Mid- and Far-Infrared Marker Bands of the Metal Coordination Sites of the Histidine Side Chains in the Protein Cu,Zn-Superoxide Dismutase

International audienceVibrational spectroscopy gives important information on the properties of ligand and metal–ligand bonds in metalloenzymes. Infrared spectroscopy is appealing for the study of metal active sites that are not amenable to Raman spectroscopy. We present a combined experimental and theoretical approach to analyze the mid- and far-IR spectra of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) as a probe of the histidine ligands. This metalloenzyme provides a unique model to identify specific IR signatures of metal–histidine coordination and to study their alterations as a function of the metal (copper/zinc), the copper valence state (+I/+II), the histidine coordination mode (Nτ and Nπ) and the histidine protonation state. DFT calculations combined with normal mode descriptions from potential energy distribution calculations were performed on two slightly different cluster models. Differences in the constraints at the side chain of one histidine Cu ligand sensibly modify the geometric parameters and vibrational properties. Electrochemically induced FTIR difference spectroscopy provided mid- and far-IR fingerprint spectra of the Cu protein in aqueous media that are sensitive to the redox state of the Cu centre at the active site. Comparisons of the DFT predictions with the experimental IR modes of the histidine ligands at the Cu,Zn-SOD active site showed that useful mid-IR markers of histidine Nτ and Nπ coordination were predicted with good accuracy. The DFT analysis further demonstrated a link between the ν(C4–C5) mode frequency of His46 and the specific properties of the His46–Cu bond in Cu,Zn-SOD. A combined theoretical and experimental approach on samples in H2O and 2H2O or 15N-labelled samples identified the contributions from the histidine side chain modes in the 669–629 cm–1 region

Fabrication and electrical integration of robust carbon nanotube micropillars by self-directed elastocapillary densification

Vertically-aligned carbon nanotube (CNT) "forest" microstructures fabricated by chemical vapor deposition (CVD) using patterned catalyst films typically have a low CNT density per unit area. As a result, CNT forests have poor bulk properties and are too fragile for integration with microfabrication processing. We introduce a new self-directed capillary densification method where a liquid is controllably condensed onto and evaporated from CNT forests. Compared to prior approaches, where the substrate with CNTs is immersed in a liquid, our condensation approach gives significantly more uniform structures and enables precise control of the CNT packing density and pillar cross-sectional shape. We present a set of design rules and parametric studies of CNT micropillar densification by this method, and show that self-directed capillary densification enhances the Young's modulus and electrical conductivity of CNT micropillars by more than three orders of magnitude. Owing to the outstanding properties of CNTs, this scalable process will be useful for the integration of CNTs as functional material in microfabricated devices for mechanical, electrical, thermal, and biomedical applications

Prognostic value of CCND1 gene status in sporadic breast tumours, as determined by real-time quantitative PCR assays

The CCND1 gene, a key cell-cycle regulator, is often altered in breast cancer, but the mechanisms underlying CCND1 dysregulation and the clinical significance of CCND1 status are unclear. We used real-time quantitative PCR and RT–PCR assays based on fluorescent TaqMan methodology to quantify CCND1 gene amplification and expression in a large series of breast tumours. CCND1 overexpression was observed in 44 (32.8%) of 134 breast tumour RNAs, ranging from 3.3 to 43.7 times the level in normal breast tissues, and correlated significantly with positive oestrogen receptor status (P=0.0003). CCND1 overexpression requires oestrogen receptor integrity and is exacerbated by amplification at 11q13 (the site of the CCND1 gene), owing to an additional gene dosage effect. Our results challenge CCND1 gene as the main 11q13 amplicon selector. The relapse-free survival time of patients with CCND1-amplified tumours was shorter than that of patients without CCND1 alterations, while that of patients with CCND1-unamplified-overexpressed tumours was longer (P=0.011). Only the good prognostic significance of CCND1-unamplified-overexpression status persisted in Cox multivariate regression analysis. This study confirms that CCND1 is an ER-responsive or ER-coactivator gene in breast cancer, and points to the CCND1 gene as a putative molecular marker predictive of hormone responsiveness in breast cancer. Moreover, CCND1 amplification status dichotomizes the CCND1-overexpressing tumors into two groups with opposite outcomes

Natural history of NF1 c.2970_2972del p.(Met992del): confirmation of a low risk of complications in a longitudinal study.

Individuals with the three base pair deletion NM_000267.3(NF1):c.2970_2972del p.(Met992del) have been recognised to present with a milder neurofibromatosis type 1 (NF1) phenotype characterised by café-au-lait macules (CALs) and intertriginous freckling, as well as a lack of cutaneous, subcutaneous and plexiform neurofibromas and other NF1-associated complications. Examining large cohorts of patients over time with this specific genotype is important to confirm the presentation and associated risks of this variant across the lifespan. Forty-one individuals with the in-frame NF1 deletion p.Met992del were identified from 31 families. Clinicians completed a standardised clinical questionnaire for each patient and the resulting data were collated and compared to published cohorts. Thirteen patients have been previously reported, and updated clinical information has been obtained for these individuals. Both CALs and intertriginous freckling were present in the majority of individuals (26/41, 63%) and the only confirmed features in 11 (27%). 34/41 (83%) of the cohort met NIH diagnostic criteria. There was a notable absence of all NF1-associated tumour types (neurofibroma and glioma). Neurofibroma were observed in only one individual-a subcutaneous lesion (confirmed histologically). Nineteen individuals were described as having a learning disability (46%). This study confirms that individuals with p.Met992del display a mild tumoural phenotype compared to those with 'classical', clinically diagnosed NF1, and this appears to be the case longitudinally through time as well as at presentation. Learning difficulties, however, appear to affect a significant proportion of NF1 subjects with this phenotype. Knowledge of this genotype-phenotype association is fundamental to accurate prognostication for families and caregivers