Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of murine juvenile myogenic progenitors.

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accretion as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.

Cell autonomous requirement of neurofibromin (Nf1) for postnatal muscle hypertrophic growth and metabolic homeostasis.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a multi-organ disease caused by mutations in neurofibromin 1 (NF1). Amongst other features, NF1 patients frequently show reduced muscle mass and strength, impairing patients' mobility and increasing the risk of fall. The role of Nf1 in muscle and the cause for the NF1-associated myopathy are mostly unknown. METHODS: To dissect the function of Nf1 in muscle, we created muscle-specific knockout mouse models for NF1, inactivating Nf1 in the prenatal myogenic lineage either under the Lbx1 promoter or under the Myf5 promoter. Mice were analysed during prenatal and postnatal myogenesis and muscle growth. RESULTS: Nf1Lbx1 and Nf1Myf5 animals showed only mild defects in prenatal myogenesis. Nf1Lbx1 animals were perinatally lethal, while Nf1Myf5 animals survived only up to approximately 25 weeks. A comprehensive phenotypic characterization of Nf1Myf5 animals showed decreased postnatal growth, reduced muscle size, and fast fibre atrophy. Proteome and transcriptome analyses of muscle tissue indicated decreased protein synthesis and increased proteasomal degradation, and decreased glycolytic and increased oxidative activity in muscle tissue. High-resolution respirometry confirmed enhanced oxidative metabolism in Nf1Myf5 muscles, which was concomitant to a fibre type shift from type 2B to type 2A and type 1. Moreover, Nf1Myf5 muscles showed hallmarks of decreased activation of mTORC1 and increased expression of atrogenes. Remarkably, loss of Nf1 promoted a robust activation of AMPK with a gene expression profile indicative of increased fatty acid catabolism. Additionally, we observed a strong induction of genes encoding catabolic cytokines in muscle Nf1Myf5 animals, in line with a drastic reduction of white, but not brown adipose tissue. CONCLUSIONS: Our results demonstrate a cell autonomous role for Nf1 in myogenic cells during postnatal muscle growth required for metabolic and proteostatic homeostasis. Furthermore, Nf1 deficiency in muscle drives cross-tissue communication and mobilization of lipid reserves.

Design and Precursor-based Solid-State Synthesis of Mixed-Linker Zr-MIL-140A.

Acetic acid, an alternative green solvent, was utilized for the solvothermal synthesis of four 2D materials of composition [Zr2O2(OAc)2(BDC-F)], [Zr2O2(OAc)2(BDC-F4)], [Zr2O2(OAc)2(BDC)], and [Zr2O2(OAc)2(NDC)] (BDC, terephthalate; BDC-F, 2-fluoroterephthalate; BDC-F4, tetrafluoroterephthalate; NDC, 2,6-naphthalenedicarboxylate). The first three compounds were subsequently reacted with terephthalic acid in solid-state reactions to form porous MIL-140A-type metal-organic frameworks and mixed-linker derivatives ([ZrO(BDC)1-x(BDC-Y)x], x = 0-0.18, Y = F, F4). The reaction kinetics of the formation of MIL-140A were investigated with the aid of time-resolved synchrotron and temperature-resolved in-house X-ray powder diffraction experiments. Thorough compositional analyses and solid-state NMR spectroscopic experiments were used to assess the crystallographic ordering of the different linker molecules. Additionally, acetic acid-based routes for the direct synthesis of MIL-140A-NO2 and a novel MIL-140A-(CH3)2 derivative were discovered.

Solvent Impact on the Properties of Benchmark Metal-Organic Frameworks: Acetonitrile-Based Synthesis of CAU-10, Ce-UiO-66, and Al-MIL-53.

Herein is reported the utilization of acetonitrile as a new solvent for the synthesis of the three significantly different benchmark metal-organic frameworks (MOFs) CAU-10, Ce-UiO-66, and Al-MIL-53 of idealized composition [Al(OH)(ISO)], [Ce6 O4 (OH)4 (BDC)6 ], and [Al(OH)(BDC)], respectively (ISO2- : isophthalate, BDC2- : terephthalate). Its use allowed the synthesis of Ce-UiO-66 on a gram scale. While CAU-10 and Ce-UiO-66 exhibit properties similar to those reported elsewhere for these two materials, the obtained Al-MIL-53 shows no structural flexibility upon adsorption of hydrophilic or hydrophobic guest molecules such as water and xenon and is stabilized in its large-pore form over a broad temperature range (130-450 K). The stabilization of the large-pore form of Al-MIL-53 was attributed to a high percentage of noncoordinating -COOH groups as determined by solid-state NMR spectroscopy. The defective material shows an unusually high water uptake of 310 mg g-1 within the range of 0.45 to 0.65 p/p°. In spite of showing no breathing effect upon water adsorption it exhibits distinct mechanical properties. Thus, mercury intrusion porosimetry studies revealed that the solid can be reversibly forced to breathe by applying moderate pressures (≈60 MPa).

Clinical symptoms and sonographic follow-up after surgical treatment of nonparasitic liver cysts.

BACKGROUND: The optimal treatment of nonparasitic liver cysts is still a topic of debate. Only symptomatic cysts are being considered as requiring treatment. Aim of this study is to evaluate our experience with this disease over the past ten years with a structured follow-up program. METHODS: From January 2000 to August 2010, 56 consecutive patients with nonparasitic liver cysts were treated at our institution. We assessed morbidity, recurrence and complication rates, quality of life as well as pre- and post-operative sonographic status of the cysts and course of clinical symptoms. RESULTS: In 84% of the patients surgery was started as a laparoscopic procedure. Conversion rate was 6.4%. Average diameter of deroofed cysts was 12 cm. Overall complication rate was 16% and overall recurrence rate 28.3% (8.7% recurrences at the surgical site, 19.6% new or enlarged cysts). One half of the patients were symptom-free after surgery and the other half had at least one persisting symptom post-operatively. In one half of these patients with persisting symptoms, symptoms were ameliorated by surgery. In the other half of patients the number of symptoms increased after surgery. Two thirds of the overall patients reported their post-operative health as being good or very good. CONCLUSIONS: Surgical deroofing is the most effective treatment option for symptomatic liver cysts. Half of our patient population retained at least one symptom from a group of more than ten abdominal symptoms.Only the minority of these cases may be attributed to true recurrence, de-novo cysts or growing pre-existing cysts. The analysis of our cases suggests that the persistent symptoms in our patients may in part be due to the fact that the association between clinical complaints and the liver cysts was not sufficiently established. A more rigid patient selection should be implemented in order to achieve better results from the treatment of cysts. Because even large cysts are frequently asymptomatic, patient selection should not primarily be based on the cyst size only. The decision should be based strictly on the correlation between cyst / cyst location and symptoms / clinical complaints. In our opinion, further diagnostic procedures may be necessary in individual cases to clarify such a correlation.

Tissue oxygenation during exercise measured with NIRS: reproducibility and influence of wavelengths.

Near-infrared spectroscopy (NIRS) is widely used for the measurement of skeletal muscle oxygenation during exercise as it reflects muscle metabolism, and most studies report a large variability between subjects. Here we assess the data quality of tissue oxygen saturation (SO2) and oxygenated (oxyHb) and deoxygenated (deoxyHb) haemoglobin concentrations recorded during an incremental cycling protocol in nine healthy volunteers. The protocol was repeated three times on the same day and a fourth session on a different day to estimate the reproducibility of the method with a broadband, spatially resolved spectroscopy (SRS) system. We found that the inter-subject variation in SO2 (standard deviation ≈ 6 %) was considerably larger than the reproducibility (≈ 1.5 %) both for the same-day and different-day tests. The reproducibility of changes in SO2 was better than 1 %.

Urinary proteomics based on capillary electrophoresis coupled to mass spectrometry in kidney disease.

Urine is an excellent sample source in the proteomic study of diseases. It is available in large quantities, is relatively stable, is not contaminated by cells or lipids, and has shown to provide information not only on the organs in contact with the urinary tract but also of more remote organs and tissues. In addition to these qualities, it can be collected by untrained personnel. For these reasons, urinary proteomic studies have escalated in recent years with the aim of identifying biomarkers that could be use for diagnosis or to predict the outcome of renal pathologies. In this chapter, we present one of the analytical platforms that has been successfully used in a number of studies for the identification and validation of biomarkers in kidney diseases. This technique is capillary electrophoresis coupled online to an electrospray ionization time-of-flight mass spectrometer (CE-MS). This technology has proven to be highly reproducible, sensitive with a quick analysis time, important features when analytical platforms have to be used in a clinical setting.

Optimal Jamar dynamometer handle position to assess maximal isometric hand grip strength in epidemiological studies.

PURPOSE: Hand grip strength is an indicator of general muscle strength that is measured using a hand dynamometer. In some studies, a subject's grip strength is taken to be the maximal grip strength achieved from measurements taken at several different dynamometer handle positions. However, little is known about the influence of these different positions on the measured grip strength. The aim of the study was to identify one standard handle position that could be used to assess the grip strength of all subjects. METHODS: Grip strength was assessed with a hand dynamometer (Jamar Plus+; Sammons Preston, Rolyon, Bolingbrook, IL). Each participant's grip strength was measured 3 times in each of 5 different handle positions with each hand. The best position for each participant was defined as the position at which they achieved maximal grip strength. RESULTS: The mean (± standard deviation) age of the 50 participants was 41 (± 13) years. Maximal grip strength was 43.7 (± 12.4) kg for all participants; 55.0 (± 10.2) kg for men and 35.4 (± 5.2) kg for women. Handle position 2 was the best position for 70% of participants. The mean difference between the grip strength achieved by each participant at handle position 2 and that achieved at each participant's best position was 0.8 (± 1.78) kg. CONCLUSIONS: Our results show that measurements taken at a single standard handle position are sufficiently accurate to assess grip strengths for all subjects. We therefore recommend handle position 2 as the standard position for measuring grip strength with the Jamar Plus+ hand dynamometer. CLINICAL RELEVANCE: The assessment of grip strength with the Jamar Plus+ dynamometer is easier and faster if a single, standard handle position is used rather than multiple different positions. As well as providing accurate results, a single, standard handle position also reduces fatigue and increases the comparability of results between subjects.

Performance of different separation methods interfaced in the same MS-reflection TOF detector: a comparison of performance between CE versus HPLC for biomarker analysis.

One of the aims in the field of proteomics is the identification of a protein or polypeptide, or a range of these compounds, that could provide pre-symptomatic indication of the onset of a disease. A number of analytical techniques have been employed to try and achieve this end. These techniques have been applied to the complete range of body fluids and tissues that are readily available from clinical studies. Of these sample sources, the urinary low molecular weight peptidome has been shown to reflect changes in the health status of the individual. The alterations that occur in the polypeptide make up of urine, which reflect changes in biological status, are known as biomarkers. To be able to determine these changes no single technique has emerged that can cope with detecting the large number of peptides present and quantifying them over the wide concentration range they exist in. In this investigation, we made use of a single reflectron time of flight (RTOF)-MS analyser to which we first connected a CE system and then a nanoflow HPLC. Two pooled male and female standard urine samples were compared on these systems. Both techniques had similar results in terms of number of peptides detected and the mass range the peptides were detected over. The major differences in terms of biomarker research were the ability in CE to calibrate the migration time of the peptides to allow comparison between samples. In addition, CE was shown not to suffer from carry over from previous samples as was seen in the LC analysis.

Human urinary peptide database for multiple disease biomarker discovery.

PURPOSE: Human urine is an ideal candidate for use in clinical diagnostics. It is easily available, as untrained personnel can collect it. It correlates well with the pathophysiology of a number of diseases, making it a useful source for clinical proteomics. EXPERIMENTAL DESIGN: In this article, we give an update of the human urinary peptide database derived from over 13,000 data sets of CE-MS by now. RESULTS: Urine samples from both patients and healthy subjects were analyzed by CE-MS; these included 47 different pathophysiological conditions. Besides defining biomarkers by their experimental parameters, information on their sequences provides fundamental data into the pathological pathways of diseases. Therefore, we have sequenced 953 urinary peptides by using state-of-the-art top-down MS/MS. Identified biomarkers of all clinical proteomic CE-MS studies including their regulation are also listed in this work. CONCLUSIONS AND CLINICAL RELEVANCE: Biomarker discovery can be used in the management of a wide range of diseases, by combining these data sets of the database. Taking this approach, we can reveal details, at a molecular level, on the pathogenesis of a number of diseases, in particular those associated with urine production and excretion.

Urinary proteomics for prediction of preeclampsia.

Preeclampsia is a major determinant of fetal and maternal morbidity and mortality. We used a proteomic strategy to identify urinary biomarkers that predict preeclampsia before the onset of disease. We prospectively collected urine samples from women throughout pregnancy. Samples from gestational weeks 12 to 16 (n=45), 20 (n=50), and 28 (n=18) from women who subsequently had preeclampsia develop were matched to controls (n=86, n=49, and n=17, respectively). We performed capillary electrophoresis online coupled to micro-time-of-flight mass spectrometry. Disease-specific peptide patterns were generated using support vector machine-based software. Candidate biomarkers were sequenced by liquid chromatography-tandem mass spectrometry. From comparison with nonpregnant controls, we defined a panel of 284 pregnancy-specific proteomic biomarkers. Subsequently, we developed a model of 50 biomarkers from specimens obtained at week 28 that was associated with future preeclampsia (classification factor in cases, 1.032 ± 0.411 vs controls, -1.038 ± 0.432; P<0.001). Classification factor increased markedly from week 12 to 16 to 28 in women who subsequently had preeclampsia develop (n=16; from -0.392 ± 0.383 to 1.070 ± 0.383; P<0.001) and decreased slightly in controls (n=16; from -0.647 ± 0.437 to -1.024 ± 0.433; P=0.043). Among the biomarkers are fibrinogen alpha chain, collagen alpha chain, and uromodulin fragments. The markers appear to predict preeclampsia at gestational week 28 with good confidence but not reliably at earlier time points (weeks 12-16 and 20). After prospective validation in other cohorts, these markers may contribute to better prediction, monitoring, and accurate diagnosis of preeclampsia.

Comprehensive human urine standards for comparability and standardization in clinical proteome analysis.

PURPOSE: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well-characterized "real life" sample that can be used as reference standard in urine clinical proteomics studies. EXPERIMENTAL DESIGN: We report on the generation of male and female urine samples that are extensively characterized by different platforms and methods (CE-MS, LC-MS, LC-MS/MS, 1-D gel analysis in combination with nano-LC MS/MS (using LTQ-FT ultra), and 2-DE-MS) for their proteome and peptidome. In several cases analysis involved a definition of the actual biochemical entities, i.e. proteins/peptides associated with molecular mass and detected PTMs and the relative abundance of these compounds. RESULTS: The combination of different technologies allowed coverage of a wide mass range revealing the advantages and complementarities of the different technologies. Application of these samples in "inter-laboratory" and "inter-platform" data comparison is also demonstrated. CONCLUSIONS AND CLINICAL RELEVANCE: These well-characterized urine samples are freely available upon request to enable data comparison especially in the context of biomarker discovery and validation studies. It is also expected that they will provide the basis for the comprehensive characterization of the urinary proteome.

Total hemoglobin mass, iron status, and endurance capacity in elite field hockey players.

The aims of this study were as follows: To evaluate total hemoglobin mass (tHbmass) in international field hockey players; to examine the correlation between tHbmass and maximum oxygen uptake (VO2max); and to assess influences of iron status on tHbmass and on VO2max. The players of the German women's (N = 17, aged 24.8 +/- 3.0 [21-31] years) and men's (N = 17, aged 24.2 +/- 2.9 [19-32] years) national field hockey team were investigated. tHbmass was measured by an optimized carbon monoxide rebreathing method. The following parameters were measured in venous blood: Hemoglobin concentration (Hbconc), hematocrit (Hct), number and percentage of reticulocytes, reticulocyte hemoglobin content, serum iron, serum ferritin, serum transferrin, unsaturated iron-binding capacity, and serum soluble transferrin receptor concentration. VO2max was determined in a treadmill test. tHbmass (women: 10.6 +/- 1.1 g/kg; men: 12.5 +/- 0.9 g/kg) correlated to VO2max (women: 46.6 +/- 2.9 mL/min/kg; men: 55.8 +/- 4.0 mL/min/kg) in women (r = 0.56, p < 0.05) and in men (r = 0.57, p < 0.05), whereas Hbconc and Hct did not. The investigated parameters of iron status showed no association to tHbmass or to VO2max. In conclusion, tHbmass can be used as an indicator for endurance capacity in elite field hockey players, whereas Hbconc may not. tHbmass or VO2max were not influenced by the actual iron status of the investigated athletes.