Pixel classification method in optical coherence tomography for tumor segmentation and its complementary usage with OCT microangiography

A novel machine‐learning method to distinguish between tumor and normal tissue in optical coherence tomography (OCT) has been developed. Pre‐clinical murine ear model implanted with mouse colon carcinoma CT‐26 was used. Structural‐image‐based feature sets were defined for each pixel and machine learning classifiers were trained using “ground truth” OCT images manually segmented by comparison with histology. The accuracy of the OCT tumor segmentation method was then quantified by comparing with fluorescence imaging of tumors expressing genetically encoded fluorescent protein KillerRed that clearly delineates tumor borders. Because the resultant 3D tumor/normal structural maps are inherently co‐registered with OCT derived maps of tissue microvasculature, the latter can be color coded as belonging to either tumor or normal tissue. Applications to radiomics‐based multimodal OCT analysis are envisioned.      

Effect of Subpressor Dose of Angiotensin II on Pain-Related Behavior in Relation with Neuronal Injury and Activation of Satellite Glial Cells in the Rat Dorsal Root Ganglia

To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied the effect of subpressor dose (150 ng/kg/min) of Ang II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRGs) after chronic constriction injury (CCI). Systemic continuous delivery of Ang II induced the tactile, heat and cold hyperlagesia, when measured at 7 days ofpost-injury. Blockade of the AT₁ receptor with losartan (2.5 mg/kg/day) prevented tactile hyperalgesia and attenuated cold hyperalgesia, but did not affect the response to noxious heat stimulus. A marked increase of large-sized injured primary afferent neurons, detected by ATF3 immunolabeling, was seen in lower lumbar DRGs on ipsilateral side after Ang II treatment. Subpressor dose of Ang II induced an increase of activated SGCs (detected by GFAP immunolabeling) enveloping large-diameter neurons. Our results suggested that Ang II through the AT₁ receptor activation is an important regulatory factor in neuropathic pain perception and plays an important role in the injury of large-sized primary afferent neurons and activation of SGCs elicited by the CCI.     

Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1

In eukaryotic ribosomes, termination of translation is triggered by class 1 polypeptide release factor, eRF1. In organisms with a universal code, eRF1 responds to three stop codons, whereas, in ciliates with variant codes, only one or two codon(s) remain(s) as stop signals. By mutagenesis of the Y-C-F minidomain of the N domain, we converted an omnipotent human eRF1 recognizing all three stop codons into a unipotent 'ciliate-like' UGA-only eRF1. The conserved Cys127 located in the Y-C-F minidomain plays a critical role in stop codon recognition. The UGA-only response has also been achieved by concomitant substitutions of four other amino acids located at the Y-C-F and NIKS minidomains of eRF1. We suggest that for eRF1 the stop codon decoding is of a non-linear (non-protein-anticodon) type and explores a combination of positive and negative determinants. We assume that stop codon recognition is profoundly different by eukaryotic and prokaryotic class 1 RFs.     

In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes

New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes.     

Micronuclei, DNA single-strand breaks and DNA-repair activity in mice exposed to 1,3-butadiene by inhalation

We investigated single-strand breaks and endonuclease III-sensitive sites in DNA along with gamma-irradiation-specific DNA-repair activity in hepatocytes and frequencies of micronuclei in polychromatic bone-marrow erythrocytes of male NMRI mice (2 months old, weight 30-35 g) during sub-acute inhalation exposure to 1,3-butadiene (28 days, 500 mg/m3) and up to 28 days after the exposure. Concentrations of 1,3-butadiene in blood, an indicator of internal exposure, moderately increased during the exposure period. The most interesting finding was that gamma-irradiation-specific DNA-repair activity gradually increased during exposure, being significantly higher compared with control levels on days 7 and 28 of exposure (P = 0.005 and 0.035, respectively), reaching a maximum on day 1 after the termination of exposure (P = 0.003) and then returning to control levels. A significant correlation between gamma-irradiation-specific DNA-repair activity and the concentration of 1,3-butadiene in blood (R = 0.866, P = 0.050) supports a possible induction of DNA-repair activity by the exposure to 1,3-butadiene and formation of its metabolites. The initial increase in micronucleus frequency (micronuclei per 1000 cells) in the exposed mice continuously decreased from 20.4 +/- 5.1 (day 3) to 15.1 +/- 3.2 (day 28) within the exposure period, and subsequently from 12.4 +/- 5.1 to 4.6 +/- 1.6 in the period following termination of the 1,3-butadiene exposure, while micronucleus frequencies in control animals were significantly lower (from 1.7 +/- 1.5 to 4.2 +/- 0.8).     

Nutritional strategies to modulate inflammation and oxidative stress pathways via activation of the master antioxidant switch Nrf2

The nuclear factor E2-related factor 2 (Nrf2) plays an important role in cellular protection against cancer, renal, pulmonary, cardiovascular and neurodegenerative diseases where oxidative stress and inflammation are common conditions. The Nrf2 regulates the expression of detoxifying enzymes by recognizing the human Antioxidant Response Element (ARE) binding site and it can regulate antioxidant and anti-inflammatory cellular responses, playing an important protective role on the development of the diseases. Studies designed to investigate how effective Nrf2 activators or modulators are need to be initiated. Several recent studies have shown that nutritional compounds can modulate the activation of Nrf2–Keap1 system. This review aims to discuss some of the key nutritional compounds that promote the activation of Nrf2, which may have impact on the human health.     

Molecular characterization of binding of calcium and carbohydrates by an early activation antigen of lymphocytes CD69

CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. Here, we show that a globular C-terminal domain of CD69 belonging to C-type lectins binds calcium through Asp 171, Glu 185, and Glu 187 with K(d) approximately 54 microM. Closure of the calcium-binding site results in a conformational shift of Thr 107 and Lys 172. Interestingly, structural changes in all of these amino acids lead to the formation of high-affinity binding sites for N-acetyl-D-glucosamine. Similarly, a structural change in Glu 185 and Glu 187 contributes to a high-affinity site for N-acetyl-D-galactosamine. Site-directed mutagenesis and molecular modeling allowed us to describe the structural details of binding sites for both carbohydrates. These studies explain the importance of calcium for recognition of carbohydrates by CD69 and provide an important paradigm for the role of weak interactions in the immune system.     

Crystal structure of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E, a key enzyme involved in polychlorinated aromatics biodegradation

Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail.     

A novel radioimmunoassay of 16alpha-hydroxy-dehydroepiandrosterone and its physiological levels

16alpha-Hydroxy-dehydroepiandrosterone (16alpha-OH-DHEA) belongs to the products of extensive DHEA metabolism in mammalian tissues. It is a precursor of 16alpha-hydroxylated estrogens, increased levels of which are associated with autoimmune disorders. A highly specific radioimmunoassay of unconjugated 16alpha-OH-DHEA was developed and evaluated. Polyclonal rabbit antisera were raised against 3beta,16alpha-dihydroxy-17,19-dione-19-O-(carboxymethyloxime) and 3beta,16alpha-dihydroxy-7,17-dione-7-O-(carboxymethyloxime) BSA conjugates. Two methods were used for preparation of the conjugates. Homologous radioiodinated derivatives with tyrosine methyl ester were prepared as tracers. While antisera to 7-CMO cross-reacted with DHEA as much as by 58%, the cross-reaction of the chosen antiserum prepared via 19-oxogroup by micellar conjugation technique with 16beta-OH-DHEA was only 0.13% and with all other structurally related steroids, including DHEA were lower than 0.01%. The detection limit was 0.017 pmol (5.7 pg)/tube, the average intra- and inter-assay coefficients of variation were 8.2 and 11.4%, respectively. Mean recovery of serum spiked with 16alpha-OH-DHEA varied between 80 and 110%, the results were independent on sample dilution. 16alpha-OH-DHEA concentrations in 18 randomly selected sera, including 6 samples from patients with thyroid cancer were compared with results obtained by earlier GC-MS method. Physiological levels of 16alpha-OH-DHEA in 316 sera (184 females and 132 males) analyzed so far varied between 0.0 and 1.86 nmol/l.