Identification of leptomeningeal metastasis-related proteins in cerebrospinal fluid of patients with breast cancer by a combination of MALDI-TOF, MALDI-FTICR and nanoLC-FTICR MS

Leptomeningeal metastasis (LM) is a devastating complication occurring in 5% of breast cancer patients. However, the current 'gold standard' of diagnosis, namely microscopic examination of the cerebrospinal fluid (CSF), is false-negative in 25% of patients at the first lumbar puncture. In a previous study, we analyzed a set of 151 CSF samples (tryptic digests) by MALDI-TOF and detected peptide masses that were differentially expressed in breast cancer patients with LM. In the present study, we obtain for a limited number of samples exact masses for these peptides by MALDI-FTICR MS measurements. Identification of these peptides was performed by electrospray FTICR MS after separation by nano-scale LC. The database results were confirmed by targeted high mass accuracy measurements of the fragment ions in the FTICR cell. The combination of automated high-throughput MALDI-TOF measurements and analysis by FTICR MS leads to the identification of 17 peptides corresponding to 9 proteins. These include proteins that are operative in host-disease interaction, inflammation and immune defense (serotransferrin, alpha 1-antichymotrypsin, hemopexin, haptoglobin and transthyretin). Several of these proteins have been mentioned in the literature in relation to cancer. The identified proteins alpha1-antichymotrypsin and apolipoprotein E have been described in relation to Alzheimer's disease and brain cancer.     

Small bowel image classification using cross-co-occurrence matrices on wavelet domain

This paper presents a novel system to compute the automated classification of wireless capsule endoscope images. Classification is achieved by a classical statistical approach, but novel features are extracted from the wavelet domain and they contain both color and texture information. First, a shift-invariant discrete wavelet transform (SIDWT) is computed to ensure that the multiresolution feature extraction scheme is robust to shifts. The SIDWT expands the signal (in a shift-invariant way) over the basis functions which maximize information. Then cross-co-occurrence matrices of wavelet subbands are calculated and used to extract both texture and color information. Canonical discriminant analysis is utilized to reduce the feature space and then a simple 1D classifier with the leave one out method is used to automatically classify normal and abnormal small bowel images. A classification rate of 94.7% is achieved with a database of 75 images (41 normal and 34 abnormal cases). The high success rate could be attributed to the robust feature set which combines multiresolutional color and texture features, with shift, scale and semi-rotational invariance. This result is very promising and the method could be used in a computer-aided diagnosis system or a content-based image retrieval scheme.     

The p76 and p100 truncated forms of the Rb protein exert antagonistic roles on cell death regulation in human cell lines

Several caspase-cleaved forms of the retinoblastoma protein have been described. Here, we compared the effect of full-length Rb versus the truncated p76^Rb and p100^Rb proteins on cell death regulation in five human cell lines. Interestingly, we observed that p76^Rb triggers cell death in all tested cell lines and that p100^Rb protects two cell lines against etoposide or TNF-α-induced cell death, whereas full-length Rb has no apoptotic effect. These results show that truncated forms of Rb can have specific activities in the regulation of cell death. They also suggest that caspase cleavage of Rb should not be simply assimilated to a degradation process. Finally, we show that cell death induced by p76^Rb is Bax-dependent and is diminished by Bcl-2 overexpression or by caspase inhibition and that p100^Rb could inhibit cell death by decreasing both p53 stability and caspase activity.     

Enhance Stability and in vitro Cell Response to a Bioinspired Coating on Zr Alloy with Increasing Chitosan Content

The aim of the present paper is to characterize bioinspired chitosan (CS) + hydroxyapatite (HA) coatings with various components ratio on a zirconium alloy with titanium.The coatings were characterized by FT-IR,SEM,hydrophilic/hydrophobic balance,adherence,roughness,electrochemical stability and in vitro cell response.Electrochemical tests,including potentiodynamic polarization curves and electrochemical impedance spectroscopy,were performed in normal saline physiological solution.Cell viability of MC3T3-E1 osteoblasts,lactate dehydrogenase,nitric oxide,and Reactive Oxygen Species (ROS) levels,as well as actin cytoskeleton morphology,were evaluated as biological in vitro tests.The results on in vitro cell response indicated good cell membrane integrity and viability for all samples,but an increased cell number,a decreased ROS level and a better cytoskeleton organization were noticed for the sample with a higher CS content.The coating with highest CS concentration indicated the best performance based on the experimental data.The highest hydrophilic character,highest resistance to corrosion and best biocompatibility as well recommend this coating for bioapplications in tissue engineering.     

GroE-dependent expression and purification of pig heart mitochondrial citrate synthase in Escherichia coli

Citrate synthase (CS) is a dimeric, mitochondrial protein, composed of two identical subunits (M(r) 48969 each). The nuclear-encoded alpha-helical protein is imported into mitochondria post-translationally where it catalyses the first step of the citric cycle. Furthermore, the pathway of thermal unfolding as well as the folding pathway was studied extensively, making CS a well-suited substrate protein for studying chaperone function. In chaperone research the quality of the substrate proteins is essential to guaranty the reproducibility of the results. In this context, we here describe the GroE-enhanced recombinant expression and purification of CS. CS was expressed in E. coli by using an arabinose regulated T7 promotor. Under standard expression conditions only insoluble, inactive CS was detected. Interestingly, the expression of soluble and active CS was possible when GroEL/GroES was co-expressed. Furthermore, a shift to lower expression temperatures increased the amount of soluble, active CS. We describe for the first time, the purification of CS in soluble and active form by following a CiPP strategy (capture, intermediate purification, polishing). After the initial capturing step on DEAE-Sephacel the protein was further purified on a Q-Sepharose column. After these two steps of anion-exchange chromatography a final size-exclusion chromatography step on a Superdex 75-pg column yields CS with a purity over 99%. Using this expression and purification strategy 1 mg CS per g E. coli wet weight were purified.     

Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche

At present, there is little information on the phylogenetic diversity of microbial species that inhabit the gastrointestinal tracts of wildlife. To increase understanding in this area, we initiated a characterization of the bacterial diversity in the digestive tracts of three wild African ruminant species namely eland (Taurotragus oryx), Thompson's gazelle (Gazella rufifrons) and Grant's gazelle (Gazella granti), together with a domesticated ruminant species, zebu cattle (Bos indicus), and a non-ruminant species, zebra (Equus quagga). Bacterial diversity was analysed by PCR amplification, sequencing and phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences. A total of 252 full-length 16S rDNA sequences averaging 1,500 base pairs (bp) in length, and an additional 27 partial sequences were obtained and subject to phylogenetic analysis. Using a 98% criterion for similarity, all except for one of the sequences were derived from distinct phylotypes. At least 24 distinct operational taxonomic units (OTU's) could be identified, with the majority of these sequences representing hitherto uncharacterized species and genera. The sequences were generally affiliated with four major bacterial phyla, the majority being members of the Firmicutes (low G+C Gram-positives) related to the genera Clostridium and Ruminococcus. By contrast, with earlier studies using 16S rDNA sequences to assess biodiversity in Bos taurus dairy cattle, Gram-negative bacteria in the Bacteroidales (Prevotella-Bacteroides group) were poorly represented. The lack of redundancy in the 16S rDNA dataset from the five African ungulate species, and the presence of novel sequences not previously described from the gastrointestinal tract of any animal species, highlights the level of diversity that exists in these ecosystems and raises the question as to the functional role of these species in the gastrointestinal tract.     

Solution structure and stability of the full-length excisionase from bacteriophage HK022.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.