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Conceived and designed the experiments: SV BDS PV. Performed the experiments: SV GE DH OS MPD MV SVM VAM. Analyzed the data: SV GE DH OS MPD MV PV. Contributed reagents/materials/analysis tools: SV VAM. Wrote the paper: SV BDS PV.
Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC); however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I–associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling.
Parkinson's disease is the most common neurodegenerative movement disorder, and mutations in several genes are known to cause the disorder. A common theme among several PD–associated genes is a link to mitochondria, organelles that use their electron transport chain to generate ATP. One of the PD–associated genes encodes a mitochondrial kinase Pink1, but it is not known what the primary role of Pink1 is within the mitochondria. Indeed, loss of Pink1 function in cells and model organisms results not only in mitochondrial morphological defects but also in an enzymatic deficit at the level of the first protein complex in the electron transport chain. Here, we express yeast Ndi1p (an enzyme that can bypass electron transport in Complex I) and sea squirt alternative oxidase (an enzyme that can bypass electron transport in Complex III/IV) in pink1 mutant fruit flies and find that supplementing the mutants with Ndi1p, but not with alternative oxidase, results in significant rescue of multiple phenotypes. Conversely, mitochondrial morphological defects in pink1 mutants are rescued by genetically improving mitochondrial fission, but this manipulation fails to improve the enzymatic deficiency at the level of Complex I. Our data thus pinpoint an important mode of action of Pink1 at the level of Complex I, and this action at least in part precedes defects at the level of mitochondrial remodeling.
Parkinson's disease (PD (OMIM #168600)) is the most common neurodegenerative movement disorder [1]. While diverse processes including autophagy, apoptosis, oxidative stress and accumulation of protein inclusions have been implicated in the etiology of the disease, mitochondrial dysfunction appears to play a central role as well [2]–[4]. Mitochondrial toxins, such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) or rotenone, that block Complex I of the mitochondrial electron transport chain (ETC) cause clinical features reminiscent of PD in humans and are commonly used to create animal models of the disease [5], [6]. Furthermore, Complex I deficiency is often observed in neurons of PD patients [7] and mutations in genes causing familial forms of PD, including pink1 (PARK6, OMIM #605909, Gene ID: 65018), parkin (PARK2, OMIM #600116, Gene ID: 5071) and DJ-1 (PARK7, OMIM #606324, Gene ID: 11315) result in defects in mitochondrial morphology and/or function in model organisms [8]–[14]. Molecular genetic analyses of PD-associated genes will thus yield important insights into the mechanisms of PD.
Pink1 (CG4523 Gene ID: 31607) is a serine/threonine kinase involved in maintaining mitochondrial integrity [8]–[10] and loss-of-function mutants show hallmark mitochondrial defects including male sterility, an inability of most flies to fly as well as an inability to maintain synaptic transmission during intense stimulation, a deficit that can be rescued by supplementing synapses with ATP [15], [16]. The observation of larger clumped mitochondria in pink1 mutants suggests a model where Pink1 is involved in the clearance of dysfunctional mitochondria [17]–[21]. This is in line with experiments that show an alleviation of pink1-associated phenotypes by over-expression of Parkin (CG10523 Gene ID: 40336), an E3 ubiquitin ligase involved in mitophagy [8]–[10] and by the notion that loss-of-function pink1 trumps Parkin recruitment to mitochondria [18]. Furthermore, the morphological defects in pink1 mutants can be modulated by altering the levels of proteins involved in mitochondrial fusion or fission, and this is thought to facilitate mitophagy [17], [19], [21], [22]. However, these studies are not conclusive as functional defects in pink1 mitochondria at the level of Complex I have been observed in the absence of severe morphological alterations [15], [23] and such functional defects can eventually result in mitochondrial morphological alterations [24]–[26]. These results suggest an alternative model where functional defects in pink1 mutant mitochondria precede morphological alterations and mitophagy.
To determine if Pink1 acts to regulate ETC function we performed genetic studies with heterologous alternative enzymes that can bypass either Complex I, or Complex III and IV. Although each of the ETC complexes in flies (and humans) comprise numerous proteins (Complex I contains more than 40 subunits in humans and in flies), Saccharomyces cerevisiae Ndi1p (Gene ID: 854919) (UAS-NDI1) constitutes an alternative NADH oxidoreductase that transfers electrons from NADH to ubiquinone, delivering electrons to downstream complexes and therefore can be used to bypass electron transport in Complex I in higher order species [27], [28]. Similarly, 'Ciona intestinalis' alternative oxidase AOX (Gene ID: 3293227) is able to bypass the cytochrome c chain and Complexes III and IV by using electrons from ubiquinol to reduce oxygen [29]. While neither Ndi1p, nor AOX themselves transfer protons across the inner mitochondrial membrane, they may add to the proton motive force by facilitating the ubiquinone cycle, thus contributing to Complex III and IV mediated proton translocation upon expression of Ndi1p, or to Complex I mediated proton translocation upon expression of AOX. Illustrating this idea, expression of Ndi1p in Drosophila can rescue partial loss-of-function mutations in Complex I components and confers rotenone resistance [30], [31], and expression of AOX can rescue partial loss-of-function mutations in Complex III and IV components and confers cyanide resistance [32]. Hence, Ndi1p and AOX allow us to genetically dissect the ETC.
Here, we assay the role of Complex I in pink1-associated defects and find that Ndi1p can rescue pink1 phenotypes while AOX cannot. Similarly, loss-of-function of a Complex I component phenocopies many of the pink1 mutant phenotypes and these defects can also be rescued by expression of Ndi1p. In contrast, expression of Ndi1p fails to rescue parkin mutants, indicating that Ndi1p acts downstream or in parallel to Pink1 but upstream of Parkin. Further supporting this model, we do not find reduced enzymatic activity of Complex I in parkin mutants, and while modulating mitochondrial remodeling using Drp1 (CG3210 Gene ID: 33445) or Opa1 (CG8479 Gene ID: 36578) can rescue the defects in mitochondrial morphology in pink1 mutants, these manipulations do not rescue decreased enzymatic activity of Complex I observed in pink1 mutants. Thus, our studies suggest that the defects at Complex I in pink1 mutants are upstream of several of the events that lead to pink1-associated phenotypes.
Electron transfer activity of the mitochondrial multi-protein Complex I, NADH:ubiquinone oxidoreductase (EC 188.8.131.52) can be recapitulated by a single yeast protein, Ndi1p [31], [33], [34]. To determine whether the reduced respiratory chain activity in pink1 mutants reported previously [15] are an upstream defect in the mutants, we generated transgenic flies that harbor a UAS-Sacharomyces cerevisiae NDI1 construct allowing expression under the control of GAL4. Quantitative RT-PCR using RNA from flies that harbor the UAS-NDI1 transgene and the ubiquitously expressing da-GAL4 driver indicates that NDI1 expression levels in such animals is similar to that of an endogenously expressed Complex I component CG3446 (Figure S1A). In addition, quantitative RT-PCR using RNA of UAS-NDI1 bearing flies, in the absence of GAL4 also shows low but significant expression of NDI1 RNA, particularly in the male reproductive organ (Figure 1A, blue). Ndi1p confers rotenone insensitive NADH oxidoreductase activity in flies as the enzymatic activity of isolated Complex I in the presence of rotenone, a Complex I inhibitor, remains high in mitochondria from flies expressing Ndi1p, but is dramatically reduced in rotenone-treated mitochondria from control flies (Figure S1B) [30], [31]. Ndi1p expression in Drosophila is benign as ubiquitous expression (da-GAL4) does not lead to obvious behavioral or developmental abnormalities (Figure S1C, S1D). Ndi1p expression rescues lethality associated with RNAi-induced systemic loss-of-function of CG18624 (Gene ID: 31697), an evolutionary conserved Complex I component, and also the mitochondrial defects associated with loss of CG12079 (Gene ID: 38378), another conserved Complex I component (below). These data confirm that Ndi1p is functional.
Next, we generated pink1 mutant animals that express Ndi1p. While pink1 mutant males are sterile, low expression of Ndi1p is sufficient to completely revert this defect (Figure 1B). Given that pink1 is located on the X-chromosome, homozygous pink1 females are never observed. However, in the presence of NDI1, fertile homozygous pink1 female flies are obtained (Figure S2A), whose genetic makeup we confirmed by genomic PCR (Figure S2B). Furthermore, while in pink1 mutants morphological defects in spermatid mitochondria are apparent [10], [31], expression of Ndi1p also rescues these mitochondrial defects (data not shown). Thus, expression of Ndi1p rescues pink1-associated male sterility and mitochondrial morphological defects in the germline to a level indistinguishable from wild type controls.
Drosophila flight muscles require large amounts of metabolic energy supplied by mitochondria. In adult pink1 mutant flies, mitochondrial deficits lead to muscle degeneration and a severe defect to fly. While expression of Ndi1p using da-GAL4 does not affect flight (Figure S1C), expression in pink1 mutants improves flight (Figure 1C). Although expression of Ndi1p does not restore the pink1 flight defect to control levels, it is important to note that previous experiments demonstrating rescue of Pink1 phenotypes using over-expressed Parkin showed very similar results [9].
Ndi1p expression also rescues degeneration of the indirect flight muscles in pink1 mutants as evaluated by decreased indentations in the thoraces of the flies (Figure S2C). As flight muscle degeneration correlates with an accumulation of enlarged mitochondria in pink1 mutants [8]–, we labeled the mitochondrial pool using mito-GFP (Figure 1E). In adult pink1 flight muscles, the mitochondria appear enlarged and clumped when compared to controls (Figure 1E, middle) and Ndi1p expression in pink1 mutants partially trumps this defect (Figure 1E, right). Finally, pink1 mutants that express Ndi1p have an increase in ATP levels compared to pink1 mutants not expressing Ndi1p (Figure 1D). Thus, enhanced ATP levels in pink1 mutants that express Ndi1p may dampen mitochondrial dysfunction and diminish muscle degeneration.
Previous data indicated that mitochondrial defects cause synaptic vesicle trafficking and neurotransmitter release defects at pink1 mutant synapses [15], [23]. We therefore measured neurotransmitter release at the Drosophila third instar neuromuscular junction (NMJ). Upon stimulation of the motor neuron at 1 Hz, pink1 mutants, pink1 mutants that express Ndi1p, as well as control animals that express Ndi1p, show normal neurotransmitter release as gauged by the amplitude of the excitatory junctional potential (EJP) (Figure S3A). In contrast, when stimulated at high frequency (10 Hz), neurotransmitter release in pink1 mutants gradually declines, in line with a defect to mobilize 'reserve pool' (RP, see below) vesicles that are only used under such 'stressed' conditions [16], [35]. Interestingly, pink1 mutants that express Ndi1p maintain normal levels of synaptic transmission during a 10 Hz 10 min stimulation paradigm (Figure S3B). These data indicate that synaptic transmission defects at pink1 mutant NMJs are also rescued by expression of Ndi1p. As previously shown, this neurotransmission deficit is likely caused by a lack of mobilization of the reserve pool synaptic vesicles within NMJ boutons [15]. We used the fluorescent dye FM 1–43 to label vesicles loaded in the RP [36] using the stimulation paradigm depicted in Figure 1F [37]. While pink1 mutants show a significant reduction in RP vesicle labeling, pink1 mutants that express Ndi1p display labeling of RP vesicles very similar to controls (Figure 1F, 1G). Thus, synaptic function deficits in pink1 mutants are alleviated by expression of yeast Ndi1p.
In pink1 mutant motor neurons, mitochondria are morphologically normal but show only partial mitochondrial membrane depolarization [15]. We assessed the mitochondrial membrane potential in pink1 mutants rescued with Ndi1p using the ratiometric dye JC-1. [16], [38]. Interestingly, compared to pink1 mutants, mitochondria at synaptic boutons of pink1 mutants that express Ndi1p are significantly more polarized and show more intense red JC-1 labeling (Figure 1H, 1I). Although Ndi1p itself is not involved in proton transfer over the inner mitochondrial membrane, our data indicate that expression of Ndi1p can restore a negative mitochondrial membrane potential in pink1 mutants. Potentially Ndi1p improves electron transfer from NADH to complex III/IV that in turn helps to restore the proton gradient in mitochondria at synapses.
To further test the specificity of Ndi1p-dependent rescue of pink1-associated phenotypes, we also tested the ability of Ciona intestinalis AOX to bypass pink1 defects. Our rationale is that AOX can also transport electrons but at a different site within the ETC: AOX uses electrons from ubiquinol to reduce oxygen and thereby bypasses both Complex III and IV [29], [32]. We confirm the functionality of AOX in flies because lethality induced by expression of RNAi to cyclope (CG14028 Gene ID 46040) that encodes a Complex IV component, is rescued upon expression of AOX (data not shown) [32]. Similar to NDI1 we also find basal expression of AOX in the male reproductive organ that is much induced by the presence of da-GAL4 (Figure 2A) and ubiquitous expression of AOX also does not affect flight, muscle degeneration or the mitochondrial membrane potential (Figure S4A, S4B; see below). In contrast to Ndi1p, expression of AOX completely fails to alleviate pink1-associated phenotypes such as male fertility, flight or mitochondrial morphology (Figure 2B, 2C, 2E Figure S4C). AOX also does not revert the RP defects in pink1 mutant animals (Figure 2F, 2G) nor does it alleviate the reduced red JC-1 labeling observed in mitochondria at boutons of pink1 mutants (Figure 2H, 2I). Finally, also the reduced ATP levels, observed in pink1 mutant animals, are not rescued by AOX (Figure 2D). Thus, AOX expression does not rescue pink1 deficiency.
Our work suggests that several pink1 mutant phenotypes stem from defects at the level of Complex I. To further test this hypothesis, we used RNAi mediated knock down of evolutionary conserved Complex I subunits (Figure 3A). As expected, down regulation of Complex I subunits results in significantly lower Complex I enzymatic activity (Figure 3B) and expression of NDI1 in these flies results in an increased ATP concentration compared to RNAi of Complex I subunits in the absence of NDI1 expression (Figure 3C). While expression of RNAi to some of the Complex I subunits results in developmental lethality, RNAi to other Complex I subunits yields adult flies, and we used one of those (CG11455; Gene ID: 33179) to assess flight upon Complex I knock down. Under 'standard testing conditions' (see methods) RNAi to this Complex I subunit does not result in a strong defect to fly (blue bar Figure 3D), however under more 'stringent conditions' (see methods) about half of the flies fail to fly, and this defect is rescued by expression of NDI1 (red bars Figure 3D). Thus, while reduced Complex I activity results in a defect to fly, the flight deficit upon knock down of this Complex I subunit is milder than that observed in pink1 mutants (Figure 3E).
Next, we determined mitochondrial morphology using Mito-GFP in third instar larval muscles upon knock down of a Complex I subunit. Similar to mitochondria in pink1 mutants, mitochondria in muscles of animals that express RNAi to a Complex I subunit are swollen and clumped, but the defects we observe are in general less severe than those seen in pink1B9 null mutants. Interestingly, expression of NDI1 significantly alleviates these defects (Figure 3F). To assess mitochondrial function in animals that express RNAi to a Complex I subunit with or without NDI1, we assessed the mobilization of RP vesicles and we quantified red JC-1 labeling intensity within neuromuscular mitochondria. As indicated in Figure 3G–3J, reduced Complex I activity results in reduced RP vesicle mobilization and less red JC-1 labeling in boutonic mitochondria, and expression of NDI1 can significantly rescue these defects as well. Thus, similar to pink1 mutants, loss-of-function of a component of Complex I results in reduced ATP and functional defects in synaptic mitochondria, and these defects are alleviated by NDI1p. Furthermore, we also find that reduced Complex I activity leads to morphological defects in muscular mitochondria and a defect to fly in adult flies, but these defects are in general milder than those observed in pink1 null mutants.
Pink1 has been suggested to act upstream of Parkin to regulate, in a linear pathway, mitophagy [8]–[10], [39], [40]. We therefore expressed Ndi1p in parkin mutant flies but we did not observe a rescue of male fertility, flight defects or muscular degeneration (Figure 4A, 4B, 4D). In line with these observations, mitochondrial morphological alterations caused by Parkin deficiency are also not rescued by expression of Ndi1p (Figure 4C). Thus, parkin mutants cannot be rescued by expression of Ndi1p.
To determine if parkin mutants also display reduced enzymatic activity of Complex I we isolated mitochondria from parkin null mutant flies and from controls and measured Complex I enzymatic activity. In contrast to pink1 mutants, the isolated enzymatic activity of Complex I in parkin mutant mitochondria is similar to controls (Figure 4E). These data are in further support of a model where Complex I defects in pink1 mutants occur upstream from the defects caused by loss of Parkin function.
Previous reports indicate that genetic manipulation of the mitochondrial remodeling machinery using over expression of drp1 or loss-of-function of opa1 alleviates mitochondrial morphological defects, muscle degeneration and flight deficits both in pink1 and in parkin mutant flies [17], [19] and we confirm these results (Figure 5A, 5B, data not shown). In contrast however, sterility of pink1 mutant males is not rescued by increased drp1 or decreased opa1 (Figure 5C), suggesting that manipulation of mitochondrial remodeling cannot rescue all pink1-related phenotypes. As mitochondrial morphological phenotypes may result from alterations in numerous biochemical pathways [26], we assessed directly whether drp1 or opa1 affect the enzymatic activity of Complex I in pink1 mutant flies. However, as shown in Figure 5D, the enzymatic activity of Complex I is still reduced to a level similar to that observed in pink1 mutants. These data thus indicate that the enzymatic deficiency at the level of Complex I in pink1 mutants precedes mitochondrial morphological deficits, or that they in part occur independently.
In this work we present compelling evidence that the mitochondrial kinase Pink1 is critically required to maintain efficient Complex I enzymatic activity in mitochondria and that this function precedes mitochondrial remodeling or mitophagy (Figure 5E). While Pink1 likely acts via multiple (phospho-) targets [41], [42], our data suggests a pathway in which many of the deficiencies in pink1 can be traced back to mitochondrial dysfunction [8]–[10], [15].
Our experiments indicate that Pink1 acts at, or in parallel to, Complex I, in line with the reduced enzymatic activity of this complex in pink1 mutant mouse cells and flies [15], [23]. Expression of Ndi1p alleviates many pink1-associated phenotypes, suggesting that more efficient electron transport between NADH and ubiquinone is mediated by Ndi1p (bypassing the endogenous Complex I deficiency) [30] in pink1 mutants that boosts formation of a proton gradient by Complex III and IV. Although AOX expression also improves ETC efficiency [32], it does not rescue pink1-associated phenotypes, in contrast to AOX rescuing DJ-1β (CG1349 Gene ID: 43652) and cyclope associated phenotypes [32]. The lack of rescue is likely not due to the fact that AOX is insufficiently activated as a result of low reduced ubiquinone concentrations [43] as expression of AOX in pink1 mutants results in premature death of pink1 animals such that only few pink1 mutants that express AOX emerge as adults (data not shown). We surmise that the lower Complex I activity in pink1 mutants, which results in reduced proton transfer across the inner mitochondrial membrane [15] is further propagated by the presence of AOX, that transfers electrons to oxygen without pumping protons [29]. These data thus argue against general mitochondrial dysfunction in pink1 and a universal failing of the ETC [23] but reveals an important role for Pink1 upstream or in parallel to Complex I enzymatic activity [15].
Previous work indicates that loss of pink1 in some cell types results in mitochondrial fragmentation, a process preceding mitophagy [44]. Several lines of evidence now indicate that some of the mitochondrial morphological defects occur downstream of functional deficits in pink1 mutants. First, we show that expression of Ndi1p in pink1 mutants alleviates part of the mitochondrial morphological defects. Second, RNAi-mediated knock down of an evolutionary conserved Complex I component results in mitochondrial dysfunction but also mitochondrial swelling and clumping, indicating that functional mitochondrial defects can lead to morphological defects [24], [26], [45]. Third, mitochondrial functional defects in pink1 mutant flies and mice have been widely observed in neuronal populations where mitochondrial morphological defects are not (yet) prevalent [15], [23]. Fourth, while facilitating mitochondrial fission in pink1 mutants alleviates mitochondrial morphological defects, a deficiency at the level of the enzymatic activity of Complex I persists. Previous results in pink1 knock out cells had also indicated that mitochondrial swelling defects in pink1 mutant cells can be rescued by modulating the levels of the mitochondrial fission machinery [46], but also this manipulation failed to rescue the defect in mitochondrial membrane potential caused by loss of Pink1 [47]. Thus, our data indicate that the upstream molecular dysfunction in pink1 mutants on Complex I is a major culprit in the development of the pink1 mutant phenotypes (Figure 5E).
Our rescue experiments indicate that expression of NDI1 can significantly rescue numerous pink1 associated phenotypes, including male sterility, vesicle trafficking and mitochondrial membrane potential. Likewise, NDI1 also alleviates mitochondrial morphological defects in pink1 mutant muscles, but rescue of this morphological defect is only partial. Similarly, mitochondrial morphological defects observed in animals that express RNAi to a Complex I subunit and flight defects in such animals are in general less severe than those seen in pink1 null mutants. These results are consistent with Pink1 also acting in parallel to its role at the level of Complex1; however, we cannot exclude the possibility that the partial rescue of morphological defects in pink1 mutants upon expression of NDI1 originates from an incomplete reconstitution of ETC activity under these conditions, and that the pink1 mutant conditions at the level of Complex I may not be exactly recapitulated by knock down of the Complex I components. Furthermore, given that Complex I dysfunction results in mitochondrial morphological defects and mild flight defects that are rescued by NDI1 expression, and the observation that the enzymatic defects at the level of Complex I in pink1 mutants are not rescued upon expression of Drp1 or loss of opa1 function, the data are consistent with the pink1-associated Complex I defects to act at least in part upstream of remodeling and suggest an important and central role for Complex I in Pink1 induced mitochondrial pathology.
Our work expands on previous genetic and cell biological studies, and indicates that Pink1 can act at a different level, upstream of Parkin, to control Complex I enzymatic activity (Figure 5E). Indeed, unlike pink1 mutants, loss of parkin in flies does not cause significantly reduced enzymatic activity of Complex I seen in pink1 mutants, and in addition, parkin loss of function is not rescued by expression of Ndi1p. Our data indicate that Complex I deficiency in pink1 mutants is specific and that this defect is not a result of abnormal mitochondrial remodeling or mitophagy. This model is consistent with a role of Pink1 in controlling mitochondrial health and also does not exclude a downstream or parallel role where Pink1 can be triggered to recruit Parkin, facilitating mitophagy (Figure 5E).
w; UAS-mito:GFP, w; da-Gal4 and w; P{lacW}opa1-like3475/CyO (opa1S3) were obtained from Bloomington stock center (Indiana, USA); w pink1B9 and w pink1RV, parkin1 and parkinRV [48] were gifts from Jongkyeong Chung (Korea, Advanced Institute of Science and Technology) [9] and parkinΔ21 mutant flies were a gift from Graeme Mardon (Baylor College of Medicine) [11]. drp1+ genomic rescue constructs were provided by Hugo Bellen (Baylor College of Medicine) [16] and w1118; UAS-AOXF6 were a gift from Howard T. Jacobs [32] (Finland, Institute of Medical Technology, Tampere University Hospital). w1118; UAS-CG12079RNAi (w1118; P{GD5910}v13856), w1118; UAS-CG11455RNAi (w1118; P{GD4800}v12838) and cyclope (CG14028) RNAi (w1118; P{GD908}v13403) were from the Vienna Drosophila RNAi Center (VDRC) [49]. Flies were raised on standard cornmeal and molasses medium.
The coding region of Saccaromyces cerevisiae NDI1 was amplified from yeast genomic DNA (Patrick Van Dijck, VIB Leuven), with the following primers; CGG AAT TCC AAA ATG CTA TCG AAG and GGC GGC CGC CTA TAA TCC TTT A using 2 X BIO-X-ACT Short Mix (BIOLINE), cloned in the EcoR1 and Not1 of pUAST-Attb (43) and sequenced. Transgenic flies were created at GenetiVision Inc. (Houston, USA) using PhiC31 mediated transgenesis in the VK1 docking site (2R, 59D3) [50].
Testis samples from w; daGal4/+ and w; UAS-NDI1 and w; UAS-NDI1/+; da-Gal4/+ and w; UAS-AOXF6 and w; UAS-AOXF6/+; da-Gal4/+ adult flies were micro-dissected in ice-cold PBS followed by snap-freezing on dry ice. Third instar larvae from w; daGal4/+ and w; UAS-CG12079RNAi/da-Gal4 were also snap-frozen on dry ice. Q-RT-PCR was performed using standard procedures, as outlined in Text S1, and relative RNA levels were calculated according to the ΔΔCt method. Primers are listed in Table S1.
Single males (30) of the genotypes indicated in the figure legends were crossed to 1–3 day old w1118 virgins and a score of 1 was assigned when offspring was detected.
For flight assays, batches of 5 freshly eclosed male flies grown at 18°C of the genotypes indicated in the figure legends were put at room temperature for 2 days and then transferred to an empty vial (5 cm D, 10 cm H). For 'standard testing conditions', flies were allowed to climb above a marked line at 9 cm height, the vial was gently tapped and visually scored for flying flies over the next minute. For more 'stringent testing conditions' flies were allowed to climb above a marked line at 9 cm height, and flying flies were scored immediately following tapping of the vial. Flies at the bottom were removed and the remaining flies were retested. Flies that fly twice were assigned a score of 1, the others a score of 0. Student's t test was used to assess the statistical differences.
Five third instar larvae or five thoraces from 2–3 days-old flies were dissected and homogenized in 50 µl of 6 M guanidine-HCl 100 mM Tris and 4 mM, EDTA, pH 7.8. These homogenates were snap-frozen in liquid nitrogen and then boiled for 3 min. Samples were then centrifuged and the supernatant was diluted (1/50) in extraction buffer, mixed with luminescent solution (ATP Determination Kit, Invitrogen) and luminescence was measured on an EnVision Multilabel Reader (Perkin Elmer). Luminiscence was normalized to protein amount (mg) (Bradford) and compared to ATP standards. n=3. Student's t test was used to assess the statistical differences.
Thoraces of adult flies were viewed under an Olympus SZX12 microscope equipped with a DF PLAPO 1X PF lens and the pictures were captured with an Olympus U-CMAD3 camera.
Mitochondrial morphology in adult flight muscles of flies grown at 18°C and reared at room temperature for 2 days or in wandering third instar larvae grown at 25°C was assessed by visualizing mitochondrial tagged GFP (mito-GFP), excited using 488 nm laser light and imaged on a Zeiss LSM 510 META confocal microscope using a 63×oil NA 1.4 lens (for adult muscles) or a 40×oil NA 1.3 lens (for larvae) using a 500–530 band pass emission filter.
RP vesicles of larvae were labeled by electrically stimulating motor neurons of third instar larval fillets in HL-3 with 2 mM Ca2+ for 10 min at 10 Hz and then leaving the preparation to rest for 5 min in the presence of the dye following stimulation. This protocol labels the entire vesicle pool; the exo-endo cycling pool (ECP) and the RP. To unload the FM 1–43 from the ECP, leaving RP labeling intact, preparations were subsequently incubated for 5 min in HL-3 with 90 mM KCl and 2 mM Ca2+ (in the absence of FM 1–43) [16]. Following washing in Ca2+ free HL-3, NMJs were imaged on a Nikon FN-1 microscope with a DS-2MBWc digital camera, 40×W NA 0.8 objective and quantification of labeling intensity was performed using NIS-Elements AR 3.10.
JC-1 (Molecular Probes) labeling was performed on wandering third instar larvae of the genotypes indicated in the figure legends as described previously [15]. Red and green fluorescence was captured on a Nikon FN-1 microscope with a Hamamatsu ORCA-R2, 40W NA 0.8 objective. Quantification of red and green labeling intensity was performed using NIS-Elements AR 3.10.
Complex I activity measurements were performed as described [15]. Data represent at least 3 independent experiments where mitochondrial preparations from 50 animals were prepared for each independent experiment. The Complex I enzymatic activity was normalized to Citrate Synthase enzymatic activity.
Expression of NDI1 is benign and rescues Complex I defects. (A) Quantitative RT-PCR using primers to CG3446, a component of ETC Complex I and to NDI1, in whole flies using tissue of the following genotypes w; da-Gal4 (green), w; UAS-NDI1 (orange) and w; UAS-NDI1; da-Gal4 (blue). Data normalized to housekeeping genes (Text S1). (B) Complex I enzymatic activity measurements in mitochondrial homogenates of control (w pink1RV; da-Gal4), w; UAS-NDI1; da-Gal4, w pink1B9; da-Gal4 and w pink1B9; UAS-NDI1; da-Gal4 in the absence of Rotenone (green) and presence of Rotenone (orange). Student's t-test: * p<0.05; ** p<0.01; ns=non-significant. Data represent the average +/− SEM of n=3 experiments with 50 flies each. (C) Quantification of flight in w; UAS-NDI1; da-Gal4 flies and w; da-Gal4/+ controls. Data represent the average +/− SEM of n=10, (5 flies per independent test assayed). ns=non-significant. (D) Quantification of thorax indentations in NDI1 expressing flies: w; UAS-NDI1; da-Gal4 and da-Gal4/+ controls. Data represent the average +/− SEM of n=6 (5 flies per independent test assayed). ns=non-significant.
Expression of NDI1 rescues sterility and indentation phenotypes of pink1 mutant flies. (A) Images of hemizygous male w pink1RV, w pink1B9 and w pink1B9; UAS-NDI1 flies and of homozygous female w pink1RV and w pink1B9; UAS-NDI1 flies. na=not applicable. (B) PCR on genomic DNA from w pink1RV males (lane 1), w pink1B9/+ heterozygous females (lane 2), w pink1B9 hemizygous males (lane 3) and w pink1B9; UAS-NDI1 homozygous females (lane 4) using primers to amplify the pink1 locus, the NDI1 transgene and male fertility factor kl3 on the Y chromosome (Text S1). Primers used are listed in Table S1. (C) Quantification of thorax indentations in control (w pink1RV; da-Gal4/+) in pink1B9 mutant (w pink1B9; da-Gal4/+) and in pink1B9 mutant flies that express Ndi1p (w pink1B9; UAS-NDI1/+; da-Gal4/+). Student's t-test: * p<0.05. Data represent the average +/− SEM of n=10 experiments with 5 flies each.
Expression of NDI1 rescues pink1 mutant defects in neurotransmitter release. (A) Quantification of the amplitude of excitatory junctional potentials measured in 2 mM external calcium in pink1RV controls (w pink1RV; da-Gal4/+), in larvae expressing NDI1 (w; UAS-NDI1/+; da-Gal4/+), in pink1B9 mutants (w pink1B9; da-Gal4/+) and in pink1B9 mutants expressing NDI1 (w pink1B9; UAS-NDI1/+; da-Gal4/+). Student's t-test: ns=non-significant. Data represent the average +/− SEM of n=4 animals (8 NMJs). (B) Relative EJP amplitudes measured in 2 mM Ca2+ during 10 min of 10 Hz stimulation in controls, pink1B9 mutants, pink1B9 mutants expressing NDI1 and controls expressing NDI1 (genotypes, see A). EJP amplitudes were binned per 30 s and normalized to the average amplitude of the first 10 EJPs. Data represent the average +/− SEM of n=4 animals (8 NMJs).
Expression of AOX is benign. (A) Quantification of flight in w; UAS-AOX/+; da-Gal4/+ and in w; da-Gal4/+ controls. Data represent the average +/− SEM of n=6 (5 flies per independent test). Student's t-test: ns=non-significant. (B) Quantification of indentations in w; UAS-AOX/+; da-Gal4/+ and in w; da-Gal4/+ controls. Data represent the average +/− SEM of n=6 (5 flies per independent test). Student's t-test: ns=non-significant. (C) Quantification of thorax indentations in control (w pink1RV; da-Gal4/+) in pink1B9 mutant (w pink1B9; da-Gal4/+) and in pink1B9 mutant flies that express AOX (w pink1B9; UAS-AOX/+; da-Gal4/+). Student's t-test: ns=non-significant. Data represent the average +/− SEM of n=6 experiments with 5 flies each.
Primers used for Q-RT-PCR and PCR from genomic DNA. List of primers used for Q-RT-PCR and of primers used for PCR of genomic DNA.
Supplemental methods.
We thank Howard Jacobs, Jeehye Park and Jongkyeong Chung, Kyoung Sang Cho, Graeme Mardon, Hugo Bellen, the Bloomington (Indiana, USA) and VDRC (Vienna, Austria) Drosophila stock centers, and the Developmental Studies Hybridoma bank (Iowa, USA) for reagents. We also thank members of the Verstreken and De Strooper labs for constructive comments.
The authors have declared that no competing interests exist.
This work is supported by a Marie Curie Excellence grant (MEXT-CT-2006-042267); an ERC Starting Grant (260678) and FWO grants to PV and BDS; the Research Fund KU Leuven; a Methusalem grant of the Flemish Government and KULeuven; the Francqui Foundation; VIB, an Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) fellowship to MV; and an FWO Vlaanderen fellowship to SV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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62 year old woman | Birmingham, MI, USA | Seeking men 48-60 within 50 miles
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Majority of healthcare organizations have recently seen 'significant' data security incident
87 percent of health information security officers and other health IT professionals said that cybersecurity has become a higher business priority within their organizations in the last year, according to a new HIMSS survey.
Health IT, Biotech, Convergence
Allscripts invests $200M in Soon-Shiong's NantHealth in software integration deal (updated)
In a joint press release, NantHealth and Allscripts said that they would jointly integrate their software via application provider interfaces
Read the MedCity News survey on corporate venture capital in healthcare
Lake Whillans, in an effort to help level the playing field for entrepreneurs in the new world of healthcare investing, has partnered with MedCity News to deliver independent research on corporate venture capital in the life sciences. MedCityNews.com reached out to its 400,000 monthly readers, and more than 450 healthcare innovators familiar with corporate firms shared their insights. What we found is unique.
Chicago mental health agency tries mobile care management with veterans
A Chicago mental health and community services agency is piloting a mobile care management platform with its population of military veterans.
Would Epocrates do better with management separate from athenahealth?
Consider it more wondering aloud than anything close to an official announcement, but athenahealth CEO Jonathan Bush publicly mused Wednesday whether Epocrates might be more successful "outside of my management sphere."
IBM Watson changing quickly to make sense of health data explosion
More than 80 percent to 90 percent of all healthcare data in existence was generated in the last 2-3 years, and 90 percent of that data is unstructured. IBM Watson wants to help.
Health IT, Patient Engagement, Payers
Innovation Summit: Video not always best for telemedicine
Video is the standard for telemedicine, right? Not so fast, said speakers on Tuesday at the third-annual National Healthcare Innovation Summit? in Chicago,
Digital health startup using hand hygiene tool to reduce HAI raises nearly $10M
SwipeSense graduated from Healthbox, which has invested in the business along with Jumpstart.
At ASCO15 meeting: Breakthrough melanoma treatment, Memorial Sloan Kettering oncologist slams cancer drug price tags (Morning Read)
Bristol-Myers Squibb revealed results of Phase 3 study of combined melanoma treatment that could be a cancer treatment breakthrough, but comes with a big price tag and side effects.
Small primary care practices can thrive with EHRs, ingenuity
With a little ingenuity, small primary care practices can improve and offer a competitive advantage to the likes of national retail clinics.
it's possible for small medical groups to thrive in the current environment. It just takes a little ingenuity and some properly implemented technology.
The changing landscape of medtech innovation
Last week in Medcity News, Tiffany Wilson Karp, the executive director of the Global Center for Medical Innovation, discussed the changing landscape for early stage funding in the medical device space. As Karp observes, a partnership between a start-up and corporate venture arm can be ideal. There are, however, potential pitfalls to such a relationship.
The Benecure platform uses gamification to encourage long-term health improvement
Chicago-based startup, Benecure, is looking to help make long-term behavior change for those with chronic […]
Video: Our interview with athenahealth CEO Jonathan Bush
Watch our video interview with athenahealth CEO Jonathan Bush (and a statue of Mick Jagger), from the Hard Rock Café in Chicago, filmed during HIMSS15.
HIMSS keeps growing, and news keeps flowing
We're just starting to recover from the week that was HIMSS15, the largest health IT […]
Athenahealth's Bush: Interoperability is 'prophylaxis against being a jerk'
The health IT landscape is shifting fast, pushed this month by a government report chastising […]
Self-insured N.H. hospital tries accountable care for employees
St. Joseph Hospital in Nashua, N.H., is making its first foray into accountable care not because of a risk-based contract with an outside payer, but because it self-insures employees, and thus was already at financial risk for the health of many of its patients.


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Recent News
June 2015: I'll be talking at The Photographers' Gallery on Friday 19th June between 3-4pm with Jonathan Simpson and James Bell about our work (I'm showing half of Covering The Carpet) in the FreshFaced+WildEyed 2015 show.
My work featured on Dazed & Confused, It's Nice That, Harper's Bazaar Brasil and The Telegraph.
I took over The Photographers' Gallery Instagram account for a week and did SuperMassiveBlackHole's Instagram challenge for 10 days.
May 2015: Covering The Carpet was shown in ETALAGE III at AWOL Gallery, Manchester from 8th to 15th May.
Covering The Carpet received an Honourable Mention in The Magenta Foundation's Flash Forward 2015.
April 2015: One of my photographs was published in Issue 8 of Der Greif magazine, Germany.
Click for the Your Dedication Worries Me A Little project
Published October 2013 - Reality of Youth Going Backwards in Vain (self published)
With an essay by Joanna L. Cresswell
Available here
(A big thank you to Clare Hewitt, Stu & Justine at SMITH Design and Joanna!)
Covering The Carpet
Your Mind & Body Is All That You've Got II
Your Mind & Body Is All That You've Got
One Is Not Like The Other
The Many Faces of JFA
Reality of Youth Going Backwards in Vain


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South Texas police catch juvenile detention center escapee in underwear
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Real Estate
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Review: 'Water by the Spoonful'
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12 Texas beaches for that sand and surf glow
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Citizen engagement crucial in a 21st century San Antonio
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2016 Acura MDX — a new nine-speed automatic transmission is the big change.
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Aetna to buy Humana as health insurer landscape shifts
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Beautiful Hill Country ranch listed for $18.5 million
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Kin Man Hui/San Antonio Express-News
Kerrville-Tivy struck early with a 79-yard strike to Johnny Manziel (03) against Brenham's Robert Felder (07) in the first quarter in the 4A football state semifinals in Georgetown, Texas on Saturday, Dec. 12, 2009. Tivy lost 31-21.
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by Paul Sanders
For average users, DVD players aren't much of a mystery. DVD players produce high-quality video, especially when compared to old VHS tapes. Additionally, you can get a whole lot more from your DVD collection if you know the details of DVD player technology and what your options are. Here are some answers to more advanced questions about DVD players to help you get the best entertainment possible.
Can DVD players produce HD video?
DVD players offer a detailed picture, but it's not HD in the sense that is used by the entertainment industry today. Typically, what is meant by "high-definition video" is 1080p resolution video quality. Most DVDs don't have this level of picture detail to offer. Up-converting DVD players can increase the picture resolution to near 1080p artificially, but the DVD players still can't quite match 1080p resolution of some HDTV broadcasts and Blu-ray discs.
How can I transfer my VHS tapes to DVDs?
DVDs have a much longer life than VHS tapes, so they're a good way to preserve home movies and irreplaceable tapes. Transferring your old tapes to DVD will require a DVD recorder connected to a VCR, or a VCR/DVD recorder combination. You can then play them on any DVD player that plays recordable discs.
Why won't my DVD player play certain discs?
DVD players have a limited number of formats they can read. If your DVD is from another country and has the wrong region code (a geographical coding to prevent piracy), or the disc is in a recordable format that your DVD player doesn't recognize, the disc may not play. You may find region-less DVD players or video players that accept multiple recording formats that will play those discs.
What do "progressive scan" and "up-conversion" mean?
Progressive scan refers to the way your TV presents video. Older video could only be displayed by TVs in lines: First, the screen would display every other line and then would fill in the rest on the next pass, refreshing many times a second. Since HDTVs came out, broadcasts and movies have switched to displaying the entire frame in one pass. This full scanning of the image is called "progressive scan." Up-conversion is the process by which advanced DVD players convert older video formats into progressive scan while enhancing the resolution of the picture.


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Penetrating, beautifully sparse, and eerie in its stillness, Gerbrand Bakker's The Twin tells the story of Helmer van Wonderen, an aging farmer whose life has been characterized by passivity, inaction, and a profound sense of isolation. Having begrudgingly taken over the family dairy farm after the accidental death of his twin brother Henk, Helmer finds himself, after 55 years, suddenly and unexpectedly the master of his own life—if only he knew what to do with it.
For years, Helmer has been "doing things by halves," living the life that was intended for his brother, a monotonous existence at the beck and call of his tyrannical father. As the story opens, however, Helmer has moved his father, close to death, to a bedroom upstairs, an act of self-assertion that pushes him to reassess his past and the decisions—or lack thereof—that have left him stagnated and alone. "Why did I let it all happen like this?," he wonders. "I could have said 'no' to Father and 'do it yourself' or just 'sell up.'"
But while Helmer may not be satisfied with the life that he has accepted for himself, it is a routine that if not fulfilling, is at least familiar. "I've milked the cows, day after day," he reflects.
In a way, I curse them, the cows, but they're also warm and serene when you lean your forehead on their flanks to attach the teat cups. There is nothing as calming, as protected, as a shed full of cows on a winter's evening. Day in, day out, summer, autumn, winter, spring.
In the absence of any truly meaningful, reciprocative human relationships, Helmer has forged quiet connections with his animals. He finds solace in the ritual of milking his cows, keeps two identical donkeys as pets, and almost drowns himself trying to save a sheep mired in an irrigation ditch. And it is through natural imagery such as this—swallows sleeping on telephone lines, a hooded crow alighting outside the kitchen window, ducks swimming in a pond—that Bakker (a former linguist who has since become a gardener) is able to not only reveal more of his taciturn protagonist's interiority, but also bring the narrative to a kind of gentle compromise between what should have been and what simply is.
On an unexpected trip to Denmark—his first holiday "in thirty-seven years of milking day and night"—Helmer walks down to a beach at sunset. "The beach is deserted," he says.
There are no hooded crows in the sky and even the busy grey sandpipers are missing. . . I am the only one for miles around making any noise . . . I know I have to get up. I know the maze of paths and unpaved roads in the shade of the pines, birches and maples will already be dark. But I stay sitting calmly, I am alone.
By the novel's close, Helmer has found some measure of peace and acceptance in his quiet life—even in his solitude.
Founded in 1960 by such creative pioneers as George Perec, Raymond Queneau and Italo Calvino, the Oulipo, shorthand for Ouvroir de littérature potentielle, came about in when a group of writers and mathematicians sought constraints to find new structures and. . .
There's little to say about a series of prose poems that willfully refuse to identify pronoun antecedents. Or perhaps there are a million things. The poems in Morse, My Deaf Friend— the chapbook by Miloš Djurdjević published by Ugly Duckling. . .
The Crimson Thread of Abandon is the first collection of short fiction available in English by the prolific Japanese writer and all-around avant-garde trickster Terayama Shūji, who died in 1983 at the age of 47. This collection would be important. . .
Last year, NYRB Classics introduced English-language readers to Catalan writer Josep Pla with Peter Bush's translation of The Gray Notebook. In that book, Pla wrote about life in Spain during an influenza outbreak soon after World War I, when. . .
"Your bile is stagnant, you see sorrow in everything, you are drenched in melancholy," my friend the doctor said.
bq. "Isn't melancholy something from previous centuries? Isn't some vaccine against it yet, hasn't medicine taken care of it yet?" I. . .
What to make of Vano and Niko, the English translation of Erlom Akhvlediani's work of the same name, as well as the two other short books that comprise a sort of trilogy? Quick searches will inform the curious reader that. . .
The opening of Jón Gnarr's novel/memoir The Indian is a playful bit of extravagant ego, telling the traditional story of creation, where the "Let there be light!" moment is also the moment of his birth on January 2nd, 1967. Then. . .


text:
This is how you do a Pride dinner.
Shop the Trend: Cross-Body Bags
The basic, form-fitting tee can be dressed up or down, tucked into pencil skirts or worn with a sleek blazer to the office. Go with a neckline that suits your personal taste, whether a v-neck like Zoe Saldana or a high crew.
Looking Back: Kate Moss


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Yael Nissan
Original recipe yields 6 servings
Amount per serving ({{servings}} total)
Based on a 2,000 calorie diet
Ready In
Yum! I used less water and pureed the carrots along with the cauliflower, and it couldn't be any better.
Damn good! As an avid cooker I can highly recommend this to anybody! What I changed a bit: I used good chicken stock (Knorr brand is the best!) instead of water and I also added Cayenne pepper l...
Good recipe...milk can be added instead or in addition to water.(also, no need to boil carrots separatly)


text:
Peter Sproul is a tax principal in PwC's insurance practice, where he provides tax services to some of the Northeast's largest and most complex multinational and regional insurance companies.
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Received on time as promised. Price was reasonable and had assurances knowing it was a Google Trusted Store.
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text:
This has been an amazing year for snowfall. According to a local television weather person, we are in the top four snowfall years since recording began and may just break the all-time record before the season is over. I like snow, but even I have reached a saturation point this year.
Last fall, I was thinking about taking down the fabric gazebo we had set up in the back yard before snow overpowered it. I ended up a day late; an unexpected overnight snowfall of wet, heavy flakes fell here in Big Lake. The gazebo came down under the weight and probably took out the two lawn chairs and folding table that were under the structure. I'll know for sure after things melt this spring.
That's not the worst. I have a small storage shed in the backyard to which I had attached two wooden racks, one on either side, to store my canoes. Again, I noticed the buildup of snow both on the canoes themselves and on the shed roof. In previous years, the building and canoes shed the snow during warm periods with no problem, so I wasn't too concerned. So much for that. After this last warm period, I noticed the snow had slid off the shed roof and took down both racks and canoes. The canoes ended up on the ground.
My Old Town canoe, the "Peaceful Journey," had been cabled to the shed to prevent theft. The cable ripped the thwart out and tore the canoe body, destroying the craft. The other canoe wasn't cabled so I think it survived. I needed snowshoes to check on the status of the canoes after they fell.
When I saw that damaged Old Town sticking out of the snow, I started thinking about some of the adventures I've had in that watercraft. I bought it new in Georgia in 1972, so it served a long and useful life. I've used that canoe to duck hunt and fish in Georgia and moose and bear hunt and fish here in Alaska. While most of the trips have been uneventful, I can think of three where I could have drowned because of circumstances.
My black Lab, Troubles, and I were going fishing one day on a lake in north Georgia. He decided he wanted out, so he jumped on top of the gunwale and pushed off with his hind legs before I even knew what he was doing. That kick-off rolled the canoe out from under me. The Old Town model I had was designed to float, even when full of water, so I just climbed in and began the slow process of paddling for shore. Troubles and I had a discussion about dog-canoe manners when we both got back to dry land.
The second time, a friend, Troubles and I were floating a small river in Georgia looking for ducks. We hit a low, unavoidable sweeper and the canoe rolled, dumping all three of us into the river current. We all survived, although for a while I thought Troubles had drowned because I had tied him into the canoe because of his jumping. I lost a shell vest and a paddle. The paddle was returned about six weeks later when one of the guys I worked with found it while floating the same river.
The third time, Troubles and I were going duck hunting on a semi-remote lake in southwestern Alaska. I had a small outboard motor mounted on the canoe's factory side-mount near the stern. As we were crossing the lake, the wind came up. I successfully turned the canoe around, but couldn't outrun the following swell and waves began breaking over the stern of the canoe. I couldn't bail fast enough to keep up with the inflow of water. The canoe eventually filled enough for the stern to sink, swamping the canoe and causing it to roll over because of the offset outboard. One of the guys at the site noticed the overturned canoe and came out in a boat to investigate. That saved both Troubles' and my bacon!
Are you noticing a trend here? I did, so I purchased a commercial set of canoe outriggers that I have used faithfully for the past dozen or more years, without further incident.
I've also had many fun trips in that canoe as well. When we lived on Afognak Island, I kept the canoe near a remote lake where my wife and I would go fishing or just paddle around to explore the area. After a brown bear chewed holes in the bow, I had to do some repair work. Since moving to the Valley, I've used the canoe to fish in our stocked lakes and check a bear bait station one year.
I've landed my biggest stocked lake rainbow ever from that canue after hooking it using a four-weight fly rod. On another trip, my wife caught a nice rainbow that was grabbed by a loon. I've hunted moose after crossing a remote lake south of Big Lake in the canoe. I have lots of fond memories involving the "Peaceful Journey."
I hadn't used the canoe all that much the last few years. Being 40-years-old, the plastic was really beginning to show its age. I'll salvage parts after the snow melts and make a point of paying closer attention to snowfalls next winter.
Howard Delo is a retired fisheries biologist with the Alaska Department of Fish and Game. You can leave him a message by emailing firstname.lastname@example.com.


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HOME NBA Philadelphia 76ers Gear Philadelphia 76ers Mens Philadelphia 76ers Mens T-Shirts Philadelphia 76ers Mens T-Shirts Short Sleeved (127 items) Product ID: 1465049
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Manufacturer direct items are shipped directly from the manufacturer. These items are not available for international or expedited shipping.
Customized items can be personalized with options such as your name, your favorite number, and/or designs. Some options may be limited by league rules.


text:
SFMTA "daylighting" in theory.
SFMTA daylighting in practice:
(I wonder if the SFMTA waives it's own $2.50 "convenience fee" when it pays the all those tickets that it surely must issue to, more or less, itself – you know, our "City Family.")
SFMTA "daylighting" in theory.
SFMTA daylighting in practice:
(I wonder if the SFMTA waives it's own $2.50 "convenience fee" when it pays the all those tickets that it surely must issue to, more or less, itself – you know, our "City Family.")
So sure, jumping the light and loitering in the street, that's a routine thing, and that's part of the reason why most* ped deaths due to traffic accidents on the streets of San Francisco last year were primarily the fault** of the pedestrians themselves.***
But to show the proper attitude, make sure to look away from the traffic you've been improperly blocking for no reason:
That's it, now you're on the trolley!
*Just a smidge above 50% for 2014, admittedly higher than the typical year.
**And if you're talking about some partial fault on behalf of peds, then this 50 something percent figure would climb into the 60's or 70's. Of course, you're not legally required to walk defensively, but, of course, it's nevertheless a good idea. This is why I'm less likely to die as a pedestrian in a traffic accident per hour of walking than you, the more typical SF ped, one assumes
***Per SFGov, your beloved SFGov, which is run**** by that guy that the urbanist-type group(s) you're a dues paying member of almost certainly endorsed for election.****
****You know, under our Strong Mayor form of government
Of course back in '49, you know, 1849, some people were still calling Kearny Street La Calle de la Fundacion – it was the start of the grid system of streets we know today.
Anyway, not much has changed since the founding of the SFPD 166 years ago – a time-traveling gold miner from back then would have no problem understanding this aspect of The Future:
Click to expand
Here's the SFMTA's official six-figure-a-year spokesperson on the topic of when pedestrians can cross a street, from just last year:
"They can start whenever they want," Rose said."
Of course this is wrong, as even Paul Rose himself would admit now, after being corrected.
So, why did he say that? Because he, like his employer, has a half-assed approach to safety, and, one supposes, he, like his employer, is mired in politics.
Now do you suppose that Paul Rose was at all interested in examining why he told the peds of San Francisco that it was A-OK for them to violate CA state law? Oh no, not at all. And do you think he checked with anyone before he spouted off? Prolly not.
Like I say, a half-assed approach.
Now we're in 2015, the era of SF VisionZero 2024, which has the goal, one that nobody actually believes in, but they have to pretend that they do believe in it, of having no more transportation deaths in San Francisco County starting in 2024 and continuing in perpetuity.
It'll look a little something like this, supposedly:
Now do you see the beauty in this? By the time SFGov fails to achieve this impossible goal, all the people who glibly made the promise will be out of office, right? How convenient.
The big problem with the approach that SFGov is taking is assuming that traffic deaths are a street design issue, as opposed to a human behavior issue. So most of the emphasis appears to be upon SFGov spending more money, which of course SFGov loves to do anyway.
And the part of VisionZero SF that's focuses on behavior seems misplaced, for political reasons.
For example, there's this:
"Focus on the Five – Using multi-year collision data, the San Francisco Police Department (SFPD) is focusing on enforcing the five violations that are most frequently cited in collisions with people walking. The goal is to have half their traffic citations be for these five violations."
So if the SFPD started handing out tickets for jaywalking, you know, in a big way, that would certainly help with traffic safety, over the long term, to at least a slight degree, but that would take the SFPD away from its "Focus On The Five" goal.
The problem with Focus On The Five is that it ignores Vehicle Code violations on behalf of pedestrians, one supposes for political reasons. In fact, the cause of most pedestrian and cyclist deaths last year in San Francisco was the behavior of the pedestrians and cyclists themselves.
And what's this talk about "automated enforcement?" How about this, how about hooking up all of the SFMTA's vehicles to an automated enforcement mechanism that would detect speed limit, stop sign and red light violations using on board sensors and GPS? Then, after Ed Reiskin parks his government-paid SFMTA car or an operator parks her bus, SFPD tickets would be issued, you know, daily. Whoo boy, what are the odds of something like that happening?
So that's SF VisionZero 2024, a buzz-phrase that means absolutely nothing.
Via Stanley Roberts of KRON-TV's People Behaving Badly comes this "viral" (just two views so far, but the day is young) video.
The driver of this ride should have his/her license pulled until somebody in authoritah can figure out what went wrong:
But that's not going happen, 'cause driving is a right, and not a privilege. In California, anyway.
I call this real red light running because it's obvious that the errant driver entered the intersection on a red light (and I'm basically saying that the color of the light when a driver exits doesn't matter and that peds need to wait for an intersection to clear whether or not they have a green. In California, anyway.) I don't know if there was any bad behavior on the part of the ped(s).
The saving grace is that a lot of people who don't know what they're doing behind the wheel drive real slow. This is a possible drunkie…
Man, you won't catch me crossing Fulton just hoping that drivers will see me and stop. Oh no.
But this woman took the plunge. See the driver signalling to others? That's what it takes sometimes:
I don't know, we had The Freeway Revolt so now we have streets like Fulton acting as substitute freeways.
The SFMTA spends its money inefficiently, so it says it can only afford to install something like five traffic lights per year. (Oh, it's so hard, it's so hard to do, they say. No it's not, but anyway…)
So, we don't want freeways and we won't tolerate any transportation-related deaths (or we won't tolerate any transportation-related deaths starting in 2024). This doesn't add up.
So, slow down Fulton if you want, but what that does is push the Outer Richmond farther and farther away from San Francisco…
This is typical, this is routine – people parking on Masonic northbound and then jaywalking across five lanes of traffic to get to Trader Joe's #100 and then jaywalking again back to their rides
Why do people do this? Well, 'cause getting from northbound Masonic to southbound, which is the only way to get into the parking lot, is a PITA. Drivers are banned from simply turning left into the parking lot because that would end up blocking half of northbound Masonic, and of course Masonic is the Great Connector betwixt The Avenues and the Place Where People Want To Be.
And even if you get yourself facing southbound, you still have to queue up to get into TJ's ridiculously small parking lot. Hey, couldn't they have built an underground garage? Well, sure, but you'd have to talk with the Planning Department about that. And hey, couldn't they have built parking on the roof? Well, sure, and actually they did but you'd have to talk with the Planning Department about that because the average shopper isn't allowed to park on the roof.
And actually, the current parking situation is better than before. Our vaunted Planning Department did a very poor job with this project and now we're left with a kludgy fix that commits part of Masonic to TJ's shoppers idling and parking and waiting.
So that's the situation, that's why people say I-don't-wanna-deal-with-all-that and simply park on northbound Masonic on the east side of the street.
And that's fine, that's legal, but then the shoppers see that northbound Masonic has long stretches when it's empty (because drivers need to wait at a red for a long time to let traffic on Geary go through) and they see a bunch of stalled traffic on southbound Masonic (because of the shoppers queuing up and also to wait at a red for a long time to let traffic on Geary go through). So they march across 30 MPH Masonic to get to the store.
How many TJs shoppers do this on a busy day? IDK, hundreds. It's their thing, it's their routine.
So can you die doing this? Sure. Does TJ's know about this situation? Sure. I don't see how they couldn't be aware. I mean, when you have journalists calling up your store asking about how somebody died, I assume that you're aware of the situation.
What's the solution? Well, people'd be safer walking down to Geary and crossing legally, but they all already know that.
You see the problem is that they don't know how dangerous it is to do what they're doing.
Hey, you know how many people die at Ocean Beach during a typical year? A lot. So many theat they have a special sign:
How about similar signs for shoppers at this unique store:
People Jaywalking Have Died Here
How about that?
Unique situations call for unique signs, right?
Are you going to do anything at all, Trader Joe's #100?
This road would be a good one to have more traffic signals and less median:
I'd plan ahead to cross at an intersection with a signal, or I'd wait for a break in traffic, as if I were a jaywalker. I wouldn't just start crossing the street just hoping drivers would notice me.
Anyway, sometimes all the drivers stop, except one, and that's when the trouble starts…
Instead of heading up the hill to get back to their ride, these gals walked downhill a few paces and then used congested traffic near Geary to their advantage:
That's better than most, of course.
What they're doing is turning a complicated, deadly game of chicken into a simpler chore of waiting for northbound traffic to get a red and then shuffling across two lanes…


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Suit up and prepare for whatever Halloween throws you in these officially licensed Iron man glasses - Tony Stark approves!
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132 Views 1 Reply Latest reply: Feb 4, 2014 1:12 PM by ETeague
ETeague Feb 4, 2014 1:12 PM
Re: I have artwork on my phone that does not match the song even though I have itunes match. How do I get the correct artwork? in response to Kristine9581
I go to amazon.com, look up the album in the search bar, the do a copy (right click, copy) on the artwork that shows up. Then you go to iTunes and right click the album you want to update with that artwork, click "get info" click in the artwork square and press the 'command and V' keys for paste (fyi right click paste does not work here) you must just click in the square and press command-V which will paste it. It should then place that artwork in the square to update all songs in that album. Hope this helps...


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Transgender , 26-30
You could call me 'different'
Although I have no problems making friends,
I have wacky ideas, and I just love having fun..
I'm very positive these days, though I still catch myself being quite cynical at times..
I guess my way of trying to find a balance between being care-free.. while also being careful,
I have a good understanding of myself and how to keep myself satisfied on a regular basis...
Last Seen Dec 15, 2014
Member Since Jan 20, 2012
Favorite Quote
- a little Scandinavian
Politics Moderate
Horoscope Scorpio
Special day 11-3
Books A Scanner Darkly, Fear and Loathing in Las Vegas, Hater
Music Classic rock, indie bands, dance
Movies Alice in Wonderland
Local Time July 5th, 11:27 PM
I Want to Know If the Person In Your Avatar Is the Real You
Clown Love.. Let me start by saying my avatar is a picture of me.. but let me explain. This picture I took last Halloween after I got back home after going trick or treating with my brother and his son... [more]
iamalexa has shared 9 Mature Experiences
The Feeling Is Back Again I have early memories of wishing that I was a girl, from when I was very very young, I wasn't really sure why that was, but I would think about it to myself at night in bed, almost all the time.… [more]
I Say I Don't, But Actually I Do Care What Others Think Of MeIt Couldn't Be More True. I have always acted differently around other people, like I've always wanted them to see me in a certain way. Cool with everyone and everything and nothing can affect me because I am solid as a … [more]
1-3 of 3 Stories


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Christian & Small LLP is pleased to announce that Jonathan Macklem has been chosen for inclusion on the Rising Stars 2014 list. The Rising Stars selection process is multi-phased and candidates must be either 40 years or younger, and in practice for 10 or fewer years. www.csattorneys.com
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text:
Missing Man
1 Arrested After Missing Man Found Dead
A missing New Castle man was found dead Wednesday night and another man has been taken into custody.
Missing Greensburg Man Found Safe At Hotel
Police have found a missing Greensburg man, just a day after his family gave an emotional appeal for him to come home.
Police Continue Search For Missing North Braddock Man With Dementia
Allegheny County Police are asking for help from the public in finding a North Braddock man who disappeared back in June.
Missing Avalon Man Found Safe
Police need help tracking down a 75-year-old man who may suffer from dementia.
Police Search For Missing North Huntingdon Man
Police are looking for a missing 29-year-old man, who was last seen about a week ago.
State Police Searching For Missing Winfield Twp. Man
State police are searching a Butler County man who disappeared over the weekend and hasn't been seen since.
Body Found In Washington Co. Field May Be Missing Elderly Man
Pennsylvania State Police in Washington County reported that a body was found in a field on Daniel's Run Road at Brady Road in Scenery Hill.
Family, Friends Hold Vigil Hoping For Missing Man's Safe Return
A $3,000 reward is now being offered for information leading to the whereabouts of an elderly man who went missing earlier this month from Washington County.
Police Search For Missing Westwood Man
Police are looking for a missing man from Westwood.
Police Searching For Missing Elderly Washington Co. Man
The search for a missing man in Washington County resumed Wednesday morning.
Police Seek Public's Help To Locate Missing North Braddock Man
Police are searching for a North Braddock man who suffers from dementia.
McDonald Man Found After Missing For 3 Weeks
Police have found a man who was reported missing from McDonald, Pa.
Missing Elderly Man's Body Found In Beaver County
Officials in Beaver County have recovered the body of an elderly man who disappeared after wandering away from his home in the middle of the night.
Pittsburgh Crime Stoppers, Family Seeking Answers In Missing Man Case
Marking the second anniversary of his disappearance, the family of a missing Penn Hills man had a large billboard put on Washington Boulevard, near Hamilton Avenue.
Police: Missing Aliquippa Man Seen On Surveillance Video
Police say a man from Aliquippa who disappeared earlier this week is safe.


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Smarter. Faster. Tougher. An Exhibition on Sports, Fashion + Tech opens tomorrow
Opening with the PanAm Games, the Design Exchange's 2015 summer/fall exhibition focuses on sportswear, and is the second offsite exhibition that will run in the heart of the PanAm Village in the Distillery District. The exhibition runs...
Jul 7, 2015 6:00PM
2015 Top Ten Endangered Places and Worst Losses List released
The Top Ten Endangered Places List is released annually to bring national attention to sites at risk due to neglect, lack of funding, inappropriate development and weak legislation. From unique 19th-century landmarks to simple vernacular...
Jul 7, 2015 3:30AM
Art by Architects call for artists
If you love architecture, this is one auction you won't want to miss! The Metro Vancouver Chapter of the Royal Architectural Institute of Canada (RAIC) is proud to launch the first Art by Architects (AxA) Auction, a group exhibition and...
Jul 7, 2015 2:25AM
Winnipeg Architecture Foundation offers free tours through September
The Winnipeg Architecture Foundation (WAF) is offerning a variety of tours through July, August and September. All tours begin at 7:00pm on Wednesday evenings; they are free of charge and fully accessible. Confirmed tours to date are: *July...
Jul 5, 2015 2:00AM
Nader Tehrani appointed Dean of Cooper Union's School of Architecture
After a nearly two-year search, the Cooper Union in New York has selected Nader Tehrani as the new Dean of the Irwin S. Chanin School of Architecture, effective immediately. Following Dean Anthony Vidler's resignation in 2013, Elizabeth...
Jul 4, 2015 2:00AM
Feature Contemporary Art Fair releases 2015 exhibitor list
Taking place from October 22-25, 2015, Feature Contemporary Art Fair is dedicated exclusively to cutting-edge contemporary art and brings together artists whose practices are outstanding for their ability to innovate, challenge and inspire.
Jul 3, 2015 11:00PM
Canadian Architect is a magazine for architects and related professionals practicing in Canada.
Teacher, Mentor, Partner
TEXT Talbot Sweetapple
Back to the Land
"Architecture is a cultivating instrument, whether you're rehabilitating a piece of urban fabric or whether you're clearing land to restore a field in the countryside," Brian MacKay-Lyons, FRAIC, said in a recent...
Walking and Working
Brian MacKay-Lyons is the 2015 winner of the RAIC Gold Medal, the highest honour bestowed by the Royal Architectural Institute of Canada. Canadian Architect recently spoke to Brian about his career trajectory, philosophy of practice, and...
Annex-Newcom Network:


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Southern Fried Rice is a comfortable guesthouse in an attractive colonial building in Kata, arguably Phuket's most... 추가 정보 »
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Driving Directions to Campus
I-79 to Exit 113 (Grove City)
Take Route 208 East into Grove City to the first traffic light.
Turn right onto Main Street (Rt. 58) and go to the second traffic light (Liberty Ave).
Turn left into the campus and follow the road to Crawford Hall. (Administration Bldg.)
I-80 to Exit 24 (Grove City / Sandy Lake)
Take Route 173 South into Grove City.
Airport Orbit
Airport transportation service (Airport Orbit) and car service (Sprocket Point-2-Point Transfers)
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Complete the application form, answering all questions that pertain to you. This information is required for your permanent record in the Registrar's Office and to assist your academic advisor.
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Grove City College has a reputation for high academic standards.
Each fall, Grove City College provides a snapshot of our current freshman class including average GPA, average standardized test scores, geographic reach and ethnic diversity.


text:
With our checklist you get a quick overview of how Projectmanager helps you to control and simplify all project-oriented operational processes.
At the moment we cannot offer you a trial site, since we are at the midst of releasing a new and improved version of the software. However, you are welcome to fill out this note of interest and we will notify you when the new version is ready.
Artisan sólo utilizará la información facilitada para instalar el sitio de prueba y ponerse en contacto con usted.


text:
Marco Rubio did his best Barack Obama impression Thursday, professing his profound belief in the American Dream.
Read a cautionary tale about the seductive and dangerous power of a charlatan sociopath, featuring goats and the American Dream.
Hear the soaring anguished cadences of Martin Luther King Jr. proclaiming an American Dream.
coined 1931 by James Truslow Adams (1878-1949), U.S. writer and popular historian (unrelated to the Massachusetts Adamses), in "Epic of America."
[The American Dream is] that dream of a land in which life should be better and richer and fuller for everyone, with opportunity for each according to ability or achievement. It is a difficult dream for the European upper classes to interpret adequately, and too many of us ourselves have grown weary and mistrustful of it. It is not a dream of motor cars and high wages merely, but a dream of social order in which each man and each woman shall be able to attain to the fullest stature of which they are innately capable, and be recognized by others for what they are, regardless of the fortuitous circumstances of birth or position. [Adams]Others have used the term as they will.
A phrase connoting hope for prosperity and happiness, symbolized particularly by having a house of one's own. Possibly applied at first to the hopes of immigrants, the phrase now applies to all except the very rich and suggests a confident hope that one's children's economic and social condition will be better than one's own.


text:
The Official Univision.com Toolbar 220.127.116.11
by alexbehrmann, CONDUIT-AMO
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- 'The Odd Couple' turns 50
- Library explores the FAQs around 'Exploring Human Origins' exhibit
- Eight-year-old boy creates Monkees video, gets nod from Micky Dolenz
- A belly full of laughter: EPAC presents 'Monty Python's Spamalot'
- Trolley'n for brews
- Pretzel Fest: twisted fun for everyone
- Armed Forces Day swing dance
- Ephrata Police caution on new smoking rules
- Pretzel Fest will feature 13 tasting stations
Lycoming art show participant Trefny participates in scholars forum Students recognized
Emily Mixell of Reinholds recently participated in Lycoming College will host its annual Senior Art Show Mixell is majoring in art with a concentration in commercial design.
Andrew Trefny, Denver, son of Paul and Terry Trefny and a 2010 graduate of Cocalico High School. A regional planning major, he presented "Storm Water Runoff Trends: A Study Employing Green Space Initiatives to Analyze the Decline of Non-point Source Pollutants in Philadelphia."
Emily Mixel, Reinholds, was honored at Lycoming College's annual leadership and service awards banquet. Lycoming College recognized co-curricular student achievement on campus during its seventh annual Leadership and Service Awards Banquet on April 10.


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1963 VW beetle bug, one owner, well restored, 32.791 miles, 4 cyl. This is an estate vehicle, and will sell to the highest bid on... More
This VW Thing is a Florida car that runs and drives great. It has a new top. Powered by the 4-cylinder VW engine with... More


text:
Crofut, William Elmer; wholesale rubber; born, Syracuse, N. Y., Sept. 5, 1874; son of Elmer Burr and Hattie A. Davis Crofut; educated in public and private schools of Syracuse and Syracuse University; married, Concord, N. H., Dec. 5, 1899, Ruth Paul; one son, William E., Jr., served in 141st N. Y., Spanish-American War; came to Cleveland, October, 1899, as manager of a mercantile agency; three years later engaged as sec'y and treas. Ohio Rubber Co., remaining with the firm until September, 1905, when he organized the Forest City Rubber Co., continuing as president-treasurer and gen. mgr. since; member Mayfield Country Club, Chamber of Commerce, Credit Men's Ass'n.
Biographical Sketch of William Elmer Crofut
Show some words
This page is part of a larger collection.
Access the full collection at The Book of Clevelanders.
Access the full collection at The Book of Clevelanders.
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Topics: Biography, Spanish-American War
Locations: Cleveland Ohio, Concord New Hampshire, Cuyahoga County OH, Merrimack County NH, Onondaga County NY, Syracuse New York
Surnames: Crofut
Topics: Biography, Spanish-American War
Locations: Cleveland Ohio, Concord New Hampshire, Cuyahoga County OH, Merrimack County NH, Onondaga County NY, Syracuse New York
Surnames: Crofut
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