Culm Brittleness of Barley (Hordeum vulgare L.) Mutants Is Caused by Smaller Number of Cellulose Molecules in Cell Wall

The physicochemical nature of the cell wall was determined in the fourth internode of three isogenic brittle mutants of barley (Hordeum vulgare L.) and corresponding nonbrittle strains. Cellulose contents of the brittle culms were 17.5 to 20.3% of those of corresponding nonbrittle strains. No major difference was found in lignin and noncellulose components (except glucose) between brittle and nonbrittle strains. Maximum bending stresses of brittle culms were 38.0 to 54.2% of those of corresponding nonbrittle strains. The degree of polymerization of cellulose, measured by viscometry, was similar between the brittle and the nonbrittle strains. Mole number of cellulose molecules in a unit length of brittle culms, calculated by dividing cellulose mass by molecular weight, was 7.7 to 17.3% of those of the nonbrittle strains. These results indicate that brittleness of mutant culms is due to fewer numbers of cellulose molecules in the cell walls.     

Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis

“Brittle culm” mutants found in Gramineae crops are suitable materials to study the mechanism of secondary cell wall formation. Through positional cloning, we have identified a gene responsible for the brittle culm phenotype in rice, brittle culm 3 (bc3). BC3 encodes a member of the classical dynamin protein family, a family known to function widely in membrane dynamics. The bc3 mutation resulted in reductions of 28–36% in cellulose contents in culms, leaves, and roots, while other cell wall components remained unaffected. Reductions of cell wall thickness and birefringence were observed in both fiber (sclerenchyma) and parenchymal cells, together with blurring of the wall’s layered structures. From promoter-GUS analyses, it was suggested that BC3 expression is directly correlated with active secondary cell wall synthesis. These results suggest that BC3 is tightly involved in the synthesis of cellulose and is essential for proper secondary cell wall construction.     

Antenatal Dexamethasone after Asphyxia Increases Neural Injury in Preterm Fetal Sheep

Background and Purpose

Maternal glucocorticoid treatment for threatened premature delivery dramatically improves neonatal survival and short-term morbidity; however, its effects on neurodevelopmental outcome are variable. We investigated the effect of maternal glucocorticoid exposure after acute asphyxia on injury in the preterm brain.

Methods

Chronically instrumented singleton fetal sheep at 0.7 of gestation received asphyxia induced by complete umbilical cord occlusion for 25 minutes. 15 minutes after release of occlusion, ewes received a 3 ml i.m. injection of either dexamethasone (12 mg, n = 10) or saline (n = 10). Sheep were killed after 7 days recovery; survival of neurons in the hippocampus and basal ganglia, and oligodendrocytes in periventricular white matter were assessed using an unbiased stereological approach.

Results

Maternal dexamethasone after asphyxia was associated with more severe loss of neurons in the hippocampus (CA3 regions, 290±76 vs 484±98 neurons/mm², mean±SEM, P<0.05) and basal ganglia (putamen, 538±112 vs 814±34 neurons/mm², P<0.05) compared to asphyxia-saline, and with greater loss of both total (913±77 vs 1201±75/mm², P<0.05) and immature/mature myelinating oligodendrocytes in periventricular white matter (66±8 vs 114±12/mm², P<0.05, vs sham controls 165±10/mm², P<0.001). This was associated with transient hyperglycemia (peak 3.5±0.2 vs. 1.4±0.2 mmol/L at 6 h, P<0.05) and reduced suppression of EEG power in the first 24 h after occlusion (maximum −1.5±1.2 dB vs. −5.0±1.4 dB in saline controls, P<0.01), but later onset and fewer overt seizures.

Conclusions

In preterm fetal sheep, exposure to maternal dexamethasone during recovery from asphyxia exacerbated brain damage.     

Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.     

L-Type Ca2+ Channel Sparklets Revealed by TIRF Microscopy in Mouse Urinary Bladder Smooth Muscle

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca²⁺, referred to as Ca²⁺ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca²⁺ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca²⁺ (0.035±0.01 vs. 0.23±0.07 Hz/mm²; P<0.05). Co-loading of smooth muscle strips with the slow Ca²⁺ chelator EGTA-AM (10 mM) confirmed that Ca²⁺ sparklets are restricted to the cell membrane. Ca²⁺ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm²; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm²; P<0.05). Ca²⁺ sparklets were unaffected by inhibition of P2X₁ receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca²⁺ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm²; P<0.05), demonstrating that Ca²⁺ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca²⁺ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman''s rank correlation 0.2, P>0.05), implying that Ca²⁺ store mediated signals regulate Ca²⁺ sparklets. Under control conditions, inhibition of store operated Ca²⁺ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca²⁺ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca²⁺ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca²⁺ concentrations.     

Anatomical and Physiological Changes after Paclitaxel-Coated Balloon for Atherosclerotic De Novo Coronary Lesions: Serial IVUS-VH and FFR Study

Aims

To assess the serial changes of de novo coronary lesions treated with paclitaxel-coated balloon (PCB) using intravascular ultrasound virtual histology (IVUS-VH) and fractional flow reserve (FFR).

Method and Results

This prospective observational study enrolled 27 patients with coronary artery disease treated with PCB who underwent coronary angiography, IVUS-VH and FFR before, immediately after intervention and at 9 months. 28 de novo lesions were successfully treated with PCB. Angiographic late luminal loss was 0.02 ± 0.27mm. Mean vessel and lumen areas showed increase at 9 months (12.0 ± 3.5mm² to 13.2 ± 3.9mm², p <0.001; and 5.4 ± 1.2mm² to 6.5 ± 1.8mm², p <0.001, respectively). Although mean plaque area was unchanged (6.6 ± 2.6mm² to 6.6 ± 2.4mm², p = 0.269), percent atheroma volume decreased significantly (53.4 ± 7.9% to 49.5 ± 6.4%, p = 0.002). The proportion of plaque compositions including fibrous, fibrofatty, dense calcium and necrotic core by IVUS-VH was unchanged at 9 months. The FFR of the treated lesion was 0.71 ± 0.13 pre-procedure, 0.87 ± 0.06 post-procedure and 0.84 ± 0.06 at follow-up.

Conclusions

De novo coronary lesions treated with PCB showed persistent anatomical and physiological patency with plaque redistribution and vessel remodeling without chronic elastic recoil or plaque compositional change during follow-up.     

Anterograde Degeneration along the Visual Pathway after Optic Nerve Injury

Purpose

To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy.

Methods

Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively.

Results

An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm², P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10² cells/mm², P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10² cells/mm², P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm², P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10² cells/mm², P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway.

Conclusions

Anterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.     

Characterization and gene mapping of a brittle culm mutant of diploid wheat (Triticum monococcum L.) with irregular xylem vessels development

Diploid wheat, Triticum monococcum L. (einkorn) is an ideal plant material for wheat functional genomics. Brittle culm mutant was identified by screening of the ethyl methane sulphonate-treated M ₂ progenies of a T. monococcum accession pau14087 by banding the plant parts manually. The brittle culm mutant with drooping leaves, early flowering, reduced tiller numbers and susceptible to lodging had also exhibited brittleness in all plant parts than the wild-type parents. Comprehensive mechanical strength, histological, biochemical, scanning electron microscopy, and Fourier transform infrared spectroscopy analyses of brittle culm mutant supplemented and complemented the findings that the mutant had defective cellulose biosynthesis pathway and deposition of cell wall materials on secondary cell wall of mechanical tissues. Microscopic studies demonstrated that the decrease in cellulose contents resulted in the irregular cell wall organization in xylem vessels of leaf vascular bundles. To map the brc5 mutant, mapping populations were developed by crossing the brittle culm mutant with wild Triticum boeoticum acc. pau5088, having non-brittle characters. The brittle culm mutation was mapped between SSR markers, Xcfd39 and Xgwm126 on 5A^mL chromosome of T. monococcum, with genetic distances of 2.6 and 4.8 cM, respectively. The brc5 mutant mapped on 5A^mL, being distinct from a previously mapped brittle culm mutant in wheat, has been designated as brc5. The work on fine mapping and map-based cloning of brc5 gene regulating synthesis and deposition of cellulose on the secondary cell wall is in progress.     

T Lymphocyte Density and Distribution in Human Colorectal Mucosa,and Inefficiency of Current Cell Isolation Protocols

Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3⁺ lymphocytes, including CD4⁺ and CD8⁺ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r²=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3⁺ T lymphocytes with 37,400 ± 2,801 cells/mm³ and 33,700 ± 4,324 cell/mm³, respectively. Sigmoid mucosa contained 57% CD3⁺CD4⁺ and 40% CD3⁺CD8⁺ T lymphocytes which calculates to 21,300 ± 1,476/mm³ and 15,000 ± 275/mm³ T lymphocytes, respectively. Rectal mucosa had 57% CD3⁺CD4⁺ and 42% CD3⁺CD8⁺ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm³, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm³ and 2,708 ± 245/mm³ CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3⁺, CD4⁺ and CD8⁺ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.     

Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml⁻¹, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.     

Stereoscopic Analysis of Optic Nerve Head Parameters in Primary Open Angle Glaucoma: The Glaucoma Stereo Analysis Study

PurposeThe Glaucoma Stereo Analysis Study (GSAS), a cross sectional multicenter collaborative study, used a stereo fundus camera to assess various morphological parameters of the optic nerve head (ONH) in glaucoma patients and investigated the relationships between these parameters and patient characteristics.ResultsPatient characteristics included refractive error of −3.38±3.75 diopters, intraocular pressure (IOP) of 13.6±2.6 mmHg, and visual field mean deviation (MD) of −4.71±3.26 dB. Representative ONH parameters included a horizontal disc width of 1.66±0.28 mm, vertical disc width of 1.86±0.23 mm, disc area of 2.42±0.63 mm², cup area of 1.45±0.57 mm², and cup volume of 0.31±0.22 mm³. Correlation analysis revealed significant negative associations between vertical cup-to-disc ratio (0.82±0.08) and MD (r = −0.40, P<0.01) and between disc tilt angle (10.5±12.5 degrees) and refractive error (r = −0.36, P<0.01). Seventy-five percent of the eyes had a positive value for rim decentering (0.30±0.42), indicating that rim thinning manifested more often as an inferior lesion than a superior lesion.ConclusionWe used stereoscopic analysis to establish a database of ONH parameters, which may facilitate future studies of glaucomatous changes in ONH morphology.     

Decreases in Molecular Diffusion,Perfusion Fraction and Perfusion-Related Diffusion in Fibrotic Livers: A Prospective Clinical Intravoxel Incoherent Motion MR Imaging Study

Purpose

This study was aimed to determine whether pure molecular-based diffusion coefficient (D) and perfusion-related diffusion parameters (perfusion fraction f, perfusion-related diffusion coefficient D*) differ in healthy livers and fibrotic livers through intra-voxel incoherent motion (IVIM) MR imaging.

Material and Methods

17 healthy volunteers and 34 patients with histopathologically confirmed liver fibrosis patients (stage 1 = 14, stage 2 = 8, stage 3& 4 = 12, METAVIR grading) were included. Liver MR imaging was performed at 1.5-T. IVIM diffusion weighted imaging sequence was based on standard single-shot DW spin echo-planar imaging, with ten b values of 10, 20, 40, 60, 80, 100, 150, 200, 400, 800 sec/mm² respectively. Pixel-wise realization and regions-of-interest based quantification of IVIM parameters were performed.

Results

D, f, and D* in healthy volunteer livers and patient livers were 1.096±0.155 vs 0.917±0.152 (10⁻³ mm²/s, p = 0.0015), 0.164±0.021 vs 0.123±0.029 (p<0.0001), and 13.085±2.943 vs 9.423±1.737 (10⁻³ mm²/s, p<0.0001) respectively, all significantly lower in fibrotic livers. As the fibrosis severity progressed, D, f, and D* values decreased, with a trend significant for f and D*.

Conclusion

Fibrotic liver is associated with lower pure molecular diffusion, lower perfusion volume fraction, and lower perfusion-related diffusion. The decrease of f and D* in the liver is significantly associated liver fibrosis severity.     

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V_max, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V_max was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min⁻¹ for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min⁻¹ (U₀ = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Chimeric cells of maternal origin do not appear to be pathogenic in the juvenile idiopathic inflammatory myopathies or muscular dystrophy

IntroductionMicrochimeric cells have been studied for over a decade, with conflicting reports on their presence and role in autoimmune and other inflammatory diseases. To determine whether microchimeric cells were pathogenic or mediating tissue repair in inflammatory myopathies, we phenotyped and quantified microchimeric cells in juvenile idiopathic inflammatory myopathies (JIIM), muscular dystrophy (MD), and noninflammatory control muscle tissues.MethodFluorescence immunophenotyping for infiltrating cells with sequential fluorescence in situ hybridization was performed on muscle biopsies from ten patients with JIIM, nine with MD and ten controls.ResultsMicrochimeric cells were significantly increased in MD muscle (0.079 ± 0.024 microchimeric cells/mm² tissue) compared to controls (0.019 ± 0.007 cells/mm² tissue, p = 0.01), but not elevated in JIIM muscle (0.043 ± 0.015 cells/mm²). Significantly more CD4+ and CD8+ microchimeric cells were in the muscle of patients with MD compared with controls (mean 0.053 ± 0.020/mm² versus 0 ± 0/mm²p = 0.003 and 0.043 ± 0.023/mm² versus 0 ± 0/mm²p = 0.025, respectively). No differences in microchimeric cells between JIIM, MD, and noninflammatory controls were found for CD3+, Class II+, CD25+, CD45RA+, and CD123+ phenotypes, and no microchimeric cells were detected in CD20, CD83, or CD45RO populations. The locations of microchimeric cells were similar in all three conditions, with MD muscle having more microchimeric cells in perimysial regions than controls, and JIIM having fewer microchimeric muscle nuclei than MD. Microchimeric inflammatory cells were found, in most cases, at significantly lower proportions than autologous cells of the same phenotype.ConclusionsMicrochimeric cells are not specific to autoimmune disease, and may not be important in muscle inflammation or tissue repair in JIIM.     

Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls

The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.     

Quantification of Tumor Vessels in Glioblastoma Patients Using Time-of-Flight Angiography at 7 Tesla: A Feasibility Study

Purpose

To analyze if tumor vessels can be visualized, segmented and quantified in glioblastoma patients with time of flight (ToF) angiography at 7 Tesla and multiscale vessel enhancement filtering.

Materials and Methods

Twelve patients with newly diagnosed glioblastoma were examined with ToF angiography (TR = 15 ms, TE = 4.8 ms, flip angle = 15°, FOV = 160×210 mm², voxel size: 0.31×0.31×0.40 mm³) on a whole-body 7 T MR system. A volume of interest (VOI) was placed within the border of the contrast enhancing part on T1-weighted images of the glioblastoma and a reference VOI was placed in the non-affected contralateral white matter. Automated segmentation and quantification of vessels within the two VOIs was achieved using multiscale vessel enhancement filtering in ImageJ.

Results

Tumor vessels were clearly visible in all patients. When comparing tumor and the reference VOI, total vessel surface (45.3±13.9 mm² vs. 29.0±21.0 mm² (p<0.035)) and number of branches (3.5±1.8 vs. 1.0±0.6 (p<0.001) per cubic centimeter were significantly higher, while mean vessel branch length was significantly lower (3.8±1.5 mm vs 7.2±2.8 mm (p<0.001)) in the tumor.

Discussion

ToF angiography at 7-Tesla MRI enables characterization and quantification of the internal vascular morphology of glioblastoma and may be used for the evaluation of therapy response within future studies.     

The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on β-Glucan

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50°C on β-glucan. Under these conditions specific activity was 239.2±9.1 U mg⁻¹ and the half-life of the enzyme was 84.6±3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the K_m was 3.2±0.5 mg mL⁻¹ and the V_max was 0.41±0.02 µmol min⁻¹. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.     

Characterization of Breast Tumors Using Diffusion Kurtosis Imaging (DKI)

Aim

The aim of this study was to investigate and evaluate the role of magnetic resonance (MR) diffusion kurtosis imaging (DKI) in characterizing breast lesions.

Materials and Methods

One hundred and twenty-four lesions in 103 patients (mean age: 57±14 years) were evaluated by MR DKI performed with 7 b-values of 0, 250, 500, 750, 1,000, 1,500, 2,000 s/mm² and dynamic contrast-enhanced (DCE) MR imaging. Breast lesions were histologically characterized and DKI related parameters—mean diffusivity (MD) and mean kurtosis (MK)—were measured. The MD and MK in normal fibroglandular breast tissue, benign and malignant lesions were compared by One-way analysis of variance (ANOVA) with Tukey''s multiple comparison test. Receiver operating characteristic (ROC) analysis was performed to assess the sensitivity and specificity of MD and MK in the diagnosis of breast lesions.

Results

The benign lesions (n = 42) and malignant lesions (n = 82) had mean diameters of 11.4±3.4 mm and 35.8±20.1 mm, respectively. The MK for malignant lesions (0.88±0.17) was significantly higher than that for benign lesions (0.47±0.14) (P<0.001), and, in contrast, MD for benign lesions (1.97±0.35 (10⁻³ mm²/s)) was higher than that for malignant lesions (1.20±0.31 (10⁻³ mm²/s)) (P<0.001). At a cutoff MD/MK 1.58 (10⁻³ mm²/s)/0.69, sensitivity and specificity of MD/MK for the diagnosis of malignant were 79.3%/84.2% and 92.9%/92.9%, respectively. The area under the curve (AUC) is 0.86/0.92 for MD/MK.

Conclusions

DKI could provide valuable information on the diffusion properties related to tumor microenvironment and increase diagnostic confidence of breast tumors.     

CHOLINESTERASE ACTIVITY OF NODAL AND INTERNODAL REGIONS OF MYELINATED NERVE FIBERS OF FROG

The distribution of cholinesterase (Ch-esterase) in isolated myelinated fibers of the frog has been investigated. Quantitative microgasometric measurements have confirmed the previous histochemical observations. Both approaches indicate that in frog nerve fibers acetylcholinesterase (ACh-esterase) is the only or the predominant enzyme when selective inhibitors and different substrates are used: acetylcholine (ACh), butyrylcholine, and acetyl-B-methylcholine (Mecholyl). By means of the microgasometric technique, a significant difference in ACh-esterase activity between axons isolated from ventral (37.2 ± 1.7 µmole x 10^-5 ACh/mm²/hr) and dorsal roots (2.0 ± 0.9 µmole x 10^-5 ACh/mm²/hr) was found. In the region of the node of Ranvier the enzyme activity (50.4 ± 4.4 µmole x 10^-5 ACh/mm²/hr) appears to be considerably higher than in the internodal area (36.6 ± 2.1 µmole x 10^-5 ACh/mm²/hr). The findings are discussed in relation to the theory of saltatory conduction and the ACh system.     

Slow Cone Reflectance Changes during Bleaching Determined by Adaptive Optics Scanning Laser Ophthalmoscope in Living Human Eyes

To investigate the changes in the reflectance of human cone photoreceptors by an adaptive optics scanning laser ophthalmoscope (AO-SLO) during photobleaching. A custom-built AO-SLO with an observation light of 840-nm was used to measure the cone densities and the reflectance changes during bleaching by 630 nm red light emitting diodes. Measurements were made at 1° and 3° temporal to the fovea within an area of 1° × 1° in 8 eyes of 8 normal subjects. After dark-adaptation, images of the cone mosaics were recorded continuously for 5-min before, 5-min during, and after 5-min of light stimulation with a sampling rate of 5-Hz. The first positive peak (P1) was observed at 72.2 ± 15.0-s and a second positive peak (P2) at 257.5 ± 34.5-s at 1°. The increase of the reflectance of P1 was significantly larger at 1° (34.4 ± 13.9%) than at 3° (26.0 ± 10.5%; P = 0.03, Wilcoxon’s signed rank test). The average cone density at 1° (51123.13 ± 1401.23 cells/mm²) was significantly larger than that at 3° (30876.13 ± 1459.28 cells/mm²; P <0.001, Wilcoxon’s signed rank test). The changes in the reflectance of the cones during bleaching by red light had two peaks. The two peaks may be caused by regeneration of cone photopigment during bleaching.