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Motor neuron degeneration and skeletal muscle denervation are hallmarks of amyotrophic lateral sclerosis (ALS), but other neuron populations and glial cells are also involved in ALS pathogenesis. We examined changes in inhibitory interneurons in spinal cords of the ALS model low-copy Gurney G93A-SOD1 (G1del) mice and found reduced expression of markers of glycinergic and GABAergic neurons, that is, glycine transporter 2 (GlyT2) and glutamic acid decarboxylase (GAD65/67), specifically in the ventral horns of clinically affected mice. There was also loss of GlyT2 and GAD67 messenger RNA-labeled neurons in the intermediate zone. Ubiquitinated inclusions appeared in interneurons before 20 weeks of age, that is, after their development in motor neurons but before the onset of clinicalsigns and major motor neuron degeneration, which starts from 25weeks of age. Because mutant superoxide dismutase 1 (SOD1) in glia might contribute to the pathogenesis, we also examined neuron-specific G93A-SOD1 mice; they also had loss of inhibitory interneuron markers in ventral horns and ubiquitinated interneuron inclusions. These data suggest that, in mutant SOD1-associated ALS, pathological changes may spread from motor neurons to interneuronsin a relatively early phase of the disease, independent of the presence of mutant SOD1 in glia. The degeneration of spinal inhibitory interneurons may in turn facilitate degeneration of motor neurons and contribute to disease progression.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, characterized by late-onset progressive degeneration of motor neurons resulting in paralysis of limb, facial, and respiratory muscles and usually leading to death within 5 years after diagnosis. In most patients, the disease is sporadic, whereas approximately 10% of cases have a monogenetic inherited form (1). Mutations associated with ALS have been identified in the gene for superoxide dismutase 1 (SOD1) (2) and, more recently, in genes encoding the DNA/RNA binding proteins, TAR-DNA-binding protein-43 (TDP-43) and FUS/TLS (1, 3). In addition, mutations in optineurin, VAPB, angiogenin, DCTN1, FIG4, senataxin, and alsin are associated with some ALS cases and ALS-like disorders (1, 4). Nuclear-to-cytoplasmic redistribution, aberrant processing and aggregation of TDP-43 are prominent pathological features in most ALS patients, independent of the presence of TDP-43 mutations (3, 5, 6). SOD1-associated ALS (SOD1-ALS) and FUS/TLS-ALS patients instead develop aggregates of SOD1 (2, 7, 8) and FUS/TLS (3, 9, 10), respectively. These and additional data point to an important role for protein aggregation in the pathogenesis of ALS, but the relationships between protein aggregation, neuronal degeneration, and clinical manifestations in different types of ALS are poorly understood (3, 7, 8, 11).
Hypotheses on ALS pathogenesis usually focus on toxic mechanisms that primarily affect motor neurons; their vulnerability has been linked to their large size, long axons, high metabolic activity, specialized physiological properties, and their dependence on skeletal muscle- and glia-derived factors (12, 13). Although motor neuron degeneration and skeletal muscle denervation represent hallmarks of ALS, cell loss and degenerative changes also occur in other neuron populations throughout the nervous system, particularly in the motor cortex and in glia (14-21). The involvement of multiple cell types favors disease models in which abnormalities in non-motor neurons or non-neuronal cells occur concurrently or sequentially with motor neuron degeneration and raises questions about the relationship between the degeneration of different cell types. For example, multiple clinicopathological studies have investigated the relationship between degeneration of spinal motor neurons and cortical (upper) motor neurons that underlie lower and upper motor neuron manifestations, respectively. These studies have provided variable results that suggest independent degeneration of cortical motor neurons and spinal motor neurons, anterograde propagation of disease from motor cortex to spinal cord, or retrograde progression of disease from spinal cord to motor cortex (14, 22-26). A linkage between upper and lower motor signs in initial stages of disease has been suggested, that is, when there are focal manifestations; this linkage may be lost in later stages of disease as neurodegeneration and clinical manifestations spread independently in the cortex and spinal cord (27). Taken together, clinicopathological studies indicate that neurons interconnected or contiguous to affected regions develop degenerative changes more readily than other cells and point to the occurrence of disease mechanisms that mediate or facilitate spreading of disease to neighboring or interconnected neurons (14).
Considerable attention has been devoted to the role of glia in SOD1-ALS (28). For example, SOD1 aggregates are present in astrocytes in the spinal cords of SOD1-ALS patients and transgenic mouse models (29). Other evidence pointing to a role of glial abnormalities in SOD1-ALS comes from studies with mutant SOD1 transgenic mice that express no or reduced levels of mutant SOD1 in glia. These mice show slower disease progression and prolonged survival compared with ubiquitous mutant SOD1-expressing mice, although disease onset is unaltered (30-33). On the other hand, expression of mutant SOD1 in neurons determines disease onset and is sufficient to cause an ALS-like disease in mice, indicating that mutant SOD1 in glia does not contribute to disease initiation but rather to disease propagation (28, 34). How mutant SOD1 in glia contributes to disease progression remains to be defined.
The intermediate zone of the spinal cord (Rexed laminae IV-VIII) contains several neuron types that play important roles in controlling the activities of motor neurons, for example, in reflexive and patterned movements and the maintenance of muscle tone (35, 36). Abnormalities in 1 or more populations of interneurons may cause overexcitation or overinhibition of motor neurons and possibly causing them excitotoxic stress (37). There are reports of loss of intermediate zone interneurons in the spinal cord of ALS patients (17, 38) and mutant-SOD1 transgenic mice (39, 40). Furthermore, abnormalities in inhibitory interneurons or inhibitory synapses may occur before motor neuron degeneration in mutant-SOD1 transgenic mice (41-43). We previously showed that spinal interneurons in a low-copy line of G93A-mutant SOD1 mice (G1del mice) (44) start to express the stress transcription factors c-Jun and ATF3 from 20 weeks of age (45, 46). This is several weeks later than the onset of c-Jun and ATF3 activation in motor neurons (at 14-15 weeks), but before the onset of motor manifestations, which starts after the age of 25 weeks in these mice (13, 45). To explore the role of non-motor neuron abnormalities in ALS further, we studied degeneration of spinal inhibitory interneurons in mutant SOD1-ALS mice.
Materials and Methods
Transgenic Mice
Ubiquitous G93A-SOD1 mice were originally derived from Gurney G1 mice, but because of a reduction in the transgene copy number (8 instead of ∼20 transgene copy numbers per haploid genome), they show a delayed disease onset and are termed G1del mice (34, 44). The G1del mice were maintained under standard housing conditions in a FVB/N background by mating hemizygote males with nontransgenic females. Nontransgenic offspring served as controls (34). Neuron-specific G93A-hSOD1 mice carrying the complementary DNA (cDNA) of G93A-mutant hSOD1 cloned into the Thy1.2 expression cassette were generated as described (34). Data from this study were obtained from homozygotes of the T3 line (T3T3 mice) generated by intercrossing T3 hemizygotes and from T3hSOD1 double transgenic mice generated by crossing T3 mice with line N29 wild-type hSOD1-overexpressing mice (34). Onset of clinical disease was determined on the basis of weight, the ability to extend the hind limbs, and the ability to hang upside down on a grid for 1 minute (34). Mice reached end-stage disease when they could not right themselves within 5 seconds when placed on their back, when they lost more than 30% of their maximal weight, or when they developed infection of 1 eye. End-stage mice also were unable to hang in the hanging grid test, which predominantly depends on performance of forelimb muscles.
GlyT2-GFP transgenic mice bred in the C57BL6/J background (47) were obtained from Dr Hanns Ulrich Zeilhofer (Institute of Pharmacology Toxicology, University of Zurich). All animal experiments were approved by the Erasmus University animal care committee and performed in accordance with the guidelines of the Principles of Laboratory Animal Care (National Institutes of Health Publication No. 86-23) and the European Community Council Directive (86/609/EEC).
Mice were anesthetized with pentobarbital and perfused transcardially with 4% paraformaldehyde. The lumbar and cervical spinal cord were carefully dissected, embedded in gelatin blocks, and sectioned at 40 μm with a freezing microtome, as described (48). Free-floating sections were processed for immunohistochemistry using a standard avidin-biotin-immunoperoxidase complex method (ABC; Vector Laboratories, Burlingame, CA) with diaminobenzidine (0.05%) as the chromogen (48). Guinea pig anti-glycine transporter 2 (GlyT2; Millipore, Billerica, MA; 1:10000) and rabbit anti-glutamic acid decarboxylase (GAD65/67; Millipore; 1:4000) were used as primary antibodies to label glycinergic and GABAergic nerve terminals, respectively. Other primary antibodies were rabbit anti-ATF3 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000), rabbit anticalbindin (Swant, Marly, Switzerland; 1:10000), goat anti-choline acetyl transferase (ChAT; Millipore; 1:500), rat anti-Mac2 (Cedarlane, Burlington, Ontario, Canada; 1:2000), rabbit anti-phospho-c-Jun (Ser63; Cell Signaling, Beverly, MA; 1:1000), mouse antiparvalbumin (Swant; 1:10000), mouse antiubiquitin (clone FK2; Affinity BioReagents, Golden, CO; 1:2000), and rabbit antiubiquitin (Dako, Glostrup, Denmark; 1:2000). Immunoperoxidase-stained sections were analyzed and photographed using a Leica DM-RB microscope and a Leica DC300 digital camera.
To examine the relative intensity of glycine transporter 2 (GlyT2) and GAD65/67 immunoperoxidase staining, lumbar L3-L5 sections were photographed using a 5× objective, and optical densities were determined from TIFF files using MetaMorph image analysis software (Molecular Devices, Sunnyvale, CA). Optical densities were determined in rectangular areas of 200 × 250 μm and 100 × 400 μm for the ventral and dorsal horns, respectively. To minimize variability resulting from the sectioning and staining procedures, these analyses were performed with spinal cord specimens embedded in a single gelatin block and processed in the same run. Gelatin blocks typically contained 12 cervical C5-C8 or lumbar L3-L5 specimens from G1del mice of 3 different ages or disease stages (e.g. 20 weeks, 30 weeks, end-stage disease, n = 3 per group) and nontransgenic littermates aged 20 to 30 weeks (n = 3). Other blocks contained specimens from 40-week-old T3T3 mice (n = 3), symptomatic (70-100 weeks old, n = 3), and 2-year-old nontransgenic or T3 hemizygote littermates (34).
In Situ Hybridization
In situ hybridization (ISH) was performed on free-floating 4% paraformaldehyde-fixed 30-μm-thick frozen sections using digoxigenin-labeled RNA probes, as described (49). Sense and antisense probes were transcribed from linearized plasmid constructs containing the partial GlyT2 or Gad67 cDNA sequences using a digoxigenin labeling kit (Roche, Indianapolis, IN). Sections were incubated overnight at 65°C with the probes diluted at 200 ng/mL. After hybridization, the digoxigenin-labeled RNA-RNA complex was detected by using alkaline phosphatase-conjugated sheep-antidigoxigenin (Roche; diluted 1:4000, incubated 48 hours at 4°C), with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium as substrate and chromogen, respectively.
Alkaline phosphatase-stained sections were analyzed and photographed using a Leica DM-RB microscope and a Leica DC300 digital camera. For quantitative analysis of the number of GlyT2 and Gad67 messenger RNA (mRNA)-labeled cells, lumbar L3-L5 sections were examined with an Olympus microscope fitted with a Lucivid miniature monitor coupled to StereoInvestigator software (version 4.37; MicroBrightField, Colchester, VT). The sections were systematically sampled across the spinal cord (each 10th section). The counting fields, 350 μm apart, were 0.0225 mm2; cells in contact with the left and lower boundaries of the counting fields were excluded. The area of the gray matter multiplied by the number of cells per squared millimeter gave the number of cells per section. Sections used for this analysis were produced in a single staining run to avoid variability in staining intensities and numbers of labeled cells resulting from differences in staining conditions between runs.
To combine GlyT2 or GAD67 ISH with immunofluorescence, hybridized digoxigenin complementary RNA was visualized using a tyramide amplification method with fluorescein isothiocyanate-labeled tyramide (50). After hybridization, sections were incubated with biotinylated-sheep antidigoxigenin antibody (Roche) (1:500; 48 hours at 4°C in phosphate-buffered saline [PBS], 2% milk powder, and 0.5% Triton X-100), followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories) and subsequently reacted with fluorescein isothiocyanate-tyramide conjugate (4 μg/mL) in the presence of H2O2 (0.001%) in PBS containing 0.1 mol/L imidazole, pH 7.6 (51). Thereafter, the sections were washed in PBS and processed for immunofluorescence with rabbit anti-ATF3 and rabbit antiubiquitin as the primary antibodies and Cy3-labeled donkey-anti-rabbit (1:200; Jackson ImmunoResearch, Bar Harbor, ME) as the secondary antibody. Double-labeled sections were analyzed with a Zeiss LSM 510 confocal laser scanning microscope using 40×/1.3 and 63×/1.4 oil-immersion objectives.
Western Blot
Spinal cord specimens were homogenized and sonicated in 20 volumes of PBS containing 0.5% Nonidet P-40 (NP-40) and protease inhibitors cocktail (Sigma) and centrifuged at 800 × g for 5 minutes at 4°C, and protein concentrations of the supernatants (S1) were determined. Samples containing 2 to 10 μg protein were electrophoresed on 8% or 10% SDS-PAGE gels and blotted on polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% nonfat dry milk in PBS with 0.05% Tween 20, incubated in primary antibody, diluted in PBS with 0.05% Tween 20 with 1% dry milk followed by an incubation in peroxidase-conjugated secondary antibody, incubated in chemiluminescence reagent (Amersham, Piscataway, NJ), and exposed to film or a Kodak Image station. Primary antibodies used for Western blot included mouse antiactin (Millipore; 1:4000), guinea pig anti-GlyT2 (Millipore; 1:5000), rabbit anti-GAD65/67 (1:5000), and rabbit antimurine SOD1 (SOD101; Stressgen, Victoria, British Columbia, Canada; 1:8000).
Reverse Transcription-Polymerase Chain Reaction
Semiquantitative reverse transcription-polymerase chain reaction (PCR) was performed as previously described (52). Total RNA was extracted from spinal cord tissue using TRIzol and treated with DNAse. The RNA (5 μg) was converted into cDNA using oligo(dT) primer and reverse transcriptase in a total reaction volume of 20 μl. Polymerase chain reaction was performed with 0.1 μL of the reverse transcriptase reaction mixture in a reaction volume of 25 μl. Primers used were as follows: ChAT, 5′-GCGAATCGTTGGTATGACAAGTC-3′ (forward) and 5′-TTGAAGTTTCTCTGCCGAGGAG-3′ (backward); G6dph, 5′-CTTTGGACCCATCTGGAATCG-3′ and 5′-CACTTTGACCTTCTCATCACGGAC-3′; Gad67, 5′-TACGGGGTTCGCACAGGTC-3′ and 5′-CCCCAGGCAGCATCCACAT-3′; GlyT2, 5′-TACCGCTACCCTAACTGGTCCATGG-3′ and 5′-ATCCACACGACTGGACTAGCACTGA; GlyT2A, 5′-ACTCTACGGTTCAATCTGTTGTCC-3′ and 5′-GGTCCTAGGTGCACGAGGACTATCCCGG-3′. The number of PCR cycles and quantity of cDNA were determined to be within the linear range of the reactions. For quantification, the PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and scanned on a Molecular Dynamics Typhoon instrument.
Statistical Analysis
Statistical analyses were done using GraphPad Prism software (San Diego, CA). Means from different age groups and different transgenic mouse lines were compared using 1-way analysis of variance and the Tukey post test.
Reduced GlyT2 and GAD65/67 Protein and mRNA Levels in the Spinal Cord of Symptomatic G1del Mice
G1del (also termed G1slow or low-copy G1) mice ubiquitously express G93A-mutant SOD1; they develop weakness in 1 or more limbs from age 24 to 30 weeks and die of fatal paralysis between 32 and 40 weeks of age (48, 53). Early pathological and molecular changes in motor neurons include swelling and vacuolization of a subset of mitochondria starting before 3 weeks (53), the appearance of ubiquitinated pathology, Golgi fragmentation, endoplasmic reticulum stress, and ATF3 activation starting from 13 to 15 weeks (13, 45). These changes precede neuromuscular denervation, loss of motor neurons, and astrogliosis and microgliosis. Abnormalities in interneurons (ATF3 and phospho-c-Jun expression) occur after 20 weeks (45). In this study, we examined G1del mice at ages 10 to 15 weeks (early presymptomatic), 20 to 25 weeks (late presymptomatic), and 30 weeks (symptomatic) and mice that had reached disease end stage.
To identify inhibitory interneurons, we used the expression of GlyT2 and GAD as markers for glycinergic and GABAergic neurons, respectively (49, 54, 55). Immunohistochemistry showed that GlyT2 was expressed throughout the spinal cord gray matter except for the most superficial part of the dorsal horn (Figs. 1A-D). At high magnification, labeling was distributed in punctae, consistent with a predominant localization in nerve terminals (Figs. 1A-D) and in accord with previous studies (54). The distribution of GlyT2 immunoreactivity in spinal cords of 10- and 20-week-old G1del mice was indistinguishable from that in nontransgenic mice (Figs. 1B, C), whereas in symptomatic and end-stage G1del mice, there was a marked reduction of GlyT2 immunoreactivity, particularly in the ventral horn motor neuronal cell groups (Figs. 1D, H). GlyT2 immunoreactivity was not altered in the dorsal horn (Figs. 1D, H). Reduced GlyT2-immunoreactivity in the ventral horn occurred in both cervical and lumbar cord but, in general, was more prominent in the latter.
GABAergic interneurons were labeled with an antibody that binds both GAD67, the predominant GAD isoform in the ventral horn, and GAD65 that is expressed in a more restricted set of GABAergic interneurons and terminals (55, 56). GAD65/67 immunoreactivity was present throughout the spinal cord gray matter with more prominent labeling in the dorsal horn compared with that of GlyT2 (Fig. 1E). As with GlyT2, no change in overall staining occurred in spinal cord sections from 10- and 20-week-old G1del mice (Figs. 1F, I). There was reduced GAD65/67 immunoreactivity in the ventral horn of symptomatic G1del mice (Figs. 1G, I).
Western blot showed reduced GlyT2 and GAD65/67 immunoreactivity in spinal cord homogenates of symptomatic and end-stage G1del mice (Figs. 2A, B). In addition, we performed semiquantitative reverse transcription-PCR analysis to examine changes at the mRNA level. Consistent with the immunohistochemistry and Western blotting, GlyT2 and GAD67 mRNA levels in the spinal cord of 20-week-old G1del mice were the same as in the spinal cord of nontransgenic mice, whereas GlyT2 and GAD67 mRNA levels were reduced in symptomatic and end-stage G1del mice (Figs. 2C, D). In the end-stage disease mice, GlyT2 and GAD67 mRNA levels were reduced by approximately 50%, which was comparable to the loss of ChAT mRNA, which is predominantly produced by motor neurons.
We studied GlyT2 and GAD67 mRNA expression using ISH. Consistent with previous reports (49, 54), GlyT2 mRNA-labeled cells were predominantly localized in the deep dorsal horn and the intermediate zone (Rexed laminae V-VIII), whereas they were detected at a low frequency in the motor columns in the ventral horn (Rexed laminae IX) (Fig. 3A) and the superficial dorsal horn. Labeled cells were small- to medium-sized (15-25 μm in diameter) and showed varying labeling intensities. The distribution of GlyT2 mRNA was the same as in nontransgenic mice in 10- and 20-week-old G1del mice, whereas 30-week-old symptomatic G1del mice showed a reduction of GlyT2 mRNA, which was due to both reduced numbers of GlyT2 mRNA-labeled cells and reduced staining intensities in remaining cells (Figs. 3A-C). Quantitative analysis indicated a 50% loss of GlyT2 mRNA-labeled cells in lumbar L3-L5 sections of 30-week-old G1del mice compared with controls and 20-week-old G1del mice (Fig. 3G).
GAD67 mRNA labeling was most prominent in dorsal lamina of the spinal cord with a high density of small intensely stained cells in Rexed laminae II-III (Fig. 3D). Labeled cells in other lamina usually were less intensely detected, whereas a very low number of GAD67 mRNA-positive cells were present in the motor columns. As with GlyT2 mRNA, there was reduced GAD67 mRNA in 30-week-old and end-stage G1del mice (Fig. 3F). Neurons in superficial dorsal horn seemed relatively spared. Quantitative analysis indicated 30% to 40% loss of labeled cells in lumbar L3-L5 sections of 30-week-old G1del mice versus control and 20-week-old G1del mice (Fig. 3G).
ATF3 Expression and Ubiquitinated Aggregates in Inhibitory Spinal Interneurons
To determine whether ATF3 was associated with inhibitory interneurons, we combined fluorescent ISH of GlyT2 or GAD67 mRNA with ATF3 immunofluorescence. Double labeling with ATF3 revealed multiple GlyT2 mRNA/ATF3- and GAD67 mRNA/ATF3 double-labeled neurons in the intermediate zone and deep dorsal horn of 30-week-old and end-stage G1del mice (Figs. 4A-D). Systematic analysis of lumbar L3-L5 sections of 30-week-old G1del mice indicated that 27% ± 5% (mean ± SE, n = 3 animals, 30-40 ATF3 cells analyzed per mouse) of ATF3-positive cells were GlyT2 mRNA-positive, whereas 33% ± 5% were Gad67 mRNA-positive. A subset of double-labeled neurons showed eccentric nuclei (Figs. 4A, C, D) (suggesting pathologic alteration), consistent with previous observations (45).
To determine whether spinal inhibitory interneurons also develop ubiquitinated SOD1 aggregates, we double-stained for GlyT2 or GAD67 mRNA and polyubiquitinated epitopes. Multiple GlyT2 or GAD67 mRNA-stained neurons containing ubiquitin-immunoreactive structures were identified in the spinal cords of symptomatic G1del mice (Figs. 4E-G). The mRNA staining did not overlap with that of ubiquitin. In lumbar L3-L5 sections (2 mice, 3 sections per mouse), approximately 50% (14/26) of intermediate zone neurons with ubiquitin immunopositivity also stained for GlyT2 mRNA. Similarly, in GAD67 mRNA/ubiquitin-stained sections, approximately 50% (12/27) of ubiquitinated neurons stained for GAD67 mRNA. These data indicate that G1del mice show loss of spinal inhibitory interneurons, which is preceded by ATF3 expression and the appearance of ubiquitinated aggregates, 2 key pathological hallmarks that also precede motor neuron loss.
Early Dendritic Ubiquitin Pathology in Inhibitory Interneurons in G1del Spinal Cord
Ubiquitinated aggregates in motor neurons of G1del mice occur initially and more frequently in dendrites than in the cell soma (34, 45). These aggregates are strongly immunoreactive for human SOD1 and first appear at 13 to 15 weeks and only in the motor columns (45). Double labeling with ChAT showed that at 15 weeks, essentially all ubiquitinated dendrites were immunoreactive for ChAT (Figs. 5A, B). However, a proportion of ubiquitinated dendrites was ChAT-negative and also occurred in the intermediate zone in 20-week-old and older mice (Fig. 5A). Thus, ubiquitin pathology also occurred in dendrites of non-motor neurons in older mice.
To determine whether non-motor neuron dendritic ubiquitin pathology also occurs in the dendrites of inhibitory interneurons, we double stained for ubiquitin and parvalbumin (Figs. 5A, C). Parvalbumin, a small calcium binding protein, was used as a substitute marker instead of GlyT2 or GAD65 because staining for GlyT2 and GAD67 protein or mRNA does not outline the dendritic compartment of interneurons. Parvalbumin is reported to be present in the somatodendritic compartment of a subset of inhibitory interneurons that project to motor neurons (57). Accordingly, we obtained evidence in GlyT2-GFP transgenic mice that more than 90% of parvalbumin-positive neurons in the intermediate zone and ventral horn of spinal cord are glycinergic (47) (Figure, Supplemental Digital Content 1, ). Double labeling for ubiquitin and parvalbumin showed that at 20 weeks, but not at 15 weeks, a subset of ubiquitinated neurites were parvalbumin positive (Figs. 5A, C). Parvalbumin-positive ubiquitinated dendrites were preferentially localized in the intermediate zone, although sometimes they occurred in the motor columns. Triple staining for ubiquitin, ChAT, and parvalbumin showed that parvalbumin was only present in a subset of ChAT-negative ubiquitinated dendrites, indicating the presence of ubiquitin in dendrites of other populations of spinal interneurons. Of note, we never observed ChAT and parvalbumin double-stained neurons and neurites, thereby excluding the possibility that parvalbumin-positive dendrites were from motor neurons.
Early Microglial Activation in the Intermediate Zone in G1del Mice
There was a low density of activated microglia, identified using an antibody against Mac2 (58), in the motor columns of 15-week-old G1del mice, whereas higher densities of Mac2-positive microglia were seen in the motor columns at older ages (Figs. 5E-H). Activated microglia appeared in the intermediate zone between 20 and 25 weeks. Activated microglia in motor columns were usually in close apposition with motor neurons. Sections from 25-week-old G1del mice stained for both Mac2 and parvalbumin revealed multiple examples of Mac2-positive microglia contacting parvalbumin-positive interneurons (Fig. 5G). Together, these data further support the relatively early pathological alterations of spinal interneurons, including a subclass of parvalbumin-positive cells.
Interneuron Abnormalities in Neuron-Specific G93A-SOD1 Mice
To determine whether neuron-specific mutant SOD1 expression also is sufficient to trigger interneuron abnormalities, we examined homozygous T3T3 mice that express G93A-mutant SOD1 in neurons throughout the CNS, including spinal motor neurons and interneurons (34). Hemizygote T3 mice do not develop clinical and pathological signs of motor abnormalities up to 2 years, whereas homozygous T3T3 mice develop a motor neuron disease strongly resembling that in G1del mice. The age of onset in T3T3 mice is higher (>54 weeks) and considerably more variable (54 to >104 weeks) than in G1del mice, likely because of lower mutant SOD1 expression levels in motor neurons (34). ATF3 immunohistochemistry revealed ATF3-immunoreactive motor neurons and interneurons in spinal cord of symptomatic (n = 2) and end-stage (n = 5) T3T3 mice (Figs. 5A, E, I). ATF3 staining was also observed in the spinal cord of 2-year-old presymptomatic T3T3 mice (n = 2) but not in 40-week-old T3T3 mice (n = 4). Hemizygote T3 mice and nontransgenic mice showed ATF3 staining in some motor neurons at 2 years (Fig. 6) but not at 40 and 70 weeks (not shown). Old T3 and nontransgenic mice did not show ATF3-positive interneurons (Fig. 6).
Immunostaining with an anti-phospho (ser63)-c-Jun antibody resulted in motor neuron and interneuron staining patterns that strongly resembled those obtained with anti-ATF3 antibody. Thus, phospho-c-Jun-positive motor neurons and interneurons occurred in spinal cord of 2-year-old, symptomatic, and end-stage T3T3 mice, whereas no or sporadic-labeled motor neurons occurred in spinal cord of nontransgenic, T3, and 40-week-old T3T3 mice (Figs. 6B, F). Immunohistochemistry showed the presence of intensely ubiquitin-immunoreactive dendrites and cell bodies in the spinal cord of symptomatic T3T3 mice and 2-year-old but not 40-week-old T3T3 presymptomatic mice. Ubiquitinated dendrites were in both the ventral horn and the intermediate zone, indicating that the ubiquitinated aggregates were in interneurons in addition to motor neurons (Fig. 6G). Direct evidence for death of interneurons in T3T3 mice was obtained by reanalyzing sections stained with a silver degeneration staining method that outlines dying neurons and their processes (34). This analysis revealed multiple argyrophilic interneurons (Figure, Supplemental Digital Content 2, ).
Symptomatic T3T3 mice showed reduced levels of GlyT2 immunoreactivity in the ventral horn but not in the dorsal horn (Fig. 6H). No changes in GlyT2 immunoreactivity occurred in spinal cord of 40-week-old T3T3 mice or T3 mice up to 2 years, indicating that reduced GlyT2 immunoreactivity correlated with the occurrence of other degenerative changes and clinical manifestations.
As in G1del mice, all symptomatic T3T3 mice showed ubiquitin-parvalbumin double-labeled dendrites and cell bodies (Figs. 7A, B), as well as parvalbumin-immunoreactive neurons with nuclear ATF3 staining (Fig. 7D). We also combined ubiquitin with GlyT2 staining, which enabled us to examine whether motor neurons with ubiquitinated aggregates were contacted by glycinergic boutons (Figs. 7B, C). Most ubiquitin-positive motor neurons (15/17 identified in lumbar sections from symptomatic T3T3 mice) were surrounded by GlyT2-immunoreactive boutons (Fig. 7C), whereas ubiquitinated motor neurons in lumbar sections from 20- to 25-week-old G1del mice generally showed a normal pattern of GlyT2-immunoreactive boutons (not shown). Quantitative analyses to identify subtle losses of GlyT2-immunoreactive boutons (as previously demonstrated for high copy G1 mice [41) were beyond the scope of this study.
Dendritic Ubiquitin Pathology and Microglia Activation in the Intermediate Zone After the Ventral Horn in Neuron-Specific G93A Mice
As indicated above, no ubiquitin pathology was observed in spinal cord of 40-week-old T3T3 mice, whereas in presymptomatic and symptomatic T3T3 mice, ubiquitinated aggregates were always in both motor neurons and interneurons. Thus, it was not possible to determine whether ubiquitin pathology appeared at an earlier time point in motor neurons than in interneurons, as in G1del mice (Fig. 5A). To address this question, we used T3hSOD1 mice, which develop a considerably more predictable disease phenotype (34). The T3hSOD1 mice take advantage of the fact that coexpression of high levels of wild-type SOD1 (via a yet poorly understood mechanism) facilitates onset an progression of disease, and lowers the threshold of the concentration of mutant SOD1 required to cause disease within the normal lifespan of mice (48, 59, 60). T3hSOD1 mice develop signs of muscle weakness starting from 1 year of age, but dendrites with ubiquitinated aggregates appear before the age of 20 weeks, long before clinical onset (34). Double labeling of ChAT and ubiquitin showed that the large majority of these ubiquitinated dendrites were cholinergic (Figs. 7E, G), whereas only a few dendrites were ChAT-negative (Figs. 7F, G). A higher level of ChAT-negative ubiquitinated dendrites occurred in a later presymptomatic stage at 35 weeks (Fig. 7G). These data indicate that in T3 neuron-specific G93A mice (as in G1del mice), dendritic ubiquitin pathology occurs first only in motor neurons, and later, albeit still in a presymptomatic stage, also in spinal interneurons. Similarly, as in G1del mice, neuron-specific G93A mice show microglia activation first in the motor columns and subsequently in the intermediate zone (Figs. 7H-K). Taken together, these data indicate that neuron-specific and ubiquitous G93A-SOD1 mice develop similar interneuronal pathological features, specifically after the onset of the disease in motor neurons.
We have shown that SOD1-ALS transgenic mice expressing G93A mutant SOD1 lose the markers of glycinergic and GABAergic inhibitory neurons GlyT2 and GAD65/67 mRNA and protein in the spinal cord, indicating their involvement in the disease. These interneurons develop 2 key pathological hallmarks that also characterize degenerating motor neurons: ubiquitinated inclusions and expression of the stress transcription factor ATF3. These findings complement previous studies showing the degeneration of spinal interneurons in sporadic ALS patients and in transgenic SOD1-ALS mice (17, 38, 39, 61). We also show that ubiquitin pathology appears first in motor neurons (before 15 weeks of age), and subsequently in interneurons (onset between 15 and 20 weeks of age). This is consistent with the sequential expression of ATF3 in motor neurons and interneurons in G93A-SOD1 mice (45).
Because ubiquitin aggregates occur first and more frequently in dendrites rather than in the cell soma of neurons (34, 45) and because GlyT2 and GAD67 mRNA and protein are not present in dendrites, we used parvalbumin as a substitute marker for the dendrites of inhibitory interneurons. Although there is compelling evidence that parvalbumin in spinal cord intermediate zone is predominantly associated with inhibitory neurons (Figure, Supplemental Digital Content 1, ) (57), it cannot be excluded that some parvalbumin-immunoreactive dendrites in the intermediate zone belong to excitatory neurons. Nevertheless, our data show that in 15-week-old G1del mice, ubiquitin staining is only associated with motor neuronal somata and dendrites (identified by ChAT staining),while at 20 weeks, approximately half of the ubiquitin-stained dendrites are ChAT-negative and are located in the intermediate zone, indicating that they are not of motor neurons. In view of our data that at least 50% of the ubiquitin-immunoreactive intermediate zone neurons express GlyT2 or GAD67 mRNA in a later phase of disease, it is likely that a significant proportion of the non-motor neuron ubiquitinated dendrites at 20 weeks belong to inhibitory interneurons.
The onset of degenerative changes in interneurons precedes the onset of behavioral motor manifestations and most of the motor neuron degeneration (45), raising the question as to whether interneuron degeneration contributes to further degeneration and motor signs. Our data indicate that the overall level of GlyT2 and GAD immunoreactivity in the ventral horn is unaltered at 20 weeks, but subtle changes at inhibitory synapses on individual motor neurons cannot be excluded. Several studies suggest that changes in synaptic inputs to motor neurons contribute to motor neuron degeneration and motor manifestations of ALS (42, 62-65). Two recent studies also indicate that there are subtle losses of inhibitory synapses innervating motor neurons in presymptomatic high-copy G1 mice (41, 43), and compensatory sprouting of inhibitory glycinergic axons may also occur (41). These and our present data support the notion that early degenerative changes in inhibitory interneurons may contribute to the degeneration of motor neurons in SOD1-ALS mice, for example, by facilitating excitotoxic stress, one of the potential factors contributing to motor neuron degeneration in SOD1-ALS (28, 37, 42).
Ubiquitin pathology and ATF3 expression appeared in interneurons several weeks after their appearance in motor neurons. This raises the possibility that interneuron degenerative changes somehow are a consequence of motor neuron degeneration. A linkage between interneuron and motor neuron degeneration is also suggested by the fact that GlyT2 and GAD67 immunoreactivity is specifically reduced in the ventral horn, indicating the selective involvement of inhibitory interneurons innervating motor neurons. Longitudinal analyses of muscle denervation in SOD1-ALS mice have shown that large motor neurons that innervate type IIB muscle fibers and form the forceful fast-fatigable (FF) units degenerate before clinical onset (66, 67). The time of appearance of ubiquitin pathology and the onset of ATF3 expression (at 13-15 weeks) coincide with the onset of early molecular changes in FF motor neurons, as identified in gene profiling experiments of motor neuron subtypes (13). Instead, fast fatigue-resistant and slow motor neurons become involved in later phase of disease (13, 66, 67). Together, these data indicate that the disease in SOD1-ALS mice starts in FF motor neurons and subsequently involves other motor neurons and at the same time spinal interneurons.
The appearance of interneuron abnormalities after the onset of motor neuron degeneration, combined with evidence that mutant SOD1 in glia contributes to disease progression after initiation of disease in motor neurons, may indicate that mutant SOD1 in glia plays an important role of the spreading of disease to interneurons. However, we found that neuron-specific G93A-SOD1 mice develop the same interneuronal abnormalities as in ubiquitous mutant SOD1-expressing G1del mice, indicating that glial mutant SOD1 expression is dispensable for triggering spinal interneuron degeneration. One proposed glial mechanism is that microglial activation triggered by motor neuron degeneration could cause or facilitate the degeneration of other neurons (28, 68). Some evidence suggests that toxic microglial actions requires microglial mutant SOD1 expression (31, 69), but mutant SOD1-independent actions of activated microglia may also occur; indeed, deleterious actions of microglia have been proposed for multiple neurodegenerative conditions (70). However, a major role of activated microglia in triggering early interneuron alterations in SOD1-ALS mice seems unlikely in view of our data that microglial activation is still limited at the time of the first appearance of interneuron dendritic ubiquitin pathology (i.e. between 15 and 20 weeks in ubiquitous G1del mice and 20 and 35 weeks in neuron-specific T3hSOD1 mice).
An alternative mechanism that could explain the appearance of interneuronal pathology subsequent to motor neuronal pathology is transcellular transmission of protein aggregation by seeds of aggregated species (71). Recent evidence indicates that such a mechanism may contribute to the spreading of pathology in neuronal protein aggregation disorders, including synucleinopathies and tauopathies (71). Furthermore, seeding-like properties have been demonstrated for mutant SOD1 (72). Hence, a possible scenario that is compatible with our data would be that mutant SOD1 aggregates released by degenerating motor neurons are taken up by interneuronal nerve endings and retrogradely transported to their parent cell to trigger or facilitate further SOD1 aggregation leading to the ubiquitinated inclusions and, eventually, cell death.
The availability of SOD1 transgenic mice models has enabled the precise dissection of the pathological progression of disease, indicating the involvement of different cell types at different disease stages as well as different cell autonomous and non-cell autonomous disease mechanisms. A central question is whether similar mechanisms also operate in sporadic ALS, which is predominantly characterized by TDP43 aggregates. The recent availability of new mouse models, such as TDP43 transgenic mice that reproduce aspects of this TDP43 pathology (73-76), may help resolve this question and further unravel the disease mechanisms underlying ALS.
The authors thank Drs N. Nelson and N.J.K. Tillakaratne for GlyT2 and GAD67 cDNA constructs and Dr H.U. Zeilhofer for providing GlyT2-GFP transgenic mice.


text:
Winch solenoid question
I want to build in-cab controls and move the winch solenoids under the hood of my '87 YJ. I was given a Superwinch LP8500 that's not working and has no controller... I know, I know, use it as a door-stop and buy a Warn. Look, I can't squeeze that kind of money out of my budget right now. So, I'm trying to fix what I got.
A local electric motor shop checked the winch out for me. The shop says the motor works well but the solenoid is bad. So far, the cheapest solenoid I have found is $75 + shipping. I have been told that I can use 2, $15, Ford style solenoids to replace the $75 factory one. Id like to go this route if I can.
I searched this forum and found some great ideas for the in-cab controls. But, I have yet to find a diagram or photo of a Superwinch motor, showing which post is which. The Superwinch website isnt much help, only an owners guide rrrr!
I know there has to be a Jeeper out there that can do this in their sleep. Looking forward to seeing your ideas!
Now if I could only sell a kidney and buy a Warn...
Seems like if you hook it up and it runs the opposite direction you want it to, hook it up the other way. Thats basically what the switch and solenoids do.
I went to the Superwinch web site and they have a solenoid for $25.35 plus shipping.
Also, I have an Engo winch and their manual has a pretty good wiring diagram (page 5), maybe this can work for you: http://mediacdn.shopatron.com/media/...pdf?1299773251
Hope this helps.
Thanks, I saw that on the Superwinch site also. But, the dead solenoid on my winch looks like the $70+ one. Are they that different? Wow, the Engo diagram is much better then Superwinch's! I'll compare it to my winch in the daylight tomorrow.
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Effects of Beech Bark Disease on Aboveground Biomass and Species Composition in a Mature Northern Hardwood Forest, 1985 to 2000
Jodi A. Forrester, Gregory G. McGee and Myron J. Mitchell
The Journal of the Torrey Botanical Society
Vol. 130, No. 2 (Apr. - Jun., 2003), pp. 70-78
Published by: Torrey Botanical Society
Stable URL: http://www.jstor.org/stable/3557531
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We examined changes in species composition and aboveground biomass of a maturing, Adirondack northern hardwood forest over a 15-yr period in order to assess the potential effects of beech bark disease on the structure and nutrient cycling processes of the stand. Aboveground biomass of living and dead stems, and annual litter production were estimated in 1985 and 2000 within a 1.96 ha area using total stem inventories (≥ 5 cm dbh). A comparison of speciescomposition and biomass of the forest, as well as mortality and growth rates between the two sampling years are presented. The aboveground biomass of living stems decreased from 209.8 Mg · ha-1 in 1985 to 195.7 Mg · ha-1 in 2000. Acer rubrum was the only species to increase in biomass. Fagus grandifolia and Acer saccharum were the most important species in 1985 and 2000, although F. grandifolia increased in importance while A. saccharum decreased in importance during this time. Large stems of F. grandifolia experienced high mortality rates, but were balanced by ingrowth of smaller stems. In 2000, 58% of F. grandifolia exhibited no to minor signs or symptoms of beech bark disease, while 28 and 13% displayed moderate and high signs of infection, respectively. Ninety-nine percent of the uninfected stems were < 15 cm dbh. Although total aboveground biomass decreased during the 15-yr period, annual litter production remained constant. Furthermore, the increase in F. grandifolia importance value did not substantially alter the lignin content of litter. We conclude that the presence of BBD is hastening the onset of uneven-aged conditions in this second-growth stand but may not lead to large changes in forest nutrient cycling processes based on the lack of change in total lignin content of the leaf litter layer. Currently it appears the canopy will continue to be dominated by shade tolerant species, although the importance of F. grandifolia and A. saccharum are changing especially with respect to the relative importance of different size classes.
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Item #: 2-54122
Our Price: $258.84
Availability: Usually leaves our warehouse within 1-2 business days
Circuit boards are shipped in sealed plastic. Once the sticker is broken, the circuit board is neither returnable nor exchangeable.
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I ordered a few items for my hot tub and was very pleased with the parts and the price and speed of delivery.I am so happy i found the hot tub works .I wanted to know how or if i...read more


text:
Blue Ridge is located in Texas.
Blue Ridge, Texas has a population of
Blue Ridge is
family-centric than the surrounding county with
46.34% of the households
containing married families with children.
The county average for households married with children is 45.86%.
The median household income in Blue Ridge, Texas is
The median household income for the surrounding county is $83,238
compared to the national median of $53,046.
The median age of people living in Blue Ridge is
39.5 years.
The average high temperature in July is 95
degrees, with an average low temperature in January of 31.9 degrees.
The average rainfall is approximately 38.8 inches
per year, with 1.4 inches of snow per year.
Real Living Real Estate Group
8920 Directors Row
Dallas, TX
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text:
When Ruchi Sanghvi arrived for her first job interview at Facebook's headquarters, no one was there. It was the fall of 2005 and when she reached the startup's graffiti-covered offices in downtown Palo Alto at noon, they were empty.
Scooped by
Giorgio Fontana
onto La scimmia nuda e Internet [ cyberantropologia ]


text:
Keizer Police Department
Media Release
4900 block 13th Avenue North Keizer, Oregon
Case Number:
16 - 1955
Date & Time:
Sunday, May 15, 2016 at 2:17 AM
The Keizer Police Department responded to a drive-by shooting that was reported at 2:17 a.m. this morning in the 4900 block of 13th Avenue North in Keizer where an unspecified number of gunshots were fired at a residence and a vehicle that was parked in the driveway. The residence was occupied by four individuals at the time of the shooting, none of whom were injured. The living room window was damaged as were the back window and front windshield of the vehicle that was parked in the driveway.
No arrests have been made and the incident remains under investigation. Anyone with information is asked to contact Keizer Police Department Detective Arsen Avetisyan at 503-390-3713 Ext. 3514.
This media release was disseminated by Deputy Chief Jeffrey Kuhns on Sunday, May 15, 2016 at 5:22 PM. If you need further information regarding this incident, please contact our Public Information Officer, Deputy Chief Jeffrey K. Kuhns, during normal weekday business hours. If this is an after hours, weekend or holiday inquiry, please contact the author or the On-Duty Supervisor at the authors phone number and extension listed below or through our dispatch center at 503-390-2000.
Deputy Chief Jeffrey Kuhns
503-390-3713 ext. 3480


text:
By Emily Bazar, USA TODAY
Imagine Archbishop Desmond Tutu in the kitchen, his apron splattered with flour and curry paste. What might the Nobel Peace Prize winner from South Africa be making?
A mix of chicken, potatoes, peppers and tomato puree that bears his name and gets its kick from a teaspoon of Tabasco.
Playwright Eve Ensler likes to make a five-ingredient, garlic-heavy dish that calls for whole wheat, not regular, pasta.
And actress Mia Farrow, who loves her "Awesome Pancakes" so much she makes them every day, credits a special ingredient: vinegar. "It works like egg whites in raising the pancakes and making them fluffy," she says.
Farrow, Tutu and Ensler reveal culinary secrets in Ingredients for Peace, a new cookbook that offers recipes from more than 60 Nobel Peace Prize laureates and peace activists.
'CLEAVING': Julie of 'Julie & Julia' writes again
The recipes range from the familiar (hollandaise sauce) to the lesser known (Timorese fish balls).
They come from well-known figures, such as Farrow, and grass-roots activists, such as West Virginia Catholic school teacher Nora Sheets and her students, who advocate against land mines and contributed a recipe for Whirled Peas.
Book to support women's initiative
"You need a little of this, a dash of that, a sprinkle of salt or whatever, to make a recipe come together and be really good," says 1997 Peace Prize laureate Jody Williams, who won for her work to ban land mines. "The same thing with peace. ... You need justice, you need equality, you need human rights and more."
Williams and her stepdaughter Emily Goose are the forces behind the cookbook, which they self-published through Lulu.com in September. The book, $18.64 on Lulu, also will be available on Amazon.com in mid-December.
Half of the sales will be donated to the Nobel Women's Initiative, which supports women working for peace globally.
The book was originally Goose's senior high project at Fredericksburg Academy in Virginia. Goose, 19, was editor of the yearbook and wanted to build on her layout experience. Williams had tried, and failed, to form a recipe group among activist friends.
It didn't hurt that they both consider themselves foodies.
"This wasn't going to be just about design," says Goose, now a first-year student at Smith College in Massachusetts. "It was me learning about all of these peace advocates ... and that one person can make a difference."
Williams solicited recipes from friends over eight months and has tried about one-third of them.
Tutu gave Williams a recipe for Tutu Chicken, but she says it may have come from his wife. "I do not get the impression that Tutu spends 'quality time' in the kitchen," Williams says.
Williams contributed two recipes: South Asian Meatloaf, seasoned with the spice blend garam masala, and Fantastic Bourbon Apple Pie. Goose contributed recipes for hollandaise sauce and chocolate mousse.
She chose hollandaise sauce because another contributor, Betty Williams, calls for it in her Salmon in Pastry recipe, but didn't provide instructions. Williams, co-winner of the Peace Prize in 1976 for her work to end sectarian violence in Northern Ireland, says she gives in to Popeyes chicken cravings on visits to the USA but cooks every day at home in Ireland.
"When you're doing this work, you're on a stage all the time, you're eating in restaurants, you're staying in hotels," she says. "The only time I get to feel normal is in my own kitchen."
Farrow guests love the pancakes
Farrow, too, cooks every day when home. She says she was "very trepidatious" before trying the vinegar-egg substitution for her pancake recipe but says it doesn't change the flavor.
"When I have guests, they all love me much more after the pancakes," says Farrow, a UNICEF goodwill ambassador who raises money for children whose lives have been affected by violence. She has made the conflict in Darfur a primary focus.
Some recipes, including Farrow's, give measurements in dollops, handfuls and even "lots of" (Ensler's Activist Pasta).
Carrie Bachman, a cookbook publicist who doesn't represent this book, says people are interested in cookbooks by famous figures, but she's cautious when recipes haven't been thoroughly tested and measurements are inexact.
This book may be more of an "armchair cookbook," one that people read, but not necessarily cook from, she says.
"If I'm going to invest the time, especially when it comes to baking, I really would only want to bake from people who are professional bakers," she says.
Jody Williams likes "the majority" of the recipes she has tried but says the book is as much about the message as the meals: "I'd like to see people think about the different people who contributed ... and understand what peace really is."
Guidelines: You share in the USA TODAY community, so please keep your comments smart and civil. Don't attack other readers personally, and keep your language decent. Use the "Report Abuse" button to make a difference. Read more.


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The integrating theme of the research proposed in this Program Project Grant (PPG) application is that depression must be disaggregated into its constituent parts to understand why it is a prognostic risk for major adverse cardiac events and/or all cause mortality (MACE/ACM) in patients after an Acute Coronary Syndrome (ACS). The emergence of genetics and genomics research over the past decade has created great excitement in its promise for increasing our ability to prevent and more powerfully treat the major chronic diseases such as CHD. In particular, genetic studies have enormous potential to improve our understanding of how depression increases risk for MACE/ACM, or to determine if there is a common genetic vulnerability for both of these complex phenotypes. This potential remains to be fully realized, in part because of our current inability to identify reliable, enduring, and conceptually distinct depression components or intermediary phenotypes that mark patients at risk. We propose in this PPG that studying depression intermediary phenotypes will advance the field by: a) improving prognostic risk prediction;b) identifying specific targets for candidate genes underlying the risk conferred by depression;and c) identifying targets for treatments designed to ultimately improve cardiac and mortality outcome. It is also essential to gain an understanding of the mechanisms through which the depression intermediary phenotypes act. We propose to test if the Melancholic Depression and Incident Depression, two intermediary phenotypes with a distinct etiology and course, are uniquely predictive of 1-year MACE/ACM (Project 1), and if promising behavioral mechanisms (Project 2) and biological mechanisms (Project 3) mediate/confound the association between these depression intermediary phenotypes and MACE/ACM. In Project 1 we will enroll 1,400 ACS patients, phenotype their depression (i.e., Melancholic, Incident, or "Other"), and follow them for major adverse cardiovascular outcomes and all-cause mortality for one year;in Project 2 we will enroll 1,134 patients from Project 1 and electronically monitor their aspirin, clopidogrel, beta blocker and statin medications for up to one year;we will also assess a number of other promising behavioral mechanisms. In Project 3 we will enroll 1,260 ACS patients from Project 1 and assess their inflammatory levels and platelet aggregation at hospitalization, when medications are known to be ingested, and one month later, when non-adherence may affect these biological mechanisms. This proposed PPG will address the NHLBI Strategic Plan, Goal #2, by identifying those depression phenotypes and mechanisms that place ACS patients at excess cardiac and mortality risk. Doing so will identify potentially more successful and personalized intervention strategies, and accelerate our ability to discover the genes implicated in depression's risk for cardiac disease and death.
|Denton, Ellen-Ge D; Shaffer, Jonathan A; Alcantara, Carmela et al. (2015) Hispanic residential ethnic density and depression in post-acute coronary syndrome patients: Re-thinking the role of social support. Int J Soc Psychiatry 61:225-35
|Edmondson, Donald; Green, Philip; Ye, Siqin et al. (2014) Psychological stress and 30-day all-cause hospital readmission in acute coronary syndrome patients: an observational cohort study. PLoS One 9:e91477
|Kronish, Ian M; Rieckmann, Nina; Burg, Matthew M et al. (2014) The psychosocial context impacts medication adherence after acute coronary syndrome. Ann Behav Med 47:158-64
|Whang, William; Peacock, James; Soliman, Elsayed Z et al. (2014) Relations between depressive symptoms, anxiety, and T Wave abnormalities in subjects without clinically-apparent cardiovascular disease (from the Multi-Ethnic Study of Atherosclerosis [MESA]). Am J Cardiol 114:1917-22
|Shaffer, Jonathan A; Diaz, Keith; Alcántara, Carmela et al. (2014) An inexpensive device for monitoring patients' weights via automated hovering. Int J Cardiol 172:e263-4
|Denton, Ellen-ge; Green, Philip; Newman, Jonathan et al. (2014) The benefits of neighborhood racial diversity: neighborhood factors and its association with increased physical activity in ACS patients. Int J Cardiol 173:517-8
|Wasson, L T; Shaffer, J; Alcántara, C et al. (2014) The association of posttraumatic stress disorder and quality of life during the first year after acute coronary syndrome. Int J Cardiol 176:1042-3
|Davis, Nicholas; Lee, Melissa; Lin, Albert Y et al. (2014) Postoperative cognitive function following general versus regional anesthesia: a systematic review. J Neurosurg Anesthesiol 26:369-76
|Alcantara, Carmela; Peacock, James; Davidson, Karina W et al. (2014) The association of short sleep after acute coronary syndrome with recurrent cardiac events and mortality. Int J Cardiol 171:e11-2
|Burg, Matthew M; Meadows, Judith; Shimbo, Daichi et al. (2014) Confluence of depression and acute psychological stress among patients with stable coronary heart disease: effects on myocardial perfusion. J Am Heart Assoc 3:e000898
Showing the most recent 10 out of 48 publications


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A spokesman said that out of 2 billion passengers screened nationwide since 9-11, there have been only 110,000 abuse complaints.What does that mean? 110,000 complaints sounds like a hell of a lot of complaints. Sure, it's a small fraction of the total screened, but what is a reasonable number for things like this? Keep in mind that most of those screened just take off their shoes and belt and walk though the metal detector. What percent of those taken aside for special attention complain? What percentage of complaints do police from interactions with the public? What percentage do private security guards get? Is the TSA number high or low with respect to these similar professions? And are there certain screeners who get a lot more complaints than others?
'Right', as it is used these days, means classical liberalism plus Nazism. It means all those things I listed (Bill-of-Rights type thinking: individualism, rule of law, separation of powers, free trade, free market, Rights-of-Man, limited government) In other words, it is a meaningless definition.
Now, why should anyone define a term to include classical liberal ideals (individual liberty, limited government, rule of law, separation of powers, etc.) with Nazism? Answer: to denigrate classical liberal ideals. ...
It would be like defining 'mammals' to mean 'warmblooded organisms with hairy skin who give birth live to their young PLUS snakes and black widow spiders.' That way, if you cannot criticize mammals for anything, you can always claim that they are coldblooded legless creatures with poisonous fangs who eat their mates and do not care for their young.
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Chicken Florentine
|Whole chicken breasts
|2 Pound, skinned, boned, and halved (Use Free Range Chicken, 2 Pieces, 1 Pound Each)
|Olive oil
|1⁄4 Cup (4 tbs)
|680 Gram, washed, dried, stemmed, and chopped (2 Bunches)
|To Taste
|Black pepper
|To Taste, freshly ground
|Monterey jack cheese
|1⁄2 Cup (8 tbs), freshly grated
|Heavy cream
|1 Cup (16 tbs)
|Fresh basil
|2 Tablespoon, minced
|Fresh chives
|2 Tablespoon, minced
1. Preheat the oven to 375° F.
2. With a sharp and pointed knife, make an incision in the thickest end of the chicken breast. Do not cut all the way through.
3. Take a sauté pan and heat 1 teaspoon of oil into it.
4. Add spinach, salt, and pepper and sauté the ingredients against a medium flame for nearly 2 minutes or until spinach is wilted.
5. Remove the wilted spinach to a plate to get it cooled against room temperature.
6. Grasp out the water out of spinach and mix cheese in it.
7. Place the chicken breasts on a work surface.
8. Fill the pockets with spinach mix.
9. Roll each of the chicken meat in a plastic warp to make sausage shape.
10. Once the meat pieces take sausage shape, remove the plastic wrap.
11. In a sauté pan, heat the left over oil.
12. Sauté the chicken breasts until the meat color turns golden brown on all sides, cook for t 5 minutes.
13. Transfer to a baking dish and bake for 10 minutes, or until done.
14. Pour off any excess oil from the pan. Deglaze the pan with the cream, cooking over medium-high flame until it is slightly reduced.
15. Lower the heat and add the herbs. Stir and cook for 1 minute more. Add salt and pepper to make taste.
16. Slice the chicken breasts in rounds on the diagonal.
17. Pour sauce on four warmed plates. Place chicken pieces on the sauce
18. Serve the food to enjoy.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size
Calories 674 Calories from Fat 381
% Daily Value*
Total Fat 44 g67.3%
Saturated Fat 19 g95.2%
Trans Fat 0.1 g
Cholesterol 228.7 mg76.2%
Sodium 492 mg20.5%
Total Carbohydrates 9 g2.8%
Dietary Fiber 4.1 g16.5%
Sugars 0.9 g
Protein 62 g124.9%
Vitamin A 354.8% Vitamin C 94.3%
Calcium 35.3% Iron 37.6%
*Based on a 2000 Calorie diet


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This review is from: BYTECC SVST-15 15 ft. SVGA w/3.5mm Stereo male to male Cable
Pros: Good quality video and sound. Works well to hookup a laptop to a HDTV.
Cons: Audio cable was not long enough to reach the audio input on my laptop. Only buy this if your SVGA input is right next to your 3.5mm audio input. If they are not on the same side of your laptop don't buy this unless you have an 3.5mm extender cord.
Other Thoughts: Should have read all the reviews. Common problem, looks like Ii'll pay another $5 for an extender.READ FULL REVIEW


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This is a small, powerful, turret pistol. Stats: Range 70 feet. True trigger Trigger guard Sturdy Compact Has a turret Looks Sick Please dont hate on this ...
Credit for the parts list go to: KnexBuilderFreak and KnexSuperBuilderFreak!!!!!THANKYOU!!!!! Connectors: Yellow: 18 Green:5 Red: 5 White: 7 Orange: 6 Light Grey: 19 Grey: 32 Blue: 2 ...
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Replace the white connector with a yellow!!!!!!! OMG
Finishing. Also don't forget to add a band that goes from the trig to some spot so that the trigg moves intothe right place
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On Mar 28, 2008, at 10:48, David Lutterkort wrote:
* there were no tests for the two new methods. In general, try to add/expand the tests in tests/tc_connect.rb when you add to the bindings, though that is highly dependent on what the test driver supports. In this case though it was pretty easy. Thanks again for doing this, and I am looking forward to the virDomainPinVcpu patch.
Attached is a patch, implementing Domain.pin_vcpu method. I have tested it locally and it works. Here is my test code: require 'libvirt' conn = Libvirt::open("") dom = conn.lookup_domain_by_name("centos52") dom.pin_vcpu(0,[2,3])After running it, I can verify via virsh that it does pin CPUs correctly:
virsh > vcpuinfo centos52 VCPU: 0 CPU: 2 State: blocked CPU time: 40.8s CPU Affinity: --yy However, when I try this method from test/tc_connect.rb it fails: # rake test (in /home/lord/src/ruby-libvirt)Loaded suite /usr/lib/ruby/gems/1.8/gems/rake-0.7.3/lib/rake/ rake_test_loader
Startedlibvir: error : this function is not supported by the hypervisor: virDomainPinVcpu
E.... Finished in 0.006319 seconds. 1) Error: test_domain(TestConnect): SystemCallError: unknown error - libvir call virDomainPinVcpu failed ./tests/tc_connect.rb:113:in `pin_vcpu' ./tests/tc_connect.rb:113:in `test_domain' 5 tests, 60 assertions, 0 failures, 1 errors rake aborted!Command failed with status (1): [/usr/bin/ruby -Ilib:ext/libvirt "/usr/ lib/...]
(See full trace by running task with --trace)I will appreciate if somebody could help me to figure out why it is happening.
I might need to implement couple more methods, so if there are any remarks on
my code, I will appreciate your feedback. Sincerely, Vadim
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Peary Or Cook: Who Discovered The North Pole?
Almost simultaneously two men claimed to have attained a goal that explorers had striven toward for centuries. There were strong hints that one of them was an impostor
April 1962 | Volume 13, Issue 3
The main party had been on the ice about two weeks when the supporting divisions finally began turning back. First to return was a party commanded by Dr. J. W. Goodsell, expedition surgeon, which on March 14 had reached latitude 84 degrees 29 minutes north. Another assistant, Donald B. MacMillan, proceeded only a short distance farther before turning back on March 15. Five days later, Borup, who had rejoined Peary with additional supplies, led a party back from 85 degrees 23 minutes north. On March 26, a party commanded by a young Cornell professor, Ross Marvin, returned from 86 degrees 38 minutes.
The last supporting group to turn back was Bartlett's. At 3 P.M. on April 1 it departed for land from 87 degrees 46 minutes 49 seconds—farther north than anyone else had yet traveled. As he left, Bartlett called out to Peary, "Good-by, good luck."
Peary watched the captain and his two Eskimos start southward and finally disappear behind ice hummocks. Then he turned again toward the north. This was the moment for which he had been waiting: the Pole was apparently within reach, and he had with him a picked group—the Negro Henson, four Eskimos, forty of the best dogs, and five sledges. After a short sleep and a generous breakfast Peary departed soon after midnight, leaving the others to load the sleds and catch up.
The morning of April a was superb for traveling. The sky was a clear, brilliant blue, the temperature minus twenty-five degrees. The journey now turned into a dash. The nearer Peary got to the Pole the more possessed he became, rousing his men after brief naps and urging them to quicken their pace.
Then, on April 4, Peary encountered a lead 100 yards wide, bridged only by young ice. He observed it bend slightly underfoot, as salt-water ice will do, but he decided to risk a crossing. He himself went first, sliding his feet, keeping them far apart to distribute his weight. The others sent their sledges and dog teams across alone; then they followed. The last two men crossed on all fours. Peary later wrote: I watched them from the other side with my heart in my mouth—watched the ice bending under the weight of the sledges and the men. As one of the sledges neared the north side a runner cut clear through the ice, and I expected every moment that the whole thing, dogs and all, would go through … and down to the bottom.
They all crossed safely, however, and by 10 A.M. April 6, 1909, Peary estimated that he had reached his destination. While Henson and the Eskimos secured the dogs and commenced building igloos Peary began unloading and unpacking several bundles. Henson saw him pull out a small package from under his coat, open it, and unfurl the silk taffeta United States flag made by Mrs. Peary in 1898, which he always carried.
Peary fastened the flag to a staff and planted it firmly on top of his igloo. Then he took an observation of the sun and calculated his latitude—89 degrees 57 minutes ii seconds, about three nautical miles from the Pole. Henson saw him square his jaw.
"I was sure that he was satisfied," Henson wrote. "Feeling that the time had come, I ungloved my right hand and went forward to congratulate him."
At that moment, however, Peary pressed his hands over his eyes. He did not see Henson's extended hand. "The accumulated weariness of all those days and nights of forced marches and insufficient sleep, constant peril and anxiety, seemed to roll across me all at once," Peary remembered. "I was actually too exhausted to realize at the moment that my life's purpose had been achieved."
Later, he and Henson corroborated the first finding, and in his diary Peary could finally exult: The Pole at last. The prize of three centuries. My dream and goal for twenty years. Mine at last! I cannot bring myself to realize it. It seems all so simple and commonplace.
On September 1, 1909, Americans thrilled to some surprising news telegraphed from Lerwick, in the Shetland Islands. " REACHED NORTH POLE APRIL 21, 1908 …" it announced. The telegram was not signed by Robert E. Peary but by Frederick A. Cookthe quiet, pleasant surgeon of Peary's 1891 expedition. Apparently, if his telegram was to be believed, Cook had not only reached the North Pole, but had beaten the famous Peary to the goal—by almost a year.
In later messages Cook described his journey and explained his sixteen-month delay in announcing his feat to the world. From the northern tip of Axel Heiberg Island, 520 miles from the Pole, he had set out across the ice on March 18, 1908, with four Eskimos (two of whom accompanied him for only three marches), twenty-six dogs, and two sledges. After arriving at the Pole he raced southward. He said he reached land again on June 13, but that he lost his way and was forced to winter at Cape Sparbo, on Devon Island, living off the bleak country. Finally, in April, 1909—about the time Peary was reaching the Pole—he and the two Eskimos returned to their base in Greenland, traveled southward to Upernavik, and there arranged sea transportation to Copenhagen.
World adulation was still being showered on Cook when, on September 6, 1909, five days after the first message, another startling telegram came, this time from Indian Harbor, Labrador. " STARS AND STRIPES NAILED TO THE POLE ," the dispatch announced. It was signed, " PEARY ."
Headlines proclaimed the astounding news: within five days of each other, two men had claimed discovery of the North Pole. So erupted one of the most vicious controversies in the annals of exploration.


text:
|Property type
|Minimum Stay
|4 - 7 nights
This two story, two bedroom Caribbean Cottage is absolutely perfect for the budget-minded traveler.
Located on the south (Beach) side of the road in peaceful Colliers, on the East End of Grand Cayman, it is literally steps away from a pristine, white powdery sand beach. Nestled amongst lush, tropical plants, palm and coconut trees, there is a large balcony off the 2nd floor for reading or sun-tanning. Please note: There is NO VIEW of the ocean; you must walk around the big yellow house get to the beach. Basic, clean facilities (no marble foyers, dishwasher, nor Jacuzzi tub in the cottage).
The cottage is just 10 minutes from the new Shetty Hospital for Cardiac Care -- which is locate in High Rock. This is the perfect, low cost option if you have family who are in the hospital and you want to stay close by.
The 'Cayman Cottage by the Sea' is fully equipped with all linens, dishes and cooking utensils. It is ideal for 4 guests, but will hold a total of 5: 2 in each bed and one on a roll-away cot upstairs. (but would still be extremely crowded). A crib & high chair is available.
The configuration is as follows: queen sized bed on the main floor, a double bed upstairs, and a roll-away cot on the 2nd floor. There are two bathrooms; one on each floor and laundry facilities.
Everything you need for a short or long stay. At a price you can afford.
NOTE: VRBO Rates listed are incorrect due to the constantly fluctuating US Dollar. VRBO will not allow us to list the prices in USD since we are not located in the USA, even though we quote in USD and charge in USD. Please refer to our own website for the correct rates.
ALSO NOTE: In February & March, arrivals on Saturday or Sunday are preferred.
This would be known as "The Gardiner's Cottage" if staff were hired to tend to the grounds. The 1 bedroom apartment, which we call "Snuggle Beach" inside the large yellow house, would be the Maid's Quarters.
The location is perfect-- the East End really allowed us get away from cruise ship crowds and tourists. Yet, when we wanted to go to 7-mile beach, it's just a 45 minute drive. It does have several great snorkeling sites, too. We would recommend renting a car to stay at the cottage, then you are free to explore the entire island.
The cottage was perfect for our family of 4. It is super clean and it has a nice lay-out. Nadine, the owner, is wonderful-- we truly enjoyed talking with her and she was very helpful. She ensured that we had everything we needed-- she even got a pizza pan for us, so we could cook pizza for our kids! She is a fun person, and her energy spread to make our visit fun.
The beach out front is gorgeous-- with very few people in the area. The old dock makes a wonderful habitat to many species of colorful fish, so we snorkeled here daily! We highly recommend the Cottage by the Sea. (Hi, Nadine! Thank you!)
My stay at this cottage was everything I had hoped for and more. I thought I had reservations at a 7 mile resort for a conference I was attending only to find out an hour before leaving for the airport that they had been erroneously canceled. One of the best things to happen to me! I emailed Sandra after seeing the cottage was available. She called me and helped work everything out, sent directions, things to do, places to eat all before I had to leave. I loved getting away from seven mile beach each day as I drove back to the cottage and watched the scenery morph from kfc/shops/and resorts to a different pace and natural beauty. I sat on the beach each evening to see the stars and snorkeled the dock each chance I could (saw ~20 species of fish). I loved how cozy the cottage was, the fully-stocked kitchen, the upstairs balcony, and getting to know Nadine. She helped with everything (I had a hard time locking the door) and is simply a really interesting, cool person. Amazing experience...already convincing my husband to return with me.
We arrived at the Cottage on April 24 for our honeymoon. We had issues with the flight which made us arrive later than expected but we had no issues finding or getting into the cottage. Nadine came over and met us as we arrived. She had put beautiful flower petals around the cottage and left us a bottle of champagne and a welcome note. We were so very pleased with this place. The cottage was very clean and cozy. It had everything we needed. You truly are a few steps from the beach and the view is amazing. If you are wanting to go be around a ton of people and party all night...this is NOT the place for you. We saw maybe 5 other people on the beach the entire 6 days we were there and they were people that had walked from a resort down the road. Our desire was to go somewhere we could relax and chill as much as possible and this place did not disappoint. There was construction being done to the big house. Nadine apologized for the noise but we honestly never heard it. Speaking of Nadine - She is WONDERFUL!!! We felt like we left there knowing a new friend. She even sent us home with an amazing wood carved fish that we will forever cherish.
We recommend this place to anyone traveling to Grand Cayman. If we ever go back we know exactly where we will be staying. :-)
We had a truly wonderful time staying at the Cayman Cottage by the Sea. Sandra responded to all of our messages promptly, was extremely helpful, professional and pleasant to communicate with. The property manager Alden was amazingly prompt, capable and really helpful, too. The cottage is very homey, clean, well-stocked with plenty of towels and is in very close proximity to the beach. The location is fantastic if you want to avoid the typical resort crowds. One of our favorite things during our stay was watching the sunrise every morning from the main deck, which has a lovely view of the ocean, just steps away from our door. Sandra provided a great list of places to eat, things to do, and other helpful information about the area. We tried a couple of the restaurants she recommended, and the food and service was terrific. Based on our all-around great experience here, we highly recommend this property.
We've been to Cayman a few times but this is the first at the cottage. We stayed in May for 14 days...loved it! If you are looking for peace and quiet...this is where you need to be.
The owners are so kind, helpful and welcoming. Never ever had any issues with the owners or anyone near the cottage. Seemed safe, as we had no problems.
Anytime we needed anything- towels, etc-the owners- Gary & Nadine were helpful to provide for us. Learned about the island, lime trees, places to see, fishing on island, etc.
It is a cute and so quiet. Cottage had a grill, which we used a lot. Also had plenty utensils, cups and things. No issues there. Stocked nicely. Yes, had some bugs here & there but that's like any place. No major concern, and I'm a neat/clean freak. There's bug spray there you can use...No biggie.
We maybe saw 2-4 people on the beach...which we liked. If you like no crowds, no cruise ships or touristy type stuff, then this is good for you. It's simple and not overdone...that makes it authentic.
This side of island is quiet..rent a car- as we did, then you can go wherever. Best part is that you get a pretty beach and quiet all the time if you want it.
We already decided to go back and visit. Can't come soon enough!
My family rented this April 2015 for 10 days. I usually don't take the time to write a review on places that we stayed before, because we mostly stayed in great places. However, I think that you should read my comments before renting this cottage. You might think twice before. Here is why : According to the information and photos of the cottage on the website and sended by the manager (Sandra Tenus), we thought that this place will be perfect for our stay. She send great information and assured me that it is a safe location to rent. When we arrived to the location, there was nobody waiting for us to give us the keys. We wait for a while and even went to the owner's house, but nobody was there. We took the initiative to enter the house and by surprise, the door wasn't locked. So we figured that we could settle our stuffs.
At first glance, the house looks very clean and convenient. However, the shower on the ground floor did not have much water to take a shower. The shower on the 2nd floor was stronger, but the bathroom is made for small people. I'm not a tall person (4,11), but I had to sit in the tub to take a shower. Otherwise, my shoulders touch the ceiling. The kitchen is very convenient, but you should clean the ustensils and accessories, as there were few cockroaches in the kitchen drawers.
Also, do not trust the pictures of the exterior of the house. They do not correspond to reality. The beach located behind the house is not well maintained nor cleaned unlike the pictures on the website.
However, it is not these drawbacks which have ruined our vacation, is the money that was stolen from one of our suitcases. If you rent this place, make sure not only to lock the door of the house, but the windows and all your suitcases. Also, beware of the cleaning lady. Our money was stolen when she was in service. After this incident, we locked our suitcases with a lock before leaving home. When we got home, the lock of the suitcase that contained our money stolen, was damaged. As if someone forced the hole to insert the key and tried to break the lock (luckily I had a solid lock). The house had been no infringement. So we assume that the thief has a duplicate key of the house. Only the owners could have those keys or the cleaning lady. Again, she was on duty that day. The owners could not do anything because we did not have evidence to identify her. My advice, if you still want to rent this cottage, just be more careful than you usually do.
Cayman Cottage by the Sea is located in the peaceful community of Colliers, on the East End of Grand Cayman. We are 2 minutes away from a small strip mall with full convenience store, pizza parlour and bakery. Also located across from the convenience store is the famous Caymanian 'Barefoot Man' dinner show -- a must see when you're in Cayman. 2 minutes in the opposite direction takes you to one of the best dive operators on the island, 'Ocean Frontiers'. There are great restaurants in BOTH directions.
|Rate Period
|Weekend Night
High Season 2017
Jan 2 2017 - Mar 4 2017
7 night minimum stay
|Notes: Rates are based on 2 guests; add $11 per night, per additional guest
Spring Break 2017
Mar 5 2017 - Apr 8 2017
6 night minimum stay
|Notes: Rates are based on 2 guests; add $11 per night, per additional guest
High Season 2017
Apr 9 2017 - Apr 15 2017
5 night minimum stay
|Notes: Rates are based on 2 guests; add $11 per night, per additional guest
Low Season 2017
Apr 16 2017 - Sep 3 2017
4 night minimum stay
|Notes: Rates are based on 2 guests; add $10 per night, per additional guest
Off Season 2017
Sep 4 2017 - Oct 28 2017
4 night minimum stay
|Notes: Rates are based on 2 guests; add $8 per night, per additional guest
Low Season 2017
Oct 29 2017 - Nov 30 2017
4 night minimum stay
|Notes: Rates are based on 2 guests; add $10 per night, per additional guest
Dec. 2017
Dec 1 2017 - Dec 16 2017
5 night minimum stay
Christmas Holidays 2017
Dec 17 2017 - Jan 2 2018
7 night minimum stay
|Notes: Rates are based on 2 guests; add $15 per night, per additional guest
High Season 2018
Jan 3 2018 - Mar 10 2018
5 night minimum stay
My Standard Rate
5 night minimum stay
|Cleaning Fee
|$114 + Tax
|Tax Rate
Children 5 and under are free. Please do NOT include them when typing in the # of guests when you are getting a quote.
Prices quoted on VRBO are an approximation as VRBO will only allow one to put in the prices in the local currency and does not give the option for inputting in USD, so there may be fluctuation in the US rates based on currency exchange rates.
Prices include taxes, electricity, cleaning and service charges.