News Article | March 9, 2016
Extended Data Table 1 summarizes all the observations made in both the initial 2013 follow-up and 2015 May/June observations (project code p2886). The ALFA beam 0 pointing positions in J2000 equatorial coordinates are summarized in Extended Data Table 2. In the p2030/p2886 observations, the major axis of the ALFA receiver was rotated 19°/90° with respect to North4. In 2015 May/June, we searched for additional bursts from FRB 121102 using a grid of six pointings using the seven-beam ALFA receiver to cover a generous ~9′ radius around the discovery beam position and side-lobe. The ALFA receiver was aligned East–West to optimize the sky coverage for this specific purpose. The centre beams of the six grid pointings are shown in red in Fig. 1, and the six outer ALFA beams are shown in blue. Each grid pointing position was observed at least four times for ~1,000 s. The beam positions of the discovery observation and 2013 follow-up gridding4 with ALFA (in that case rotated 19° with respect to North) are also indicated using the same colour scheme. The outer six ALFA beams in the multiple grid observations are only at roughly the same position because the projection of the ALFA beams on the sky depends on the position of the telescope feed with respect to the primary reflecting dish, and these do not overlap perfectly between independent observations. Two bursts on May 17 (bursts 2 and 3) and two on June 2 (bursts 4 and 5) were detected at a single grid position: FRBGRID2b in ALFA beam 6, which had positions of α = 05 h 32 min 01 s, δ = +33° 07′ 56′′ and α = 05 h 32 min 01 s, δ = +33° 07′ 53′′ (J2000) at the two epochs—that is, only a few arcseconds apart. Six more bursts (bursts 6–11) were detected on June 2 at a neighbouring grid position, FRBGRID6b in ALFA beam 0, ~1.3′ away at α = 05 h 31 min 55 s, δ = +33° 08′ 13′′. In all cases bursts were detected in only one beam of the seven-beam ALFA receiver at any given time. This shows that the bursts must originate beyond Arecibo’s Fresnel length of ~100 km (ref. 19). The intermittency of FRB 121102 makes accurate localization more challenging. Nonetheless, the detection in adjacent grid positions is informative, and to refine the position of FRB 121102, we simply take the average position between FRBGRID2b ALFA beam 6 and FRBGRID6b ALFA beam 0, which gives: α = 05 h 31 min 58 s, δ = +33° 08′ 04′′ (J2000) and, equivalently, Galactic longitude and latitude l = 174.89°, b = −0.23°. The approximate uncertainty radius of ~3′ is based on the amount of overlap between the two detection beam positions and the ALFA beam width at half power, which is ~3.5′. The distance from the initially reported burst 1 position is 3.7′, consistent with the interpretation that this burst was detected in a side-lobe. Although FRB 121102 bursts have been detected in beams with different central sky positions, all detections are consistent with a well defined sky position when one considers the imprint of the ALFA gain pattern on the sky during each observation4. Noteworthy is the fact that FRB 121102 lies directly in the Galactic plane, whereas the other claimed FRBs lie predominantly at high Galactic latitudes. The PALFA survey is only searching in the Galactic plane, however, and no comparable FRB survey at 1.4 GHz with Arecibo has been done at high Galactic latitudes. Therefore, this difference may simply be a consequence of where Arecibo has most deeply searched for FRBs and does not necessarily suggest that FRB 121102 is of Galactic origin. Furthermore, FRB 121102 was found in the Galactic anti-centre region of the PALFA survey, whereas searches in the inner-Galaxy region have thus far found no FRBs14. This may be because the Galactic foregrounds in the anti-centre region are comparatively low, so the deleterious effects of DM smearing and scattering, which may reduce our sensitivity to FRBs, are less important in the outer Galaxy than the inner Galaxy. The low Galactic latitude of FRB 121102 also contributes to its low DM excess factor β ≈ 3 compared to the β ≈ 1.2–40 range seen for the other 15 FRBs in the literature. Only FRB 010621 (ref. 27), with β ≈ 1.2, has a lower β than FRB 121102, and it has been proposed to be Galactic28. We note, however, that six of 16 FRBs have DMs comparable to or lower than FRB 121102. Furthermore, its total Galactic DM excess pc cm−3 is larger than that of the first-discovered FRB1. Lastly, within a generous 20-degree radius of FRB 121102, the highest-DM pulsar known is the millisecond pulsar PSR J0557+1550 (ref. 29; also a PALFA discovery), which has DM = 103 pc cm−3and β = 0.6, as well as the highest DM-inferred distance15 of any pulsar in this region, d = 5.7 kpc. FRB 121102’s DM is clearly anomalous, even when compared to this distant Galactic anti-centre pulsar. At an angular offset of 38°, we note the existence of PSR J0248+6021, with DM = 370 pc cm−3 and β = 1.8. Although the DM of this young, 217-ms pulsar is in excess of the maximum Galactic contribution in the NE2001 model15, this can be explained by its location within the dense, giant H ii region W5 in the Perseus arm30 at a distance of 2 kpc. A similar association for FRB 121102 has been sought to explain its β ≈ 3, but multi-wavelength investigations have as yet found no unmodelled Galactic structure4, 19. In summary, FRB 121102’s comparatively low β does not strongly distinguish it from other FRBs, or necessarily suggest it is more likely to be Galactic. Here we provide a brief description of the Arecibo Mock spectrometer data and search pipeline14 used for our follow-up observations of FRB 121102. The 1.4-GHz data were recorded with the Mock spectrometers, which cover the full ALFA receiver bandwidth in two subbands. Each 172-MHz subband was sampled with 16 bits, a time resolution of 65.5 μs, and frequency resolution of 0.34 MHz in 512 channels. The data were later converted to 4-bit samples to reduce the data storage requirements. Before processing, the two subbands were combined into a single band of 322 MHz (accounting for frequency overlap between the two subbands), which was centred at 1,375 MHz and spans 1,214.3–1,536.7 MHz. We used the PALFA PRESTO-based16 search pipeline14 to search for astrophysical signals in the frequency and time domains. These data were processed using the McGill University High Performance Computing Centre operated by Compute Canada and Calcul Québec. The presence of RFI can have a detrimental effect on our ability to detect bursts. We therefore applied PRESTO’s rfifind software tool to identify contaminated frequency channels and time blocks. Flagged channels and time blocks were masked in subsequent analyses. Time blocks contaminated by RFI are identified using data that are not corrected for dispersive delay (that is, DM = 0 pc cm−3), in order to avoid removing astrophysical signals. The data were corrected for dispersion using 7,292 trial DMs in the range 0–9,866.4 pc cm−3, generating a time series at each trial. We performed Fourier analyses of all the time series to look for periodic signals using PRESTO’s accelsearch software tool and detected no significant signal of a plausible astrophysical origin. We searched for single pulses in each dispersion-corrected time series by convolving a template bank of boxcar functions with widths ranging from 0.13 ms to 100 ms. This optimizes the detection of pulses with durations longer than the native sample time of the data. Single-pulse events at each DM were identified by applying a signal-to-noise ratio (S/N) threshold of 5. These single-pulse events were grouped and ranked using the RRATtrap sifting algorithm17. An astrophysical pulse is detected with maximum S/N at the signal’s true DM and is detected with decreasing S/N at nearby trial DMs. This is not generally the case for RFI, whose S/N does not typically peak at a non-zero trial DM. The RRATtrap algorithm ranks candidates based on this DM behaviour and candidate plots are produced for highly ranked single-pulse groups. These plots display the S/N of the pulse as a function of DM and time as well as an image of the signal as a function of time and observing frequency (for example, Fig. 2). The resulting plots were inspected for astrophysical signals, and pulses were found at a DM of ~559 pc cm−3 at a sky position consistent with the discovery position of FRB 121102 (ref. 4). It is possible that the analysed data contain weaker bursts, which cannot be reliably identified because their S/N is too low to distinguish them from RFI or statistical noise. If, in the future, the bursts are shown to have an underlying periodicity, then this would enable a deeper search for weak bursts. Using several approaches, we searched for an underlying periodicity matching the arrival times of the eight bursts detected in the 2015 June 2 observing session. There are no significant periodicities detected through a standard fast Fourier transform of the time series. We then carried out a similar analysis to that routinely used to detect periodicities in sporadically emitting radio pulsars31. In this analysis, we calculate differences between all of the burst arrival times and search for the greatest common denominator of these differences. We found several periods, not harmonically related, that fitted different subsets of bursts within a tolerance of 1% of the trial period, but none that fitted all of the bursts. We subsequently calculated residuals for the times-of-arrival for the eight bursts detected on 2015 June 2 for a range of trial periods using the pulsar timing packages TEMPO and PINT (see ‘Code availability’ section). We found that some of the periods returned by the differencing algorithm also resulted in residuals with root-mean-square value of less than 1% of the trial period. However, there were many non-harmonically related candidate periods resulting in residuals of a comparable root-mean-square value. Furthermore, given the number of trials necessary for this search, none of these trial periods was statistically significant. In addition, owing to the small number of detected bursts, and the widths of the pulses, we were not sensitive to periodicities much shorter than ~100 ms because our tolerance for a period match (or acceptable root-mean-square value) becomes a large fraction of the period and there are many possible fits. The 16-day gap between the 2015 May and June detections precluded us from including the May bursts in any search for periodicity in the single pulses. To produce the spectra shown in the right panels of Fig. 2, we corrected each spectrum for the bandpass of the receiver. We estimated the bandpass by taking the average of the raw data samples for each frequency channel. We then median-filtered that average bandpass with a width of 20 channels to remove the effects of narrow-band RFI and divided the observed spectrum of each burst by this median-filtered bandpass. The band-corrected burst spectra shown in the right sub-panels of Fig. 2 are still somewhat contaminated by RFI, however. The bottom and top ten channels (3.4 MHz) of the band were ignored owing to roll-off in the receiver response. To characterize the bandpass-corrected spectrum of each burst, we applied a power-law model using least-squares fitting. The power-law model is described by S ∝ να, where S is the flux density in a frequency channel, ν is the observing frequency, and α is the spectral index. These measured spectral indices and their uncertainties are shown in Table 1. We do not include a spectral index value for burst 6 because of the RFI in the lower half of the band. For bursts 7 and 9, we exclude data below 1,250 MHz, because of RFI contamination. For bursts 8 and 10, the power-law model was not a good descriptor, and therefore no value is reported in Table 1. We verified this technique by applying the bandpass correction to PALFA data of pulsar B1900+01. The measured spectral index was calculated for ten bright single pulses, and the values are consistent with the published value. We measured the DM for 10 of the 11 bursts and additionally the dispersion index ξ (from the dispersive delay Δt ∝ ν−ξ) for the brightest two. The DM and the dispersion index were calculated with a least-squares routine using the SIMPLEX and MIGRAD functions from the CERN MINUIT package (http://www.cern.ch/minuit). The user specifies the assumed form of the intrinsic pulse shape, which is then convolved with the appropriate DM smearing factor. For these fits a boxcar pulse template was used. Subbanded pulse profiles for each burst were generated by averaging blocks of frequency channels. The number of subbands generated depended on the S/N of the burst to ensure that there was sufficient S/N in each subband for the fit to converge. Subbands with no signal were excluded from the fit. Furthermore, the data were binned in time to further increase the S/N and reduce the effects of frequency-dependent flux evolution. As the true intrinsic pulse width is not known, each burst was fitted with a range of boxcar widths. The parameters corresponding to the input template yielding the cleanest residuals are reported. The DM value was fitted keeping the DM index fixed at 2.0. We note that burst 7 was too weak and corrupted by RFI to obtain reasonable fits. Additionally, for the brightest two bursts (8 and 11), we also did a joint fit of DM and dispersion index. The resulting dispersion index fits were 2.00 ± 0.02 and 1.999 ± 0.002 for bursts 8 and 11, respectively. These values are as expected for radio waves travelling through a cold, ionized medium. Frequency-dependent pulse profile evolution introduces systematic biases into the times of arrival in each subband. These biases in turn bias the DM determination. These systematics cannot be mitigated without an accurate model for the underlying burst shape versus frequency, which is not available in this case, and is further complicated by the fact that the burst morphology also changes randomly from burst to burst. We estimated the systematic uncertainty by considering what DM value would produce a delay across our observing band that is comparable to half the burst width in each case. Table 1 presents the results of the fits with the statistical and systematic uncertainties both quoted. The DM estimates do not include barycentric corrections (of the order of 0.01–0.1 pc cm−3). Although FRB 121102 is close to the ecliptic, the angular separation from the Sun was always much larger than 10°, and any annual contribution to the DM from the solar wind was small (<10−3 pc cm−3)32, 33. These effects are, therefore, much smaller than the aforementioned systematics in modelling the DMs of the bursts. The ±1σ range of DMs for the ten new bursts is 558.1 ± 3.3 pc cm−3, consistent with the discovery value4, 557.4 ± 2.0 pc cm−3. The DM values and dispersion indices reported here and previously4 were calculated using different methods. These two approaches fitted for different free parameters, so different co-variances between parameters may result in slightly different values. Also, different time and frequency resolutions were used. Nonetheless, the burst 1 parameters quoted here and previously4 are consistent within the uncertainties. The consistency of the DM values is conclusive evidence that a single source is responsible for the events. Some FRBs have shown clear evidence for multi-path propagation from scattering by the intervening interstellar or extragalactic material along the line of sight2, 6, 7, 8. However, the burst profiles from FRB 121102 show no obvious evidence for asymmetry from multi-path propagation. An upper bound4 on the pulse broadening time from burst 1 is 1.5 ms at 1.5 GHz. Using the NE2001 model for a source far outside the Galaxy, the expected pulse broadening is ~20 μs × ν−4.4 with ν in gigahertz, an order of magnitude smaller than the ~2-ms pulse widths and ~0.7-ms intra-channel dispersion smearing. The features of the spectra cannot be explained by diffractive interstellar scintillations; the predicted scintillation bandwidth for FRB 121102 is ~50 kHz at 1.5 GHz, which is unresolved by the 0.34-MHz frequency channels of our data. We would, therefore, also not expect to observe diffractive interstellar scintillation in our bursts. Additional scattering occurring in a host galaxy and the intergalactic medium is at a level below our ability to detect. However, observations at frequencies below 1.5 GHz may reveal pulse broadening that is not substantially smaller than the upper bound if we use as a guide the observed pulse broadening from other FRBs2, 6, 7, 8. Future observations that quantify diffractive interstellar scintillations can provide constraints on the location of extragalactic scattering plasma relative to the source, as demonstrated for FRB 110523 (ref. 8). The upper bound on pulse broadening for FRB 121102 implies that the apparent, scattered source size for radio waves incident on the Milky Way’s interstellar medium is small enough that refractive interstellar scintillation (RISS) from the interstellar medium is expected. For the line of sight to FRB 121102, we use the NE2001 model to estimate an effective scattering-screen distance of ~2 kpc from Earth and a scattering diameter of 6 milliarcseconds. The implied length scale for phase-front curvature is then l ≈ 2 kpc × 6 milliarcseconds = 12 au. For an effective, nominal velocity, V = 100 × V km s−1, the expected RISS timescale is days. At 1.5 GHz and with an effective velocity due to Galactic rotation of about 200 km s−1 in the direction of FRB 121102, RISS timescales of 20–40 days are expected. Modulation from RISS can be several tens of per cent34. This level of modulation could play a part in the detections of bursts in 2015 mid-May and 2015 June and their absence in 2015 early-May and at other epochs. However, the Solar System and the ionized medium have the same Galactic rotation, so the effective velocity could be smaller than 100 km s−1, leading to longer RISS timescales. The beam positions used in Fig. 1 and the data of the bursts used to generate Fig. 2 are provided as Source Data files (available online with the figures). The code used to analyse the data are available at the following sites: PRESTO (https://github.com/scottransom/presto), RRATtrap (https://github.com/ckarako/RRATtrap), TEMPO (http://tempo.sourceforge.net/), and PINT (http://github.com/nanograv/PINT).
News Article | February 15, 2017
While studying age-associated dendritic restructuring in C. elegans neurons7, we noticed that fluorescent signals originating from neurons sometimes appeared situated outside of the cell in defined vesicle-like structures that we call exophers (Fig. 1a–c, Extended Data Figs 1a–c, 2g). We first characterized exophers associated with the six gentle touch receptor neurons, for which cell bodies and dendrites are easily visualized. We found that exophers are comparable in size (average diameter 3.8 μM) to neuronal somas (Extended Data Fig. 1d). The size of the vesicles, the morphological stages in their biogenesis (Fig. 1a–c), and the genetic requirements for their production (Extended Data Table 1a) distinguish them from much smaller exosomes (around 30–100 nm; Extended Data Table 2 compares exophers to characterized extracellular vesicles). Neuronal exophers do not seem to result from classical cell division: (1) exophers did not stain with the nuclear DNA indicator DAPI (Fig. 1b); (2) cell division-inhibiting hydroxyurea8 did not change exopher levels (n > 30 per trial, three trials); and (3) RNA interference (RNAi)-mediated disruption of cell cycle genes did not change exopher detection (Extended Data Table 1b). We found that exopher production is not restricted to a specific transgene reporter or line (examples in Fig. 1, Extended Data Fig. 1). Amphid neurons that are dye-filled via openings to the outside environment9 (Extended Data Fig. 1e, f) can produce exophers, confirming that exophers can form under native physiological cellular conditions. Exopher production differs markedly among the six touch receptor neurons, with ALMR neurons producing exophers most frequently (Fig. 1d). Many neuronal types can produce exophers, including dopaminergic PDE and CEP neurons (Extended Data Fig. 1g, h), FLP neurons (not shown) and sensory ASER neurons (Extended Data Fig. 1i). Time-lapse analyses (Supplementary Videos 1, 2) revealed that exophers typically arise from the soma by asymmetrically amassing labelled protein to create a balloon-like extrusion via a pinching off event; the exopher compartment then moves outward from the neuronal cell body (extrusion approximately 15–100 min; Fig. 1a, Extended Data Fig. 1a). The plasma membrane reporter P PH(plcDelta)::GFP (Extended Data Fig. 2a) and electron microscopy data (Extended Data Fig. 2) confirm that exophers are membrane-bound. Exophers can initially remain connected to the soma by a thin thread-like tube (Fig. 1c) that allows the transfer of tagged proteins and calcium into the attached exopher compartment (Extended Data Figs 1a, 3, Supplementary Video 2). Exophers ultimately disconnect from the originating neuronal soma (Extended Data Fig. 3). Time-lapse studies indicated that aggregating mCherry often appeared preferentially concentrated into exophers, and neurons expressing the huntingtin (Htt) protein with a neurotoxic polyglutamine tract of 128 repeats (Htt-Q128) could also concentrate and extrude this aggregating protein in exophers (Fig. 2a, b). We therefore further queried the relationship of aggregating or toxic protein expression to exopher production. Strains expressing Q128 (toxic, with high levels of apparent aggregation10, 11) produced significantly more exophers than strains that did not express polyQ or that expressed Htt-Q19 (non-toxic and low aggregation) (Fig. 2c). Likewise, aggregating mCherry lines exhibited higher average exopher numbers over adult life than lines expressing soluble green fluorescent protein (GFP) (see Fig. 2d). High aggregate load in individual neurons was predictive of increased exopher production on the following day (Fig. 2e). Conversely, mCherry RNAi reduced the number of exophers by approximately one-half in a line producing aggregating mCherry (Fig. 2f). Although our studies cannot determine the relative contribution of aggregate load from protein expression levels, they suggest that proteostatic challenges increase exopher production. Consistent with a potential role for exophers in the elimination of potentially harmful neuronal contents, the expression of amyloid-forming human Alzheimer’s disease fragment amyloid-β in ASER neurons increases exopher numbers (Fig. 2g). Our combined observations on exopher formation, contents and frequency of detection suggest that exophers preferentially include aggregated, excess, or otherwise neurotoxic proteins for removal. To address the hypothesis that aggregation-prone proteins might be selectively extruded in exophers, we constructed a line that expressed both an aggregation-prone mCherry (Is[P mCh1]) and a non-aggregating GFP (Is[P GFP]) and compared the red and green fluorescence distribution between exophers and somas (example in Fig. 2h, data in Fig. 2i). In 22 out of 23 exophers, we found higher relative levels of mCherry in the exopher, and higher relative levels of GFP in the soma. Neurons appear to extrude aggregation-prone mCherry preferentially compared with soluble GFP, suggesting that deleterious materials are identified and sorted for export during exopher-genesis. To investigate whether proteostatic challenges enhance the exopher production response, we manipulated the in vivo protein-folding milieu. We found a roughly sixfold increase in exopher production in an hsf-1(sy441) mutant deficient in the core proteostasis transcription factor HSF-1 (and therefore deficient in chaperone expression) (Fig. 3a). We impaired autophagy by treating animals with the pharmacological inhibitor spautin-1 and by RNAi knockdown (lgg-1, atg-7, bec-1, lgg-1/2) in a strain expressing aggregation-prone mCherry, and measured a significant increase in exopher incidence (Fig. 3b, c). Impairment of proteasome activity with the inhibitor MG132 on strain Is[P mCh1] also increased exopher production (Fig. 3d). Given that inhibiting several facets of proteostasis increases exopher extrusion, we suggest that exophers may constitute a previously undescribed component of the proteostasis network, which may function as a backup or alternative response to rid cells of neurotoxic aggregates/proteins when proteostasis becomes overwhelmed by mounting intracellular proteotoxicity. Exopher production occurs with a notable bimodal distribution throughout adult life: exophers are most commonly observed at adult days A2–A3, diminish in abundance at A4–A8, and then reappear again later in life at approximately A10–A11 (Fig. 2d; similar young adult pattern with dye-filled amphid neurons, Extended Data Fig. 1f; and with a 1-day earlier onset in an hsf-1 mutant, Extended Data Fig. 1j). The distinctive temporal production profile suggests that conditions permissive for exopher production exist in young adulthood but can then be limited or remain below a threshold until late adulthood. The coincidence of the early peak with a transition in C. elegans young adult proteostasis management12, 13, 14 suggests that the first wave of exopher-genesis may serve as a normal component of an orchestrated proteostasis reset in young adulthood that involves the removal of neuronal debris generated during development; the later adult increase in exopher production may be the consequence of age-associated decline in proteostatic robustness. Rather than inducing neuronal death or dysfunction, exopher-genesis seems to be beneficial. First, in hundreds of longitudinal observations, we did not observe neuronal loss after exopher production: exophers are distinct from apoptotic bodies in their biogenesis (Fig. 1a, Extended Data Fig. 1a), and the soma of an exopher-producing neuron retains normal ultrastructural features (Extended Data Fig. 2e). Second, the relative functionality of proteotoxically stressed neurons that have generated exophers is increased compared with neurons that did not extrude exophers. In blinded studies of a line expressing cyan fluorescent protein (CFP)-tagged Q128, which progressively impairs touch sensation10, we found that midlife touch sensitivity is greater when ALMR had definitely produced an exopher at A2, as compared to age-matched siblings in which ALMR had not produced an exopher (Fig. 3e). Third, we identified pod-1 and emb-8 as polarity genes required in adults for exopher-genesis (Fig. 3f), and found that adult RNAi knockdown impaired midlife touch sensitivity (Fig. 3g). Although we cannot rule out that pod-1 and emb-8 RNAi interventions might generally disrupt adult neuronal function, taken together our data are consistent with a model in which adult neurons that do not make exophers become functionally compromised compared to those neurons that extruded offending contents. Overall, adult neurons seem to be healthier after a considerable expulsion of potentially toxic contents. Considering the large apparent volume of exophers, we proposed that they might include organelles. Indeed, both lysosomes (Extended Data Fig. 4) and mitochondria (Fig. 4a, b, Extended Data Fig. 5) can be extruded in exophers. Mitochondrially localized GFP reporters revealed mitochondrial inclusion in budding and dissociated exophers, with punctate or filamentous morphology typical of adult mitochondrial networks (Fig. 4a, Extended Data Fig. 5a–c). To address whether impairing mitochondrial quality enhances the production of exophers, we genetically manipulated the mitophagy mediator dct-1 (homologue of mammalian BNIP3), the human Parkinson’s disease homologues pink-1 (PINK)15 and pdr-1 (PARK2)16 implicated in mitochondrial maintenance, and the mitochondrial unfolded protein response gene ubl-5 (ref. 17) (Fig. 4c, d). We conclude that several genetic approaches that impair mitochondria can increase exopher-genesis. To address the hypothesis that stressed or damaged mitochondria might be preferentially segregated to exophers, we used the mitochondrial reporter mitoROGFP, which changes its peak excitation wavelength from around 405 nm (oxidized) to 476 nm (reduced) according to the local oxidative environment18, 19. We find a significant increase in the 405 nm (oxidized)/476 nm (reduced) excitation ratio of mitochondria in exophers compared to those in somas (Fig. 4e), roughly equivalent to the redox excitation ratio observed in C. elegans neurons subjected to H O -induced oxidative stress19. We confirmed higher oxidation scores using MitoTimer, an alternative reporter of mitochondrial matrix oxidation20 (Extended Data Fig. 5d). In addition, touch neurons of juglone-treated21 bzIs166[P mCherry]; zhsEx17[P mitoLS::ROGFP] animals had significantly higher numbers of mitochondria-including exophers than matched controls (Extended Data Fig. 5e). Although compromised mitochondrial health may impair neuronal proteostasis, thus increasing exopher production, our data establish that touch neurons can eject mitochondria via exophers, which raises the intriguing possibility that exopher-genesis may constitute a previously unappreciated removal-based mechanism of mitochondrial homeostasis. We next sought to determine the fate of the extruded exopher and its contents. With time, exopher fluorescence intensity diminishes or disappears (persistence times 1–12 h), possibly as exopher contents are degraded internally or digested by the neighbouring hypodermis that fully surrounds the touch neuron and has degradative capabilities. Disruption of the C. elegans apoptotic engulfment genes ced-1 (homologue of mammalian CD91, LRP1 and MEGF10, and fly Draper), ced-6 (GULP) and ced-7 (ABC1) increases the detection of ALMR neurons that have extruded several exophers (Fig. 5a, Extended Data Fig. 6a); however, the genetic manipulation of a parallel engulfment pathway comprising ced-2 (Crk-II), ced-5 (DOCK180), ced-10 (RAC1), ced-12 (ELMO) and psr-1 (PSR) did not change the frequency of exopher generation or the detection of multiple exophers. Moreover, we did not detect the apoptotic ‘eat-me’ signal phosphatidylserine on the exopher surface using a widely expressed phosphatidylserine-binding annexinV::GFP (0 out of 43 exophers; Extended Data Fig. 6b). Our data suggest that hypodermal recognition/degradation of exophers and their contents occurs by mechanisms that are at least in part distinct from the classical removal of apoptotic corpses, but involve the CED-1, CED-6 and CED-7 proteins. Electron microscopy studies also show that the hypodermis may mediate the degradation of at least some exopher contents (Extended Data Fig. 2d–f, h). The lack of a detectable phosphatidylserine signal on exophers raised the question as to whether at least some exopher contents might be destined to elude hypodermal degradation. Indeed, fluorescent mCherry protein that was originally expressed specifically in touch neurons, or fluorescent DiI loaded into dye-filling neurons, appeared later in distant scavenger coelomocytes (Fig. 5b–d, Extended Data Fig. 6c). Blocking coelomocyte uptake capacity by cup-4 mutation22 caused fluorescent particles to accumulate outside neurons, possibly within the pseudocoelom (body cavity; Extended Data Fig. 6d, e). We conclude that some exopher contents transit the hypodermal tissue to be released into the pseudocoelomic fluid, from which materials can later be taken up by distant coelomocytes. Exophers can therefore mediate transfer of neuronal materials to remote cells. Considerable excitement in the neurodegenerative disease field has been generated by the findings that mammalian neurons can extrude conformational disease proteins, including in Alzheimer’s, Parkinson’s and prion disease23. The production of exophers in C. elegans constitutes a newly identified mechanism by which neurons can transfer cellular material (preferentially neurotoxic species) to other cells. Notably, in a C. elegans muscle model of prion toxicity, offending prion proteins were transferred among muscle cells and ultimately localized to coelomocytes24. We speculate that the basic mechanism we document here may correspond to a conserved pathway for the transfer of toxic contents out of many cell types. In this regard, it may be noteworthy that mammalian aggregated poly-Q-expanded huntingtin can transfer between neurons via tunnelling nanotubes25, 26, 27 that resemble thin connections between C. elegans somas and exophers, and that neuronal polyQ in Drosophila is transferred to glia via a process that requires the CED-1 homologue, Draper28. Recent reports show that mitochondria can transfer out of specific cells to contribute positive roles (mesenchymal stem cells via tunnelling nanotubes29; astrocytes to neurons in a stroke model30), but our study underscores a generally underappreciated option for mitochondrial quality control: mitochondrial expulsion. The mitochondrial expulsion we report in C. elegans touch neurons has a notable mammalian counterpart: mouse mitochondria originating in retinal ganglion cells can be extruded into neighbouring astrocytes for degradation6 (with some similar morphology to C. elegans exophers; see fig. 1e of ref. 6). Although further study will be required to establish definitively the health status and fates of transferred mitochondria in the C. elegans model, it is tempting to speculate that transcellular degradation of mitochondria may be a more broadly used mechanism of mitochondrial quality control than currently appreciated, with associated potential importance in neuronal health. Overall, although further experiments are needed to determine the detailed mechanisms at play and validate the proposed functions of exophers in proteostasis and the removal of damaged organelles, we suggest that exopher production is a previously unrecognized mechanism for clearing out accumulating protein aggregates and dysfunctional organelles that threaten neuronal homeostasis (Extended Data Fig. 7). The analogous process in mammals could enable the transfer of misfolded protein and/or dysfunctional mitochondria to neighbouring cells, promoting human pathology in neurodegenerative disease if compromised. Mechanistic dissection of this new aspect of proteostasis and mitochondrial homeostasis should thus inform on fundamental mechanisms of neuronal maintenance and suggest targets for intervention in neurodegenerative disease.
News Article | February 16, 2017
NEW YORK--(BUSINESS WIRE)--Mantle Ridge LP (“Mantle Ridge”), an investment firm that owns approximately 4.9% of the outstanding common shares of CSX Corp. (NASDAQ:CSX) (the “Company”), today sent the following letter to the CSX Board of Directors. I read with interest your press release announcing a special meeting to poll shareholders on their views about our proposals. Hunter and I are committed exclusively to finding ways to maximize shareholder value here. We know that the Board shares this objective. Because every day’s delay in beginning to implement Precision Scheduled Railroading (PSR) under conditions that enable its success costs the Company dearly, it is in the shareholders’ best interest that we promptly resolve this through negotiation rather than wait months. Continuing the exploration for common ground makes sense. As I have said in my earliest communications with you, and in each correspondence since, I am convinced there is ample room for agreement. We have been standing by since last week for constructive counterproposals and we are ready to discuss them as they arrive. Early on in this process, I explained that we were looking to you to help us shape a solution that the Board would embrace, and that would best advance the shareholders’ objective of a swift and certain transformation. I made that clear in my January 24th letter to the Board. The relevant passage is below: “You Are Necessarily the Leader of this Process For a variety of reasons, you are uniquely positioned to lead: you know the Company, the people, and the dynamics at the Board and in the executive ranks; you have had dealings with me, and you know Hunter by reputation; you have engineered countless deals; and you have been through a somewhat similar (but the differences are greater than the similarities) experience in the past. We seek your guidance in supporting your leadership.” We remain hopeful, Ned, that you and we can together craft a solution that works for all. If so the shareholder wins big. In the spirit of continuing to engage with you pending the special meeting, I thought it would be useful for me to give you some high-level input on what you said in the press release, to discuss process, and a proposed path forward. The press release suggests that Mantle Ridge is looking for “substantial board representation.” In fact, we are requesting only a single Mantle Ridge representative on the Board (me). Neither Hunter nor the other Board slots we discussed represents Mantle Ridge in any way. The press release goes on to say that Mantle Ridge wants to designate six directors and suggests that granting our ask would grant us effective control. In fact, you and I were engaged in a process to identify high quality independent people that the Board and Mantle Ridge could embrace. As part of that process, I gave you a list (including lengthy bios) of 11 exceptional individuals with broad and relevant experience. None of them has any relationship with me outside of my efforts to recharge the CSX Board. Each one is entirely independent of me and is committed to acting in the best interests of all of the shareholders. Each one would make a superb board member. You indicated to me that you were impressed with the list. You agreed that the Board would not consider any of the people on the list surrogates of mine, or anything but truly independent and qualified. I offered to arrange for you and the governance committee to meet any or all of the candidates. You assured me that there was no need to do that, since you were confident that together we could settle on people from that list once we settled on the other issues. Why are we asking that new directors be added? As we’ve discussed, Precision Scheduled Railroading requires dramatic operational and cultural change. Change like that starts at the top, with significant new blood on the Board not wed to the old ways or legacy decisions and with no ties to any previous strategy or any one. The messaging to all concerned constituencies that the external change agent – Hunter – is coming in with very substantial support empowers Hunter. Conversely, without enormous board support, the outcome and rate of the transformation will be at risk. As Hunter said in our own press release, “if we create the right conditions for success, we have the best chances for success.” When I mentioned “conditions for success,” you asked what that means. I explained to you and the rest of the Board that the conditions at CP were ideal (from early on during the transformation, new independent directors comprised a majority of the board). But I also said I think that something less than that would be workable at CSX and that I was open to exploring any proposals that you suggested could work. More generally, I have said repeatedly, that my view of how well a proposed governance deal will affect Hunter’s ability to succeed is based on an assessment of roll-offs, additions, and board roles/committees. No one of these dimensions can be considered on its own. They are a package. It is a balancing act. And different packages and permutations can work. The question before us is which package or permutation is acceptable to the Board, and sufficiently empowers Hunter to deliver for the shareholders. I must confess, however, I was made somewhat cautious during this process. The Board’s letter to Hunter proposed that he be hired for only two years, not the four we explained was necessary. This raised concerns relating to the Board’s commitment to the transformation. That lack of commitment could create material risk to the timely and successful implementation of PSR. Having lived this, I cannot overemphasize the risk this brings to our delivering to shareholders the swift and certain transformation they want and expect. With that in mind, I told you that the addition of three independent directors the Board offered was better than its initial offer of two. I suggested you take four back for consideration. I also made clear that I was open to different approaches concerning how quickly the Board size comes down. I note that Tuesday’s press release could be read to say we were looking for the Board to drop down to 12 after John Breaux ages out in 2018. I never made that ask, and in fact I had just assumed that the reconstituted Board would find a replacement when he chooses to step down. I said that I considered Mr. Breaux a valuable member of the Board and explicitly said more than once that a retirement age cap should not get in the way of his continuing to serve for many years if that was his preference. I continue to hope he stays for a while. Similarly, when we discussed committee composition and leadership, I gave you my thoughts but made clear that when the time comes we would sit down and agree on a list. I said that it made sense for the new directors to occupy a minority of the committee chairs. I explained that the committees that were most important for the transformation to succeed were Governance and Compensation and that it made sense that two of the independents occupy those. For avoidance of doubt, Mantle Ridge has been seeking to add to the Board only one representative, plus Hunter, plus independent people. The sole prism through which we should evaluate the number of new directors that should be added to the Board is the best interests of the Company. That the board restructuring we are discussing comes in connection with an activist whose holdings are 5% of the Company, or 1%, or 15% is irrelevant. The size and nature of the restructuring should be solely a function of the shareholders’ best interests, which I think you agree requires installing Hunter and creating the conditions for him to succeed. No other consideration – the size of the proposing shareholder or otherwise – should be a factor. Mantle Ridge is in no way seeking or gaining control, and remains open to discussions about the details. This is not a “battle for control”. The press release suggests that Hunter is asking for a $300 million compensation package. In our view, that’s a major mischaracterization. Regrettably, it has confused the shareholders. Hunter’s compensation package adds up to approximately $32 million per year for four years, of which approximately $20 million per year is explicitly performance based and should therefore be discounted in the customary and substantial way performance based grants normally get discounted for compensation and accounting purposes. In addition to Hunter’s compensation package, there is a one-time cost of extraction from CP (i.e., the value Hunter forfeited in order to free him up from his two-year non-compete and allow him to work at CSX), which should not be viewed as compensation. That cost is $84 million plus a gross up of somewhere between $0 and $23 million (depending on Hunter’s tax position). Importantly, as you know, in no way does Mantle Ridge receive any economic gain from this arrangement. As a matter of fact, Mantle Ridge’s investors assumed this liability to effect the release of Mr. Harrison from his non-compete and make him available to solve CSX’s succession problem. This just covers that cost. Aside from our different ways of characterizing the one-time cost of extraction, the main difference between our number and yours is the value of the option grant. The $160 million referenced in the Company’s press release confuses the matter. That figure does not fairly represent the economic cost of this package to the Company, or its value to Hunter. And it should not be the basis for evaluating the package. As you and I discussed, in our view the proper way to think about that value is to view it against the unaffected stock price of $36.88 rather than today’s “Hunter rally” price. The market has already baked in anticipated strong execution by Hunter worth $10 per share and, since his strike price will be based on the inflated date-of-grant price, Hunter will receive no in-the-money value for that first $10 per share of his value creation. For this reason, we believe the economically correct view is that he was granted an option that is $10 per share out of the money on the day of grant. Viewed that way, the Black-Scholes value of the grant is only $78 million, and half of that grant is performance based (based on goals that no other CEO in this industry has reached) and as such should be accounted for at a large discount. It only has meaningful value if Hunter knocks the cover off the ball, in which case the magnitude of the package would be de minimis relative to the value he uniquely could create. And it would certainly be deserved. This is the correct characterization of the economic cost of the package to shareholders and the economic value to Hunter. We had discussed this verbally a couple of times and you seemed to follow the analysis and accept the conclusion. Importantly, because under the proposal Hunter’s options are so far out of the money relative to the pre-Hunter price, it is important that we never lose sight of the fact that his package is worth very little unless he performs spectacularly. As with the governance proposal, we are looking for your guidance in shaping a deal that works for all. Ned, a lot of the points you raised in the press release could easily have been addressed through continued back and forth, but the back and forth stopped. Hunter and I (and our counsel) repeatedly asked for a counter on the compensation and governance terms, but we never received one. Instead, we have a special meeting that will distract current management and – more importantly – will delay roll out of PSR and disrupt the Company’s operations. When you compare this cost with the cost of the questions at issue, it becomes clear that the Board should redouble its effort to get a settlement. Again, you are clearly the best party to get us there, and we continue to look to you for guidance on proposals that would work. With that in mind, as you and I gear up for the special meeting, I remain open to engaging constructively with you in a way that could bring this to a more rapid and satisfactory close for the shareholders. Over the last 24 hours, I have spoken to many shareholders of the Company. Without exception, they share our eagerness to get Hunter into the seat as quickly as possible under conditions for success that would enable him replicate the CP transformation. They agree that the cost to the Company of delaying this outcome until a Special Meeting gets scheduled in April, or possibly later, is dear. None of us wants to wait. I would ask the Board to consider the clarification of the underlying economics of Hunter’s compensation package we provide above and to reconsider whether they can consider accepting it, or at least providing a counter proposal. Concerning the governance proposal, this is a soft and tricky exercise in judgment that balances additions, roll-offs, and roles/committees. If the Board can commit to a process that promptly gets us to a four-year deal with Hunter, that will in my mind be a sufficient gesture of commitment that we can go forward with the addition of me and Hunter and three of the independent directors (rather than the four in our last proposal), with continued discussion concerning roll-offs, committee composition, and roles. Ned, we have come a long way. We are close. We owe it to the shareholders to get a deal done promptly. Let’s do it. If you are willing, we are glad to meet in person and hammer this out this weekend, hopefully delivering good news to the shareholders early next week. As before, I've taken the liberty of copying the other members of the Board as well as Ms. Fitzsimmons. Ms. Fitzsimmons, please pass on copies of this letter to every other Board member. Very truly yours, Mantle Ridge is a private investment firm founded by Paul Hilal. Mantle Ridge seeks to help create enduring value, and believes that constructive and cooperative engagement between boards, management teams, and engaged shareholders is the best means to achieving that goal. Mantle Ridge’s approach is informed by extensive research and a depth of experience in value investing, activist engagements, corporate governance, and business operations, in the context of diverse sector expertise. For more information, go to www.mantleridge.com. This press release contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934 (as amended, the “Exchange Act”), regarding, among other things, Mantle Ridge’s plans to distribute a definitive proxy statement, as well as the benefits of Precision Scheduled Railroading (and the cost of delaying its implementation). Such forward-looking statements involve known and unknown risks, uncertainties and other factors that may cause actual results, performance or achievements to be materially different from any future results, performance or achievements expressed or implied by such forward-looking statements. Readers are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date thereof. Mantle Ridge undertakes no obligation to publicly release the result of any revisions to these forward-looking statements that may be made to reflect events or circumstances after the date hereof or to reflect the occurrence of unanticipated events. Mantle Ridge LP (“Mantle Ridge”) and certain of its affiliates intend to file with the Securities and Exchange Commission (the “SEC”) a proxy statement and accompanying proxy card to be used to solicit proxies in connection with the upcoming special meeting of shareholders (the “Special Meeting”) of CSX Corporation (the “Company”) announced on February 14, 2017 and the election of a slate of director nominees at the 2017 annual shareholders meeting of the Company (the “Annual Meeting”). The participants in the solicitation are anticipated to include MR Argent Offshore AB Ltd., MR Argent Offshore BB Ltd., MR Argent Offshore CB 01 Ltd., MR Argent Offshore CB 02 Ltd., MR Argent Offshore CB 03 Ltd., MR Argent Offshore CB 04 Ltd., MR Argent Offshore CB 05 Ltd., MR Argent Offshore CB 07 Ltd. (collectively, the “MR Funds”), MR Argent Fund CE LP, MR Argent Advisor LLC (the “Advisor”), Mantle Ridge GP LLC, Mantle Ridge LP, MR Argent GP LLC, MR S and P Index Annual Reports LLC (“Shareholder”), Mr. Hilal (collectively with the MR Funds, MR Argent Fund CE LP, the Advisor, Mantle Ridge GP LLC, Mantle Ridge LP, MR Argent GP LLC and Shareholder, the “Mantle Ridge Parties”) and E. Hunter Harrison. MANTLE RIDGE STRONGLY ADVISES ALL SHAREHOLDERS OF THE COMPANY TO READ THE DEFINITIVE PROXY STATEMENTS AND OTHER DOCUMENTATION RELATED TO THE SOLICITATION OF PROXIES BY MANTLE RIDGE AND ITS AFFILIATES FROM SHAREHOLDERS OF THE COMPANY FOR USE AT THE SPECIAL MEETING AND ANNUAL MEETING WHEN THEY BECOME AVAILABLE, BECAUSE THEY WILL CONTAIN IMPORTANT INFORMATION, INCLUDING INFORMATION RELATING TO THE PARTICIPANTS IN SUCH PROXY SOLICITATION. SUCH DEFINITIVE PROXY STATEMENTS, OTHER PROXY MATERIALS AND ANY OTHER RELEVANT DOCUMENTATION WILL BE AVAILABLE AT NO CHARGE ON THE SEC’S WEB SITE AT HTTP://WWW.SEC.GOV. IN ADDITION, MANTLE RIDGE WILL PROVIDE COPIES OF THE DEFINITIVE PROXY STATEMENT AND OTHER MATERIALS WITHOUT CHARGE, WHEN AVAILABLE, UPON REQUEST. REQUESTS FOR COPIES SHOULD BE DIRECTED TO D.F. KING & CO., INC., 48 WALL STREET, 22ND FLOOR, NEW YORK, NEW YORK 10005 (CALL COLLECT: (212) 269-5550) OR EMAIL: INFO@DFKING.COM. As of the date hereof, the Mantle Ridge Parties beneficially own (within the meaning of Rule 13d-3 under the Exchange Act) an aggregate of approximately 44,352,597 shares of common stock, $1.00 par value, of the Company (the “Shares”). Of those approximately 44,352,597 Shares, MR S AND P Index Annual Reports LLC owns approximately 6 Shares in record name. As of the date hereof, MR Argent Fund CE LP possesses economic exposure to an aggregate of 570,600 Shares due to certain cash-settled total return swap agreements. MR Argent Fund CE LP also holds American-style call options to purchase cash-settled total return swaps referencing an aggregate of 105,232 Shares. Mantle Ridge LP, as the sole member of the Advisor, which is in turn the advisor to the MR Funds, may be deemed to have the shared power to vote or direct the vote of (and the shared power to dispose or direct the disposition of) the approximately 44,352,590 Shares held for the accounts of the MR Funds and the approximately 6 Shares directly owned by Shareholder and, therefore, may be deemed to be the beneficial owner of such Shares. MR Argent Advisor LLC, as the advisor to, and holder of 100% of the noneconomic voting interests in, the MR Funds, may be deemed to have the shared power to vote or direct the vote of (and the shared power to dispose or direct the disposition of) the approximately 44,352,590 Shares held for the accounts of the MR Funds and, therefore, may be deemed to be the beneficial owner of such Shares. By virtue of his position as the managing member of Mantle Ridge GP LLC, the general partner of Mantle Ridge LP, which is in turn the sole member of both Shareholder and MR Argent Advisor LLC, the advisor to the MR Funds, Mr. Hilal may be deemed to have the shared power to vote or direct the vote of (and the shared power to dispose or direct the disposition of) of the approximately 44,352,590 Shares held for the accounts of the MR Funds and the approximately 6 Shares directly owned by Shareholder and, therefore, may be deemed to be the beneficial owner of such Shares.
News Article | October 5, 2016
The following fly strains were used: NP1–Gal4 and FRT19A–δ-COPG0051 (from DGRC); esg–Gal4 and tsh–Gal4 (from S. Hayashi); wg–Gal4 (from J.-P. Vincent); UAS–upd (generated in our laboratory); UAS–NDN(2X) (from M. Fortini); UAS–bmm (from R. P. Kuhnlein); UAS–Hnf4 and GAL4–dHFN4 ; UAS–nlacZ (from C. Thummel); mira–GFP (from F. Schweisguth); pmCherry–Atg8a (from E. Baehrecke); UAS–GAP43–mCherry (from T. Lecuit); UAS–drprRNAi and UAS–drpr (from M. Freeman); UAS–DJunAsp (from M. Mlodzik); UAS–hepCA, UAS–RasV12, UAS–scu, UAS–rpr, UAS–p53, UAS–p35, UAS–Cat, UAS–Sod, UAS–Sod2, UAS–RacDN(N17), UAS–RacV12, UAS–bskDN, hopTum-l, puc–lacZ (pucE69), UAS–2XEYFP, tub–Gal80ts, and fly lines used for MARCM clones (FRT19A, tub–Gal80; FRT82B, tub–Gal80; SM6, hs–flp; MKRS, hs–flp; act>y+>Gal4, UAS–GFP; FRT82B–γ-COP10; and UAS–γ-COP FRT82B–γ-COP10) were obtained from the Bloomington Drosophila Stock Center at Indiana University. An upstream activating sequence (UAS)-regulated double-stranded inverse-repeat construct was designed to target Arf79F: (UAS–Arf79FRNAi)-VDRC Transformant ID: 23082 (v23082). The RNA level was reduced to 39.0% in the Act–Gal4–UAS–Arf79FRNAi flies (ref. 16), and the phenotypes were confirmed by two independent RNAi lines (v103572 and v23080). The other RNAi lines used were: δ-COP–v41551; RNA level was reduced to 14.3% in Act–Gal4–UAS–δ-COPRNAi flies (ref. 16), and phenotypes were confirmed by an independent RNAi line (Bloomington stock number 31764 (BL31764 (TRiP ID HM04076)). β-COP–BL31709 (TRiP ID HM04016); RNA level was reduced to 13.3% in Act–Gal4–UAS–β-COPRNAi flies (ref. 16), and phenotypes were confirmed by two independent RNAi lines (v109641 and v15418). β’-COP–BL31710 (TRiP ID HM04017); RNA level was reduced to 3.2% in Act–Gal4–UAS–β’-COPRNAi flies (ref. 16). ζ-COP-BL28960 (TRiP ID HM05171); RNA level was reduced to 47.0% in Act–Gal4–UAS–ζ-COPRNAi flies (ref. 16), and phenotypes were confirmed by two independent RNAi lines (v34768 and v104405). garz-BL31232 (TRiP ID JF01013); RNA level was reduced to 52.4% in the Act–Gal4–UAS–garzRNAi flies (ref. 16). Acsl–BL27729 (TRiP ID JF02811); RNA level was reduced to 25.5% in Act–Gal4–UAS–AcslRNAi flies (ref. 16). bgm-v34854; RNA level was reduced to 56.2% in Act–Gal4–UAS–bgmRNAi flies (ref. 16), and phenotypes were confirmed by two independent RNAi lines (v105635 and BL28639 (TRiP ID JF03054)). γ-COP–BL28889 (TRiP IDHM05099); Atg5–BL34899 (TRiP ID HMS01244); Atg12RNAi (from E. Baehrecke, ref. 28); mbc–BL32355 (TRiP ID HMS00346); PSR–BL33700 (TRiP ID HMS00576); mys–BL33642 (TRiP ID HMS00043); CycT–BL32976 (TRiP ID HMS00776). The sequences used for each VDRC knock-down strain are available at https://stockcenter.vdrc.at) and for each Bloomington knock-down strain at http://flystocks.bio.indiana.edu. The data presented in all figures were generated by using the first RNAi line for all genes. To induce MARCM clones, three- or four-day-old adult female flies were heat-shocked twice with an interval of 8–12 h at 37 °C for 60 min. The flies were transferred to fresh food daily after the final heat shock and their posterior midguts were processed for staining at the indicated times. To target the expression of UAS-linked genes in the cell types of interest, we used specific drivers. The posterior midgut of adult Drosophila is maintained by multipotent ISCs2, 3, which differentiate into secretory enteroendocrine cells and absorptive enterocytes through immature enteroblasts. Enterocytes are polyploid and express the transcription factor Pdm1. Enteroendocrine cells are diploid and express the transcription factor Prospero (Pros). UAS-linked genes can be targeted to enterocytes by the MyolAGal4 (NP1–Gal4) driver8 or to ISCs and enteroblasts by the escargot (esg)–Gal4 driver2. To target expression of UAS-linked genes in RNSCs, we also used esg–Gal4 (ref. 4); for the quiescent HISCs, the wingless (wg)–Gal4 driver was used5, 6. To investigate the response of the different cells to cell-death effectors, we first overexpressed reaper (rpr, an inhibitor of Death-associated inhibitor of apoptosis 1; Diap-1) in them, using the cell-type-specific Gal4 drivers combined with the temperature-sensitive Gal4 repressor tub–Gal80ts (ref. 29). We used the inducible NP1–Gal4; tub–Gal80ts–UAS–rpr; UAS–GFP to express rpr in enterocytes (NP1ts > rpr), esg–Gal4; tubGal80ts–UAS–rpr; UAS–GFP (esgts > rpr) in ISCs, enteroblasts and RNSCs, and wg–Gal4; tubGal80ts–UAS–rpr; UAS–GFP (wgts > rpr) in HISCs. The NP1ts > rpr flies were raised to adulthood at 18 °C and shifted to 29 °C for 24 h to induce rpr expression. Four male UAS–RNAi transgene flies were crossed with 8 female virgins of NP1ts, esgts, and wgts at 18 °C. Three- to five-day-old adult flies with the appropriate genotype were transferred to new vials at 29 °C for the indicated times before dissection. For p53, we did not find a significant change in esg+ progenitors and enteroendocrine cell numbers after the flies (esgts > p53) were cultured for 7 days at 29 °C, although a previous study found that a 15-day induction ablated nearly all esg+ cells and reduced enteroendocrine cell numbers8. Fly intestines were dissected in PBS and fixed in PBS containing 4% formaldehyde for 30 min. After three 5-min rinses with 1× PBT (PBS + 0.1% Triton X-100), the samples were blocked in 1× PBT containing 5% normal goat serum overnight at 4 °C and incubated first with primary antibody overnight at 4 °C or at room temperature for 2 h, and then with a fluorescence-conjugated secondary antibody for 2 h at room temperature. Samples were mounted in Vectashield mounting medium with DAPI (Vector Laboratories). The following antibodies were used: rabbit polyclonal anti-β-Gal (1:1,000; Cappel); mouse anti-Dl (Delta, 1:20; DSHB); mouse monoclonal anti-Prospero (Pros, 1:50; DSHB); rabbit polyclonal anti-Pdm1 (1:1,000, a gift from X. Yang); mouse monoclonal anti-Arm N27A1 (1:20; DSHB); Rabbit monoclonal anti-Phospho-SAPK/JNK (1:200; Cell Signaling); rabbit-polyclonal anti-GFP (1:500, Invitrogen); mouse monoclonal anti-GFP (1:100; Invitrogen), and chicken polyclonal anti-GFP (1:3,000; Abcam). Secondary antibodies were goat anti-mouse, anti-chicken, and goat anti-rabbit IgG conjugated to Alexa488 or Alexa568 (1:400; Invitrogen). DAPI (Sigma) was used to stain DNA. CellMask Deep Red plasma membrane dye (Life Technologies, C10046) was used to visualize the plasma membrane. Midguts were labelled with CellMask Deep Red Plasma Membrane Stains (1:2,000) for 7 min30. DHE staining was performed as described previously31. In brief, guts were dissected in 1× PBS, incubated in 30 μM DHE (Invitrogen) in PBS for 5 min at room temperature in the dark, washed twice, mounted and immediately imaged by confocal microscopy. Guts were dissected in 1× PBS and then stained without fixation in 0.5 μM Lysotracker Red DND-99 (Invitrogen) for 3 min at room temperature. They were then washed three times in 1× PBS, fixed for 20 min in 4% formaldehyde, washed three times in 1× PBT, rinsed twice with 1× PBS, mounted in Vectashield with DAPI and analysed on a confocal microscope. Apoptosis was detected by TUNEL with the ApopTag Red In situ Apoptosis Detection Kit (Chemicon International) according to the manufacturer’s instructions. Guts were dissected in 1× PBS and then stained in 1.5 μM PI (Invitrogen) for 15 min at room temperature. The guts were then fixed for 20 min in 4% formaldehyde, washed three times in 1× PBT, rinsed twice with 1× PBS, mounted in Vectashield with DAPI and analysed on a confocal microscope. Oil Red O staining was performed as described previously32. In brief, Drosophila midguts were dissected in 1× PBS and fixed in 4% formaldehyde for 30 min. Midguts were washed three times in 1× PBS, double-distilled water and a 60% isopropanol solution. From the stock solution of Oil Red O (Sigma-Aldrich; 0.1% solution in isopropanol), a working solution was prepared by mixing 6 ml of 0.1% Oil Red O in isopropanol and 4 ml of double-distilled water. Midguts were incubated for 20 min in this solution and then washed in 60% isopropanol and water. The midguts were mounted in Vectashield mounting medium with DAPI (Vector Laboratories) and were imaged by confocal microscopy. Images were captured with the Zeiss LSM 510 confocal system and processed with LSM Image Browser and Adobe Photoshop. To determine the percentage of GFP+ cells, the GFP+ cells and total cells were counted in a 5,000-μm2 area of a single confocal plane. In esgts samples, cells were counted in the posterior midgut and Malpighian tubules; in wgts samples, they were counted in the hindgut–midgut junction; and in esgtswgts samples, they were counted in the hindgut–midgut junction, the posterior midgut and the Malpighian tubules. The number of Pros+ nuclei was counted in a 0.08-mm2 surface area of a microscopic image from a similar region of each posterior midgut33. Cells per tumour were determined by counting the total number of nuclei within GFP+ tumours. All of the images were taken with the LSM5 Image Browser using the same confocal settings (Zeiss). Statistical analyses were performed using GraphPad Prism. Sample sizes (n) reported reflect the number of individual midguts. All experiments were performed in triplicate. P values were obtained between two groups using the Student’s t-test. For all statistical analysis, differences were considered to be statistically significant at values of P < 0.05. Cell surface markers were analysed by flow cytometry. Cultured or treated cells were dissociated by 0.05% trypsin-EDTA and centrifuged. Single cells were resuspended with PBS containing 2% FBS and fluorescent-conjugated antibodies FITC-CD44 (clone G44-26, BD Biosciences) and PE-CD24 (clone ML5, Biolegend), and incubated on ice for 30 min. After washing three times with PBS containing 2% FBS, cells were resuspended with PBS containing 2% FBS and analysed by BD FACS Caliber (BD Biosciences). Arf1 inhibitors are BFA (brefeldin A)34 from Sigma, GCA (golgicide A)35 from Santa Cruz, secin H336 from Cayman chemical, LM1137 from A. Chavanieu, LG838 from L. Frigerio. 2-deoxy-d-Glucose (2-DG)39; JNK inhibitor SP 60012540; FAO inhibitors:triascin C41, etomoxir42, and mildronate43 from Cayman chemical, Enoximone44 from Tocris. Rac1 inhibitor from Santa Cruz. N-Acetyl-L-cysteine (NAC) 45 from Sigma. For control experiments, a DMSO control (100 μl in 10 ml food) was used. All inhibitors were mixed in the fly food with following concentrations; Arf1 inhibitors: BFA (50 ng ml−1 and 200 ng ml−1), GCA (5 μM), LM11 (50 μM), LG8 (100 μM), and secin H3 (50 μM); JNK inhibitor: Sp600125 (50 μM); Rac1 inhibitor (100 μM); ROS inhibitor: NAC (10 mM); FAO inhibitors: triacsin C (5 μM), mildronate (100 μM), etomoxir (100 μM), enoximone (100 μM); and glycolysis inhibitor 2-DG (50 mM). We mixed each inhibitor in fly food and tested different concentrations of these inhibitors. We used the concentration in which the inhibitors could kill tumour cells. At the beginning of experiments, to find out whether the flies would eat the inhibitors, we added green food dye to fly food. Flies were fasted for 1 h and then 8–10 flies were transferred to a vial containing coloured fly food mixed with inhibitors. We found that within 1 h of feeding the green dye could be seen through the abdomen of each fly, which suggest that the fly food mixed with inhibitors was edible to the flies. However, we excluded the food dye from the food used in the main experiments. For the main experiments, we fed flies with food containing inhibitors for 4 days. We repeated each inhibitor treatment three times. The human prostate cancer cell line DU145, colon cancer cell line HT29, and breast cancer cell lines MCF7, MDA-MB-231 (provided by the DCTD Tumour Repository) were cultured in RPMI1640 supplemented with 10% fetal bovine serum and 100 units per ml penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO . Cells were seeded at 2 × 105 cells per well in 6-well plates. Treatments with the indicated chemicals were started the next day, and cells were incubated for 2 more days. The surviving cells were stained with Crystal Violet (EMD Millipore). For quantification, the stained cells were solubilized in 1% SDS, and the absorbance at 595 nm was determined with a microplate reader. Single cells were cultured in a Corning Costar Ultra-Low attachment 24-well plate (Sigma-Aldrich) in sphere culture medium, consisting of DMEM/F12 (1:1), B27 (Invitrogen), and 20 ng ml−1 EGF (Invitrogen), with the indicated chemicals. The number of spheres was counted after 10 days of culture. An RNeasy Mini Kit (Qiagen) was used to extract the total RNA from human cancer cells. The cDNA was synthesized from 1 μg RNA from each sample using a reverse transcription kit (Promega). Real-time PCR was performed in a 15-μl reaction system using SYBR Advantage qPCR Premix (Clontech). All of the reactions were performed in triplicate in a RealPlex 2 system (Eppendorf). The relative gene expression was quantified as described previously46. The sequence of each primer was as follows: ACTB, 5′-GATCATTGCTCCTCCTGAGC-3′ and 5′-ACTCCTGCTTGCTGATCCAC-3′;CDH1, 5′-ACCAGAATAAAGACCAAGTGACCA-3′ and 5′-AGCAAGAGCAGCAGAATCAGAAT-3′; CD44, 5′-GAGCATCGGATTTGAGA-3′ and 5′-CATACTGGGAGGTGTTGG-3′.
News Article | February 23, 2017
NEWS RELEASE REGULATED INFORMATION INSIDE INFORMATION Record revenue of $30 million, up 70% from 2015 Total test volume up 57% over 2015 Conference call for analysts and investors today at 15:00 CET / 09:00 EST, details below IRVINE, CA, and HERSTAL, BELGIUM - 7:00 AM, February 23, 2017 - MDxHealth SA (Euronext: MDXH.BR), or the "Company", today announced its financial results for the financial year ended December 31, 2016. Dr. Jan Groen, Chief Executive Officer of MDxHealth commented: "The strong progress made in 2016 with the accelerated adoption of ConfirmMDx and the successful launch of our first liquid biopsy test, SelectMDx, demonstrates the significant potential of MDxHealth's world leading urological oncology franchise." "With the ongoing roll out of SelectMDx, together with the planned launch of our second liquid biopsy test AssureMDx for Bladder Cancer this year, the Company continues to make rapid and meaningful progress in the expansion of its portfolio of products that address unmet medical needs for cancer patients, while leveraging the commercial infrastructure we have built over the last several years. We are pleased with our progress in 2016 and believe MDxHealth is well positioned to build strong, sustainable growth in 2017 and beyond." Strong growth of ConfirmMDx driven by wide market adoption and continued expansion of reimbursement coverage NCCN Guideline inclusion and increasing adoption has enabled MDxHealth to secure 19 reimbursement contracts, while 28 payors issued positive medical coverage policies. The test is now also covered under the California Medical Assistance Program (Medi-Cal), the single largest state-run public health program in the US with nearly 12 million enrollees. This positive trend will continue in 2017 with Horizon Blue Cross Blue Shield recently issuing positive policy coverage for more than 3.8 million members. The recently obtained unique ConfirmMDx CPT code, effective January 2018, is expected to further streamline the Company's reimbursement efforts and significantly reduce collection periods. Further to the period end, MDxHealth was awarded a US Government Services Administration (GSA) contract. This contract is critical to making ConfirmMDx widely accessible to authorized government customers. The Company's continued investments in demonstrating clinical utility and improved patient care from use of ConfirmMDx has resulted in publication in 10 clinical scientific publications and presentations, including in the journal The Prostate. Scientific data were also presented at several urology conferences across the US, including the Annual Meeting of the American Urology Association (AUA) in San Diego, and the Symposium of the American Society of Clinical Oncology Genitourinary Cancers Symposium (ASCO GU) in San Francisco. Successful global commercial launch of first liquid biopsy test SelectMDx. Second liquid biopsy test AssureMDx for Bladder Cancer set for 2017 full commercial launch Following the European launch in late 2015, the Company's proprietary liquid biopsy test SelectMDx was commercially launched in the US in March 2016. This non-invasive urine-based test identifies men at risk for clinically significant prostate cancer who may benefit from an initial prostate biopsy or magnetic resonance imaging (MRI). The validation study published in European Urology confirms the superior performance compared to other commonly used biomarker tests and risk calculators. The enrollment of the prospective 4M clinical study on 600 subjects evaluating the synergy of MRI and SelectMDx is complete and results are expected to be published in the course of 2017. In the US, SelectMDx is marketed as a Laboratory Developed Test (LDT) through MDxHealth's sales force. Outside the US, the Company pursues a direct sales strategy in the Benelux, Germany and Italy, complemented by European and global distributors and commercial lab partners. The Company concluded several agreements for the distribution of SelectMDx, covering various countries in Europe, Central and Latin America, Israel and most recently Hong Kong and Macao China. Outside the US, the test is currently performed at the Company's ISO 13485:2016 certified lab in Nijmegen, The Netherlands. Volumes for SelectMDx since its US launch in 2016 were nearly 3.5 times higher compared to the first year for ConfirmMDx, representing 17% of the total test volume for 2016. The fast uptake of this test by urologists and their patients has already resulted in 11 payor contracts in the first year. In the last quarter of 2016, the Company beta launched its second liquid biopsy test, AssureMDx for Bladder Cancer. The launch followed the publication of two clinical validation studies underscoring the test's clinical validity. AssureMDx has a very high negative predictive value of 99% and may significantly reduce unnecessary invasive and costly cystoscopy procedures. With 700,000 cystoscopy procedures performed in the US every year, AssureMDx addresses a potential $300 million market in the US, and is scheduled for commercial launch in the US as an LDT by the end of the first half of 2017. MDxHealth has built a robust portfolio of proprietary biomarkers for molecular assays for areas such as colorectal, lung and brain cancers. In addition to its proprietary offering in uro-oncology, the Company leverages this non-core portfolio of biomarkers and technologies through various licensing agreements. In 2016, MDxHealth granted a non-exclusive worldwide license for its methylation-specific PSR (MSP) technology to QIAGEN for use in its diagnostic cervical cancer assay, QIAsure. Key unaudited consolidated figures for the financial year ended December 31, 2016 (thousands of US dollars, except number of shares and per share data): Total revenues for the year ended December 31, 2016 amounted to $30 million, an increase of 70% compared to total revenues of $17.6 million a year earlier and included $25 million of product and service income. The strong growth in the US contributed $24.4 million or 82% of total revenue. Non-US revenues included initial sales of SelectMDx, milestone payments and royalties from license deals, and came in at $5.5 million, up 133% or $2.4 million compared to 2015. Total net billings for 2016 amounted to $49.3 million. However, the Company only recognizes revenue when there is reasonable evidence that the test will effectively be reimbursed. As a result, a significant portion of the revenue is only recognized when the payment is collected, leaving a significant portion of invoiced amounts unrecognized in 2016. The deferral of part of the revenue gradually decreases as the Company concludes firm agreements for reimbursement with a growing number of payors. Cost of goods sold for 2016 came in at $10.1 million, compared to $6.9 million in 2015. Increased revenues resulted in a gross profit of $19.9 million, while a sustained focus on operational efficiencies yielded an improvement of the gross profit margin from 60.9% in 2015 to 66.3% in 2016. Operating expenses for the year ended December 31, 2016 amounted to $32.7 million, an increase of $7.6 million or 30.2%. The year-on-year increase is mainly attributable to the acquisition of NovioGendix (renamed to MDxHealth BV), which was only included for one quarter in 2015. Furthermore, MDxHealth invested in the build-out of the organisation to support the global commercial launch of SelectMDx. MDxHealth adopts a direct sales strategy for SelectMDx in Benelux, Germany and Italy, supported by European and global distributors and commercial lab partners. The Company appointed a global commercial team to cover business development and direct sales. Finally, the Company continued to validate the clinical utility of its expanded offering through clinical trials and publications. R&D spending remained level with last year despite the increased pace of newly developed product launches. EBITDA for the year improved by $2.5 million as the loss was reduced from $13.6 million in 2015 to $11.1 million. This improvement was partly offset by increased amortization charges, bringing the Company's net loss for 2016 to $13.2 million, $1.3 million better than in 2015. The increased amortization resulted from scheduled amortization of intangible assets associated with the acquisition of NovioGendix in 2015. Cash and cash equivalents stood at $30.8 million at the end of 2016 after having successfully raised $21.7 million (€20.4 million) in a private placement of 4,526,962 new shares at €4.50 ($4.99) per share. The number of outstanding shares at December 31 was 49,796,595. Increased private payor adoption and a sustained focus on reimbursement has helped to improve working capital throughout 2016. Cash used by operations amounted to $16.6 million, compared to $14.4 million in 2015, and included cash collections of $19.7 million, a 61% increase year-on-year. The award of a unique CPT code by the AMA, which will become effective January 1, 2018, is expected to significantly shorten collection periods from both Medicare and private payors. MDxHealth is committed to maintaining its focus on its strategic priorities during 2017. These include: MDxHealth will host a conference call today at 15:00 CET / 14:00 GMT / 09:00 EST / 06:00 PT to discuss its Financial Year 2016 results. To access the conference call, please dial one of the appropriate numbers below quoting the conference ID 69886101. The call will be conducted in English and a replay will be available for 30 days. The presentation will be made available on the investors section of the MDxHealth website shortly before the call and can be accessed at: http://mdxhealth.com/investors. The Company's statutory auditor, BDO Bedrijfsrevisoren Burg. Ven. CBVA, has confirmed that its audit procedures with respect to the Company's consolidated financial statements, prepared in accordance with the International Financial Reporting Standards as adopted in the European Union, have been substantially completed, that these procedures have not revealed any material adjustments that would have to be made to the accounting information derived from the Company's consolidated financial information that is included in this press release, and that it intends to issue an unqualified opinion. The condensed Consolidated Statement of Comprehensive Income may be found on the Company's website at www.mdxhealth.com. The full Annual Report is expected to be made available to the public via the Company's website in April 2017. MDxHealth is a multinational healthcare company that provides actionable molecular diagnostic information to personalize the diagnosis and treatment of cancer. The Company's tests are based on proprietary genetic, epigenetic (methylation) and other molecular technologies and assist physicians with the diagnosis of urologic cancers, prognosis of recurrence risk, and prediction of response to a specific therapy. MDxHealth's European headquarters are in Herstal, Belgium, with laboratory operations in Nijmegen, The Netherlands, and US headquarters and laboratory operations based in Irvine, California. For more information visit mdxhealth.com and follow us on Twitter at: twitter.com/mdxhealth. This press release contains forward-looking statements and estimates with respect to the anticipated future performance of MDxHealth and the market in which it operates. Such statements and estimates are based on assumptions and assessments of known and unknown risks, uncertainties and other factors, which were deemed reasonable but may not prove to be correct. Actual events are difficult to predict, may depend upon factors that are beyond the company's control, and may turn out to be materially different. MDxHealth expressly disclaims any obligation to update any such forward-looking statements in this release to reflect any change in its expectations with regard thereto or any change in events, conditions or circumstances on which any such statement is based unless required by law or regulation. This press release does not constitute an offer or invitation for the sale or purchase of securities or assets of MDxHealth in any jurisdiction. No securities of MDxHealth may be offered or sold within the United States without registration under the US Securities Act of 1933, as amended, or in compliance with an exemption therefrom, and in accordance with any applicable US securities laws. NOTE: The MDxHealth logo, MDxHealth, ConfirmMDx, SelectMDx, AssureMDx and PredictMDx are trademarks or registered trademarks of MDxHealth SA. All other trademarks and service marks are the property of their respective owners.
News Article | October 26, 2016
Each year, NASA's Chandra X-ray Observatory helps celebrate American Archive Month by releasing a collection of images using X-ray data in its archive. The Chandra Data Archive is a sophisticated digital system that ultimately contains all of the data obtained by the telescope since its launch into space in 1999. Chandra's archive is a resource that makes these data available to the scientific community and the general public for years after they were originally obtained. Each of these six new images also includes data from telescopes covering other parts of the electromagnetic spectrum, such as visible and infrared light. This collection of images represents just a small fraction of the treasures that reside in Chandra's unique X-ray archive. From left to right, starting on the top row, the objects are: Westerlund 2: A cluster of young stars - about one to two million years old - located about 20,000 light years from Earth. Data in visible light from the Hubble Space Telescope (green and blue) reveal thick clouds where the stars are forming. High-energy radiation in the form of X-rays, however, can penetrate this cosmic haze, and are detected by Chandra (purple). 3C31: X-rays from the radio galaxy 3C31 (blue), located 240 million light years from Earth, allow astronomers to probe the density, temperature, and pressure of this galaxy, long known to be a powerful emitter of radio waves. The Chandra data also reveal a jet blasting away from one side of the central galaxy, which also is known as NGC 383. Here, the Chandra X-ray image has been combined with Hubble's visible light data (yellow). PSR J1509-5850: Pulsars were first discovered in 1967 and today astronomers know of over a thousand such objects. The pulsar, PSR J1509-5850, located about 12,000 light years from Earth and appearing as the bright white spot in the center of this image, has generated a long tail of X-ray emission trailing behind it, as seen in the lower part of the image. This pulsar has also generated an outflow of particles in approximately the opposite direction. In this image, X-rays detected by Chandra (blue) and radio emission (pink) have been overlaid on a visible light image from the Digitized Sky Survey of the field of view. Abell 665: Merging galaxy clusters can generate enormous shock waves, similar to cold fronts in weather on Earth. This system, known as Abell 665, has an extremely powerful shockwave, second only to the famous Bullet Cluster. Here, X-rays from Chandra (blue) show hot gas in the cluster. The bow wave shape of the shock is shown by the large white region near the center of the image. The Chandra image has been added to radio emission (purple) and visible light data from the Sloan Digital Sky Survey showing galaxies and stars (white). RX J0603.3+4214: The phenomenon of pareidolia is when people see familiar shapes in images. This galaxy cluster has invoked the nickname of the "Toothbrush Cluster" because of its resemblance to the dental tool. In fact, the stem of the brush is due to radio waves (green) while the diffuse emission where the toothpaste would go is produced by X-rays observed by Chandra (purple). Visible light data from the Subaru telescope show galaxies and stars (white) and a map from gravitational lensing (blue) shows the concentration of the mass, which is mostly (about 80%) dark matter. CTB 37A: Astronomers estimate that a supernova explosion should occur about every 50 years on average in the Milky Way galaxy. The object known as CTB 37A is a supernova remnant located in our Galaxy about 20,000 light years from Earth. This image shows that the debris field glowing in X-rays (blue) and radio waves (pink) may be expanding into a cooler cloud of gas and dust seen in infrared light (orange). NASA's Marshall Space Flight Center in Huntsville, Alabama, manages the Chandra program for NASA's Science Mission Directorate in Washington. The Smithsonian Astrophysical Observatory in Cambridge, Massachusetts, controls Chandra's science and flight operations. For more Chandra images, multimedia and related materials, visit: http://www.nasa.gov/chandra Please follow SpaceRef on Twitter and Like us on Facebook.
News Article | February 15, 2017
Like cosmic lighthouses sweeping the universe with bursts of energy, pulsars have fascinated and baffled astronomers since they were first discovered 50 years ago. In two studies, international teams of astronomers suggest that recent images from NASA's Chandra X-ray Observatory of two pulsars -- Geminga and B0355+54 -- may help shine a light on the distinctive emission signatures of pulsars, as well as their often perplexing geometry. Pulsars are a type of neutron star that are born in supernova explosions when massive stars collapse. Discovered initially by lighthouse-like beams of radio emission, more recent research has found that energetic pulsars also produce beams of high energy gamma rays. Interestingly, the beams rarely match up, said Bettina Posselt, senior research associate in astronomy and astrophysics, Penn State. The shapes of observed radio and gamma-ray pulses are often quite different and some of the objects show only one type of pulse or the other. These differences have generated debate about the pulsar model. "It's not fully understood why there are variations between different pulsars," said Posselt. "One of the main ideas here is that pulse differences have a lot to do with geometry -- and it also depends on how the pulsar's spin and magnetic axes are oriented with respect to line of sight whether you see certain pulsars or not, as well as how you see them." Chandra's images are giving the astronomers a closer than ever look at the distinctive geometry of the charged particle winds radiating in X-ray and other wavelengths from the objects, according to Posselt. Pulsars rhythmically rotate as they rocket through space at speeds reaching hundreds of kilometers a second. Pulsar wind nebulae (PWN) are produced when the energetic particles streaming from pulsars shoot along the stars' magnetic fields, form tori -- donut-shaped rings -- around the pulsar's equatorial plane, and jet along the spin axis, often sweeping back into long tails as the pulsars' quickly cut through the interstellar medium. "This is one of the nicest results of our larger study of pulsar wind nebulae," said Roger W. Romani, professor of physics at Stanford University and principal investigator of the Chandra PWN project. "By making the 3-D structure of these winds visible, we have shown how one can trace back to the plasma injected by the pulsar at the center. Chandra's fantastic X-ray acuity was essential for this study, so we are happy that it was possible to get the deep exposures that made these faint structures visible." A spectacular PWN is seen around the Geminga pulsar. Geminga -- one of the closest pulsars at only 800 light-years away from Earth -- has three unusual tails, said Posselt. The streams of particles spewing out of the alleged poles of Geminga -- or lateral tails -- stretch out for more than half a light-year, longer than 1,000 times the distance between the Sun and Pluto. Another shorter tail also emanates from the pulsar. The astronomers said that a much different PWN picture is seen in the X-ray image of another pulsar called B0355+54, which is about 3,300 light-years away from Earth. The tail of this pulsar has a cap of emission, followed by a narrow double tail that extends almost five light-years away from the star. While Geminga shows pulses in the gamma ray spectrum, but is radio quiet, B0355+54 is one of the brightest radio pulsars, but fails to show gamma rays. "The tails seem to tell us why that is," said Posselt, adding that the pulsars' spin axis and magnetic axis orientations influence what emissions are seen on Earth. According to Posselt, Geminga may have magnetic poles quite close to the top and bottom of the object, and nearly aligned spin poles, much like Earth. One of the magnetic poles of B0355+54 could directly face the Earth. Because the radio emission occurs near the site of the magnetic poles, the radio waves may point along the direction of the jets, she said. Gamma-ray emission, on the other hand, is produced at higher altitudes in a larger region, allowing the respective pulses to sweep larger areas of the sky. "For Geminga, we view the bright gamma ray pulses and the edge of the pulsar wind nebula torus, but the radio beams near the jets point off to the sides and remain unseen," Posselt said. The strongly bent lateral tails offer the astronomers clues to the geometry of the pulsar, which could be compared to either jet contrails soaring into space, or to a bow shock similar to the shockwave created by a bullet as it is shot through the air. Oleg Kargaltsev, assistant professor of physics, George Washington University, who worked on the study on B0355+54, said that the orientation of B0355+54 plays a role in how astronomers see the pulsar, as well. The study is available online in arXiv. "For B0355+54, a jet points nearly at us so we detect the bright radio pulses while most of the gamma-ray emission is directed in the plane of the sky and misses the Earth," said Kargaltsev. "This implies that the pulsar's spin axis direction is close to our line-of-sight direction and that the pulsar is moving nearly perpendicularly to its spin axis." Noel Klingler, a graduate research assistant in physics, George Washington University, and lead author of the B0355+54 paper, added that the angles between the three vectors -- the spin axis, the line-of-sight, and the velocity -- are different for different pulsars, thus affecting the appearances of their nebulae. "In particular, it may be tricky to detect a PWN from a pulsar moving close to the line-of-sight and having a small angle between the spin axis and our line-of-sight," said Klingler. In the bow-shock interpretation of the Geminga X-ray data, Geminga's two long tails and their unusual spectrum may suggest that the particles are accelerated to nearly the speed of light through a process called Fermi acceleration. The Fermi acceleration takes place at the intersection of a pulsar wind and the interstellar material, according to the researchers, who report their findings on Geminga online and in the current issue of Astrophysical Journal. Although different interpretations remain on the table for Geminga's geometry, Posselt said that Chandra's images of the pulsar are helping astrophysicists use pulsars as particle physics laboratories. Studying the objects gives astrophysicists a chance to investigate particle physics in conditions that would be impossible to replicate in a particle accelerator on earth. "In both scenarios, Geminga provides exciting new constraints on the acceleration physics in pulsar wind nebulae and their interaction with the surrounding interstellar matter," she said. * "Deep Chandra Observations of the Pulsar Wind Nebula Created by PSR B0355+54," Noel Klingler et al., 2016 Dec. 20, Astrophysical Journal [http://iopscience.iop.org/article/10.3847/1538-4357/833/2/253, preprint: https://arxiv.org/abs/1610.06167]. * "Geminga's Puzzling Pulsar Wind Nebula," B. Posselt et al., 2017, to appear in Astrophysical Journal [http://apj.aas.org, preprint: https://arxiv.org/abs/1611.03496]. Other team members include George C. Pavlov, senior scientist in astronomy and astrophysics, Penn State; Pat O. Slane, lecturer and senior astrophysicist, Harvard Smithsonian Center for Astrophysics; Roger Romani, professor of physics, Stanford University; Niccolo Bucciantini, permanent researcher, INAF Osservatorio Astorfisico di Arcetri; Andrei M. Bykov, head of the Laboratory for High Energy Astrophysics, Ioffe Physical-Technical Institute; Martin C. Weisskopf, project scientist, NASA/Marshall Space Flight Center; Stephen Chi-Yung Ng, assistant professor of physics, University of Hong Kong. Additional team members for the study on B0355+54 include Blagoy Rangelov, postdoctoral researcher, George Washington University; Tea Temim, JWST Support Scientist, Space Telescope Science Institute; Douglas A. Swartz, research scientist, Marshall Space Flight Center and Rolf Buehler, staff scientist, DESY Zeuthen. NASA and the Russian Science Foundation supported this work. Please follow SpaceRef on Twitter and Like us on Facebook.
News Article | January 7, 2016
After decades of studies and research, scientists have estimated the age of the observable universe to be roughly 13.8 billion years old. The connection between distance and the speed of light -- explained by Albert Einstein's theory of relativity -- has allowed scientists to look at different regions of the vast outer space which lie 13.8 billion light-years away. The age and distance of the universe -- are these small hints to the possible existence of alien life? Scientists have yet to form a firm conclusion, but in late November last year, some experts were able to detect five mysterious radio bursts which may have all come from outside the Milky Way galaxy. These radio signals were discovered after an "alien megastructure" was reported to be orbiting around a distant star known as KIC 8462852. "It almost doesn't matter where you point your telescope, because there are planets everywhere. If there's somebody out there, there are going to be so many of them out there that I do think there's a chance," explained astronomer Seth Shostak of the Search for Extraterrestrial Intelligence (SETI) Institute in California. Now, a new study presented at the annual meeting of the American Astronomical Society in Florida suggests that an old, densely-packed and isolated group of stars located within the Milky Way may possibly sustain extraterrestrial life. These stars, collectively called globular clusters, may be a cradle of advanced civilizations, experts said. The Possibility Of Alien Life In Globular Star Clusters Scientists from the Harvard-Smithsonian Center for Astrophysics (CfA) and the Tata Institute of Fundamental Research in Mumbai believe that globular star clusters may be the first place in our galaxy to contain intelligent life beyond Earth. What exactly are globular star clusters? These are densely-packed and tight groups that contain thousands or millions of stars. These balls of star clusters may be about 100 light-years across each other on average, and are as old as the Milky Way galaxy itself. Our galaxy is home to about 150 globular star clusters, where most of them orbit the galactic outskirts. On average, these star clusters may be 10 to 12 billion years old, just a couple billion years younger than the observable universe. But Houston, We Have A Problem The stars within globular clusters have fewer of the essential elements considered as "building blocks" of planets, such as silicon (Si) and iron (Fe), because these elements must be formed in earlier generations of stars. This lack in heavy elements has led other scientists to argue that globular star clusters are less likely to contain planets. In fact, only one planet has been found within globular clusters: the oldest known exoplanet called PSR B1620-26 b or Methuselah. Still, astronomers Rosanne DiStefano and Alak Ray said these views are "too pessimistic." "It's premature to say there are no planets in globular clusters," said Ray. The duo explained that a lot of exoplanets have been discovered around host stars that are only one-tenth as rich with metals as our Sun. While planets that are Jupiter-sized are found more around stars that contained higher levels of Fe and Si, planets that are Earth-sized show no such bias. Another main problem: because globular clusters are too close-knit, this specific environment could threaten the possible formation and existence of planets within it. Scientists said a neighboring star could wander too close to a planetary system, consequently disrupting the gravitational forces and resulting to the unfortunate hurling of worlds into interstellar space. What Could Be the Right Clue? DiStefano and Ray explained that the habitable zone or the "Goldilocks" zone of a star varies greatly. The Goldilocks zone is the right distance at which planets would be not too warm or not too cold to have liquid water. Brighter stars have more distant Goldilocks zones, and have shorter life spans. Because globular clusters are old, these extremely bright stars have died out. In contrast, planets that orbit around dimmer stars huddle closer to each other. These dimmer stars are faint and closer, but they also live long enough to become red dwarfs. Potentially habitable planets that these faint stars host would orbit nearby and be relatively safe from stellar interactions. "Once planets form, they can survive for long periods of time, even longer than the current age of the universe," said DiStefano. What If Planets Within Globular Clusters Evolve? If livable planets could form within globular star clusters and survive for billions of years, extraterrestrial life in said planets would have enough time to become complex and even develop intelligence. The alien civilization would truly be different from our own. In our solar system, the nearest star is about four light-years (24 trillion miles) away. In a globular cluster, the nearest star may be 20 times closer or only one trillion miles apart. Interstellar exploration and communication, as well as space travel, would definitely be easier. DiStefano and Ray call this potential theory the "Globular Cluster Opportunity." "Sending a broadcast between the stars wouldn't take any longer than a letter from the U.S. to Europe in the 18th century," said DiStefano. Space missions would definitely take less time. NASA's Voyager probes are 100 billion miles away from our planet. In terms of globular cluster distance, this is one-tenth as far as it would take to reach the nearest star. A civilization at Earth's current technological level could easily send interstellar probes within the realm of a globular star cluster. DiStefano said the nearest globular cluster to our planet is thousand light-years away. This is why it is difficult for us to find planets, particularly in a space environment with a crowded core. However, it is possible to detect globular cluster planets on galactic outskirts. Through gravitational lensing, scientists might even spot free-floating planets or planets whose gravity magnifies light from a star. Lastly, scientists say that using SETI search methods to target globular clusters is an intriguing idea. SETI uses arrays of radio telescopes called Allen Telescope Array (ATA) to look for laser or radio broadcasts. Astronomer Frank Drake used the Arecibo radio telescope to broadcast the first deliberate message from our planet to outer space, a message directed to globular cluster Messier 13 (M13) or the Hercules Globular Cluster.
News Article | November 7, 2016
The Crab Nebula seen in the optical by the Hubble Space Telescope. The Crab is an example of a pulsar wind nebula. Astronomers have modeled the detailed shape of another pulsar wind nebula to conclude, among other things, that the pulsar’s spin axis is pointed almost directly towards us. Credit: NASA/ Hubble Space Telescope Neutron stars are the detritus of supernova explosions, with masses between one and several suns and diameters only tens of kilometers across. A pulsar is a spinning neutron star with a strong magnetic field; charged particles in the field radiate in a lighthouse-like beam that can sweep past the Earth with extreme regularity every few seconds or less. A pulsar also has a wind, and charged particles, sometimes accelerated to near the speed of light, form a nebula around the pulsar: a pulsar wind nebula. The particles' high energies make them strong X-ray emitters, and the nebulae can be seen and studied with X-ray observatories. The most famous example of a pulsar wind nebula is the beautiful and dramatic Crab Nebula. When a pulsar moves through the interstellar medium, the nebula can develop a bow-shaped shock. Most of the wind particles are confined to a direction opposite to that of the pulsar's motion and form a tail of nebulosity. Recent X-ray and radio observations of fast-moving pulsars confirm the existence of the bright, extended tails as well as compact nebulosity near the pulsars. The length of an X-ray tail can significantly exceed the size of the compact nebula, extending several light-years or more behind the pulsar. CfA astronomer Patrick Slane was a member of a team that used the Chandra X-ray Observatory to study the nebula around the pulsar PSR B0355+54, located about 3400 light-years away. The pulsar's observed movement over the sky (its proper motion) is measured to be about sixty kilometer per second. Earlier observations by Chandra had determined that the pulsar's nebula had a long tail, extending over at least seven light-years (it might be somewhat longer, but the field of the detector was limited to this size); it also has a bright compact core. The scientists used deep Chandra observations to examine the nebula's faint emission structures, and found that the shape of the nebula, when compared to the direction of the pulsar's motion through the medium, suggests that the spin axis of the pulsar is pointed nearly directly towards us. They also estimate many of the basic parameters of the nebula including the strength of its magnetic field, which is lower than expected (or else turbulence is re-accelerating the particles and modifying the field). Other conclusions include properties of the compact core and details of the physical mechanisms powering the X-ray and radio radiation. Explore further: Astrophysicists conduct very high energy studies of a highly extended pulsar wind nebula More information: Deep Chandra Observations of the Pulsar Wind Nebula Created by PSR B0355+54. arxiv.org/abs/1610.06167
News Article | December 9, 2016
With a project pipeline of more than 20GW and six turbine makers, financing and transmission will be the industry's main challenges next year, according to Lauro Fiuza, the chairman of the board of the Brazilian wind power association ABEEólica. “We worked very hard to convince the government to hold a tender this year so the important thing is that the tender will be held which is signal that the wind programme will continue,” he said. Sector activity has been slow this year. On 19 December, developers will have the last chance to contract wind generation capacity after earlier tenders were cancelled due to political and economic problems, which have slashed demand for power. Signals are still not as positive as investors would like for the tender, which aims to contract projects for 2019. Analysts caution it could be one of the slowest years for wind power since 2012 when less than 300MW of new wind were contracted. Turbine manufacturers badly need new supply contracts. They have 8GW of potential orders through 2019, but this is not enough all factories operating at high capacity. The outlook doesn't look good. Although more than 21GW of wind projects had been registered for the tender, only about 3GW will compete after the government declined to clear the rest due to transmission capacity shortfalls. “We are contributing to the solution for the transmission problem,” says Fiuza. “We hired PSR consulting firm to carry out a study for transmission for the wind sector”. Although the original government plan to contract 2GW of capacity annually is unlikely to be met, the administration of President Michel Temer is trying to improve planning, while hinting it will guarantee returns for investors. He took office in August following the impeachment of Dilma Rousseff. “We saw a change in the transmission tender [in October] which was a success because the government has understood it cannot restrict profitability,” says Fiuza. ABEEolica supports a more market-friendly approach. A reduction of the role of the National Development Bank (BNDES) in financing the sector could open new opportunities for wind power investors. “Financing from the BNDES has gone down from 80% to below 50% and this has a negative impact. But it forces us to seek new financing options and adjust the tenders to allow for new types of financing,” he says ABEEólica has been involved in discussions about financing – promoting some of them in the past year – and aims to continue those talks in 2017. Officials at the trade group like the idea of dollar-denominated PPAs. This would allow investors to tap international markets, drastically reducing financing costs from BNDES’ 12% annually to single digits. Fiuza recognises dollar-denominated PPAs are a complex issue in Brazil given the turbulent economy and currency exchange rate fluctuations. He notes, however, that Brazil already has some energy contracts linked to the US dollar involving the 14GW nameplate capacity Itaipu hydro facility on the Brazilian-Paraguyan border and others involving natural gas. “Our economy is a little more complex [compared to other Latin American countries] so we would not able to have the whole PPA linked to the dollar,” he says. Temer's administration has offers some support for the idea but has not taken a final decision. The economic problems and exchange rate uncertainty will take a toll in the upcoming tender, analysts say. They expect contracts totaling between 500MW and 2GW. Fiuza notes that wind will have an important role to play in the country's energy mix over the ong term. ABEEolica has commissioned studies that showed that Brazil has only 1.7GW of excess nameplate generation capacity, an indication that the government should contract power now in anticipation the economy will improve. “Historically in Brazil, when the economy rebounds, power demand rises twice as fast as the economy”, he says, noting that ABEEólica is already lobbying for the government to hold a tender in the first half of 2017. “It is clear companies that will take part in this tender will be the big groups that have a lot of cash. But even if we do not contract what we want it won’t be too bad. It’s like a diet: it doesn’t kill you, but its an opportunity to streamline things and adjust the machinery," he says.