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A technician of the U. Geological Survey uses a mass spectrometer to determine the proportions of neodymium isotopes contained in a sample of igneous rock. Cloth wrappings from a mummified bull Samples taken from a pyramid in Dashur, Egypt. This date agrees with the age of the pyramid as estimated from historical records. Charcoal Sample, recovered from bed of ash near Crater Lake, Oregon, is from a tree burned in the violent eruption of Mount Mazama which created Crater Lake. This eruption blanketed several States with ash, providing geologists with an excellent time zone. Charcoal Sample collected from the “Marmes Man” site in southeastern Washington. This rock shelter is believed to be among the oldest known inhabited sites in North America.
Potassium-Argon Dating Methods
Slideshows Videos Audio. Here of some of the well-tested methods of dating used in the study of early humans: Potassium-argon dating , Argon-argon dating , Carbon or Radiocarbon , and Uranium series. All of these methods measure the amount of radioactive decay of chemical elements; the decay occurs in a consistent manner, like a clock, over long periods of time.
Thermo-luminescence , Optically stimulated luminescence , and Electron spin resonance. All of these methods measure the amount of electrons that get absorbed and trapped inside a rock or tooth over time. Since animal species change over time, the fauna can be arranged from younger to older.
As potassium decays, it turns into argon. But unlike radiocarbon dating, the older the sample, the more accurate the dating — researchers.
How Accurate is K-Ar Dating? Email: laurence unmaskingevolution. Webpage: www. Messel, “A Modern Introduction to Physics” vol. The radiogenic argon that builds up in potassium-rich minerals after they have crystallized, therefore, furnishes a good measure of the age of the sample. The rubidium-strontium and uranium-lead techniques are very difficult to use with such samples, because the slow decay rates of the parent isotopes have not allowed a significant increase in the daughter isotopes.
What can potassium argon dating be used for
Some updates to this article are now available. The sections on the branching ratio and dating meteorites need updating. Radiometric dating methods estimate the age of rocks using calculations based on the decay rates of radioactive elements such as uranium, strontium, and potassium. On the surface, radiometric dating methods appear to give powerful support to the statement that life has existed on the earth for hundreds of millions, even billions, of years.
SITE. ROCK TYPE. DATE OF FORMATION. K-Ar. ASSESSED AGE. Haualalai (Hawaii). Basalt. AD million years old. Mt Etna (Sicily). Basalt.
Some of the common mistakes we make in evaluating claims are resisting contrary evidence, looking for confirming evidence, and preferring available evidence. To counteract these tendencies, we need to take deliberate steps to examine critically even our most cherished claims, search for disconfirming evidence as well as confirming, and look beyond evidence that is merely the most striking or memorable. I heard about a group of people yes they happen to be creationists with an agenda, but this should be irrelevant to the question I am posing!
They claim that the rocks they obtained were from a lava flow which came out of the volcano in They sent these rocks to 2 labs and had them dated by potassium-argon dating to be between , and 1 million years old. Since the real age of the rocks was around 50 years, does this demonstrate that K-Ar dating is inaccurate? I can think of several possibilities in response to this question:. I haven’t heard of this being done, however if you have some evidence to this effect please share it.
Radioactive dating is a method of dating rocks and minerals using radioactive isotopes. This method is useful for igneous and metamorphic rocks, which cannot be dated by the stratigraphic correlation method used for sedimentary rocks. Over naturally-occurring isotopes are known. Some do not change with time and form stable isotopes i. The unstable or more commonly known radioactive isotopes break down by radioactive decay into other isotopes.
Radioactive decay is a natural process and comes from the atomic nucleus becoming unstable and releasing bits and pieces.
Potassium argon dating methods – Is the number one destination for online dating with Both methods date smaller samples of absolute dating in accuracy.
Radiometric dating of rocks and minerals using naturally occurring, long-lived radioactive isotopes is troublesome for young-earth creationists because the techniques have provided overwhelming evidence of the antiquity of the earth and life. Some so-called creation scientists have attempted to show that radiometric dating does not work on theoretical grounds for example, Arndts and Overn ; Gill but such attempts invariably have fatal flaws see Dalrymple ; York and Dalrymple Other creationists have focused on instances in which radiometric dating seems to yield incorrect results.
In most instances, these efforts are flawed because the authors have misunderstood or misrepresented the data they attempt to analyze for example, Woodmorappe ; Morris HM ; Morris JD Only rarely does a creationist actually find an incorrect radiometric result Austin ; Rugg and Austin that has not already been revealed and discussed in the scientific literature.
The creationist approach of focusing on examples where radiometric dating yields incorrect results is a curious one for two reasons. First, it provides no evidence whatsoever to support their claim that the earth is very young. If the earth were only —10 years old, then surely there should be some scientific evidence to confirm that hypothesis; yet the creationists have produced not a shred of it so far.
Where are the data and age calculations that result in a consistent set of ages for all rocks on earth, as well as those from the moon and the meteorites, no greater than 10 years?
Potassium-argon dating method
Though we assess the age, over different thermal. K-Ar radiometric dating methods of beta decay of the most accurate in the s, is required to more accurately determine the importance of each. If it gave an older the method – for decades, measured was developed in accuracy and argon-argon, years old volcanic rocks based on.
Where excess argon is required to three unprovable. Reliability and accurate numerical ages under test conditions is K-Ar dating is capable of years old material is a radiometric dating is specialized and as.
A disadvantage to this technique is that in order to get accurate results, the Potassium-argon dating has been used to date volcanic layers above and below.
Most of the chronometric dating methods in use today are radiometric. That is to say, they are based on knowledge of the rate at which certain radioactive isotopes within dating samples decay or the rate of other cumulative changes in atoms resulting from radioactivity. Isotopes are specific forms of elements. The various isotopes of the same element differ in terms of atomic mass but have the same atomic number. In other words, they differ in the number of neutrons in their nuclei but have the same number of protons.
The spontaneous decay of radioactive elements occurs at different rates, depending on the specific isotope. These rates are stated in terms of half-lives. In other words, the change in numbers of atoms follows a geometric scale as illustrated by the graph below. The decay of atomic nuclei provides us with a reliable clock that is unaffected by normal forces in nature. The rate will not be changed by intense heat, cold, pressure, or moisture. Radiocarbon Dating. The most commonly used radiometric dating method is radiocarbon dating.
It is also called carbon and C dating.
Dating Fossils in the Rocks
Radiometric dating, often called radioactive dating, is a technique used to determine the age of materials such as rocks. It is based on a comparison between the observed abundance of a naturally occurring radioactive isotope and its decay products, using known decay rates. It is the principal source of information about the absolute age of rocks and other geological features, including the age of the Earth itself, and it can be used to date a wide range of natural and man-made materials.
The best-known radiometric dating techniques include radiocarbon dating, potassium-argon dating, and uranium-lead dating. By establishing geological timescales, radiometric dating provides a significant source of information about the ages of fossils and rates of evolutionary change, and it is also used to date archaeological materials, including ancient artifacts.
The K/Ar dating method applied to Holocene volcanic eruptions in Southern Italy. ). Measuring an accurate age, especially in the case of old samples (>.
Potassium, an alkali metal, the Earth’s eighth most abundant element is common in many rocks and rock-forming minerals. The quantity of potassium in a rock or mineral is variable proportional to the amount of silica present. Therefore, mafic rocks and minerals often contain less potassium than an equal amount of silicic rock or mineral. Potassium can be mobilized into or out of a rock or mineral through alteration processes. Due to the relatively heavy atomic weight of potassium, insignificant fractionation of the different potassium isotopes occurs.
However, the 40 K isotope is radioactive and therefore will be reduced in quantity over time. But, for the purposes of the KAr dating system, the relative abundance of 40 K is so small and its half-life is so long that its ratios with the other Potassium isotopes are considered constant. Argon, a noble gas, constitutes approximately 0. Because it is present within the atmosphere, every rock and mineral will have some quantity of Argon.
Argon can mobilized into or out of a rock or mineral through alteration and thermal processes. Like Potassium, Argon cannot be significantly fractionated in nature. However, 40 Ar is the decay product of 40 K and therefore will increase in quantity over time. The quantity of 40 Ar produced in a rock or mineral over time can be determined by substracting the amount known to be contained in the atmosphere.
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This page has been archived and is no longer updated. Despite seeming like a relatively stable place, the Earth’s surface has changed dramatically over the past 4. Mountains have been built and eroded, continents and oceans have moved great distances, and the Earth has fluctuated from being extremely cold and almost completely covered with ice to being very warm and ice-free.
These changes typically occur so slowly that they are barely detectable over the span of a human life, yet even at this instant, the Earth’s surface is moving and changing. As these changes have occurred, organisms have evolved, and remnants of some have been preserved as fossils. A fossil can be studied to determine what kind of organism it represents, how the organism lived, and how it was preserved.
What dating method did scientists use, and did it really generate reliable results? More. Potassium-Argon and Argon-Argon Dating of Crustal Rocks.
The potassium-argon K-Ar isotopic dating method is especially useful for determining the age of lavas. Developed in the s, it was important in developing the theory of plate tectonics and in calibrating the geologic time scale. Potassium occurs in two stable isotopes 41 K and 39 K and one radioactive isotope 40 K. Potassium decays with a half-life of million years, meaning that half of the 40 K atoms are gone after that span of time. Its decay yields argon and calcium in a ratio of 11 to The K-Ar method works by counting these radiogenic 40 Ar atoms trapped inside minerals.
What simplifies things is that potassium is a reactive metal and argon is an inert gas: Potassium is always tightly locked up in minerals whereas argon is not part of any minerals. Argon makes up 1 percent of the atmosphere. So assuming that no air gets into a mineral grain when it first forms, it has zero argon content.
Potassium-argon (K-Ar) dating
Potassium—argon dating , abbreviated K—Ar dating , is a radiometric dating method used in geochronology and archaeology. It is based on measurement of the product of the radioactive decay of an isotope of potassium K into argon Ar. Potassium is a common element found in many materials, such as micas , clay minerals , tephra , and evaporites. In these materials, the decay product 40 Ar is able to escape the liquid molten rock, but starts to accumulate when the rock solidifies recrystallizes.
Over time, the unstable potassium isotope (40K) from the rocks can be dated more accurately because they are found in the sedimentary rock.
Potassium-argon dating , method of determining the time of origin of rocks by measuring the ratio of radioactive argon to radioactive potassium in the rock. This dating method is based upon the decay of radioactive potassium to radioactive argon in minerals and rocks; potassium also decays to calcium Thus, the ratio of argon and potassium and radiogenic calcium to potassium in a mineral or rock is a measure of the age of the sample.
The calcium-potassium age method is seldom used, however, because of the great abundance of nonradiogenic calcium in minerals or rocks, which masks the presence of radiogenic calcium. On the other hand, the abundance of argon in the Earth is relatively small because of its escape to the atmosphere during processes associated with volcanism. The potassium-argon dating method has been used to measure a wide variety of ages. The potassium-argon age of some meteorites is as old as 4,,, years, and volcanic rocks as young as 20, years old have been measured by this method.
Potassium-argon dating. Info Print Cite. Submit Feedback. Thank you for your feedback. The Editors of Encyclopaedia Britannica Encyclopaedia Britannica’s editors oversee subject areas in which they have extensive knowledge, whether from years of experience gained by working on that content or via study for an advanced degree See Article History. Read More on This Topic.


GM Patents a New Noise-Canceling Sound System
Photo Credit: Chevrolet.com
Remember the good old days when you simply turned up the stereo volume when you had the T-tops out in your C3 Corvette?
Well, those simplistic days are long gone. Witness the space-age patent for a new noise-canceling sound system that’s recently been filed by General Motors with the U.S. Patent and Trade Office, according to a post on CarBuzz.
The new system uses a combination of several sensors to determine how the sound should be adjusted for the passengers amidst a sea of ever-changing conditions.
The ultimate goal is to make sure the sounds in the vehicle are at their optimal levels. In the case of a Corvette, for example, the system could use multiple sensors – including ones picking up sound and even light changes – to determine if, say, the roof panel has been removed and the stereo needs to be louder or noise-canceling needs to be turned on because of wind noise.
But hearing your favorite music isn’t the only concern of this new system. It also aims to make sure the car’s occupants are able to clearly hear warning chimes and alerts.
By using multiple sensors and processor modules instead of just one, GM can hone each sensor to look for specific frequencies and sound levels, and the processors can decide on the best plan of action, sometimes even deciding that no change is needed.
If all this seems like a little overkill, the high-tech system aims to keep passengers from missing important warning chimes or perhaps critical sounds like an approaching fire truck, ambulance, or police car.
No word yet on when the new system might be available
Where is that ‘Clutch-By-Wire’ Manual Transmission for the C8 Corvette?
The C8 Corvette E-Ray’s Hybrid DCT Transmission is Potentially Revealed in Patent by Tremec
[PICS] GM Receives Design Patent for the C8 Corvette Coupe


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The time has come for Wale’s More About Nothing to debut on streaming platforms around the World Wide Web. Earlier this afternoon, the Washington, D.C. native announced that his classic mixtape will arrive on a streaming platform near your phone, laptop or tablet on April 29, 2022. Just as all listeners remembered it back in the day of Facebook and iPods, the mixtape comes complete with tracks like “The Soup” and “The MC.” Not to mention, it also feature commentary from legendary comedian Jerry Seinfeld and NBA Champion Kevin Durant.
“In the end, it’s just about as good as the first Seinfeld mixtape. And that means that it’s pretty damn good,” critic Adam Kivel of Consequence wrote about the project when it dropped in 2010.
More than a decade later, Wale’s released a number of incredible projects, captivated audiences with records like “On Chill” and earned a Grammy nomination for his work on “Lotus Flower Bomb” with Miguel. Not to mention, he recently made his major motion picture debut in Ambulance. It’s amazing what a decade change can do, but it will always be fun to look back at the early days. Check out the trailer below and get ready for More About Nothing to ease its way into TIDAL, Apple Music, Spotify, YouTube Music, Audiomack, Pandora, etc. on April 29, 2022.

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Thanks for the ride report rockinrog. My wife and I did a similiar ride last summer as well. We rode the entire BRP northbound and continued on Skyline Drive and hung a left and headed for West Virginia after about 70 miles of Skyline. Skyline was a bit too laid back but still beautiful. It helped that the traffic was light on Skyline.
Ride well and often

domain: geography


As Epic Games slowly makes its way to Season 10 of Fortnite this summer, the company is going to update the required specs to play the game. The developers posted a new notice this week, letting PC players know of a change where their graphics cards will need to run Microsoft DirectX 11 in order to keep playing the game starting in July.
Starting with Season 10, the PC version of Fortnite will require a graphics card capable of running Microsoft DirectX 11.
While a DirectX 11-capable graphics card has always been one of Fortnite's system requirements, we've kept support online for players with older graphics cards – so they could keep playing!
However, when Season 10 begins, Fortnite will no longer be compatible with these older graphics cards. This allows us to focus our development and testing efforts to further optimize the Fortnite experience for all players.
That news may be a little troublesome for gamers with an older PC model and force many people to get an upgrade or leave Fortnite until they do. But for the vast majority of gamers, unless you own a PC made before 2011, chances are everything you need will be compatible with what you own. Unless, you know, you cheapened out on parts. But that's another topic for yourself.


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Slot machines can get you the largest jackpot and change your fortune forever. Here are some proven slot strategies for beginners that can make them richer.
If you want to try out simple casino games, slot machines will be the right match. The best part of slots is that you will not need any real strategy here. All you need to do is place money in the slot machine; hit the spin button ET Voila! Your road to riches opens. Suppose you are looking for the right spot to try your luck on the slot machines, you can try out G2G1BET. This website will provide you with a plethora of options for slot games.
The graphic intense slot games can also act as a source of great entertainment. The special features, bonus games, and the games inside games make the slot a favorite of many users. However, if you are a beginner, you may read through the below tips to maximize your chances of winning.
Slots are Random
Yes, you heard that right! Slot machines are indeed random, and hence you can choose any slot machines that fit within your bankroll and provide you with maximum entertainment.
Never Miss Out on the Progressive Jackpots
This is an important rule for novice players. You can win millions of dollars in seconds if you get blessed by lady luck and hit the progressive jackpot button.
Check for the Bonus Rounds
The bonus rounds can make you win more, and some slots can have more than one type of bonus round. Hence, it will be prudent to check for the bonus rounds of slots before commencing.
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Enviro completes Michelin share offer
Recycling firm Scandinavian Enviro Systems AB (Enviro) has completed a share issue worth 116 million Swedish Krone to Michelin. The issue is part of the letter of intent announced on 15 April 2020 and signals a “long-term strategic partnership between the two companies”.
Michelin’s investment in Enviro was made by way of a directed share issue (private placement) of a total of 116,165,223 shares at a subscription price of Swedish Krone (SEK) 0.28 per share, whereby, the share capital was increased by SEK 4,646,608.92. In total, the share issue generated proceeds of approximately SEK 32.5 million before transaction costs. As a result 20 per cent of the Enviro is held by Michelin and Michelin the largest shareholder.
The next steps include: a project to build a factory and to industrialize the technology (the location of the plant will be confirmed at a later date); a joint supply agreement between Michelin and Enviro; and a final agreement for a strategic partnership following the letter of intent, which is expected to be completed by mid 2020.

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Selection of Books, Manuscripts, Engravings, and Autograph Letters Remarkable for their Interest and Rarity. Five Hundreth Catalogue.
Click thumbnails for expanded view.
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Catalogue 500. An incredible bookseller's catalogue to commemorate a milestone in Maggs' history with many examples of fine bindings, and great books and manuscript material from all periods. Each item has a illustration associated with it. 375 pages with 225 plates. Some pages in 2 colors. Original wrappers, defective at the top.
Alan Wofsy Fine Arts, LLC.
- San Francisco, California
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This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1−/−) was found. Majority of Tie2-Cre/Tsc1−/− embryos died at embryonic day 14.5 in utero. Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1−/− embryos. Endothelial cells in Tsc1-deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.
Tuberous sclerosis complex (TSC) is an autosomal-dominant hamartoma disorder (1). The clinical phenotypes of TSC are characterized by abnormal vascularization in multiple organs, including skin angiofibroma, retinal hamartomas, cardiac rhabdomyomas, pulmonary lymphangioleiomyomatosis (LAM), renal angiomyolipoma (AML) and liver hemangiomas (2). The underlying genetic etiology of TSC involves mutations in one of two tumor suppressor genes, TSC1 and TSC2, which encode the hamartin and tuberin proteins, respectively (3,4). The hamartin–tuberin complex functions as an inhibitor of a key regulator of cell growth and proliferation, mechanistic target of rapamycin (mTOR) (5–9). mTOR modulates anabolic processes that promote cell growth and is exquisitely sensitive to cellular growth conditions. TSC1-TSC2 complex turns off mTOR under perturbed cellular growth conditions. Potentiated by various growth stimuli, AKT inactivates TSC1-TSC2 complex and consequently the activated mTOR promotes a subset of protein synthesis (10).
Animal models have been established to investigate the pathogenic role of mTOR signaling in TSC (11). A homozygous Tsc1 knockout (KO) mouse model was found to be lethal at embryonic (E) days 10.5–11.5 (7). And a conditional KO model of the Tsc1 gene in neuroprogenitor cells in the brain based on a nestin promoter-driven Cre allele was shown to be lethal within 24 h after birth, and reduced pup–maternal interaction was implicated as the major cause of death (12). Although ventricular myocyte-specific deletion of Tsc1 does not result in embryonic lethality, these mice die at approximately postnatal month 6, showing severe myocyte hypertrophy, with morphology similar to that observed in human rhabdomyoma which is the first sign of fetal TSC (13). Thus, the expression of Tsc1 is essential for proper embryogenesis.
Aneurysms in the brain, chest and abdomen have been reported in TSC patients and are associated with increased mobility and mortality (14–16). AMLs are composed of anomalous vessels, immature smooth muscle cells and adipocytes (17). They are associated with a high risk of spontaneous hemorrhage, arterial hypertension and kidney failure. Currently, little is understood about the underlying vascular pathogenesis of TSC. To investigate whether malfunction of Tsc1 contributes to TSC pathogenesis through its influence on vascularization, possibly involving the PI3 K/Akt/mTOR signaling pathway, a vascular endothelial cell (EC)-specific Tsc1 KO mouse model was generated and analyzed.
Generation of endothelial Tsc1 homozygous mice
To address the potential role of Tsc1 in the homeostasis of vascular ECs, mice containing a floxed Tsc1 allele were crossed with transgenic mice expressing Cre under the control of the endothelial-specific Tie2 promoter (18). The resultant Cre-mediated deletion of Tsc1 exons 17 and 18 leads to a frame-shift mutation that produces a non-functional protein (Fig. 1A). To verify the efficiency and specificity of Tie2-Cre-mediated recombination in ECs, we bred Tie2-Cre mice with mT/mG mice, a double fluorescent reporter mice, which express membrane-targeted red fluorescent tdTomato (mT) prior to Cre excision and membrane-targeted EGFP (mG) following Cre excision in non-recombined and recombined cells, respectively (19,20). The pattern of mT and mG fluorescence was observed in the yolk sacs of Tie2-Cre;mT/mG and mT/mG embryos yolk sacs by a fluorescent microscope. The results revealed only strong red fluorescence in mT/mG yolk sac vessels. In contrast, Tie2-Cre-induced green fluorescent protein (GFP) expression was observed in vessels, as demonstrated by a red-to-green fluorescence color switch following crossing with mT/mG mice (Fig. 1B). When PCR analysis of the yolk sacs was performed with primers targeting the wildtype (WT), floxed and mutant Tsc1 alleles, the various genotypes were observed in PCR gels as expected (Fig. 1C). We localized the vascular ECs with the CD31 marker and then found that the expression of Cre was colocalized with CD31 in vascular ECs in mutant embryos tissues (mT/mG+/−; Tie2-Cre+/−) (Supplementary Material, Fig. S1). Immunofluorescent staining indicated that Tsc1 (hamartin) protein expression was decreased in the mutant embryo endothelia, labeled by endothelial marker, endomucin (Fig. 1D) (21,22). Furthermore, western blotting analysis demonstrated that Tsc1 was dramatically decreased in yolk sacs of the EC Tsc1 KO (Tie2-Cre/Tsc1−/−) mice compared with the controls (Fig. 1E). Taken together, the results showed that Tsc1 deficiency was mediated by Cre-recombinase in ECs.
ECs-specific deletion of Tsc1 activates mTOR signaling
We examined whether loss of the Tsc1 allele led to hyperactivation of mTOR. Phosphorylation of S6 (pS6) or 4EBP-1 (p-4EBP-1) reflects mTOR activity. The phosphorylation levels of both S6 and 4EBP-1 were elevated in the mutant yolk sacs (Tie2-Cre/Tsc1−/−) compared with control littermates (Fig. 2A). Phospho-Akt was decreased in the mutant yolk sacs, indicating hyperactive mTOR-mediated negative feedback regulation of Akt signaling (Fig. 2A) (7,23,24). Additionally, the Tsc1 mutant embryos displayed enhanced pS6 expression in vascular ECs compared with control littermates in immunostaining analysis (Fig. 2B). Collectively, the results revealed excessive mTOR activity in yolk sacs of Tie2-Cre/Tsc1−/− embryos.
The endothelial Tsc1 homozygous mutation is embryonic lethal
Genotyping of tail DNA revealed that no endothelial Tsc1 KO (Tie2-cre/Tsc1−/−) offsprings were obtained from the mating of Tie2-Tsc1+/− and Tsc1fx/fx mice, indicating in utero lethality due to loss of Tsc1 expression (Table 1). To determine the developmental stage at which lethality occurred, embryos were collected at different gestational times and analyzed. No obvious differences in morphology could be detected between WT and mutant embryos from E9.5 to 11.5 (Supplementary Material, Fig. S2). At E12.5, the blood vessels in the heads were very pale in 60% of the Tie2-Cre/Tsc1−/− embryos, indicating reduced amounts of red blood cells or a lack of head vasculatures (Fig. 3Aa and b). Furthermore, the majority of Tsc1 mutant embryos began to die at E13.5, as demonstrated by the lack of a heartbeat or the presence of necrotic tissue. Hemorrhage was observed at this stage, and the yolk sacs of mutant embryos were pale, whereas those of WT embryos displayed well-formed vessels filled with red blood cells. Up to E14.5, the Tsc1 mutant embryos showed various and more severe vascular defects, including edema and subcutaneous hemorrhage at multiple sites (Fig. 3Ac and d). After E14.5, no living Tsc1 mutant embryos were observed, and all homozygous mutant embryos began to degenerate. Intriguingly, there was no obvious difference in the size of the mutant embryos compared with WT littermates, indicating that the mutation did not cause obvious developmental delay in utero. The data suggest that the Tsc1-deficient embryos died during mid-gestational development.
E, embryonic day; P, postnatal day.
aGenotyping was precluded by resorption.
bThe values in parentheses indicate the numbers of embryos that died.
Tsc1 deficiency in endothelium causes severe defects in angiogenesis
To discern whether the lethality of ECs Tsc1-null embryos was attributable to defects in angiogenesis, we analyzed the vascular morphology of the embryo proper as well as the yolk sac. Vascular remodeling into small and large vessels was significantly reduced in the head region of mutant embryos compared with WT littermates (Fig. 3A). Whole-mount immunostaining of CD31 revealed obvious vascular remodeling defects in the E9.5 and 10.5 mutant embryos (Fig. 3B). Vascular remodeling into small and large vessels was significantly reduced in the head region of mutant embryos compared with WT littermates. The mutant embryos exhibited large branching vessels, but the formation of the capillary network was perturbed. At E11.5, the primary head veins were less elaborated, with thinner vessel branches (Fig. 3C). The yolk sac vasculature was also affected by EC-specific Tsc1 deletion, as revealed through visualization of the embryonic vasculature by staining with an antibody against endomucin. The WT yolk sacs showed a well-organized hierarchical structure of large and small vessels. In contrast, the mutant yolk sacs displayed decreased sprouting angiogenesis and a much smaller number of branched vessels. Furthermore, although the primary capillary plexuses formed in the mutant yolk sacs, the major vitelline vessels were irregularly shaped (Fig. 3D; Supplementary Material, Fig. S3). We also observed abnormalities in the placenta when Tsc1 was deleted. At E14.5, the placentas of the mutant embryos exhibited a dilated labyrinth (La), while the spongiotrophobast (Sp) layer appeared to be unaffected (Fig. 3E). The thickness of the placental La was increased compared with littermates, whereas there was no significant difference in the Sp layer detected between the placentas of Tie2-Cre/Tsc1−/−mice and their littermates (Fig. 3F). No difference was observed in vasculogenesis between mutant and WT embryos. Blood smears from the mutant embryos and their liver tissues showed no difference in the proportion of hemangioblasts compared with WT mice (Supplementary Material, Fig. S4). These data suggested that disruption of Tsc1 was not required for vasculogenesis but prevented later angiogenesis.
EC-specific deletion of Tsc1 induces cell apoptosis, decreases cell proliferation and disrupts the expression of angiogenic factors
The expression of alpha-smooth muscle actin (α-SMA) was significantly reduced in mutant vessels. Consistent with the observed vessel formation defects, the expression of the CD31 was also markedly decreased (Fig. 4A). Quantitative detection of CD31 and α-SMA revealed an obvious reduction in the number of arteries in the Tie2-cre/Tsc1−/−embryos (Fig. 4B and C). To test whether this vessel remodeling was attributable to a difference in cell growth between WT and mutant mice, we next examined apoptosis in the vessels using terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end-labeling (TUNEL) assays. Apoptosis in ECs was increased in mutant compared with WT embryos. Moreover, the proliferation of ECs was significantly lower in mutant embryos, as determined by Ki67 immunostaining (Fig. 4D). The results demonstrated that cell growth was abnormal during vessel development in the mutant embryos. Finally, the mRNA levels of angiogenic factors were evaluated by qPCR analysis of the yolk sacs in mutant embryos and their WT littermates. The yolk sacs of mutant embryos showed significantly elevated levels of transforming growth factor-β1 (Tgf-β1), platelet-derived growth factor-b (Pdgf-b), Tek and Egfr mRNA, but significantly decreased levels of vascular cell adhesion molecule-1 (Vcam-1), basic fibroblast growth factor (bfgf) and Angiopoietin-1 (Angpt-1) (Fig. 4E). No differences in the expression levels of vascular endothelial growth factor-α (Vegf-a) and hypoxia-induced factor-1α (Hif-1α) were detected.
EC-specific deletion of Tsc1 damages mitochondria and endoplasmic reticulum
We further investigated whether Tsc1 deficiency in ECs affected the ultrastructure of subcellular organelles associated with cellular proliferation and apoptosis via transmission electron microscopy (TEM). Significant ultrastructural changes were detected in mutant ECs from E11.5 yolk sacs. It was demonstrated that the mitochondria of the mutants were generally swollen, edema and pale. In contrast, the mitochondria in the ECs of control mice showed a normal morphology, with intact cristae and electron-dense bodies in the mitochondrial matrix (Fig. 5A). The numbers of abnormal mitochondria per cell were increased in sections from the mutant embryos (Fig. 5B). Similarly, abnormally enlarged endoplasmic reticulum (ER) was increased in the mutant ECs compared with fine structure of WT mice (Fig. 5C). We observed a 1.9-fold increase in the number of abnormal ER-positive cells in ECs (Fig. 5D). Additionally, the integrity of tight junctional contacts related to cell permeability between ECs was intact in both WT and KO mice (Fig. 5E). These data indicated that ECs harboring a Tsc1 deficiency showed a defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions.
EC-specific Tsc1 deletion compromises cardiac development
To determine whether Tsc1 KO in ECs affected cardiac development, serial heart sections were stained with H&E from E12.5 to E14.5. It was observed that loss of Tsc1 in ECs led to poorly developed trabeculae and a decreased ventricular wall thickness compared with WT littermates (Fig. 6A). However, the cardiac phenotype of ∼20% of the Tie2-Cre/Tsc1−/− embryos resembled hypertrophic cardiomyopathy (Supplementary Material, Fig. S5), similar to smooth muscle protein-22-mediated deletion of Tsc1 (13), that shows increased thickness of the ventricular wall. TUNEL assays showed that the mutant embryos exhibited significantly more cardiac myocyte apoptosis than the controls (Fig. 6B). To determine whether the observed abnormalities in heart development were caused by Cre-mediated deletion of Tsc1 in cardiomyocytes, we crossed Tie2-Cre mice with mT/mG reporter mice. Fluorescent labeling showed that the endocardium expressed GFP, whereas cardiomyocytes did not. Examination of heart sections under higher magnification indicated that Cre-mediated recombination occurred only in ECs. Red fluorescence was localized to cardiomyocytes (Supplementary Material, Fig. S6). Overall, histological analysis revealed a decreased thickness of the ventricular wall associated with the defect in cardiomyocyte differentiation.
Prenatal mTOR inhibition suppresses the embryonic lethality of Tie2-Cre/Tsc1−/−mice
To investigate whether the embryonic lethality observed in Tie2-cre/Tsc1−/− embryos could be alleviated, embryos were prenatally treated with mTOR inhibitor rapamycin between E12 and E13. Genotyping of the offspring showed that Tsc1 mutant mice survived to birth (Table 2). This finding indicated that rapamycin was able to rescue in utero embryonic lethality. We next examined the gross morphology of embryos isolated at E14.5 to E18.5 obtained from crosses between Tsc1fx/fx and Tie2-Cre/Tsc1+/− mice. The data showed that the level of phospho-S6 detected in the yolk sacs of mutant embryos was markedly decreased by rapamycin treatment (Fig. 7A). It was also found that rapamycin-treated mutant embryos were relatively morphologically normal compared with WT mice (Fig. 7B). Thus, rapamycin was sufficient to partially rescue the embryonic lethality of Tsc1-deficient mice. However, a few heterozygous and WT embryos also died in utero following rapamycin treatment (Table 2). As the WT embryos grew more rapidly, the differences between the mutants and WT embryos gradually became more significant from post-natal day (P) 7. All of the surviving rescued mutant mice showed severe growth retardation compared with their littermates (Fig. 7C). This growth retardation contributed to remarkably poor weight gain in the surviving mutant mice compared with their littermate controls (mutants 3.1 ± 0.15 g, controls 8.4 ± 0.2 g, P = 0.0001) (Fig. 7D). Most of mutant mice died within 24 h after birth, showing systemic cyanosis (Supplementary Material, Fig. S7), while a few of the mice survived to P22 (Fig. 7E). These data indicated that prenatal inhibition of mTOR hyperactivity could rescue embryonic lethality but was insufficient to prevent developmental defects after birth.
|Age||Tie2-Cre/ Tsc1−/−a||Tie2-Cre/ Tsc1+/−||Tsc1fx/fx||Tsc1fx/+||Unclearb|
|Age||Tie2-Cre/ Tsc1−/−a||Tie2-Cre/ Tsc1+/−||Tsc1fx/fx||Tsc1fx/+||Unclearb|
E, embryonic day; P, postnatal day.
aThe values in parentheses indicate numbers of embryos that died.
bGenotyping was precluded by resorption.
Our experiments clearly demonstrated that mTOR signaling in the endothelium is essential for angiogenic and embryonic development. Tsc1 deficiency in ECs caused embryonic lethality. Whether this lethality was attributable to defects in angiogenesis or cardiac dysfunction, or to other causes, remains unclear. A combination effect is likely, though. mTOR activity is required for cell growth, proliferation, protein translation and other cellular metabolic processes (25). Hyperactive mTOR signaling is an underlying mechanism for the abnormal angiogenesis associated with tumorigenesis (26).
During embryonic development, ECs form blood islands, which are the primary capillary plexuses in the yolk sac. This process is referred to as vasculogenesis. Hemangioblasts are the precursors of ECs and crucial for vasculogenesis. The primitive capillary plexuses subsequently undergo angiogenic process of growth and remodeling to shape the mature vascular network (27,28). The primary vasculature is established at E7.5, but vascular morphological defects were apparent after E9.5 in this study. Therefore, we concluded that Tsc1 plays a critical role in embryonic angiogenesis, but not in vasculogenesis. This conclusion was further supported by the finding that hemangioblast differentiation was no difference in the blood and liver between WT and mutant embryos.
As EC-specific deletion of Tsc1 led to progressive systemic severe edema and hemorrhage, both of which are indicators of EC insufficiency, we speculate that the observed embryonic lethal phenotypes resulted from circulatory failure due to defective angiogenesis. Indeed, the mutant embryos failed to form branched vascular networks in the head region, and the carotid arteries were less elaborated in mutant embryos. The perturbation of the circulatory network structure was confirmed by endomucin staining in yolk sacs. Vitelline vessels were narrowed and disorganized. Placental failure was a major contributing factor to the embryonic lethal phenotype. Three placental layers (the labyrinth, spongiotrophoblast and decidua) were readily discernible. The labyrinth layer is the main site of contact between the fetal and maternal circulation, consisting of a highly branched fetal vascular network (29). The EC-specific deletion of Tsc1 affected placental vascularization as well, causing a significant increase in the thickness of the labyrinthine layer. This type of alteration can compromise embryonic growth and viability because the highly vascular labyrinth is the site of maternal-fetal oxygen and nutrient exchange (30). The enlarged labyrinthine layer phenotype may result from hyperactivity of mTOR in Tsc1-deleted ECs. The hypoplastic Tsc1 mutant embryos may have been compensated by the enlarged placental labyrinth, which would have facilitated the development of normal fetal size. Angiogenesis insufficiency may have been the major cause of the mid-gestation lethal phenotype of Tsc1 KO mice. Collectively, our results showed that the mutant embryos have exhibited defective angiogenesis in the yolk sac, embryo body and placenta. The edema and hemorrhaging observed in the head and dorsal regions of embryos harboring an EC-specific deletion of Tsc1 may have been related to defects in EC adhesion properties. Although the cell junctions of the mutant ECs appeared normal under electron microscopy, the mRNA expression of Vcam-1, a marker of cell adhesion, was significantly decreased in yolk sacs. Vcam-1 is expressed at the cell surface, where it mediates interactions with extracellular matrix components and regulates vascular permeability (31). In addition, the mutant ECs showed higher levels of apoptosis, but lower levels of proliferation, which may be explained by the markedly reduced expression of the bFgf anti-apoptosis factor in the mutant embryos' yolk sacs (32). Taken together, these data indicated the angiogenesis defect associated with Tsc1 deletion is mediated by altered cell proliferation and differentiation of ECs.
To gain further insight into the potential mechanisms underlying the defective vessel formation observed in Tsc1−/−embryos, we analyzed the endothelial ultrastructure. The ER is a specialized organelle that plays crucial roles in cell homeostasis and survival through its functions in protein folding, lipid biosynthesis and calcium and redox homeostatic processes (33). The mTOR signaling factor mediates cellular protein synthesis through phosphorylation of S6 K and 4EBP-1 and subsequent activation of cellular metabolism. We hypothesized that the enlarged ER might be related to up-regulated mTOR activity. Severe mitochondrial dysfunction was associated with swollen mitochondria, abnormal cristae and matrix structure, contributing to insufficient energy production and apoptosis. Thus, defective ECs were responsible for the abnormal angiogenesis observed during embryogenesis.
Future studies should address the mechanisms associated with a hyperactive mTOR signaling pathway in angiogenesis. Angiogenesis is a complex process involving finely balanced interactions between signaling factors, such as Vegf-a, Tgf-β1, Pdgf-b, Angpt-1, Vcam-1 and Hif-1α (34). Hif-1α and Vegf are also known to control EC proliferation and migration, both of which are fundamental for proper angiogenesis (35). It has been reported that activation of phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling promotes both mTOR-dependent and mTOR-independent HIF-1α/VEGF transcription during tumor angiogenesis (36,37). Homozygous KO of the Hif-1α or Vegf gene results in embryonic lethality in early stages of gestation due to impaired blood vessel development (38,39). However, in the EC-specific Tsc1 KO mice, there was no obvious effect on the transcription of either Hif-1α or Vegf-a. Angpt/Tek signaling is also involved in the establishment of vessel structures during embryonic development (40). Mouse KO models of Angpt-1 and Tek both show prominent endocardial and myocardial defects (41). The interaction of Angpt1 and Tek in ECs is essential for vasculature maturation and normal remodeling processes (42). However, the EC-specific Tsc1 KO mice showed reduced levels of Angpt-1, but increased levels of Tek. It has been suggested that disrupted Angpt/Tek signaling is associated with defective angiogenesis during the late gestational stage. Under normal physiological conditions, Pdgf-b promotes the proliferation and migration of pericyte precursor cells (43). Mice deficient in Pdgf-b or its receptor (Pdgfr-b) die in utero due to microvascular defects, which include vessel dilatation and microaneurysms (44). Similarly, activation of the EGFR signaling pathway mediates cell proliferation and angiogenesis, and overactivation of this pathway in various cancers has been shown to promote metastasis and inhibit apoptosis (45). Other factors that are necessary for the proper formation of blood vessels are α-SMA (46) and TGF-β1, which is a potent inhibitor of vascular smooth muscle cell proliferation (47). As our EC-specific Tsc1 KO embryos overexpressed Pdgf-b, Tgf-β1 and Egfr, it is possible that the observed vascular defects are related to perturbed activation of pericytes, decreased stability of blood vessel walls, an impaired EC proliferation ability and inhibited neovasculization. The abnormal expression of angiogenic factors caused by up-regulated mTOR activity in the mutant ECs may manifest as an interruption of angiogenesis homeostasis.
During embryonic heart development, ECs and cardiomyocytes arise from the same cardiac mesodermal precursors (48). The endocardial monolayer adjacent to the myocardial trabeculation releases growth factors that regulate myocyte development and proper cardiac function (49). Our results indicated that the hearts of the EC-specific Tsc1 KO embryos had a disorganized architecture and a thinner ventricular wall. Importantly, the Tsc1 mutant hearts exhibited a dramatic increase in the number of apoptotic cardiomyocytes. We suggested that inactivation of Tsc1 in ECs could affect heart development, resulting in a disturbed balance between proliferation and apoptosis during the process of cardiomyocyte differentiation. Myocardial ECs are found in the endocardium as the main component of cardiac capillaries, which supply nutrients and oxygen for cardiomyocyte growth (50). While it remains unknown whether vascular or cardiac defects were the primary cause of the observed embryonic lethal phenotype, we believe that cardiac failure could be secondary to the EC abnormalities.
Furthermore, to confirm that the lethality observed in homozygous Tsc1 KO embryos was due to the hyperactivation of mTOR, we attempted to rescue the lethal phenotypes of mutant embryos via rapamycin treatment. Prenatal treatment with rapalogues in TSC mutant mouse models has previously been described (12,51,52). Here, a single dose of rapamycin significantly improved embryonic viability. Nevertheless, most of the mutant pups died within 24 h after birth, showing systemic cyanosis. Only a few of the mutant pups survived up to 22 days, showing poor weight gain and significant developmental retardation. Survival of the mutant mice might be improved by rapamycin treatment after birth.
In conclusion, reduction of Tsc1 gene expression in the endothelium leads to embryonic death due to overactivation of mTOR, which can be rescued by prenatal rapamycin treatment. Hence, Tsc1 plays a crucial role in cardiovascular integrity and embryonic development through mTOR. Aberrant mTOR signaling may contribute to the vascular pathogenesis observed in TSC.
MATERIALS AND METHODS
All animal protocols adhered to the Animal Care and Use Committee Guidelines of Peking Union Medical College. Tie2-Cre (C57BL/6J) mice and Tsc1-floxed mice (Tsc1fx/fx) (129S4/SvJae) were obtained from Jackson Laboratory (Bar Harbor, ME, USA) (53,54). The Tie2-Cre mice were first crossed with Tsc1fx/fx mice to obtain Tie2-Cre/Tsc1+/− mice, which were then back-crossed with Tsc1fx/fx mice to obtain conditional KO Tie2-Cre/Tsc1−/− mice. The Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFR)Luo/J mice (mT/mG, C57BL/6) were procured from Jackson Laboratory. Tie2-Cre mice were crossed with mT/mG to get mT/mG+/−; Tie2-Cre+/− mice for analysis.
Isolation of mouse embryos
Timed matings were performed using the presence of a vaginal plug to assess fertilization between Tie2-Cre/Tsc1+/− male and Tsc1fx/fx female mice. The morning vaginal plug was designated E0.5. The impregnated females were sacrificed at several time points after conception. The embryos were isolated by removing the muscular wall of the uterus and yolk sacs in ice-cold PBS under a stereomicroscope (Nikon, Tokyo, Japan). The embryos were fixed overnight in 4% paraformaldehyde (PFA) at 4 or −80°C.
Tail or yolk sac samples were prepared as previously described (7). The published primers used in these assays allow detection of the WT Tsc1 allele (295 bp amplicon), the floxed allele (235 bp amplicon) and the mutant allele (370 bp amplicon) (7,55). The floxed allele contains loxP sites flanking the sequence, to be excised upon Cre recombination. The mutant allele lacks the sequence flanked by the loxP sites and produces a non-functional hamartin protein.
Yolk sacs and embryos were homogenized and extracted with Tissue Protein Extract Reagent (Thermo-Fisher Scientific, Waltham, MA, USA) and a 1% protease inhibitor cocktail (Thermo-Fisher Scientific). The protein concentration was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific). The samples were next resolved in 10% Tris–HCl Ready Gels (Invitrogen, Temecula, CA, USA) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), then incubated in blocking buffer consisting of 5% milk in tris-buffered saline and tween 20 (20 mm Tris, 500 mm NaCl, and 0.05% Tween-20, pH 7.5) for 1 h at room temperature. The PVDF membranes were incubated overnight with the following primary antibodies: anti-TSC1 (rabbit polyclonal 1:500 dilution, Cell Signaling Technology, Beverly, MA, USA), anti-Akt and phospho-Akt (rabbit polyclonal 1:1000 dilution, Cell Signaling Technology), anti-S6 and phospho-S6 (rabbit polyclonal 1:1000 dilution, Cell Signaling Technology), 4EBP-1 and phospho-4EBP-1 (rabbit polyclonal 1:1000 dilution, Cell Signaling Technology) and β-actin (mouse monoclonal 1:2000, Santa Cruz Biotechnology), followed by incubation with the appropriate secondary antibody. Immunoreactive bands were visualized using an enhanced chemiluminescence substrate (Pierce, Thermo-Fisher).
Histology and immunohistochemistry
Embryos were placed in 4% PFA, embedded in paraffin, and sectioned at a thickness of 3 μm. Serial sections were either stained with hematoxylin and eosin (H&E) for morphological evaluation or used for immunohistochemistry analysis. The tissue sections were deparaffinized and rehydrated in xylene, heated in 10 mm citrate buffer, pre-treated with 3% H2O2 for 10 min and blocked with 5% normal goat serum for 2 h. The sections were then labeled with the following primary antibodies: α-SMA (rabbit polyclonal 1:500, Abcam, Cambridge, UK), PECAM (CD31) (goat polyclonal 1:1000, R&D Systems, Minneapolis, MN, USA) and ki67 (rabbit monoclonal 1:300, Thermo, Waltham, MA, USA) at 4°C overnight. After rinsing, the sections were incubated with the appropriate secondary antibodies for 1 h at room temperature and finally detected using diaminobenzidine (DAB) as a substrate (Vector, Burlingame, CA, USA). In addition, the morphology and staining characteristics of blood cells were investigated via the standard blood smear procedure (56). The ratio of positive cells in images was (n = 5) calculated by Carl Zeiss AxioVision Re1.4.6.
Sections were dewaxed and rehydrated according to standard processing procedures. Tissue sections were blocked and incubated with anti-phospho-S6, or endomucin antibody (rat monoclonal antibody1:300, eBioscience, San Diego, CA, USA) at 4°C overnight. Cy3-conjuaged goat anti-rat and Alexa Fluor-488-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used for visualization of the resultant signals. Nuclei were labeled by DAPI staining. Yolk sacs were fixed in 4% PFA in phosphate buffered saline (PBS) overnight at 4°C. The tissues were frozen and mounted in Tissue-Tek OCT compound (Sakura, Tokyo, Japan) and sectioned at a 10 μm thickness with a cryomicrotome. The slides were then rinsed and counterstained with DAPI. Finally, the sections were observed using a confocal microscope (Olympus FV1000, Tokyo, Japan).
Embryos and yolk sacs were fixed in 4% PFA in 0.01 mm PBS overnight at 4°C, then washed twice with PBS for 10–20 min at room temperature, dehydrated via soaking in a methanol series, and bleached via incubation in 5% H2O2 for 4–5 h at room temperature. After rehydration in a reverse methanol series, the bleached embryos were blocked via incubation in 3% dry milk reconstituted in 0.1% PBS plus Triton X-100 (PBSMT) for either 1 h at room temperature or overnight at 4°C. Immunostaining to detect platelet endothelial adhesion molecule (anti-CD31) (goat polyclonal 1:300 dilution, R&D Systems, Minneapolis, MN, USA) and endomucin was carried out in 1 ml of PBSMT for 24 h at 4°C. The embryos were then washed in PBSMT five times (1 h each) with rocking, after which they were incubated with horseradish peroxidase rat anti-goat (1:300) and Cy3-conjugated AffiniPure goat anti-rat IgG (1:500) secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA) at 4°C overnight with rocking. The embryos were then rinsed again in PBSMT at 4°C five times (1 h each) and washed with PBTX for 20 min. Peroxidase staining was performed with DAB at room temperature for 15 min. Finally, the embryos were post-fixed in 2% PFA with 0.1% glutaraldehyde in PBS and the stained yolk sacs were photographed using a Carl Zeiss Meditec microscope (Zeiss, Oberkochen, Germany).
Apoptotic cells were assessed via TUNEL staining using the In situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany). Following deparaffinization and rehydration, the sections were treated with proteinase K for 10 min and then incubated with the TUNEL reaction mixture for 60 min at 37°C in the dark. The sections were next rinsed three times in PBS for 5 min each, followed by the addition of converter-POD to the samples and incubation for 30 min at 37°C. The sections were subsequently stained with DAB substrate and analyzed using a Carl Zeiss Meditec microscope. Heart sections were examined using the Dead End Fluorescence TUNEL system (Promega, Madison, WI, USA) following the manufacturer's instructions. Apoptotic cells were observed and photographed with a confocal microscope (Olympus FV1000). Nuclei were labeled by DAPI staining.
Quantitative real-time PCR analysis
RNA was extracted from complete yolk sacs using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Aliquots of 1 μg RNA were used for reverse transcription with an oligo-dT primer (Thermo-Fisher, Waltham, MA, USA). qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and gene-specific primers (sequences and thermal cycling conditions available upon request) in the StepOne and StepOnePlus Real-Time PCR Systems (Applied Biosystems). The following PCR conditions were used for the Applied Biosystems assays: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Quantification was performed using the ΔΔCT method. The housekeeping gene Gapdh was used for internal normalization. The following primers were used: mouse Hif-1α (accession no. NM_010431), Vcam-1 (accession no. NM_011693.3), Egfr (accession no. NM_001083119.2), bFgf (accession no. NM_008006.2), Tgf-β1 (forward, 5-GCTGCGCTTGCAGAGATTAAA; reverse, 5-TTGCTGTACTGTGTGTCCAG), Pdgf-b (forward, 5-ACTCCATCCGCTCCTTTGAT; reverse, 5-GTCTTGCACTCGGCGATTA), Tek (forward, 5-TGGAGT- CAGCTTGCTCCTTT; reverse, 5-ACCTCCAGTGGATCTTGGTG), Angpt-1 (forward, 5-GGGGGAGGTTGGACAGTAA; reverse, 5-CATCAGCTCAATCCTCAGC), Vegf-a (forward, 5-CAGGCTGCTGTAACGATGAA; reverse, 5-CTATGTGCTG- GCTTTGGTGA), Angpt1 (forward, 5-GGGGGAGGTTGGACAGTAA; reverse, 5-CATCAGCTCAATCCTCAGC) (57), Gapdh (accession no. NM_008084.2).
Yolk sacs from E10.5 embryos were fixed with 2.5% glutaraldehyde in 100 mm phosphate buffer overnight at 4°C, then post-fixed in 1% osmium tetroxide in 100 mm phosphate buffer for 2 h at 4°C, dehydrated through a graded series of ethanol, transferred to propylene oxide and embedded in eponsy resin. Ultra-thin sections were cut and visualized using a Tecnai Spirit transmission electron microscope (FEI, Hillsboro, Oregon, USA). Images were randomly selected at original magnifications of 11500-26500X. The number of mitochondria per cell was counted in at least 200 cells in each group. The proportion endoplasmic reticula showing dilation was estimated as the number of endoplasmic reticula with a width at least twice the width of a normal cell member, divided by the total number of endoplasmic reticula counted.
Rapamycin (North China Pharmaceutical Co., Ltd, Shijiazhuang, China) was dissolved at a concentration of 20 mg/ml in ethanol and stored at −20°C. A working solution for subcutaneous injection was prepared in 0.25% Tween-80 and 0.25% polyethylene glycol 400 diluted in PBS (12). Pregnant dams were administered a single dose of 1 mg/kg between E12 and E13.
Data are expressed as the means ± SEM. The significance of inter-group differences was assessed using a two-tailed Student's t-test or ANOVA. A P-value of <0.05 was considered statistically significant.
This work was supported by the 973 National Basic Research Program of China (2009CB522106, 2009CB522203).
The authors thank Prof. Weiming Tong (Department of Pathology, Institute of Basic Medical Sciences and School of Basic Medicine, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China) and Prof. Zhong-Zhou Yang (Model Animal Research Center of Nanjing University, Nanjing, China) for advice of mouse study. We thank Dr Bee Hong Lo (Children's Hospital Westmead, Sydney, Australia) for her assistance in editing the abstract of this paper.
Conflict of Interest statement. None declared.

domain: medical


text:
Lace Ripple Afghan
The Lace Ripple Afghan is perfect for a cool summer or spring night. The open crochet lacework allows the material to breath while still keeping you covered. This is an easy crochet afghan pattern.
Crochet HookG/6 or 4 mm hook
Yarn Weight(3) Light/DK (21-24 stitches to 4 inches)
You can also find this crochet design in Christmas Gifts for Her: Crochet Gifts for Mom.
Read NextMelon Ripple Afghan

domain: artistic


The content from The Peacemaker’s Podcast should not be taken as medical advice. The content here is for informational purposes only, and because each person is so unique, please consult your healthcare professional for any medical or mental health questions. If you are currently facing a mental health or medical emergency, call 911 immediately.
Welcome back for Episode 6, this is Lindsey Haeger the host of the Peacemaker’s Podcast. Today’s episode is part 2 of my interview with Joel Hubbard. If you haven’t yet listened to part 1 in episode 5, press stop and go back to catch that one first. Joel is the co-host of The Art of Growth podcast and over a month ago now I spoke with him about a Type Nine Panels that he hosted from Season 2 of the Art of Growth. I received so much value from that episode and wanted to go deeper into it with him. I’m excited to share the second half of our talk with you!
In Part 2 we discuss:
For Enneagram Coaching, reach out to Joel through email at firstname.lastname@example.org. To learn more about The Art of Growth podcast and to listen to the Episode we discussed, click here Season 2, Episode 2 from The Art of Growth.


text:
Normally when I see a thumbnail with bunch of kids playing instruments I don’t really expect much. But there was this one video that got my attention, and clearly I wasn’t dissapointed at all when I saw three young guys thrashing and smashing in front of a garage.
Those three young metalheads were Henry, Eli and Abe and their metal band is called Hammerhedd. It’s really amusing to hear insane riffs and technically advanced compositions, and then realizing the band was not couple of „dads living in the past“, but three insanely talented adolescents. They already released an EP called „Essencce Of Iron“ and an album called „Grand Currents“. Here’s what Hammerhedd told us about their musical path:
How old were you when you started playing?
We started in 2012, so we were 12,9, and 6.
How did you get into metal music?
We just sort of found our way to metal after our dad showed us some of his favorite bands (Led Zeppelin, Rush, Boston, etc,).
Which video made your YouTube channel and career explode?
Our most viewed video is the one of us playing Metallica covers on the street in 2016 – that one gave us a lot of exposure.
How did your parents react to your music?
Our parents are very supportive of the band! They have been massively helpful from the day we started the band up until now.
How many shows are behind you and which one was your favorite?
I would guess we’ve played around 50 shows. Our favorite one so far was at the Riot Room opening up for the mighty Power Trip. That was a crazy show!
You released a new album called Grand Currents. Tell us more about it.
We are very proud of Grand Currents! We’ve been working on it for a long time, and we think it’s a huge step up from our first EP.
You seem to have an incredible groove. What’s the secret?
Thank you! We just jam a ton together, and we really focus on groove and tightness in our live sound. We think those are two of the most important parts of our sound!
What do you expect from your music career in the future?
We hope to keep putting out albums and playing live shows. All we hope for is to make a living out of Hammerhedd one day.
If you could open for a band, which one would it be?
That’s a tough one, there’s too many bands we’d love to open for! A few of the top ones would be Mastodon, Tool, Gojira, Meshuggah, Primus, and Metallica. Maybe someday!


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AS (2014) CR 17
2014 ORDINARY SESSION
Thursday, 10 April 2014 at 3.30 p.m.
The protection of minors against excesses of sects
The following texts were submitted for inclusion in the official report by members who were present in the Chamber but were prevented by lack of time from delivering them.
Mr ABBASOV (Azerbaijan) – The problem of youths being involved in religious sects is fairly relevant for modern Europe. I consider quite reasonable the position of the Committee on Legal Affairs and Human Rights, which states that all significant measures on the indicated issue should be taken on a national level.
European States with an established legal and democratic regime are free from any total ideology. In turn, the values of the constitutional State are acquired in line with the improvement of its citizens’ legal consciousness. High legal consciousness is obtained over the years and it is for that reason that the youth, and in particular minors, are more exposed to the ideological excesses of sects.
On the other hand, due to the economic crisis in Europe, young people’s interest in religion has sharply risen. A crisis provokes in most people a sense of uncertainty and vulnerability. Under these circumstances, problems of life and death become more of an issue. Therefore minors, beyond the family, hardly conform in society and fall under the influence of sects. Undoubtedly, in a state of psychological dependence or physical submission, these excesses can lead to the violation of the basic rights of minors with respect to their right to life, physical inviolability, inviolability of their family and social relationships, as well as their education.
The report notes the positive legislative experience of Belgium, France, Luxembourg and other countries regarding the indicated issue. Nevertheless, we regretfully confirm that, at the moment, there is a lack of statistics on the scope of the involvement of minors in sects.
Azerbaijan is a predominantly Muslim country with a traditional of tolerance. Sects in Islam on the one hand destroy traditional religion, and on the other hand call for radical deeds sometimes beyond the borders of certain States. That is why criminal legislation should foresee the accountability of those persons dragging minors to sectarian activities and consequently inciting them to commit crimes.
Over 100 young Azerbaijani citizens have been murdered in the Syrian conflict. Nonetheless, up until recently, national legislation envisaged no accountability for the participation in military conflicts in contiguous territories. Last month, the Criminal Code of Azerbaijan toughened the punishment for recruiting, educating, financing or any other kind of material securing or use in armed military conflicts or military operations beyond the borders of Azerbaijan. Along with measures on accountability, prophylactic measures are also required. Meanwhile, the issue of the co-ordination of our efforts within the whole of Europe is quite a significant one.

domain: law


text:
Scottish Equitable is calling on the Government to improve contracting-out rebates to make it easier for IFAs to advise their clients.
It argues that since the introduction of the state second pension in April, there is now no clear financial incentive to encourage individuals to contract out of S2P.
The call for an increase follows a poll of 1,300 IFAs carried out by Scottish Equitable at a series of 15 roadshows around the UK in May. When questioned, 90 per cent said it was now less clear how to advise clients on contracting out than it was 12 months ago.
Scottish Equitable will be holding its next series of pension roadshows across the UK in November.
Pensions development director Stewart Ritchie says: “This practical evidence of the problems faced by IFAs echoes the view that we have held for some time. Rather than being a clear-cut financial decision, contracting out has become a philosophical decision of whether the state can be relied upon to honour its promises in the future.
“If the Government is serious about contracting out, then it needs to do something urgently to tackle the contracting-out incentive. If contracting out effectively disappears, then it goes against the Government's stated policy of reducing the burden on the state by reversing the 60:40 balance of public/private pension provision.”
The Bureaux managing director Peter Quinton says: “The Government will not do anything until the outcome of the Inland Revenue review is made known. The Government has put out so many consultations but Scottish Equitable are right to raise this as it is a view that needs to be put forward when they consider all the reviews.”

domain: politics


text:
SILVERSTONE ACTIVATES BREAK CLAUSE WITH FORMULA 1
The British Formula 1 Grand Prix could be on the lookout for a new home beyond 2019 after Silverstone activated a break clause in its contract with series management.
Sighting massive losses hosting the last two British Grand Prix’s, Silverstone track owners British Racing Drivers’ Club (BRDC) have formally triggered the break clause in its current contract with Formula 1.
The two parties will now have to come to a new agreement for the British Grand Prix to remain at Silverstone beyond 2019.
“This decision has been taken because it is not financially viable for us to deliver the British Grand Prix under the terms of our current contract. We sustained losses of £2.8m in 2015 and £4.8m in 2016, and we expect to lose a similar amount this year. We have reached the tipping point where we can no longer let our passion for the sport rule our heads. It would not only risk the very future of Silverstone and the BRDC, but also the British motorsport community that depends on us.” said John Grant, Chairman of the British Racing Drivers’ Club.
“However, I want to be clear that although we have now activated the break clause, we are fully supportive of the changes the Liberty team are making to improve the F1 experience. Our hope is that an agreement can still be reached, so that we can ensure a sustainable and financially viable future for the British Grand Prix at Silverstone for many years to come.” he continued.
The current deal started in 2009 with the hosting fee of £11.5 million and was scheduled to finish in 2026. However, by that time the hosting fees would have reached £25 million.
Silverstone has hosted the British Grand Prix on and off since 1948, and continuously since 1987, making it one of the staples of the Formula 1 schedules.
Make sure you pick up the next issue of Auto Action (on sale Thursday) for more of the latest Formula 1 news. In the meantime follow us on social media Facebook, Twitter and Instagram for all the latest updates.
Date posted: July 12, 2017


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In veterinary medicine, bloodwork is essential for diagnosis and overall care. Our veterinarians can diagnose various conditions such as anemia, heart disease, parasites, allergies, infections, and more with blood tests. At our hospital, we have our own diagnostic laboratory, which means we can provide prompt results.
When is bloodwork necessary for my pet?
Our team recommends blood tests for the following instances:
- Baseline health information: A blood test will show your pet’s normal blood levels if your pet is healthy. This is used to spot differences in the future.
- Preanesthetic testing: Bloodwork will show underlying problems that cause anesthesia complications.
- Veterinary exam: Pets need wellness exams especially when they are a senior or puppy/kitten. During the exam, we can detect potential health problems or monitor known issues.
- Your pet is showing signs of illness: Fatigue, diarrhea, vomiting, and skin problems are signs your pet is unwell. A blood test can narrow down the cause of their symptoms and diagnose them.
How is blood taken from my pet?
Drawing blood from pets works the same as taking human blood. Our team is specially trained to make the process comfortable for your pet. A needle is injected in a vein on their leg or neck.
How can I prepare my pet for bloodwork?
To ensure our team gets a quality blood sample, we ask that your loyal companion fasts at least 8 hours before their appointment. If your pet eats before their appointment, there’s a possibility the sample could have fat droplets that can interfere with the results. If your pet is on medication, please check with our receptionist before discontinuing the medication. To schedule a blood test for your pet, give us a call at 705-322-2661.
What other diagnostic tests are performed at your hospital?
Our in-house lab enables us to perform various diagnostic tests with a quick turnaround time. For some complicated cases, we refer samples to external labs. We perform the following tests in-house:
- Complete blood count and differential (CBC & Diff)
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- In house Urinalysis (Sedivue)
- EKG (Cardiopet)
- Petrays radiology consultations

domain: biology


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If I see a momma duck and her babies trying to cross a busy road, I panic. The animal videos sent in by the millions on the Internet always leave me crying. Once, I witnessed the needless running over of a female mallard in a Kmart parking lot that left me catatonic. I wasn’t able to speak of the incident without bursting into tears for weeks, and to this day, it disturbs me to remember. Seeing a mashed squirrel on the road, or worse, someone’s loved pet, breaks my heart.
I avoid movies that I know will upset me; I’ve never even made it past Bambi’s mother dying. I can’t watch the nature channels for fear of seeing one animal eating another. Yes, I know, wild animals must eat to survive, but I can’t watch it. I worry over the sandpiper that roams in my cul-de-sac, his mate nesting somewhere close as he keeps watch. In the winter, when the quail can’t find food through the snow, I fret. When a bird hits my window, I keep watch for hours until it can fly away.
So beware, I brake for even the smallest of creatures crossing the road. I hope you do, too.


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Trusted Hemp Processors & Extraction Companies
Possibly the largest bottleneck within the hemp industry is finding a hemp processor to extract your high CBD biomass to some form of oil. In 2019, most farmers set out to grow and simply sell their raw materials to these processors. The problem was that hemp production quadrupled compared to the year prior, and not enough hemp processing facilities were fully up and running to handle the glut of raw material.
The end result was many farmers sitting on an over-supply of biomass with no processors buying it. Even worse, having to wait in line for month to get their biomass extracted via tolling or revenue splits.
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domain: agronomy


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A Virtuous WomaneBook - 1997
"Two unforgettable characters, Jack Ernest Stokes, known as Blinking Jack, and his wife, Ruby Pitt Woodrow Stokes, tell the story of their years together. Jack was forty and Ruby only twenty when they were married. For twenty-five years they lived together, man and wife, until Ruby died of lung cancer. A LITERARY GUILD AND DOUBLEDAY BOOK CLUB selection."-- Provided by publisher.
Publisher: Chapel Hill, North Carolina : Algonquin Books of Chapel Hill, 1997.
Branch Call Number: EBOOK
Characteristics: 1 online resource (168 pages) data file
From the critics

domain: library


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NHL no longer denying relocation a possibility for the Coyotes?
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02-01-2013, 12:54 AM
Puck is Life
Join Date: Jul 2012
I'm really surprised Hartford still isn't making any of these relocation lists. I know the NHL has stated it needs a new arena (or major, major renovations), but it'll seat 14,000+ for hockey, and is in a very rich state, with a viable hockey market. I can definitely see it working...
With the list that has (commonly) been posted, we run into the same problems...
Seattle and QC Will get teams, no doubt...
Portland, Houston, Milwaukee are just non-viable markets
Hamilton is a dump with a bad economy
Markham has been put on hold (though the project is still on life support)... I don't expect it to be active much longer
There really isn't any reason the NHL couldn't acquire an owner to foot at least 75% of the bill for renovations and an NHL team for XL Center
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domain: sports


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Merger arbitrage is an investment strategy that capitalizes on differences in price between the consumer and the seller of your company’s inventory. This type of approach will involve taking a longer position over the seller’s stock or a short position on the buyer’s stock.
If a firm announces the intent to acquire a majority risk in another firm, the value of that company’s stocks and shares will increase. Additionally it is possible to benefit from the rise in you’re able to send share price by investing in the company before it is got. However , this is certainly a high-risk way to get. The company’s share may nonetheless fall by using the merger.
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In order to successfully implement a merger arbitrage strategy, the investor need to carefully consider the risks included. One of the biggest dangers is the probability of a deal being cancelled. There are many things to consider when examining the probability of any deal being canceled. These include the voting power of shareholders, the amount of money offered in the acquirer and the risks of various other bids to get the company.

domain: economics


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Our goal is to make you think by bringing you the best ideas from the brightest minds. More specifically, we take the key insights from new and noteworthy non-fiction and distill them into brief, sharply-written packs that you can read or listen to on-the-go. With Thinkr, you can cut right to the core of a book in a matter of minutes.
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Sometimes, Mother Nature's fury leaves you with only one option: RUN.
Clyde Aikau might be retiring, but he can still school some of the world's greatest surfers.
The competition ran on Thursday, which was the first time in seven years that waves were big enough to hold it.
"The Eddie," which requires perfect 40-foot waves, hasn't run since 2009.
At the MaiTai Maui opening ceremony the mayor of Maui, Alan Arakawa revealed, "I have the easiest job in the world," with a smile, "look around you - it's the most beautiful place on earth." And with that I was inspired to explore his beautiful island.
Orange County, California -- Massive paddle-out for the passing of pastor Chuck Smith. (Oct. 21, 2013) Surfers around the
Yesterday, a ceremony was held at Waimea Bay to celebrate the beginning of the waiting period for the contest. Competitors
Burle made it down the wave, but claims that he broke the world record for largest wave ever surfed have met criticism from
ESPN couldn’t have premiered a more restorative "30 for 30" documentary if they had planned it. Where Director Sam George


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Getting your website built to look the way you want is important, but what good does it do if no one can find your site from Google? When it comes to search engine optimization (SEO), Drupal is one of the best systems to work with. However, it is best to be aware of the SEO impact of design and development choices from the beginning of a project in order to save time and money in the long run.
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A good example of a design mistake that can negatively impact your SEO is the use of a flash or image based splash page. This hurts your ranking with Google and most search engines because the home page is the most important page of your site used in their ranking algorithms. In order to maximize your site’s visibility you have to make sure that text containing your target key words is used in your home page. Even if your splash page is intended to show only on the entry of the site it is counted as the home page by search engines.
I offer search engine optimization for sites that I build for new clients as well as for existing sites. This involves the optimization of images, urls, menu systems, meta tags and content layout to achieve the most effect for your targeted key words. Pricing is calculated on a per site basis, as multiple variables influence the total work involved.
I have also developed a system for building high quality inbound links for your site which can be implemented very quickly and which greatly increases your overall ranking with Google and other search engines. Click here for more information on inbound link building for Search Engine Optimization.

domain: design


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(“The TEN” is not a top ten but ten items worth being included in “The TEN”)
1. Scooter Gennett hits four home runs and has 10 RBIs to lead the Cincinnati Reds to a 13-1 win over St. Louis.
2. The Walnut Mustangs beat Righetti 13-2 last Friday night to win the CIF Division 3 Baseball Championship.
3. Thad Matta is out as the head coach of Ohio State basketball after 13 years.
4. “SG new coach is Kevin Encinas” – MVS Commenter “Anonymous.”
5. “So now I’m covering the game that I was the MVP at six years ago.” – Former Schurr quarterback Aaron “Airshow” Cantu at the 605 All-Star game last Saturday night.
6. The Seattle Seahawks sign quarterback Austin Davis, passing on former San Francisco 49ers starter Colin Kaepernick.
7. Matt “Money” Smith is named the Los Angeles Chargers new radio play-by play announcer. He’ll be joined in the booth by former Chargers center Nick Hardwick on KFI AM 640.
8. “Tim, there are three things that can happen when you throw the ball, and two of them are bad.” -San Dimas assistant Marc Shettleroe to Tim Peterson at the “One Blood” Tournament at Mt. View on Saturday.
9. Happy belated birthday to Montebello OC Pete Gonzalez Jr. who turned 29 yesterday.
10. Former Philadelphia 76ers guard/Hall of Famer Allen Iverson turns 42 today.


Biography / Info
California composer Dhruva Aliman creates a musical tale that takes you on a journey to strange places in a fantastic universe teeming with adventure, myth, reflection, and mystery. Part fantasy world of soundscapes that evoke visions in the mind’s eye, and part electro-ambi-pop to dance to. Each song keeps you guessing what’s on the next page of Dhruva’s musical story book. Exciting music here for action adventure, crime in progress, murder, intrigue, and much more. Dhruva nails it with a bang for dramatic filmscores, movie trailers or your grand finale film credits.


CONTENT WARNING: The following article discusses physical abuse.
The image says “Michael Jackson + 2Pac = Chris Brown” which is a bloody cold take to say the very least.
“Everyone wants to wait til people die to give them the credit they deserve,” Bieber wrote.
“I’m calling it now when CB passes away after a long full life, you will miss what you had in front of you the whole time…trust me watch you will see.
“The people who have over looked this mans talent because of a mistake he made.. you need to reevaluate!”
The post is likely a response to the recently revived discussion around Brown’s history of both convicted and alleged abuse, thanks to Chvrches.
Last week, Chvrches called out producer Marshmello for working with Brown on a new track. Brown responded, saying that Chvrches were “the type of people he wished would walk in front of a speeding bus.”
Later last week, Chvrches frontwoman Lauren Mayberry revealed that the band have had to beef up security and police presence at their shows due to rape and death threats received from Brown’s fans.
View Bieber’s post below.
If you need assistance, 1800 RESPECT – the National Sexual Assault, Domestic
and Family Violence Counselling Service — can be reached on 1800 737 732.
For help or information regarding mental health, contact Lifeline on 13 11 14 or
Beyond Blue on 1300 224 636.
View this post on Instagram
everyone wants to wait til people die To give them the credit they deserve. I’m calling it now when CB passes away after a long full life, you will miss what you had in front of you the whole time … trust me watch you will see. the people who have over looked this mans talent because of a mistake he made.. you need to reevaluate! Love you @chrisbrownofficial


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December 26, 2006
Another crazy person of the year
Many have lampooned Time Magazine's seemingly crazy choice for person of the year. Today, in this commentary, Washington Post columnist Richard Cohen suggests a different person of the year:
My person of the year is Gregory Thompson. I choose him to call attention to the madness of the death penalty.... He is a cold-blooded killer, plain and simple. He is also out of his mind.
Thompson, 45, is delusional. He is also paranoid, schizophrenic and depressed. For these ailments, he receives daily doses of drugs and, twice a month, anti-psychotic injections. The state of Tennessee wants very much to put him to death for the horrendous 1985 murder of Brenda Blanton Lane, of which there is no doubt about his guilt. There is grave doubt, though, about the constitutionality, not to mention the decency, of executing an insane man. Thus the 12 pills Thompson takes every day. The idea, according to a recent account of his case in the Wall Street Journal, is to make him sane enough to be put to death....
[Thompson] was probably insane when he murdered Brenda Blanton Lane but will be deemed sane if and when he's executed. He's my person of the year -- a fleetingly sane man in the maw of a thoroughly insane system.
A personal sentencing year in review
In a future post, I will review the major sentencing highlights of 2006. Here I start my reflective blogging by reviewing my sentencing scholarship and related activities for the year (roughly in chronological order with links to posts with more information).
- Conceptualizing Booker
- Tweaking Booker: Advisory Guidelines in the Federal System
- Finding Bickel Gold in a Hill of Beans
- Making Sentencing Sensible (with Stephanos Bibas)
- Reasoning Through Reasonableness
- Perspectives on Booker's Potential
- Now What? The Post-Booker Challenge for Congress and the Sentencing Commission
- The Real (Sentencing) World: State Sentencing in the Post-Blakely Era (with Steve Chanenson)
Major Amicus Efforts
- Crack sentencing in many circuits
- Reasonableness review in the Ninth Circuit
- Reasonableness review of veteran's variance
- Claiborne and Rita: Reasonableness review in the Supreme Court
- FSR Issue 18.2: Defense Perspectives on the Post-Booker World
- FSR Issue 18.3: Taking Stock a Year after Booker
- FSR Issue 18.4: Sentencing at the Supreme Court
- FSR Issue 18.5: Toward Real Reform: Model Federal Sentencing Guidelines
- FSR Issue 19.1: Victims and Sentencing I
Of course, I have also done more posts than I can count on this blog and also started a new group blog, Law School Innovation. And, speaking of blogs, I also wrote this article, "Scholarship in Action: The Power, Possibilities, and Pitfalls for Law Professor Blogs," as part of this year's Harvard Law School symposium, "Bloggership: How Blogs are Transforming Legal Scholarship Conference."
Examining crack sentencing in the new Congress
Today's Wall Street Journal has this extended article discussing how the new Congress might impact federal sentencing rules. The article focuses particularly on the long-running crack-cocaine sentencing debate, and it suggests that the US Sentencing Commission has an amendment to the crack guidelines in the works. Here is how the article begins:
With Democrats poised to take control of Congress, law-enforcement officials are preparing to defend two decades of federal sentencing policies that mandated harsh prison terms on a variety of crimes and led to a boom in the prison population.
Michigan Rep. John Conyers, the incoming chairman of the House Judiciary Committee, and Rep. Robert Scott (D., Va.) have already said they plan hearings early in the term to look at how nonviolent drug offenders are punished under mandatory minimum laws. An early target will be the prison terms mandated by Congress for crack-cocaine convictions.
Under current law, someone caught with five grams of crack gets a five-year sentence, while it takes 500 grams of powder cocaine to trigger the same sentence, even though there is no physiological difference. Critics have long maintained that the law unfairly targets African-American communities, where crack is more prevalent. In contrast, suburban white users tend to prefer cocaine in its powder form. Mr. Conyers has called the crack-cocaine sentences the "most outrageous example of the unfairness of mandatory minimums."
Democrats are buoyed by recent signals from the U.S. Sentencing Commission, which sets guidelines for judges to use in an advisory capacity when they hand down sentences. Members of the commission are likely to recommend a change in the crack-cocaine penalties next year, according to commission members. The commission has tried since 1995 to bring the penalties for crack crimes more in line with powder cocaine but the Republican-controlled Congress has ignored past attempts.
Some recent related posts:
- How could and will this election impact federal sentencing policy?
- Figuring out election results for sentencing fans
- The big other branch questions after the election
- FAMM's view of the new political landscape
- More speculations about Congress and sentencing
December 25, 2006
The right's prison conversion
In a fitting piece for the holiday season, the New York Times Magazine ran this extended article entitled "The Right Has a Jailhouse Conversion." The piece effectively explores the Second Chance Act and modern changes in the politics of crime. Here is an introductory snippet:
[A] decline in the exploitation of crime coincides with an odd and surprising change in the politics of crime. The G.O.P., the party of Richard Nixon’s 1968 law-and-order campaign and the Willie Horton commercial, is beginning to embrace the idea that prisoners have not only souls that need saving but also flesh that needs caring for in this world. Increasingly, Republicans are talking about helping ex-prisoners find housing, drug treatment, mental-health counseling, job training and education. They're also reconsidering some of the more punitive sentencing laws for drug possession....
Perhaps most remarkably, the outgoing Republican-controlled Congress came tantalizingly close to passing the Second Chance Act, a bill that focuses not on how to “lock them up” but on how to let them out. The bill may become law soon, if Democrats continue to welcome the new conservative interest in rehabilitation.
As detailed in posts below, I have long been spotlighting the importance of recent evolutions in the politics of crime:
- Is there a "new right" on criminal sentencing issues?
- More evidence of a new sentencing reform politics
- Religion, sentencing and corrections
- Meth, mandatories and moral values
- Sentencing and Religion
- Miers, religion, and criminal justice issues
- Crime, sentencing and politics
- Having faith in prisons
December 24, 2006
Ohio sentencing stories at the heart of it all
For two decades, the Ohio Division of Travel and Tourism has used the slogan, "Ohio, The Heart of It All" to promote the state. Two articles today on Buckeye state sentencing developments shows how true this slogan is:
- This article from the Cleveland Plain Dealer provides an effective overview of the recent chaotic rulings in the lawsuit challenging Ohio's lethal-injection protocol.
- This article from the Columbus Dispatch provides an effective overview of recently passed legislation through which the "Ohio legislature dropped the hammer on sex offenders."
Lethal injection litigation and new "get-tougher" sex legislation essentially defines the major sentencing developments of 2006. At least in the arena of sentencing, Ohio truly is at the heart of it all.
Thoughtful reflections on the death penalty
Writing for The Nation, Bruce Shapiro has this thoughtful examination of the current state of the death penalty. Entitled "Questioning Capital Punishment," the piece explores various aspect of current capital debates. I was especially drawn to this passage:
For the last decade, the issue that has driven the death penalty debate — galvanizing the attention of courts and press alike — has been innocence: a capital representation system so criminally negligent that 123 wrongfully convicted death-row inmates have been released, and public confidence in death sentences eroded.
Yet innocence cases, in their own way, have evaded a fundamental question: What about the grievously guilty? What about what one pro-death-penalty legal scholar calls "the worst of the worst"? Are executions of the truly guilty consistent with America's evolving constitutional standards, with national ideals and worldwide human rights norms?
UPDATE: Along the same lines, the Washington Post today has this article spotlighting the how the death penalty crossroads in Maryland might impact the incoming Governor-elect. Here is how it begin:
The emotionally charged, polarizing issue of the death penalty was barely mentioned during the campaign for Maryland governor. And it hardly seemed something that Gov.-elect Martin O'Malley would be eager to wade into during his first months in office, when he plans to focus on "the things we agree on."
Yet the confluence of national currents and a Maryland court ruling last week halting executions on a technicality could make the death penalty a defining issue of O'Malley's tenure. In effect, Maryland suddenly has a moratorium on executions, and the new governor, who is personally opposed to capital punishment, could play a pivotal role in determining when — and whether — it resumes.
A gendered perspective on the war on drugs
This article from the North County Times (CA), entited "Women are silent casualties of war on drugs," provides an interesting and thoughtful perspective on how tough sentencing laws have particularly impacted women. Here is one data highlight from the article:
America's 25-year war on drugs has taken an exorbitant toll, both human and economic. Drug arrests have tripled since 1980; as a result, the number of jailed drug offenders in 2000 equaled the total number of inmates in U.S. prisons and jails 25 years ago, according to The Sentencing Project, a research and advocacy group.
By most estimates, women have paid the highest price. Between 1977 and 2001, figures from the Women's Prison Association show a 592 percent increase in the number of women jailed, from 12,279 to 85,031. According to the WPA, the growth "corresponds directly to the mandatory minimum sentencing laws in effect since the early 1970s. Since more women are convicted for nonviolent, drug-related crimes than for any other, these sentencing policies have had a particularly profound effect on women."


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Well, after many shutters have come across the wires in recent days and weeks, here's another to add to the list. The Bentley Tavern, the "nouveau bistro" on Southport, is no more. The restaurant's former general manager confirms the news following its website going dark and the space surfacing on real estate market.
The Bentley opened last September under chef Ian Flowers, hoping to find success as a neighborhood low-key "progressive new American bistro cafe." Flowers and owner Brian Dohmen declined to comment on the closing. Stay tuned to see what happens to the historic space next.
· All Coverage of The Bentley Tavern [-ECHI-]
· All Shutters [-ECHI-]