a benefit for CABG regarding relevant secondary endpoints such as death from cardiovascular cause and hospitalization for cardiovascular reasons and a statistically significant mortality benefit in the prespecified long-term analysis2. Patients in the STICH trial were treated with renin angiotensin system inhibitors and beta blockers in almost 90% of the cases, but dosages of heart failure specific drugs were not reported.The Surgical Treatment for Ischemic Heart Failure (STICH) trial enrolled patients with reduced left ventricular ejection fraction (LVEF) and coronary artery disease who were candidates for coronary artery bypass grafting (CABG)3. The results of STICH should be interpreted such that medical and surgical options are not competitors, but rather complementary strategies. Hence, not only the prescribed drugs but also their dosages could have important effects on outcome.It has been shown that adherence to guideline-specific medical therapy is a strong predictor of outcome in chronic heart failure4. Data for patients with heart failure undergoing cardiac surgery are completely lacking. Here, we sought to evaluate the intensity, i.e. the percentage of recommended target dose, and the effect of medical therapy with heart failure specific drugs in a STICH-eligible population under “real world” conditions at a tertiary care university center.Guidelines recommend early discharge management in patients with acute heart failure with an emphasis on initiation and planning of therapyConsecutive patients suffering from ischemic heart failure with a LVEF of ≤35% who underwent elective CABG at a tertiary care center between 2009 and 2013 were included in this study. Exclusion criteria were acute surgery and concomitant valve replacement or reconstruction. Baseline evaluation was performed on the day of admission early in the morning. Patients had taken their morning medication, vital signs were taken and blood was drawn after patients had rested in a supine position.Mortality status was assessed through the Austrian statistic agency (Statistics Austria) as well as by telephone follow-up and review of patient files in hospitals of the same city.5. Group 1: not prescribed; group 2: 1–49% of recommended target dosage; group 3: 50–99% of recommended target dosage; group 4: 100% of recommended target dosage. For this study, optimal medical therapy (OMT) was defined as at group 3 or 4 for (1) beta blocker (BB) and for (2) renin angiotensin system (RAS) antagonists plus (3) any prescription of a mineralocorticoid receptor antagonist (MRA). This is based on the current guidelines and a prospective trial showing that while higher dosages are beneficial, there was no difference between 50–99% and 100% of target dosage6. It was measured by analyzing the prescribed dosages at admission and discharge. Continuous variables are presented as mean and standard deviation and compared by the independent samples t-test. Total numbers and proportions are reported for categorical outcomes and tested by the Chi-square test. Heart failure therapy prescription at admission and discharge was compared by the Wilcoxon signed-rank test. The effect of heart failure therapy prescription on survival was assessed with a Cox-regression analysis including therapy prescription at discharge from primary hospital stay  and the logistic EuroSCORE. SPSS Statistics  was used for statistical analysis. A two-sided p-value less than 0.05 was considered as significant.The analysis of heart failure specific medical therapy was based on a prospectively maintained database. Heart failure specific medication was grouped according to the dosage prescribed as percentage of the dosage recommended in the guidelines for the treatment of chronic heart failure by the European Society of CardiologyThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.The study population consisted of 128 consecutive patients. Baseline demographic data, clinical characteristics and risk factors are reported  died within 30 days after surgery. One patient was lost to follow-up due to geographical reasons. After a mean follow-up period of 47 ± 22 months, 34 patients (27%) died. The one and five year survival was 91% and 74%, respectively. A Cox-regression analysis including medication at discharge and preoperative logistic EuroSCORE, revealed the use of MRA or BB as well as the preoperative EuroSCORE as independent predictive factors . The need to obtain informed consent was waived by the EC.


DNMT3B: rs2424909, DNMT1: rs4804490, DNMT3A: rs1550117 and DNMT3L: rs7354779). Eight hundred and thirty three idiopathic infertile males and four hundred and ten fertile controls from the hospitals affiliated to Nanjing Medical University between 2010 and 2012 were recruited in the study. We demonstrated a significantly increased risk of idiopathic infertility with abnormal semen parameters in association with the heterozygous genotype of variant rs4804490. Moreover, the AA genotype of variant rs4804490 was associated with significantly decreased risk for male infertility with abnormal semen parameters. A decreased risk of idiopathic infertility with abnormal semen parameters was associated with the homozygous genotype of variant rs2424909. These results suggested that variants in different DNMT genes have different relationships with idiopathic male infertility, and Chinese men carrying these variants have an increased or decreased risk of abnormal semen parameters.The purpose of this study was to investigate the association between male infertility and single-nucleotide polymorphisms (SNPs) of DNA methyltransferases (DNMT) genes ( Most studies published to date support the view that abnormal epigenetic changes also resulted in male infertility. Lande-Diner and other researchers have concluded that DNA methylation is an essential epigenetic modification specifically related to gene silencing3. Aberrant DNA methylation has been proposed as a possible mechanism compromising male fertility6. The aberrant DNA methylation found in infertile males may be a consequence of a dysfunction in the machinery involved in establishing and maintaining normal DNA methylation. Accumulating evidence has indicated that male infertility results from gene mutations and single-nucleotide polymorphisms (SNP)8.Male infertility is a heterogeneous disorder that contributes to the impairment of spermatogenesis. Clinical investigations have confirmed that a significant proportion of male infertility is idiopathic, the molecular mechanisms underlying the defects have not been elucidatedDNMTs may cause spermatogenic defects12. DNA methyltransferases (DNMTs) are a family of proteins responsible for transferring methyl groups specifically to cytosine in CpG dinucleotides14. Genes involved in the epigenetic pathways are likely to affect DNA methylation and are associated with male infertility. Researchers have analyzed the genetic variants within the genes to investigate the epigenetic processes in the aetiology of diseases15.Mouse models have shown that aberrant DNA methylation patterns resulting from gene targeting of the DNMT116, DNMT3A, DNMT3B19, and DNMT3L21) have been extensively characterized in mammals. DNMT1, which is often referred to as the maintenance methyltransferase, is responsible for maintaining pre-existing methylation patterns during DNA replication23. DNMT3A and DNMT3B are considered to be de novo DNA methyltranferases, which are critical in the dynamic DNA methylation process during embryogenesis and pathogenesis24. In addition to these active enzymes, the DNMT3L protein, catalytically inactive by itself, also contributes to de novo methylation by interacting with the catalytic domains of DNMT3A and DNMT3B and enhancing their enzymatic activity27. DNA methylation, mediated by DNMTs, is required for proper embryonic development28 and for the formation of mature functional germ cells29. Dysregulation of the DNMTs may lead to various conditions30, including spermatogenic defects12, autoimmune disorders31 and cancer32. To better understand the relationships between polymorphisms in DNMTs and idiopathic male infertility, we studied the associations between genetic variation in DNMT genes and altered semen parameters. We show that variants in the DNMT3B and DNMT1 may have different relationships with idiopathic male infertility. Furthermore, we compared the transcriptional factors between C allele and T allele of rs2424909, C allele and the A allele of rs4804490.Four DNMTs . The inclusion criteria consisted of five terms: (i) a normal 46, XY karyotype; (ii) absence of Y chromosomal microdeletions of AZF region; (iii) lack of hypogonadotropic hypogonadism; (iv) normal sexual and ejaculatory functions and no seminal tract obstruction, varicocele; (v) no history of infection or other diseases that could affect fertility. Finally, 833 men are included as the study subjects. The controls were healthy men who had fathered at least one healthy child, without assisted reproductive measures during the same period as those of the cases recruited in the same hospital. A scheduled interview was arranged for each subject to collect the basic information, including personal background, lifestyle factors, occupational and environmental exposures, sexual and reproduction status, genetic risk factors, medical history and physical activity (e.g. exercise status). Then, each subject donated a 5-ml peripheral blood sample and a semen sample for genetic testing. These patients and healthy donors were all ethnically Han Chinese east China.Semen samples were obtained in private by masturbation into a sterile wide-mouth and metal-free glass container after a recommended at least 3-day sexual abstinence. After liquefaction at 37 °C for 30 minutes, conventional semen analysis was conducted in accordance with guidelines of the WHO Laboratory Manual for the Examination of Human Semen, including semen volume, sperm number per ejaculum, sperm concentration, motility, progression and motion parameters by using Micro-cell slide and computer-aided semen analysis . Each sample was assessed twice with strict quality control.DNMT1 (rs4804490); DNMT3A (rs1550117); DNMT3B (rs2424909); DNMT3L (rs7354779). We selected only variants where the frequency of the minor allele was greater than 5%. For quality control, all genotyping assays were performed without knowing the case or control status of the subjects, and a random 5% of cases and controls were genotyped twice by different individuals.Genomic DNA was extracted from leukocyte pellets of the venous blood by traditional proteinase K digestion, followed by phenol-chloroform extraction and ethanol precipitation. Genotypes were detected by the TaqMan allelic discrimination Assay on an ABI 7900HT real-time PCR system . The gene variants studied were: AliBaba2 is a program for predicting binding sites of transcription factors. We used the program to compare putative transcriptional factor-binding sites of rs2424909 and rs4804490, rs1550117 and rs7354779. Firstly, we searched the base sequences of rs2424909, rs4804490, rs1550117 and rs7354779 (51 bases). Then, by respectively entering C allele and T allele of rs2424909, C allele and A allele of rs4804490, A allele and G allele of rs1550117, C allele and T allele of rs7354779, the different binding sites of transcription factors emerged.6/ml), sperm number per ejaculate (<40 × 106/ml) and sperm motility (<50% motile sperm). The control group consisted of proven fertile men with all four semen parameters at or above the WHO reference value. The subjects with idiopathic infertility were first divided into two groups according to semen parameters: case Group I was defined as idiopathic infertile men with all four semen parameters at or above the WHO reference value and the subjects in case Group II had at least one of the abnormal semen parameters. Case Group II was divided into four subgroups: Subgroup I (semen volume <2 ml), Subgroup II (sperm concentration <20 × 106/ml), Subgroup III (sperm number per ejaculate <40 × 106/ml) and Subgroup IV (sperm motility <50% motile sperm). Analysis of variance was used to compare the mean age, body mass index (BMI) and pack-years of cigarette smoking between case and control groups. The χ2 test was used to evaluate the difference in smoking status and alcohol status between the two groups. Multivariate logistic regression analysis was performed to obtain the odds ratios (OR) for male infertility and 95% confidence intervals (95% CI) with adjustment for age, BMI, smoking status and alcohol drinking. All statistical analyses were carried out using Stata , and p value of less than 0.05 was considered to be statistically significant.Semen parameters were dichotomized based on WHO reference values  for semen volume (<2 ml), sperm concentration (<20 × 106/ml (n = 340); Subgroup III: patients with sperm number per ejaculum <40 × 106/ml (n = 311); and Subgroup IV: patients with sperm motility <50% motile sperm (n = 611). Demographic categories by fertility and semen quality are described in Table The final study consisted of 1243 men, 833 idiopathic infertile patients and 410 fertile controls. All subjects were Han Chinese, with a mean (±SD) age of 29.38 (±4.15) years. Eight hundred and thirty three men were idiopathic infertile men, 191 men  with normal semen quality and 642 men  with abnormal semen quality according to WHO standard. In addition, patients in the Case Group II were further divided into four subgroups: Subgroup I: patients with semen volume < 2 ml (n = 122); Subgroup II: subjects with sperm concentration <20 × 10DNMT genes among the cases and controls are shown in Tables DNMT1 CC genotype was used as the reference group, the CA genotype was significantly associated with increased risk for male infertility in Case Group I  and Case Group II . Under the recessive model of inheritance, the DNMT1 AA genotype was associated with significantly decreased risk for male infertility with abnormal semen parameters , compared with other genotypes. In addition, compared to the DNMT3B CC genotype, the association between the CT genotype and male infertility was not significantly different , but the TT homozygous genotype was associated with significantly decreased risk for male infertility with abnormal semen parameters . Under the recessive model of inheritance, the DNMT3B variant homozygote was associated with significantly decreased risk for male infertility with abnormal semen parameters , compared with other genotypes, after adjustment for age, BMI, smoking and drinking status in the multivariate logistic regression analysis . Under the recessive model of inheritance, subjects in Subgroup IV had a significantly decreased frequency of the DNMT1 variant homozygote compared with other genotypes . When the DNMT3B CC genotype was used as the reference group, the carrier of TT genotype in Subgroup IV had a significantly decreased risk of male infertility . The DNMT3B variant homozygote had a significantly decreased risk of male infertility in the Subgroup IV  compared with other genotypes (Table DNMT3A (rs1550117) and DNMT3L (rs7354779) between the cases and controls . In addition, under the recessive model of inheritance, the AA genotype of variant rs4804490 and TT genotype of variant rs2424909 were significantly associated with decreased risk for male infertility with abnormal semen parameters. Among men with normal semen parameters, there were no significant differences in risks associated with these genotypes. Furthermore, no significant associations with risk of idiopathic infertility associated with either normal or abnormal semen parameters were found for the rs1550117 and rs7354779 variants. A recent study by Huang et al. investigated the association between DNMT3L polymorphisms and male infertility with azoospermia33. They found that the presence of rs2070565 allele A increased the risk of azoospermia in males. Additionally, they did not report any significant difference for rs7354779 between the infertile patients and fertile controls, which similar to our findings. These results suggest that different variants in the genes involved in epigenetic pathways may have different relationships with idiopathic male infertility and men with these variants have an increased or increased risk of abnormal semen parameters associated with male infertility.This case-control study analyzed DNMT1 and DNMT3B nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%34. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumor suppressor gene p16, and growth suppression34. Rhee and colleagues demonstrated that these two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells34. The precise mechanism of the variant rs4804490 and rs2424909 in male infertility remains unclear, as there is no direct functional data available. A single-base substitution may change the binding of transcription factors and consequently influence their function. We therefore raised hypotheses that these variations may be associated with changes of transcription factors. Changes of the binding of transcription factors were observed with both of the variations. Variants of DNMT1 and DNMT3B are risk factors for idiopathic male infertility.Genetic disruption of both DNMT1 CA genotype had a significantly increased risk of idiopathic male infertility compared with DNMT1 CC genotype carriers, although no significant difference was found in DNMT3A and DNMT3L. Our results support the hypothesis that DNMT1 polymorphism may be associated with an increased risk of idiopathic male infertility in a Han-Chinese population. These SNPs may alter a transcription factor binding site in the alternative promoter, and the variant genotype (s) may be in linkage disequilibrium with other untyped susceptibility loci. Moreover, the variants may alter the binding of a regulatory miRNA or contribute to the differential expression of alternatively spliced DNMT3B variants.In conclusion, our study demonstrates that some representative variants of the DNMT genes may modulate the risk of male infertility associated with abnormal semen parameters. Confirmation of similar findings in larger study groups would be needed. In summary, in this case-control study, we found that the carrier of 36. AP-1 transcription factor, known to play a vital role in cell proliferation and neuronal activation, is also involved in cell apoptosis in response to stress, lack of survival signals or DNA damaging agents37. Nuclear factor-κB (NF-κB) transcription factor regulates a wide array of genes mediating numerous cellular processes such as proliferation, differentiation, motility and survival38. In Fig. 40. Transcription factor Specificity protein 1 (Sp1) plays a role in promoting oncogenes required for tumor survival, metastasis and progression. c-Jun, a component of the transcription factor AP-1, regulates gene expression and cell function.As shown in Fig. DNMT3B and DNMT1 could regulate gene expression by the creation or deletion of transcription factors and influence the sperm maturation process and male fertility. Microarray analysis and real time quantitative PCR has shown that the differential gene expression of DNMT1 between various forms of testicular cancer was consistent41. Adiga et al. documented that DNMT3B is differentially expressed42 in the preleptotene/zygotene and pachytene spermatocytes from fertile and infertile men. Future mechanistic studies about functional significance of the variation at DNMT1 and DNMT3B will help fully flesh out the relationship between polymorphism of DNMTs, abnormal DNA methylation patterns, and spermatogenic failure.At present, there is no evidence that these transcription factors interact with DNMTs to control their DNA binding specificity. We suggest that the genetic variants Supplementary Information


Tumor metastases are the major cause of cancer morbidity and mortality. A subpopulation of tumor cells with stem-like properties is assumed to be responsible for tumor invasion, metastasis, heterogeneity and therapeutic resistance. This population is termed cancer stem cells (CSCs). We have developed a simple method for identification and characterization of circulating cancer stem cells among circulating epithelial tumor cells (CETCs).CETCs were cultured under conditions favoring growth of tumorspheres from 72 patients with breast cancer, including a subpopulation of 23 patients with metastatic disease. CETCs were determined using the maintrac® method. Gene expression profiles of single CETCs and tumorspheres of the same patients were analyzed using qRT-PCR.Sphere formation was observed in 79 % of patients. We found that the number of tumorspheres depended on stage of disease. Furthermore, the most important factor for growing of tumorspheres is obtaining chemotherapy. Patients with chemotherapy treatment had lower numbers of tumorspheres compared to patients without chemotherapy. Patients with HER2 positive primary tumor had higher number of tumorspheres. Analysis of surface marker expression profile of tumorspheres showed that cells in the spheres had typical phenotype of cancer stem cells. There was no sphere formation in a control group with 50 healthy donors.This study demonstrates that a small fraction of CETCs has proliferative activity. Identifying the CETC subset with cancer stem cell properties may provide more clinically useful prognostic information. Chemotherapy is the most important component in cancer therapy because it frequently reduces the number of tumorspheres. Breast cancer is the most common cancer in women worldwide and development of distant metastases is a main reason for cancer mortality. Distant metastasis formation is due to spread of cancer cells from the primary location through circulation , 2. PresTranscription factors such as Oct4, Sox2 and Nanog are required to maintain pluripotency and self-renewal capacity of cancer stem cells and play an important role in the uncontrolled proliferation of cancer cells .+/CD24−, overexpress neoangiogenic and cytoprotective factors and genes of pluripotency [A functional approach, tumorsphere formation assay, based on the unique property of stem cells to survive and grow in suspension culture, is successfully applied to enrich stem cells from invasive tumor samples or cancer cell lines. Tumorspheres have been reported to display typical stem cell surface markers like CD44ipotency , 20.The aim of the present study was to develop a new approach based on functional, pheno-and genotypic features of CSCs for detection and characterization of cells with proliferative activity and cancer stem cell properties among circulating epithelial tumor cells.Patients characteristics are given in Table During culture in non-adhesive, suspension culture the CETCs with proliferative activity grew as spherical tumorspheres. After 3-5 days of culture first signs of sphere formation were observed. Spheres grew gradually over time and after 21 days of culture all spheroids had a diameter between 30 and 60 μm (median 40)   Figure . FurtherThe frequency of spheroids cultured from CETCs was monitored in patients without and with distant metastases in order to test whether the number of tumorspheres correlated with disease progression. The total amount of tumorspheres was higher in patients with metastatic disease as compared to patients without metastases  Figure . PatientSurprisingly, patients who received no chemotherapy (56.9%) had higher numbers of tumorspheres compared to patients who had received chemotherapy (34.7%)  Figure . MoreoveIn contrast to the number of CETCs the number of tumorspheres was an independent factor of the presence of metastases  in patients with breast cancer. The receiver operating characteristic (ROC) curve shows that the number of spheres that can be grown from a patient's blood is a predictor of the presence of distant metastases. The AUC for number of spheres was higher than for the number of CETCs and it is 0.71 (p=0.005) Figure . The cut2 in conventional cell culture flasks. Only a small subpopulation of epithelial tumor cells circulating in patients' blood had the capacity to form spheres under these conditions and no spheres could be grown from normal subjects. In contrast to Yu et al we were able to successfully culture CETCs from breast cancer patients in early stage as well as in advanced stage of disease [Culturing blood white cells comprising CETCs from patients with breast cancer we were able to identify a subpopulation of CETCs with clonal proliferative activity growing into tumorspheres. These tumorspheres can be cultured in special medium supplemented with growth factors during 21 days at 37°C and 5% CO disease . Sphere- disease . In our Based on the type of growth, staining properties and genotypic characterization of the spheres this subpopulation had cancer stem cell (CSC) properties and these cells may be progenitors of metastases leading to relapse and treatment failure in patients with breast cancer.Immunostaining showed both CD44 high/CD24 low expression and ALDH1 activity which are CSC markers. CD44 normally takes part in cell-cell and cell-matrix adhesion interactions, which is involved in cancer cell migration, proliferation and metastasis. Current experimental evidence indicates that the CD24 negative subpopulation might represent a more drug resistant phenotype . Our finThe expression of pluripotency- associated factors playing a crucial role in the development and malignant progression of various types of cancer including breast cancer , Aktas eThe number of tumorspheres was significantly higher in patients with HER2 positive tumors compared to HER2 negative tumors consistent with the finding of Korkaya et al. that HER2 overexpression increases the mammary stem cell population and also increases the capacity for sphere formation from tissue cultures. Furthermore, overexpression of HER2 in a series of human cancer cell lines increases the percentage of cells with ALDH activity, leading to increased amounts of spheres  suggestiIn the adjuvant situation where there is no residual disease present monitoring of CETCs has been shown to provide a valuable tool to routinely monitor response to therapy  and is cIn conclusion, we are able to culture a subpopulation of circulating tumor cells and pheno- and genotypically characterize them for putative stem cell markers. We postulate that tumorspheres are a surrogate of distant metastasis in breast cancer patients. Further studies could provide new insights into the tumorigenic process and assess the potential of these spheroids in breast carcinomas as therapeutic target.5 cells/ml in RPMI-1640 medium supplemented with L-glutamine, HEPES, penicillin/streptomycin and growth factors such as EGF, insulin and hydrocortisone. Cells were maintained at 37°C and 5% CO2 without movement of the culture flasks. Fresh medium was added every five days and formation of tumorspheres was observed under an inverted light microscope  at 40x magnification every 7 days. After 21 days tumorspheres were collected and prepared for immunostaining. Samples were centrifuged at 250 g for 7 min. The supernatant was discarded and the pellet resuspended with 500 μl PBS. 50 μl of cell suspension was transferred into 1.5 ml reaction tubes and prepared for the phenotypic characterization. Tumorspheres were incubated with 5 μl of fluorescein-isothiocyanate (FITC)-conjugated mouse anti-human EpCAM antibody  and 5 μl of phycoerythrin (PE)-conjugated mouse anti-human CD-44 antibody  or with 5 μl of phycoerythrin (PE)-conjugated mouse anti-human CD-24 antibody  for 15 min in cold. The samples were subsequently diluted with 430 μl PBS-EDTA and then stored overnight at 4°C. 100 μl of cell suspension was transferred to 96-wells plates . Analysis of red and green fluorescence of the tumorspheres was performed using a fluorescence scanning microscope . Tumorspheres were stained with propidium iodide to evaluate their viability prior to analysis. Finally, only vital tumorspheres with intact morphology were counted. ALDH1 activity of spheroids was determined using an ALDEFLUOR assay kit  according to the manufacturer's protocol. In short, tumorspheres were suspended in ALDEFLUOR assay buffer containing ALDH1 substrate and incubated 45 min at 37°C. In parallel, a sample was treated with an ALDH1-specific inhibitor, as negative control for background fluorescence. Stained spheroids were analyzed with fluorescence scanning microscope . Moreover we examined 50 healthy donors aged from 20 to 53 years for determination of specificity and sensitivity and we observed no sphere formation in all cases.72 patients with histologically confirmed breast cancer, 49 with primary breast cancer and 23 patients with metastatic disease were included in the study. Blood samples were drawn into normal blood count tubes with ethylene diaminetetraacetic acid (EDTA) for enumeration and cultivation of CETCs. The maintrac® approach was used for detection and quantification of CETCs, as reported previously . In brieIn some cases we performed dissociation of spheroids with StemPro®Accutase®  for 3 min at room temperature to obtain a second generation of tumorspheres.TM Whole Transcriptome Amplification kit (Takara Bio Inc./Mobitec). Subsequently, PCR was carried out with Light Cycler® 480 SYBR Green I Master (Roche). The following primers were used in this study: rpl13a: AGC TCA TGA GGC TAC GGA AA (forward) and CTT GCT CCC AGC TTC CTA TG (reverse); epcam: GGG AAA TAG CAA ATG GAC ACA (forward) and CGA TGG AGT CCA AGT TCT GG-5 (reverse); vimentin: TCC GCA CAT TCG AGC AAA GA (forward) and ATT CAA GTC TCA GCG GGC TC (reverse); nanog: GGA TCC AGC TTG TCC CCA AA (forward) and TGC ACC AGG TCT GAG TGT TC (reverse); oct4: GGC CAC ACG TAG GTT CTT GA (forward) and ATA CCT TCC CAA ATA GAA CCC C (reverse); sox2: GCG GAA AAC CAA GAC GCT C (forward) and TCA TGT GCG CGT AAC TGT CC (reverse); cd133: GTC CTG GGG CTG CTG TTT AT (forward) and TCT GTC GCT GGT GCA TTT CT (reverse); aldh1 CTG AGC CAG TCA CCT GTG TT (forward) and GGA CAG GTA AGT CTG GCG TG (reverse). Relative expression levels were calculated after normalization to the reference gene ribosomal protein L13a (RPL13a) by using the ΔΔCT method.For quantitative RT-PCR CETCs and tumorspheres were isolated individually using a semi-automated capillary approach and deposited one by one into micro cups. cDNA was prepared with CellAmpData were analyzed using Student's t test for comparison of two variables or ANOVA for three and more variables. If the sample distribution was asymmetrical nonparametric tests  were performed. Relationships between parameters were evaluated using Pearson or Spearman's rank correlation.Diagnostic performance of tumorsphere count was assessed by constructing a ROC curve, and was evaluated by calculating the area under the ROC curve.


Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium’s other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response. They lack kinase domains, suggesting these proteins must target others with enzymatic activity. CaMs modulate transcription by binding to Calmodulin-binding Transcription Activators (CAMTAs) (2+-Dependent Protein Kinases (CPKs) and Calcineurin-B Like proteins (CBLs). The latter target CBL-Interacting Protein Kinases (CIPKs) to effect cellular responses  while Phospholipase D catalyses production of phosphatidic acid, thus [Ca2+]cyt would have the capacity to trigger distinct lipid signals depending on the location and Ca2+-sensitivity of the phospholipases. Targets of lipid signals have been reviewed recently by Plant roots are exposed to a variety of abiotic stresses as they navigate the soil, foraging for nutrients and water. Cytosolic free calcium  . Other mesponses . Changesfamilies . Phospho2+]cyt measurements are from Arabidopsis thaliana seedlings and guard cells, achieved using the luminescent Ca2+-interacting aequorin protein. Far fewer studies have focused specifically on roots or utilized the greater sensitivity and spatial resolution of ratiometric fluorescent dyes. The genetically encoded YC3.6 Ca2+ reporter is now being used for both Arabidopsis and rice roots  or depolarize (render it less negative). Manipulating PM voltage elicits distinct [Ca2+]cyt signatures and resultant transcriptional responses (Hyperpolarization-Activated Ca2+ Channels (HACCs); Depolarization-Activated Ca2+Channels (DACCS); Voltage-Independent Ca2+Channels (VICCs) (2+ influx routes is afforded by reactive oxygen species (ROS) that are produced during development and stress responses . This regulation depends on the specific ROS, its position, the cell type and the cell’s developmental state. In Arabidopsis roots, sensitivity of PM Ca2+ channel activation by extracellular H2O2 decreases as epidermal cells mature but is still greater than that of the cortex  have advanced our understanding of the Caesponses . Studies (VICCs) . Thus che cortex . A similericycle . In epidlar H2O2 , 2007. T2+]cyt elevation in roots remains poorly understood in terms of the genes encoding the PM or endo membrane Ca2+ channels involved. Plants have multi-gene families of Glutamate Receptor-Like channels  and Cyclic Nucleotide-Gated channels , with each gene encoding a potential subunit of a potentially tetrameric channel. Some members have been characterized as having Ca2+ channel forming ability  thus leading to the threat of hypoxic challenge from waterlogged soil . Salinit+ influx into the root are not fully known but include the PM cyclic nucleotide-gated channels CNGC3 . Both channels types are present in Arabidopsis root epidermal PM  was sensitive to inhibition of soluble guanylyl cyclase activity and the signal evoked by an equivalent osmotic stress was insensitive . This channel mediates the [Ca2+]cyt response to hyperosmotic stress , with [Ca2+]cyt activating these enzymes through their EF hands . In accordance with this, Arabidopsis root cortical cells lacking RBOHD and F have much lower PM hyperpolarization-activated Ca2+ activity in response to NaCl challenge than wild type  acts to limit NaCl-induced [Ca2+]cyt increase, partly by limiting Ca2+ release from mitochondria. In the arp2/3 mutant, the [Ca2+]cyt increase is greater than wild type and so is the extent of mitochondrial-driven cell death  and inositol trisphosphate (InsP3) suggested involvement of the InsP3 pathway in Arabidopsis roots  are firmly implicated in salt stress responses  in Arabidopsis leaves  would bind Ca2+, and open to release K+ to the cytosol to maintain a favorable Na+/K+ ratio  acts to promote Na+ efflux  would restore [Ca2+]cyt to pre-stimulus levels  which can then react with the serine/threonine protein kinase CIPK24 (SOS2) (+ antiporter SOS1 at its auto-inhibitory C-terminus to achieve activation of Na+ efflux (Arabidopsis by Mitogen Activated Protein Kinase6 (MPK6) and loss of MPK6 function impairs root growth under salt stress  pathway lies downstream of the root cyt increase activates the root PM Phospholipase C2  as a diagnostic of a UPR response  cells containing the Fura-2 Ca2+-reporting dye lead to the identification of Calcium-permeable Stress-gated cation Channel1   . PERK4’songation . This imlevation . Anotherlevation .. GLRs abidopsis . The [Cass genes . Droughtss genes . In maizss genes .2+]cyt and is relevant to waterlogged soils. In Arabidopsis, root PM harbors two mechanosensitive Ca2+-permeable channels, MCA1 and MCA2  PM voltage as H+ efflux is curtailed. The extent and duration of membrane depolarization varies with sensitivity to O2 deprivation and cell type. Values of -70 to -80 mV have been reported for O2-deprived barley root cells  are activated  or absence (anoxia) causes transient increases in root cyt with clearance to the apoplast by ACA10-13 and sequestration to the vacuole by CAX3. How they are regulated remains to be determined, as does the involvement of the structurally compromised wall and the consequence of this higher level of apical [Ca2+]cyt. The area of higher [Ca2+]cyt reported appears to correspond with the zone of inhibition of primary root elongation and the induction of cell death , as were three CPK genes  all suggesting a distinct change in intracellular Ca2+ signaling (WRKY transcription factors (TF) , two three MYB family TF  and downregulation of two BZIP family TFs   . B depriroot tip . It has  nucleus  and this nucleus , suggest3 and above). Although some of these metals  are required as micronutrients they can be damaging in excess whilst others (such as Cd) have no physiological role and can be deleterious even at low concentrations, often impairing mineral nutrition. The consequences of heavy metal exposure have been reviewed recently by 2+), intersects with hormonal responses and detoxification.At the opposite end of nutritional deprivation is heavy metal stress. Industrial activity, mining and modern agricultural practices can lead to soil contamination by heavy metals (defined here as 7 g/cm2+-based processes because of its similar size. A recent review by 2+ a key question is whether Cd (as a Ca2+ “substitute”) generates a signal in its own right or whether its impairment of Ca2+ homeostasis will be, in effect, the signal. Certainly Cd depolarises the rice root epidermal PM, which would impair Ca2+ influx and results in inhibition of root elongation  and activate a possible DACC in wheat root PM  were identified in Arabidopsis due to their sequence similarity to the bacterial Mechanosensitive channels of small conductance (MscS)  . These aArabidopsis mechano-stimulated [Ca2+]cyt increase and downstream transcriptional regulation  may be influenced by mechano-sensing. At this point, it is unclear whether FERONIA also plays a role in the mechanical induction of lateral root formation   have been reviewed by es e.g., . By usinn leaves . Using ae CRLK1; . This woe CRLK1; . This pae CRLK1; . [Ca2+]cignaling . Part ofFactor2) .Triana bogotensis  and elevation of root [Ca2+]cyt (aequorin and cameleon determinations). This indicates that heterotrimeric G proteins lie upstream of PM Ca2+ channels in the cold response. In common with other stress responses, NADPH oxidase activity also appears in cold stress and needs to be placed in relation to Ca2+ channels. Arabidopsis roots exposed to cold stress use AtSRC2 (Soybean gene Regulated by Cold2) to enhance Ca2+ activation of the AtRBOHF NADPH Oxidase (2+ channel activity. Curtailing cold-induced [Ca2+]cyt elevation could involve vacuolar CAX1 activity in Arabidopsis (Cold stress has been found to depolarise the PM of root cells from cucumber and gotensis , consistof roots . Cold haof roots  but this PM DACC , this cotoplasts . Actin don cells . This inon cells . It woulolerance . Converseedlings . The posubunit α . It is r Oxidase . This subidopsis .2+]cyt increases, and by extension those of organelles. Many members of channel gene families still await characterization. The identification of new families of channels is challenging and will require different approaches linking forward and reverse genetics to electrophysiology. Targets of Ca2+-binding and interacting proteins also require further study. Components common to different abiotic stresses are emerging such as Arabidopsis CIPK23 in K+ and nitrate deprivation. These common regulatory components are likely to represent critical steps where complex stress signals encountered in the soil are integrated in unified responses. Receptor like kinases such as FERONIA or PERK4 have emerged as new components in [Ca2+]cyt signaling and perhaps other related proteins will be found to have a role in abiotic stress signaling. Remodeling of calcium signaling machinery after stress is also apparent with the possibility of components common to different stresses. For example, Arabidopsis CNGC19 is upregulated under B limitation and salinity stress (2+]cyt-dependent transcriptional responses can be delineated and future work could include the impact of stress-induced calcium signaling on epigenetic inheritance (A repeated message from this review is how incomplete our knowledge is of the channels mediating stress-induced [Cay stress . Finallyeritance .All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. KW, EM, SS, and JD wrote the review. KW produced the figures.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.


We note that the nomenclature is generic, extendable to stable isotope-labeled lipid molecules and applicable to automated annotation of fragment ions detected by most contemporary lipidomics platforms, including LC-MS/MS-based routines.Advances in mass spectrometry-based lipidomics have in recent years prompted efforts to standardize the annotation of the vast number of lipid molecules that can be detected in biological systems. These efforts have focused on cataloguing, naming and drawing chemical structures of intact lipid molecules, but have provided no guidelines for annotation of lipid fragment ions detected using tandem and multi-stage mass spectrometry, albeit these fragment ions are mandatory for structural elucidation and high confidence lipid identification, especially in high throughput lipidomics workflows. Here we propose a nomenclature for the annotation of lipid fragment ions, describe its implementation and present a freely available web application, termed ALEX Advances in mass spectrometry (MS)-based lipidomics have enabled comprehensive lipidome analysis at high throughput with generation of large amounts of spectral data that can be harnessed to identify and quantify several hundred lipid molecules in a single sample –7. Appli2 or MS/MS) where an intact lipid precursor ion is isolated by a mass analyzer, subjected to collision-induced dissociation (CID) and generated fragment ions are subsequently detected using either a low or a high mass resolution detector system +, respectively. Similarly, association with positive and negative adduct ions such as sodium and formate are denoted as [M+Na]+ and [M+HCOO]-, respectively. For lipids this convention is often adapted by substituting M with shorthand notation for intact lipid molecules (e.g. [PC 34:1+H]+). In comparison, the annotation of lipid fragment ions is much more inconsistently implemented and ranges from graphical displays of complex MSn spectra without any shorthand notation to use of chemical formulas and nominal masses to signify structures of fragment ions . While chemical formulas can serve as unique identifiers of fragment ions and nominal masses can help indicate fragmentation pathways, these shorthand notations often make it unintuitive and complicated to relate fragment ions back to the structure of the intact lipid molecule. A more informative approach is to denote fragment ions in reference to structural attributes of the intact lipid molecule. For example, many glycerophospholipids, including phosphatidylethanolamine (PE) and phosphatidylserine (PS), lose their head groups to yield fragment ions with structures that resemble “biological” lipids such as DAG and PA, that in turn can be denoted using shorthand notation resembling intact lipids (e.g. [DAG 36:1+H-18]+ and [PA 38:4-H]-). Similarly, fragmentation of many lipids yield FA carboxylate anions analogous to deprotonated non-esterified fatty acids (e.g. [NEFA 16:0-H]-). However, using names of intact lipids to annotate lipid fragment ions is misleading as, for example, FA carboxylate fragment ions are not produced by simple deprotonation but instead derive from a charge-mediated fragmentation process. Notably, implementation of nomenclature for lipid fragment ions could help novice and experienced lipidomists to more easily read fragment ion spectra and assess the merits of lipid identifications. Moreover, it would also help to implement reporting standards that secure the quality of high throughput lipidomics resource data that is being produced at an accelerated rate nowadays [Annotation of mass spectra of intact precursor ions is based on the tradition that uncharged molecules are represented by the symbol M and that charged derivatives corresponding to loss or gain of a proton are denoted as [M-H]nowadays ,37. In aFig 1). This classification has yet to be implemented into a cheminformatics framework (as we do herein), but exists today on a more unintuitive practical level that is implicit in the guidelines for appropriate shorthand notation of intact lipid molecules N),N),S1 TexFig 7 showing fragment ion spectra from four representative lipid molecules labeled with different configurations of heavy nuclei. Of note, LCFs are denoted by the lipid class abbreviation followed first by specification of stable isotopes and then by the nominal mass in parentheses. For example, “PC(+[2]H13)(197)” and “PI(+[2]H6)(247)” indicate charged phosphocholine and phosphoinositol structures labeled with thirteen 2H atoms and six 2H atoms, respectively . Similarly, MLFs are denoted by HCA abbreviation followed first by specification of stable isotopes and then by any chemical modifications in parentheses. For example, the fragment ion with m/z 499.3572 in Fig 8B is denoted as “-FA 16:0(+[2]H3)(-H)” to indicates neutral loss of an FA 16:0 moiety having three 2H atoms as a ketene. Moreover, the fragment ion with m/z 268.2344 in Fig 8D is denoted as LCB 18:0;3(+[13]C2[15]N)(-[13]CH8[15]NO) to indicate a charged LCB fragment originally having two 13C and one 15N incorporated but after fragmentation having lost a chemical feature corresponding to one 13C, eight H, one 15N and one O.For annotation of fragment ions we subsequently added the naming convention for stable isotopes into the rule sets for LCFs, MLFs, iMLFs and DBFs. This extension is exemplified in n analysis. We note that the ALEX123 lipid calculator at the present features 28 lipid classes labeled with stable isotopes that can be generated when feeding cells or animals with the commercially available metabolic precursors 2H9-choline, 2H3-methionine, 2H6-inositol, 13C315N-serine and their combinations (S1 Table).In summary, the ability of the fragment ion nomenclature to consistently account for shorthand notation of also stable-isotope labeled lipid fragment ions demonstrates its generic nature and highlight that it can readily be extended to describe a wide range of fragment structures and accurately match these to structure-specific fragment ions detected by MSm/z values in mass spectra. We have demonstrated that the nomenclature is able to systematically and consistently describe structural details of fragment ions released upon CID of unlabeled and stable isotope-labeled molecular lipid species encompassing 47 lipid classes and five different lipid categories. Furthermore, we have shown that the nomenclature can be computerized and made searchable in the online ALEX123 lipid calculator to support both manual and automated high confidence lipid identification in biological sample matrices. Notably, the fragment nomenclature framework also provides an avenue to develop new algorithms for automated high confidence lipid identification in high throughput lipidomics studies. As such, the text-based fragment nomenclature and “substrings” thereof can be queried using algebraic string-based operators available in all programming languages . This text-based information can, for example, be harnessed for counting the frequency of specific fragment types across large number of samples and also for implementing complementarily filters to secure high confidence lipid identification . Based on its systematic design and its ability to be easily computerized we deem that our proposed fragment nomenclature can become a valuable addition to the expanding palette of cheminformatics tools that are being developed to assist the characterization of lipid molecules in biological systems. Finally, we note that the nomenclature is applicable to annotation of MSn spectra of lipid molecules acquired by both direct infusion-based (i.e. shotgun) and LC-based lipidomics techniques, and also mass spectrometry imaging approaches.In this report we have outlined a generic framework for shorthand notation of lipid fragment ions. The framework consists of a three-step procedure that systematically recapitulates lipid fragmentation using mass-balanced chemical reactions showing charged and neutral fragment structures, uses defined rules for annotating specific types of fragments, and uses a decision tree-based routine for implementing shorthand notation of detected fragment ion S1 Text(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.


To develop a cerebrospinal fluid (CSF) miRNA diagnostic biomarker for glioblastoma.® Human MicroRNA Panel. CSF miRNA profiles from glioblastoma patients and controls were created from three discovery cohorts and confirmed in two validation cohorts.Glioblastoma tissue and matched CSF from the same patient (obtained prior to tumor manipulation) were profiled by TaqMan OpenArrayp=0.008). When prospectively applied to cisternal CSF, the sensitivity and specificity of the ‘signature’ for glioblastoma detection were 67% and 80%, respectively. For lumbar CSF, the sensitivity and specificity of the signature were 28% and 95%, respectively. Comparable results were obtained from analyses of CSF extracellular vesicles (EVs) and crude CSF.miRNA profiles from clinical CSF correlated with those found in glioblastoma tissues. Comparison of CSF miRNA profiles between glioblastoma patients and non-brain tumor patients yielded a tumor “signature” consisting of nine miRNAs. The “signature” correlated with glioblastoma tumor volume (We report a CSF miRNA signature as a “liquid biopsy” diagnostic platform for glioblastoma. Glioblastoma, defined by the World Health Organization (WHO) glioma classification as grade IV astrocytoma, is the most common form of primary brain cancer in adults , 2. DiagCSF is an appealing and accessible bio-fluid for glioblastoma “liquid biopsy”. The bio-fluid lies in close proximity to tumor tissue, often bathing tumor or its associated microenvironment . The CSFExtracellular Vesicles (EVs) are cell-secreted vesicles that range 30-2000 nm in size that mediate critical biologic functions, including cellular remodeling and intracellular communication . Cancer Here, we examined miRNA profiles of the CSF EVs and “crude” CSF derived from glioblastoma patients. miRNA is an attractive biomarker platform given its stability in bio-fluids , selectiT cut-off of 35, we found that 200-400 miRNAs were detected in the glioblastoma specimens . Between 30-50% of these miRNAs were detected in the matched EV CSF , significant variation in miRNA profiles were found between CSF derived from the first three cohorts . To account for this variability, we used miRNA profiles derived from all three cohorts in our signature development. Details of the analysis can be found in We then used LASSO  to develP < 0.0001, Figure Pre-operative MRI was available for 11 of the patients in Cohort 1. We created tumor volumes based on the Agfa CD Viewer and related these to the CSF miRNA gene signature scores. A positive correlation was observed between miRNA signatures and tumor volumes Figure . GlioblaWe tested the performance of the 9-miRNA signature in a prospective manner. Since most EV miRNAs are also detected in crude CSF, we opted to validate our signature using unfractionated crude CSF. We prospectively collected and profiled cisternal CSF from an additional 22 patients (Cohort 4: 10 glioblastoma and 12 non-oncologic patients). Using the cutoff (0.4) established during the discovery process, the signature correctly identified 8/10 subjects with glioblastoma and 8/12 non-oncologic subjects, yielding a sensitivity of 80% and specificity of 67%. The AUC was 0.75  Figure .We also prospectively collected and profiled lumbar CSF from 18 glioblastoma and 20 non-oncologic patients (Cohort 5). Using the same coefficients and cutoff score, the 9 miRNA signature correctly identified 5/18 glioblastoma subjects and 19/20 non-oncologic subjects, yielding a sensitivity of 28% and specificity of 95%. The AUC was 83% . Figure . Notably13 lumbar glioblastoma CSF samples were collected as a part of Cohort 1. We had compared the performance of our miRNA signature in these samples in order to afford direct comparison to that seen in the cisternal samples. In the cohort 1 lumbar CSF samples, the 9 miRNA signature correctly identified glioblastoma subjects in 3/13 glioblastoma samples yielding a sensitivity of 23%. These results were comparable to those observed in the validation cohorts, confirming our observation that the diagnostic utility of the 9-miRNA signature is optimal when applied to cisternal CSF .miR-21  play a pIn current clinical practice, CSF sampling is not routinely performed in glioblastoma patients. The sensitivity of CSF cytology as a diagnostic tool for glioblastoma is ∼10%  and belovs. lumbar). However, even after correcting for site of collection, this variability remained. It is worthwhile noting that the CSF samples were collected in our study through a Standard Operating Procedure (SOP) and processed identically post-collection. The variability observed between these cohorts, in the context of the published literature, suggests that CSF miRNA profiles are likely influenced by physiologic factors or perturbation that was not accounted for by the SOP . As such, the robustness of the CSF miRNA signatures are largely a function of the sample size, since larger sample sizes afford a greater likelihood of minimizing the undue influence of any particular perturbation/physiology. Our study is particularly important in this context, since our study design is the only one in the literature that derived the signature through three independent cohorts, summing to 135 CSF samples. We subsequently validated our results in another 60 prospectively collected CSF. The scale of our study as well as the meticulous effort devoted to validation is notable in the reported literature of glioblastoma CSF biomarkers.There has been significant variability in the reported miRNA profiles in CSF derived from glioblastoma patients –23. We oAn important finding in this study is that the miRNA contents of cisternal and lumbar CSF differ. We found that less than half of the miRNAs detected in cisternal CSF were detected in lumbar CSF , likely EVs have been touted as platforms for diagnostic and prognostic biomarker interrogation –28. The The literature that examined altered miRNA regulation in glioblastoma has expanded over the past decade . It is nWhile our miRNA signature performed well as a diagnostic tool in cisternal CSF, opportunities for obtaining these samples are admittedly limited. Such samples can be obtained only from patients with an Ommaya reservoir or a ventriculo-peritoneal shunt. Because these procedures involve placement of an indwelling catheter that is in direct communication to cisternal CSF, serial samples can be safely acquired in this patient population. As such, clinical testing of the cisternal CSF signature is feasible in the subpopulation of glioblastoma patients with an indwelling shunt system. Moreover, serial sampling of cisternal CSF from this patient population may afford a minimally invasive platform for tracking glioblastoma disease burden. We are in the process of collecting and testing CSF from recurrent glioblastoma patients to further test the utility of our miRNA signature.In sum, our study provides a proof-of-principle study demonstrating the plausibility of CSF miRNA profiling as a “liquid biopsy” platform for glioblastoma diagnosis and provides the basis of future validation of this platform.Five cohorts of patients totaling 195 subjects provided CSF for these studies . Manufacturer's cartridges consisted of 818 TaqMan qPCR assays arranged on 384 well plates, with primers targeting 754 miRNA species, and 16 replicate wells of one negative and 3 positive RNA controls. Megaplex™ RT Primers, Human Pool A v2.1 and Megaplex™ RT Primers, Human Pool B v3.0 were used for the reverse transcription step. Megaplex™ PreAmp Primers, Human Pool A v.2.1 and Megaplex™ PreAmp Primers, Human Pool B v3.0 were used for the PreAmp step. The samples within each of the 3 discovery cohorts were assayed on the same date using the same reagents. The validation samples (cohorts 4 and 5) were assayed in two different batches on two different dates and data was combined for analysis.RNA was extracted from each sample using the miRCURY™ RNA Isolation Kit (Exiqon). Samples assayed were EV, supernatant and tissue from cohort 1; EV and supernatant from cohort 2; EV from cohort 3; and whole CSF from cohorts 4 and 5. Four microliters of RNA extract (4-20ng/μl) was used as input for microRNA profiling on the TaqManT value ≥35 were considered below the detection threshold. In tumor tissue samples, CT values for the query miRNAs were normalized using the mean of the positive controls . For the CSF samples, the positive control miRNAs were not uniformly expressed at high levels across samples. For the discovery cohorts global mean normalization was performed in which normalized CT values were calculated as the raw CT value minus the arithmetic mean of all expressed miRNAs in the sample [miRNA species with Ce sample . For thee sample  with assp-value < 0.05  and the same direction of differential expression; this test of replication has overall Type I error rate ∼1%. This candidate selection plan was pre-specified and documented.The classifier was trained using the three discovery cohorts . When both supernatant and EV miRNA was available within a cohort, we used the fraction with the higher median number of detected miRNA species for analysis within that cohort. For cohorts with low detection rates, we used both fractions with a Bonferroni correction for the two comparisons. Differentially expressed CSF miRNAs between glioblastoma and non-oncologic subjects were identified using the limma Bioconductor package , with FDCandidate miRNAs were carried forward to a multivariate model to discriminate glioblastoma from non-oncologic controls using L1-penalized logistic regression . The mod4 cells in 4 μl HBSS) were stereotactically injected into the brains of nude mice at age 6 weeks old. The coordinates were: 1.8 mm to the right of bregma and 3 mm deep from the dura. Aged-matched nude mice were used as controls. Four weeks after injection, CSF samples were collected from the cisterna magna as previously described [Dissociated glioblastoma stem cells JVJ , followed by qPCR using SsoAdvanced™ Universal Probes Supermix (Bio-Rad) and miRNA specific Taqman assay on a Bio-Rad CFX96 instrument. For the detection of miRNA from murine CSF, collected CSF was lysed directly in buffer containing 50mM Tris pH 8, 140mM NaCl, 1.5mM MgClTaqman miRNA assay for miR-10b, miR-16, and miR-21 were purchased from ThermoFisher Scientifics.


The aims of this paper are to examine: (1) the relationship between high pre-pregnancy BMI and antenatal depression; (2) whether BMI and antenatal depression interact to predict diet and gestational weight gain (GWG).Data came from the Avon Longitudinal Study of Parents and Children (ALSPAC). Underweight women were excluded. Pre-pregnancy BMI was self-reported and antenatal depression was assessed using the Edinburgh Postnatal Depression Scale at 18 and 32 weeks’ gestation to identify persistently elevated depressive symptoms (EPDS>12). Dietary patterns were calculated from food frequency questionnaires at 32 weeks’ gestation. GWG was categorised using the USA Institute of Medicine guidelines.This study included 13,314 pregnant women. Obese women had significantly higher odds of antenatal depression than normal weight controls after adjusting for socio-demographics and health behaviours . Every unit increase in pre-pregnancy BMI was associated with approximately 3% higher odds of antenatal depression . Antenatal depression was not meaningfully associated with dietary patterns after adjusting for confounders and was not associated with inadequate or excessive GWG. There was no evidence for an interaction of depression and BMI on either diet or GWG.Healthcare professionals should be aware of the dose-response relationship between high pre-pregnancy BMI and antenatal depression.The online version of this article (doi:10.1007/s00737-016-0635-3) contains supplementary material, which is available to authorized users. The prevalence of obesity is rising globally, and approximately 20% of women in the UK and USA are now estimated to be obese when they become pregnant , but the literature is very inconsistent . First, the relationship between pre-pregnancy BMI and antenatal depression was investigated; including adjustment for confounders and examination of the interaction of BMI category and SES on the risk of antenatal depression. The associations between antenatal depression and (1) dietary patterns during pregnancy; and (2) GWG were then examined, and the interactions of antenatal depression and pre-pregnancy BMI on these outcomes were tested.http://www.bris.ac.uk/alspac/researchers/data-access/data-dictionary/). All pregnant women living in the former Avon Health Authority, South West England, with due dates between 1st April 1991 and 31st December 1992 were eligible to participate. The ALSPAC study initially enrolled 14,541 women. An estimated 85–90% of the women approached agreed to participate  were excluded from the sample for these analyses, as were women who were underweight at the start of pregnancy  who were not the focus of this study. 91.6 % of women in the original cohort were included in the current study.Women in the ALSPAC cohort who experienced pregnancy or neonatal loss (2), overweight (BMI 25–30 kg/m2) and obese (BMI ≥30 kg/m2).In the ALSPAC study, women self-reported their height and retrospectively self-reported their pre-pregnancy weight as part of a postal questionnaire sent to women after enrolment. This was used to calculate pre-pregnancy BMI, classified using the World Health Organisation  categoriAntenatal depression was assessed using the Edinburgh Postnatal Depression Scale  and early pregnancy health behaviours  were included as covariates in this study. More detail on the measurement and coding of these variables is given in Online Resource n = 8165) of participants had missing data for at least one of the analysis variables but only 20.5% (n = 2981) of participants had missing data for more than five variables. All variables had some missing data; the proportion of missing data per variable ranged from 0.5 to 31.2% . The unadjusted and fully adjusted analyses were also repeated using pre-pregnancy BMI as a continuous predictor.Linear regression was used to examine the associations between antenatal depression and dietary patterns in the third trimester. The interaction of antenatal depression and pre-pregnancy BMI category on dietary pattern scores was tested. Multiple logistic regression models were used to examine the association between antenatal depression and dietary patterns adjusted for (1) socio-demographic factors and (2) socio-demographic factors, health behaviours and BMI.Finally, the relationship between antenatal depression and risk of inadequate or excessive GWG was examined using multinomial logistic regression, with recommended GWG as the base outcome. Each analysis compared the odds of (1) inadequate and (2) excessive GWG with the odds of recommended GWG, for women with antenatal depression compared to those without antenatal depression. The interaction of antenatal depression and pre-pregnancy BMI category on GWG was tested. The effect of adjusting for confounders was examined in two adjusted models, the first controlling for socio-demographic factors only and the second additionally controlling for health behaviours and BMI.n = 652) as well as women who were underweight at the beginning of pregnancy (n = 575).In total, 14,541 women were recruited into the ALSPAC cohort during pregnancy; 13,314 of these women were included in the current study following multiple imputation of missing data and exclusion of women with pregnancy or neonatal loss ; 14.3 % of women scored >12 at this time. At 32 weeks’ gestation, the median score on the EPDS was 7 (interquartile range 3–11) and 15.7 % of women scored >12. Overall, 7.9 % of the study sample had persistently elevated symptoms of depression (EPDS>12 at both 18 and 32 weeks’ gestation) and were defined as having antenatal depression for this study. Antenatal depression was most prevalent among women who were younger, of non-white ethnicity, multiparous and unmarried, as well as those with lower levels of education, manual or unskilled jobs and low social support  but the association between pre-pregnancy overweight and antenatal depression was not statistically significant . After adjusting for socio-demographic factors , the association between obesity and antenatal depression reduced in magnitude but remained significant . The association between overweight and antenatal depression remained non-significant . Additionally adjusting for health behaviours had little effect on the magnitude of the associations . The unadjusted and adjusted associations are shown in Fig. p = 0.429 for overweight and p = 0.952 for obese).The prevalence of antenatal depression was lowest among women who were normal weight when they became pregnant (7.6%), intermediate among overweight women (8.5%) and highest among women who were obese when they became pregnant 10.7%). Women who were obese at the start of pregnancy had significantly higher odds of antenatal depression than normal weight women . This remained significant in the fully adjusted model, with approximately 3% higher odds of antenatal depression for every unit increase in pre-pregnancy BMI .There was a significant unadjusted association between pre-pregnancy BMI (as a continuous variable) and risk of antenatal depression . After adjusting for socio-demographic confounders, as well as in the fully adjusted model, antenatal depression was significantly associated with higher confectionary dietary pattern scores only . There was also no evidence for an interaction of antenatal depression and pre-pregnancy BMI category on risk of inadequate or excessive GWG (interaction terms: p = 0.328 to p = 0.906). The association between depression and GWG did not change substantially after adjusting for socio-demographic factors  or in the fully adjusted analyses .The proportion of women with GWG within the recommended range was very similar among those with and without antenatal depression (27.9 and 28.1% respectively), whilst inadequate GWG was slightly more common among women with antenatal depression than those without (14.0 vs. 12.0% respectively) and excessive GWG was slightly less common (58.1 vs. 59.9% respectively). Women with antenatal depression did not have significantly higher odds of either inadequate or excessive GWG than women without antenatal depression in the unadjusted analyses  but the association was not significant for overweight women , both compared with normal weight controls (based on 9915 women with complete data for these variables). Neither association was significant in the fully adjusted complete case analyses, which included 7464 participants . The association between continuous pre-pregnancy BMI and antenatal depression was statistically significant in both the unadjusted  and fully adjusted complete case analyses . There were no meaningful differences in findings for the associations of antenatal depression with dietary patterns or GWG between the complete case and imputed analyses  than women who were normal weight, after adjusting for socio-demographic factors and health behaviours. There was also evidence for a small but statistically significant increase in the odds of antenatal depression for each unit increase in pre-pregnancy BMI. Hence there is a dose-response relationship between BMI and depression in pregnancy that is not explained by socio-demographic or lifestyle risk factors and health professionals should ensure to ask about depressive symptomatology in obese pregnant women. Women with antenatal depression reported less healthy dietary patterns during the third trimester than women without depression, but these associations were almost entirely accounted for by socio-demographic confounding. Finally, antenatal depression was not associated with either inadequate or excessive GWG, and there was no evidence for an interaction of pre-pregnancy BMI category and antenatal depression status on either dietary patterns or GWG.The unadjusted association between pre-pregnancy obesity and antenatal depression found in this study was similar to the pooled estimate for this association obtained in a recent systematic review and meta-analysis , although ethnic minority women were underrepresented, and married or cohabiting women, owner-occupiers and those with a car in their household were overrepresented  among women who are obese when they become pregnant. The adjusted association indicated approximately 40% higher odds of antenatal depression for obese women compared with normal weight controls. This association may have public health significance given the high prevalence of both obesity and depression during pregnancy. However, there were no meaningful associations between antenatal depression and dietary patterns after adjusting for socio-demographic confounders and no associations between antenatal depression and increased risk of inadequate or excessive GWG in this sample. Future research is needed to replicate these findings in a more recent cohort and to extend the evidence base to examine the relationships between antenatal depression and adverse pregnancy outcomes  among women with high pre-pregnancy BMI.ESM 1Online Resource 1 Assessment of socio-demographic and health behavioural variables (PDF 55 kb)ESM 2Online Resource 2 Variables included in the multiple imputation model and proportion of missing data for each variable (PDF 30 kb)ESM 3Online Resource 3 Associations between participant characteristics and missing data (PDF 59 kb)ESM 4Online Resource 4 Characteristics of the ALSPAC dataset before and after multiple imputation (PDF 19 kb)ESM 5Online Resource 5 Complete case analyses for the relationships between probable antenatal depression and diet and GWG (PDF 33 kb)Below is the link to the electronic supplementary material.


Parkinson’s disease (PD) is a common neurodegenerative disease whose pathological hallmark is the accumulation of intracellular α-synuclein aggregates in Lewy bodies. Lipid metabolism dysregulation may play a significant role in PD pathogenesis; however, large plasma lipidomic studies in PD are lacking. In the current study, we analyzed the lipidomic profile of plasma obtained from 150 idiopathic PD patients and 100 controls, taken from the ‘Spot’ study at Columbia University Medical Center in New York. Our mass spectrometry based analytical panel consisted of 520 lipid species from 39 lipid subclasses including all major classes of glycerophospholipids, sphingolipids, glycerolipids and sterols. Each lipid species was analyzed using a logistic regression model. The plasma concentrations of two lipid subclasses, triglycerides and monosialodihexosylganglioside (GM3), were different between PD and control participants. GM3 ganglioside concentration had the most significant difference between PD and controls . Next, we used a collection of 20 GM3 and glucosylceramide (GlcCer) species concentrations normalized to total lipid to perform a ROC curve analysis, and found that these lipids compare favorably with biomarkers reported in previous studies . Our results suggest that higher plasma GM3 levels are associated with PD. GM3 lies in the same glycosphingolipid metabolic pathway as GlcCer, a substrate of the enzyme glucocerebrosidase, which has been associated with PD. These findings are consistent with previous reports implicating lower glucocerebrosidase activity with PD risk. GBA) are strongly associated with PD [GBA encodes the lysosomal enzyme β-glucosidase (GCase), which hydrolyzes glucosylceramide and glucosylsphingosine to ceramide and sphingosine, respectively, and plays a key role in the glycosphingolipid metabolic pathway. Diminished GCase activity as a result of homozygous or compound heterozygous mutations in GBA leads to Gaucher disease, the most common genetic lysosomal storage disease. In addition to GBA, other lysosomal proteins have been associated with PD, e.g., acid sphingomyelinase [SMPD1, deficient in Niemann-Pick disease types A and B) and LIMP-2 [LRRK2 has been linked to endolysosomal/autophagic function [Parkin and PINK1 have been associated with mitochondria, the major cellular stations controlling lipid synthesis and metabolism [Parkinson’s disease (PD) is a common neurodegenerative disease, the burden of which is expected to grow as the current population ages. Numerous studies have described the contribution of aberrant lipid metabolism in a variety of neurodegenerative diseases, such as Alzheimer’s disease  and Hunt with PD . GBA encyelinase   were included. Subjects were non-fasting. Information on demographics, medical history, medication, family history of PD [The study included 150 PD participants and 100 controls who participated in the ‘Spot’ study ,22. In bry of PD , the Uniry of PD  were colPlasma lipid extracts were prepared by modifying previously published lipid extraction protocols . BrieflyQuantification of endogenous lipid species was accomplished using multiple reaction monitoring (MRM) transitions that were developed in earlier studies ,26 in coThe concentrations of individual lipid species are presented as pmol/μl of plasma sample. As an alternate quantification, we also calculated lipid concentrations as mol% of the total lipid measured to correct for the differences in total lipid amount between samples. Mol% values were calculated by dividing individual lipid concentration by the total lipid concentration (excluding cholesterol and cholesterol ester) of each sample. Both values are presented in Glycerophospholipids, lyso phospholipids and diacylglycerol (DAG) were annotated as <lipid abbreviation> <total fatty acyl chain length>:<total number of unsaturated bonds>. Sphingolipids were annotated as <lipid abbreviation> <sphingoid base residue>/<fatty acyl residue>. Triacylglycerol (TAG) was annotated as <TAG> <total fatty acyl chain length>:<total number of unsaturated bonds>(<diacylglycerol residue>/<fatty acyl residue>). CE was annotated as <CE>-<fatty acyl residue>.2-test . We then evaluated q-values, controlling for the false discovery rate at a level of 0.05  [Demographics and clinical characteristics were compared between PD and control participants by Student's t test, Chi Square and the Fisher Exact test as appropriate. The lipid measures in both pmol/μl and mol% were quantile normalized, and each lipid was in-turn fitted with a logistic regression model  with PD status as the dependent variable, lipid measure as independent variables while controlling for age, sex, body mass index (BMI), and statin usage. For each lipid, we evaluated the statistical significance of residuals for predicting PD status based on Wald’s χqvalue’) . It shouIn the classification analysis, the samples were randomly separated in a 90/10 split into development and validation cohorts. Quantile normalization was applied separately to the lipid measures of both cohorts and a support vector machine with a radial basis kernel function (R package ‘e1071’) was trained with the development cohort and evaluated with the validation cohort. Features included 20 selected lipid species that lie in the GCase pathway, including GM3 and GlcCer, as well as the controlled variables listed above. We used a random resampling procedure to estimate the expected area under the receiver operating characteristic (ROC) curve.Demographics and clinical characteristics of PD and control participants are compared in The extracted plasma lipid samples were analyzed blinded to clinical characteristics, using the LC-MS methodology described above. Of the 39 subclasses of lipids tested, including 520 specific lipid species, we identified two lipid subclasses, triglycerides and GM3, and 33 lipid species that were significantly different between PD patients and healthy controls. The list of all significant lipid species is included in The most striking differences between controls and PD were in GM3  analysis using non-linear classifier was conducted to evaluate the utility of 20 selected lipid species , consisting of GM3 and GlcCer , in discGBA or LRRK2 G2019S mutations. Our key finding is the significantly higher GM3 plasma concentration in PD cases compared to controls.In this study, we report a comparison of 520 plasma lipid species from 39 different lipid subclasses in 150 PD cases and 100 controls, non-carriers of ,[GM3 lipids are considered the simplest members of the ganglioside family, containing three monosaccharide groups attached to a ceramide backbone. They serve as precursors to complex gangliosides of the a- and b-series that are abundant in the brain . GM3 and,.Our data partially corroborates the work of Mielke et al. , where oWhile we cannot determine the relationship between GM3 levels in plasma and the brain, our findings of elevated GM3 in PD are consistent with the neuropathological literature. A study of 118 autopsies of subjects age 20 to 100, demonstrated increasing proportions of GM3 with age, and twoThe potential mechanism by which altered GM3 levels may be associated with PD was investigated in a few models. Di Pasquale and colleagues identified a ganglioside-binding domain (GBD) in α-synuclein, which has marked preference to GM3. This GBD includes residue E46, which is mutated in a familial form of PD (E46K). The deleterious effect of the mutation (in forming channels) was reversed in vesicles enriched with GM3. ConversGBA or LRRK2 G2019S mutations.Our study has some limitations. First, the degree of natural variation in blood lipids between and within individuals and the reference population is not well understood or documented. Plasma lipid composition variability is driven by a complex interplay of factors including genetics, diet, environment, and even autonomous circadian clocks ,46,47, aIn summary, our findings highlight the potential role of GM3 in idiopathic PD. Future studies should not only examine the lipid panel used in this study, but also include other complex gangliosides like GM2 and GD3, which are derived from GM3 in its biosynthetic pathway. Exploring interactions between these glycosphingolipids and α-synuclein as well as replicating our findings in neuropathological samples is necessary to better understand the pathophysiology of GM3 in idiopathic PD.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.