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PubMed2006

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis - electrospray - time-of-flight mass spectrometry.

Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N-glycans has been developed. The TOF MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH(2) are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O-glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms.

Acetylation↗

PubMed2002

Molecular characterization of FRAXB and comparative common fragile site instability in cancer cells.

The common fragile site, FRA3B, has been shown to be a site of frequent homozygous deletions in some cancers, resulting in loss of expression of the associated FHIT gene. It has been proposed that FHIT is a tumor suppressor gene that is inactivated as a result of the instability of FRA3B in tumorigenesis. More recently, deletions at other common fragile sites, FRA7G and FRA16D, have been identified in a small number of cancer cell lines. Here, we have mapped and molecularly characterized the frequently observed common fragile site FRAXB, located at Xp22.3. Like other common fragile sites, it spans a large genomic region of approximately 500 kb. Three known genes, including the microsomal steroid sulfatase locus (STS), map within the fragile site region. We examined FRAXB and four other fragile sites (FRA3B, FRA7G, FRA7H, FRA16D), and several associated genes, for deletions and aberrant transcripts in a panel of cancer cell lines and primary tumors. Deletions within FRAXB were seen in 4/27 (14.8%) of the primary tumors and cell lines examined. Three of the 21 (14.3%) cell lines examined were characterized by loss of expression of one or more FRAXB-associated genes. Moreover, all of the fragile sites examined were characterized by genomic deletions within the fragile site regions in one or more tumors or cell lines, including FRAXB, which is not associated with any known tumor suppressor genes or activity. Our results further support the hypothesis that common fragile sites and their associated genes are, in general, unstable in some cancer cells.

Adenocarcinoma↗

PubMed2004

Phenotype-genotype characterization of 10 families with severe a subunit factor XIII deficiency.

Factor XIII (FXIII) deficiency is a very rare severe autosomal bleeding disorder with a frequency of 1:2,000,000 in the general population and only a few patients have been genetically characterized so far. We report a phenotype-genotype characterization of 10 unrelated Iranian patients. Two FXIII (transglutaminase) activity assays showed no FXIII activity, except a conserved residual activity in patients receiving prophylactic substitution treatment. FXIII antigen concentrations measured by two immunoassays were comparable. Genotype characterization identified four novel mutations (2 missense and 2 small deletions) and two previously reported missense mutations in the FXIII A subunit gene (F13A). Molecular modeling was carried out to reveal the structural consequences of the missense mutations, that caused the replacement of an arginine residue involved in the formation of structurally important extensive hydrogen-bonded network. The replacements [c.320G>A (p.Arg77His) in the beta-sandwich, c.868C>T (p.Arg260Cys), c.869G>A (p.Arg260His) and c.1236G>T (p.Arg382Ser) in the core domain] resulted in the loss or impairment of such H-bonded network. Energy decomposition analysis demonstrated that this situation leads to the instability and perhaps to the incorrect folding of the A subunit, that would explain the development of severe FXIII deficiency.

Adult↗

PubMed2004

Characterization of seven novel mutations in seven patients with GAMT deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive error of creatine synthesis characterized by cerebral creatine deficiency, accumulation of guanidinoacetate, mental retardation, epilepsy and extrapyramidal signs. So far, six mutations have been identified in seven patients. We investigated seven new patients by screening the promoter, 3'UTR, and six exons and exon/intron boundaries using direct sequencing and denaturing gradient gel electrophoresis. The clinical and biochemical phenotype was characterized by scoring the degree of main clinical manifestations and by determination of urinary guanidinoacetate concentrations and of GAMT activity in fibroblasts / lymphoblasts, respectively. We identified 7 novel mutations, including c.64dupG (exon 1; 4/14 alleles); c.59G>C (exon 1; 3/14 alleles); c.491delG (exon 5; 2/14 alleles); c.160G>C (exon 1; 2/14 alleles); and c.152A>C (exon 1; 1/14 alleles); c.526dupG (exon 5; 1/14 alleles); c.521G>A (exon 5; 1/14 alleles), and two polymorphisms c.626C>T (exon 6) and c.459+71G>A (intron 4). Frameshift and missense mutations in exon 1 were prevalent in the 4 patients with the severe phenotype, however a clear genotype-phenotype correlation has not been established in the limited number of patients characterized so far.

Adolescent↗

PubMed2005

Genetic variant characterization in intron 4 of the surfactant protein B gene.

Genetic variants in intron 4 of the surfactant protein B gene SFTPB have been associated with pulmonary morbidity in newborn infants and adults. Genetic variant discovery in intron 4 requires high fidelity polymerase amplification due to a variable number of intermotif dinucleotide repeats and reliable characterization of alleles genetically distinct due to insertion, deletion, or both of 11 conserved sequence motifs. To optimize genetic variant discovery, we selected a high fidelity polymerase enzyme by replicate amplification and compared automated sequencing with agarose gel electrophoresis for all variant alleles (N=68) in a cohort of Missouri infants with (N=187) and without (N=53) respiratory distress. We identified and characterized six new alleles based on sequence motifs and two pairs of variant alleles with similar mobilities by agarose gel electrophoresis but distinct motif sequences. We confirmed uniformity of invariant alleles by sequencing (N=77). Logistic regression using race and intron 4 genotype for infants > or = 35 weeks gestation suggested that the invariant allele was independently associated with increased risk of respiratory distress (OR for the invariant allele 2.7, 95% CI 1.0-7.2). Reliable characterization of genetic variants in intron 4 requires both a high fidelity polymerase and sequencing of variant alleles.

Base Sequence↗

PubMed1991

Oncofetal mucin M1 epitope family: characterization and expression during colonic carcinogenesis.

The gastric mucin M1 antigens, markers associated with colonic carcinogenesis, have been characterized by new antimucin monoclonal antibodies (MAbs). These MAbs, obtained against mucins isolated from a human ovarian mucinous cyst (MAbs 19M1, 21M1 and 45M1) and from a pancreatic adenocarcinoma (MAb 96RA), were compared with 5 other anti-M1 mucin MAbs described previously, which characterized the a, b, c, d and e mucin M1 epitopes. Using immunoperoxidase, these new MAbs exclusively stained the surface gastric epithelium of normal human gastro-intestinal tract and reacted with fetal, precancerous and cancerous colonic mucosa, but not with normal colon. Immunoradiofixation studies showed that these new MAbs are directed against 3 epitopes (f, g and h) which are different from the a, b, c, d and e mucin M1 epitopes, though present on the same a immunoreactive high-molecular-weight components (greater than 1,000 kDa) with a density of 1.4 by CsCl-density-gradient ultracentrifugation. M1 antigenicity is characterized by a family of 8 different M1 epitopes which were destroyed with beta-mercaptoethanol (except for the f epitope), sensitive to a 5 hr trypsin treatment and resistant to 5 mM periodate (except for the h epitope). Some epitopes (b, c and d) showed increasing immunoreactivity after 20 mM periodate treatment, suggesting cryptic location. In rat-colon adenocarcinomas, M1 mucin epitopes were masked but could be decrypted using high periodate treatment, similar to normal rat gastric mucosa, thus suggesting the absence of drastic changes in the saccharide coat of the peptide mucin portion bearing M1 epitopes. Cryptic location, periodate resistance, sensitivity to protease and conformational behavior strongly suggest that the peptidic core of gastric (or fetal colonic) mucin plays a role in M1 immunoreactivity. Indeed, the resurgence of M1 antigens during colonic carcinogenesis is due to re-expression of the peptide core of gastric (or fetal colonic) mucins.

Animals↗

PubMed1993

Biochemical and immunological characterization of the human carcinoma-associated antigen MH 99/KS 1/4.

We have characterized the 38-kDa transformation-associated membrane glycoprotein MH 99, whose expression is highly elevated in many epithelial malignancies. A spontaneous cleavage of MH 99 into a 32- and a 6-kDa chain in some carcinoma cell lines was recently shown. Sequence homologies to nidogen, a matrix-adhesion molecule, support the suggestion of a receptor-like function. In this study, we characterized biochemical and immunogenic aspects of MH 99. Transformed epithelial cell lines which do not spontaneously cleave MH 99 were exposed to 8 proteases with distinct specificities. Each of the enzymes produced similar specific fragmentation into chains of about 30 to 32 and 6 kDa, indicating a characteristic cleavage site of MH 99. The fragments were not distinguishable from those in carcinoma cells showing spontaneous cleavage of MH 99. The specific fragmentation depends on the localization in intact membranes and is not shared by other membrane proteins. N-glycosylation of MH 99 of about 4 kDa was exclusively found on the 32-kDa fragment. Characterization of antigenic epitopes was performed using 16 different monoclonal antibodies (MAbs). Only 2 independent determinants were found. One is located on the 32-kDa chain and is recognized only by the MM 104 MAb. The other 15 antibodies bind to a dominant epitope on the 6-kDa fragment which can be divided into 3 overlapping sub-epitopes. The unique features of MH 99 indicate that its immunogenic epitopes are mainly located at its 6-kDa chain, and support the suggestion of a transformation-associated cell-surface receptor which might be proteolytically regulated.

Animals↗

PubMed1987

Characterization of the tissue proliferated at the blood interface of carbon/ceramic composites.

The present study focuses on cell adhesion/differentiation and material stability of surfaces of the three carbon/ceramic composites implanted in intra-atrial position in dogs for 1 year. Before implantation their surface was characterized by scanning electron microscopy. After harvesting, the tissue proliferated on the blood interface was examined by histology, scanning, and transmission electron microscopy, wavelength dispersive and x-ray spectrometry, electrophoretic and enzymatic characterization of glycosaminoglycans (GAGs) which were compared to endocardiac tissue as control samples. One year after implantation, the pattern of GAGs in the newly developed tissue was characterized by: 1) a constant increase of the total GAGs present on all carbon composites, 2) a significant increase of dermatan sulfate (p less than 0.05), 3) a significant increase of chondroitin sulfate (p less than 0.05), 4) a significant decrease of heparan sulfate in Group 1, whereas this GAG fraction was increased in Groups 2 and 3. Cellular surface differentiation towards endothelial-like cells occurred in places particularly in groups 1 and 3, whereas only fibrous tissue was found covering the implants in Group 2. Fibroblastic cells with dense intracellular deposits, which produced emission of Si, Ca, and C energy as well as extracellular lipidic containing inclusions were observed. The macromolecular modifications were associated with 1) the absence of endothelial lining, 2) the migration of carbon and silicon particles, and 3) the occurrence of calcifications and lipidic inclusions. These results suggest that the relative smoothness of these materials could be responsible for the development of a tissue that did not adhere to the biomaterial, indicating that cell adhesion and functional differentiation are in intimal relationship with the physical-chemical structure of the material surface.

Animals↗

PubMed2005

Surface characterizations and corrosion resistance of nickel-titanium orthodontic archwires in artificial saliva of various degrees of acidity.

This study investigated the surface characterizations and corrosion resistance of as-received commercial nickel-titanium (NiTi) dental orthodontic archwires from different manufacturers using a cyclic potentiodynamic test in artificial saliva with various acidities. An atomic force microscope was used to evaluate the surface topography of the NiTi wires. The surface chemical analysis of the passive film on the NiTi wires was characterized using X-ray photoelectron spectroscopy and Auger electron spectroscopy. A scanning electron microscope, together with an energy-dispersive spectrometer, was used to analyze the surface characterizations of the NiTi wires before and after the corrosion tests. Two-way analysis of variance was used to analyze the corrosion-resistance parameters with the factors of wire manufacturer and solution pH. The results showed that the surface structure of the passive film on the tested NiTi wires were identical, containing mainly TiO2, with small amounts of NiO. A different surface topography was observed on the NiTi wires from various manufacturers. The corrosion tests showed that both the wire manufacturer and solution pH had a statistically significant influence on the corrosion potential, corrosion rate, passive current, breakdown potential, and crevice-corrosion susceptibility. The difference in the corrosion resistance among these NiTi dental orthodontic archwires did not correspond with the surface roughness and pre-existing defects.

Corrosion↗

PubMed2005

Injectable gels of anionic collagen:rhamsan composites for plastic correction: preparation, characterization, and rheological properties.

The present article describes the preparation and characterization of anionic collagen gels obtained from porcine intestinal submucosa after 72 h of alkaline treatment and in the form of rhamsan composites to develop injectable biomaterials for plastic reconstruction. All materials were characterized by SDS/polyacrylamide gel electrophoresis, infrared spectroscopy, thermal stability, potentiometric titration, rheological properties, and fluidity tests. Biocompatibility was appraised after the injection of anionic collagen: rhamsan composites at 2.5% in 60 North Folk rabbits. Independently of processing, the collagen's secondary structure was preserved in all cases, and after 72 h of hydrolysis the collagen was characterized by a carboxyl group content of 346+/-9, which, at physiological pH, corresponds to an increase of 106+/-17 negative charges, in comparison to native collagen, due to the selective hydrolysis of asparagine and glutamine carboxyamide side chain. Rheological studies of composites at pH 7.4 in concentrations of 2, 4, and 6% (in proportions of 75:1 and 50:1) showed a viscoelastic behavior dependent on the frequency, which is independent of concentration and proportion. In both, the concentration of the storage modulus always predominated over the loss modulus (G'>G'' and delta<45 degrees ). The results from creep experiments confirmed this behavior and showed that anionic collagen:rhamsan composites at pH 7.4 in the proportion of 50:1 are less elastic and more susceptible to deformation in comparison to gels in the proportion of 75:1, independent of concentration. This was further confirmed by flow experiments, indicating that the necessary force for the extrusion of anionic collagen:rhamsan composites, in comparison to anionic collagen, was significantly smaller and with a smooth flow. Biocompatibility studies showed that the tissue reaction of anionic collagen:rhamsan composites at 2.5% in the proportion of 75:1 was compatible with the application of these gels in plastic reconstruction. These results suggest that the association of collagen with rhamsan may be a good alternative in the replacement of glutaraldehyde to stabilize the microfibril assembly of commercial collagen gel preparations.

Administration, Intravesical↗

PubMed2007

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse.

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.

ATP Binding Cassette Transporter 1↗

PubMed2004

Biophysical and pharmacological characterization of spermatogenic T-type calcium current in mice lacking the CaV3.1 (alpha1G) calcium channel: CaV3.2 (alpha1H) is the main functional calcium channel in wild-type spermatogenic cells.

Mammalian acrosome reaction (AR) requires successive activation of three different types of calcium channels (T-type channels, Inositol-3-phosphate (InsP3) receptors, and TRPC2 channels). All the calcium signaling is under the control of the activation of the first-one, a T-type calcium channel. The molecular characterization of the T-type calcium channel is still a matter of debate, previous reports showing the presence of transcripts for Ca(V)3.1 and Ca(V)3.2 subunits. Using mice deficient for Ca(V)3.1 subunit, we show that the T-type current density in spermatogenic cells is not reduced in deficient mice versus control mice. We characterized the biophysical and pharmacological properties of T-type current in spermatogenic cells from Ca(V)3.1 deficient mice. Biophysical and pharmacological properties of spermatogenic T-type current from wild-type and Ca(V)3.1 deficient mice demonstrate that Ca(V)3.3 does not contribute to T-type current. Moreover, nickel and amiloride inhibit T-type currents in deficient and wild-type mice with similar potencies. These results demonstrate that T-type currents in spermatogenic cells is due to Ca(V)3.2 subunit and that Ca(V)3.1 contributes to a very negligible extent to the T-type currents. Thus, the deficient Ca(V)3.1 mouse model allows the characterization of native Ca(V)3.2 currents in spermatogenic cells. Spermatogenic Ca(V)3.2 currents present specific feature in comparison to the cloned Ca(V)3.2 current so far. More particularly, the time-dependence of recovery from short-term inactivation of native spermatogenic Ca(V)3.2 is close to 100 millisecond, a value expected for Ca(V)3.1 current.

Amiloride↗

PubMed1995

Ultrastructural characterization of neurons recorded intracellularly in vivo and injected with lucifer yellow: advantages of immunogold-silver vs. immunoperoxidase labeling.

Immunoperoxidase labeling of lucifer yellow provides a sensitive method for morphological characterization of neurons recorded intracellularly in vitro or in vivo. However, the reaction product is often so dense that it obscures ultrastructural details necessary for the analysis of synaptic contacts onto individually filled neurons. In the present study, we describe a silver intensification procedure using 1 nm gold labeling of lucifer yellow as an optimal means for immunocytochemically identifying single physiologically characterized neurons at the ultrastructural level. Single neurons in the frontal cortex of anesthetized rats were impaled in vivo and filled with lucifer yellow. The brains were then perfused with an acrolein fixative. Single vibratome sections through the recording site were reacted with a rabbit antibody directed against lucifer yellow followed by goat anti-rabbit 1 nm gold-labeled IgG and silver intensified. For comparison, additional sections were processed for immunoperoxidase detection of lucifer yellow. Labeled sections were processed for light microscopy or embedded in plastic for electron microscopy. The immunogold-silver label as well as peroxidase reaction product of lucifer yellow was readily detected in cell bodies, proximal and distal dendrites, and spines. However, in contrast to immunoperoxidase, the immunogold-silver reaction did not obscure subcellular organelles. Most importantly, the synaptic junctions formed by afferents to the filled neuron were more easily identifiable following the immunogold-silver procedure. This clear visualization of postsynaptic densities is essential for examining synaptic circuitry between afferents and physiologically characterized neurons.

Animals↗

PubMed2004

Application of scanning probe microscopy to the characterization and fabrication of hybrid nanomaterials.

Scanning probe microscopy (SPM) is a widely used experimental technique for characterizing and fabricating nanostructures on surfaces. In particular, due to its ability to spatially map variations in materials properties with nanometer spatial resolution, SPM is particularly well suited to probe the subcomponents and interfaces of hybrid nanomaterials, i.e., materials that are made up of distinct nanometer scale components with distinguishable properties. In addition, the interaction of the SPM tip with materials can be intentionally tuned such that local surface modification is achieved. In this manner, hybrid nanostructures can also be fabricated on solid substrates using SPM. This report reviews recent developments in the characterization and fabrication of hybrid nanomaterials with SPM. Specific attention is given to nanomaterials that consist of both organic and inorganic components including individual biomolecules mounted on inorganic substrates. SPM techniques that are particularly well suited for characterizing the mechanical and electrical properties of such hybrid systems in atmospheric pressure environments are highlighted, and specific illustrative examples are provided. This review concludes with a brief discussion of the remaining challenges and promising future prospects for this field.

Animals↗

PubMed2006

Atomic force microscopy characterization of Xenopus laevis oocyte plasma membrane.

We used atomic force microscopy (AFM) to characterize the plasma membrane of Xenopus laevis oocytes. The samples were prepared according to novel protocols, which allowed the investigation of the extra- and intracellular sides of the membrane, both of which showed sparsely distributed spherical-like protrusions. Regions with comparably sized and densely packed structures arranged in an orderly manner were visualized and dimensionally characterized. In particular, two different arrangements, hexagonal and square packing, were recognizable in ordered regions. The lateral dimension of structures visualized on the external side had a normal distribution centered on 25.5 +/- 0.3 nm (mean value +/- SE), whereas that on the intracellular side showed a normal distribution centered on 30.2 +/- 0.8 nm. The height of the protrusions was 2-5 nm on the external side and 1-3 nm on the intracellular side. The mean number of structures on the external and intracellular sides of the plasma membrane was about 1000 microm(-2) and 850 microm(-2) respectively. Trypsin treatment greatly decreased the size of the membrane protrusions, thus confirming the proteic nature of the structures. These results show that AFM is a useful tool for structural characterization of proteins in a native eukaryotic membrane.

Animals↗

PubMedDate not supplied

Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography.

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

MRI tissue characterization of experimental cerebral ischemia in rat.

PURPOSE: To extend the ISODATA image segmentation method to characterize tissue damage in stroke, by generating an MRI score for each tissue that corresponds to its histological damage. MATERIALS AND METHODS: After preprocessing and segmentation (using ISODATA clustering), the proposed method scores tissue regions between 1 and 100. Score 1 is assigned to normal brain matter (white or gray matter), and score 100 to cerebrospinal fluid (CSF). Lesion zones are assigned a score based on their relative levels of similarities to normal brain matter and CSF. To evaluate the method, 15 rats were imaged by a 7T MRI system at one of three time points (acute, subacute, chronic) after MCA occlusion. Then they were killed and their brains were sliced and prepared for histological studies. MRI of two or three slices of each rat brain (using two DWI (b = 400, b = 800), one PDWI, one T2WI, and one T1WI) was performed, and an MRI score between 1 and 100 was determined for each region. Segmented regions were mapped onto the histology images and scored on a scale of 1-10 by an experienced pathologist. The MRI scores were validated by comparison with histology scores. To this end, correlation coefficients between the two scores (MRI and histology) were determined. RESULTS: Experimental results showed excellent correlations between MRI and histology scores at different time points. Depending on the reference tissue (gray matter or white matter) used in the standardization, the correlation coefficients ranged from 0.73 (P < 0.0001) to 0.78 (P < 0.0001) using the entire dataset, including acute, subacute, and chronic time points. This suggests that the proposed multiparametric approach accurately identified and characterized ischemic tissue in a rat model of cerebral ischemia at different stages of stroke evolution. CONCLUSION: The proposed approach scores tissue regions and characterizes them using unsupervised clustering and multiparametric image analysis techniques. The method can be used for a variety of applications in the field of computer-aided diagnosis and treatment, including evaluation of response to treatment. For example, volume changes for different zones of the lesion over time (e.g., tissue recovery) can be evaluated.

Animals↗

PubMedDate not supplied

MRI in characterization of focal liver lesions: comparison of T2 weighting by conventional spin-echo and turbo spin-echo sequences.

Forty-one patients with 61 proved focal liver lesions underwent MRI of the liver at 1.0 T, with the aim of evaluating the usefulness of turbo spin-echo (TSE) sequences in characterizing focal liver lesions, by comparing them with conventional spin-echo (CSE) sequences. Two different TSE protocols were employed. with constant echo time and varying repetition time: TSE-S (3000 msec) and TSE-L (5100 msec). All images were evaluated quantitatively (signal-to-noise ration 'SNR') and qualitatively: because benign lesions were all liquid (12 cysts and 10 hemangiomas), they were well characterized morphologically on the basis of signal intensity. Mean SNR was significantly different between metastases and benign lesions (P < .0001) with all T2 sequences. Among the single T2 sequences tested, logistic regression analysis showed TSE-L to have the best predictive ability of the nature of focal lesions, with a G value of 42.02, compared to 29.87 of TSE-S and 25.55 of CSE second echo (SE II). The combination of TSE-L with TSE-S did not modify these results, whereas the combination of TSE-L with CSE only resulted in slight improvement (G = 46.95). Comparison of the receiver operating characteristic (ROC) curves showed only SE II (area under the ROC curve of .8312) to be significantly inferior to the best single sequence, or TSE-L (area under the ROC curve of .9176; P = .027). All sequences were equivalent in qualitative evaluation with good reproducibility, sensitivity ranging from .94 to 1.0 and specificity ranging from .86 to .93. This study confirms the value of TSE sequences in characterization of focal liver lesions. Time of acquisition is strongly reduced with these sequences, whereas results are fairly similar to those obtained with CSE. TSE sequences could therefore replace CSE for the study of focal liver lesions.

Adult↗