Integrative metabolomics-genomics analysis identifies key networks in a stem cell-based model of schizophrenia.

Schizophrenia (SCZ) is a neuropsychiatric disorder, caused by a combination of genetic and environmental factors. The etiology behind the disorder remains elusive although it is hypothesized to be associated with the aberrant response to neurotransmitters, such as dopamine and glutamate. Therefore, investigating the link between dysregulated metabolites and distorted neurodevelopment holds promise to offer valuable insights into the underlying mechanism of this complex disorder. In this study, we aimed to explore a presumed correlation between the transcriptome and the metabolome in a SCZ model based on patient-derived induced pluripotent stem cells (iPSCs). For this, iPSCs were differentiated towards cortical neurons and samples were collected longitudinally at various developmental stages, reflecting neuroepithelial-like cells, radial glia, young and mature neurons. The samples were analyzed by both RNA-sequencing and targeted metabolomics and the two modalities were used to construct integrative networks in silico. This multi-omics analysis revealed significant perturbations in the polyamine and gamma-aminobutyric acid (GABA) biosynthetic pathways during rosette maturation in SCZ lines. We particularly observed the downregulation of the glutamate decarboxylase encoding genes GAD1 and GAD2, as well as their protein product GAD65/67 and their biochemical product GABA in SCZ samples. Inhibition of ornithine decarboxylase resulted in further decrease of GABA levels suggesting a compensatory activation of the ornithine/putrescine pathway as an alternative route for GABA production. These findings indicate an imbalance of cortical excitatory/inhibitory dynamics occurring during early neurodevelopmental stages in SCZ. Our study supports the hypothesis of disruption of inhibitory circuits to be causative for SCZ and establishes a novel in silico approach that enables for integrative correlation of metabolic and transcriptomic data of psychiatric disease models.

Treatment With Hydrolyzed Diet Supplemented With Prebiotics and Glycosaminoglycans Alters Lipid Metabolism in Canine Inflammatory Bowel Disease.

Canine inflammatory bowel disease (IBD) is a chronic, immunologically mediated intestinal disorder, resulting from the complex interaction of genetic, environmental and immune factors. Hydrolyzed diets are used in dogs with food-responsive diarrhea (FRD) to reduce adverse responses to immunostimulatory proteins. Prebiotics (PRBs) and glycosaminoglycans (GAGs) have previously been demonstrated to show anti-inflammatory activity in the intestinal mucosa. Notably, hydrolyzed diets combined with the administration of PRBs and GAGs offer a promising approach for the treatment of canine IBD. Our aim was to investigate the effects of hydrolyzed diet and GAG+PRB co-treatment on the serum metabolomic profile of IBD dogs. Dogs with IBD randomly received either hydrolyzed diet supplemented with GAGs and PRBs (treatment 1) or hydrolyzed diet alone (treatment 2) for 10 weeks. A targeted metabolomics approach using mass spectrometry was performed to quantify changes in the serum metabolome before and after treatment and between treatment 1 and 2. Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA) and univariate statistics were used to identify differences between the treatment groups. PCA, PLS-DA, and HCA showed a clear clustering of IBD dogs before and after hydrolyzed diet, indicating that the treatment impacted the serum metabolome. Univariate analysis revealed that most of the altered metabolites were involved in lipid metabolism. The most impacted lipid classes were components of cell membranes, including glycerophospholipids, sphingolipids, and di- and triglycerides. In addition, changes in serum metabolites after GAG+PRB co-treatment suggested a possible additional beneficial effect on the lipid metabolism in IBD dogs. In conclusion, the present study showed a significant increase in metabolites that protect gut cell membrane integrity in response to hydrolyzed diet alone or in combination with GAG+PRB co-treatment. Administration of such treatment over 70 days improved selected serum biomarkers of canine IBD, possibly indicating improved intestinal membrane integrity.

Targeted metabolomic analysis of serum amino acids in the adult Fontan patient with a dominant left ventricle.

Growing interest lies in the assessment of the metabolic status of patients with a univentricular circulation after Fontan operation, especially in changes of amino acid metabolism. Using targeted metabolomic examinations, we investigated amino acid metabolism in a homogeneous adult Fontan-patient group with a dominant left ventricle, seeking biomarker patterns that might permit better understanding of Fontan pathophysiology and early detection of subtle ventricular or circulatory dysfunction. We compared serum amino acid levels (42 analytes; AbsoluteIDQ p180 kit, Biocrates Life Sciences, Innsbruck, Austria) in 20 adult Fontan patients with a dominant left ventricle and those in age- and sex-matched biventricular controls. Serum concentrations of asymmetric dimethylarginine, methionine sulfoxide, glutamic acid, and trans-4-hydroxyproline and the methionine sulfoxide/methionine ratio (Met-SO/Met) were significantly higher and serum concentrations of asparagine, histidine, taurine, and threonine were significantly lower in patients than in controls. Met-SO/Met values exhibited a significant negative correlation with oxygen uptake during exercise. The alterations in amino acid metabolome that we found in Fontan patients suggest links between Fontan pathophysiology, altered cell energy metabolism, oxidative stress, and endothelial dysfunction like those found in biventricular patients with congestive heart failure. Studies of extended amino acid metabolism may allow better understanding of Fontan pathophysiology that will permit early detection of subtle ventricular or circulatory dysfunction in Fontan patients.

Targeted metabolomic analysis of serum phospholipid and acylcarnitine in the adult Fontan patient with a dominant left ventricle.

BACKGROUND: Patients with a Fontan circulation have altered cholesterol and lipoprotein values. We analysed small organic molecules in extended phopsholipid and acylcarnitine metabolic pathways ('metabolomes') in adult Fontan patients with a dominant left ventricle, seeking differences between profiles in baseline and Fontan circulations. METHODS: In an observational matched cross-sectional study, we compared phosphatidylcholine (PC), sphingomyelin (SM), and acylcarnitine metabolomes (105 analytes; AbsoluteIDQ® p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) in 20 adult Fontan patients having a dominant left ventricle with those in 20 age- and sex-matched healthy controls. RESULTS: Serum levels of total PC (q-value 0.01), total SM (q-value 0.0002) were significantly lower, and total acylcarnitines (q-value 0.02) were significantly higher in patients than in controls. After normalisation of data, serum levels of 12 PC and 1 SM Fontan patients were significantly lower (q-values <0.05), and concentrations of 3 acylcarnitines were significantly higher than those in controls (q-values <0.05). CONCLUSION: Metabolomic profiling can use small specimens to identify biomarker patterns that track derangement in multiple metabolic pathways. The striking alterations in the phospholipid and acylcarnitine metabolome that we found in Fontan patients may reflect altered cell signalling and metabolism as found in heart failure in biventricular patients, chronic low-level inflammation, and alteration of functional or structural properties of lymphatic or blood vessels. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier NCT03886935.

Prophylaxis of chemotherapy-induced neutropenia and febrile neutropenia with lipegfilgrastim in 2489 cancer patients: final results from the non-interventional study NADIR.

Objectives: The non-interventional study (NIS) NADIR (DRKS00005711) evaluated the effectiveness and safety of prophylaxis with lipegfilgrastim, a glycopegylated granulocyte-colony stimulating factor, in 2500 patients undergoing chemotherapy in routine clinical practice. Primary objective was the incidence of chemotherapy-induced severe neutropenia, febrile neutropenia (FN), and neutropenia-associated complications. Methods: NADIR was a prospective NIS conducted in 201 study centers in Germany. Results: The analysis included 2489 patients. Main tumor types were breast cancer (n = 1198, 48.1%), lung cancer (n = 303, 12.2%), non-Hodgkin lymphoma (NHL; n = 337, 13.5%), and prostate cancer (n = 111, 4.5%). Nine hundred and ten (36.6%) patients were aged ≥65 years (regarded as "elderly" patients). Severe neutropenia (CTCAE grade 3/4) was reported in 26.8% (n = 666) and 25.2% (n = 229) of the total population and elderly patients, respectively. FN was documented in 2.7% (n = 68) of the total population vs 3.0% (n = 27) of elderly patients. Primary prophylaxis with lipegfilgrastim among patients with high risk of FN (>20%) was documented in 83.5% of the total population and 75.1% of elderly patients. Infections (CTCAE grade 3/4) were documented in 99 patients (4.0%) in the total population vs 47 (5.1%) elderly patients. Fatal infections were reported in 14 (0.6%) patients in the total population vs 11 (1.2%) elderly patients. Overall, most frequent lipegfilgrastim-related adverse events (AEs) included bone pain (8.0%), anemia (3.2%), leucocytosis (2.7%), and thrombocytopenia (2.5%). Of the patients, 18.0% had ≥1 documented serious AE; none of the fatal events (2.7%) was lipegfilgrastim-related. Conclusions: Lipegfilgrastim administered to patients with solid tumor/NHL undergoing chemotherapy in routine clinical practice showed similar effectiveness and safety compared to the pivotal trials.

The metabolomic plasma profile of myeloma patients is considerably different from healthy subjects and reveals potential new therapeutic targets.

INTRODUCTION: Multiple myeloma (MM), a malignant plasma cell disorder, is still an incurable disease. Thus, the identification of novel therapeutic targets is of utmost importance. Here, we evaluated the peripheral blood-based metabolic profile of patients with MM. MATERIAL & METHODS: Peripheral blood plasma levels of 188 endogenous metabolites, including amino acids, biogenic amines, acylcarnitines, glycerophospholipids, sphingomyelins, and hexoses were determined in patients with plasma cell dyscrasias: monoclonal gammopathy of undetermined significance, a precursor stage of MM (MGUS, n = 15), newly diagnosed MM, (NDMM, n = 32), relapsed/refractory MM (RRMM, n = 19) and in 25 healthy controls by mass spectrometry. RESULTS: Patients with NDMM, RRMM and MGUS have a substantially different metabolomic profile than healthy controls. The amount of eight plasma metabolites significantly differs between the NDMM and MGUS group: free carnitine, acetylcarnitine, glutamate, asymmetric dimethylarginine (ADMA) and four phosphatidylcholine (PC) species. In addition, the levels of octadecanoylcarnitine, ADMA and six PCs were significantly different between RRMM and MGUS patients. 13 different concentrations of metabolites were found between RRMM and NDMM patients (free carnitine, acetylcarnitine, creatinine, five LysoPCs and PCs). Pathway analyses revealed a distinct metabolic profile with significant alterations in amino acid, lipid, and energy metabolism in healthy volunteers compared to MGUS/MM patients. CONCLUSION: We identified different metabolic profiles in MGUS und MM patients in comparison to healthy controls. Thus, different metabolic processes, potentially the immunoregulation by indoleamine 2,3 dioxygenase-1 (IDO), which is involved in cancer development and progression supporting inflammatory processes in the tumor microenvironment and glutaminolysis, can serve as novel promising therapeutic targets in MM.

Binding of MBD proteins to DNA blocks Tet1 function thereby modulating transcriptional noise.

Aberrant DNA methylation is a hallmark of various human disorders, indicating that the spatial and temporal regulation of methylation readers and modifiers is imperative for development and differentiation. In particular, the cross-regulation between 5-methylcytosine binders (MBD) and modifiers (Tet) has not been investigated. Here, we show that binding of Mecp2 and Mbd2 to DNA protects 5-methylcytosine from Tet1-mediated oxidation. The mechanism is not based on competition for 5-methylcytosine binding but on Mecp2 and Mbd2 directly restricting Tet1 access to DNA. We demonstrate that the efficiency of this process depends on the number of bound MBDs per DNA molecule. Accordingly, we find 5-hydroxymethylcytosine enriched at heterochromatin of Mecp2-deficient neurons of a mouse model for Rett syndrome and Tet1-induced reexpression of silenced major satellite repeats. These data unveil fundamental regulatory mechanisms of Tet enzymes and their potential pathophysiological role in Rett syndrome. Importantly, it suggests that Mecp2 and Mbd2 have an essential physiological role as guardians of the epigenome.

Efficacy and safety of lipegfilgrastim compared with placebo in patients with non-small cell lung cancer receiving chemotherapy: post hoc analysis of elderly versus younger patients.

PURPOSE: Lipegfilgrastim, a glycoPEGylated recombinant granulocyte colony-stimulating factor (G-CSF), reduces neutropenia duration and febrile neutropenia (FN) incidence in patients with cancer receiving myelosuppressive chemotherapy. A phase 3 trial of lipegfilgrastim was conducted in patients with advanced non-small cell lung cancer (NSCLC) receiving cisplatin/etoposide (which produces mild-to-moderate myelosuppression). Because patients aged >65 years are at higher risk for FN versus younger patients, this post hoc analysis compared outcomes in elderly (>65 years) versus younger participants in this trial. METHODS: Patients were randomized 2:1 to receive a once-per-cycle single subcutaneous injection of lipegfilgrastim 6 mg or placebo, with up to 4 cycles of every-3-week cisplatin (day 1) and etoposide (days 1-3). The primary end point was FN incidence during cycle 1. Outcomes were compared across treatment groups and by age groups (≤65 and >65 years). RESULTS: For patients aged ≤65 years, FN incidence during cycle 1 was similar in the lipegfilgrastim and placebo groups (3.0 vs 3.2 %, respectively), whereas for elderly patients, there was a reduction in FN incidence with lipegfilgrastim (0 vs 13.3 %, respectively). In both age subgroups, lipegfilgrastim showed a propensity to reduce the incidence and duration of severe neutropenia, time to absolute neutrophil count (ANC) recovery, and depth of ANC nadir. Adverse events were generally similar between groups. CONCLUSIONS: This analysis suggests that in patients with a higher FN risk, such as the elderly patients of this study, lipegfilgrastim reduces not only the duration of severe neutropenia but also the incidence of FN.

Chemotherapy-associated treatment burden in breast cancer patients receiving lipegfilgrastim or pegfilgrastim: secondary efficacy data from a phase III study.

PURPOSE: Lipegfilgrastim is a once-per-cycle glycoPEGylated granulocyte colony-stimulating factor (G-CSF). Noninferiority of lipegfilgrastim versus pegfilgrastim was demonstrated in a phase III trial in chemotherapy (CTx)-naïve breast cancer patients. Secondary outcomes relating to treatment burden are reported here. METHODS: Patients with high-risk stage II, III, or IV breast cancer were randomized to receive lipegfilgrastim 6 mg (n = 101) or pegfilgrastim 6 mg (n = 101) subcutaneously on day 2 of each CTx cycle. Doxorubicin 60 mg/m(2) plus docetaxel 75 mg/m(2) commenced on day 1, for up to four cycles. Secondary end points included days in the hospital or intensive care unit (ICU), use of intravenous antibiotics for febrile neutropenia (FN) or related infections, and measures of CTx delivery (dose delays, reductions, and omissions). RESULTS: One lipegfilgrastim recipient and two pegfilgrastim recipients were hospitalized in cycle 1 because of FN or associated infection. The lipegfilgrastim-treated patient spent 1 day in the ICU for FN, and the two pegfilgrastim-treated patients were hospitalized for FN for 5 and 6 days, respectively. All hospitalized patients received antibiotics. An additional pegfilgrastim-treated patient received antibiotics but was not hospitalized. Most patients received CTx as scheduled; over 98% received their planned doxorubicin and docetaxel doses in all cycles. In the lipegfilgrastim group, no patients had a CTx dose reduced or omitted; eight patients in the pegfilgrastim group had a CTx dose reduced or omitted during cycles 2-4. CONCLUSIONS: The burden of treatment associated with myelosuppressive CTx was similar in breast cancer patients treated with lipegfilgrastim or pegfilgrastim.

Phase III, randomized, double-blind, placebo-controlled, multicenter study of lipegfilgrastim in patients with non-small cell lung cancer receiving myelosuppressive therapy.

PURPOSE: The aim of this study was to demonstrate lipegfilgrastim superiority versus placebo in adults with non-small cell lung cancer receiving myelosuppressive chemotherapy. METHODS: This phase III, double-blind study randomized chemotherapy-naive patients to receive cisplatin and etoposide with either lipegfilgrastim 6 mg or placebo. Because of the placebo control, patients at individual high risk for febrile neutropenia (FN; ≥20%) were excluded. Study drug was administered on day 4 (24 h after chemotherapy) of a 21-day cycle for ≤4 cycles. Primary efficacy measure was FN incidence in cycle 1. Secondary assessments included duration of severe neutropenia (DSN), absolute neutrophil count (ANC) profile, and adverse events (AEs). RESULTS: The study included 375 patients (lipegfilgrastim, n = 250; placebo, n = 125). Lipegfilgrastim superiority for FN incidence in cycle 1 was not achieved but incidence was lower (2.4%) versus placebo (5.6%). Cycle 1 mean DSN was significantly shorter for lipegfilgrastim (0.6 ± 1.1 days) versus placebo (2.3 ± 0.5 days; p < 0.0001). Incidence of severe neutropenia was significantly lower for lipegfilgrastim versus placebo overall and in each cycle (all, p < 0.0001). Mean ANC nadir was lowest in cycle 1 but significantly higher for lipegfilgrastim (1.60 ± 1.64) than placebo (0.67 ± 0.85; p < 0.0001). Mean time to ANC recovery was shorter with lipegfilgrastim in each cycle. Treatment-emergent AEs were similar between treatment groups. CONCLUSIONS: Lipegfilgrastim was not statistically superior to placebo for incidence of FN in cycle 1, but was more effective in reducing incidence of severe neutropenia, DSN, and time to ANC recovery, with an acceptable safety profile. Controlled-trials.com identifier: ISRCTN55761467.

EGFR/ERBB receptors differentially modulate sebaceous lipogenesis.

The roles of the epidermal growth factor receptor (EGFR) in sebaceous glands remain poorly explored. We show that human sebocytes express EGFR and lower levels of ERBB2 and ERBB3, all receptors being downregulated after the induction of lipid synthesis. Nile red staining showed that siRNA-mediated downregulation of EGFR or ERBB3 increases lipid accumulation, whereas ERBB2 downregulation has no effect. Spectrometry confirmed induction of triglycerides after EGFR or ERBB3 downregulation and revealed induction of cholesteryl esters after downregulation of EGFR, ERBB2 or ERBB3. Thus, EGFR/ERBB receptors differentially modulate sebaceous lipogenesis, a key feature of sebaceous gland physiology and of several skin diseases.

First-in-human, phase I/IIa dose-escalation and safety study of balugrastim in breast cancer patients receiving myelosuppressive chemotherapy.

PURPOSE: To evaluate safety of balugrastim, a recombinant human serum albumin and granulocyte colony-stimulating factor (G-CSF), administered over a range of therapeutic doses in women with breast cancer receiving doxorubicin plus docetaxel chemotherapy. METHODS: The phase I, sequential dose-escalation first segment compared subcutaneous balugrastim 50, 150, 300, and 450 µg/kg during chemotherapy cycles 0-2. The randomized (2:2:1), open-label, phase IIa second segment compared balugrastim 300 or 450 µg/kg with pegfilgrastim 6 mg during chemotherapy cycles 1 and 2. RESULTS: In the phase I segment, balugrastim was escalated to 450 µg/kg in 13 patients without dose-limiting toxicity. Three (9.7 %) of the 31 adverse events (AEs) reported in nine patients were grade 3 (agranulocytosis, vomiting, hypertension); none was grade 4. In the open-label phase IIa segment (N = 51), the majority of the 64 AEs reported in 31 (75.6 %) balugrastim-treated patients were grade 1 (59.4 %), with 39.1 % grade 2, 1.6 % grade 3 (one AE of vomiting), and none grade 4. Of the 16 AEs reported in seven (70.0 %) pegfilgrastim-treated patients, 87.5 % were grade 1, 6.3 % were grade 2, 6.3 % were grade 3 (one AE of thrombocytopenia), and none were grade 4. Overall, there were six bone pain AEs reported, one in the balugrastim 300 µg/kg group and five in the balugrastim 450 µg/kg group. No AEs in either study necessitated treatment interruption/discontinuation. The incidence and duration of grade 3-4 neutropenia were similar between balugrastim- and pegfilgrastim-treated patients. CONCLUSIONS: Balugrastim was well tolerated in this small population of breast cancer patients.

Phosphorylation of TET proteins is regulated via O-GlcNAcylation by the O-linked N-acetylglucosamine transferase (OGT).

TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli.

Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis.

Lipid metabolism depends on lipid droplets (LD), cytoplasmic structures surrounded by a protein-rich phospholipid monolayer. Although lipid synthesis is the hallmark of sebaceous gland cell differentiation, the LD-associated proteins of sebocytes have not been evaluated systematically. The LD fraction of SZ95 sebocytes was collected by density gradient centrifugation and associated proteins were analyzed by nanoliquid chromatography/tandem mass spectrometry. 54 proteins were significantly enriched in LD fractions, and 6 of them have not been detected previously in LDs. LD fractions contained high levels of typical LD-associated proteins as PLIN2/PLIN3, and most proteins belonged to functional categories characteristic for LD-associated proteins, indicating a reliable dataset. After confirming expression of transcripts encoding the six previously unidentified proteins by qRT-PCR in SZ95 sebocytes and in another sebocyte line (SebE6E7), we focused on two of these proteins, ALDH1A3 and EPHX4. While EPHX4 was localized almost exclusively on the surface of LDs, ALDH1A3 showed a more widespread localization that included additional cytoplasmic structures. siRNA-mediated downregulation revealed that depletion of EPHX4 increases LD size and sebaceous lipogenesis. Further studies on the roles of these proteins in sebocyte physiology and sebaceous lipogenesis may indicate novel strategies for the therapy of sebaceous gland-associated diseases such as acne.

Lipegfilgrastim: pharmacodynamics and pharmacokinetics for body-weight-adjusted and 6 mg fixed doses in two randomized studies in healthy volunteers.

OBJECTIVE: Two phase I, single-blind (subject blinded to treatment), randomized studies were conducted to assess the pharmacodynamics, pharmacokinetics, safety, and tolerability of lipegfilgrastim compared with pegfilgrastim in healthy adult volunteers. METHODS: Study 1 consisted of a pilot safety phase (N = 8) during which subjects received a single body-weight-adjusted subcutaneous dose of lipegfilgrastim 25 µg/kg and a dose escalation phase (N = 45) wherein subjects received lipegfilgrastim 50 or 100 µg/kg or pegfilgrastim 100 µg/kg. Study 2 was a single-blind, fixed-dose study (N = 36) comparing subcutaneous lipegfilgrastim 6 mg and pegfilgrastim 6 mg. RESULTS: Cumulative exposure (AUC0-t last and AUC 0-∞) and peak exposure (Cmax) were higher for lipegfilgrastim than pegfilgrastim after both weight-adjusted and fixed dosing. In both studies, the terminal elimination half-life of lipegfilgrastim was 5-10 hours longer than the terminal elimination half-life for pegfilgrastim at the maximum dose, and the time to maximum serum concentration (tmax) was observed later for lipegfilgrastim than for pegfilgrastim. The area over the baseline effect curve (AOBEC) for absolute neutrophil count (ANC) was approximately 30% greater after lipegfilgrastim dosing compared with the same dose of pegfilgrastim at the maximum dose. Both drugs were well tolerated, with a similar occurrence of adverse events between treatment groups. Key limitations of these studies include the small numbers of subjects and differences in dosage regimens between the two studies. CONCLUSIONS: In these studies, lipegfilgrastim provided a longer-lasting increase in ANC compared with pegfilgrastim at an equivalent dose, without increasing the peak ANC values. This may reflect the higher cumulative exposure and slower clearance (therefore longer body residence) of lipegfilgrastim. These data support the use of single-dose lipegfilgrastim 6 mg in subsequent phase III trials as prophylactic treatment for patients receiving myelosuppressive chemotherapy.

TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation.

The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes.

Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine-serine-rich (RS) domains of SR- and SR-like proteins.

The Fe(II) and 2-oxoglutarate dependent oxygenase Jmjd6 has been shown to hydroxylate lysine residues in the essential splice factor U2 auxiliary factor 65 kDa subunit (U2AF65) and to act as a modulator of alternative splicing. We describe further evidence for the role of Jmjd6 in the regulation of pre-mRNA processing including interactions of Jmjd6 with multiple arginine-serine-rich (RS)-domains of SR- and SR-related proteins including U2AF65, Luc7-like protein 3 (Luc7L3), SRSF11 and Acinus S', but not with the bona fide RS-domain of SRSF1. The identified Jmjd6 target proteins are involved in different mRNA processing steps and play roles in exon dependent alternative splicing and exon definition. Moreover, we show that Jmjd6 modifies splicing of a constitutive splice reporter, binds RNA derived from the reporter plasmid and punctually co-localises with nascent RNA. We propose that Jmjd6 exerts its splice modulatory function by interacting with specific SR-related proteins during splicing in a RNA dependent manner.

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

The polyserine domain of the lysyl-5 hydroxylase Jmjd6 mediates subnuclear localization.

Jmjd6 (jumonji-domain-containing protein 6) is an Fe(II)- and 2OG (2-oxoglutarate)-dependent oxygenase that catalyses hydroxylation of lysine residues in proteins involved in pre-mRNA splicing. Jmjd6 plays an essential role in vertebrate embryonic development and has been shown to modulate alternative splicing in response to hypoxic stress. In the present study we show that an alternatively spliced version of Jmjd6 lacking the polyS (polyserine) domain localizes to the nucleolus, predominantly in the fibrillar centre. Jmjd6 with the polyS domain deleted also interacts with nucleolar proteins. Furthermore, co-immunoprecipitation experiments and F2H (fluorescent 2-hybrid) assays demonstrate that Jmjd6 homo-oligomerization occurs in cells. In correlation with the observed variations in the subnuclear distribution of Jmjd6, the structure of Jmjd6 oligomers in vitro changes in the absence of the polyS domain, possibly reflecting the role of the polyS domain in nuclear/nucleolar shuttling of Jmjd6.

Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.

Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.