Ageing is a progressive decline of intrinsic physiological functions. We examined the impact of ageing on the ultrastructure and function of mitochondria in mouse and fruit flies (Drosophila melanogaster) by electron cryo-tomography and respirometry. We discovered distinct age-related changes in both model organisms. Mitochondrial function and ultrastructure are maintained in mouse heart, whereas subpopulations of mitochondria from mouse liver show age-related changes in membrane morphology. Subpopulations of mitochondria from young and old mouse kidney resemble those described for apoptosis. In aged flies, respiratory activity is compromised and the production of peroxide radicals is increased. In about 50% of mitochondria from old flies, the inner membrane organization breaks down. This establishes a clear link between inner membrane architecture and functional decline. Mitochondria were affected by ageing to very different extents, depending on the organism and possibly on the degree to which tissues within the same organism are protected against mitochondrial damage.
Mitochondria produce most of the ATP in non-photosynthetic eukaryotes, providing the energy to drive a multitude of cellular processes. Mitochondria have an inner membrane, which surrounds the matrix, and an outer membrane, which surrounds the inner membrane and separates the mitochondrial compartments from the cytoplasm. In addition to a multitude of soluble enzymes and ribosomes, the matrix houses the mitochondrial genome (mtDNA), which in humans and flies encodes 13 hydrophobic subunits of the oxidative phosphorylation system. Thus nearly all mitochondrial proteins are nuclear-encoded and imported into the different mitochondrial compartments by a set of protein translocases (Pfanner et al., 2014). The oxidative phosphorylation (OXPHOS) system consists of the respiratory chain complexes I to IV and the mitochondrial F1Fo-ATP synthase, sometimes referred to as complex V. Complexes I to IV transfer electrons from soluble electron donors to molecular oxygen. In this process, complexes I, III and IV generate an electrochemical proton potential across the mitochondrial inner membrane that is used by the ATP synthase to produce ATP by rotary catalysis (Leslie et al., 1999). Oxidative phosphorylation occurs mostly, if not entirely, in the deeply invaginated cristae of the inner mitochondrial membrane (Gilkerson et al., 2003; Vogel et al., 2006). The ATP synthase forms long rows of dimers at the tightly curved cristae ridges, while the respiratory chain complexes are confined to the remaining membrane regions (Davies et al., 2012, 2011; Paumard et al., 2002).
Ageing has long been linked to mitochondrial dysfunction. In mammals, the age-related weakening of physiological functions frequently goes along with a decline in health. While there has been considerable progress in the study of age-associated diseases, the biological mechanisms of ageing remain elusive. Ageing is attributed to either 'programmed' or 'damage-based' processes (de Magalhaes, 2011). More than 50 years ago, Harman postulated that ageing results from the accumulation of molecular damage caused by oxygen radicals, often called reactive oxygen species (ROS) (Harman, 1956). Oxygen radicals are side products of electron transfer reactions in respiratory chain complexes I and III (Dröse and Brandt, 2012). Later refinements of Harman's free radical theory of ageing postulated that an impairment of respiratory chain function increases ROS production, resulting in greater mtDNA damage, which in turn would compromise the turnover of damaged respiratory chain complexes, resulting in a vicious circle of damage and decline (Harman, 1972). The mitochondrial DNA mutator mouse model has provided experimental evidence of a causative link between mtDNA mutations and an ageing phenotype in mammals (Trifunovic et al., 2004), although mitochondrial defects in this model were found not to be associated with an increase in ROS production (Trifunovic et al., 2005). Several other key predictions of the free-radical theory (Stuart et al., 2014) have also not been substantiated. For example, in some model organisms, such as the nematode Caenorhabditis elegans, moderately elevated levels of ROS, induced either chemically or through genetically engineered defects in the respiratory chain, actually increase lifespan (Heidler et al., 2010; Lee et al., 2010; Yang and Hekimi, 2010). In the same organism, removal of the ROS-scavenging superoxide dismutase (SOD) does not increase oxidative damage to mtDNA and has no apparent effect on lifespan (Doonan et al., 2008; Gruber et al., 2011; Van Raamsdonk and Hekimi, 2009). In the fly, SOD knockouts do have a decreased lifespan, but they do not accumulate mtDNA mutations more quickly than wildtype (Itsara et al., 2014).
In addition to energy conversion and ATP production, the oxidative phosphorylation system also has a key role in shaping the inner membrane cristae (Davies et al., 2012, 2011; Paumard et al., 2002). Accumulating oxidative damage and OXPHOS dysfunction might, therefore, be expected to affect mitochondrial ultrastructure, which should be visible by electron microscopy. Recent analyses of yeast and mouse mitochondria show that a defect in the supra-molecular organisation of the ATP synthase results in aberrant cristae morphology (Davies et al., 2012; Mourier et al., 2014). In aged cultures of the short-lived filamentous fungus Podospora anserina, the inner membrane strikingly vesiculates and ATP synthase dimers break down (Brust et al., 2010; Daum et al., 2013). However, little is known about the impact of ageing on mitochondrial morphology and membrane structure in metazoans. Previous studies of the ultrastructure of mitochondria in mouse liver by electron microscopy of thin plastic sections revealed anomalous cristae in a subpopulation of the organelles (Wilson and Franks, 1975). A study of mouse skeletal muscle mitochondria found that mitochondria from animals that had exercised in an electrically-driven treadmill occasionally lost their cristae, whereas no differences were observed in non-trained animals (Ludatscher et al., 1983). Mitochondria from aged D. melanogaster flight muscle were found to have cristae 'swirls' that were attributed to oxidative damage (Walker and Benzer, 2004), and mitochondria in aged Drosophila repleta heart muscle were enlarged (Sohal, 1970).
We chose D. melanogaster (average lifespan >50 days depending on environmental conditions [Linford et al., 2013]) and mouse (average life span 107 weeks for females and 114 weeks for males [Turturro et al., 2002]) as two well-established metazoan ageing models (Cho et al., 2011) for a systematic study of mitochondrial ultrastructure, respiration and ROS production in young and old animals. Our results reveal clear tissue- and organism-related age-specific differences, establishing an apparent link between mitochondrial ultrastructure and function.
To find out how ageing impacts mitochondrial function and ultrastructure, mitochondria from heart, liver and kidney of young (20 weeks) and old (80–96 weeks) mice were isolated for electron cryo-tomography (cryoET) and high-resolution respirometry. Isolated mitochondria were incubated with respiratory substrates that deliver electrons at the level of complex I (pyruvate, glutamate and malate) or complex II (succinate and rotenone). The mitochondrial oxygen consumption was recorded under phosphorylating (addition of ADP and Pi; state 3), non-phosphorylating (addition of oligomycin to inhibit ATP synthase; state 4) and uncoupled conditions (addition of CCCP). Mitochondrial respiratory rates normalised to protein content were found to be highest in heart mitochondria, ~2–3 fold lower in kidney and ~5 fold lower in liver (Figure 1A). In line with previous reports (Mulligan et al., 2014; Weindruch et al., 1980; Wilson et al., 1975), our investigation showed no significant differences in respiratory rates between mitochondria isolated from 20-week-old and 80-week-old heart tissue (Figure 1A), and no age-related change in the rate of ATP production was observed (Figure 1C). Moreover, maximal activities of key mitochondrial enzymes remained unchanged (Figure 1E). These results prompted us to investigate how ageing impacts mitochondrial ROS homeostasis. We first assessed the peroxide yield, defined as the hydrogen production rate normalized to the mitochondrial respiration assessed under the same conditions (Votyakova and Reynolds, 2001). Surprisingly, no age-dependent increase in hydrogen peroxide release relative to the amount of oxygen consumed was observed in mitochondria isolated from heart, liver or kidney (Figure 1B). To investigate ROS homeostasis further, we analysed the steady-state levels and activity of the antioxidant enzymes superoxide dismutase (SOD1, SOD2) and catalase in mitochondria from young and old heart, liver or kidney by quantitative western blot electrophoresis and gel densitometry (Figure 1D). This revealed a tendency towards lower levels of antioxidant enzymes in aged mice. Catalase activity was reduced by 30% in old mouse liver (Figure 1F) and SOD activity was almost halved in old mouse kidney (Figure 1—figure supplement 1).
Bioenergetic and functional analysis of mitochondria from heart, liver and kidney from young (20 weeks old; white bars) and old (80–96 weeks old; black bars) mice.
(A) Oxygen consumption rate of mitochondria isolated from young (n = 7–8) or old (n = 8) animals. Isolated mitochondria were incubated with electron donors to complex I (pyruvate, glutamate, malate) or complex II (succinate, complex I inhibited with rotenone). Each set of substrates was successively combined with ADP (to assess the phosphorylating respiration, state 3), oligomycin (to measure non-phosphorylating respiration, state 4) and finally uncoupled by adding increasing concentrations of CCCP. (B) Mitochondrial peroxide yield assessed in mitochondria from young (n = 3–4) and old (n = 3–4) animals. (C) Mitochondrial ATP synthesis rate in heart mitochondria from young (n = 4) and old (n = 3) animals. Error bars indicate mean ± standard error of the mean (SEM). (D) Steady-state levels of different antioxidant enzymes in heart, liver and kidney mitochondria isolated from young (y) and old (o) mice were quantified by western blot analyses. Long (LE) and short exposure (SE) times are presented for catalase and SOD1 detection. (E) Citrate synthase (CS) and respiratory chain enzyme activity (complex I, II and IV) measurements in heart, liver and kidney tissue extracts from young (n = 4) and old (n = 4) animals. Error bars indicate mean ± SEM. (F) Catalase enzyme activity measured in heart, liver and kidney tissue extracts from young (n = 4) and old (n = 4) animals.
Vitrified samples of the mitochondrial preparations used for high-resolution respirometry were analysed by electron cryo-tomography (cryo-ET). Mitochondria isolated from young mouse heart showed morphologies typical of tissues with a high energy demand. Stacks of parallel, thin and flat lamellar cristae were embedded in a dense matrix (Figure 2, left, Video 1). Cristae frequently appeared discontinuous in 2D slices, but 3D volumes indicated that this was due to fenestration of the lamellar disks rather than to disconnected cristae vesicles (Figure 2—figure supplement 1A). Crista junctions were circular (70%, average diameter 15 ± 2 nm) or slightly elongated (30%, 16 ± 1 × 29 ± 2 nm, dimensions ± standard deviations measured in the typical tomogram of Figure 2, top row, second from left). Most cristae were branched and connected to the inter-membrane space by more than one junction. They were also highly interconnected through narrow apertures (Figure 2—figure supplement 1B). In 20% of the mitochondria examined, membranes of two or more neighbouring cristae were so closely appressed that there was almost no matrix between them. These regions often showed membrane 'swirls' of high and variable membrane curvature, involving several neighbouring crista lamellae (Figure 2—figure supplement 1C). There was no apparent difference between the structures of isolated sub-sarcolemmal and interfibrillar mitochondria. Comparing mitochondria from young and old mouse hearts indicated similar morphologies (Figure 2, right), except that about 24% of mitochondria from aged heart tissue had some exceptionally wide cristae (Table 1; Figure 2—figure supplement 1D).
Tomographic volume and 3D segmentation of the mitochondrion from young mouse heart shown in Figure 2, upper left, right hand panel.
Cryo-ET of heart mitochondria from young (left, 20 weeks old) and old (right, 80–96 weeks old) mice.
Upper panel: slices through tomographic volumes (scale bars, 250 nm). Lower panels: segmented 3D volumes of two typical mitochondria with closely stacked, roughly parallel cristae (blue). The outer membrane (omitted for clarity in the right panel) is yellowish grey. Cristae are connected to the intermembrane space by well-defined, multiple crista junctions.
Overview of organisms and tissues analyzed. #number of animals dissected (mouse) or number of mitochondrial preparations (fly) vs number of individual mitochondria examined tomographically.
The morphology of mitochondria isolated from mouse liver was conspicuously different from that of mouse heart (Figure 3, left; Video 2). The cristae were more heterogeneous, less regular and not arranged in parallel stacks. They were generally wider and did not span the entire mitochondrion. Also, the cristae were less interconnected to one another and to the inner boundary membrane. As in cardiac mitochondria, fenestration was observed, although it was less frequent. The matrix was very dense, making it difficult to segment larger mitochondria. In one sample, the matrix contained dense granules up to 50 nm in diameter (Video 2). No such granules were found in heart mitochondria. In two instances, mitochondria had central low-density compartments that we refer to as voids (see below).
Tomographic volume and 3D segmentation of the mitochondrion from young mouse liver shown in Figure 3, upper left.
Cryo-ET of liver mitochondria from young (left, 20 weeks old) and old (right, 80 weeks old) mice.
Upper panel: slices through tomographic volumes (scale bars, 250 nm). Lower panels: segmented 3D volumes. About 25% of the mitochondria from old animals have large central voids (red). The voids were connected to the inter-membrane space (IMS) by openings of variable size.
Tomograms of mouse liver mitochondria from aged animals revealed two different phenotypes. While the majority of mitochondria looked similar to those isolated from young mouse liver, 32% (n = 31) had conspicuous low-density compartments, or voids, in the centre of the organelle (Figure 3, right, Video 3; Table 1). Segmentation of 3D volumes revealed that the membrane delineating these central voids was continuous with the inner membrane. In three segmented mitochondria, the voids accounted on average for 22% of the total cristae surface. To estimate their volumes, voids and mitochondria were approximated as simple geometrical shapes (spheres or cylinders for voids, cylinders for mitochondria). In the three segmented organelles, the voids occupied on average 6% of the total mitochondrial volume. The voids were not internal vesicles, but were connected to the inter-membrane space via apertures of varying size. In some cases two such connections were observed on opposite ends of the voids, resulting in a toroid or doughnut-shaped matrix. In other cases, the outer membrane appeared to protrude into the inner membrane voids. The cristae extending from the boundary membrane into the matrix looked normal, but none protruded into the matrix from the membrane defining the voids. In two tomograms, dense granules measuring up to 100 nm in diameter were observed in the matrix.
Tomographic volume and 3D segmentation of the mitochondrion from old mouse liver shown in Figure 3, upper right, central panel.
Kidney mitochondria resembled heart mitochondria more closely than those from liver (Figure 4A). Mitochondria from young and old mouse kidney were similar, except that dense matrix granules were found in 18% of samples from old animals compared to only one (3%) found in the samples from young animals. Cristae were lamellar but generally wider than those in cardiac tissue and less stringently arranged in parallel stacks. Occasionally cristae formed membrane swirls. 18% of kidney mitochondria from old mice and 12% from young mice had the characteristic morphology described for apoptotic cells (Scorrano et al., 2002; Sun et al., 2007). In these mitochondria, the cristae were irregular, not arranged in any discernible pattern, and the membrane curvature was locally inverted. Crista junctions were very wide or not discernible, resembling those of prohibitin-deficient mouse mitochondria, in which OPA1 (which is essential for cristae junction formation) is incorrectly processed (Merkwirth et al., 2008). The inner membrane enclosed a convoluted but apparently continuous volume. The width of the inter-membrane space between the inner boundary and outer membrane was largely the same as in normal heart, liver or kidney (Figure 4B), indicating that the mitochondria were intact and had not suffered from osmotic shock during isolation (Wrogemann et al., 1985). Results are summarized in Table 1.
Cryo-ET of kidney mitochondria from young (20 weeks old) and old (80 weeks old) mice.
(A) About 80% of the kidney mitochondria from young (left) or old (right) animals had lamellar and locally parallel cristae, not unlike heart mitochondria, except that the cristae were less tightly packed. (B) About 20% of the mitochondria from young and old kidney showed an inner membrane morphology resembling that in apoptotic cells (Scorrano et al., 2002), without any discernible pattern and wide or irregular junctions. Scale bars, 250 nm.
Next, we compared the function and ultrastructure of wild-type mouse mitochondria to those from the heart and liver of mtDNA mutator mice, a strain with a premature ageing phenotype (Trifunovic et al., 2004). A minor respiratory defect was found under phosphorylating and uncoupling conditions in heart from mtDNA mutator mice (Figure 5A), consistent with previous observations (Trifunovic et al., 2004, 2005). The morphology of heart mitochondria from the mtDNA mutator mice resembled wildtype heart mitochondria, except for a high incidence (40%) of ellipsoidal peripheral voids connected to the cristae or the inter-membrane space that were delineated by a double membrane (Figure 5B). The matrix space between the two membranes was minimal. In one mtDNA mutator mouse heart mitochondrion, the outer membrane was punctured by small openings (Figure 5B, left).
Activity and ultrastructure of mtDNA mutator mouse mitochondria.
(A) Oxygen consumption rate assessed in heart mitochondria from control (white bars, n = 8, 30 weeks old) and mutator (black bars, n = 8, 30 weeks old) mice. Mitochondria were isolated and analysed as in Figure 1. (B–C) Cryo-ET. (B) Mutator mouse heart mitochondria had lamellar, parallel cristae that were similar to those of wild-type heart mitochondria (see Figure 2), but with occasional peripheral voids at the inner boundary membrane. (C) About 40% of liver mitochondria from mutator mice had low-density central voids, as in old liver (see Figure 3).
Mitochondria isolated from mtDNA mutator mouse liver showed the same central low-density voids (Figure 5C) as liver mitochondria from old wild-type mice (Figure 3), but at greater prevalence (40%; Table 1). In one case, cristae with apparently normal junctions extended from the central void into the matrix, which was not observed in wild-type mice (Figure 5C). About 20% of mtDNA mutator mouse liver mitochondria contained dense matrix granules (Table 1).
We also examined the ultrastructure and function of mitochondria from young and old D. melanogaster, a non-mammalian metazoan. The flies showed standard sigmoidal survival curves, with a mean lifespan of 69.5 days and a maximum lifespan of 78.5 days (Figure 6A). Life span correlated with climbing ability, as reported previously (Rhodenizer et al., 2008). Climbing ability remained constant for 25 days and then dropped rapidly (Figure 6A). We investigated the respiratory activity of mitochondria from young and old flies (15 and 70 days, respectively; Figure 6A). The respiration rate in old flies decreased by up to 60% (Figure 6B), and the peroxide yield increased by 40–100% (Figure 6C), indicating that mitochondrial function is severely compromised, confirming an earlier report (Ferguson et al., 2005).
Activity and ultrastructure of D. melanogaster mitochondria.
(A) Survival rates (n = 150; solid line) and climbing ability (n ≥ 25; dashed line) of wDah wild-type flies. Error bars represent SEM and arrows indicate sampling points for young (15 days old) and old (70 days old) flies. (B) Oxygen consumption rate assessed in mitochondria from young (white bars, n = 3) and old (black bars, n = 3) flies. Mitochondria were isolated and analysed as in Figure 1. Succinate and glycerol-3-phosphate (AS), and finally rotenone were added (GS) for comparison to complex I-driven respiration (CPI). (C) Mitochondrial peroxide yield in mitochondria from young (white bars, n = 3) and old (black bars, n = 3) flies. (D–E) Cryo-ET of typical mitochondria from young (D) or old (E) flies. Mitochondria from young flies had lamellar, mostly parallel cristae, similar to those from mouse heart. Mitochondria from old flies had highly variable shapes and cristae organisation, with the following main types (clockwise from top left): elongated and branched morphology; round, lacking cristae; small disconnected cristae; concentric narrow cristae (note that the innermost vesicle, shown in a darker shade of blue, appears to be unconnected to other membranes); irregular, wide cristae.
We performed cryo-ET on three sample preparations per age group to find out whether and how this functional decline is reflected in mitochondrial morphology. Results are summarized in Table 1. Mitochondria from young flies (n = 29; Figure 6D, Video 4) looked similar to those from mouse heart, with a dense matrix and thin, lamellar, mostly parallel cristae. All cristae had ridges indicative of ATP synthase dimer rows (Davies et al., 2011; Strauss et al., 2008) and several junctions to the intermembrane space. Wide cristae were observed in 10% of the mitochondria, similar to mouse heart. A small subpopulation of mitochondria (10%) was unusually elongated (axial ratio above 3:1) but looked otherwise normal. By contrast, the structure of mitochondria from old D. melanogaster (n = 39) was heterogeneous (Figure 6E). Approximately 75% of mitochondria had normal, well-developed cristae, although in 15% the cristae were wider (not shown). Some of these mitochondria were unusually long and thin (18%), or branched (5%) (Figure 6E, top left). In the remaining 25% of old fly mitochondria, the inner membrane assumed a variety of non-standard shapes. Several organelles that were identified as mitochondria by their characteristic double membrane and dense matrix appeared to lack cristae entirely (Figure 6E, top right) or the cristae were minimally developed (10%). Many cristae were not connected to the intermembrane space and were, therefore, small vesicles completely surrounded by matrix (Figure 6E, lower right, Video 5). In one mitochondrion, the cristae were spherical (Figure 6E, centre left). In two others, they were concentric (Figure 6E, lower left, Video 6), lacking the membrane ridges associated with ATP synthase dimer rows (Davies et al., 2011; Strauss et al., 2008). These observations establish a strong link between mitochondrial inner membrane organisation and respiratory function in D. melanogaster.
Tomographic volume and 3D segmentation of the mitochondrion from young fly shown in Figure 6D, upper panel.
Tomographic volume and 3D segmentation of the mitochondrion from old fly shown in Figure 6E, lower right.
Tomographic volume and 3D segmentation of the mitochondrion from old fly shown in Figure 6E, lower left.
Our study provides a systematic assessment of changes in mitochondrial function and inner membrane structure upon ageing in two common metazoan ageing model organisms, mouse and D. melanogaster. 3D volumes of entire mitochondria at an estimated resolution of 3 to 5 nm were generated by cryo-ET. As far as possible, mitochondria from different organisms and tissues were treated in the same way. The fact that there are clear differences between mitochondria from young and old mouse liver, for example, and that these mitichondria look different from heart and kidney mitochondria allows us to conclude that the isolation process itself does not affect mitochondrial membrane structure significantly. We conclude further that the different appearance of the young mouse liver mitochondria is not an artefact of isolation, but a genuine feature.
We found a clear correlation between OXPHOS capacity and the number of inner membrane cristae per unit volume. Mitochondrial respiration was highest in mouse heart and young fly mitochondria, which were entirely filled with closely stacked, parallel cristae. This arrangement, which is characteristic for tissues with high respiratory activity, maximises the membrane area available for oxidative phosphorylation (Davies et al., 2011). In mouse heart and young fly mitochondria, we frequently observed cristae fenestration, which is a morphological characteristic of tissues with high energy demand (Slautterback, 1965; Smith, 1963). Fenestration increases the total length of cristae ridges that harbour the ATP synthase, and hence the potential for ATP production. By comparison, mouse liver mitochondria had fewer cristae and the matrix was both denser and more voluminous, in line with the lower respiratory rate and higher metabolic activity of liver cells. Mouse kidney mitochondria were structurally more diverse. Remarkably, a significant proportion of kidney mitochondria from both young and old mice had a morphology typical of apoptotic cells (Scorrano et al., 2002; Sun et al., 2007). This correlates well with the recently reported continuous turnover and short lifespan of kidney cells of about 30–60 days (Rinkevich et al., 2014), compared to the 200–400 day lifespan of rat hepatic cells (Macdonald, 1961) and the very low turnover rates for cardiomyocytes of 1.3–4% per year (Malliaras et al., 2013). The peroxide yield correlated inversely with respiratory activity and was highest in mouse liver and lowest in mouse heart, suggesting that liver mitochondria may be affected by oxidative damage more severely than mitochondria from cardiac tissue.
Unexpectedly, levels of mitochondrial respiration, respiratory chain activity, hydrogen peroxide production or steady-state levels of antioxidant enzymes did not vary greatly with age, indicating moderate changes at most in respiratory rates and hydrogen peroxide yield in ageing mice. This contravenes the free-radical theory of ageing, which postulates a major age-related increase in ROS production and respiratory defects. In line with our results on mitochondrial function, the morphology of mouse heart mitochondria did not change much with age. By contrast, one out of four mitochondria from old mouse livers had a striking, age-specific phenotype characterized by a central matrix void. We found the same feature in mitochondria of mtDNA mutator mouse livers, where it was considerably more prevalent. Although we do not yet know what causes the voids and how they affect mitochondrial function, they evidently reflect a specific reorganisation rather than a random breakdown of the inner membrane. The lack of sharp cristae ridges and crista junctions in the void membranes suggests that these mebranes do not contain ATP synthase dimers and hence do not contribute to oxidative phosphorylation. The fact that mitochondria with these central voids have otherwise normal cristae may explain why no age-related reduction in overall respiratory activity was evident. If the majority of mitochondria are normal, they can apparently compensate for a loss of inner membrane area available for oxidative phosphorylation in 25% of the total population. Similar ring- or cup-shaped mitochondria with central cavities have been reported (Ghadially, 1988) and ascribed to the effects of drugs, toxins (David and Kettler, 1961; Stephens and Bils, 1965) or oxidative damage (Ding et al., 2012). Although the cavities found in these earlier studies were lined by both the outer and inner mitochondrial membranes, the central voids described here may likewise result from such damage, as damage to mtDNA is known to accumulate in liver tissues (Barazzoni et al., 2000; Yen et al., 1991). If denatured respiratory chain complexes are cleared from the membrane, the lipids left behind would be expected to form such featureless membrane regions expanding into the matrix interior. The matrix granules that we found in old liver and kidney mitochondria may consist at least in part of denatured respiratory chain complexes, consistent with their absence from old heart mitochondria, in which we did not observe matrix granules. Indeed, it has been shown that these granules contain complex IV subunits (Hertsens et al., 1986) in addition to lipids, glycoproteins and calcium (Ghadially, 2001; Jacob et al., 1994).
Interestingly, changes in the membrane structure of mouse mitochondria were organ-dependent, in a way that suggests different degrees of resilience against ageing. Mouse cardiac mitochondria showed the highest respiratory rates but appeared to be protected most effectively from oxidative damage, as indicated by their low and unchanged peroxide yield. Again, these findings contradict the classical free-radical theory, which would predict that cardiomyocytes with their high density of highly active mitochondria produce more ROS and thus age faster (Stuart et al., 2014). An analysis of DNA methylation has shown that heart tissue ages more slowly than would be predicted chronologically (Horvath, 2013). This observation is in line with our findings, which thus imply that mitochondria in vital organs with slow regeneration, such as the heart, are protected from damage more effectively than mitochondria in other organs. The effect may be genetically controlled through the expression levels of superoxide dismutase or catalase. Overexpression of catalase targeted to mitochondria has been shown to attenuate ageing effects in the murine heart (Dai et al., 2009). However, ROS signalling (Lenaz, 2012) requires a fine equilibrium of ROS production and sequestration that is probably tissue-dependent.
The most striking differences in our study, both with respect to morphology and activity, were found between mitochondria from young and old D. melanogaster. About half of the mitochondria from old flies had lost the standard organization of the inner membrane into boundary membranes with well-developed cristae and crista junctions. Many had round or concentric cristae that lacked sharp ridges. In extreme cases, cristae or crista junctions were completely absent. Measurements of respiratory activity in old fly mitochondria indicated that oxygen uptake was decreased by a factor of almost two and peroxide yield was increased by more than 50%, in line with increased mitochondrial H2O2 production in live ageing Drosophila (Cochemé et al., 2011). These results suggest strongly that about 50% of the old fly mitochondria are inactive, consistent with the observation that about 25% of the mitochondria in old flies lack normal cristae or crista junctions and that an additional 18% deviate from the standard morphology. Such a drastic breakdown of mitochondrial structure and function would result in the death of the organism within a short period.
It is interesting to compare our results on mouse and fruit fly mitochondria to similar studies on the filamentous fungus P. anserina (Brust et al., 2010; Daum et al., 2013), another well-characterized ageing model (Scheckhuber and Osiewacz, 2008). P. anserina has an average lifespan of only 18 days, three times shorter than D. melanogaster, and almost 50 times shorter than mouse. Morphological changes in P. anserina mitochondria were both more homogenous and more extreme than those in fruit flies, affecting about 80% of organelles from senescent populations (Brust et al., 2010). Cryo-ET of aged P. anserina mitochondria or inner membrane vesicles indicated that the cristae had receded into the inner boundary membrane and that ATP synthase dimers dissociated into monomers (Daum et al., 2013). In terms of inner membrane morphology, old P. anserina mitochondria resembled the quasi-apoptotic subpopulation in mouse kidney, the tissue with the highest turnover rate in our study, suggesting similar mechanisms of programmed ageing. On the one hand, the higher proportion of functional mitochondria in old flies indicates that the decline is less complete and slower than that in P. anserina. On the other hand, it is much more rapid in D. melanogaster than in mouse, suggesting a link between the complexity of an organism and the rate of ageing.
The increasing complexity of organisms goes along with an increasingly complex ageing process. For the primitive multicellular eukaryote P. anserina, a straightforward correspondence between age, mitochondrial ultrastructure and organization of the mitochondrial ATP synthase has been shown (Brust et al., 2010; Daum et al., 2013). In Drosophila, we now establish a link between inner membrane morphology and functionality, which correlates closely with age and agility. In mouse, the relationship between inner membrane ultrastructure, function and age is less clear-cut and evidently tissue-dependent. While mouse heart mitochondria show little if any change with age, a quarter of liver mitochondria display a severe, age-related phenotype that does not seem to result in an overall reduction of oxidative phosphorylation. A relatively high proportion of kidney mitochondria in young and old mice resemble those observed during apoptosis, consistent with the high turnover of kidney cells. Our data thus indicate major differences in how ageing relates to mitochondrial morphology and function in metazoans. In mouse, we find no evidence of age-related progressive impairment of the oxidative phosphorylation system or increase of mitochondrial H2O2 production, whereas both effects are evident in Drosophila.
All mice mutations in this study were on an inbred C57Bl/6N nuclear background. Mutator mice were generated as previously described (Ross et al., 2013). Briefly, PolgAWT/mut mice were generated by crossing a PolgAWT/WT female with a PolgAWT/mut male, and subsequently inter-crossing the progeny to generate PolgAmut/mut (mutator mice). Mice were maintained on a standard mouse chow diet and sacrificed at different time points by cervical dislocation in strict accordance with the recommendations and guidelines of the Federation of the European Laboratory Animal Science Association (FELASA). Protocols were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen, Germany (Permit ref: 84–02.05.20.12.086).
wDah wild-type flies were maintained at 25°C and fed a standard sugar/yeast/agar diet (SYA). Once mated, females, raised at controlled larval densities, were used. Adult flies were kept in SYA food vials containing 10 or 25 flies per vial for survival and climbing analysis, respectively. Climbing ability was assessed as previously described (Greene et al., 2003).
Mice were sacrificed by cervical dislocation, and heart, liver and kidneys were quickly collected in ice-cold DPBS (Gibco), minced and homogenized with a few strokes of a Potter S homogenizer (Sartorius) in 5 ml (for heart and kidney) or 20 ml (for liver) of ice-cold mitochondria isolation buffer (MIB; 310 mM sucrose, 20 mM Tris-HCl, 1 mM EGTA, pH 7.2). Mitochondria were purified by differential centrifugation (1200 g for 10 min) and the supernatants were then centrifuged at 12,000 g for 10 min. The crude mitochondrial pellet was resuspended in an appropriate volume of MIB. Mitochondrial concentration was determined using the Protein DC Lawry based assay (Bio-Rad).
Fruit flies were homogenized with a few strokes of a loose Potter S homogenizer (Sartorius) in 5 ml of ice-cold mitochondria isolation buffer (MIB; 310 mM sucrose, 20 mM Tris-HCl, 1 mM EGTA, pH 7.2). After filtration through a 100 µm nylon mesh filter, mitochondria were further homogenized in a tight Potter S homogenizer (Sartorius) and purified by differential centrifugation (800 g for 10 min) and the supernatant was then centrifuged at 4500 g for 15 min. The crude mitochondrial pellet was resuspended in an appropriate volume of MIB. Mitochondrial concentration was determined using the Protein DC Lowry based assay (Bio-Rad).
The rate of mitochondrial oxygen consumption was measured as previously described (Freyer et al., 2012) at 37°C using 65–125 μg of crude mitochondria in 2.1 ml of mitochondrial respiration buffer (120 mM sucrose, 50 mM KCl, 20 mM Tris-HCl, 4 mM KH2PO4, 2 mM MgCl2, 1 mM EGTA, pH 7.2) in an Oxygraph-2k (Oroboros Instruments). Oxygen uptake was measured using either pyruvate/glutamate/malate (10 mM pyruvate, 5 mM glutamate and 5 mM malate) or 10 mM succinate and 10 nM rotenone. Oxygen consumption was assessed under phosphorylating conditions with 1 mM ADP (state 3) or non-phosphorylating conditions by adding 2.5 μg/ml oligomycin (pseudo state 4). Respiratory control ratios (Brand and Nicholls, 2011) were above 10 with pyruvate/glutamate/malate and above 5 with succinate-rotenone. Respiration was uncoupled by successive addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) up to 3 μM to reach maximal respiration. The same procedure was used for D. melanogaster, except that isolated mitochondria were first incubated with pyruvate/glutamate/malate/proline (10 mM pyruvate, 5 mM glutamate, 5 mM malate, 10 mM proline) for state 3, state 4 and the uncoupled state. Succinate (10 mM), glycerol-3-phosphate (10 mM) and finally rotenone (10 nM) were then added to determine the maximal respiration driven by succinate and glycerol-3-phospate versus complex I driven-respiration.
Isolated mitochondria (65 µg/ml) were suspended in respiration buffer (see above). After addition of 2 mM succinate, 10 nM rotenone and 1 mM ADP, oxygen consumption and ATP synthesis rates were measured as previously described (Mourier et al., 2010). Aliquots were collected every 20 s and precipitated in 7% HClO4/25 mM EDTA, centrifuged at 16,000 g for 10 min and then neutralized with 2 M KOH, 0.3 M MOPS. The ATP content in these samples was determined with the ATPlite 1step (PerkinElmer). In a parallel experiment, oligomycin (2.5 μg/ml protein) was added to the mitochondrial suspension to determine the rate of non-oxidative ATP synthesis.
The rate of H2O2 production was determined by monitoring the fluorescence emission at 590 nm upon oxidation of the indicator dye Amplex Red (5 U/ml) in the presence of horseradish peroxidase (1 µM) with excitation at 560 nm. A standard curve was obtained by adding known amounts of H2O2 to the assay medium in the presence of the reactants. Mitochondria (65 µg protein ml−1) were incubated in respiratory medium (see above) at 37°C. H2O2 production was initiated by substrate addition, and the rate was determined by monitoring the fluorescence change with time (Votyakova and Reynolds, 2001).
Tissue proteins (15–50 μg) were diluted in phosphate buffer (50 mM KH2PO4, pH 7.4) followed by spectrophotometric analysis of isolated respiratory chain complex activities at 37°C using a Hitachi UV-3600 spectrophotometer. Citrate synthase activity was measured at 412 nm (ε = 13,600 M−1cm−1) after addition of 0.1 mM acetyl-CoA, 0.5 mM oxaloacetate and 0.1 mM 5,5-dithiobis-2-nitrobenzoic acid (DTNB). NADH dehydrogenase activity was determined at 340 nm (ε = 6220 M−1cm−1) after addition of 0.25 mM NADH, 0.25 mM decylubiquinone and 1 mM KCN and monitoring rotenone sensitivity. Succinate dehydrogenase (SDH) activity was measured at 600 nm (ε = 21000 M−1cm−1) after addition of 40 mM succinate, 35 μM dichlorphenol indophenol (DCPIP) and 1 mM KCN. COX activity was assessed using a classical TMPD/ascorbate assay. Briefly, homogenized tissue (65 µg/ml) was suspended in mitochondrial respiration buffer (see above). Oxygen consumption was assessed in the presence of TMPD (0.2 mM), ascorbate (1 mM) and antimycin A (0.5 µM). After a few minutes of stationary respiration, KCN (2 mM) was injected into the chamber. COX activity corresponds to the KCN-sensitive respiration. Catalase activity was assessed using an Oroboros oxygraph. Catalase activity of homogenized tissues (65 µg/ml) was followed by recording the oxygen production in the presence of 0.01% H2O2. All chemicals were obtained from Sigma Aldrich.
Proteins from tissue lysates were separated by SDS-PAGE and blotted onto PVDF membranes (GE Healthcare). The following primary antibodies were used: rabbit anti-superoxide dismutase 1 (1:1000, Abcam-ab16831) and rabbit monoclonal anti-superoxide dismutase 2 (1:1000, Millipore-06–984). The following HRP-conjugated secondary antibodies were used: donkey anti-rabbit IgG (Amersham, NA9340V) and sheep anti-mouse (Amersham, NXA931). For chemiluminescence detection, samples were incubated with ECL (GE Healthcare). Densitometry analyses were performed using the FIJI software.
Mitochondria were washed twice with 320 mM trehalose, 20 mM Tris pH 7.3, and 1 mM EGTA. Samples were mixed 1:1 with fiducial gold markers (10 nm gold particles conjugated to protein A, Aurion, The Netherlands) and immediately plunge-frozen in liquid ethane on Quantifoil holey carbon grids (Quantifoil Micro Tools, Germany). Single tilt series (typically ±60°, step size 1–1.5°) were collected at 300 kV with an FEI Polara electron microscope equipped with a post-column Quantum energy filter and an Ultrascan 4 × 4 k CCD camera (Gatan, USA), or with a post-column Tridiem energy filter and a 2 × 2 k CCD camera (Gatan). Alternatively, tilt series were recorded with an FEI Titan Krios electron microscope equipped with a Quantum energy filter and a K2 summit direct electron detector (Gatan). Underfocus was 8–9 µm and the magnification was chosen to give an object pixel size between 4.3 Å and 7.3 Å. The total electron dose per tilt series was 120–150 e-/Ų. Tilt series were aligned to gold fiducial markers and tomograms were reconstructed by back-projection with the IMOD software package (Kremer et al., 1996). A final filtering step applying non-linear anisotropic diffusion (Frangakis and Hegerl, 2001) was performed to increase contrast. Tomograms were manually segmented with the program AMIRA (FEI).
Journal of Biological Chemistry 275:3343–3347.
Zeitschrift Fur Zellforschung Und Mikroskopische Anatomie 53:857–866.
I Stuart-Hamilton, editors. Cambridge: Cambridge University Press.
Journal of Biological Chemistry 287:42373–42378.
Advances in Experimental Medicine and Biology 748:145–169.
Mitochondrial changes in ageing Caenorhabditis elegans--what do we learn from superoxide dismutase knockouts?
Journal of the American Geriatrics Society 20:145–147.
Journal of Ultrastructure and Molecular Structure Research 94:1–15.
Microscopy Research and Technique 27:307–318.
Mitochondria and reactive oxygen species. which role in physiology and pathology?
Advances in Experimental Medicine and Biology 942:93–136.
Journal of Visualized Experiments : JoVE, 10.3791/50068, 23328955.
Archives of Internal Medicine 107:335–343.
Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1797:255–261.
The Journals of Gerontology: Series A 69:799–809.
Molecular Genetics and Genomics 280:365–374.
The Journal of Cell Biology 24:1–22.
The Journal of Cell Biology 19:115–138.
The Journal of Cell Biology 24:500–504.
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 57:B379–B389.
Mechanisms of Ageing and Development 12:375–392.
Biochimica Et Biophysica Acta (BBA) - Bioenergetics 806:1–8.
Biochemical and Biophysical Research Communications 178:124–131.
Thank you for submitting your article "Changes of mitochondrial ultrastructure and function during ageing in mice and Drosophila" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Sriram Subramaniam as Reviewing Editor and Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Luigi Ferrucci and Terrence Frey.
Brandt et al., present a thorough study of the effects of aging in two model systems, mice (including mtDNA mutator mice) and Drosophila melanogaster that combines careful measurements of critical mitochondrial function, enzyme content, and the production of damaging reactive oxygen species (in the form of hydrogen peroxide) on purified mitochondria with state of the art cryo-electron tomorgraphy to elucidate changes in inner membrane topography. The goal is to investigate the hypothesis that aging is manifest in cellular mitochondria and mediated by increased production of reactive oxygen species. The study includes comparison of aging on the structures of mitochondria in three critical mouse tissues; heart, liver and kidney. Drosophila mitochondria were isolated from whole organisms. Except for the aged Drosophila mitochondria, the physiological measurements and enzyme contents do not change dramatically (or in many cases even significantly) with aging, but there are dramatic ultrastructural changes. Overall, the study combines careful measurements of critical mitochondrial function, enzyme content, and the production of damaging reactive oxygen species (in the form of hydrogen peroxide) on purified mitochondria with state of the art cryo-electron tomorgraphy to elucidate changes in inner membrane topography.
1) Some additional references to include: Sun et al., 2007 who studied inner membrane remodeling extensively in a HeLa cell culture model of apoptosis that is relevant to the changes observed in this paper. These changes were also observed by Merkwirth et al., 2008 who manipulated the protein OPA1 that regulates crista junction structure.
2) One of the reviewers appeared surprised that the authors did not detect a reticular structure of mitochondria network in the heart data, since this type of structure has been identified in other studies, or substantial differences between the sub-sarcolemmal and inter-myofibrillar mitochondria, since clear morphological differences have been identified with much less sophisticated imaging. The authors should comment on whether such structures may have been present but missed, given the isolation techniques used.
3) The analysis of the tomographic data is largely descriptive, with percentages given for the number of mitochondria falling into each category. In the majority of places there is no indication of the number of mitochondria considered, nor the number of animals from which they came. This makes the percentages hard to assess. The authors should provide a table detailing the number of animals and the number of mitochondria analyzed for each condition. Extracting as many quantitative parameters as possible to describe the morphological phenotypes would also be a good idea. Adjectives such as "several", "some", etc. should be replaced by numbers wherever possible. Additionally, the authors should clarify what fraction of inner membrane is lost to the "voids". Circular or slightly elongated crista junctions are mentioned but without sizes specified. Please provide measurements of crista junction sizes of a representative sample.
4) Since purified mitochondria are being analyzed, discussion of possible changes in structure during purification should be included. Also, in most of the tomograms, the cristae look fine, but in the young mouse liver, the cristae seem enlarged (bloated) and the matrix dense as if the mitochondrion is in a condensed state.
Thank you for bringing this point to our attention. We have included these citations in subsection "Tomography of mouse kidney mitochondria" and the Discussion section of the revised manuscript, and have added a sentence referring to the OPA1 mutants in subsection "Tomography of mouse kidney mitochondria".
The reviewer may be thinking of plastic sections of cardiac tissue, where mitochondrial networks in the sarcolemma or in myofibrils may indeed look substantially different. We would like to point out that cryo-tomography of isolated mitochondria cannot visualize intact mitochondrial networks, because they are necessarily disrupted during isolation. The isolated mitochondria from both cell types would look very similar, even if the networks may look different in plastic sections.
3) The analysis of the tomographic data is largely descriptive, with percentages given for the number of mitochondria falling into each category. In the majority of places there is no indication of the number of mitochondria considered, nor the number of animals from which they came. This makes the percentages hard to assess. The authors should provide a table detailing the number of animals and the number of mitochondria analyzed for each condition.
Thank you for this suggestion. We have now added such a table (Table 1).
Extracting as many quantitative parameters as possible to describe the morphological phenotypes would also be a good idea.
Adjectives such as "several", "some", etc. should be replaced by numbers wherever possible.
The adjectives have been replaced by exact numbers and standard deviations wherever possible.
Additionally, the authors should clarify what fraction of inner membrane is lost to the "voids".
This information is now included in a paragraph in subsection "Tomography of mouse liver mitochondria" of the revised manuscript.
Circular or slightly elongated crista junctions are mentioned but without sizes specified. Please provide measurements of crista junction sizes of a representative sample.
These measurements are now included in the first paragraph in subsection "Tomography of mouse heart mitochondria" of the revised manuscript.
As far as possible, mitochondria from different organisms and tissues were treated in the same way. The fact that there are clear differences e.g. between young and old mouse liver, and that these look different from heart and kidney mitochondria allows us to conclude that the isolation process itself does not affect mitochondrial membrane structure significantly. We can further conclude that the different appearance of the young mouse liver mitochondria is not an artefact of isolation, but a genuine feature. These points are now discussed in the first paragraph in the Discussion of the revised manuscript.
We acknowledge funding from the Max Planck Society and the Bundesministerium für Bildung und Forschung (SyBACol 0315893A-B) (to LST). NGL received additional support from the Swedish Research Council (2015–00418) and the Knut and Alice Wallenberg Foundation.
Animal experimentation: This study was performed in strict accordance with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). The protocol was approved by the Landesamt für Natur, Umwelt und Verbraucherschutz in Nordrhein-Westfalen, in Germany (Permit ref: 84-02.05.20.12.086).
© 2017, Brandt et al.


This month, Kathryn and Kenzi talk about what economics can teach you about uneaten peas and matcha lattes…Surprisingly a lot! Curious about Kahnemann and Tversky's paper, click here to find "The framing of decisions and the psychology of choice" mentioned in the podcast.
If you like what you hear, rate our podcast and subscribe to hear Kathryn and Kenzi talk about how economics relates to everything from dating to free things…and even uneaten peas. Leave your praise below (you can keep your critiques to yourself).


text:
Type of site
Independent filmmaking news
Penske Media Corporation
Slogan(s)
filmmakers. biz. fans.
indiewire.com
4,867 (April 2014)[1]
Newsletter:15 July 1996 (1996-07-15)
Website:January 12, 1998 (1998-01-12)
All rights reserved. Use permitted with copyright notice intact.
Established in 1996, IndieWire (sometimes stylized as indieWIRE or Indiewire) is a film industry and review website. As of January 19, 2016, Indiewire is a subsidiary of Penske Media.[2] It has a staff of about 20, including publisher James Israel, and Editor-in-Chief Dana Harris.
The indieWIRE newsletter launched on July 15, 1996, billing itself as "the daily news service for independent film." Following in the footsteps of various web- and AOL-based editorial ventures, indieWIRE was launched as a free daily email publication in the summer of 1996 by New York and Los Angeles based filmmakers and writers Eugene Hernandez, Mark Rabinowitz, Cheri Barner, Roberto A. Quezada and Mark L. Feinsod.[3] Initially distributed to a few hundred subscribers, the readership grew rapidly, passing 6,000 in the fall of 1997.[4]
In January 1997, indieWIRE made its first appearance at the Sundance Film Festival to begin their coverage of film festivals. It offered indieWIRE: On The Scene print dailies in addition to online coverage. Printed on site, in low tech black and white style, the publication was able to scoop traditional Hollywood trade dailies Variety and The Hollywood Reporter due to the delay these latter publications had for being printed in Los Angeles. Due to a zealous staff that was willing to print and distribute said dailies at all hours of the day and night, often handing them out to audiences waiting on line for films, indieWIRE was soon dubbed The School Paper. While the style and look of the print dailies improved over the years, the nickname stuck.
The website indieWire.com launched on January 12, 1998, and indieWIRE announced it would be charging for services. While met with cautious optimism by Wired magazine,[4] the experiment failed and indieWIRE returned to a free service less than a year later.
The site was acquired by Snagfilms in July 2008. On January 8, 2009, indieWIRE editor Eugene Hernandez announced that the site was going through a re-launch that has been "entirely re-imagined."
In 2011, with the launch of a redesign, the site changed the formal spelling of its name from indieWIRE to Indiewire.
In 2012, Indiewire won the Webby Award in the Movie and Film category.
indieWIRE is said to cover lesser-known film events ignored from the mainstream perspective. In Wired, Janelle Brown wrote in 1997:
"Currently, indieWIRE has little to no competition: trades like Hollywood Reporter and Daily Variety may cover independent film, but from a Hollywood perspective, hidden by a huge amount of mainstream news. As filmmaker Doug Wolens points out, indieWIRE is one of the few places where filmmakers can consistently and reliably keep on top of often-ignored small film festivals, which films are opening and what other filmmakers are thinking."[4]
In 2002, Forbes magazine recognized IndieWire, along with 7 other entrants in the "Cinema Appreciation" category, as a "Best of the Web Pick".[5] describing its best feature as "boards teeming with filmmakers" and its worst as "glacial search engine."[6]
indieWIRE has been praised by Roger Ebert,[7] Kevin Smith, James Schamus, and Tom Bernard.
Critics Poll
2006 The Death of Mr. Lazarescu Helen Mirren for The Queen
2007 There Will Be Blood Daniel Day-Lewis for There Will Be Blood
2008 Flight of the Red Balloon Mickey Rourke for The Wrestler
2009 Summer Hours Tilda Swinton for Julia
2010 The Social Network Édgar Ramírez for Carlos
2011 The Tree of Life Michael Fassbender for Shame
Michael Shannon for Take Shelter
2012 Holy Motors Denis Lavant for Holy Motors
2013 12 Years a Slave Chiwetel Ejiofor for 12 Years a Slave
Best Lead Actor
2014 Boyhood Richard Linklater for Boyhood Marion Cotillard for Two Days, One Night Ralph Fiennes for The Grand Budapest Hotel
2015 Mad Max: Fury Road George Miller for Mad Max: Fury Road Charlotte Rampling for 45 Years Michael Fassbender for Steve Jobs
↑ "Indiewire.com Site Info". Alexa Internet. Retrieved 2014-04-01.
↑ "Penske Media Acquires Indiewire". Variety. January 19, 2016.
↑ "Indiewire at 10 and Counting". July 15, 2006. (Press release)
1 2 3 Brown, Janelle (December 22, 1997). "Indie Film News Service No Longer Free". Wired. Retrieved 2011-05-22.
↑ "Forbes Best of the Web - Cinema Appreciation". Forbes. March 25, 2002. Retrieved 2010-05-21.
↑ "Forbes Best of the Web - IndieWire". Forbes. March 25, 2002. Retrieved 2010-05-21.
↑ Ebert, Roger (June 1999). "Rule of Thumb: Best Indie Crossroads". Yahoo Internet Life - Summer Movies Guide. ZDNet. 5 (6). Archived from the original on 1999-11-13. Retrieved 2011-05-22.
Snagfilms, the parent company

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: No implementation for List extends Vehicle> annotated with @Named(value="vehicles") was bound I want to inject a specific list of implementation classes to my Service class in a specific order via guice. I cannot use @ProvidesIntoSet because that will inject all implementations of my interface and I don't want that. I want to inject only some implementations and they should be in order. So, in my Module, I have this
    @Provides
    @Named(value = "vehicles")
    fun getVehicles(
        car: Car,
        bus: Bus,
    ): List<Vehicle> {
        return listOf(
            car,
            bus
        )
    }

Now, when I try to inject this list in my Service class like
class VehicleService @Inject constructor(
     @Named("vehicles") private val vehicles: List<Vehicle>,
...

I get an error like
     No implementation for List<? extends Vehicle> annotated with @Named(value="vehicles") was bound

How can I fix this issue?

A: You are running into this issue (https://github.com/google/guice/issues/1282.) with implied wildcard types.
You need to add @JvmSuppressWildcards as described in https://github.com/google/guice/issues/1641.

redpajama_set_name: RedPajamaStackExchange


text:
from django.db import models
from django.contrib import admin

class Entry(models.Model):
	nick=models.CharField(max_length=50)
	url=models.URLField(blank=True)
	date=models.DateTimeField(auto_now_add=True)
	text=models.TextField()
try:
	admin.site.register(Entry)
except admin.sites.AlreadyRegistered:
	pass

redpajama_set_name: RedPajamaGithub


The quality of the colour reproduction of lamps is described with the colour rendering index. The higher the Ra value is, the less the artificial light distorts the colour of objects. The maximum Ra value is 100.


text:
Hans H. Gattermann (24 December 1931 – 27 January 1994) was a German politician of the Free Democratic Party (FDP) and former member of the German Bundestag.

Life
In the 1976 Bundestag elections he was elected to the German Bundestag via the North Rhine-Westphalia state list, of which he was a member until his death. Here he had been chairman of the Finance Committee since 1983.

Literature

References

1931 births
1994 deaths
Members of the Bundestag for North Rhine-Westphalia
Members of the Bundestag 1990–1994
Members of the Bundestag 1987–1990
Members of the Bundestag 1983–1987
Members of the Bundestag 1980–1983
Members of the Bundestag 1976–1980
Members of the Bundestag for the Free Democratic Party (Germany)

redpajama_set_name: RedPajamaWikipedia


9 out of 10 based on 264 ratings. 3,875 user reviews.
Remove and Replace an Old Fuse Box Get Permission. You can do a lot of the electrical work around your home yourself,..Remove the Old Fuse Box. Have the mains electrical power shut off by your supplier.Add the New Fuse Box. You can now start to add the new fuse box.
Fuse Box Replacement. Depending on the age of your home and its current wiring system, it may be time to upgrade from a fuse box to breaker box to avoid damage to your home or electrical equipment.


text:
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    [autoFocus]="true" sidebarClass="sidebar" ariaLabel="Accessibility FTW" [animate]="true" [showBackdrop]="backdropEnabled">

Styling can be altered in the _sidebar.scss file in the global styling section. All Sidebar styling must be done here. Example

  </ng-sidebar>

Full usage guide found at: https://github.com/arkon/ng-sidebar

      <button class="custom-button" (click)="toggleSidebar()">Toggle sidebar</button>
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Price is after all rebates and incentives and does not include Tax, Title, License, Registration and Documentation Fees. Customer may qualify for additional incentives. See Dealer for Details $2,000 - TMS Customer Cash. Exp. 04/30/2019 Toyota of Bellingham is pleased to offer this. LE Plus Blizzard Pearl 3.5L V6 iPhone Integration, Back up Camera, AWD / 4x4 / Four Wheel Drive, 3rd Row Seat / 7 passenger/ Third Row, Bluetooth, MP3, All Weather Floor Liners, All-Weather Floor Liner Package (TMS), AM/FM radio: SiriusXM, Brake assist, Cargo Liner, Electronic Stability Control, Front Fog & Driving Lamp, Fully automatic headlights, LE Plus Package, Satellite Radio, Wheels: 18 Split 5-Spoke Machined-Face Alloy. 20/27 City/Highway MPG Toyota of Bellingham Excellent selection of New and Used vehicles, Financing Options!


An Abington school committee member who was defeated in Saturday�s annual election by five votes says he is weighing a possible recount.
�I haven�t decided yet. I am evaluating my options,� Wayne K. Rogiers said.
Town Clerk Linda C. Adams said Thomas E. McNulty defeated Rogiers, 827 to 822. Alex G. Kokoros placed third with 555 votes.
Rogiers has 10 days after the election to file a petition for a recount signed by 10 registered voters from each of the town�s four electoral precincts.
�He just asked me what he needed to do, if he chose to do it,� Adams said.
Adams said a hand recount of the electronic tally will carry additional costs to the town for the use of town counsel and ballot counters.


text:
Victor Oladipo Thinks Aaron Gordon Will Win Tonight's Slam Dunk Contest
by Abe Schwadron February 13, 2016
Minnesota's Zach LaVine is the overwhelming favorite to win tonight's NBA Slam Dunk Contest, after his incredible performance in 2015.
The man he defeated in the Finals of that dunk contest, though, is predicting a different winner tonight. Magic guard Victor Oladipo told SLAM this weekend that he likes his teammate, 20-year-old Aaron Gordon, to win it.
"I think he's going to do really well. He's going to give Zach a run for his money," Oladipo told us from courtside at the Jordan Brand Invitational inside the Mattamy Athletic Centre in Toronto. "I'm going with my man to win."
After putting on a show opposite LaVine at last year's Slam Dunk Contest but ultimately finishing in second place, Oladipo says he never really gave much thought to returning to the Dunk Contest this year.
"I'mma let my young boy go, I'm passing the torch," Oladipo said. "I told him to have fun, man, and enjoy the moment. Not a lot of people get the opportunity to be in the Dunk Contest."
Of fellow Jordan Brand athlete Andre Drummond—also participating in tonight's Dunk Contest—Oladipo guaranteed that Dre would "do something that's going to make the crowd go ooh and ahh."
Who do you think will win the Dunk Contest tonight: LaVine, Gordon, Drummond or Will Barton?
2016 Slam Dunk Contest Aaron Gordon nba all-star weekend 2016 Slam Dunk Contest Victor Oladipo
Victor Oladipo Denies Asking Out of Indiana
Report: Bucks, Pacers Discussed Victor Oladipo Trade
Victor Oladipo, Pacers Could Re-Visit Extension Talks
Victor Oladipo Looking to Move On From Pacers
Aaron Gordon Releases New Single 'LVL UP'


I am devoted to encouraging you to do excellent work in a coherent though challenging manner. I do so that you may practice your ability to analyze opposing arguments and decide for yourself, based on evidence the best means of revealing your academic strengths.
• Complete the assigned reading before coming to class and ask questions in class based on our readings.
• Turn in drafts of essays before the due date, and I can return it to you suggested improvements.
All papers must be based on books and discussions closely related to this course and assigned reading from the syllabus.
Web pages should be based on those papers.
of new knowledge and applying newly acquired information to old ideas.
Insert some text to support your interpretations of the authors.
"Life is but a walking shadow."
Citations: these are explicit footnotes, or end notes that identify to any and all readers where you got your ideas from. A citation answers the question: "So how do you know that?"
Plagiarism-- Just Don't do it.
You must never take another writer's words and use them as if they were your own. If you do that you are guilty of the crime of plagiarism. A person guilty of the crime is a plagiarist and is untrustworthy. Doing so will earn you an F in my or other classes.
G. P. Marsh, Man and Nature, 1864.
Content is judged by criteria and by the way you say things based on evidence for your arguments.
You must always refer to the sources of your information in your bibliography at the end of the paper.
Always remember that people have a lot on their minds, so be sure to say why, what you have to say is important!
Format is as important as what you say in the content, be sure to explain how your ideas fit together.
Web pages are really a way to present the essential information in an essay, in some graphically imaginative manner to best get across the key ideas you have discovered.
Links come in three kinds!
to the web: is National Public Radio's URL.
to another place on the same page: Takes you to a spot on a long page called an anchor!
If its easier to follow then its better!
Speed in loading is great!
Short filenames [8 letters of less!] can be explained in a site-map.


Did this lyrics for 19 ninety now theme transmission help you?
Now what's the freshest (freshest?) flow you ever heard in your life?
There is no comments for the 19 ninety now theme transmission lyrics sheet music yet. Please post one now!


There are two important differences between used to and would.
The first difference is that Would should not be used unless it has already been established that the time frame is in the past, while Used to does not require this.
The second difference between Used to and Would is that Would is not used with stative verbs such as love, be, understand, and feel.
1. I used to would I don't know read a lot of books in my college days.
2. When I was a kid, I used to would I don't know often visit my grandmother.
3. She used to would I don't know be a store manager, but now she is a doctor.
4. She used to would I don't know speak French when she was in Paris.
5. When he was young, Harry used to would I don't know get up early.
6. My mom used to would I don't know make me do my homework, but now she doesn't.
7. The cat used to would I don't know lie on her lap while she was working.
8. A few years ago, they used to would I don't know practise cricket over there, but now they don't.
9. I used to would I don't know go to my sister's to play chess, but now I don't.
10. He used to would I don't know play with his friends in abandoned houses when he was a child.


She was experiencing a clasic case of sciatica; the pain started in her back and traveled from her back all the way down her leg into her foot.
I have a video of the 5 main causes of sciatica, and she had all 5. She was in bad shape and was definitely getting worse.
She had seen 4 separate specialists before entering our clinic including a consultation with a surgeon who recommended surgery for her back. Six months before coming into our clinic she had 3 injections into her spine to help eliminate her pains. The shots were effective for a few weeks before the pain came back.
She had diminished reflexes in her leg as a result of her pinched nerve, and was also showing signs of weakness. Her core was weak and she lacked endurance with simple activities.
When she started therapy with our program her pain was a 9 and her ODI was a 42%. Because she was experiencing the pain for over 5 years, in conjunction 5 different therapies and her ODI was still over 40% she was a prime candidate for surgery.
Her goal was avoid the surgery, but we were difinitely her last hope.
After her first 12 treatments her pain was down to a 2 from a 9, she no longer had pain and numbness in her foot. Her reflexes were back to normal and her ODI score was 22. She was making better progress then she had in the last 5 years.
After 30 treatments her pain was at a 0, she no longer had any numbness and pain down her leg. Her core strength was better and her ODI was a 16. ODI below 20 is considered the safe zone in therapy.
ODI 16 when she finished, into the safe zone.
We saved her from and inevitable surgery. Any patient that has back pain for more than a year and receives therapy and still has an ODI score above 40 is a candidate for surgery, and this was the case with our patient. We were able to get her out of pain and create functional stability so she can live pain free.


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When I saw these sitting on my counter the yesterday, I knew something needed to be done.
You know how I feel about wasting precious ingredients.
Since I had plans last night to go to a Passover dinner at my sister's, I wanted to make something that would be kosher-friendly. Then I realized cheesecake is almost entirely flour-free. The only real issue is the typical cookie crust.
My mom made a non-fat banana cheesecake a few years ago that was really delicious. I remember being so impressed by the creaminess despite using fat-free cream cheese. This time around, I used light cream cheese for half to prevent the cheesecake from being too dense.
Instead of using graham crackers, I made an almond crust. So not only is this cheesecake Passover friendly, but it is also gluten-free.
What is your favorite light dessert?
I am absolutely enthralled by this cheesecake and cannot wait to make it!
I don't know what else to say but yum. And I want this immdiately. Ok I guess I knew what to say.
The cheesecake looks great. I love the subs you did for the cream cheese filling.
Wow this cheesecake looks and sounds amazing! Love the Fage in there too!
I love cheesecakes, but have never had one that is banana flavored before. Such good thinking! This looks fantastic :) Happy Easter :) Wishing you a fantastic week!
Kosher for passover, gluten free, low fat…is there anything that this cheesecake CAN'T do? Sounds amazing!
I LOVE a great almond crust. Has so much flavor especially paired with bananas. Lovely cheesecake!
Now THIS looks like a perfect dessert! I would feel good serving this to company anytime! I love that you topped it with a simple drip of berry sauce. What a beautiful presentation!
I may just have to let my bananas sit a little too long this week!
Ah! i have a couple family members who are gluten free and this would be so nice at holidays! thanks for sharing!
That is gorgeous, Lauren! ANd I love the crust made with almonds.
Thanks, Carolyn! You could probably make it low(ish) carb pretty easily by swapping out the sugar.
This is a must make recipe for me! Love light, fluffy cheesecakes!
As much as I love your decadent desserts, I think I love this lightened up dessert the most! Looks like an amazing take on banana pudding (my absolute favorite dessert). Genius!
Thanks! I used to make lighter desserts much more often… I should bring them back for summer.
Iwill definitely have to try making this for my husband soon! He loves cheesecake!
Lovin' the Fage, although I'll admit, I'm lovin it most when it's full fat (Fage Total).
I guess I don't often make light desserts. I made a cake yesterday that had five sticks of butter between the cake, lemon curd and frosting. That makes me a bit sick to think of now. But it sure was deeeeelicious!
I'm usually morally opposed to light desserts (that's an oxymoron in my kitchen, LOL!) but I swear, I would totally make this. At least you didn't go too far and use Splenda, otherwise I might have to slap your hand with a ruler. ;) It looks soooo good! I like the description of it being lightly textured–that sounds wonderful!
Hahaha. You really can't tell that it is low fat when you eat it.
I love the idea of a light banana cheesecake!
Wow! This looks divine! The almond crust really sets it over the top for me.
This looks great! I love the addition of an almond crust.
I got the pleasure of trying this and it was that delicious! Very light & gluten free!
Wow, this looks fantastic! I love how many light switches you made to the cheesecake itself and the almond crust sounds delicious. I'm definitely going to give this a try!
This looks delicious. I'd probably pair this with a late harvest Riesling, perhaps a Sauternes.
I bet your husband will be a happy man! Let me know how it turns out.
OMG this looks so good. Love that it's low fat too. I have just the right amount of browning bananas that need to be used too! Buzzed this!
banana.. cheesecake.. oh my! Two of my favorite things in one :D Fage in there too. You're a genius !
The best cheesecake ever! So fluffy and perfect! I made it and it was a total success!!!
I'm so happy to hear that! Thank you for the nice comment, Alejandra!!
How long would I bake this in a mini Cheesecake pan?
Hi Andromeda- Unfortunately I'm not sure because I haven't tried it. If you are using a small muffin pan you could try 15 minutes and check at that point. If you do try it out, please let me know!
So, Just checking. It's a total of 32oz block cream cheese, right? Planning on making it for my hubby's birthday. Also, what size springform pan did you use?
That's right, 32 ounces total of cream cheese. This recipe works with a 9-inch spring-form pan.


Does your child complain of having difficulty doing their assignments in school because they can't concentrate or pay attention properly? Maybe they have to read the same material all over again, and still can't grasp what they are reading, and it can be frustrating to them. Then no doubt, there is a problem. It could be your child has a disability that they cannot help. Symptoms of learning disabilities include delayed language skills, trouble rhyming words, habitual mispronunciation, inability sounding out letters in simple words, difficulty learning letters and numbers, confusion in using words that sound alike, and difficulty following instructions.
What can you do if your child seems to have a learning disability?
Have his vision and hearing tested to rule out these causes.
Take advantage of any special program your child's school might have for children with disabilities.
Your child mirrors what he or she sees in you. So it is good if you always show a positive Outlook in learning and life as a whole.
Different approaches are used to different disabilities. be very careful on what you may want to use for it cold either encourage or discourage them. Below are some tips on how to handle different disabilities.
Kids who have hearing impairment will have hard time catching up with fast-paced speaking when they are under a traumatic event. Anyone caring for kids with this impairment should be very sensitive. Be mindful of your descriptions and the words that you use. Visual aides can be helpful when you want to convey something to them.
Kids with visual impairment are unable to pick or catch facial expressions and some other visual cues. A good way to let them understand what you are trying to say or describe with verbal cues. Describe verbally as accurate as possible all the things that they hear.
Of course we want our kids with special needs to live like the other kids in the neighborhood. We do not want them to feel they are different and needs to stay away from others but let's admit that special kids need special care and attention. As parents, it is our responsibility to make them feel loved and valued despite the disability but seeking the help of some special needs organization would even help us carry out those responsibilities. If you are considering of looking for one, below are some suggestions that you may want to look on.
The organization focuses on the needs of teenagers with disabilities by helping them cope up with the disability they have and be able to fit in a suitable workplace for them in the future.
The organization is located in Boston assisting both parents and children with special needs. Parents and kids are allowed to live in an environment where they can have a full participation to community works. This will help the kids realize the potentials they have.
In here, parents with kids of the same disability pairs up and serve as a support system to each other. They share some ideas on how the situation should be dealt on and what to do to fully help the kids realize their worth. Not only do parents and kids receive a good support system but new friendships built as well.
Project or Making Opportunities Reality Granting Assistance Nationwide. This group gives full support to families with special kids may they be biological or adopted. Besides reading materials that they provide in education parents with special kids, the organization also shoulders medical and travel expenses which are not under the insurance policy of parents.
The organization disseminate information about child and teen disabilities. Part of its aim in educating people about proper special child care is coming up with services and programs on how such special needs be addressed. The organization does not focus on a single or several disability but they have wide resources on the different ways to handle these children and teens.
Some indicators of learning disabilities may also be more prevalent during specific developmental stages such as difficulty in learning the alphabet during preschool, difficulty in telling time during ages 5 to 9 and poor handwriting during ages 10 to 13. Parents and other significant caregivers are expected to be familiar with and closely monitor developmental milestones so that they could immediately delays and lags. When you have observed quite a number of developmental difficulties and lags in your child, it would be best to consult a pediatrician or a psychologist. Whether or not your child has a learning disability, seeking expert opinion will help you understand what your child is really going through and what you can do about it.
Learning disabilities are classified into different types according to the specific skill or developmental milestone. Learning disabilities are termed as dyspraxia for problems in movement and coordination, dysphasia and aphasia for impairment in spoken and written communication, dyscalculia for problems with arithmetic and numbers, dysgraphia for difficulties with handwriting and spelling, auditory processing disorder referring to the inability to process differences between sounds and visual processing disorder referring to the difficulty in interpreting visual information. In addition, other psychological disorders also seem to have a major adverse effect in a child's learning such as attention deficit hyperactivity disorder (ADHD) and autism.
You, as a parent, should know that your child needs you more in times like these than ever. You should take active involvement in his or her struggles by keeping yourself well-informed and educated about your child's specific condition or learning disability. Never isolate your child from other kids. Give them opportunities to develop their emotional and social skills - areas that are not hampered by their learning disability.
A social welfare health and medical services program initiated by the government, Medicaid is intended to provide security and financial assistance to individuals and families in the lower-income bracket during health and medical emergencies. Millions of elderly individuals and families find Medicaid benefits to be such a relief in times of need and uncertainty. Even non-elderly individuals, both employed and unemployed, could still count on the assistance from Medicaid benefits. Although Medicaid is predominantly initiated by national government, the management and implementation of benefits availment is under the jurisdiction of state governments. Different states may wish to add implementing rules and regulations to the standard ones mandated by federal institutions.
What makes Medicaid an even better news is that it does not exempt people with disability from reaping the benefits of this insurance and financial assistance. Even other insurance programs that are supposedly tagged to be specific for disabled individuals and their families seem to have a narrower scope of specific disabilities that will be covered. Even Social Security disability benefits have been elusive of some disabled people. Fortunately, those who have been denied from availing these Social Security disability benefits could already count on the opportunity to take advantage of Medicaid benefits for people with disabilities. Important requirements to be eligible to receive Medicaid benefits include residency, immigration status and proof of US citizenship.
However, one should also bear in mind that Medicaid is not simply a dole-out charity program. Governments have strictly highlighted that families or individuals who could no longer rely on personal assets will priority recipients for Medicaid benefits. Implementing and monitoring institutions will strictly do background check on Medicaid applications to ensure that there will no fraud in the transactions. It has been noted that some Medicaid applicants have resorted to transferring their personal assets to the name of other individuals to establish or ascertain their economic status or income level. In fact, applicants who still have properties that could be passed on to their next generations will most likely be denied from Medicaid benefits. Low income and very limited assets are the top requirements for Medicaid availment.
Likewise, Medicaid disability benefits will not automatically be handed to you in a silver platter. Medicaid programs, facilities and services could vary from one state to another. Thus, it would be best to research what facilities and services are being made available by Medicaid in your state. The services you need may not be the ones offered or the ones available may not fit your need. Some facilities also choose not to accept Medicaid for payments for certain issues that have not been disclosed to the public.
Nevertheless, the Medicaid disability benefits program features several advantages that are standard across all states. Medicaid covers long-term care services both at home or at accredited nursing facilities. Unfortunately, home-care services have minimal coverage only in some states. You will no longer need to shell out your hard-earned money to avail of health and medical services. Yet, every program or initiative is not free from disadvantages and drawbacks. You can only use your Medicaid disability benefits with accredited providers, facilities and professionals. This may pose a problem if you have already maintained regular consultations with a medical specialist who is not accredited by Medicaid. The continuity of providing medical assistance and the consistency of the quality of care may be significantly compromised.
Financial experts always emphasize that financial stability goes beyond making your finances grow. To be financially smart, one should also invest in his or her future. You should also invest in protection for whatever you have - your house, your car and, most specially, your life. One aspect that is often overlooked in insurance is one's earning power. It is for this reason that disability insurance is considered to be a good buy even by the most skeptical consumer. Even if your company provides you with a certain disability coverage, it is too risky to assume a false sense of security. You will most likely find a crowd of disability insurance providers and agents offering you their policies. Here is a rundown of tips to help you find your way through different policies and get yourself the best disability insurance coverage you truly deserve.
This type of policy ensures that the coverage will protect you during those times you will no longer be able to perform or function at your current job. On the other hand, an 'any occupation' type of disability can only provide you with coverage when you do not have any job at all. If you land in any job, even if it is a case of underemployment, an 'any occupation' policy will not have you covered even with a single cent.
In most states, retirement benefits are provided when an individual reaches the age of 65. Some insurance policies only offer coverage of up to five or ten years after the date of signing up for the policy. If there is a long gap between the end of your disability insurance policy coverage and your eligibility for retirement benefits, it would mean that you will not be receiving financial assistance for those years. Insurance policies with five or ten-year coverage may have lower premium rates but may even be more expensive in the long run. Disability insurance policies with more years of coverage may seem more costly upfront but may be more practical and cost-efficient in a long-term perspective.
Make sure you can financially endure the long haul. An emergency fund will surely be helpful during these situations. The lack of an emergency fund or a contingency plan to sustain your expenses when you are off from work might lure you into debts.
The premiums you have to pay are computed based on how much benefits you would want to receive in the future. Some policies offer coverage that would equal to around 60% of your gross income. However, be careful about choosing a policy coverage with premium rates that may be too high for you to maintain.
Certain occupational classes such as contractors and seafarers face greater risks and threats than others like managers and office-based workers. You have to bear in mind that your insurance policy provider will also calculate your premium rates based on your occupational class. If you belong to high-risk professions, you may be required to pay higher premium rates. Likewise, self-employed and work-at-home professions also signal greater risk to the insurance policy provider because of the difficulty in verifying income.
Regardless of the profession you belong to, having supplemental disability insurance will surely be a lifesaver during the rainy days. Disability insurance serves as a protection for your earned income when certain disabilities disable you from fulfilling your work responsibilities. When the core functions of your work are already compromised due to the disability, you will surely appreciate having supplemental disability insurance within your reach. Disabilities can be brought about by psychological problems, illnesses or injuries. By availing of supplemental disability insurance, you will be receiving periodic payments of benefits during a specified period of time. In most instances, coverage of supplemental disability insurance policies could range from 40% to 65% of your gross income.
Nevertheless, specific coverage may vary from one policy to another. Computation of premium rates that should be paid by an applicant for supplemental disability is also based on several factors such as occupational classification. In addition, choosing a supplemental disability insurance to get for yourself is not as easy as it sounds to be because there are different types of disability insurance offered by a crowd of policy providers. Here are tidbits of what makes each type different from the others.
Individuals who are self-employed or who work at home are usually denied of their typical disability insurance because of the difficulty in verifying their income. If you want a wider coverage that will encompass a broader category of circumstances, your policy may come with high premium rates. Aside from the scope of the coverage, your specific work, the risk involved and your place of residence will also be considered in computing your insurance policy's premium rate.
This specific type of policy is most highly recommended for businessmen and entrepreneurs. This policy will cover overhead expenses of the business when the owner suffers from any type of disability. Disability of the owner could certainly take its toll on the business finances. With business overhead disability insurance, rent or mortgage costs, maintenance fees and even salaries and wages could still be taken care of.
You may not own the business but you hold a critical position or role in it. Thus, your absence could have a significant impact on the company you are working for. This could be a major headache for the business owner, your boss. This type of insurance helps ensure the continuity of business transactions even in the absence of a key official in the business. Thus, the risk for losses due to hampering of business operations is minimized.
Regardless of your income bracket, you can still enjoy individual disability benefits at 65% by availing of high-limit disability insurance. This specific insurance policy is still offered on top of the individual's disability insurance policy.
A benefit provided by employers to their employees, this specific type of policy aims to provide insurance in cases of on-the-job injuries and job-related disabilities. This may or may not be part of an employee's standard compensation package. Likewise, the coverage is only valid throughout the employment of the individual with the particular company.
This government-provided insurance coverage is intended to provide financial assistance for the citizens during times of uncertainties and financial instability. Through these government-funded programs, no citizen of these particular countries could claim to be empty-handed.
Make sure your child has a healthy diet of whole grains, fruits, and vegetables.
Have them get plenty of sleep for learning.
Love your child and support them with their challenges they face.
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