GDF15 is a dynamic biomarker of the integrated stress response in the central nervous system.

AIM: Characterize Growth Differentiation Factor 15 (GDF15) as a secreted biomarker of the integrated stress response (ISR) within the central nervous system (CNS). METHODS: We determined GDF15 levels utilizing in vitro and in vivo neuronal systems wherein the ISR was activated. Primarily, we used the murine model of vanishing white matter disease (VWMD), a neurological disease driven by persistent ISR in the CNS, to establish a link between levels of GDF15 in the cerebrospinal fluid (CSF) and ISR gene expression signature in the CNS. GDF15 was also determined in the CSF of VWM patients. RESULTS: GDF15 expression was increased concomitant to ISR activation in stress-induced primary astrocytes as well as in retinal ganglion cells following optic nerve crush, while treatment with 2Bact, a specific eIF2B activator, suppressed both the ISR and GDF15. In the VWMD model, CSF GDF15 levels corresponded with the magnitude of the ISR and were reduced by 2BAct. In VWM patients, mean CSF GDF15 was elevated >20-fold as compared to healthy controls, whereas plasma GDF15 was undifferentiated. CONCLUSIONS: These data suggest that CSF GDF15 is a dynamic marker of ISR activation in the CNS and may serve as a pharmacodynamic biomarker for ISR-modulating therapies.

p53 is regulated by the lysine demethylase LSD1.

p53, the tumour suppressor and transcriptional activator, is regulated by numerous post-translational modifications, including lysine methylation. Histone lysine methylation has recently been shown to be reversible; however, it is not known whether non-histone proteins are substrates for demethylation. Here we show that, in human cells, the histone lysine-specific demethylase LSD1 (refs 3, 4) interacts with p53 to repress p53-mediated transcriptional activation and to inhibit the role of p53 in promoting apoptosis. We find that, in vitro, LSD1 removes both monomethylation (K370me1) and dimethylation (K370me2) at K370, a previously identified Smyd2-dependent monomethylation site. However, in vivo, LSD1 shows a strong preference to reverse K370me2, which is performed by a distinct, but unknown, methyltransferase. Our results indicate that K370me2 has a different role in regulating p53 from that of K370me1: K370me1 represses p53 function, whereas K370me2 promotes association with the coactivator 53BP1 (p53-binding protein 1) through tandem Tudor domains in 53BP1. Further, LSD1 represses p53 function through the inhibition of interaction of p53 with 53BP1. These observations show that p53 is dynamically regulated by lysine methylation and demethylation and that the methylation status at a single lysine residue confers distinct regulatory output. Lysine methylation therefore provides similar regulatory complexity for non-histone proteins and for histones.

Repression of p53 activity by Smyd2-mediated methylation.

Specific sites of lysine methylation on histones correlate with either activation or repression of transcription. The tumour suppressor p53 (refs 4-7) is one of only a few non-histone proteins known to be regulated by lysine methylation. Here we report a lysine methyltransferase, Smyd2, that methylates a previously unidentified site, Lys 370, in p53. This methylation site, in contrast to the known site Lys 372, is repressing to p53-mediated transcriptional regulation. Smyd2 helps to maintain low concentrations of promoter-associated p53. We show that reducing Smyd2 concentrations by short interfering RNA enhances p53-mediated apoptosis. We find that Set9-mediated methylation of Lys 372 inhibits Smyd2-mediated methylation of Lys 370, providing regulatory cross-talk between post-translational modifications. In addition, we show that the inhibitory effect of Lys 372 methylation on Lys 370 methylation is caused, in part, by blocking the interaction between p53 and Smyd2. Thus, similar to histones, p53 is subject to both activating and repressing lysine methylation. Our results also predict that Smyd2 may function as a putative oncogene by methylating p53 and repressing its tumour suppressive function.

Biomarker context-of-use: how organizational design can impact the implementation of the appropriate biomarker assay strategy.

Since 2011, the European Bioanalysis Forum has been discussing the topic of context-of-use for biomarker assays, in support of a cross-industry implementation of its principles. The discussions have led to the acknowledgement of the challenges that we face as an industry in implementing these principles. In addition to scientific recommendations, the European Bioanalysis Forum has addressed these challenges by providing recommendations on organizational design, and what works in both sponsor and contract research organizations, to support and enable context-of-use across biomarker strategies. Here, we highlight the key considerations for organizational design to help ensure that biomarker assays are characterized and validated according to the right context-of-use, to ensure that the right decisions based on the biomarker data can be made during drug development.

Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy.

Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the repressive heterochromatin state. To elucidate its mobility and interactions, we conducted a comprehensive analysis on different time and length scales by fluorescence fluctuation microscopy in mouse cell lines. The local mobility of HP1alpha and HP1beta was investigated in densely packed pericentric heterochromatin foci and compared with other bona fide euchromatin regions of the nucleus by fluorescence bleaching and correlation methods. A quantitative description of HP1alpha/beta in terms of its concentration, diffusion coefficient, kinetic binding, and dissociation rate constants was derived. Three distinct classes of chromatin-binding sites with average residence times t(res)

Comparison of continuous blood glucose measurement with conventional documentation of hypoglycemia in patients with Type 2 diabetes on multiple daily insulin injection therapy.

BACKGROUND: The primary objective of this study was to investigate the difference in the proportion of patients with conventionally detected hypoglycemia compared with continuous glucose monitoring system (CGMS, Medtronic MiniMed, Sylmar, CA)-detected glucose values < or = 60 mg/dL (< or = 3.3 mmol/L), during the 72-h CGMS measurement period after 8 weeks' treatment with insulin glargine. METHODS: This was a multicenter (n = 125), open-label, single-arm study in patients with Type 2 diabetes mellitus (T2DM) on multiple daily injections. Patients received NPH insulin (2-week run-in) followed by glargine (8-week treatment phase). Glucose levels were measured by CGMS and self-monitored blood glucose (SMBG) profiles over the 72-h pre- and post-treatment phase. RESULTS: The full analysis set contained 367 patients [male 59%; mean age 59.2 years; mean body mass index 31.7 kg/m(2); mean hemoglobin A1c (HbA1c) 6.9%]. At end point, 209 patients (56.9%) experienced hypoglycemia according to CGMS; 97 (26.4%) recorded hypoglycemia by conventional methods. CGMS- and SMBG-determined mean daytime glucose levels were similar at baseline and end point; however, nocturnal glucose levels were significantly lower with CGMS versus SMBG at baseline [130.2 vs. 145.0 mg/dL (7.2 vs. 8.1 mmol/L)] and at end point [123.3 vs. 137.3 mg/dL (6.8 vs. 7.6 mmol/L)]. Glucose levels measured by CGMS and SMBG decreased, and HbA1c levels decreased from 6.90% at screening to 6.67% at end point (P < 0.001). CONCLUSIONS: This study demonstrates that CGMS can be successfully employed in large clinical trial settings in patients with T2DM. This easy-to-implement method may provide additional insights into glucose levels and valuable information regarding the time patients spend within the preferred glucose range.

Personality Styles of German-Speaking Psychotherapists Differ from a Norm, and Male Psychotherapists Differ from Their Female Colleagues.

Variables pertaining to the person of the psychotherapist have been neglected in psychotherapy research for some time. Concerning personality in particular, however, research has mostly focused on its relation with the psychotherapist's choice of method, or differences between the various major therapy approaches. That is, psychotherapists were compared to each other without specifying how exactly psychotherapists are in comparison to "ordinary people." We wanted to know: Are there specific personality styles that distinguish psychotherapists from the norm? A sample of 1,027 psychotherapists from Germany, Austria, and Switzerland filled out the short version of the Personality Style and Disorder Inventory (PSDI-S) via online survey. The PSDI-S is a self-report questionnaire that assesses 14 personality styles, partly related to the non-pathological equivalents of classifiable personality disorders. The psychotherapists were compared to a normative sample of 3,392 people of different professions. The results could be divided into three groups: (1) Large differences in four personality styles that might contribute to relationship skills and may enable psychotherapists to put their own personal opinion aside, show empathy and appreciation, open themselves to the emotional experience of the patient, and provide a trusting relationship. (2) Moderate differences in seven personality styles that are equally indicative of the professional social skills of the psychotherapists, i.e., they were neither submissive nor passive, not excessively helpful, but also not too self-assertive. (3) Hardly any or no differences regarding a charming (histrionic) style, optimism, and conscientiousness. Gender-specific results revealed that male psychotherapists differed from their female colleagues, but they did so differently than men and women in the normative sample do. The main limitations were that we relied on self-report and did not statistically control for gender, age, and education, when comparing to the norm. As a conclusion, German-speaking psychotherapists show personality styles that we interpret as functional for psychotherapeutic practice but this needs corroboration from studies that use different methods and measures.

SEC Based Method for Size Determination of Immune Complexes of Therapeutic Antibodies in Animal Matrix.

Therapeutic monoclonal antibodies (mAbs) represent a milestone in pharmacological development. Their superiority is based on the combination of high specificity, low toxicity, and long half-life that characterizes biologics. If biologics have Achilles' heel, it is their potential immunogenicity. To better understand the impact of the size of immune complexes of mAbs on anti-drug antibody (ADA) dependent adverse reactions in Macaca fascicularis, we developed an efficient high-throughput size exclusion chromatography- (SEC-) based methodology that enables analysis of the size, size distribution, and ratio of free and ADA-complexed mAb in serum allowing for assessment of formation and clearance of circulating ADA-mAb immune complexes (CIC).