Synergistic Tissue Counterstaining and Image Segmentation Techniques for Accurate, Quantitative Immunohistochemistry

Quantitative analysis of digitized IHC-stained tissue sections is increasingly used in research studies and clinical practice. Accurate quantification of IHC staining, however, is often complicated by conventional tissue counterstains caused by the color convolution of the IHC chromogen and the counterstain. To overcome this issue, we implemented a new counterstain, Acid Blue 129, which provides homogeneous tissue background staining. Furthermore, we combined this counterstaining technique with a simple, robust, fully automated image segmentation algorithm, which takes advantage of the high degree of color separation between the 3-amino-9-ethyl-carbazole (AEC) chromogen and the Acid Blue 129 counterstain. Rigorous validation of the automated technique against manual segmentation data, using Ki-67 IHC sections from rat C6 glioma and β-amyloid IHC sections from transgenic mice with amyloid precursor protein (APP) mutations, has shown the automated method to produce highly accurate results compared with ground truth estimates based on the manually segmented images. The synergistic combination of the novel tissue counterstaining and image segmentation techniques described in this study will allow for accurate, reproducible, and efficient quantitative IHC studies for a wide range of antibodies and tissues. (J Histochem Cytochem 56:873–880, 2008)     

Effects of Ethanol Exposure During Early Pregnancy in Hyperactive, Inattentive and Impulsive Behaviors and MeCP2 Expression in Rodent Offspring

Prenatal exposure to alcohol has consistently been associated with adverse effects on neurodevelopment, which is collectively called fetal alcohol spectrum disorder (FASD). Increasing evidence suggest that prenatal exposure to alcohol increases the risk of developing attention deficit/hyperactivity disorder-like behavior in human. In this study, we investigated the behavioral effects of prenatal exposure to EtOH in offspring mice and rats focusing on hyperactivity and impulsivity. We also examined changes in dopamine transporter and MeCP2 expression, which may underlie as a key neurobiological and epigenetic determinant in FASD and hyperactive, inattentive and impulsive behaviors. Mouse or rat offspring born from dam exposed to alcohol during pregnancy (EtOH group) showed hyper locomotive activity, attention deficit and impulsivity. EtOH group also showed increased dopamine transporter and norepinephrine transporter level compared to control group in the prefrontal cortex and striatum. Prenatal exposure to EtOH also significantly decreased the expression of MeCP2 in both prefrontal cortex and striatum. These results suggest that prenatal exposure to EtOH induces hyperactive, inattentive and impulsive behaviors in rodent offspring that might be related to global epigenetic changes as well as aberration in catecholamine neurotransmitter transporter system.     

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL^?1, respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k _cat/K _m?=?1,440 mg?mL^?1?s^?1) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.     

Insular Organization of Gene Space in Grass Genomes

Wheat and maize genes were hypothesized to be clustered into islands but the hypothesis was not statistically tested. The hypothesis is statistically tested here in four grass species differing in genome size, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, and Aegilops tauschii. Density functions obtained under a model where gene locations follow a homogeneous Poisson process and thus are not clustered are compared with a model-free situation quantified through a non-parametric density estimate. A simple homogeneous Poisson model for gene locations is not rejected for the small O. sativa and B. distachyon genomes, indicating that genes are distributed largely uniformly in those species, but is rejected for the larger S. bicolor and Ae. tauschii genomes, providing evidence for clustering of genes into islands. It is proposed to call the gene islands “gene insulae” to distinguish them from other types of gene clustering that have been proposed. An average S. bicolor and Ae. tauschii insula is estimated to contain 3.7 and 3.9 genes with an average intergenic distance within an insula of 2.1 and 16.5 kb, respectively. Inter-insular distances are greater than 8 and 81 kb and average 15.1 and 205 kb, in S. bicolor and Ae. tauschii, respectively. A greater gene density observed in the distal regions of the Ae. tauschii chromosomes is shown to be primarily caused by shortening of inter-insular distances. The comparison of the four grass genomes suggests that gene locations are largely a function of a homogeneous Poisson process in small genomes. Nonrandom insertions of LTR retroelements during genome expansion creates gene insulae, which become less dense and further apart with the increase in genome size. High concordance in relative lengths of orthologous intergenic distances among the investigated genomes including the maize genome suggests functional constraints on gene distribution in the grass genomes.     

Consistency analysis of similarity between multiple alignments: prediction of protein function and fold structure from analysis of local sequence motifs

A new method to analyze the similarity between multiply aligned protein motifs (blocks) was developed. It identifies sets of consistently aligned blocks. These are found to be protein regions of similar function and structure that appear in different contexts. For example, the Rossmann fold ligand-binding region is found similar to TIM barrel and methylase regions, various protein families are predicted to have a TIM-barrel fold and the structural relation between the ClpP protease and crotonase folds is identified from their sequence. Besides identifying local structure features, sequence similarity across short sequence-regions (less than 20 amino acid regions) also predicts structure similarity of whole domains (folds) a few hundred amino acid residues long. Most of these relations could not be identified by other advanced sequence-to-sequence or sequence-to-multiple alignments comparisons. We describe the method (termed CYRCA), present examples of our findings, and discuss their implications.     

Anterior segment mesenchymal dysgenesis: Probable linkage to the MNS blood group on chromosome 4

Thirty-seven blood samples were analyzed for linkage from members of a single family with an anterior segment mesenchymal dysgenesis (ASMD1) with variable expressivity affecting members of at least six generations. Maximum-likelihood analysis for linkage between ASMD1 and 14 biochemical and serological markers in the family showed a probable linkage between ASMD1 and the MNS blood group on the long arm of chromosome 4 (Z = 2.36 at a recombination fraction of .09).     

New Bacterial Polysaccharide from Arthrobacter

A bacterial strain (NRRL B-1973) isolated from soil at Guatemala City and tentatively identified as an Arthrobacter species produced a polysaccharide with unusual properties. Conditions were studied for the production of this microbial gum in shaken flasks and 20-liter fermentors. Suitable nutrients for optimal polysaccharide production included 3% glucose, 0.3% enzyme-hydrolyzed casein, magnesium sulfate, manganese sulfate, and potassium phosphate buffer (pH 7.0). Polysaccharide yields ranged from 40 to 45%, based on initial dextrose in the medium in 3- or 4-day fermentations. The gum was readily recovered from culture fluid by alcohol precipitation in the presence of an electrolyte. The Arthrobacter gum exhibited characteristics unique for a polyelectrolyte. Viscosity of solutions was not decreased by heating in the presence of salt, and the gum withstood a temperature of 121 C for 30 min. At polysaccharide levels above 0.75%, gels were formed when solutions were autoclaved with KCl. There was no significant change in viscosity over a pH range of 5.0 to 10.0.     

食管癌三维适形放疗前后肺功能、生活质量的变化及放射性肺炎的影响因素分析

目的:探讨食管癌三维适形放疗前后肺功能、生活质量的变化及放射性肺炎的影响因素.方法:收集2017年11月～2019年11月在我院进行三维适形放疗的食管癌患者102例,对患者放疗前后的肺功能进行检测对比,并采用生活质量评价简袁(QLQ-C30)对患者放疗前后的生活质量进行评估对比.统计患者放疗后放射性肺炎的发生率,根据患...     

Physiological and metabolomic responses of bermudagrass (Cynodon dactylon) to alkali stress

Bermudagrass (Cynodon dactylon) is a widely used warm‐season turfgrass species with superior stress tolerance except for cold. In this study, a comparative analysis of the responses to alkali stress in bermudagrass at the physiological and metabolomic levels were performed. Mild alkali with relatively low pH slightly inhibited growth of bermudagrass as evidenced by lower electrolyte leakage, more rapid growth and higher survival rate when compared to moderate and severe alkali treatments. Moreover, the amount of 37 metabolites including amino acids, organic acids, sugars and sugar alcohols were modulated by the alkali treatments. Among them, 15 metabolites were involved in carbon and amino acid metabolic pathways. Under mild alkali stress, bermudagrass possibly slowed down metabolisms to maintain basic growth. However, moderate and severe alkali‐stressed plants accumulated significantly higher amount of carbohydrates which might result in carbon starvation. Taken together, alkali stress had severely inhibitory effect partially due to combined ionic stress and high pH stress. These results suggested that bermudagrass employed different strategies in response to alkali stresses with different pH and ionic values.     

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.