Phase III, randomised, multicentre trial of maintenance immunotherapy with low-dose interleukin-2 and interferon-α for metastatic renal cell cancer

This is the first phase III randomised trial to evaluate maintenance immunotherapy in metastatic renal cell cancer (mRCC). Patients were randomised to receive treatment with a 4-week cycle of subcutaneous low doses IL-2 + IFN in months 1, 3 and 5, and then every 3 months until the first documented disease progression (arm A, suspension), or the same regimen, with chronic maintenance of immunotherapy, regardless of tumour response, until death or intolerable toxicity (arm B, maintenance). The primary endpoint was overall survival (OS); secondary endpoints were time from first progression to death (TFPTD) and tolerability. One hundred and eighty-three patients were enrolled between January 1998 and November 2003. After a median follow-up of 53.9 months, response rate, median OS and median TFPTD were 14.7% (6.3% CR) versus 11.3% (5.5% CR), 14 versus 14 months, 6 versus 5 months, in arms A and B, respectively with no significant differences between the groups. Cox regression analysis showed that the use of chemotherapy after first progression (HR 0.54; 95% CI 0.35–0.86; p = 0.008), PS = 0 (HR 0.53; 95% CI 0.35–0.81; p = 0.001) and female gender (HR 0.63; 95% CI 0.41–0.98; p = 0.038) were significantly associated with a longer TFPTD; treatment arm was not significant (HR 0.88; 95% CI 0.60–1.31; p = 0.54). Toxicity was mainly limited to WHO grades 1 or 2. Chronic maintenance immunotherapy after disease progression is feasible, but does not significantly increase OS or the TFPTD.     

Production of recombinant cholesterol oxidase containing covalently bound FAD in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).     

A graph-theoretical approach in brain functional networks. Possible implications in EEG studies

Background

Recently, it was realized that the functional connectivity networks estimated from actual brain-imaging technologies (MEG, fMRI and EEG) can be analyzed by means of the graph theory, that is a mathematical representation of a network, which is essentially reduced to nodes and connections between them.

Methods

We used high-resolution EEG technology to enhance the poor spatial information of the EEG activity on the scalp and it gives a measure of the electrical activity on the cortical surface. Afterwards, we used the Directed Transfer Function (DTF) that is a multivariate spectral measure for the estimation of the directional influences between any given pair of channels in a multivariate dataset. Finally, a graph theoretical approach was used to model the brain networks as graphs. These methods were used to analyze the structure of cortical connectivity during the attempt to move a paralyzed limb in a group (N=5) of spinal cord injured patients and during the movement execution in a group (N=5) of healthy subjects.

Results

Analysis performed on the cortical networks estimated from the group of normal and SCI patients revealed that both groups present few nodes with a high out-degree value (i.e. outgoing links). This property is valid in the networks estimated for all the frequency bands investigated. In particular, cingulate motor areas (CMAs) ROIs act as “hubs” for the outflow of information in both groups, SCI and healthy. Results also suggest that spinal cord injuries affect the functional architecture of the cortical network sub-serving the volition of motor acts mainly in its local feature property.In particular, a higher local efficiency E _l can be observed in the SCI patients for three frequency bands, theta (3-6 Hz), alpha (7-12 Hz) and beta (13-29 Hz).By taking into account all the possible pathways between different ROI couples, we were able to separate clearly the network properties of the SCI group from the CTRL group. In particular, we report a sort of compensatory mechanism in the SCI patients for the Theta (3-6 Hz) frequency band, indicating a higher level of “activation” Ω within the cortical network during the motor task. The activation index is directly related to diffusion, a type of dynamics that underlies several biological systems including possible spreading of neuronal activation across several cortical regions.

Conclusions

The present study aims at demonstrating the possible applications of graph theoretical approaches in the analyses of brain functional connectivity from EEG signals. In particular, the methodological aspects of the i) cortical activity from scalp EEG signals, ii) functional connectivity estimations iii) graph theoretical indexes are emphasized in the present paper to show their impact in a real application.

     

Exploring the cupin-type metal-coordinating signature of acetylacetone dioxygenase Dke1 with site-directed mutagenesis: Catalytic reaction profile and Fe binding stability of Glu-69 → Gln mutant

Glu-69 belongs to a proposed active-site consensus motif His⁶²-X-His⁶⁴-X₄-Glu⁶⁹ (where X is any amino acid) that acetylacetone dioxygenase Dke1 from Acinetobacter johnsonii shares with structurally related non-heme metal enzymes of the cupin protein superfamily. We report functional consequences of the site-directed replacement Glu-69 → Gln based on a detailed biochemical and kinetic characterization of the purified Dke1 mutant. Perturbations of the free energy profile of the wild-type caused by the mutation were surprisingly small, with key points of the reaction pathway such as β-diketone substrate binding, the rate-limiting reduction of dioxygen, and CC bond cleavage essentially left unaltered. Release of Fe²⁺ from the mutant active site occurred at twice the wild-type rate, and the thermal stability of β-sheet secondary structure in Fe²⁺-depleted apo-proteins was lower in the mutant. The substitution Glu-69 → Gln is thus remarkably silent regarding Dke1 function. These results do not support a unified catalytic or metal-coordinating role of Glu-69 (and its positional homologues) in O₂-dependent cupin-fold enzymes.     

Distill: a suite of web servers for the prediction of one-, two- and three-dimensional structural features of proteins

Background  

We describe Distill, a suite of servers for the prediction of protein structural features: secondary structure; relative solvent accessibility; contact density; backbone structural motifs; residue contact maps at 6, 8 and 12 Angstrom; coarse protein topology. The servers are based on large-scale ensembles of recursive neural networks and trained on large, up-to-date, non-redundant subsets of the Protein Data Bank. Together with structural feature predictions, Distill includes a server for prediction of C _α traces for short proteins (up to 200 amino acids).     

Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

Background  

The field of proteomics involves the characterization of the peptides and proteins expressed in a cell under specific conditions. Proteomics has made rapid advances in recent years following the sequencing of the genomes of an increasing number of organisms. A prominent technology for high throughput proteomics analysis is the use of liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS). Meaningful biological conclusions can best be made when the peptide identities returned by this technique are accompanied by measures of accuracy and confidence.     

Effects of nutrient availability and Ochromonas sp. predation on size and composition of a simplified aquatic bacterial community

Predation and competition are two main factors that determine the size and composition of aquatic bacterial populations. Using a simplified bacterial community, composed of three strains characterized by different responses to predation, a short-term laboratory experiment was performed to evaluate adaptations and relative success in communities with experimentally controlled levels of predation and nutrient availability. A strain with a short generation time (Pseudomonas putida), one with high plasticity in cell morphology (Flectobacillus sp. GC5), and one that develops microcolonies (Pseudomonas sp. CM10), were selected. The voracious flagellate Ochromonas sp. was chosen as a predator. To describe adaptations against grazing and starvation, abundance, biomass and relative heterogeneity of bacteria were measured. On the whole, the strains in the predation-free cultures exhibited unicellular growth, and P. putida represented the largest group. The presence of Ochromonas strongly reduced bacterial abundance, but not always the total biomass. The activity of grazers changed the morphological composition of the bacterial communities. Under grazing pressure the relative composition of the community depended on the substrate availability. In the presence of predators, P. putida abundance declined in both high and low nutrient treatments, and Pseudomonas CM10 developed colonies. Flectobacillus was only numerically codominant in the nutrient-rich environments.     

Effect of the C-terminus of murine S100A9 protein on experimental nociception

Calcium-binding protein S100A9 induces antinociception in mice evaluated by the writhing test. Similarly, a peptide identical to the C-terminus of murine S100A9 (mS100A9p) inhibits the hyperalgesia induced by jararhagin, a metalloprotease. Thus, we investigated the effect of mS100A9p on different models used to evaluate nociception. mS100A9p induced a dose-dependent inhibitory effect on the writhing test, and on mechanical hyperalgesia induced by carrageenan. mS100A9p inhibited thermal hyperalgesia induced by carrageenan. mS100A9p did not modify the nociceptive response in hot plate or tail-flick tests. These data demonstrate that the C-terminus of S100A9 protein interferes with control mechanisms of inflammatory pain.     

Expression in Escherichia coli and in vitro refolding of the human protein pLG72

Recently, genes coding for pLG72 and d-amino acid oxidase have been related to schizophrenia, a widespread psychiatric disorder that affects about 1% of population. pLG72 is a puzzling, novel protein present only in primates and proposed to be an activator of d-amino acid oxidase. Here we report on the overexpression of wild-type and His-tagged pLG72 in Escherichia coli. Both variants form inclusion bodies and have been refolded and purified to homogeneity: the acquisition of secondary and tertiary structure was demonstrated by CD spectroscopy. A figure of approximately 70 mg of pure protein per liter of fermentation broth was achieved.