We compared activation maps of professional and amateur violinists during actual and imagined performance of Mozart's violin concerto in G major (KV216). Execution and imagination of (left hand) ...fingering movements of the first 16 bars of the concerto were performed. Electromyography (EMG) feedback was used during imagery training to avoid actual movement execution and EMG recording was employed during the scanning of both executed and imagined musical performances. We observed that professional musicians generated higher EMG amplitudes during movement execution and showed focused cerebral activations in the contralateral primary sensorimotor cortex, the bilateral superior parietal lobes, and the ipsilateral anterior cerebellar hemisphere. The finding that professionals exhibited higher activity of the right primary auditory cortex during execution may reflect an increased strength of audio–motor associative connectivity. It appears that during execution of musical sequences in professionals, a higher economy of motor areas frees resources for increased connectivity between the finger sequences and auditory as well as somatosensory loops, which may account for the superior musical performance. Professionals also demonstrated more focused activation patterns during imagined musical performance. However, the auditory–motor loop was not involved during imagined performances in either musician group. It seems that the motor and auditory systems are coactivated as a consequence of musical training but only if one system (motor or auditory) becomes activated by actual movement execution or live musical auditory stimuli.
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Biopharmaceutical products are subject to in depth analysis to ensure and improve their safety and efficacy. As part of this effort the stability and aggregation mechanisms of the ...therapeutic protein is characterized over the whole life cycle. The stability and aggregation behavior of single charge variants present in biopharmaceuticals were hardly investigated. In this study we applied a previously established methodology to assess the charge variants of the drug substance (DS) of human growth hormone (hGH). We assessed the stability and aggregation propensity of an acidic variant which forms in DS at a larger extent during short time storage at elevated temperatures. We developed a semi-preparative method to separate and analyze the charge species. Thermal and colloidal stability of this variant was analyzed by light scattering methods and a stability testing in different buffer formulations. The acidic variant showed slightly attractive self-interaction at lower pH. Thermal stress did not result in increased aggregation propensity or decreased stability compared to the DS. Thus, the methodology enabled to assess the risk of a single protein variant within the DS of hGH. The approach can also be utilized for other protein drugs as previously shown for a monoclonal antibody.
Abstract Currently, histological techniques are used to analyse implant-tissue-interactions. However, these methods are destructive and time-consuming. Furthermore, they require a large number of ...animals as longitudinal observations in one individual are not possible. The evaluation by non-destructive imaging techniques provides the opportunity to study the osseous integration with a reduced number of animals and a decreased biological variability. The present study examined the suitability of magnetic resonance imaging (MRI) to assess peri-implant bone formation exemplarily for a dental implant in a minipig model. Due to its compatibility to MR imaging polyetheretherketone (PEEK) coated with a thin layer of titanium was applied as implant material. Osseointegration was analysed within different peri-implant regions quantifying bone volume density and soft tissue content, which were assessed by MRI and histology, likewise. It could be proven that the examined regions showed differences in bone formation; the region adjacent to the implant apex turned out to be the most dynamic. Both methods led to comparable results with no significant differences regarding to the assessed parameters. Moreover, it was demonstrated that titanium coated PEEK showed a sufficient osseointegration and MRI provides a promising application in monitoring bone formation.
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Biopharmaceutical products contain conformational and chemical variants, that are typically well characterized regarding identity and activity. However, little is known about their ...self-interaction propensity and tendency to unfold, which are critical characteristics for drug stability and safety. This study aimed to separate and compare charge variants of a monoclonal antibody (mAb) and to identify aggregation prone species. We show a semi-preparative cation exchange method, that we developed to separate the individual acidic and basic variants from the naïve mAb. Additionally, we demonstrate, that the yield and purity of the fractionated charge species, extracted by that method, were sufficient for subsequent analysis of aggregate content, conformation stability and self-interaction. Our analysis revealed a differently behaving acidic variant and confirmed its increased aggregation propensity by molecular modeling. During a stability study, the potentially aggregation prone charge variant posed a limited risk to the drug substance (DS). We are the first to look at the stability of single charge variants of biopharmaceuticals, and thus present manufacturers and regulatory authorities with a method to enhance drug safety.
Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae ...family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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Monoclonal antibodies (mAbs) are valuable tools both in therapy and in diagnostic. Their tendency to aggregate is a serious concern. Since a mAb drug substance (DS) is composed of ...different variants, it is important for manufacturers to know the behavior and stability not only of the mAb as a whole, but also of the variants contained in the product.
We present a method to separate hydrophobicity variants of a mAb and subsequently analyzed these variants for stability and aggregation propensity. We identified a potentially aggregation prone hydrophilic variant which is interrelated with another previously identified aggregation prone acidic charge variant. Additionally, we assessed the risk posed by the aggregation prone variant to the DS by spiking hydrophobicity variants into DS and did not observe an enhanced aggregation propensity. Thus we present an approach to separate, characterize and analyze the criticality of aggregation prone variants in protein DS which is a step forward to further assure drug safety.
As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded ...affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC–ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its β-galactosidase activity found 8.3 and 9.8 ng/μg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/μg are obtained for top-down analysis, while 14.5 and 15.9 ng/μg were found in the bottom-up analysis.
Hexagonal Aluminium nitride (h-AlN) is an important wide-bandgap semiconductor material which is conventionally fabricated by high temperature carbothermal reduction of alumina under toxic ammonia ...atmosphere. Here we report a simple, low cost and potentially scalable mechanochemical procedure for the green synthesis of nanostructured h-AlN from a powder mixture of Aluminium and melamine precursors. A combination of experimental and theoretical techniques has been employed to provide comprehensive mechanistic insights on the reactivity of melamine, solid state metal-organic interactions and the structural transformation of Al to h-AlN under non-equilibrium ball milling conditions. The results reveal that melamine is adsorbed through the amine groups on the Aluminium surface due to the long-range van der Waals forces. The high energy provided by milling leads to the deammoniation of melamine at the initial stages followed by the polymerization and formation of a carbon nitride network, by the decomposition of the amine groups and, finally, by the subsequent diffusion of nitrogen into the Aluminium structure to form h-AlN.
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