AIP Conference Proceedings | Year: 2014
High precise measurements of quasars spectra provide a powerful tool for investigation the possible spatial and temporal variations of the fine-structure constant a α = e2/(4πε0hc) in the Universe. It is demonstrated that high sensitivity to α variation can be obtained from a comparison of the quasars and laboratory spectra. For this purpose, a new method of using OIII strong nebular doublet emission lines in Sloan Digital Sky Survey (SDSS) Early Data Release quasar spectra with a derived analytical expression for the error analysis was applied to compute the α-variation. The method provides Δα/α = (-0.52 ± 0.77) × 10-5 with spanning redshift 0.16 ≤ z ≤ 0.80 over the cosmological time scales t∼1010yr. This presents a factor of ∼14 improvements on the constraint on Δα/α compared with the result using the same data published in the literature. © 2014 AIP Publishing LLC. Source
To understand the cellular and molecular mechanisms that underlie CCM formation, we first examined the temporal course of lesion formation in mice with induced, endothelial-specific deletion of Krit1 immediately after birth (iECre;Krit1fl/fl, hereafter termed Krit1ECKO mice). Vascular malformations were first detected at postnatal day 6 (P6) as dilated venules in the cerebellar white matter of Krit1ECKO mice, with numerous mature lesions present by P11 (ref. 13) (Fig 1a–c and Extended Data Fig. 1). We recently demonstrated that loss of KRIT1, CCM2 or PDCD10 in endothelial cells of the developing mouse heart upregulates expression of KLF2, KLF4, ADAMTS4 and ADAMTS5 owing to increased activity of the MEKK3 signalling pathway12. Analysis of isolated cerebellar endothelial cells from neonatal Krit1ECKO and littermate control mice at P6 revealed increased Adamts4, but not Adamts5, messenger RNA and protein, in addition to increased levels of both Klf2 and Klf4 (Fig. 1d, f). ADAMTS4 cleaves the proteoglycan versican to expose a neo-epitope (DPEAAE) that was detected immediately adjacent to the endothelial cells of both early and late CCM lesions (Fig. 1e). Increased levels of nuclear KLF4 protein and Klf2 mRNA were also detected in the endothelial cells of CCM lesions and other vessels in the cerebellum (Fig. 1e, g). These findings reveal increased levels of KLF2, KLF4 and ADAMTS4 during the earliest phase of CCM lesion formation in vivo. Recent studies have implicated numerous signalling mechanisms as causal for CCM formation, including the Rho3, 4, 14, TGF-β/BMP5, Wnt/β-catenin6 and Notch15 pathways. To determine whether changes in Rho activity are an early event in CCM pathogenesis, we examined the ROCK substrate phosphorylated-myosin light chain (pMLC). pMLC levels were markedly increased in the brain capillary and venous endothelial cells of P6 neonatal Krit1ECKO mice compared with littermate controls, including those lining the earliest detectable CCM lesions (Extended Data Fig. 2a). TGF-β/BMP signalling through SMAD1 and SMAD3 and a mechanism of EndMT has been proposed to explain gene expression changes in KRIT1-deficient brain endothelial cells, including increased KLF4 (refs 5, 16). A recent study by the same group suggested that increased β-catenin signalling is a primary event that culminates in later EndMT6. To assess endothelial β-catenin signalling during CCM lesion formation, we generated neonatal Krit1ECKO mice on a TCF/Lef:H2B-GFP Wnt/β-catenin reporter background17. Immunostaining for green fluorescent protein (GFP) revealed β-catenin signalling in white matter vascular endothelium that was not increased in P6 CCM lesions (Extended Data Fig. 2b), while the levels of both GFP protein and the β-catenin target genes Axin2 and Lef1 were unchanged in cerebellar endothelial cells isolated from P6 or P11 neonatal Krit1ECKO mice (Extended Data Fig. 2c, d). Immunostaining of brain sections and immunoblotting of cerebellar endothelial cell protein also revealed no changes in pSMAD3 (Extended Data Fig. 2e, f). Finally, analysis of cerebellar endothelial gene expression analysis revealed no change in the expression of Notch target genes at P6, although an increase in Hes1 was noted at P11 (Extended Data Fig. 3). These studies reveal that primary CCM lesion formation is associated with increases in Klf2, Klf4 and Adamts4 expression and Rho/ROCK activity, but not in TGF-β/BMP, Wnt/β-catenin or Notch signalling. The above findings suggested that changes in KLF2/4 and ADAMTS4 expression may be causal for CCM formation. The CCM complex directly binds MEKK3 (refs 7, 8, 9, 10, 11), a MAP3 kinase known to regulate KLF2 and KLF4 expression in the embryonic heart in cultured endothelial cells12, and we previously found that Map3k3 haploinsufficiency rescues the loss of cardiac jelly associated with endocardial loss of CCM signalling12. Map3k3 haploinsufficiency was also found to rescue the early embryonic lethality conferred by pan-endothelial loss of KRIT1 (Extended Data Fig. 4a and ref. 18), suggesting that excess endothelial MEKK3 signalling may have a broad role in the cardiovascular phenotypes associated with loss of CCM signalling. To determine whether this model underlies CCM formation, we generated iECre;Krit1fl/fl;Map3k3fl/+ mice (termed Map3k3HetRSQ mice). Visual inspection of the hindbrains of P11 Map3k3HetRSQ mice compared with neonatal Krit1ECKO littermate controls revealed a notable reduction in the number and size of vascular lesions (Fig. 2a). To quantify CCM formation, we imaged P11 hindbrains using contrast-enhanced, high resolution X-ray micro-computed tomography (microCT), and measured actual lesion volumes using semi-automated software. Map3k3HetRSQ mouse hindbrains exhibited nearly complete prevention of the lesion phenotype compared with neonatal Krit1ECKO littermates, as assessed by hindbrain microCT imaging (Fig. 2b), blinded measurement of total CCM lesion volume (Fig. 2c), and measurements of Klf2, Klf4 and Adamts4 in P6 cerebellar endothelial cells (Fig. 2d). While almost all neonatal Krit1ECKO mice were dead by P30, all Map3k3HetRSQ animals remained alive (Fig. 2e), and exhibited normal growth and development. Finally, partial loss of MEKK3 also fully rescued CCM lesion formation in Ccm2ECKO (iECre;Ccm2fl/fl) mice (Extended Data Fig. 4b). These genetic findings support the conclusion that CCM lesions arise from gain of MEKK3 signalling and altered downstream gene expression in the endothelium. Increased endothelial pMLC is coincident with increased Klf2, Klf4 and Adamts4 expression in the earliest CCM lesions (Fig. 1e, g and Extended Data Fig. 2a), suggesting either that changes in Rho/ROCK activity are downstream of changes in MEKK3 activity or vice versa. The Rho-inhibiting agents hydroxyfasudil, tempol and vitamin D3 (ref. 19) failed to reverse the increase in KLF2 and KLF4 expression conferred by loss of KRIT1 in cultured human endothelial cells (Fig. 2f), suggesting that Rho is not upstream of the KLF2/4 expression changes associated with loss of CCM function. By contrast, P6 Map3k3HetRSQ mice exhibited a complete normalization of endothelial pMLC staining (Fig. 2g), indicating that increased Rho activity arises secondary to increased MEKK3 signalling during CCM formation. To test the roles of KLF2 and KLF4 in CCM pathogenesis, we measured lesion formation in Klf2HetRSQ (iECre;Krit1fl/fl;Klf2fl/+), Klf2HomoRSQ (iECre;Krit1fl/fl;Klf2fl/fl) and Klf4HetRSQ (iECre;Krit1fl/fl;Klf4fl/+) mice compared with littermate neonatal Krit1ECKO controls at P11. Klf2HetRSQ and Klf4HetRSQ mice exhibited a marked but incomplete prevention of lesion formation (80% and 75% rescue for Klf2HetRSQ and Klf4HetRSQ mice, respectively) based on visual inspection of cerebellar lesions and quantification of CCM lesion volume after microCT imaging (Fig. 3a–d). Remarkably, Klf2HomoRSQ mice exhibited 99% rescue (Fig. 3a, c), with only a small amount of venule dilatation visible histologically (Extended Data Fig. 5). pMLC staining was normalized in P6 Klf2HetRSQ mice (Fig. 3e), indicating that increased Rho/ROCK activity arises secondary to increased KLF2 expression. These findings identify gain of KLF2 and KLF4 function as causal for CCM formation, and suggest that these transcription factors are the primary downstream targets of MEKK3 in this disease model. They also highlight the notable molecular conservation of this endothelial pathway: from zebrafish to mammals, and from embryonic vascular endothelium and endocardium to postnatal brain endothelium. To determine whether MEKK3–KLF2/4 signalling is increased in human CCMs, we examined resected lesions from two patients with familial CCM bearing KRIT1 and PDCD10 germline mutations, and two sporadic CCM patients lacking any previous genetic or molecular data. Markedly increased nuclear KLF2 and KLF4 expression was observed in the endothelial cells of both familial and sporadic human CCM lesions (Fig. 4a, b), consistent with increased MEKK3 signalling and studies performed using the mouse model. MEKK3 binds CCM2 through the carboxy-terminal helical harmonin domain of CCM2, and CCM2 truncation mutants lacking this domain do not bind MEKK3 (Extended Data Fig. 6a, b and refs 10, 11, 20, 21). A literature search identified a familial CCM patient with a four-nucleotide duplication in the last exon of CCM2 (CCCTdup) predicted to delete most of the helical harmonin domain22 (Fig. 4c). CCM2 CCCTdup expressed normally in HEK293T cells and bound KRIT1 and PDCD10 in a manner indistinguishable from wild-type CCM2, but failed to interact with MEKK3 (Fig. 4c–e and Extended Data Fig. 6c). These results suggest that specific disruption of the CCM2–MEKK3 interaction is sufficient to cause familial CCM disease, and that human CCMs also arise due to loss of MEKK3 regulation and increased expression of KLF2 and KLF4. How gain of MEKK3–KLF2/4 signalling confers CCM formation is unclear. It has been proposed that EndMT underlies CCM pathogenesis5, 6, 23, but we detect no evidence of a change in phenotype from endothelial to mesenchymal with loss of CCM signalling, and demonstrate that the loss of the non-mesenchymal transcription factor KLF2 is sufficient to rescue CCM formation fully. Our studies identify two effector pathways downstream of MEKK3–KLF2/4 signalling that may drive early CCM pathogenesis: Rho signalling and ADAMTS proteolytic activity. Increased Rho activity has been linked to increased stress fibre formation, loosened junctions and decreased tube formation in cultured endothelial cells3, 14, 24, and loss of vascular integrity in mice4, 25, but whether and how these changes might cause CCM formation is not yet clear. Increased ADAMTS activity is conferred by gain of KLF2 and KLF4 function (Extended Data Fig. 7), and may confer CCM formation through breakdown of a proteoglycan matrix that is required specifically for the CNS vasculature (for example, versican; Extended Data Fig. 1b). A proteolytic mechanism would also explain the autosomal dominant inheritance of this disease, as CCM-deficient endothelial cells generated by a rare second-hit mechanism26, 27 could degrade the matrix supporting an entire vessel, thereby creating a cavernous malformation. Finally, these studies may help to identify new therapies for CCM disease. Since MEKK3 upregulates KLF2 and KLF4 through the MEK5 and ERK5 downstream MAPKs12, 28, we evaluated the effects of available inhibitors on CCM formation. BIX02189 (anti-MEK5) and XMD17-109 (anti-ERK5) reversed the increase in KLF2 and KLF4 expression associated with loss of CCM signalling in cultured endothelial cells (Extended Data Fig. 8), but failed to affect CCM formation at the low doses tolerated by neonatal Krit1ECKO mice (not shown). Therapies targeting Rho (for example, fasudil29 or other recently identified agents19) or the ADAMTS proteases may prove more effective, but future studies that rigorously test the causal role of these putative downstream effectors remain essential for the rational development of CCM disease therapies.
Ahead of the April 26 anniversary, AFP looks at the steps taken since 1986 to improve nuclear safety around the world and—as Fukushima showed in 2011—the challenges that remain. Only in the USSR? Experts say a big factor behind the disaster was the unusual and poor design of the reactor, known as RMBK, particularly its propensity to sudden power surges—as happened at Chernobyl. In addition, and unlike elsewhere outside the Soviet Union, there was no containment structure shielding the reactor to stop radioactivity escaping. But there was also human error. According to the World Nuclear Association, the accident was also due to "the violation of operating procedures and the absence of a safety culture". The aftermath was also poorly handled, with officials slow to evacuate locals and Moscow sending 600,000 "liquidators" with little or no protective gear to put out a fire that raged for 10 days. The first alarm was raised on April 28, 1986, not by Russia but by Sweden after it detected an unexplained rise in radiation levels. Soviet leader Mikhail Gorbachev did not admit the disaster had occurred until May 14. With enormous public outrage around the world Chernobyl, suddenly a household name, spurred an international push— even overcoming Cold War divisions—to improve atomic safety and reassure the public. One of the most important steps was the 1989 creation of the World Association of Nuclear Operators (WANO), which carries out "peer reviews" of 430 reactors around the world to detect problems. "The industry has undoubtedly learned the lesson that we are stronger together," WANO chief executive Peter Prozesky told AFP. The demise of the Soviet Union and the end of its Cold War isolation has also removed barriers to international cooperation. Ex-communist eastern European countries, many now EU members, have also been helped to adapt their Soviet-built plants. Of the 17 RMBK reactors in operation in 1986, six have been permanently shut down. In addition the role of the UN nuclear watchdog, the International Atomic Energy Agency was beefed up. It expanded and revised safety standards and member states were required to report swiftly any incidents with potential cross-border effects. A number of international agreements were signed, the most important being the IAEA Convention on Nuclear Safety (CNS). Others covered nuclear waste and early warning systems for accidents. But any belief that enough had been done was swamped by the tsunami that knocked out the power supply and cooling systems of three reactors at the Fukushima Daiichi nuclear plant on March 11, 2011. "It was the belief in Japan at that time that this facility was robust... and even that it was not a good idea to conduct upgrades or changes to the facility because this could demonstrate in terms of public communication some weaknesses," Juan Carlos Lentijo, head of nuclear safety at the IAEA, told AFP. "This was an acute error, a huge mistake." This, the worst nuclear accident since Chernobyl, also caused an outcry, further international cooperation and agreements, and an even bigger role for WANO and the IAEA. Nuclear plant operators again say that they have made more technical improvements, including through better shielding of the nuclear material and more reliable "passive" safety systems in newer reactors. But for critics, dangerous risks remain, not least because no matter how many technical and regulatory improvements are made, the risk of human error—the common factor at Chernobyl and Fukushima—remains. According to the Union of Concerned Scientists, there were 10 "near misses" at US reactors in 2015, events that potentially increase the risk of a meltdown by at least 10 times, most due to human error. For Shawn-Patrick Stensil, a nuclear expert at Greenpeace, the biggest risk is that most reactors, particularly in the West, are decades old, their designs dating back to the 1960s and 70s. "We are now in the wear-out stage for the majority of the reactors in the world," Stensil told AFP. In addition, these reactors were all built before another risk that has reared its head in recent years—nuclear terrorism—"was even thought about," Stensil said. There are also lingering concerns about Russia—there are still 11 RMBK reactors, albeit with new safety features—particularly with Russia being a big exporter of reactors to the developing world. But Lentijo of the IAEA, whose raison d'etre is to promote nuclear technology, is more positive. "Safety has been improved, and I would say that the level is appropriate in general terms," he said. Explore further: No 'business as usual' on nuclear after Fukushima: IAEA
A protein found in neurons helps to limit inflammation in the central nervous system (CNS), contributing to the system's specialized immune environment. The CNS can stave off excessive inflammation. This 'immune privilege' has been attributed to the blood–brain barrier that restricts the entry of certain immune cells, but recent work has suggested a role for other cells and molecules. Lieping Chen at Yale University in New Haven, Connecticut, and his colleagues found that SALM5, a protein involved in neuronal growth and development, inhibits inflammation in the mouse CNS. In animals with an autoimmune CNS disease, blocking a receptor for SALM5 or treating with an antibody against SALM5 aggravated symptoms. Applying SALM5 to certain immune cells in a lab dish suppressed their response to a pro-inflammatory molecule. The findings could lead to treatments for inflammatory neurological diseases, the authors suggest.
Neurophysiologist Stephen Morairty, Ph.D., first got his feet wet in sleep research as an undergraduate performing experiments on his rugby team as part of a thesis project, encouraged by professor who was a sleep researcher himself. In graduate school Morairty watched a talk by Professor Dennis McGinty, Ph.D., a basic sleep researcher from the University of California, Los Angles, and was so inspired he followed McGinty to UCLA to receive his Ph.D. in neuroscience and work in his lab, and then continued on to do his post doc work at Harvard. When Morairty, now director of in Vivo Physiology and Pharmacology at SRI International’s Center for Neuroscience, started his lab at SRI 16 years ago with Thomas Kilduff, it was a pretty straight-forward sleep and circadian rhythms lab, he told Bioscience Technology. But new information over the last 15 years that links sleep disruptions to virtually every central nervous system (CNS) disorder, has expanded the scope of his work greatly to involve neurodegenerative or neurodevelopmental diseases such as Alzheimer’s, autism, and Huntington’s disease. “We’re finding out that the systems involved in sleep regulation appear to be attacked in many of these disorders and sleep disruption becomes a primary symptom of many of these things,” he told Bioscience Technology. In some cases, one of the very first signs of a disorder is a disruption in sleep/wake patterns. Along with looking at sleep as in indication for disease, another thing that has propelled Moriarty’s work into this sphere is the familiarity with electroencephalogram (EEG) recordings, which is a standard technique used in humans and animals to define the sleep and wakefulness electrophysiology, and has translational potential. In a recent study, published in February in the journal Sleep, Morairty and colleagues examined EEG in mouse models as a potential highly sensitive biomarker for Huntington’s disease that could predict the onset and progression of the disease before any symptoms began to show. The need for a translational biomarker Huntington’s disease, which impacts thinking, movement, and cognitive abilities, is unique among neurodegenerative diseases because it is the only one where there is a known cause. It is a single inherited gene mutation caused by the mutant huntingtin protein that causes the progressive degeneration of nerve cells in the brain. People who have the disease can get a blood test and know from birth if they have the mutation, and based on how many CAG repeats a person has, Morairty said, the earlier symptoms will occur, though it is not exact. Beyond that there is no other tracker for the disease. “We have no other way of testing if the disease course has changed if we develop a potential therapeutic and give it to people who have Huntington’s, there’s no good marker for testing whether the new treatment is having the desired effect,” Morairty said. Read more: Potential Huntington’s Treatment Successful in Animals, Moves to Clinical Testing He explained that this puts people who are developing potential therapies in a difficult place, because it may involve clinical trials that last 10 to 20 years to see if there is an effect. “It’s a really expensive clinical trial for a small population of people and it’s not very viable,” Morairty said. Pharmaceutical companies and lab tech companies working on CNS disorders have a difficult time with translational assays from preclinical to clinical models, according to Morairty. “One of the main reasons why there’s such a dearth of new medications coming out for CNS indications, is because there is very little translational – it’s like playing in the dark a lot of times.” That is where the EEG comes in. An EEG is an objective electrophysiological measure that is a read out of the network underneath the electrode, or a summation of all of the inhibitory and excitatory currents that are going on. It is collected regularly in humans, is noninvasive and is relatively inexpensive, compared to techniques such as MRI or PET scans. “The EEG has been collected in basically the same way for 80 years, with a few very small enhancements,” Morairty said. “So we had hope that by using new mathematical techniques to deconstruct it, looking at quantitative EEG analysis that we might be able to see changes in patterns that are similar in animal models and in human models.” He explained that while the behavior of a rat, monkey, or a human is quite different overall, that the structures of the brain are remarkably similar. “In gross areas of the brain, like what the hypothalamus does in a rat, is very similar to what it does in a non-human primate, and very similar to what it does in a human,” Morairty said. Similarities are also seen in the function of the cortex, and there is similar gross EEG patterns during active waking animals as active human models. When humans go to sleep their EEG changes in a very characteristic way that is the same in animal models. So, Morairty and colleagues hypothesized, why wouldn’t there be translational changes with disease progression? With Huntington’s disease it’s known that there are changes occurring along the way, as the mutant protein builds up over decades of life, and eventually reaches a tipping point where a cell becomes sick and eventually dies off. At a cellular level there are changes that are occurring but there is no way to monitor the activity of a million individual cells in human patients. Morairty said that the EEG will not give any kind of single cell readout, but the benefit is that it tells you what is going on with the network. So as cells become sick, their electrophysiological properties change, and eventually there will be a change in that network that comes before any noticeable changes in behavior. Looking through quantitative EEG analytical techniques, Morairty tested his hypothesis in the most popular Huntington disease mouse models and wild-type mice and was very pleased with the results. “The EEG patterns in these rodent models, the changes preceded any measurable changes in motor, in movement, in cognition, or in sleep/wake patterns,” Morairty said. If translational to humans this could be a big advance. The team found that although the Huntington’s disease mice and the wild-type mice start off very similar, their EEG began to change in early age, and continued to progress until it was extremely different from the wild-type, even before behavioral changes. One cautionary note is that when EEG recordings are taken from mice, the electrode is implanted under the skin, unlike on the surface of the scalp for humans. This means there is much better signal to noise ratio on the EEG output, so there are very sensitive areas that scientists are able to detect changes in the rodents. It would still be a work in progress to see how researchers could enhance those signals for humans. One thing the team hasn’t shown, and what Morairty considers to be especially important, it what would happen to the EEG if the mice were given a treatment to cause the disease to either plateau or reverse. “The EEG should show that, so if it really is a useful biomarker then we have to show that when you modulate progression of the disease that the EEG represents that.” There are a few potential new therapies, one developed by Ionis Pharmaceuticals that is meant to knock down levels of the mutant huntingtin protein in the brain with hopes of stopping, or abating progression of the disease. The treatment is in Phase 1 human trials right now, and Morarity has a proposal in with the foundation, and associated with the pharmaceutical company to see if he can get his hands on the treatment to test in the same animal model that he just published on. If approved, he will examine what happens to the EEG when levels of mutant protein are lowered in mice, which he predicts will change with the course of disease progression. “If it doesn’t then it’s not a very useful biomarker and my hypothesis is wrong,” he said. “But if it does, that shows that the EEG can be used clinically to measure the effectiveness of this new treatment.” Stay tuned for more from Stephen Morairty in an upcoming article that delves into new technologies being utilized in sleep research, such as optogenetics, DREADs, and miniaturized endomicroscopes.