U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in ‘circulating miRNA''. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.     

Biophysical applications

Hyperfine Interactions - Nuclear forward scattering (NFS) of synchrotron radiation was applied to investigate the electronic and magnetic properties of (i) the diamagnetic...     

Iron sulfur clusters in benzoyl–CoA reductase

The enzyme benzoyl-CoA reductase (BCR) has been studied by Mössbauer spectroscopy in fields up to 7 T and at temperature down to 4.2 K. It has been shown that the oxidized BCR contains three diamagnetic [4Fe-4S]²⁺ centers. Treatment of BCR with dithionite or deazaflavin reduces only one of the three centers from [4Fe-4S]²⁺ to [4Fe-4S]¹⁺. The latter exhibits Mössbauer spectra at 4.2 K in applied fields characteristic for Fe^2.5+Fe^2.5+ (S=9/2) and Fe²⁺Fe²⁺ (S=4) pairs, both coupled antiferromagnetically to total spin S=1/2.     

Molecular properties of FeCO as derived from AB initio molecular orbital calculations

We have performed ab initio all-electron and pseudopotential MO calculations on FeCO with varying degrees of sophistication in the basis sets as well as in the calculational procedures. Basis sets with at least double zeta representation for valence orbitals as well as inclusion of correlation corrections in the MO calculations are the minimum requirements in order to arrive at a clear decision about the electronic ground state of FeCO. In contradiction to the suggestion given by Guenzburger et al. [23] we derive from total energy considerations and from comparing experimental and calculated C−O stretching force constants that the ground state is a spin-quintet (⁵∑⁻) and not a spin-triplet. Our calculated quadrupole splitting for MO equilibrium geometry of FeCO⁵∑⁻ deviates by about 25% from the value which was observed by Peden et al. [1] for FeCO in solid noble gas. However, the calculated electronic configuration of iron within FeCO⁵∑⁻ of about 3d^6.44s^0.8 is far from 3d⁶4s², which approximately is expected for FeCO when comparing isomer shifts of FeCO and Fe, both matrix-isolated in solid noble gas, i.e. −0.60 mm/s and −0.75 mm/s, respectively. Thus, at the moment the question remains open to what extent the molecular properties of FeCO are affected by the solid noble gas matrix.     

Hyperfine and magnetic properties of 19e− (electron reservoir) sandwiches of Fe(I), C5R5FeC6R6 (where R=H or CH3) and (C6(CH3)6)2Fe+: Mössbauer experiments and molecular orbital calculations

The four compounds C₅H₅FeC₆H₆, C₅H₅Fe(CH₃₎₆, C₅(CH₃₎₅FeC₆(CH₃₎₆ and (C₆(CH₃₎₆₎₂Fe⁺ were studied by Mössbauer spectroscopy on powders. On the basis of semi-empirical molecular orbital calculation (Iterative Extended Hückel with self-consistence of the charge) in which parameters derived from X-ray data are used, we have modelized the thermal behaviour of the electric field gradient (EFG) tensor from 4 K to 300 K. The reduction in magnitude of electronic observables (e.g. spin orbit coupling constant and EFG magnitude) gave evidence for a dynamic Jahn-Teller effect. Spectra in a high magnetic field (6 teslas) confirmed the paramagnetic behaviour of the compounds. The sign of the EFG tensor, the magnetic hyperfine field tensor and its orientation with respect to the EFG tensor were determined.     

Size determination of metallic iron particles by temperature dependent Mössbauer and ferromagnetic resonance studies

The thermal decomposition products of NaX·Fe(CO)₅ after various steps of heat treatment, in vacuum as well as under gas atmosphere (Ar, H₂), have been investigated by temperature-dependent Mössbauer and ferromagnetic resonance measurements. The measured spectra of both techniques consistently are analyzed using superparamagnetic relaxation behavior of magnetic particles. From this analysis information concerning composition and size of magnetic iron particles is derived.     

CONTROLLING THE 3D NANOSCALE ORGANIZATION OF BULK HETEROJUNCTION POLYMER SOLAR CELLS

In this study,the three dimensional nanoscale organization in the photoactive layers of poly(3-hexylthiophene) (P3HT) and a methanofullerene derivative (PCBM) is revealed by transmission electron tomography.After annealing treatment,either at elevated temperature or during slow solvent evaporation,nanoscale interpenetrating networks are formed with high crystalline order and favorable concentration gradients of both components through the thickness of the photoactive layer.Such a tailored morphology acco...     

Mössbauer- and EPR-Snapshots of an Enzymatic Reaction: The Cytochrome P450 Reaction Cycle

Hyperfine Interactions - In this communication we present a complimentary Mössbauer- and EPR-study of the time dependance of the reaction of substrate free P450cam with peracetic acid within a...     

Tin–DNA complexes investigated by nuclear inelastic scattering of synchrotron radiation

Nuclear inelastic scattering (NIS) of synchrotron radiation has been used to investigate the dynamics of tin ions chelated by DNA. Theoretical NIS spectra have been simulated with the help of density functional theory (DFT) calculations using 12 models for different binding sites of the tin ion in (CH₃)Sn(DNA_Phosphate)₂. The simulated spectra are compared with the measured spectrum of the tin–DNA complex.     

Study of the changes in the magnetic properties of stainless steels under mechanical treatment

Six types of stainless steels (SS) were studied for changes in its structure and magnetic properties under mechanical treatment. Depending on intensity and duration of the process of plastic deformation and the SS type the paramagnetic austenite structure transforms partially to completely into ferrite structure with ferromagnetic behaviour. Some of the SS tested were found slightly modified yet in the process of its manufacturing. Only one SS type with high Ni content preserved its structure and paramagnetic properties even after very intense mechanical treatment.