Method for three-dimensional visualization of neurodegeneration in cupric-silver stained serial rat brain slices

The spatial distribution of neurodegeneration in brains is difficult to visualize when working from 2-D serial slices. In studies where repetitive operant behavior measurements are made over several weeks following organic solvent exposure, definitive evidence of degeneration in brain structures may have been significantly cleared by the time the tissue is prepared histologically. The only remaining evidence that injury has occurred may be nothing more than neuronal and cellular debris. By choosing stains that are specific for this type of residual and/or indicative of specific pathology, a 3-D representation of the spatial distribution of the neuronal and cellular debris fields within the organ can be highlighted and displayed. We present a method for visualizing the spatial distribution of neuronal degeneration that can result from low-level organic solvent exposure scenarios. A cupric-silver stain highly specific for neuronal degeneration is used to identify neuronal debris fields in 73 serial slices of brains of rodents that were exposed to toluene vapors. Serial brain sections stained with cupric-silver are scanned at 600 dpi using a gray-scale protocol. Using commercially available software, scans are assembled into 3-D images showing both topographical and internal anatomical details. The reassembled images are further processed into stereo pairs. Gray-scale scans are compared to the original sections to establish gray-scale ranges for healthy and damaged tissue and artifact staining.     

Endogenous levels of serum estradiol and sex hormone binding globulin determine bone mineral density, bone remodeling, the rate of bone loss, and response to treatment with estrogen in elderly women

A total of 489 elderly women aged 65-75 yr who participated in a 3-yr, randomized, blinded osteoporosis trial underwent measurements of serum estradiol, bioavailable estradiol, and SHBG. At baseline, bone mineral density (BMD) was lower at the femoral sites (7-19%, P < 0.05), total body (6-8%, P < 0.05), and spine (5-9%, P = 0.2) in women in the lowest tertile for serum total estradiol [<9 pg/ml (33 pmol/liter)], serum bioavailable estradiol [<2.4 pg/ml (8.8 pmol/liter)], or highest tertile for serum SHBG (>165 nmol/liter), compared with women in the highest tertiles of total estradiol [>13.3 pg/ml (49 pmol/liter)] and bioavailable estradiol [>4 pg/ml (14 pmol/liter)] or lowest tertile for SHBG (<113 nmol/liter). Bone markers were increased in women in the lowest tertile for serum total estradiol (not significant) and bioavailable estradiol (P < 0.05) and highest tertile for SHBG (P < 0.05).In the longitudinal study, the rate of bone loss in the placebo group was significantly higher in total body (P < 0.05) and spine (P < 0.05) in women in the lowest tertile, compared with the highest tertile of serum bioavailable estradiol.After treatment with conjugated equine estrogens 0.625 mg/d, the increase in BMD was 4-6% higher at the femoral sites (P < 0.05), total body (P < 0.05), and spine (not significant), in the lowest tertile, compared with the highest tertile of serum bioavailable estradiol or highest tertile, compared with the lowest tertile of serum SHBG.In summary, small variations in endogenous serum estradiol and high serum SHBG determine differences in BMD and rate of bone loss in elderly women and also affect the response to treatment with estrogen. Women with a serum estradiol level of less than 9 pg/ml (33 pmol/liter) are optimal candidates for estrogen therapy for osteoporosis prevention.     

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.     

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both “water” and “membrane” conformations of the central loop (loop-2) were determined by X-ray crystallography. The “water” conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The “membrane” conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step—the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their “water” NMR models. We found that the both toxins transform their “water” conformation of loop-2 into the “membrane” one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.     

Low Cost Embedded Copper Mesh Based on Cracked Template for Highly Durability Transparent EMI Shielding Films

Embedded copper mesh coatings with low sheet resistance and high transparency were formed using a low-cost Cu seed mesh obtained with a magnetron sputtering on a cracked template, and subsequent operations electroplating and embedding in a photocurable resin layer. The influence of the mesh size on the optoelectric characteristics and the electromagnetic shielding efficiency in a wide frequency range is considered. In optimizing the coating properties, a shielding efficiency of 49.38 dB at a frequency of 1 GHz, with integral optical transparency in the visible range of 84.3%, was obtained. Embedded Cu meshes have been shown to be highly bending stable and have excellent adhesion strength. The combination of properties and economic costs for the formation of coatings indicates their high prospects for practical use in shielding transparent objects, such as windows and computer monitors.     

Progression of visual field loss in untreated glaucoma patients and glaucoma suspects in St. Lucia,West Indies

PURPOSE: A 1986-1987 survey found 8.8% prevalence of open-angle glaucoma in the black population of St. Lucia, West Indies. This follow-up study assessed visual field loss progression in untreated glaucoma patients and glaucoma suspects 10 years later. DESIGN: Cohort study. METHODS: Subjects were 205 glaucoma patients and suspects; 1987 data included age, sex, visual acuity, and visual fields measured by automated threshold perimetry (Humphrey C 30-2 test), and 1997 data included intraocular pressure, visual acuity, and visual fields measured by the same test. Exclusion criteria included field unreliability, field improvement due to vision improvement, nonglaucomatous vision deterioration, glaucoma treatment since 1988, and scoring of a visual field as end stage in 1987. Visual fields were scored by algorithms for the Advanced Glaucoma Intervention Study (AGIS) and Collaborative Initial Glaucoma Treatment Study (CIGTS). RESULTS: By AGIS criteria, 55% of 146 right eyes and 52% of 141 left eyes showed progression of visual field loss. In linear regressions, progression severity was unassociated with sex, intraocular pressure, or baseline visual field score, but was positively associated with age (P <.001, right; P =.002, left). The cumulative probability of reaching end stage in 10 years in at least one eye was approximately 16% by AGIS criteria. By CIGTS criteria, 73% of 146 right eyes and 72% of 141 left eyes progressed. CONCLUSIONS: These data provide a unique opportunity to study progression of untreated glaucoma. The percentage of eyes showing visual field loss progression and the percentage reaching end stage were considerably higher than in studies of visual field progression in treated eyes.     

A novel method for encapsulation of poorly water-soluble drugs: precipitation in polyelectrolyte multilayer shells

A novel method to include poorly water-soluble substances into the polyelectrolyte capsules of defined size, stability, composition and affinity properties is proposed. Encapsulation explores the polarity gradient across the capsule wall. Capsules creation makes use of electrostatic interaction and can involve many substances as layer constituents, such as synthetic polyelectrolytes, proteins, nucleic acids, lipids and multivalent dyes. Using capsules made of synthetic polyelectrolytes as a model system was demonstrated how to prepare, to measure and to use this gradient for low molecular weigh materials encapsulation.     

The 1.9-A crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link

Triple-helical collagen IV protomers associate through their N- and C-termini forming a three-dimensional network, which provides basement membranes with an anchoring scaffold and mechanical strength. The noncollagenous (NC1) domain of the C-terminal junction between two adjacent collagen IV protomers from human placenta was crystallized and its 1.9-A structure was solved by multiple anomalous diffraction (MAD) phasing. This hexameric NC1 particle is composed of two trimeric caps, which interact through a large planar interface. Each cap is formed by two alpha 1 fragments and one alpha 2 fragment with a similar previously uncharacterized fold, segmentally arranged around an axial tunnel. Each monomer chain folds into two structurally very similar subdomains, which each contain a finger-like hairpin loop that inserts into a six-stranded beta-sheet of the neighboring subdomain of the same or the adjacent chain. Thus each trimer forms a quite regular, but nonclassical, sixfold propeller. The trimer-trimer interaction is further stabilized by a previously uncharacterized type of covalent cross-link between the side chains of a Met and a Lys residue of the alpha 1 and alpha 2 chains from opposite trimers, explaining previous findings of nonreducible cross-links in NC1. This structure provides insights into NC1-related diseases such as Goodpasture and Alport syndromes.