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understandBPMN: Calculator of Understandability Metrics for BPMN
Calculate several understandability metrics of BPMN models. BPMN stands for business process modelling notation and is a language for expressing business processes into business process diagrams. Examples of these understandability metrics are: average connector degree, maximum connector degree, sequentiality, cyclicity, diameter, depth, token split, control flow complexity, connector mismatch, connector heterogeneity, separability, structuredness and cross connectivity. See R documentation and paper on metric implementation included in this package for more information concerning the metrics. |
Life After OOP: C++ is not just an object-oriented language - adambyrtek
http://cplusplus-soup.com/2010/11/22/life-after-oop/
======
adambyrtek
> Think about the way Python or Ruby deals with lists and you don’t know what
> the elements of the list are — and it’s too late to find a bug when you
> accidentally append a string to a list that was only meant to contain
> integers — and then you’d wonder why these are “modern” programming
> languages.
He seems like a C++ expert, but this quote suggests that he doesn't really get
dynamic languages. Sure, this is a classic argument against dynamic typing,
but the practice shows the sky doesn't fall because of that, and it makes
programmers much more productive. Also dynamic languages put more focus on
unit testing, which is necessary for C++ code as well. The fact that a program
passes through the compiler doesn't necessarily mean that it is correct.
~~~
tgflynn
I think dynamic languages, Python in particular, are great for small scale
and/or throw-away code like data analysis projects or algorithm prototyping.
However I'd personally be very wary of using them for large scale software
systems that need to be maintained over long periods of time, because of this
lack of compile time type safety.
~~~
adambyrtek
This is a long-standing debate and I don't thing that it makes sense to dive
too deep into that in this thread. I'll just say that I don't agree with that
and point you to Bruce Eckel's essay Strong Typing vs. Strong Testing[1]. Not
to mention that there are numerous large (not huge) systems written in dynamic
languages that prove otherwise.
<https://docs.google.com/View?id=dcsvntt2_25wpjvbbhk>
------
iwr
OK, so generic, policy-based programming is a possible alternative to classic
OO. Can you recommend a few free thematic resources?
~~~
darwinGod
Modern C++ Design by Andrei Alexandrescu, for one.
~~~
fbcocq
How is this free?
|
Q:
How to call other functions from within Elixir GenServer handler?
I have a GenServer that implements functionality for a single item, e.g:
def handle_call({:sync, id}, _from, state) do
## update data
{:reply, data, sync}
end
Now I want to handle this functionality for multiple ids, e.g.:
def handle_call({:sync_all, ids}, _from, state) do
## call sync for each id
data = Enum.map(ids, fn(id) ->
GenServer.call(self(), {:sync, id})
end)
## Further reduce down data to stats
{:reply, data, sync}
end
This however does not work telling me that the process attempted to call itself.
I assume this must be due to the blocking nature of call. The same however happens if I use cast in the sync_all version.
So my question is: How can I call other GenServer tasks from within a handle_call or handle_cast function?
A:
What you would normally do in such cases is to extract the common logic to a separate function:
def handle_call({:sync, id}, _from, state) do
{data, state} = do_sync(id, state)
{:reply, data, state}
end
def handle_call({:sync_all, ids}, _from, state) do
{data, state} = Enum.map_reduce(ids, state, &do_sync/2)
{:reply, data, state}
end
defp do_sync(id, state) do
# do something
{data, new_state}
end
|
Decriminalization of marijuana offers positive outcome
Last year, Governor Rauner was presented with the notion of decriminalizing marijuana for the state of Illinois. He opposed since the original presented amount of fifteen grams, which is the decriminalized amount in Chicago, was too high of an amount with too low of a fine.
Currently, it has been a week since possessing a small amount of the controversial plant has been decriminalized in the state of Illinois. On July 29, Governor Bruce Rauner passed the senate bill 2228 which makes possession of a minor amount of marijuana a civil offense.
Now, possession of up to 10 grams of marijuana no longer means jail time, rather a fine of $100 to $200 will be issued instead.
Previous to July 29, possessing up to 2.5 grams meant facing up to a month of jail time and a hefty fee of $1,500 Those who possessed any amount between 2.5 to 10 grams bumped up the jail sentence to six months with still the $1,500 fee issued. Offenders who posses the same amount of 10 grams will now be charged a fine of nearly the amount of an illegal parking ticket.
While in the city of Chicago possession of 15 grams or more of marijuana means time behind bars, it is respectable to see the rest of the state catch up to a reasonable punishment. Rauner has added Illinois to be the 21st state in the nation to erase jail from being an outcome.
Marijuana Policy Project (MPP), a lobbyist organization based in Washington, D.C., who also lobbied for Rauner to pass the bill, released a press release which highlighted the positive outcomes of the recent passage.
“We applaud Gov. Rauner and the legislature for replacing Illinois’s needlessly draconian marijuana possession law with a much more sensible policy,” Chris Lindsey, a senior legislative counsel for the MPP said. “This commonsense legislation will prevent countless citizens from having their lives turned upside down by a marijuana possession arrest.”
Among those lives whose lives have been turned upside down from being arrested for minor possession of marijuana are college students using the plant for recreational purposes. Although this new bill, offers leeway on the possessed amount and removes the threat of jail time over their heads for holding small amounts, college students everywhere in the state should not view this as a hall pass.
“Nobody should face a lifelong criminal record and potential jail time for possessing a substance that is safer than alcohol. Serious criminal penalties should be reserved for people who commit serious crimes, not low-level marijuana offenses.”
No college student should face jail time, but no college student should perceive this new law as an incentive to use more of it or become carefree about its possession. Being a responsible young adult is still more valuable than paying any fines for holding its possession. |
The men and women that go to war are incredibly brave, after all war is absolute hell, far worse than most of us could ever even imagine. Just because soldiers are selflessly brave doesn’t mean they don’t need love–in fact, they need as much love and support as they can get given the circumstances they are put through.
Homeless animals are often caught in the turmoil of war, and just like the soldiers they are looking for love, comfort and some sense of security. Naturally, soldiers and animals gravitate to one another, forming unbreakable bonds. It’s a win-win situation for all, the soldiers get a new friend to comfort them on the toughest days, and the homeless animals can be adopted and given the care, food and love they need.
Animals are incredibly therapeutic, this fact has been proven time and time again. Case and point, animals provide soldiers with the support necessary to get through some truly difficult times. These photos of soldiers and animals that became best friends overseas will warm your heart.
1. Marines gather together and feed breakfast to an adorable stray puppy
Photo Credit: petfriendsmagazine.com
2. This soldier’s friend took this photo and captioned it: “Stray kitten sleeping on my buddy, Afghanistan 2009”
Photo Credit: imgur.com
3. This soldier has a lot of animal friends who want to share that small bag of chips with him!
Photo Credit: imgur.com
4. Solider befriends a little puppy overseas
Photo Credit: reddit.com
5. Soldier posted this photo to imgur with the caption, “My shy friend in Afghanistan.”
Photo Credit: imgur.com
6. Soldier with his rescued puppy tucked inside his backpack
Photo Credit: patch.com
7. Soldier hangs out with his loving and loyal dog
Photo Credit: unknown
8. This photo was shared through imgur with the caption, “My friend rescuing a dog in the streets of Afghanistan”
Photo Credit: imgur.com
9. A French soldier feeding his adopted kitten during the Vietnam War
Photo Credit: swallowthesky.org
10. All the love you could ever need!
Photo Credit: wildini.blogspot.com
11. Soldier troops on with kitten in tow
Photo Credit: unknown
12. Body warmth! Russian soldiers cuddle up with a puppy and take a snooze during WWII
Photo Credit: Georgy Lipskerov
13. Photo shared to reddit with the caption, “When I was in Iraq, we had a pet donkey.”
Photo Credit: reddit.com
14. Marine gives his four-legged working partner a lift back to the kennels after a rough two-hour search
Dogs work as soldiers on the front line too. Dogs are often trained to use their super powerful nose to smell out things humans could never find alone.
Photo Credit: Lucca K458
15. A dog sadly says his goodbyes to his good soldier friend
Photo Credit: imgur.com
16. Solider feeds a pack of hungry puppies
Photo Credit: unknown
17. Adorable kittens found and befriended in Afghanistan
Photo Credit: imgur.com
18. This adopted puppy was known for finding any hiding spot he could in order to stay out of the heat
Photo Credit: imgur.com
19. Tired soldier and puppy take a quick nap
Photo Credit: imgur.com
20. This adorable puppy kept soldiers company on a mountaintop north of Kabul, Afghanistan
Photo Credit: imgur.com
21. Soldier and kitty making friends
Photo Credit: imgur
22. Soldier loving on his silly little kitty
Photo Credit: imgur.com
23. Soldier in Yemen holding two cute puppies
Photo Credit: imgur.com
24. This is what true friendship looks like
Photo Credit: Christy Bormann
25. Free kisses for soldiers!
Photo Credit: Andy Masson
26. Israel Defense Forces (IDF) soldier shares a moment with her dog
Photo Credit: imgur
27. Image shared through reddit with caption, “My buddy is stationed in Bahrain. He found this guy in the trash and he’s been taking care of him.”
Who saved who?
Photo Credit: reddit.com
28. Friends at first sight, cat crawled up on soldier’s shoulders and stayed there
Photo Credit: wingless7
Next Up: 22 Soldiers On Their Days Off |
The Islamic State terror group's destruction of the oldest Christian monastery in Iraq represents "a battle of savagery against decency," U.S. Col. Steve Warren told Fox News from Baghdad.
Satellite photos obtained exclusively by The Associated Press confirm the worst fears of church authorities and preservationists -- St. Elijah's Monastery of Mosul has been completely wiped out.
For 1,400 years the compound survived assaults by nature and man, standing as a place of worship recently for U.S. troops. In earlier centuries, generations of monks tucked candles in the niches and prayed in the cool chapel. The Greek letters chi and rho, representing the first two letters of Christ's name, were carved near the entrance.
"This enemy has proven time and again its ruthlessness, its barbarity, its willingness to destroy everything from human life to civilian supporting infrastructure, to, you know, cultural artifacts, with absolute disregard for history, for humanity, or for anything that approaches decency," Col. Warren added.
In his office in exile in Irbil, Iraq, the Rev. Paul Thabit Habib, 39, stared quietly at before- and after-images of the monastery that once perched on a hillside above his hometown of Mosul. Shaken, he flipped back to his own photos for comparison.
"I can't describe my sadness," he said in Arabic. "Our Christian history in Mosul is being barbarically leveled. We see it as an attempt to expel us from Iraq, eliminating and finishing our existence in this land."
The Islamic State group, which broke from al-Qaida and now controls large parts of Iraq and Syria, has killed thousands of civilians and forced out hundreds of thousands of Christians, threatening a religion that has endured in the region for 2,000 years. Along the way, its fighters have destroyed buildings and ruined historical and culturally significant structures they consider contrary to their interpretation of Islam.
Those who knew the monastery wondered about its fate after the extremists swept through in June 2014 and largely cut communications to the area.
Now, St. Elijah's has joined a growing list of more than 100 demolished religious and historic sites, including mosques, tombs, shrines and churches in Syria and Iraq. The extremists have defaced or ruined ancient monuments in Nineveh, Palmyra and Hatra. Museums and libraries have been looted, books burned, artwork crushed -- or trafficked.
"A big part of tangible history has been destroyed," said Rev. Manuel Yousif Boji. A Chaldean Catholic pastor in Southfield, Michigan, he remembers attending Mass at St. Elijah's almost 60 years ago while a seminarian in Mosul.
"These persecutions have happened to our church more than once, but we believe in the power of truth, the power of God," said Boji. He is part of the Detroit area's Chaldean community, which became the largest outside Iraq after the sectarian bloodshed that followed the U.S. invasion in 2003. Iraq's Christian population has dropped from 1.3 million then to 300,000 now, church authorities say.
At the Vatican, spokesman Rev. Federico Lombardi, noted that since the monastery dates back to the time Christians were united, before the break with Orthodox and Catholics, the place would be a special one for many. He said it was the first news he had had of the destruction.
"Unfortunately, there is this systemic destruction of precious sites, not only cultural, but also religious and spiritual. It's very sad and dramatic," Lombardi told the AP.
The destruction of the monastery is a blow for U.S. troops and advisers who served in Iraq and had tried to protect and honor the site, a hopeful endeavor in a violent place and time.
Suzanne Bott, who spent more than two years restoring St. Elijah's Monastery as a U.S. State Department cultural adviser in Iraq, teared up when the AP showed her the images.
"Oh no way. It's just razed completely," said Bott. "What we lose is a very tangible reminder of the roots of a religion."
Army reserve Col. Mary Prophit remembered a sunrise service in St. Elijah where, as a Catholic lay minister, she served communion.
"I let that moment sink in, the candlelight, the first rays of sunshine. We were worshipping in a place where people had been worshipping God for 1,400 years," said Prophit, who was deployed there in 2004 and again in 2009.
"I would imagine that many people are feeling like, `What were the last 10 years for if these guys can go in and destroy everything?"' said Prophit, a library manager in Glenoma, Washington.
This month, at the request of AP, satellite imagery firm DigitalGlobe pulled a series of images of the same spot from their archive of pictures taken globally every day.
Imagery analyst Stephen Wood, CEO of Allsource Analysis, reviewed the pictures for AP and identified the date of destruction between Aug. 27 and Sept. 28, 2014. Before it was razed, images show a partially restored, 27,000-square-foot religious building. Although the roof was largely missing, it had 26 distinctive rooms including a sanctuary and chapel. One month later, "the stone walls have been literally pulverized," said Wood.
"Bulldozers, heavy equipment, sledgehammers, possibly explosives turned those stone walls into this field of gray-white dust. They destroyed it completely," he said. "There's nothing to rebuild."
The monastery, called Dair Mar Elia, is named for the Assyrian Christian monk -- St. Elijah -- who built it between 582 and 590 A.C. It was a holy site for Iraqi Christians for centuries, part of the Mideast's Chaldean Catholic community.
In 1743, tragedy struck when as many as 150 monks who refused to convert to Islam were massacred under orders of a Persian general, and the monastery was damaged. For the next two centuries it remained a place of pilgrimage, even after it was incorporated into an Iraqi military training base and later a U.S. base.
Then in 2003 St. Elijah's shuddered again -- this time a wall was smashed by a tank turret blown off in battle. Iraqi troops had already moved in, dumping garbage in the ancient cistern. The U.S. Army's 101st Airborne Division took control, with troops painting over ancient murals and scrawling their division's "Screaming Eagle," along with "Chad wuz here" and "I love Debbie," on the walls.
A U.S. military chaplain, recognizing St. Elijah's significance, kicked the troops out and the Army's subsequent preservation initiative became a pet project for a series of chaplains who toured thousands of soldiers through the ruin.
"It was a sacred place. We literally bent down physically to enter, an acquiescence to the reality that there was something greater going on inside," remembered military chaplain Jeffrey Whorton. A Catholic priest who now works at Ft. Bragg, he had to collect himself after viewing the damage. "I don't know why this is affecting me so much," he said.
The U.S. military's efforts drew attention from international media outlets including the AP in 2008. Today those chronicles, from YouTube videos captured on the cell phones of visiting soldiers to AP's own high resolution, detailed photographs, take on new importance as archives of what was lost.
One piece published in Smithsonian Magazine was written by American journalist James Foley, six years before he was killed by Islamic State militants.
St. Elijah's was being saved, Foley wrote in 2008, "for future generations of Iraqis who will hopefully soon have the security to appreciate it."
Fox News' Lucas Tomlinson and The Associated Press contributed to this report. |
kind: ClusterRoleBinding
apiVersion: rbac.authorization.k8s.io/v1
metadata:
name: cass-operator
subjects:
- kind: ServiceAccount
name: cass-operator
namespace: "cass-operator"
roleRef:
kind: ClusterRole
name: cass-operator-cluster-role
apiGroup: rbac.authorization.k8s.io
|
Insects usually lack the capacity for *de novo* biosynthesis of ten amino acids (aliphatic: leucine, isoleucine, valine and threonine; aromatic: phenylalanine, tryptophan and histidine; sulfur containing: methionine; basic: arginine and lysine), and must absorb them from food[@b1]. Insect midgut cells actively absorb these essential amino acids by transporters[@b2] that mainly belong to the SoLute Carrier (SLC) superfamily[@b3]. In total, 9 out of 10 SLC families participate in amino acid transport in insects[@b1]. Among the 9 SLC families, SLC6 transporters act in the apical membrane of the alimentary canal, and mediate Na^+^/K^+^ ion motive force-coupled transport of amino acids against large chemical gradients[@b4]. In contrast, the contributions of SLC7, SLC15, SLC36, SLC38 and SLC43 members, to the essential amino acid absorption in midgut, may be secondary or dispensable[@b1].
SLC6 family comprises two clearly identifiable subfamilies. The basal subfamily consists of Nutrient Amino acid Transporters (NATs) that mainly absorbs large neutral essential amino acids and proline, known as the B0 ("B" and "0" define the Na^+^-coupled broad substrate spectra for neutral amino acids) and IMINO transporters respectively. The more evolutionary subfamily is the animal-specific neurotransmitter transporters which combine several orthologous clusters of catecholamine (dopamine, norepinephrine and octopamine), indolamine (serotonin) and GABA porters[@b1].
In insects, several SLC6 NATs have been cloned from Lepidopteran species *Manduca sexta*[@b5][@b6][@b7][@b8][@b9][@b10], and Dipteran species *Aedes aegypti*[@b11], *Anopheles gambiae*[@b12] and *Drosophila melanogaster*[@b13]. When expressed in *Xenopus* oocytes, these NATs can absorb a subset of neutral amino acids[@b14]. However, *in vivo* data have not yet been reported.
A high concentration of free amino acids in insects triggers the insulin/target of rapamycin (TOR) signaling pathways[@b15][@b16]. Subsequently, insulin/TOR signaling modifies the titers of 20-hydroxyecdysone (20E) and juvenile hormone (JH) to tune larval growth and metamorphosis[@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24]. In *Leptinotarsa decemlineata* (Say), a notorious defoliator of potato, several cytochrome P450 monooxygenases such as Spook, Phantom, Disembodied, Shadow, and Shade (SHD) have been documented to be involved in the biosynthesis of ecdysone in the prothoracic gland, and in the production of 20E in the peripheral tissues[@b25][@b26]. JH has been reported to be produced in the corpora allata. JH acid methyltransferase (JHAMT) participates in JH biosynthesis[@b27][@b28]. During molting of *L. decemlineata* larvae, either JH or 20E initiates a specific gene expression cascade. Among the activated genes, *Krüppel homolog 1 (LdKr-h1)* is a JH early-inducible gene[@b28][@b29], whereas *LdE75* and Ld*FTZ-F1* are 20E-inducible[@b29][@b30]. During mid instar stage, *L. decemlineata* larvae gnaw a large quantities of potato foliage. At the end of each instar, the larvae stop feeding, and shed their cuticle to allow for further growth. For economical reasons, it appears plausible that, during the molting periods when protein digestion and amino acid absorption are shut down, transcription of relative genes should be downregulated concomitantly, regulated probably by 20E/JH.
In the present paper, we identified a putative *NAT1* gene (*LdNAT1*) in *L. decemlineata*. For the first time in insect species, we tested the induction of *NAT1* by 20E and JH, we knocked down *LdNAT1* by RNA interference (RNAi) to investigate the *in vivo* contributions of *Ld*NAT1 to amino acid absorption in the midgut, as well as to overall beetle biology.
Methods and Materials
=====================
Insects rearing and chemicals
-----------------------------
*L. decemlineata* larvae and adults were routinely reared in an insectary according to a previously described method[@b28], and were supplied with potato foliage at vegetative growth or young tuber stages in order to assure sufficient nutrition. At this feeding protocol, *L. decemlineata* larvae progressed through four distinct instars, with the approximate periods of the first-, second-, third- and fourth-instar stages being 2, 2, 2 and 4 days, respectively. Upon reaching full size, the fourth larval instars spent an additional 4--7 days as non-feeding prepupae. The prepupae then dropped to the soil and burrowed to a depth of 3--5 cm to pupate.
An ecdysteroid agonist halofenozide (Hal) (ChemService, West Chester, USA), 20-hydroxyecdysone (20E) (Sigma-Aldrich, USA), juvenile hormone (JH) (Sigma-Aldrich, USA) and a JH analog pyriproxyfen (Pyr) (Ivy Fine Chemicals Corporation, USA) were purified by reverse phase high performance liquid chromatography before experiments.
Molecular cloning and phylogenetic analysis
-------------------------------------------
Expressed sequence tags of putative *LdNAT1*, *LdInR*, *Ld4EBP*, *LdFOXO*, *LdTOR* and *LdE75* were obtained from *L. decemlineata* transcriptome[@b31] and genome data (<https://www.hgsc.bcm.edu/arthropods/colorado-potato-beetle-genome-project>). The correctness of the sequences was substantiated by polymerase chain reaction (PCR) using primers in [Table S1](#S1){ref-type="supplementary-material"}. This was followed by 5′- and 3′-RACE to complete the sequence, with SMARTer RACE cDNA amplification kit (Takara Bio., Dalian, China) and SMARTer RACE kit (Takara Bio.). The antisense/sense gene-specific primers corresponding to the 5′-end and 3′-end of the sequences were listed in [Table S1](#S1){ref-type="supplementary-material"}. After obtaining the full-length cDNA, six primer pairs ([Table S1](#S1){ref-type="supplementary-material"}) were designed to verify the complete open reading frames. The resulting sequences *LdNAT1*, *LdInR*, *Ld4EBP*, *LdFOXO*, *LdTOR*, *LdE75A*, *LdE75B and LdE75C* were submitted to GenBank with the accession number of AHH29249, KP331063, KP331062, KR075829, KR075825, KP340510, KP340511 and KT246474 respectively. Transmembrane domains of *Ld*NAT1 were predicted using TMHMM 2.0 ([www.cbs.dtu.dk/services/TMHMM](http://www.cbs.dtu.dk/services/TMHMM)).
The representative NAT sequences were retrieved from NCBI, and were aligned with the predicted *Ld*NAT1 using ClustalX (2.1)[@b32]. The neighbor-joining (NJ) tree was constructed using MEGA6[@b33] under the Poisson correction method. The reliability of NJ tree topology was evaluated by bootstrapping a sample of 1000 replicates.
Preparation of dsRNA
--------------------
The same method as previously described[@b28] was used to express ds*SHD*, ds*E75*, ds*FTZ-F1*, ds*JHAMT*, ds*AS-C*, ds*NAT1-*1, ds*NAT1-*2 and ds*egfp* derived from a 141 bp fragment of *LdSHD*, a 361 bp common fragment in the three *LdE75* isoforms, a 319 bp common fragment in both *LdFTZ-F1-1* and *LdFTZ-F1-2* cDNAs, a 261 bp fragment of *LdJHAMT*, a 206 bp fragment of *LdAS-C*, a 665 bp and a 357 bp fragments of *LdNAT1,* and a 414 bp fragment of *enhanced green fluorescent protein* gene. These dsRNAs were individually expressed with specific primers in [Table S1](#S1){ref-type="supplementary-material"}, using *Escherichia coli* HT115 (DE3) competent cells lacking RNase III. Individual colonies were inoculated, and induced to express dsRNA by addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside. The expressed dsRNA was extracted and confirmed by electrophoresis on 1% agarose gel (data not shown), and quantified using a spectrophotometer (NanoDrop Technologies, Wilmington, DE). Bacteria cells were centrifuged at 5000 ×g for 10 min, and resuspended in 0.05 M phosphate buffered saline (PBS, pH 7.4) at the ratio of 1:1. The bacterial suspensions (at dsRNA concentration of about 0.5 μg/ml) were used for bioassay.
Bioassay
--------
Our preliminary results revealed that feeding the second-instar larvae with 0.1 μg/mL of 20E-, Hal-, JH- or Pyr-immersed foliage did not affect larvae growth, pupation and adult emergence. In this survey, nine independent bioassays were carried out as previously described[@b25][@b29], using newly-ecdysed second- and third-instar larvae. The first bioassay was planned to test the effects of 20E and Hal on *LdNAT1* expression. For 1 day, ten third-instar larvae were fed leaves which have been immersed in: (1) water (control), (2) 0.1 μg/mL 20E, or (3) 0.1 μg/mL Hal. Each treatment was replicated three times, and was collected to extract total RNA. The second to fourth bioassays were to knock down *LdSHD*, *LdE75* and *LdFTZ-F1* and each bioassay had three treatments: (1) PBS-, (2) ds*egfp*-, (3) ds*SHD*-, ds*E75*- or ds*FTZ-F1*-immerged leaves. Each treatment (ten larvae) was replicated six times and was fed for 3 days. Three replicates were collected to extract total RNA and the other three replicates were used to extract 20E. The fifth bioassay was intended to determine the effects of JH and Pyr on *LdNAT1* expression by confining ten third-instar larvae for 1 day in petri dishes with: (1) water (control)-, (2) 0.1 μg/mL JH-, or (3) 0.1 μg/mL Pyr-immersed leaves. Three replicates in each treatment were collected to extract total RNA. The sixth and seventh bioassays were intended to silence *LdAS-C* and *LdJHAMT* and had three treatments: (1) PBS-, (2) ds*egfp*-, (3) ds*AS-C*- or ds*JHAMT*-immerged leaves. Six replicates in each treatment (ten larvae) were fed for 3 days to extract total RNA and JH respectively. The eighth and ninth bioassays were planned to knock down *LdNAT1* by allowing ten second- and third-instar larvae to ingest: (1) PBS-, (2) ds*egfp*-, (3) ds*NAT1*-1-, (4) ds*NAT1-*2-dipped leaves. Each treatment was repeated 15 times. For extraction of total RNA, free amino acid, 20E and JH, twelve replicates were respectively collected after continuously fed for 3 days. The remaining 3 replicates were used to measure the larval weight, and to observe the larval developing period and the pupation using the methods as described previously[@b28]. The surviving larvae were individually weighed 3, 4, 5 and 6 days after treatment. Their development was observed at 4-hr intervals. Larval instars were identified by head capsule width, the appearance of exuviae, the black color of the pronotum, and the anterior beige and posterior black stripe visible on the pronotum of the 3^rd^ and 4^th^ instars respectively. Prepupae were distinctive from larvae by their disappeared black pigmentation, their relative inactivity and their curved body shape. The initiation of pupation was indicated by the soil-digging behavior. The pupation and the adult emergence were recorded during a 4-week trial period. For each bioassay, three biological replicates were carried out.
Real-time quantitative PCR (qPCR)
---------------------------------
For tissue expression analysis, RNA samples were extracted from epidermis, foregut, midgut, hindgut, Malpighian tubules, fat body, hemocytes and brain-corpora cardiaca-corpora allata complex of the day 1 fourth-instar larvae. For temporal expression analysis, RNA templates were derived from the first, second, third larval instars at the interval of one day, and from fourth larval instars at the interval of eight hours. For analysis the effects of bioassay, total RNA was extracted from treated larvae. Each sample contained 5--30 individuals and repeated 3 times. The RNA was extracted using SV Total RNA Isolation System Kit (Promega). Purified RNA was subjected to DNase I to remove any residual DNA according to the manufacturer's instructions. Quantitative mRNA measurements were performed by qRT-PCR, using internal control genes (*LdRP4*, *LdRP18*, *LdARF1* and *LdARF4*, the primers listed in [Table S1](#S1){ref-type="supplementary-material"}) according to our published results[@b31]. An RT negative control (without reverse transcriptase) and a non-template negative control were included for each primer set to confirm the absence of genomic DNA and to check for primer-dimer or contamination in the reactions, respectively. Each sample was repeated three times. Data were analyzed by the 2^−ΔΔCT^ method, using the geometric mean of internal control genes for normalization. All methods and data were confirmed to follow the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines[@b34].
Free amino acid analysis
------------------------
The larvae were collected from 3 replicates, whereas the feces were collected from all 15 replicates after continuously ingested ds*NAT1*-1 and ds*NAT1*-2 for 3 days. The foliage samples repeated three times. The collected samples were immediately ground in liquid nitrogen and stored at −80 °C until assayed. Hydrolytic amino acids in the foliage were extracted by acid hydrolysis method. Free amino acids in the bodies and feces were extracted with 80% (v/v) ethanol at 25 °C for 10 min. Samples were centrifuged for 15 min at 10000 ×g and 4 °C. Free amino acid contents in the bodies, feces and foliage were analyzed with a Beckman 6300 Amino Acid Analyzer (Beckman Instruments Inc., Fullerton, CA, USA). The amino acid contents were given as μm per gram.
Quantitative determination of JH and 20E
----------------------------------------
Hemolymph was collected and JH was extracted following the methods described previously[@b28]. A liquid chromatography tandem mass spectrometry was used to quantify JH titers (ng per ml hemolymph)[@b35].
20E was extracted according to an ultrasonic-assisted extraction method[@b29], and its titer (ng per g body weight) was analyzed by a liquid chromatography tandem mass spectrometry-mass spectrometry system using a protocol the same as described[@b36].
Data analysis
-------------
The data were given as means ± SE, and were analyzed by analyses of variance (ANOVAs) followed by the Tukey-Kramer test, using SPSS for Windows (SPSS, Chicago, IL, USA). Pupation rate and amino acid composition were subjected to arc-sine transformation before ANOVAs.
Results
=======
Identification of a putative nutrient amino acid transporter transcript
-----------------------------------------------------------------------
We sequenced a full-length transcript encoding a putative nutrient amino acid transporter in *Leptinotarsa decemlineata*, and provisionally designated *LdNAT1*. The correctness of the cDNA was confirmed by end-to-end amplification and sequencing. It was submitted to GenBank with the accession number of AHH29249.
*LdNAT1* had a 1920 bp open reading frame encoding a 640-amino acid protein. The *Ld*NAT1 protein was predicted to have 12 transmembrane domains and intracellular C and N termini using TMHMM 2.0 ([Figure S1A](#S1){ref-type="supplementary-material"}), which was in agreement with the general structure of transporters in the SLC6 family[@b11]. Based on the sequence alignment with selected characterized NATs and a crystallized bacterial NAT[@b13][@b37], the possible substrate-binding moieties and first and second sodium-binding sites of *Ld*NAT1 were also predicted ([Figure S1A](#S1){ref-type="supplementary-material"}).
The evolutionary relationship of NAT-like representatives derived from 6 insect species was evaluated ([Figure S1B](#S1){ref-type="supplementary-material"}). As expected, *LdNAT1* belongs to the Coleopteran clade. It was first grouped with that from *Tribolium castaneum* (XP_973741), with 100% of bootstrap value, and then the two and *T. castaneum* XP_008196787 joined together, supported by 97% of bootstrap value ([Figure S1B](#S1){ref-type="supplementary-material"}).
The expression of LdNAT1
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The tissue expression patterns of *LdNAT1* were tested by quantitative real-time PCR (qRT-PCR). *LdNAT1* was detectable in the epidermis, foregut, midgut, hindgut, Malpighian tubules, fat body, hemocytes and brain-corpora cardiaca-corpora allata complex of the day 1 fourth-instar larvae. *LdNAT1* mRNA was high in the midgut and moderate in the foregut and hindgut, whereas it was expressed at low levels in the Malpighian tubules, epidermis, fat body, hemocytes and brain-corpora cardiaca-corpora allata complex ([Fig. 1A](#f1){ref-type="fig"}).
The temporal expression profiles of *LdNAT1* were also determined in the larvae. *LdNAT1* was expressed throughout all larval stages. Within the first, second and third larval instars, the expression levels of *LdNAT1* were high right after the molt than just before the molt. In the fourth larval instar, a peak of *LdNAT1* occurred 24 hours after ecdysis. Moreover, *LdNAT1* showed two troughs 80 and 96 hours after ecdysis ([Fig. 1B](#f1){ref-type="fig"}).
Juvenile hormone activates the expression of LdNAT1
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The expression patterns showed that *LdNAT1* mRNA levels were correlated with circulating JH. To determine whether JH induces *LdNAT1 in vivo*, *LdNAT1* mRNA level in newly-ecdysed *L. decemlineata* third-instar larvae were tested after ingestion of water (control)-, JH- and Pyr-immersed foliage for 1 day. Compared with control specimens, *LdNAT1* expression levels were significantly increased by 3.4 and 4.0 folds in the larvae that had ingested JH and Pyr ([Fig. 2A](#f2){ref-type="fig"}).
Moreover, *LdNAT1* transcription was significantly enhanced by 3.6 fold in the *LdAS-C* RNAi hypomorphs, in which JH titer was significantly increased ([Fig. 2B--D](#f2){ref-type="fig"}).
Furthermore, JHAMT plays a major role in JH biosynthesis[@b27][@b28]. In this study, we knocked down *LdJHAMT* by RNAi ([Fig. 2E](#f2){ref-type="fig"}) to lower JH titer ([Fig. 2F](#f2){ref-type="fig"}), and found that *LdNAT1* mRNA level was significantly diminished by 61.1% in the *LdJHAMT* RNAi larvae ([Fig. 2G](#f2){ref-type="fig"}).
20-Hydroxyecdysone inhibits the expression of LdNAT1
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The expression patterns suggest that 20E pulse at the end of each instar inhibits *LdNAT1*. To verify the suggestion, *LdNAT1* mRNA levels in *L. decemlineata* third-instar larvae were tested after ingestion of water (control)-, Hal- and 20E-immersed foliage for 1 day. Compared with control specimens, *LdNAT1* expression levels were significantly decreased by 68.4% and 78.8% in the larvae that had ingested Hal and 20E ([Fig. 3A](#f3){ref-type="fig"}).
Since RNAi of *LdSHD* reduced 20E titer in *L. decemlineata*[@b25][@b29], *LdNAT1* mRNA level was tested in the third-instar larvae in which *LdSHD* had been silenced by RNAi ([Fig. 3B](#f3){ref-type="fig"}). As expected, 20E titer had been reduced ([Fig. 3C](#f3){ref-type="fig"}), whereas *LdNAT1* mRNA level was significantly increased by 6.1 fold in the *LdSHD* RNAi hypomorphs, compared with specimens that had ingested ds*egfp* ([Fig. 3D](#f3){ref-type="fig"}).
*Ld*E75 and *Ld*FTZ-F1 mediated 20E signaling in *L. decemlineata*[@b29][@b30]. In this study, we found three *LdE75* isoforms. We knocked down all these isoforms by dietary introduction of a dsRNA derived from a common fragment in the three *LdE75* isoforms ([Fig. 3E](#f3){ref-type="fig"}). 20E titer was significantly decreased ([Fig. 3F](#f3){ref-type="fig"}), whereas *LdNAT1* transcription was significantly enhanced in the resulting larvae ([Fig. 3G](#f3){ref-type="fig"}). Moreover, knockdown of *LdFTZ-F1* by RNAi ([Fig. 3H](#f3){ref-type="fig"}) also lowered 20E titer ([Fig. 3I](#f3){ref-type="fig"}) but enhanced *LdNAT1* transcription by 3.2 fold ([Fig. 3J](#f3){ref-type="fig"}).
Knockdown of LdNAT1 affects the absorption of several neutral amino acids
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Hydrolytic amino acids in potato foliage, and free amino acids in bodies and feces of the day 1 third- and fourth-instar larvae were analyzed (Tables S2--S4). The compositions of nineteen proteinogenic amino acids (tryptophan is undetectable) were listed in [Table 1](#t1){ref-type="table"}. The compositions of cysteine, histidine, lysine, methionine, serine, tyrosine and valine in the feces of the third- and fourth-instar larvae were significantly lower than those in the body and the potato foliage. Similarly, the compositions of glutamate, isoleucine, leucine and phenylalanine in the feces of the fourth-instar larvae were significantly lower than those in the body and the potato foliage ([Table 1](#t1){ref-type="table"}). These data suggest that a total of 11 amino acids (cysteine, histidine, lysine, methionine, serine, tyrosine, valine, glutamate, isoleucine, leucine, phenylalanine) may be actively absorbed by the larval gut.
After the day 1 second- and third-instar larvae had ingested foliage treated with PBS-, ds*egfp*-, ds*NAT1-*1-, ds*NAT1-*2-dipped leaves for three days, the larvae reached to the day 1 of third- and fourth-instar stage respectively. The expression levels of *LdNAT1* in ds*NAT1-*1- and ds*NAT1-*2-fed third-instar larvae were reduced by 87.3% and 91.6% respectively, the transcript levels in ds*NAT1-*1- and ds*NAT1-*2-fed fourth-instar larvae were decreased by 88.8% and 77.4% respectively, compared with those in PBS- and ds*egfp*-fed third- and fourth-instar larvae ([Figs 4](#f4){ref-type="fig"}A and [5](#f5){ref-type="fig"}A).
The consumed foliage areas per day were measured 3 days after the bioassays, the larvae having been fed on PBS-, ds*egfp*-, ds*NAT1-*1-, ds*NAT1-*2-dipped leaves consumed similar amounts of food ([Figs 4](#f4){ref-type="fig"}B, [5](#f5){ref-type="fig"}B).
The contents of the nineteen proteinogenic amino acids in the bodies and feces of the *LdNAT1* RNAi larvae were measured ([Table 2](#t2){ref-type="table"}, Tables S2, S3). The contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the bodies of the *LdNAT1* RNAi hypomorphs were significantly lower than those in the bodies of PBS- and ds*egfp*-fed larvae, whereas the contents of these amino acids in the feces of the *LdNAT1* RNAi hypomorphs were significantly higher ([Table 2](#t2){ref-type="table"}, Tables S2, S3). These data indicate that knockdown of *LdNAT1* reduces the absorption of the seven amino acids by the larval gut.
Knockdown of LdNAT1 retards larval growth and impairs pupation
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When the larvae were weighed after 3, 4, 5 and 6 days of the initiation of the bioassays, the fresh weights were significantly reduced in the *LdNAT1* RNAi larvae ([Figs 4](#f4){ref-type="fig"}C and [5](#f5){ref-type="fig"}C).
Exposure to ds*NAT*-immersed foliage significantly delayed larval developing stage. The mean periods of those on water-, ds*egfp-,* ds*NAT1-*1-, ds*NAT1-*2-dipped leaves were 14.0, 13.8, 16.0 and 16.3 days in the larvae that had been treated at the second instar stage, and were 12.0, 12.1, 15.0 and 15.0 days in the larvae having been treated at the third instar stage. In addition, the average periods of 2^nd^, 3^rd^ and 4^th^ instars, and prepupae were further measured. Exposure to ds*NAT1-*1-, ds*NAT1-*2-dipped foliage significantly delayed the developing periods of the fourth-instar and prepupae ([Table 3](#t3){ref-type="table"}).
Moreover, the pupation rates were significantly decreased in the *LdNAT1* RNAi larvae, compared with those in the PBS- and ds*egfp*-fed larvae ([Figs 4](#f4){ref-type="fig"}D and [5](#f5){ref-type="fig"}D).
Knockdown of LdNAT1 disrupts insulin/TOR, JH and 20E signaling pathways
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InR, 4EBP, FOXO and TOR are core components in insulin/TOR signaling pathway[@b38]. We sequenced cDNAs encoding InR, 4EBP, FOXO and TOR from *L. decemlineata*. After the initiation of the bioassay for 3 days, the expression levels of the four genes in the fourth-instar alive larvae were tested. *LdInR, LdFOXO* and *Ld4EBP* mRNA levels in ds*NAT1*-1- and ds*NAT1*-2-fed larvae were significantly upregulated. In contrast, *LdTOR* mRNA level was significantly downregulated in the *LdNAT1* RNAi larvae ([Fig. 6A--D](#f6){ref-type="fig"}).
In the *LdNAT1* RNAi hypomorphs, *LdAS-C* expression levels were significantly increased ([Fig. 6E](#f6){ref-type="fig"}), whereas *LdJHAMT* mRNA levels were dramatically reduced ([Fig. 6F](#f6){ref-type="fig"}) and JH titers were significantly decreased ([Fig. 6G](#f6){ref-type="fig"}). As a result, *LdKr-h1* transcript levels were diminished ([Fig. 6H](#f6){ref-type="fig"}).
Finally, ingestion of ds*NAT1*-1 and ds*NAT1*-2 by the third-instar larvae significantly reduced *LdSHD* expression levels ([Fig. 6I](#f6){ref-type="fig"}), decreased 20E titers ([Fig. 6J](#f6){ref-type="fig"}), and diminished the transcript levels of *LdE75* and *LdFTZ-F1-1* ([Fig. 6K,L](#f6){ref-type="fig"}).
Discussion
==========
In this survey, a putative *LdNAT1* were cloned from *L. decemlineata. Ld*NAT1 had a high amino acid similarity to homologs from other insect species. The phylogenetic result indicated that *Ld*NAT1 was distantly related to other insect NAT1-like proteins. Moreover, *Ld*NAT1 was predicted to have 12 transmembrane domains, which was in agreement with the general structure of transporters in the SLC6 family[@b11]. It appears that *Ld*NAT1 may be among insect NAT-SLC6 members, and may play a principal role in active absorption and distribution of essential amino acids in *L. decemlineata*.
Tissue expression of LdNAT1
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In insects, amino acids taken up from midgut need to traverse at least three membranes to reach their intracellular site of use: (1) uptake into an epithelial gut cell, (2) basolateral efflux from the gut cell into the hemolymph, and (3) uptake into somatic cell. In general, SLC6 transporters potentially mediate the uptake steps (1) and (3), whereas other SLC members are involved in the efflux step (2)[@b1][@b39].
In this survey, tissue expression profiles revealed that *LdNAT1* mRNA levels were high or moderate in the larval gut, and lower in other surveyed tissues. Similarly, *NAT* genes in mammalians, other insect species and nematodes are highly expressed in the apical membranes of the alimentary canal, as well as in other organs with elevated requirements for essential amino acids[@b1][@b11][@b12][@b13][@b37][@b39]. For example, *MsKAAT1* in *M. sexta*[@b7], *AeAAT1*[@b11] and *AeNAT5*[@b14] in *A. aegypti*, *AgNAT6*[@b37] and *AgNAT8*[@b12] in *A. gambiae* were highly expressed in the larval guts. Moreover, insect *NAT*s showed unique expression patterns in neurons of the central ganglia and sensory system, which suggest their role as substrate providers for the synthesis of monoamine neurotransmitters[@b12][@b37].
Therefore, the tissue expression patterns of *LdNAT1* are compatible with the common idea that NATs function in: (1) active epithelial uptake of amino acids from the lumen of the gut to support systemic amino acid requirements; (2) active uptake of amino acids into specific cells to support their specialized metabolism or growth; and (3) control of the extracellular concentration of neurotransmitter amino acids in the context of synaptic transmission[@b39].
JH triggers whereas 20E represses the expression of LdNAT1
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Essential amino acids are absorbed by midgut in insects[@b11][@b12][@b13]. In *L. decemlineata* midgut, several cysteine proteases such as intestain A through E have been reported to digest foliage protein[@b40][@b41]. Vacuolar H^+^-ATPases energize Na^+^ and/or K^+^/H^+^ antiport[@b42] to actively transport Na^+^/K^+^ from the hemolymph into the midgut lumen. Na^+^/K^+^ ions are expected to subsequently trigger Na^+^/K^+^-dependent NATs to absorb amino acids in *L. decemlineata*, similar to the NAT-SLC6 members reported in other insect species[@b43].
In some insect species, genes encoding proteases, ATPase subunits and some NAT members are regulated by JH/20E. In *Spodoptera litura* larvae, for example, a putative serine protease gene *Slctlp2* was induced by JH III but not 20E[@b44]. In *Helicoverpa armigera*, the expression of a trypsin-like serine protease gene *HaTLP* was upregulated by a JH analog methoprene and downregulated by 20E *in vivo*[@b45]. In *A. aegypti*, the transcription of a serine-type protease gene *JHA15* is activated by JH in the newly emerged female adults[@b46]. Moreover, the expression of V-ATPase subunit genes were repressed by 20E in *M. sexta*[@b47]. Furthermore, two SLC7 genes, *JhI-21* and *minidiscs* (*mnd*), were JH-inducible in *D. melanogaster*[@b48]. However, whether *NAT-SLC6* genes in insects respond to JH/20E remains unproven.
In several insect species, JH peaks and troughs are observed at each ecdysis. In a termite *Cryptotermes secundus*, for example, within each instar the JH titer rose shortly before or right after the molt, and then dropped sharply[@b49]. Similar phenomenon was observed in *L. decemlineata*[@b27] and *M. sexta*[@b47]. In this study, temporal expression patterns indicate that *LdNAT1* transcript level appears to be positively correlated with the titer of JH. Thus, we determined whether the correlation had any biological significance. As expected, we discovered that either JH or Pyr induced the expression of *LdNAT1* in an *in vivo* bioassay. Moreover, we found that knockdown of *LdAS-C* to increase JH titer[@b27] activated *LdNAT1* expression. Conversely, silencing *LdJHAMT* to inhibit JH biosynthesis reduced *LdNAT1* transcript in *L. decemlineata* final instar larvae.
Temporal expression patterns also imply that 20E inhibits *LdNAT1* transcription. As expected, we found that *LdNAT1* expression was dramatically decreased in *L. decemlineata* specimens having ingested 20E or an ecdysteroid agonist Hal. Conversely, a decrease in 20E in the *LdSHD* RNAi hypomorphs activated the expression of *LdNAT1*. Thus, 20E represses the transcription of *LdNAT1* in *L. decemlineata.* In holometabolous insects such as *D. melanogaster*, 20E signal directly induces transcription of early 20E-response genes such as *DmBR-C*, *DmE74A* and *DmE75A*, and upregulates an early-late gene *DmHR3* during larval-pupal transition[@b50]. *Dm*HR3 then induces *DmβFTZ-F1* expression in mid-prepupae[@b51]. In this survey, knockdown of either *LdE75* or *LdFTZ-F1* reduced 20E titer, as our previously reported results[@b29], the expression levels of *LdNAT1* in both *LdE75* and *LdFTZ-F1* RNAi larvae were significantly increased. Therefore, our results suggested that *LdNAT1* repression required complete 20E signaling pathway.
In response to JH and 20E, *LdNAT1* transcription is activated right after the molt, and is repressed just before the molt. Thus, its protein may function in absorption of amino acids at the mid instar stage when *L. decemlineata* larvae are actively feeding.
Involvement of LdNAT1 in uptake of neutral amino acids
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*Xenopus* oocyte-expressed insect transporters, such as *M. sexta Ms*KAAT1[@b5] and *Ms*CATCH1[@b10], *A. aegypti Aa*NAT1[@b11] and *D. melanogaster Dm*NAT1[@b13], shared relatively broad substrate spectra. *Ms*KAAT1 absorbed phenylalanine, tryptophan, isoleucine, leucine, valine, methionine and alanine[@b5][@b7][@b8][@b9], whereas *Ms*CAATCH1 preferred threonine in the presence of K^+^ but preferred proline in the presence of Na^+^ [@b10]. *Ae*AAT1 from *A. aegypti* had notable apparent affinities and transport velocities for phenylalanine, cysteine, histidine, alanine, serine and methionine[@b11]. *Dm*NAT1 in *D. melanogaster* transported threonine, isoleucine, leucine, valine, histidine, phenylalanine, tyrosine, tryptophan, methionine, cysteine, alanine, proline, serine, asparagine and glycine, with virtually equal apparent affinities and transport velocities[@b13]. In contrast, several Dipteran NAT-SLC6 members showed narrow specialization for absorption of essential amino acids[@b12][@b14][@b37]. However, their orphan orthologs are absent in *D. melanogaster* or outside Diptera species[@b14].
In this survey, by comparing the compositions of nineteen proteinogenic amino acids (tryptophan is undetectable) in potato foliage, and in bodies and feces of the day 1 third- and fourth-instar larvae, we found that a total of 11 amino acids (cysteine, histidine, lysine, methionine, serine, tyrosine, valine, glutamate, isoleucine, leucine, phenylalanine) may be actively absorbed by *L. decemlineata* larval gut. In the *LdNAT1* RNAi larvae, the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the bodies were significantly lower, whereas the contents of these amino acids in the feces were significantly higher, compared with those in the bodies and feces of PBS- and ds*egfp*-fed larvae. Therefore, *Ld*NAT1 in *L. decemlineata* appears an SLC6-NAT transporter belonging to the B(0) system, like most of its homologs in Dipteran and Lepidopteran insect species[@b5][@b10][@b11][@b13].
However, among 11 amino acids being suggested active absorption by the larval gut in *L. decemlineata* in this survey, lysine, tyrosine, valine and glutamate can not be absorbed by *Ld*NAT1. This indicates that there are other functional NATs in *L. decemlineata*. Consistent with our results, a total of 9 SLC families are present in insects out of 10 SLC families participating in mammalian amino acid transport [@b1]. Work is in progress to identify these transporters.
Knockdown of LdNAT1 impaired larval development
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Silencing *LdNAT1* caused obvious negative effects: larval growth was retarded; development period was lengthened, and pupation was impaired. Similarly, RNAi of *Ae*NAT5 increased larval mortality during ecdysis and dramatically suppressed adult emergence in *A. aegypti*[@b14]. Moreover, SLC6 was prominent in bacteria and archaea, and often served in environmental absorption of tryptophan, phenylalanine, tyrosine and methionine. Depriving the organism of such amino acids, or knocking out bacterial SLC6 transporters, limited the exponential growth of bacterial populations[@b52][@b53][@b54][@b55][@b56]. In *Caenorhabditis elegans*, *snf-5* encodes a SLC6 family NAT. A loss-of-function mutation in *snf-5* increased dauer formation and reduced dauer maintenance upon starvation[@b39].
In insect, nutritional deprivation inhibited insulin/TOR pathway[@b38] to modulate insect growth and metamorphosis[@b22][@b23][@b24]. Thus, we measured the expression levels of *LdInR*, *Ld4EBP*, *LdFOXO* and *LdTOR*. As expected, the transcription of *LdInR*, *Ld4EBP* and *LdFOXO* was upregulated in *L. decemlineata*. In contrast, the mRNA level of *LdTOR* was reduced.
Our results further revealed that exposure to ds*NAT1-*1-, ds*NAT1-*2-dipped foliage significantly delayed the developing periods of the fourth-instar and prepupae, whenever we treated the larvae at the second-instar or at the third-instar stages. Thus, NAT is required for dietary intake of amino acids supporting growth and development to critical weight at the fourth (final) instar stage. Moreover, we further discovered that knockdown of *LdNAT1* reduced the mRNA level of an ecdysteroidogenesis gene *LdSHD*, decreased 20E titer, and lowered the expression of two 20E-response gene *LdE75* and *LdFTZ-F1*[@b29]. Furthermore, silencing *LdNAT1* increased the mRNA level of *LdAS-C*, reduced the expression of a JH biosynthesis gene *LdJHAMT*, decreased JH titer, and diminished the transcription of a JH early-inducible gene *LdKr-h1* in *L. decemlineata* larvae.
There are at least two alternative, but not mutually exclusive, explanations for the results. Firstly, delay of the onset of critical weight and reduced insulin/TOR signaling change the timing of 20E pulse and the level of circulating JH in the *LdNAT1* RNAi hypomorphs. Alternatively, the reduced insulin/TOR signaling decreases circulating 20E and JH[@b22][@b23][@b24]. Disturbed 20E and JH signals subsequently delay larval development and impaired pupation. Accordingly, a model for the influence of knockdown of *LdNAT1* on the larval-pupal metamorphosis is proposed in *L. decemlineata* ([Fig. 7](#f7){ref-type="fig"}).
Additional Information
======================
**How to cite this article**: Fu, K.-Y. *et al.* Knockdown of a *nutrient amino acid transporter* gene *LdNAT1* reduces free neutral amino acid contents and impairs *Leptinotarsa decemlineata* pupation. *Sci. Rep.* **5**, 18124; doi: 10.1038/srep18124 (2015).
Supplementary Material {#S1}
======================
###### Supplementary Information
This research was supported by the National Natural Science Foundation of China (31272047 and 31360442), and the Fundamental Research Funds for the Central Universities (KYTZ201403).
**Author Contributions** K.Y.F. performed all experiments, analyzed the data. W.C.G. and T.A. participated in the experiments. G.Q.L. conducted the study and wrote the manuscript. All authors approved the final manuscript.
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###### The compositions of amino acid (%) in foliage, the bodies and feces of the day 1 third- and fourth-instar larvae.
The third-instar larvae The fourth-instar larvae
--- ------------------------- -------------------------- ---------------- ---------------- ----------------
A 8.70 ± 0.41 a 7.75 ± 0.43 a 8.58 ± 0.52 a 7.60 ± 0.37 ab 6.45 ± 0.54 b
R 4.89 ± 0.27 a 4.42 ± 0.33 a 4.44 ± 0.35 a 4.50 ± 0.29 a 3.34 ± 0.26 b
D 1.23 ± 0.11 a 0.91 ± 0.05 a 0.95 ± 0.07 a 0.92 ± 0.08 a 0.71 ± 0.06 a
N 9.79 ± 0.81 a 7.47 ± 0.54 b 8.58 ± 0.43 a 8.25 ± 0.74 ab 10.81 ± 1.12 a
C 0.25 ± 0.02 b 1.36 ± 0.12 a 0.08 ± 0.00 c 0.88 ± 0.07 ab 0.06 ± 0.00 c
E 0.96±0.10 ab 1.38 ± 0.11 a 1.06 ± 0.13 a 1.34 ± 0.12 a 0.80 ± 0.09 b
Q 9.73 ± 0.85 a 12.33 ± 1.02 a 9.72 ± 0.81 a 11.65 ± 1.12 a 12.79 ± 1.23 a
G 9.62 ± 0.81 c 9.04 ± 0.75 c 18.07 ± 1.52 b 9.16 ± 1.04 c 24.60 ± 2.33 a
H 1.92 ± 0.15 b 2.56 ± 0.14 a 1.58 ± 0.12 b 2.74 ± 0.16 a 1.19 ± 0.09 c
I 4.71 ± 0.22 a 5.09 ± 0.23 a 5.22 ± 0.36 a 5.04 ± 0.42 a 3.92 ± 0.28 b
L 8.87 ± 0.52 a 7.17 ± 0.48 a 6.88 ± 0.54 ab 8.35 ± 0.57 a 5.94 ± 0.34 b
K 6.49 ± 0.33 a 7.62 ± 0.35 a 2.81 ± 0.23 b 7.52 ± 0.48 a 2.93 ± 0.23 b
M 1.35 ± 0.12 a 0.21 ± 0.01 b 0.01±0.00 c 0.20 ± 0.03 b 0.03 ± 0.00 c
F 4.22 ± 0.34 a 3.38 ± 0.30 ab 3.91 ± 0.20 a 3.37 ± 0.18 ab 2.94 ± 0.18 b
P 6.42 ± 0.53 a 6.26 ± 0.50 a 7.39 ± 0.64 a 5.63 ± 0.59 a 5.56 ± 0.53 a
S 5.59 ± 0.34 a 6.81 ± 0.42 a 4.49 ± 0.35 b 6.79 ± 0.32 a 4.15 ± 0.27 b
T 5.32 ± 0.34 bc 4.65 ± 0.42 c 8.31 ± 0.72 a 4.62 ± 0.38 c 6.26 ± 0.46 b
Y 3.07 ± 0.22 a 3.19 ± 0.17 a 2.61 ± 0.14 b 3.23 ± 0.21 a 1.96 ± 0.15 c
V 6.79 ± 0.37 b 8.31 ± 0.42 a 5.22 ± 0.25 c 8.12 ± 0.45 a 5.45 ± 0.30 c
Note: A, alanine; R, arginine; D, aspartic acid; N, asparagine; C, cysteine; E, glutamate; Q, glutamine; G, glycine; H, histidine; I, isoleucine; L, leucine; K, lysine; M, methionine; F, phenylalanine; P, proline; S, serine; T, threonine; Y, tyrosine; V, valine. Data within each line are subjected to arc-sine transformation and analyzed by ANOVAs followed by the Tukey-Kramer test. Different letters indicate significant difference at P value \< 0.05.
###### The contents of seven amino acids in the bodies and feces of the *Leptinotarsa decemlineata* larvae fed on PBS, ds*egfp*, ds*NAT1-*1 and ds*NAT1-*2-immersed foliage for 3 days.
Amino acid Origin Concentration (μmol/g)
-------------------------- ---------------- ------------------------ ---------------- ---------------- ----------------
The third-instar larvae
C Bodies 11.12 ± 0.51 a 13.12 ± 0.84 a 3.21 ± 0.21 b 2.67 ± 0.14 b
Feces 0.25 ± 0.02 b 0.33 ± 0.03 b 2.34 ± 0.14 a 3.15 ± 0.13 a
H Bodies 20.88 ± 1.24 a 20.02 ± 1.11 a 10.70 ± 0.52 b 11.17 ± 0.47 b
Feces 4.69 ± 0.20 b 4.06 ± 0.28 b 8.84 ± 0.41 a 7.87 ± 0.52 a
I Bodies 41.49 ± 2.22 a 42.16 ± 3.04 a 26.77 ± 1.29 b 28.13 ± 1.38 b
Feces 15.41 ± 0.84 b 12.68 ± 0.61 b 27.22 ± 1.15 a 29.39 ± 1.74 a
L Bodies 58.48 ± 2.41 a 58.76 ± 1.98 a 32.45 ± 2.07 b 34.68 ± 2.15 b
Feces 20.31 ± 1.06 b 19.75 ± 1.19 b 37.55 ± 1.24 a 33.17 ± 2.03 a
M Bodies 1.74 ± 0.38 a 4.26 ± 0.25 a 0.64 ± 0.04 b 0.13 ± 0.01 b
Feces 0.02 ± 0.00 b 0.03 ± 0.00 b 0.31 ± 0.02 a 0.25 ± 0.02 a
F Bodies 27.55 ± 1.07 a 28.64 ± 1.13 a 19.20 ± 1.23 b 15.73 ± 1.19 b
Feces 11.56 ± 0.81 b 9.62 ± 0.57 b 23.69 ± 1.04 a 18.96 ± 1.07 a
S Bodies 55.50 ± 2.11 a 55.86 ± 4.08 a 41.88 ± 2.58 b 34.26 ± 2.14 b
Feces 13.27 ± 1.01 b 11.67 ± 0.72 b 31.84 ± 1.22 a 29.62 ± 1.57 a
The fourth-instar larvae
C Bodies 6.89 ± 0.51 a 10.50 ± 0.67 a 2.10 ± 0.16 b 0.25 ± 0.02 c
Feces 0.25 ± 0.02 b 0.25 ± 0.02 b 4.58 ± 0.37 a 7.70 ± 0.51 a
H Bodies 21.32 ± 0.71 a 19.86 ± 0.54 a 9.44 ± 0.35 b 10.50 ± 0.49 b
Feces 4.69 ± 0.57 b 4.66 ± 0.41 b 8.64 ± 0.37 a 12.06 ± 0.61 a
I Bodies 39.21 ± 1.13 a 45.54 ± 2.02 a 21.79 ± 1.51 b 20.38 ± 1.44 b
Feces 15.40 ± 0.87 b 13.72 ± 0.74 b 30.73 ± 1.57 a 27.28 ± 1.63 a
L Bodies 65.00 ± 3.54 a 63.12 ± 4.13 a 38.40 ± 2.33 b 37.77 ± 1.79 b
Feces 23.30 ± 1.47 b 26.91 ± 1.56 b 46.79 ± 3.64 a 48.52 ± 4.11 a
M Bodies 1.60 ± 0.11 a 1.54 ± 0.08 a 0.65 ± 0.02 b 0.41 ± 0.01 b
Feces 0.15 ± 0.01 b 0.40 ± 0.02 b 2.64 ± 0.15 a 1.51 ± 0.11 a
F Bodies 26.25 ± 1.47 a 29.72 ± 1.55 a 11.51 ± 0.71 b 14.34 ± 0.57 b
Feces 11.55 ± 0.57 b 12.97 ± 0.69 b 22.34 ± 1.21 a 24.58 ± 1.54 a
S Bodies 52.88 ± 3.74 a 59.52 ± 3.69 a 26.64 ± 1.27 b 28.63 ± 1.34 b
Feces 16.27 ± 0.79 b 15.34 ± 0.85 b 31.11 ± 1.45 a 42.19 ± 1.85 a
Free amino acid contents in the bodies and feces are analyzed with a Beckman 6300 Amino Acid Analyzer. Difference of amino acid content within each line is analyzed by ANOVA followed by the Tukey-Kramer test. Data that do not share the same letters are significantly different at P values of 0.05.
###### The developing stage of *L. decemlineata* surviving larvae subjected to dietary dsRNA introduction.
Larval instar 2^nd^ 3^rd^ 4^th^ Prepupae
------------------------------------------------------------- ------------- ------------- ------------- ------------- --------------
Initiation of the bioassay at the early second instar stage
CK 2.0 ± 0.1 a 2.1 ± 0.2 a 4.1 ± 0.2 a 5.8 ± 0.3 a 14.0 ± 0.4 a
ds*egfp* 2.2 ± 0.2 a 2.0 ± 0.1 a 4.0 ± 0.2 a 5.6 ± 0.3 a 13.8 ± 0.5 a
ds*NAT1*-1 2.1 ± 0.1 a 2.1 ± 0.2 a 5.1 ± 0.2 b 6.7 ± 0.4 b 16.0 ± 0.6 b
ds*NAT1*-2 2.1 ± 0.1 a 2.2 ± 0.2 a 5.2 ± 0.3 b 6.8 ± 0.4 b 16.3 ± 0.5 b
Initiation of the bioassay at the early third instar stage
CK 2.0 ± 0.1 a 4.0 ± 0.2 a 6.0 ± 0.2 a 12.0 ± 0.3 a
ds*egfp* 2.1 ± 0.2 a 4.2 ± 0.1 a 5.8 ± 0.4 a 12.1 ± 0.4 a
ds*NAT1*-1 2.0 ± 0.1 a 5.4 ± 0.2 b 7.6 ± 0.3 b 15.0 ± 0.5 b
ds*NAT1*-2 2.2 ± 0.1 a 5.3 ± 0.2 b 7.5 ± 0.3 b 15.0 ± 0.4 b
The larval growth is checked at 4-hr intervals. See text for detail explanation for the identification of instars and prepupae. The data are given as means ± SE, and are subjected one-way ANOVA and followed by the Tukey--Kramer test. Means on the same column followed by the same letters are not significantly different at P \< 0.05.
|
---
author:
- 'Victor P. Debattista'
- Ortwin Gerhard
- 'Maartje N. Sevenster'
title: 'A Pattern Speed in the Galaxy’s OH/IR Stars'
---
Introduction
============
The Milky Way Galaxy (MWG) contains both a bar and spirals. The pattern speed/rotation frequency, $\om$, of these components have been measured with a variety of models. For the bar, models have found values $40 \ltsim \om \ltsim 60$ (Binney et al. 1991; Fux 1999; Englmaier & Gerhard 1999; Weiner & Sellwood 1999; Dehnen 1999; Bissantz et al. 2002). The spiral arm $\om$ is even more uncertain, with values in the range $13.5 \ltsim \om \ltsim 27$ reported (Morgan 1990; Amaral & Lépine 1997; Mishurov & Zenina 1999).
A model-independent method, based on the continuity equation, for measuring pattern speeds in external galaxies has been developed by Tremaine & Weinberg (1984). This method can be extended to the MWG (Kuijken & Tremaine 1991; Debattista et al. 2002). For discrete tracers in the MWG, the Tremaine-Weinberg (TW) method is contained in the following expression: $$\begin{aligned}
\DV & \equiv & \om R_0 - V_{\rm LSR} \equiv \
\frac{\kin}{\pin} - u_{\rm LSR} \frac{\smt}{\pin} \nonumber \\
& = & \frac{ \sum_{i} f(r_i)v_{r,i}}{\sum_{i} f(r_i)\sin l_i
\cos b_i} - u_{LSR}
\frac{ \sum_{i} f(r_i)\cos l_i \cos b_i}{\sum_{i} f(r_i)\sin l_i \cos b_i}
\label{eqn1}\end{aligned}$$ where $R_0$ is the Sun-MWG center distance, $V_{\rm LSR}$ is the tangential velocity of the local standard of rest (LSR), $u_{\rm LSR}$ is the radial velocity of the LSR, $f(r_i)$ is the observational detection probability (which need not be known), $v_{r}$ is the heliocentric radial velocity of a discrete tracer, and $(l,b)$ are its Galactic coordinates. Eqn. \[eqn1\] assumes only one pattern speed and a low amplitude for any rapidly growing structure. Moreover, the tracer population needs to be sampled uniformly; one such survey is the ATCA/VLA OH 1612 MHz survey (Sevenster et al. 1997a,b & 2001), covering $|l| \leq 45\degrees$ and $|b| \leq 3\degrees$.
Pattern Speed of the OH/IR Population
=====================================
We extracted from the ATCA/VLA OH 1612 MHz survey a sample of $\sim 250$ OH/IR stars which are relatively old (older than 0.8 Gyr) and bright (flux density greater than 0.16 Jy). These selection criteria give OH/IR stars between 4 and 10 kpc away from the Sun. We applied the TW analysis of Eqn. \[eqn1\] to this sample, obtaining the results shown in Fig. \[fig1\] (Debattista et al. 2002). Note that the value of $\kin/\pin$ has converged, within the errors, for $|l| > 30\degrees$.
Simple tests with models show that a measurement of $\DV$ with a sample of this size should give an average accuracy of $\sim 17\%$ and always better than $40\%$, when the asymmetry signal is as large as the one we find. From re-sampling experiments, we find $\DV = 252 \pm 41$ . If we assume $V_{\rm LSR}/R_0 = 220/8$ (from SgrA$^*$ motion, Backer et al. 1999; Reid et al. 1999 and Cepheid proper motions, Feast & Whitelock 1997) and $u_{\rm LSR} = 0$ (from SgrA$^*$ HI absorption spectrum, Radhakrishnan et al. 1980), we obtain $\om = 59 \pm 5$ . We estimate systematic error to be $\sim 10$ .
The signal we found is concentrated close the plane ($|b| \leq 1\degrees$) and at large longitude, suggesting a spiral is responsible for it (possibly the Scutum arm). The high $\om$ suggests this spiral arm is driven by the bar. Alternatively, the non-axisymmetric structure involved is an inner ring (Sevenster & Kalnajs 2001).
[8.]{}
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Binney J., Gerhard O. E., Stark A. A., et al. 1991, MNRAS, 252, 210
Bissantz N., Englmaier P., Gerhard O. 2002, MNRAS, [*submitted*]{}
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|
Emma Watson attends special pre-Orange British Academy Film Awards party, hosted by Lancome at The Savoy Hotel on February 10, 2012 in London, England.(February 09, 2012 - Source: Tim Whitby/Getty Images Europe) see more angles » |
Planet 9: If it exists, how did it end up at the edge of our Solar System?
There is evidence to suggest there is a mystery planet lurking at the edge of our Solar System – but if it does exist, how did it end up there? Researchers from the Harvard-Smithsonian Center for Astrophysics (CfA) have studied several different scenarios for where Planet 9 came from, all of which are unlikely.
Scientists have long speculated there is an unknown planet sitting at the edge of the Solar System. However, earlier this year researchers at the California Institute of Technology said they had found evidence to support this idea. They said the unusual orbital patterns of a group of Kuiper belt objects could best be explained by a giant planet gravitationally dominating that region of space.
The unusual orbits of objects around Planet 9Caltech/R. Hurt IPAC
With mounting evidence to suggest Planet 9 does indeed exist, scientists have put forward four scenarios to explain where it came from. "The evidence points to Planet 9 existing, but we can't explain for certain how it was produced," says CfA astronomer Gongjie Li, lead author on one of the three studies submitted to the Astrophysical Journal Letters.
Planet 9 orbits the Sun at between 40 billion to 140 billion miles. This is far beyond any of the other planets in the Solar System. To explain its position, scientists ran computer simulations to look at three ways the planet ended up where it has.
In the first, Planet 9 was pulled outwards by a passing star, which had a stronger gravitational pull on it than the Sun. This would have tugged the planet into a wider and more elliptical orbit. As the Sun was born in a cluster of stars, the likelihood of another star passing nearby in the Solar System's early history is the most plausible explanation. However, the simulations revealed the chance of this happening is only 10%, as a star would probably pull Planet 9 out of the Solar System completely.
Researchers find a possible ninth planet beyond NeptuneIBTimes UK
In another simulation, run by CfA's Scott Kenyon, scientists said Planet 9 may have formed much closer to the Sun. After interactions with Jupiter and Saturn, the planet was effectively kicked out to its current position. A third scenario involves Planet 9 forming at the far reaches of the solar system in the first place. The team said under the right conditions, the hypothetical passing star (mentioned earlier) would be enough for it to be pushed into its current orbit.
A final, and very unlikely scenario, involves Planet 9 being an exoplanet that either pulled into the orbit by a passing star, or drifted into the orbit by accident. This likelihood of this scenario is 2%, however.
Whether or not the planet even exists is still in question, however. As Jim Green, director of Nasa's Planetary Science Division, said earlier this year: "The possibility of a new planet is certainly an exciting one for me as a planetary scientist and for all of us. This is not, however, the detection or discovery of a new planet. It's too early to say with certainty there's a so-called Planet X. What we're seeing is an early prediction based on modelling from limited observations. It's the start of a process that could lead to an exciting result." |
Show HN: Hashnode – A network for software developers to learn and grow - prank7
https://hashnode.com/
======
fazlerocks
Co-Founder here.
Hashnode is a place for software developers to hang out and talk programming.
Rather than focusing on bugs and issues, Hashnode lets you ask opinion based
questions. We want to help beginners / intermediate developers connect with
experts and get helpful feedback for their projects.
Super excited to know what you guys think of Hashnode. We would love to have
your feedbacks/suggestions. :)
We're also Hunted on Product Hunt today.
[https://www.producthunt.com/tech/](https://www.producthunt.com/tech/)
------
lnalx
What are the advantages compared to:
\-
[https://programmers.stackexchange.com/](https://programmers.stackexchange.com/)
\- [https://stackoverflow.com/](https://stackoverflow.com/)
\- [https://www.quora.com/](https://www.quora.com/)
~~~
meekins
Judging from the description I guess the main difference is that hashnode is
open to more open-ended questions and opionated discussions where there is no
single valid answer.
Let's hope this won't become a battlefield of endless flamewars and we'll see
some quality discussions comparable to the ancient c2 wiki instead.
|
This invention relates to improvements in shoes which can eliminate the use of buckles and other rigid-type fasteners that only provide a predetermined, fixed and limited number of adjustments available to the wearer when fastening the shoe.
Some shoes available currently have a strap and buckle closure which offer only limited tautness because of the predetermined position of the holes in the strap for insertion of the rigid stud or latch of the buckle. To fasten buckle type closures, both hands are needed. When the shoe does not fit properly, the foot may shift back and forth, sideways and up and down in the shoe which can cause blisters, bunions, callouses and other problems which are painful and harmful to the feet.
In my prior U.S. Pat. No. 4,079,527 and 4,126,951 and other copending application, I disclosed various Velcro type closures for fastening shoes to maintain the desired tautness specifically across the instep.
Other U.S. patents, particularly the patent to Shaw No. 3,845,769, show the use of a Velcro fastener in footwear. Shaw discloses a therapeutic boot of essentially unyielding material shaped to fit a limb using a plurality of bands with D-rings located adjacent the split in the bottom which receive the Velcro fastener. This structure pulls the sides of the boot together toward the center split.
However, the shoes utilizing the closure assemblies referred to above are not constructed to function in combination with a non-adjustable elastic gore. |
Radioresistance of cancer stem-like cell derived from canine tumours.
Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells. |
New Beginnings HypnotherapyWare and Hertford
Weight Management
Have you been on every diet under the sun and ended up weighing more than you were when you started? Have you successfully dieted time and time again, only to put all the weight back on as soon as you stop?
Maybe it's time to give Hypnotherapy a try?
Hypnotherapy works with the subconscious to help you to change the way you think about food. That feeling of being deprived will disappear, making it gradually feel less like a diet and more like a way of life.You will begin to crave healthy, nutritious foods and your unhealthy ways of eating will be a thing of the past.The best part of all is that it WILL be a new way of life, because having changed your internal beliefs surrounding your eating habits, you will find that there is no going back - the weight will stay off effortlessly. And as the weight comes off you will find that your confidence and self-esteem will soar, spurring you on to achieve your perfect shape!
Of course Hypnotherapy also works wonderfully well by helping you to keep to a healthy way of eating, by boosting your motivation and willpower. So, if you are following a diet, either on your own or with a slimming club, and you are finding it difficult to stick to at the moment, a session or two of hypnotherapy will put you back on course!
The Other Side of the Coin
Of course, when we mention weight management, the natural thought is that of weight loss,but for some people the opposite is true.For some the uphill battle is to gain weight and Hypnotherapy can help with this too.So whether you have a poor apatite or just a busy lifestyle, hypnotherapy can set you on your way to choosing the right way of eating.
Contact Information
Share, Email & Print
DIAGNOSTIC PRIVATE ULTRASOUND SCAN SPECIALIST IN HERTFORD
Ultrasound Direct Hertford part of the UK’s leading diagnostic private ultrasound scan specialist, has been diagnosing and reassuring men and women since 1998.
Ultrasound Direct is a healthcare provider for private ultrasound scans and blood tests for all stages of pregnancy, women’s and men’s health. We offer convenient scan appointments at more than 80 clinics conveniently located across the UK.
We also offer a range of specialist musculoskeletal scans for tendons, muscles and ligaments investigation.
WHY CHOOSE ULTRASOUND DIRECT HERTFORD
High Standards - Registered with the Care Quality Commission – the independent regulator of health and social care in England.Qualified Health Professionals - All our sonographers are fully qualified to provide diagnostic ultrasounds for pregnancy, women’s and men’s health and follow the latest safety guidelines.Choice and Convenience - UK wide coverage with over 80 locations, largest range of diagnostic scans and able to book an appointment 24/7 online.
BE REASSURED…. BOOK A SCAN TODAYWhether you’re looking for reassurance or diagnostic results. Book online 24/7 for real-time appointment availability of Ultrasound Direct Hertford |
Foreign Affairs Committee Chairman Rep. Eliot Engel (D-N.Y.) on Monday issued a joint statement with his counterparts from several European allies condemning both President Donald Trump’s decision to withdraw U.S. troops from northeast Syria and the Turkish government’s subsequent military offensive in the region.
“We, the chairs of the Foreign Affairs Committees of the Parliaments of Germany, France, the United Kingdom, the European Parliament and the House of Representatives of the United States of America, jointly condemn in the strongest terms the Turkish military offensive in northeastern Syria,” the statement said. “We consider the intrusion as a military aggression and a violation of international law. The Turkish offensive is causing suffering for the local people who are forced to flee and a further instability in Syria and the neighboring region. We consider the abandonment of the Syrian Kurds to be wrong.”
“We deeply regret the decision of the President of United States to withdraw American troops from northeastern Syria which marks another landmark in the change of American foreign policy in the Near and Middle East. The turmoil caused by the Turkish offensive may contribute to a resurgence of Islamic terrorism and undermines years of effort and investment to bring stability and peace in this part of the world. Therefore, we hope the United States will take up its responsibility in Syria again,” the statement continued. “We consider the abandonment of the Syrian Kurds to be wrong. The Syrian Democratic Forces, our partner in the Global Coalition, massively contributed to the successful yet unfinished fight against Da’esh in Syria and incurred heavy losses by doing so.”
A joint statement with foreign leaders is a rare occurrence, and follows bipartisan criticism of Trump’s decision to abandon America’s Kurdish allies.
The statement was co-signed by chairman of the U.K. House of Commons Committee on Foreign Affairs Tom Tugendhat, chairman of the European Parliament Committee on Foreign Affairs David McAllister, chairman of the German Bundestag Committee on Foreign Affairs Norbert Rottgen, and Marielle de Sarnez, the chairwoman of the French National Assembly Committee on Foreign Affairs.
“This horrible war touches and affects the peoples of our countries in such an enormous way. For that reason, we, as members of our parliaments, feel compelled to making our common position clear. We unite across parties and nationalities to demonstrate our commitment to our common values, responsibility and interests,” the statement concluded.
President Trump defended the troop withdrawal during a cabinet meeting in front of reporters at the White House earlier Monday, saying he never agreed to protect the Kurds indefinitely.
“We never agreed to protect the Kurds for the rest of their lives,” Trump said. “We have a good relationship with the Kurds, but we never agreed to protect the Kurds. We have supported them for three and half to four years. We never agreed to protect the Kurds for the rest of their lives.”
[image via Drew Angerer/Getty Images]
Have a tip we should know? [email protected] |
1. Field of the Invention
The present invention generally relates to semiconductor integrated circuit devices and, more particularly, to a semiconductor integrated circuit device equipped with a test mode selecting circuit that detects a test mode voltage applied to a predetermined external-connection terminal and which test mode voltage is higher than a normal voltage applied thereto in a normal operation mode and sets internal circuits to a test mode.
2. Description of the Prior Art
An SRAM (Static Random Access Memory) device is known having a built-in test mode selecting circuit as described above. A conventional built-in test mode selecting circuit includes a plurality of MOS transistors. The operation of the test mode selecting circuit greatly depends on the characteristics of the MOS transistors, more particularly, the threshold voltages thereof. If there is a difference in the threshold voltages between SRAM devices, the respective test mode selecting circuits will operate in different manners. If the threshold voltages of the MOS transistors deviate from the designed threshold voltages, the selecting circuit formed by these MOS transistors will malfunction. A test mode voltage, higher than a normal voltage, applied to a predetermined terminal will fail to cause the test mode selecting circuit to set the internal circuits to the test mode. |
Q:
PHP MySQLi query - "Permission denied"
Im using PHP MySQLi to connect to MySQL and sometimes doing query i get error:
"Permission denied" error code: 2002.
The strange thing is that it happens for different query's and totally unpredictable. For example it might happen on third query after first two executed correctly.
I know it is not problem with MySQL because i moved it from one server to another and problem still persists. Most likely problem with PHP or interconnection between PHP and MySQL servers (they're on different machines)
Anyone got ideas?
EDIT:
query what gets "Permission denied" works if i restart script - its not permissions problem
ERROR:
[23-Apr-2011 19:00:02] PHP Warning:
mysqli::mysqli() [mysqli.mysqli]:
[2002] Permission denied (trying to
connect via
tcp://xxx.xxx.xxx.xxx:3306) in
/home/.../DB.php on line 19
[23-Apr-2011 19:00:02] PHP Warning:
mysqli::mysqli() [mysqli.mysqli]:
(HY000/2002): Permission denied in
/home/.../DB.php on line 19
A:
Same things happened on my environment. And the cause was SELinux.
You might be able to connect the database by executing php from command line
while you might not by executing on the web server.
In my case, I turned off SELinux.
# setenforce 0
Or you have to set up SELinux correctly.
|
Perinatal mental illness: definition, description and aetiology.
Perinatal mental illness is a significant complication of pregnancy and the postpartum period. These disorders include depression, anxiety disorders, and postpartum psychosis, which usually manifests as bipolar disorder. Perinatal depression and anxiety are common, with prevalence rates for major and minor depression up to almost 20% during pregnancy and the first 3 months postpartum. Postpartum blues are a common but lesser manifestation of postpartum affective disturbance. Perinatal psychiatric disorders impair a woman's function and are associated with suboptimal development of her offspring. Risk factors include past history of depression, anxiety, or bipolar disorder, as well psychosocial factors, such as ongoing conflict with the partner, poor social support, and ongoing stressful life events. Early symptoms of depression, anxiety, and mania can be detected through screening in pregnancy and the postpartum period. Early detection and effective management of perinatal psychiatric disorders are critical for the welfare of women and their offspring. |
{
"id": "garden-bench",
"name": "Garden Bench",
"games": {
"nh": {
"orderable": false,
"customizable": true,
"sellPrice": {
"currency": "bells",
"value": 4440
},
"recipe": {
"wood": 12,
"iron-nugget": 4
},
"variations": {
"black": "Black",
"green": "Green",
"navy-blue": "Navy blue",
"red": "Red",
"silver": "Silver",
"white": "White"
}
}
},
"category": "Furniture"
} |
ose 18*c + 1175 = 3929. Calculate the greatest common divisor of 17 and c.
17
Suppose z - 13 = -5*v + 1, -5*v = -5*z - 20. Suppose -v*b + 10 + 14 = 0. Calculate the greatest common factor of b and 8.
8
Let v(x) = x**2 - 14*x + 17. Let m be v(13). Suppose 0 = -m*a + 189 + 171. Calculate the highest common factor of a and 36.
18
Suppose 0 = -0*x - 2*x - 3*a + 4, -x - 2*a = -2. Suppose -2*j - x*r = -88, 2*j - 84 + 2 = r. What is the highest common factor of 105 and j?
21
Let z = -21 + 57. Let d = 0 + z. What is the greatest common factor of 24 and d?
12
Let s = -28 + 22. Let w be (-45)/(-51) + s/(-51). Calculate the greatest common factor of w and 8.
1
Let u be ((-384)/(-15))/(11 + (-3064)/280). What is the greatest common divisor of u and 7?
7
Let o be (-1 - -32) + (-20 - -13). Calculate the highest common factor of o and 78.
6
Suppose 33 = -5*b - 7. Let c be (2 + -6)*2/b. Let f be -1*(3 - 12/c). What is the greatest common factor of f and 3?
3
Let b be 55 - 0/(9 + -7). What is the highest common divisor of b and 55?
55
Suppose -17*a = -6093 + 5430. Suppose 0 = -5*v + 3 + 12. Calculate the highest common factor of v and a.
3
Let h be (-27)/(-1 + 2 + (-85)/70). Let o = 326 + -298. Calculate the highest common factor of o and h.
14
Let x(j) = 6*j**2 - 4*j + 8. Suppose 6*k = 5*k + 4. Let h be x(k). Calculate the greatest common factor of h and 22.
22
Let k = 89 + -81. Suppose 0 = -4*q + k*q - 12. What is the greatest common factor of q and 21?
3
Suppose 0 = -5*r + 3*z - 2 + 8, z - 3 = 0. Let k(t) = -t**2 + 17*t - 15. Let b be k(14). What is the greatest common divisor of b and r?
3
Let c(s) = 4*s - 1. Let p(a) = a**2 - 9*a. Let u be p(9). Suppose u = -4*d + 10 + 14. Let t be c(d). What is the greatest common divisor of 23 and t?
23
Let d = -4 + 5. Let v(o) = 11*o**3 + o. Let h be v(d). Suppose -j = 3*j - h. What is the highest common divisor of 33 and j?
3
Suppose 0*r + 12*r = 864. Calculate the greatest common factor of r and 8.
8
Let g = -1 + 1. Suppose f - 3*n + 5 = g, n - 4*n + 17 = 2*f. Suppose -45 = -s - f*s. Calculate the highest common factor of s and 9.
9
Suppose -17*r + 168 = -19. What is the highest common divisor of r and 7?
1
Suppose 7*m - 5 = 16. Calculate the highest common factor of m and 27.
3
Suppose 6 = 4*m - 30. Let i = 4097 - 4094. What is the highest common divisor of i and m?
3
Let d(o) = o**2 - o - 39. Let x be d(0). Let b be 1/((-3)/(x*1)). Let g be 1/4 + 415/4. What is the highest common factor of b and g?
13
Let k be (-110)/(-7) - 8/(-28). Let z(p) = -27*p - 94. Let d be z(-10). Calculate the highest common factor of k and d.
16
Let j be (-9)/6 - 365/(-10). Let t = j + -28. Calculate the highest common divisor of 49 and t.
7
Let r be (2/(-6))/(2/(-222)*1). Let l = 180 - r. Calculate the greatest common divisor of 13 and l.
13
Let k(s) = -3*s - 21. Let t be k(-22). What is the highest common factor of 180 and t?
45
Let r be 566/6 - (-4)/6. Suppose -2*l + 2*s + 3*s = -48, 0 = 3*l + 4*s - r. What is the highest common divisor of 116 and l?
29
Let s = 10 + 224. Let m be s/12 - 1/(-2). Suppose -4*i = i - 25. Calculate the greatest common divisor of m and i.
5
Suppose 3*h - q - 10 = 0, -4*q + 38 = 4*h - 2. Suppose 308 = -2*t + 3*t + 4*z, 0 = -h*t - 2*z + 1486. Calculate the greatest common divisor of 37 and t.
37
Suppose -3*t + 77 + 280 = 0. What is the highest common divisor of t and 63?
7
Let p be ((-27)/6)/(-9)*(3 - 1). Let y be (-3)/(p/(-208)*3). What is the highest common divisor of 13 and y?
13
Let s(u) = 3*u**2 - u. Let j be s(3). Suppose 0 = 2*r - 6*r - j. Let q(p) = -p**2 - 8*p + 6. Let i be q(r). What is the greatest common divisor of 45 and i?
9
Let k(d) = -d**3 - 18*d**2 + 45*d + 1. Let g be k(-20). Let h be (-32)/(-3) - (g/(-27) + -3). What is the greatest common divisor of h and 90?
10
Let n(x) = -3*x**2 + 4. Let b be n(-6). Let s = -60 - b. Let f(m) = m**3 + 18*m**2 - 10*m + 182. Let o be f(-19). What is the highest common divisor of o and s?
11
Let f(d) = 13*d + 24. Let p be f(3). Calculate the highest common divisor of 35 and p.
7
Let i(j) be the first derivative of j**4/4 + 22*j**3/3 + 2*j**2 + 48*j + 21. Let o be i(-21). Calculate the greatest common divisor of 45 and o.
45
Let w = -362 - -373. What is the highest common divisor of w and 2?
1
Let w = 1659 - 1652. What is the greatest common factor of w and 931?
7
Let f(s) = -2*s - 4. Let p be f(-4). Suppose 0*o + 118 = 2*o. Suppose w = -5*g + o + 125, 0 = 2*g + 5*w - 92. What is the highest common factor of p and g?
4
Let s(w) = 84*w**2 - w - 8. Let v be s(-2). Calculate the greatest common factor of 33 and v.
33
Let h = 25 - -13. What is the highest common divisor of 152 and h?
38
Suppose 73 = l - 2*u + 4*u, u + 247 = 4*l. What is the greatest common divisor of l and 84?
21
Suppose 4*s - 119 - 187 = 3*o, 0 = -2*o + 4*s - 200. Let w = 39 - o. Calculate the greatest common divisor of 29 and w.
29
Let u be ((-68)/(-10))/((-6)/120*-4). Suppose u*a - 100 = 33*a. Calculate the greatest common factor of 25 and a.
25
Let f = -22 - -286. What is the greatest common divisor of f and 66?
66
Let l = -320 - -344. Let r be 39 - 2 - (2 - 1). What is the highest common divisor of r and l?
12
Suppose -33*i + 29*i = -7276. What is the greatest common factor of 17 and i?
17
Suppose 6 = -2*i, 6 = 2*t - 2*i - 4. Suppose 4*s + 13 = t*n - 3, -6 = 3*s. Let b be (-36 - n)*1/(-2). What is the highest common factor of b and 60?
20
Suppose 3*x - 5*x - 250 = 5*z, -4*x = 4*z + 188. Let y = -38 - z. Suppose 3*p - 74 = -11. What is the greatest common factor of p and y?
7
Suppose -15*y = -17*y + 10. Suppose 0 = -m - 5*d + 52, -3*m = -0*m + y*d - 106. Suppose 242 = w - 55. Calculate the highest common divisor of w and m.
27
Let q be 2 + 22/(-10) + 615/75. Calculate the greatest common divisor of 26 and q.
2
Let g = 524 + -521. Let p(l) = 6*l**2 + l + 2. Let j be p(-2). What is the greatest common divisor of g and j?
3
Let o(z) = 23*z**2 + 7*z - 10. Let y be o(2). Calculate the highest common factor of y and 3.
3
Suppose -5*h - l - 103 = 0, l - 4 = -2*h - 47. Let d be (-664)/(-22) - h/(-110). What is the highest common divisor of d and 40?
10
Suppose 37*w + 8 = 41*w. Let z be (-7)/2*(-2 - 6 - w). What is the highest common factor of 315 and z?
35
Suppose -51 = 14*a - 275. What is the highest common divisor of 1 and a?
1
Let d = 15 + -21. Let v(m) = 4*m**2 - m + 12. Let k be v(d). Let h = -45 + k. What is the highest common factor of h and 13?
13
Let b be -2*3/27 + 3089/9. Suppose 0 = -3*y - 2*a + 155, 23*y - 21*y + 4*a - 114 = 0. Calculate the greatest common divisor of b and y.
49
Let z be (-10)/(-15) + 28/18*33. Let j = 67 - z. What is the highest common factor of 60 and j?
15
Let c(m) = 110*m + 718. Let q be c(-6). What is the highest common divisor of q and 551?
29
Suppose -4*p = -0*p + 4*h - 64, 3*h - 65 = -4*p. Suppose -18*t + 19 = -p*t. What is the greatest common divisor of 95 and t?
19
Let f be 4*(-1 + 6/4). Suppose -126 = -f*w - 16. Suppose -4*d + 126 = 2*j + 3*j, -4 = -d. Calculate the highest common factor of w and j.
11
Let a = 4467 - 4465. Suppose -2*o - 25 + 93 = 0. What is the highest common divisor of a and o?
2
Suppose -3529 = -4*l - 3*b, -5*l + b + 2069 = -2328. Calculate the greatest common factor of l and 165.
55
Suppose -4*z = -0*z - 616. Let p = -73 - -87. Calculate the highest common factor of z and p.
14
Let p = -11 - -25. Suppose -392 = -12*z - 2*z. Calculate the greatest common divisor of z and p.
14
Let u be (-262)/(-4) + (-2)/4. Let f(y) = -y**2 - 12*y - 10. Let z be f(-6). What is the greatest common divisor of u and z?
13
Let m be 14/(-70) - (-6904)/20. Calculate the highest common divisor of 138 and m.
69
Let o(s) = 3*s**2 - 9*s - 16. Let b be o(7). Suppose a - 16 = -3*a - x, 3*a - 3*x - 12 = 0. Calculate the highest common divisor of a and b.
4
Suppose y = 2*w + 9 - 3, -2*y + 48 = 5*w. Calculate the greatest common factor of y and 1442.
14
Suppose 4*t - 3*u = 2*u + 166, -t = 4*u - 31. Let z = -19 + t. Let d(c) = c**2 - 6*c - 8. Let p be d(8). What is the highest common divisor of p and z?
4
Suppose 4*w = 4 + 92. Suppose 14*b = 18*b - 12. What is the greatest common factor of w and b?
3
Let s(g) = 5*g + 38. Let t = 96 + -96. Let z be s(t). What is the hig |
-- boundary3.test
--
-- db eval {
-- SELECT t1.a FROM t1 JOIN t2 ON t1.rowid < t2.r
-- WHERE t2.a=37
-- ORDER BY x
-- }
SELECT t1.a FROM t1 JOIN t2 ON t1.rowid < t2.r
WHERE t2.a=37
ORDER BY x |
Laboratory simulations of the mixed solvent extraction recovery of dominate polymers in electronic waste.
The recovery of four dominant plastics from electronic waste (e-waste) using mixed solvent extraction was studied. The target plastics included polycarbonate (PC), polystyrene (PS), acrylonitrile butadiene styrene (ABS), and styrene acrylonitrile (SAN). The extraction procedure for multi-polymers at room temperature yielded PC, PS, ABS, and SAN in acceptable recovery rates (64%, 86%, 127%, and 143%, respectively, where recovery rate is defined as the mass ratio of the recovered plastic to the added standard polymer). Fourier transform infrared spectroscopy (FTIR) was used to verify the recovered plastics' purity using a similarity analysis. The similarities ranged from 0.98 to 0.99. Another similar process, which was denoted as an alternative method for plastic recovery, was examined as well. Nonetheless, the FTIR results showed degradation may occur over time. Additionally, the recovery cost estimation model of our method was established. The recovery cost estimation indicated that a certain range of proportion of plastics in e-waste, especially with a higher proportion of PC and PS, can achieve a lower cost than virgin polymer product. It also reduced 99.6%, 30.7% and 75.8% of energy consumptions and CO2 emissions during the recovery of PC, PS and ABS, and reduced the amount of plastic waste disposal via landfill or incineration and associated environmental impacts. |
Q:
Cannot read property 'type' of undefined (ngrx)
I am using ngrx. I got error
Cannot read property 'type' of undefined
This is part of my codes:
@Effect() foo$ = this.actions$
.ofType(Actions.FOO)
.withLatestFrom(this.store, (action, state) => ({ action, state }))
.map(({ action, state }) => {
if (state.foo.isCool) {
return { type: Actions.BAR };
}
});
A:
This issue is little tricky since it is not easy to locate the issue based on the error.
In this situation, when state.foo.isCool is false, no action is returned.
So changing to something like this will solve the issue:
@Effect() foo$ = this.actions$
.ofType(Actions.FOO)
.withLatestFrom(this.store, (action, state) => ({ action, state }))
.map(({ action, state }) => {
if (state.foo.isCool) {
return { type: Actions.BAR };
}
return { type: Actions.SOMETHING_ELSE };
});
|
package com.jomeslu.sample;
import android.content.Context;
import android.support.test.InstrumentationRegistry;
import android.support.test.runner.AndroidJUnit4;
import org.junit.Test;
import org.junit.runner.RunWith;
import static org.junit.Assert.*;
/**
* Instrumentation test, which will execute on an Android device.
*
* @see <a href="http://d.android.com/tools/testing">Testing documentation</a>
*/
@RunWith(AndroidJUnit4.class)
public class ExampleInstrumentedTest {
@Test
public void useAppContext() throws Exception {
// Context of the app under test.
Context appContext = InstrumentationRegistry.getTargetContext();
assertEquals("com.jomeslu.sample", appContext.getPackageName());
}
}
|
926 P.2d 1168 (1996)
Robert Patrick KELLY, Appellant,
v.
Lisa Ann KELLY, Appellee.
No. S-7376.
Supreme Court of Alaska.
November 22, 1996.
William T. Ford, Anchorage, for Appellant.
James J. Hanlon, Taylor & Hanlon, P.C., Anchorage, for Appellee.
Before COMPTON, C.J., and RABINOWITZ, MATTHEWS, EASTAUGH and FABE, JJ.
OPINION
MATTHEWS, Justice.
I. FACTS & PROCEEDINGS
Robert and Lisa Kelly were married on May 3, 1992. Both were long time residents of Dutch Harbor. The only child of the marriage, Ryan Patrick Kelly, was born on December 18, 1992.
Robert filed for divorce in May 1994. He requested sole custody of Ryan or, in the alternative, joint physical and legal custody.
Robert currently works as a longshoreman and earns over $60,000 per year. Lisa's employment has been sporadic and significantly less remunerative. Following the divorce, Robert intends to remain in Alaska, while Lisa intends to move out of state.
Trial in this case was held for three days. On September 6, 1995, Judge Card issued revised findings of fact and conclusions of law regarding child custody and visitation. Judge Card found that Robert and Lisa both are "good parents." He also found that Lisa cannot, consistent with her current lifestyle, provide Ryan with a stable home. On September 6, 1995, the superior court issued the following custody order:
Until Ryan begins Kindergarten [approximately three years after the trial *1169 date], he shall reside with each parent for alternating six month periods, beginning with the father, Robert Kelly, on July 1, 1995, and extending through Christmas of 1995 until approximately December 31. At that time, and provided Lisa Kelly has established a stable environment in which to care for Ryan, her six months visitation shall commence. .. .
....
After Ryan begins Kindergarten, and if the parents are living greater than 200 miles apart and provided Lisa Kelly can provide Ryan with a stable environment, then Lisa Kelly shall [have custody except during the Christmas break, spring break, and the summer months].
The superior court also ordered Robert to pay $10,000 of Lisa's $13,418.65 in outstanding attorney's fees. Robert contests the superior court's decisions regarding child custody and attorney's fees.
II. DISCUSSION
A. Divided Custody
Although some courts have awarded divided physical custody, those cases, in contrast to the situation in this case, typically involve parents who reside in the same community.[1] Where parents reside in different parts of the country, an award of divided physical custody may foreclose "a stable environment and the development of permanent associations...." John P. McCahey et al., Child Custody and Visitation Law and Practice, § 13.04(2)(1993). Against this backdrop, Robert argues that the divided custody award in this case was an abuse of the court's discretion.
Although we regard the question as close, we do not find an abuse of discretion on this record. No evidence was presented that the arrangement was likely to prove harmful to Ryan. Robert requested divided custody, in preference to primary custody in Lisa. Finally, divided custody will terminate when Ryan reaches school age.[2]
B. Permanent Custody
Robert argues that the superior court erred in ordering that when Ryan enters school Lisa will have custody during the school year and Robert will have custody during the summer months. A review of the superior court's findings and conclusions reveals certain findings which could be regarded as a basis for preferring Robert as primary custodian (greater stability, presence of extended family network in Dutch Harbor), a number of findings which apply equally to Robert and Lisa, but no findings which support the court's award of primary custody to Lisa. Accordingly, the court's award of permanent custody to Lisa after Ryan enters school is vacated and this aspect of the case is remanded. The court is instructed to enter findings supporting the award of primary custody to Lisa, or, if the court determines that its decision cannot be supported on the present record, the court may award primary custody to Robert, or conduct such additional proceedings as the court determines to be appropriate. We retain jurisdiction with respect to this issue.
*1170 C. Attorney's Fees
The trial court's award of attorney's fees to Lisa accurately recognizes the substantial disparity between the earning capacity and economic situations of the parties. The award was thus in accordance with our cases[3] and was not an abuse of discretion.
III. CONCLUSION
This case is AFFIRMED in part, VACATED in part, and REMANDED for further proceedings in accordance with this opinion.
NOTES
[1] See, e.g., Lapp v. Lapp, 293 N.W.2d 121 (N.D. 1980) (Affirming an alternating six-month arrangement for a seven-year-old girl. The court specifically noted that both parents lived in the same community.); Beck v. Beck, 86 N.J. 480, 432 A.2d 63, 69 n. 7 (1981) (Affirming an alternating four-month arrangement. The court stated that the "geographic proximity of the two homes is an important factor" in awarding alternating custody. Id. 432 A.2d at 72.); Peyton v. Peyton, 457 So.2d 321, 323 (La. Ct. App. 1984) (Affirming an order of joint custody in which a four-year-old girl alternated three-month periods with her mother and father. The court noted that the child would benefit from contact with both parents and said the degree of possible disruption to the child would be relatively low since the parents lived across the street from each other.); In re Marriage of Ryan, 222 Mont. 188, 720 P.2d 691 (1986) (Affirming an order of joint custody in which the child alternated weeks between the parents' homes. The order also provided that if one parent moved away, the child would remain with the parent who stayed in the community. The order was affirmed, even though the mother wanted to move with the child.).
[2] These reasons also lead us to conclude that this is an inappropriate case in which to decide whether to adopt a general presumption disfavoring divided custody when the parents do not reside in the same community.
[3] We have repeatedly stated that the cost and fee awards in a divorce are to be based primarily on the relative economic situations and earning powers of the parties. See, e.g., Lone Wolf v. Lone Wolf, 741 P.2d 1187, 1192 (Alaska 1987); Cooke v. Cooke, 625 P.2d 291, 293 (Alaska 1981); Johnson v. Johnson, 564 P.2d 71, 76-77 (Alaska 1977).
|
Smart contracts are one of technology’s current buzz words. Much is said about them, much is written about them and there is an increasing acceptance that they will soon officially confirm their position as the next big thing. However, who really understands them. Who has taken the step back and asked what their real purpose is, what they really are and who can benefit from them?
As with all new ideas, a lot of what’s being said and written isn’t actually correct. We thought it may be beneficial if we used our experience in this very specialist area to shatter five of the most common myths surrounding smart contracts.
A smart contract is a contract
No it isn’t. Although the term ‘smart contract’ is now in common parlance, it’s totally misleading. It would be much more accurate to call it a smart automated computer code. The use of the term smart contract has led many people to believe this is something that can assist their business, could be a contract set up on the blockchain to be used as a precedent for terms of business etc. This is not what a smart contract is (at this stage in any event).
A smart contract is smart
Sorry, that’s another no. In fact we should revise the previous point; if we were being pedantic we’d also drop the smart and just call it an automated computer code. Smart contracts aren’t smart in the true sense. They don’t rely on artificial intelligence. They rely totally on the architecture put in place by the developers and their solicitors. Maybe ‘potentially useful automated computer code’ would be a more accurate description.
Arguably smart contracts are fit for purpose
OK, there is lots of buzz about smart contracts but, in their current form, can they do what they’re set up to do? If you are considering using smart contracts then we’d assume you are in a position where you need to enter into a series of commercial contracts with a number of customers and/or suppliers. If so those contracts must afford you the highest levels of legal protection so, at this stage at least, a traditional contract may actually be the smart option.
Smart contracts can’t be enforced in a court of law
Any contract can be enforced in law. As explained above, the definition of smart contract is misleading. It is hard initially to understand how or why anyone would want to sue in relation to automated computer code. However, when thinking deeper, each smart contract is based upon a heads of agreement (presumably drafted by a lawyer or the parties to the agreement) the structure will clearly set out how it is to perform. If the computer code does not perform, then of course, their would be a breach of contract between the developer and the client who required the smart contract. Another point, if automated, how does it stop? There have to be mechanisms in place for the client or the developer to pull the plug. If there are not (or if for example, the developer was the party with the power to stop the smart contract from working and refused to) then the client would have the right to go to court for injunctive relief.
Therefore, whilst computer code itself cannot be sued, the parties to the initial agreement for development certainly can.
Similarly in order to work, each contract will also need to identify the parties involved, their jurisdictions and their responsibilities. All of this information can be used by a solicitor to resolve a dispute should it arise.
Smart contracts will continue unlegislated and unregulated
As with any new technology or idea, ultimately, legislation and regulations will follow. There is much talk in the industry now about regulating digital currency exchanges. To this end, get ready for the Smart Contracts Act 2025 now! With so much potential revenue at stake the framework will have to be supported by legislation in the same way as every other part of a commercial vehicle is. It is also highly likely this legislation will reflect legal precedents being established now.
However attractive bringing new technology into your business may be, smart contract may not be the answer in their current state or, indeed, under its current name. This technology is new. It will develop, it will change. Will it be widely adopted though? The answer is unknown at this stage,
Selachii are recognised as one of the leading dispute resolution specialists within the fields of smart contracts, Bitcoin and blockchain technology. Partner Richard Howlett regularly provides commentary for the sector’s leading publications and for leading media outlets including the BBC and speaks on the legal issues affecting these growing commercial technologies. |
174 P.3d 11 (2007)
In the Matter of the DEPENDENCY OF A.K.
In the Matter of the Dependency of M.H.-O.
In the Matter of the Dependency of Y.H.
No. 78426-4.
Supreme Court of Washington, En Banc.
Argued February 13, 2007.
Decided December 20, 2007.
*13 Gregory Charles Link, Washington Appellate Project, Seattle, for Petitioners.
Sheila Malloy Huber, Stephen H. Hassett, Office of the Attorney General, Olympia, for Respondent.
Nancy Lynn Tainer, Sherri Wolson, ACLU of Washington Foundation, Seattle, for Amicus Curiae (ACLU).
Stephen Alan Smith, Kari Lee Vander Stoep, Kirkpatrick & Lockhart Preston Gates Ell, Seattle, Tammy Seltzer, Bazelon Center for Mental Health Law, Washington, DC, for Amicus Curiae (American Academy of Child and Adolescent Psychiatry).
Stephen Alan Smith, Kari Lee Vander Stoep, Kirkpatrick & Lockhart Preston Gates Ell, Seattle, Jennifer Mathis, Bazelon Center for Mental Health Law, Washington, DC, for Amicus Curiae (Bazelon Center for Mental Health Law).
Justin Dolan, Garvey Schubert Barer, Seattle, for Amicus Curiae (Children's Alliance).
Beth Ann Colgan, Casey Trupin, Columbia Legal Services/Institutions Project, Seattle, Patricia J. Arthur, National Center for Youth Law, Oakland, CA, Anne Aiping Lee, Brent M. Pattison, TeamChild, Kimberly Dawn Ambrose, University of Washington School of Law, Seattle, for Amicus Curiae (Columbia Legal Services).
Stephen Alan Smith, Kari Lee Vander Stoep, Kirkpatrick & Lockhart Preston Gates Ell, Seattle, for Amicus Curiae (Federation of Families for Children's Mental Health, Mental Health America, National Alliance on Mental Illness, and National Council for Community Behavioral Healthcare).
Patricia J. Arthur, National Center for Youth Law, Oakland, CA, for Amicus Curiae (National Center for Youth Law).
Nancy Lynn Sapiro, Seattle, for Amicus Curiae (Northwest Women's Law Center).
Beth Ann Colgan, Casey Trupin, Columbia Legal Services/Institutions Project, Seattle, for Amicus Curiae (TeamChild).
*14 ALEXANDER, C.J.
¶ 1 Petitioners M.H.-O. and Y.H. are teenage girls who ran away multiple times from foster homes in which they had been placed. The juvenile court found each of them in contempt of court for running away and used its "inherent contempt power" to order each of them to spend 30 to 60 days in detention. Petitioners ask this court to reverse the Court of Appeals decision affirming the juvenile court orders. They argue that the Court of Appeals erred in concluding that the juvenile court has the inherent power to impose punitive sanctions on a youth for indirect contempt. Although we disagree with petitioners' assertion, we conclude that the juvenile court improperly resorted to the use of its inherent power in this case. We, therefore, reverse the Court of Appeals.
I
A.Y.H.
¶ 2 In 2001, Y.H. was found by the Yakima County Juvenile Court to be a dependent child. Consequently, she was placed in foster care. Y.H. ran away from the foster home at least six times in 2003 and 2004. The first time she ran away, Yakima County Juvenile Court Commissioner Robert Inouye warned her that she needed to stay in the foster home in which she had been placed. After subsequent runs, Y.H. was found in contempt and sentenced to three to seven days in detention, with the option to purge her contempt by writing an essay and promising not to run away again. After her fourth disappearance, Y.H. was also moved to a new foster home. The fifth time Y.H. ran away, Commissioner Inouye warned her that if she ran again he might have to resort to the court's inherent contempt power in order to impose greater sanctions.
¶ 3 Finally, after the sixth time Y.H. ran away, respondent, Department of Social and Health Services (DSHS), asked the juvenile court to exercise its inherent contempt power and impose a punishment greater than the statutory remedy of up to seven days in juvenile detention with an option to gain earlier release by purging the contempt. Commissioner Inouye conducted a hearing on DSHS's request and heard testimony from witnesses. He subsequently sentenced Y.H. to 30 days in detention for contempt, without the opportunity to purge the contempt. He found:
If we continue to use [the] Becca procedure,[[1]] [Y.H.] will continue to make empty promises and continue to run and place her self at serious risk.
. . . .
[Y.H.]'s disobedience to the court orders has escalated in severity over time, rather than lessening in response to the Becca contempt sanctions.
[Y.H.]'s mother believes that the Becca sanctions are inadequate to change [Y.H.]'s behavior, and that something different should be tried, if another run is to be avoided.
There is reason to believe that an inherent contempt disposition will likely have coercive effect on [Y.H.]. It will become clear to [Y.H.] that continued future decisions to violate court orders may have much more serious consequences. It will give her a period of time to stabilize without the adverse influences which she seeks while one [sic] the run. It will not give her the opportunity to run again the next day after her contempt hearing (as she did on 8-30-03).
The stakes are high at this point[. Y.H.] appears headed for a very dangerous life style which includes gangs, drugs and sex to the exclusion of stability, safety and education. . . . We are risking a catastrophe with her future if we are unable to *15 formulate an adequate response to her bad choices.
Clerk's Papers (CP) (23252-2-III) at 73-74.
¶ 4 Y.H. moved for revision of the order. A judge of the Yakima County Superior Court upheld the commissioner's use of inherent contempt power to impose a 30-day sentence. Y.H. then appealed to the Court of Appeals.
B.M.H.-O.
¶ 5 Like Y.H., M.H.-O. was found by the Yakima County Juvenile Court to be a dependent child and placed in foster care. She also ran away from her placement at least six times in 2003 and 2004, two of these times within a day of promising the court she would not run again. After each of the first four of these disappearances, the juvenile court found M.H.-O. in contempt and sentenced her to four to seven days in detention, with the option to purge her contempt. Once, she was released after merely promising not to run again. After the third time M.H.-O. ran, Commissioner Inouye warned her that he might have to resort to the court's inherent contempt power to impose greater sanctions if she ran again.
¶ 6 The fifth time M.H.-O. ran away, DSHS moved for the juvenile court to exercise its inherent contempt power. Commissioner Inouye set a trial date, advised the parties that the contempt must be proved beyond a reasonable doubt, and warned M.H.-O. that detention imposed under inherent contempt power could last until she turned 18 and carried no option to purge the contempt. M.H.-O. stipulated "that she had run as alleged," in exchange for a recommended sentence of 30 days. CP (23211-5-III) at 86. Sentencing M.H.-O. to 30 days, with no option to purge the contempt, Commissioner Inouye found:
[M.H.-O.] has repeatedly promised not to run, and repeatedly broken those promises. Given this recent history, a new purge promise not to run could not be believed.
There is reason to believe that an inherent contempt consequence with no purge option could achieve what a purgeable 7 days of civil contempt consequence could not. It will afford [M.H.-O.] a longer period of time to stabilize under the influence of a "home" where she is not on the streets and on the run. It will give her an opportunity to reflect and become more accustomed to a lifestyle which includes school and continuity.
Id. at 87.
¶ 7 A week after M.H.-O. was released from detention, she ran away again. DSHS again moved for the court to use its inherent contempt power to impose an appropriate sanction upon M.H.-O. After being advised of the "potential consequences" of the contempt motion "and of her rights," M.H.-O. again stipulated that she had violated a court order by running away. Id. at 76. In determining a proper sanction for M.H.-O.'s sixth contempt, Commissioner Inouye stated:
This court has attempted to persuade [M.H.-O.], through use of the usual civil contempt remedies, to begin following court orders and live in a safe manner. These efforts have failed, repeatedly.
. . . .
[M.H.-O.] has repeatedly demonstrated that this limited consequence does not deter her from choosing to run. . . .
. . . .
Hopefully [M.H.-O.] will grow out of this phase, before she suffers further serious or irreparable harm. Eventually a civil contempt sanction may have some actual coercive effect for her. . . .
At present, the court is unable to assure the basic safety of [M.H.-O.] without resort to the inherent contempt powers[.] The legislatively provided tools have proven inadequate.
Id. at 78. Commissioner Inouye went on to explain that there was "a reasonable basis for believing that some other specific period of detention will achieve what seven days will not," because M.H.-O. was "asking for help with in-patient drug treatment," and he believed that "[a] more extended period of time under the auspices of juvenile detention would give a more significant opportunity for her to experience being drug free in a more structured environment including an education *16 component." Id. at 79. Commissioner Inouye decided to give M.H.-O. a "more extended sentence" than he had prior to that time, because "that prospect is likely to have a greater deterrent effect." Id. Accordingly, he sentenced M.H.-O. to 60 days in detention, with no purge option.
¶ 8 M.H.-O. moved for revision of both orders. A judge of the Yakima County Superior Court upheld the commissioner's use of inherent contempt power to impose both the 30-day and 60-day sentences. M.H.-O. appealed to the Court of Appeals.
C. Court of Appeals Decision
¶ 9 The Court of Appeals essentially consolidated Y.H.'s and M.H.-O.'s appeals, along with a similar appeal by a third juvenile, A.K.[2] That court concluded that the juvenile court possesses the inherent power to impose a punitive sanction, such as the determinate sentences in this case, for indirect contempt of court. In re Dependency of A.K., 130 Wash.App. 862, 867, 125 P.3d 220 (2005). It ruled, however, that this power can be used only when the juvenile court finds (1) "that the statutory remedy is inadequate to meet the juvenile's needs" and (2) "that a different period of detention is necessary." Id. The Court of Appeals further determined that criminal due process protections must be afforded in a punitive contempt proceeding, including
notice of the charges, a reasonable opportunity to respond, the presumption of innocence, the right to have guilt proved beyond a reasonable doubt, the right to refuse to testify, the right to call witnesses and to cross-examine, the assistance of counsel, and the right to a trial before an unbiased judge.
Id. at 878, 125 P.3d 220 (citing Young v. United States ex rel. Vuitton et Fils S.A., 481 U.S. 787, 798-99, 107 S.Ct. 2124, 95 L.Ed.2d 740 (1987); In re Winship, 397 U.S. 358, 368, 90 S.Ct. 1068, 25 L.Ed.2d 368 (1970)).
¶ 10 Applying these standards, the Court of Appeals concluded that the order relating to A.K. was deficient, because it failed to "specifically state the reasons why the juvenile court decided that the statutory civil remedial sanctions were inadequate" and why "a determinate sentence without the opportunity to purge would better address [her] contempt." Id. at 886, 125 P.3d 220. Accordingly, A.K.'s order was vacated.[3] The Court of Appeals also vacated one of the orders relating to M.H.-O., on the basis that she stipulated to facts without being informed of all her due process rights. The Court of Appeals affirmed the other two orders, which are now before us on review.
II
A. Mootness
¶ 11 This case is technically moot, petitioners having each served the sentence imposed for contempt. In re Det. of Swanson, 115 Wash.2d 21, 24, 793 P.2d 962, 804 P.2d 1 (1990). Consequently, effective relief cannot be afforded to either of them. Moreover, petitioners are now over the age of 18 and no longer subject to the jurisdiction of the juvenile court.
¶ 12 However, "[t]his court may decide a moot case if it involves matters of continuing and substantial public interest." Id. To determine "whether or not a sufficient public interest is involved," this court looks at three criteria: "`(1) the public or private nature of the question presented; (2) the desirability of an authoritative determination which will provide future guidance to public officers; and (3) the likelihood that the question will recur.'" Id. at 24-25, 793 P.2d 962, 804 P.2d 1 (quoting Dunner v. McLaughlin, 100 Wash.2d 832, 838, 676 P.2d 444 (1984)).
¶ 13 This consolidated case meets each of the three criteria. Although the due *17 process rights of juveniles are individual rights, the public has a great interest in the care of children and the workings of the foster care system. See, e.g., In re Interest of M.B., 101 Wash.App. 425, 433, 3 P.3d 780 (2000). The authority of the courts is similarly a public matter. In re Cross, 99 Wash.2d 373, 377, 662 P.2d 828 (1983). A determination of how the courts' inherent power interacts with the statutory contempt scheme will provide useful guidance to judges. Finally, the Court of Appeals noted in this case that the "exercise of inherent contempt authority to force compliance with placement orders is likely to recur," making "[c]larification of the court's authority to exercise inherent contempt power . . . a matter of continuing public interest." A.K., 130 Wash.App. at 870 n. 4, 125 P.3d 220. We agree. This case alone involved four such exercises of inherent contempt power in less than two months. The fact that we have been presented with a number of amicus curiae briefs speaks to the substantial public interest. Thus, we consider it appropriate to review this case.
B. Inherent Contempt Power of the Juvenile Court
¶ 14 "Contempt of court" is intentional:
(a) Disorderly, contemptuous, or insolent behavior toward the judge while holding the court, tending to impair its authority, or to interrupt the due course of a trial or other judicial proceedings;
(b) Disobedience of any lawful judgment, decree, order, or process of the court;
(c) Refusal as a witness to appear, be sworn, or, without lawful authority, to answer a question; or
(d) Refusal, without lawful authority, to produce a record, document, or other object.
RCW 7.21.010(1). Contempt may be direct, occurring in the court's presence, or indirect, occurring outside of court. Int'l Union, United Mine Workers of Am. v. Bagwell, 512 U.S. 821, 827 n. 2, 114 S.Ct. 2552, 129 L.Ed.2d 642 (1994). A court's authority to impose sanctions for contempt is a question of law, which we review de novo. See M.B., 101 Wash.App. at 454, 3 P.3d 780.
¶ 15 Because contempt of court is disruptive of court proceedings and/or undermines the court's authority, courts are vested with "an inherent contempt authority, as a power `necessary to the exercise of all others.'" Bagwell, 512 U.S. at 831, 114 S.Ct. 2552 (quoting United States v. Hudson, 11 U.S. (7 Cranch) 32, 34, 3 L.Ed. 259 (1812)) (citations omitted). Inherent contempt power is separate from statutorily granted contempt power. State v. Ralph Williams' N.W. Chrysler Plymouth, Inc., 87 Wash.2d 327, 335, 553 P.2d 442 (1976); Keller v. Keller, 52 Wash.2d 84, 86, 323 P.2d 231 (1958). It is "created by the constitution, . . . comes into being upon the very creation of . . . a court and remains with it as long as the court exists." Blanchard v. Golden Age Brewing Co., 188 Wash. 396, 423, 63 P.2d 397 (1936). The inherent contempt power "is lodged permanently with [the court], and the legislature may not, by its enactments, deprive the court of that power or curtail its exercise." Id. at 424, 63 P.2d 397; see also Mead Sch. Dist. No. 354 v. Mead Educ. Ass'n, 85 Wash.2d 278, 287, 534 P.2d 561 (1975); State v. Estill, 55 Wash.2d 576, 579, 349 P.2d 210 (1960). The legislature may only "regulate that power," and only "as long as it does not diminish it so as to render it ineffectual." Mead Sch. Dist., 85 Wash.2d at 287, 534 P.2d 561 (citing Carter v. Commonwealth, 96 Va. 791, 32 S.E. 780 (1899)). This inherent authority allows courts to impose sanctions upon the contemnor, after appropriate due process protections are provided.
¶ 16 Due process requirements vary depending on whether the contempt is direct or indirect and whether the sanctions imposed are remedial or punitive in nature. See Bagwell, 512 U.S. at 831, 114 S.Ct. 2552. A "remedial sanction" is one that is "imposed for the purpose of coercing performance when the contempt consists of the omission or refusal to perform an act that is yet in the person's power to perform." RCW 7.21.010(3). It is considered civil, rather than criminal, in nature. Bagwell, 512 U.S. at 827, 114 S.Ct. 2552. A "punitive sanction," on the other hand, is "imposed to punish a *18 past contempt of court for the purpose of upholding the authority of the court," RCW 7.21.010(2), and it is considered criminal in nature, Bagwell, 512 U.S. at 828, 114 S.Ct. 2552. In determining whether sanctions are punitive or remedial, courts look not to the "stated purposes of a contempt sanction," but to whether it has a coercive effect whether "the contemnor is able to purge the contempt and obtain his release by committing an affirmative act." Id.
¶ 17 Due process requirements do not prevent the use of inherent contempt power; they merely limit its exercise. In delineating the process required when exercising this authority, the United States Supreme Court has differentiated between three types of use: (1) imposition of remedial sanctions for direct contempt, (2) imposition of remedial sanctions for indirect contempt, and (3) imposition of punitive sanctions for direct or indirect contempt. Id. at 832-33, 114 S.Ct. 2552. Different procedural protections are required for each of these three types of cases,[4] but due process does not prevent the court from exercising its inherent contempt power in any of those three ways. See id. Contrary to petitioners' claim, a court may use its inherent power to impose punitive sanctions for indirect contempt without violating the due process clauses of the United States Constitution.
¶ 18 In the present case, a juvenile court commissioner exercised this power. The juvenile court is a division of the superior court. State v. Werner, 129 Wash.2d 485, 492, 918 P.2d 916 (1996); RCW 13.04.021(1). As such, it possesses the inherent power granted to the superior court under our constitution. See Const. art. IV, §§ 5-6; see also State v. Martin, 36 Wash. App. 1, 4, 670 P.2d 1082 (1983), rev'd on other grounds, 102 Wash.2d 300, 684 P.2d 1290 (1984). Thus, the juvenile court, like other courts, possesses inherent power to sanction direct or indirect contempt by punitive or remedial sanctions. In Washington's court system, a juvenile court commissioner has "the power, authority, and jurisdiction, concurrent with a juvenile court judge, to hear all cases under this chapter and to enter judgment and make orders with the same power, force, and effect as any judge of the juvenile court." RCW 13.04.021(1); see also Const. art. IV, § 23. Consequently, the court commissioner issuing the inherent contempt orders in this case had the inherent power that is possessed by a superior court judge.
C. Limitations on the Exercise of Inherent Contempt Power
¶ 19 While inherent contempt authority is a critical component of judicial power, its use is only appropriate in limited situations. We have long held that courts may not exercise their inherent contempt power "[u]nless the legislatively prescribed procedures and remedies are specifically found inadequate." Mead Sch. Dist., 85 Wash.2d at 288, 534 P.2d 561 (citing State ex rel. Curtiss v. Erickson, 66 Wash. 639, 642, 120 P. 104 (1912), aff'd on other grounds by Carlson v. Washington, 234 U.S. 103, 34 S.Ct. 717, 58 L.Ed. 1237 (1914); State ex rel. Dye v. Rielly, 40 Wash. 217, 220, 82 P. 287 (1905)); see also State v. Boatman, 104 Wash.2d 44, 48, 700 P.2d 1152 (1985); State v. Browet, Inc., 103 Wash.2d 215, 218, 691 P.2d 571 (1984). "Only under the most egregious circumstances should the juvenile court exercise its contempt power to incarcerate a status offender in a secure facility. If such action is necessary, the record should demonstrate that all less restrictive alternatives have failed." State v. Norlund, 31 Wash.App. 725, 729, 644 P.2d 724, review denied, 98 Wash.2d 1013 (1982); see also In re Pers. Restraint of King, 110 Wash.2d 793, 802, 756 P.2d 1303 (1988).
¶ 20 In this case, the juvenile court commissioner did not specifically find that one of the statutory remedies available to him was *19 inadequate: criminal contempt of court, under RCW 7.21.040. Under that statute, a court may impose punitive sanctions of up to $5,000, up to one year imprisonment, or both on adult contemnors, after certain procedures are followed. RCW 7.21.040(5). Juvenile status offenders[5] can also be sanctioned criminally for contempt. State v. A.L.H., 116 Wash.App. 158, 162, 163-64, 64 P.3d 1262 (2003) (citing In re Interest of Rebecca K., 101 Wash.App. 309, 2 P.3d 501 (2000)). When juveniles are found guilty of a nonenumerated offense equivalent to an adult gross misdemeanor, such as contempt, see RCW 9A.20.010(2)(b), .021(2), the conviction is classified as a category D juvenile offense. RCW 13.40.0357. Category D offenses are punishable by confinement in a juvenile detention facility for up to 30 days, up to 12 months' community supervision, up to 150 hours' community restitution and/or a fine up to $500. Id. Under RCW 7.21.040(5), Commissioner Inouye could have sentenced petitioners to 30 days in juvenile detention, without a purge condition, after finding the remedial RCW 7.21.030(2)(e) sanction inadequate and affording proper criminal due process protections.
¶ 21 We recognize that the holding of Division Two of the Court of Appeals in A.L.H. may be inconsistent with our conclusion that criminal contempt sanctions may be imposed on juveniles violating a placement order in a dependency case. In A.L.H., the court held that only civil contempt sanctions may be imposed on a juvenile for violating an at-risk youth (ARY) order. The ARY statutes constitute a separate chapter of Title 13 RCW from the dependency statutes. As amended in 1998, the ARY contempt statute provided, "Failure by a party to comply with an order entered under this chapter is a civil contempt of court as provided in RCW 7.21.030(2)(e), subject to the limitations of subsection (3) [which limits sanctions to $100 and/or seven days' confinement] of this section." RCW 13.32A.250(2). The Court of Appeals interpreted this statute as "expressly limit[ing]" sanctions that may be sought for contempt to the remedial sanctions laid out in RCW 7.21.030(2)(e). A.L.H., 116 Wash.App. at 164, 64 P.3d 1262. "If contempt charges are brought against a juvenile in violation of an ARY order, the State must seek civil contempt remedies," the court concluded, but "any juvenile offender in contempt of court on some other basis may be subject to criminal, civil, or summary contempt under the general contempt statutes." Id. at 163-64, 64 P.3d 1262.
¶ 22 The wording of the dependency contempt statute the statute at issue here underwent the same 1998 amendments and is essentially identical to the ARY contempt statute: "Failure by a party to comply with an order entered under this chapter is civil contempt of court as provided in RCW 7.21.030(2)(e)." RCW 13.34.165(1). This subsection, like the ARY subsection, is followed by a subsection limiting "remedial sanction[s]" to seven days' confinement, RCW 13.34.165(2). Thus, when Commissioner Inouye made the orders at issue here, he specifically found that criminal contempt sanctions under RCW 7.21.040 were unavailable, basing that finding on the A.L.H. decision.
¶ 23 We disagree with Commissioner Inouye. First, we note that A.L.H. concerned a different statute than the one we are interpreting: the ARY contempt statute. Although the wording of the two statutes is similar, the purposes behind the statutes are somewhat different. The ARY statutes were designed to provide parents of at-risk youth with tools to assist them in raising their children and keeping their children safe. RCW 13.32A.010. The legislature specifically stated that services were to be offered "on a voluntary basis whenever possible . . . and that the courts [should] be used as a last resort." Id. The dependency statutes, on the other hand, were intended to protect the health and safety of children when "the rights of basic nurture, physical and mental *20 health, and safety of the child and the legal rights of the parents are in conflict." RCW 13.34.020. These statutes appear to contemplate greater court involvement, while the ARY statutes were partially aimed at providing interventions to keep children out of detention.
¶ 24 In addition, we do not find the A.L.H. decision entirely persuasive. Division Two of the Court of Appeals provided little to no reasoning for its decision limiting sanctions in particular cases to civil contempt remedies. In fact, the other Court of Appeals ARY cases cited in A.L.H. M.B. and Rebecca K. can be read as suggesting the opposite conclusion: that criminal sanctions can be imposed for violation of an ARY order, so long as the proper due process is afforded. Both of those cases addressed whether the legislature had, by declaring the RCW 7.21.030(2)(e) sanction to be "remedial," constitutionally transformed criminal sanctions into civil sanctions, allowing determinate sentences to be imposed without purge conditions and without criminal due process protections. See M.B., 101 Wash.App. 425, 3 P.3d 780; Rebecca K., 101 Wash.App. 309, 2 P.3d 501. Both opinions concluded that confinement in juvenile detention without a purge condition remained a punitive sanction requiring criminal due process, regardless of what the legislature called it. M.B., 101 Wash.App. at 445-46, 3 P.3d 780; Rebecca K., 101 Wash.App. at 316-17, 2 P.3d 501. Division Three of the Court of Appeals further stated in Rebecca K. that "[c]riminal contempt proceedings must be initiated by a criminal information filed by the State in order to comply with due process." Rebecca K., 101 Wash.App. at 317, 2 P.3d 501 (citing A.D.F. v. State, 88 Wash.App. 21, 26, 943 P.2d 689 (1997), superseded by statute on other grounds by State v. A.L.H., 116 Wash. App. 158, 64 P.3d 1262). Concluding that the sanctions in the Rebecca K. case were punitive in nature, the court reversed the orders of contempt, because the requirements of RCW 7.21.040 were not followed. Similarly, Division One "emphasize[d]" in M.B. that "due process prohibits a court from using either statutory or inherent power to justify its actions if the contempt sanctions are themselves punitive, unless the contemnor is afforded criminal due process protections." M.B., 101 Wash.App. at 453, 3 P.3d 780 (emphasis added). We infer from this language that Divisions One and Three of the Court of Appeals consider statutory criminal contempt sanctions to remain available in ARY cases after the 1998 amendments.
¶ 25 Finally, we interpret statutes so as to give effect to legislative intent. Campbell v. Dep't of Soc. & Health Servs., 150 Wash.2d 881, 894, 83 P.3d 999 (2004). The "chief objective" of the legislature's 1998 amendments to the contempt statutes "was to make detention available as a coercive tool for juvenile courts." M.B., 101 Wash.App. at 446, 3 P.3d 780; see also Laws of 1998, ch. 296, § 35 ("[i]t is the intent of the legislature to authorize a limited sanction of time in juvenile detention independent of chapter 7.21 RCW for failure to comply with court orders in . . . dependency cases for the sole purpose of providing the courts with the tools necessary to enforce orders in these limited types of cases because other statutory contempt remedies are inadequate"). The legislature did not expressly designate this new tool the exclusive remedy, instead noting that it "may be imposed in addition to, or as an alternative to, any other remedial sanction authorized by this chapter." RCW 7.21.030(2)(e). We have previously stated, "[b]ecause civil and criminal contempt sanctions employ different procedures and are applied for fundamentally different purposes, statutes providing for one kind of contempt cannot be read to circumscribe statutes providing for the other." King, 110 Wash.2d at 800, 756 P.2d 1303. We conclude that the legislature did not intend, by amending the dependency contempt statute, to abrogate the availability of criminal contempt sanctions under RCW 7.21.040 in dependency cases. Instead, as the legislature stated, it intended to merely create a new alternative sanction.
¶ 26 The dissent points out that the legislature, when creating the Becca sanctions, intended to discourage the filing of criminal charges against status offenders. Dissent at 24. Our holding in no way undermines this goal. We do not hold that criminal *21 sanctions should be sought before Becca sanctions and other civil statutory sanctions; we do not speak to the order in which statutory remedies should be utilized. Instead, we merely adhere to our previous jurisprudence requiring courts to utilize all the tools the legislature has seen fit to provide before exercising broader inherent powers. The legislature carefully crafted the new tool they intended to provide, limiting it to seven days in detention. We do not infer from this careful limitation an intent to allow courts to disregard other statutes and sentence juveniles to whatever time in detention they felt was reasonable.
¶ 27 Because we conclude that statutory criminal contempt sanctions are available for violation of a dependency order, it follows that a juvenile court must find those sanctions inadequate before exercising its inherent contempt power.[6] In the present case, Commissioner Inouye failed to do this. Consequently, his resort to inherent authority was premature and improper. Accordingly, we reverse the Court of Appeals' decision to the contrary. As a result, we need not consider petitioners' other claims for relief.
III
¶ 28 A juvenile court commissioner possesses the inherent power to impose punitive or remedial sanctions for contempt of court, whether that contempt occurs in or outside of the courtroom. However, before exercising that power, the court must specifically find all statutory contempt remedies inadequate. Because the commissioner did not do so in this case, we reverse the Court of Appeals' decision on the two inherent contempt orders before us and vacate those orders.
C. JOHNSON, SANDERS and J.M. JOHNSON, JJ., concur.
MADSEN, J. (concurring).
¶ 29 I agree with the majority that before a dependency court may exercise its inherent authority to hold a juvenile in contempt and impose a punitive sanction, it first must find that the statutory remedies for criminal contempt under RCW 7.21.040 are not adequate. However, to the extent that the majority may be read to require a dependency court to resort to criminal contempt before exercising its inherent authority to impose a coercive contempt sanction, I disagree. Criminal contempt and remedial contempt sanctions are aimed at different issues, and a judge who is concerned with coercing compliance with a court order will have no reason to consider the adequacy of criminal contempt sanctions.
¶ 30 Under RCW 13.34.165(2), a dependency court may impose up to seven days as a remedial sanction when a party fails to comply with an order entered under that chapter. However, a contempt sanction is only remedial if the contemnor is allowed an opportunity to purge the contempt and gain release. Int'l Union, United Mine Workers of Am. v. Bagwell, 512 U.S. 821, 827 n. 2, 114 S.Ct. 2552, 129 L.Ed.2d 642 (1994). Thus, as long as a dependency court employing the sanctions allowed under RCW 13.34.165(2) provides an opportunity for a juvenile to purge the contempt, the sanction is remedial. If a dependency judge concludes that seven days is an insufficient amount of time to coerce compliance, then the judge has the *22 inherent power to impose a longer detention period as long as the juvenile has the power to end detention by complying at any time. As the Court of Appeals has observed in analogous circumstances: "On the rare occasion when a juvenile court decides it must disregard the statutory seven-day limit and resort to its inherent contempt powers, the court must enter a finding as to why the statutory remedy is inadequate and articulate a reasonable basis for believing why some other specific period of detention will achieve what seven days will not." In re Interest of M.B., 101 Wash.App. 425, 453, 3 P.3d 780 (2000).
¶ 31 In both of the cases before this court, the dependency courts imposed determinate, punitive sanctions without providing for a purge mechanism. Accordingly, the sanctions imposed were criminal, and the dependency courts in each case committed error by failing to provide due process protections, including initiation of charges by information, appointment of counsel, trial, and proof beyond a reasonable doubt. See Bagwell, 512 U.S. at 826, 114 S.Ct. 2552 ("`[C]riminal penalties may not be imposed on someone who has not been afforded the protections that the Constitution requires of such criminal proceedings.'" (quoting Hicks v. Feiock, 485 U.S. 624, 632, 108 S.Ct. 1423, 99 L.Ed.2d 721 (1988))).
¶ 32 Perhaps of more concern, however, is the use of contempt proceedings in dealing with chronic runaways. As the Court of Appeals has observed,
[o]nly under the most egregious circumstances should the juvenile court exercise its contempt power to incarcerate a status offender in a secure facility. If such action is necessary, the record should demonstrate that all less restrictive alternatives have failed.
State v. Norlund, 31 Wash.App. 725, 729, 644 P.2d 724, review denied, 98 Wash.2d 1013 (1982).
¶ 33 A court should consider the mental health needs of the dependent child before resorting to a contempt sanction.[1] Many children in foster case suffer from mental health problems that lead to their runaway behavior. When considering whether less restrictive alternatives exist, the question is not merely whether a seven day purgeable sanction has proved ineffective, but whether needed mental health services or chemical dependency treatment have been provided.
¶ 34 As stated by amicus American Academy of Child and Adolescent Psychiatry, "The failure to address the underlying problems and stressors that lead to runaway behavior is compounded by punishing the young people, making it more likely that they will continue to run away from their foster care placements and encounter the very dangers from which the courts are obligated to protected them." Mem. of Amicus Curiae Am. Acad. of Child & Adolescent Psychiatry in Supp. of Pet'r's at 5.
¶ 35 Amicus calls this court's attention to numerous studies indicating that detention does not have an ameliorative effect on runaway behavior, and, in fact, often exacerbates the problem.[2] The record in this case bears this out: repeated detention of these children did not stop them from running away.
¶ 36 According to the records, after Y.H.'s placement in foster care she ran away several times. Three times Y.H. was sentenced to seven days in detention for contempt. After her fourth disappearance she was moved to a *23 different foster home, but no additional services were provided to assist in making her placement more successful or to address her running behavior. Instead, when Y.H. ran again the dependency court sentenced her to a 30 day period of detention. Similarly, M.H.-O. ran away from her placement at least six times. After the first four times, M.H.-O. was sentenced to seven days in detention. The fifth time she ran, the dependency court sentenced her to 30 days in detention. One week later, M.H.-O. ran again. Prior to her incarceration M.H.-O. made several requests for mental health services, but those services were not provided. Instead, after M.H.-O. ran again, the dependency court sentenced M.H.-O. to 60 days in detention. While in detention, her mental health worsened and she heard voices, rocked back and forth, and began cutting herself.
¶ 37 Detention should not be used as a substitute for access to basic services, treatment, and care. The repeated use of contempt proceedings is often ineffective and offers little opportunity to address the underlying problems that result in runaway behavior. In contempt proceedings, the focus is on deterring the child's misbehavior rather than ensuring the State is upholding its responsibility to provide an individualized response to the runaway behavior.
¶ 38 Another reason detention proves ineffective as a deterrent to runaway behavior is that children in foster care often run because of their desire to connect with family, friends, and familiar surroundings. Here, for instance, Y.H. repeatedly sought out her mother while on the run, while M.H.-O. ran to her father in Nebraska. Punishing these children with detention, where they must adjust to a new set of peers and authority figures in an unfamiliar environment, only increases their desire to run.
¶ 39 Children in foster care who suffer from mental health disorders present difficult challenges. However, incarceration in a locked detention facility punishes rather than rehabilitates these children. As amicus The Children's Alliance and Columbia Legal Services points out, there are alternatives to incarceration that are available by statute, including evaluation and treatment in secure facilities under RCW 70.96A.140 and .245 (chemical dependency involuntary treatment act) and RCW 71.34.600 (parent initiated mental health involuntary treatment act). Incarceration before fully exploring such alternatives is not a proper use of the court's inherent contempt powers, criminal or remedial.
BRIDGE, J., concurs.
OWENS, J. (dissenting).
¶ 40 The majority holds that a juvenile court must exhaust the remedies under the criminal contempt statute, RCW 7.21.040, before exercising its inherent contempt power. Under the criminal contempt statute, the State must file a criminal information to initiate the contempt proceeding. Under the civil contempt statute, RCW 7.21.030, the court may detain juveniles governed by the dependency statutes for up to seven days for civil contempt of court. RCW 7.21.030(2)(e). The majority's holding requires the State to initiate criminal contempt proceedings if the court determines that the statutory civil remedy is inadequate and a detention of more than seven days is needed to coerce the contemnor to comply with the court's orders. This result conflicts with long-standing jurisprudence confirming a court's authority to impose a coercive detention for indirect contempt within a civil proceeding and contravenes the legislature's intent to reduce the number of criminal charges against juveniles. I would hold that a juvenile court may exercise its inherent authority to order a coercive detention after finding the civil contempt statute inadequate without first exhausting the criminal statutory remedy. Thus, I respectfully dissent.
¶ 41 The majority's holding diminishes the court's inherent contempt power to enforce its orders and ignores long-standing precedent allowing a judge to impose coercive detention in a civil proceeding. The power of courts to punish contempt "is a necessary and integral part of the independence of the judiciary, and is absolutely essential to the performance of the duties imposed on them by law." Gompers v. Buck's Stove & Range *24 Co., 221 U.S. 418, 450, 31 S.Ct. 492, 55 L.Ed. 797 (1911); see also Int'l Union, United Mine Workers of Am. v. Bagwell, 512 U.S. 821, 831, 114 S.Ct. 2552, 129 L.Ed.2d 642 (1994) (holding that courts have independent power to impose submission to their orders) (citing Anderson v. Dunn, 19 U.S. (6 Wheat.) 204, 5 L.Ed. 242 (1821)); Shillitani v. United States, 384 U.S. 364, 370, 86 S.Ct. 1531, 16 L.Ed.2d 622 (1966) ("There can be no question that courts have inherent power to enforce compliance with their lawful orders through civil contempt."). The United States Supreme Court has consistently held that a court can impose a conditional fixed term of detention in a civil proceeding to coerce a contemnor into complying with the court's orders. Bagwell, 512 U.S. at 828, 114 S.Ct. 2552; accord Shillitani, 384 U.S. at 368, 86 S.Ct. 1531 (upholding a two-year civil contempt sanction); Gompers, 221 U.S. at 442, 31 S.Ct. 492 (holding that imprisonment for civil contempt is intended to coerce the defendant "to do what he had refused to do"). Requiring a court to first turn to the criminal contempt statute before exercising its inherent power abdicates the court's inherent power to impose detention in civil contempt proceedings because the court will have to rely on the executive branch to enforce its orders, which it may refuse to do.
¶ 42 The majority's holding also disregards the legislature's intent to keep juveniles out of the criminal justice system. Under the majority's holding, a court wishing to impose eight days of confinement must seek a criminal charge. The legislature has consistently and repeatedly enacted statutes aimed at decriminalizing juvenile proceedings. State v. Schaaf, 109 Wash.2d 1, 15, 743 P.2d 240 (1987) ("For more than 70 years, this state has been trying to avoid accusing and convicting juveniles of crimes."). Contrary to the majority's assertions, majority at 20, the legislature has made clear that courts should impose detention without the filing of criminal charges: "[i]t is the intent of the legislature to avoid the bringing of criminal charges against youth who need the guidance of the court rather than its punishment." Laws of 1998, ch. 296, § 35 (emphasis added).
¶ 43 The noninclusion of the juvenile chapters in the criminal contempt statute further demonstrates the legislature's intent to decriminalize juvenile offenders. The majority concludes that courts should more readily impose criminal sanctions in dependency cases reasoning that, unlike at-risk-youth cases, dependency cases "contemplate greater court involvement." Majority at 20. The legislature makes no similar distinction. Failure to comply with an order entered under either the at-risk-youth statutes or the dependency statutes is civil contempt of court as defined in RCW 7.21.030(2)(e). See RCW 13.32A.250(2); RCW 13.34.165(1). The legislature amended RCW 7.21.030 in 1998 to provide a civil contempt remedy of commitment to juvenile detention for seven days in both at-risk-youth and dependency cases. See RCW 7.21.030(2)(e). The legislature did not likewise amend the criminal contempt statute to include youth governed by the at-risk-youth and dependency statutes. See RCW 7.21.040.
¶ 44 Any concerns that a court using this inherent power will hold juveniles in detention indefinitely is unfounded. A judge may not exercise inherent contempt power without limit. The exercise of inherent contempt power must comport with due process to protect against any arbitrary exercise of official power. Bagwell, 512 U.S. at 830-34, 114 S.Ct. 2552. In a civil contempt proceeding, the court must also afford the contemnor the opportunity for release from a fixed term of detention by satisfying a purge condition. See id. at 828, 114 S.Ct. 2552 (holding that a detention is coercive and civil where the contemnor is able to purge the contempt and thus "`carries the keys of his prison in his own pocket'") (internal quotation marks omitted) (quoting Gompers, 221 U.S. at 442, 31 S.Ct. 492); Shillitani, 384 U.S. at 370-71, 86 S.Ct. 1531 ("The conditional nature of the imprisonment . . . justifies holding civil contempt proceedings."); In re Pers. Restraint of King, 110 Wash.2d 793, 805, 756 P.2d 1303 (1988) ("The incarcerated contemnor must be afforded the opportunity to purge himself of the contempt."). In the instant case, the judge erred to the extent he did not include a purge option for Y.H. In the case of Y.H., the judge ordered 30 days of detention without *25 an option to purge. Clerk's Papers (CP) (23252-2-III) at 73-74. In the case of M.H.-O., the judge ordered 60 days of detention but scheduled a hearing 20 days later to review M.H.-O.'s participation and progress in services. If M.H.-O. progressed to the court's satisfaction, she would earn her release from detention a purge option. CP (23211-5-III) at 80-81.
¶ 45 I would hold that a judge has the inherent power to impose detention in a civil contempt proceeding and need not expose youth to criminal proceedings before exercising this power. This holding is most consistent with the inherent authority of courts to enforce their orders and the intent of the legislature to reduce the number of criminal charges brought in juvenile proceedings. Holding otherwise will tie the hands of trial courts by eliminating their authority to effectively enforce their orders. Accordingly, I respectfully dissent.
FAIRHURST and CHAMBERS, JJ., concur.
NOTES
[1] In 1995, the Washington Legislature passed a bill known as the "Becca Bill," which amended the Family Reconciliation Act (chapter 13.32A RCW) to provide parents of runaway children a tool to control them through the legal system. See Alison G. Ivey, Comment, Washington's Becca Bill: The Costs of Empowering Parents, 20 Seattle U.L.Rev. 125 (1996); Laws of 1995, ch. 312. A later amendment, known as the "Becca Too" bill added the current "remedial" contempt sanction to RCW 13.34.165. See Ivey, supra; Laws of 1996, ch. 133. Because of this, the imposition of remedial contempt sanctions with a purge condition is known as the "Becca procedure" or "Becca sanctions."
[2] The Court of Appeals opinion addressed four orders: one involving Y.H., two involving M.H.-O., and one involving the third juvenile, A.K. Commissioner Inouye had sentenced A.K. to 60 days in detention, without a purge option, for running away from her placement a fifth time. That sentence, like the others, was imposed pursuant to the juvenile court's inherent contempt power.
[3] Because the Court of Appeals vacated A.K.'s order, only Y.H. and M.H.-O. petitioned this court for review.
[4] In the first scenario, summary adjudication is appropriate. Bagwell, 512 U.S. at 832, 114 S.Ct. 2552; Keller, 52 Wash.2d at 87, 323 P.2d 231. In the second, the contemnor must be given notice, a reasonable time to prepare a defense, and hearing before sanctions are imposed. Bagwell, 512 U.S. at 832, 114 S.Ct. 2552; In re Marriage of Nielsen, 38 Wash.App. 586, 589, 687 P.2d 877 (1984). Before punitive sanctions may be imposed, the contemnor must receive full criminal due process. Bagwell, 512 U.S. at 833, 114 S.Ct. 2552; see also In re Pers. Restraint of King, 110 Wash.2d 793, 800, 756 P.2d 1303 (1988).
[5] Status offenders are juveniles "who are before the court because their behavior endangers their welfare," including runaways such as M.H.-O. and Y.H.M.B., 101 Wash.App. at 434, 3 P.3d 780 (citing Jan C. Costello & Nancy L. Worthington, Incarcerating Status Offenders: Attempts to Circumvent the Juvenile Justice and Delinquency Prevention Act, 16 Harv. C.R.-C.L. L.Rev. 41, 42-46 (1981)).
[6] Contrary to the dissent's contention, this does not "abdicate [] the court's inherent power" by leaving enforcement of its orders to the discretion of the executive branch of government. Dissent at 24. The United States Supreme Court has previously recognized that courts are not stripped of their authority or inherent contempt power as a result of having to exercise that power through a separate criminal trial. See Bagwell, 512 U.S. at 838-39, 114 S.Ct. 2552 (holding that the imposition of some procedural burdens, such as the requirement of a jury trial, on inherent judicial contempt power does not prevent the courts from exercising that authority through a criminal trial); Gompers v. Buck's Stove & Range Co., 221 U.S. 418, 451, 31 S.Ct. 492, 55 L.Ed. 797 (1911) (holding that "a separate and independent proceeding at law for criminal contempt" can "vindicate the authority of the court"). Courts are also not constrained by the criminal contempt statute to wait for a prosecutor to decide to take action. RCW 7.21.040(2)(c) allows the judge whose order was violated to request that an action be commenced and "appoint a special counsel to prosecute [the] action," if "required for the administration of justice." This is consistent with the United States Supreme Court's determination that the contempt power of the courts "necessarily encompasses the ability to appoint a private attorney to prosecute the contempt." Young, 481 U.S. at 793, 107 S.Ct. 2124.
[1] Studies indicate that up to 80 percent of children in foster care require mental health services. See American Academy of Child & Adolescent Psychiatry, Policy Statement: Psychiatric Care of Children in the Foster Care System (2001), available at http://aacap.org/cs/root/policy_statements/psychiatric_care_of_children_in_the _foster_care_system (last visited Dec. 10, 2007).
[2] See, e.g., Kelly Dedel, Office of Community Oriented Policing Services, U.S. Dep't of Justice, Juvenile Runaways (Feb.2006), available at http://www.popcenter.org/Problems/PDFs/Juvenile Runaways.pdf; Marni Finkelstein et al., Youth Who Chronically AWOL From Foster Care: Why They Run, Where They Go, and What Can Be Done, Vera Institute of Justice (Aug.2004), available at http://www.vera.org/publication_pdf/244_460.pdf; Kevin M. Ryan, Stemming the Tide of Foster Care Runaways: A Due Process Perspective, 42 Cath. U.L.Rev. 271, 279 (1993); Caren Kaplan, Children Missing from Care: An Issue Brief, Child Welfare League of America (2004), available at http://www.cwla.org/programs/fostercare/childmiss.htm (last visited Dec. 10, 2007).
|
With one Toronto man estimating he has up to 1,000 half-siblings, some fertility-treatment experts are calling on Canada to legally restrict how many children can be born from a single donor’s semen.
The growing families of donor offspring could cause unusual spread of genetic malformations, raise the risk of inadvertent incest between biological brothers and sisters and prove emotionally taxing to the children, critics say.
Although medical groups and others already recommend restrictions in the number of pregnancies per donor, legislation is needed to ensure sperm banks and their suppliers follow the proper limits, said Juliet Guichon, a bio-ethics professor at the Unviersity of Calgary.
“It [self regulation] is not working,” said Prof. Guichon. “There’s no incentive. It’s the market economy: why would you limit business?”
Various reports on the infertility industry, including the 1993 federal Royal commission on new reproductive technology and an earlier B.C. commission, have actually been recommending limits of as few as six pregnancies per donor for the last 30 years, she said.
Britain, some Australian states, New Zealand, the Netherlands and a handful of other European countries already have laws that restrict the number of children per donor, Prof. Guichon noted.
The issue came to the fore again this week, however, with reports from the U.S. — which has no legislated limits — that one donor there has 150 offspring. The genetic siblings have been catalogued on an unofficial but widely used American web site — the Donor Sibling Registry — that brings together such relatives, sometimes based on the number assigned to the donor by his sperm bank.
The Colorado-based registry’s director says another group of 75 offspring includes several Canadians. About 95% of sperm used in artificial insemination and in-vitro fertilization treatments here actually comes from the States.
Barry Stevens, a Toronto filmmaker, said he was born in the U.K. in 1952 with sperm from a donor who supplied his semen over about three decades, and probably produced 500 to 1,000 children, now spread through Britian, Canada and other countries.
“There should be limits, because if some offspring want to find their relatives, and want to meet their donor … it’s kind of daunting when it’s in the hundreds,” he said. “For some, it becomes kind of creepy and freaky.”
The fertility industry has restricted its practices considerably since the start of artificial-insemination around the middle of the last century, but critics say the lack of regulation or monitoring of what happens to donor sperm means the real-life practice is still largely unknown.
At ReproMed, which runs Canada’s only sperm bank, administrators do impose restrictions, said Dr. Alfonso Del Vaille, its director. Donors are limited to three live births per 100,000 population in a given geographic area, though that could mean as many as 75 offspring in a city the size of Toronto.
Dr. Del Vaille said he would support legislated limits, so long as they are based on good science.
The chief concern stemming from large donor families is the risk that half-siblings unknowingly enter sexual relationships, upping the risk of birth defects in any resulting children.
Mr. Stevens said he knows of donor offspring who have married or had sex with half siblings, and said the chance of that happening is greater than it might seem. He said he has talked to a U.S. donor who moved to Toronto and ended up by sheer coincidence living next door to a lesbian couple whose children were born with the use of his sperm.
Experts also worry about the possibility that a genetic flaw, which would be passed on to only a couple of children in a natural family, could be spread to dozens more through sperm or egg donation.
And there is also the less tangible effect of a child learning that their direct biological family extends far and wide. Wendy Kramer, the donor-registry’s director, recalls one mother who came to the web site anxious to find one or two genetic siblings to her daughter, an only child, only to discover the donor had sired 18 offspring.
“The mother was upset: ‘Oh, my God, I didn’t expect this … How do I create a connection with 18 families?’ ”
Even some of those in the fertility industry itself say there is a need to legislate limits, though doing so would likely further restrict the supply of donor sperm. “It’s one of those big picture areas that as a society and as a culture we really need to sit down and think about what we are doing,” said Roger Pierson, a University of Saskatchewan fertility expert and spokesman for the Canadian Fertility and Andrology Society.
Which government would enact such laws in Canada is another question, though, since the federal Assisted Human Reproduction Act, designed to govern such matters, was declared mostly unconstitutional by the Supreme Court last year.
It could be that the provinces would have to band together and bring in legislation jointly, said Ms. Guichon.
Other experts say the federal government, which still does regulate the importation of sperm, could impose rules allowing only sperm that has produced a limited number of offspring to be brought in to the country from elsewhere.
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Compact shoot and leafy head 1, a mutation affects leaf initiation and developmental transition in rice (Oryza sativa L).
The shoot apical meristem (SAM) produces lateral organs in a regular spacing (phyllotaxy) and at a regular interval (phyllochron) during the vegetative phase. In a Dissociation (Ds) insertion rice population, we identified a mutant, compact shoot and leafy head 1 (csl1), which produced massive number of leaves (~70) during the vegetative phase. In csl1, the transition from the vegetative to the reproductive phase was delayed by about 2 months under long-day conditions. With a reduced leaf size and severe dwarfism, csl1 failed to produce a normal panicle after the transition to reproductive growth. Instead, it produced a leafy panicle, in which all primary rachis-branches were converted to vegetative shoots. Phenotypically csl1 resembled pla mutants in short plastochron but was more severe in the conversion of the reproductive organs to vegetative organs. In addition, neither the expression nor the coding region of PLA1 or PLA2 was affected in csl1. csl1 is most likely a dominant mutation because no mutant segregant was observed in progeny of 67 siblings of the csl1 mutant. CSL1 may represent a novel gene, which functions downstream of PLA1 and/or PLA2, or alternatively functions in a separate pathway, involved in the regulation of leaf initiation and developmental transition via plant hormones or other mobile signals. |
Inhibition of SOX9 Promotes Inflammatory and Immune Responses of Dental Pulp.
The process of pulpitis is characterized by extracellular matrix imbalance and inflammatory cell infiltration. As an essential transcription factor, sex-determining region Y-box 9 (SOX9) is significantly inhibited by tumor necrosis factor alpha in inflammatory joint diseases. The aim of this study was to explore the role of SOX9 in extracellular matrix balance, cytokine expression, and the immune response in dental pulp. The expression of SOX9 in normal and inflamed pulp tissue/human dental pulp cells (HDPCs) was detected by immunohistochemistry, Western blot, and quantitative polymerase chain reaction (qPCR). SOX9 small interfering RNA was used to knock down SOX9 expression of dental cells in vitro; extracellular matrix imbalance was analyzed by qPCR, Western blot, and gelatin/collagen zymography, and the secretion of cytokines was scanned by antibody arrays. The immune response of THP-1 was investigated by cell migration assay, cell attachment assay, phagocytosis assay, and enzyme-linked immunosorbent assay. The interaction of SOX9 with target genes was explored by chromatin immunoprecipitation (ChIP). SOX9 was strongly expressed in normal dental pulp tissue and HDPCs and reduced in inflamed pulp. SOX9 knockdown could inhibit the production of type I collagen, stimulate the enzymatic activities of MMP2 and MMP13, and regulate the production of interleukin (IL) 8 of HDPCs. SOX9 knockdown also effectively suppressed the differentiation and functional activities of THP-1. ChIP showed that the binding of the SOX9 protein with matrix metalloproteinase (MMP)-1, MMP-13, and IL-8 gene promoters was reduced after being treated with recombinant human tumor necrosis factor alpha. SOX9 was inhibited in inflamed dental pulp and may participate in the regulation of extracellular matrix balance, the inflammatory process, and the immune response. |
In conventional AM (amplitude-modulation) optical transmission techniques a D.C. level, or pedestal, is transmitted in conjunction with an amplitude-modulated carrier. The D.C. level, however, does not contribute any information to the transmitted AM-signal. Furthermore, transmission of the D.C. level increases the amount of power necessary for transmission of the AM-signal, thereby having an adverse impact on the efficiency of the conventional AM scheme.
FIG. 1A illustrates the waveform of the output signal of a conventional AM modulator. A D.C. level 101 is implicit in this signal. As a consequence, the power transmitted is unnecessarily increased and the efficiency of the conventional AM system is degraded. FIG. 1B illustrates one modulation cycle of a signal transmitted from a conventional AM transmitter. Cross-hatched area 100 represents the energy (E) transmitted, which is expressed as: ##EQU1## The transmission of unnecessary energy may prove disadvantageous in many applications, and it is thus an object of the invention to provide an energy-efficient modulation technique, and also a corresponding demodulation technique. |
The Story: On Sunday Bubba Watson, one of the most untraditional golfers on the PGA Tour, was the surprise winner of the 2012 Masters Tournament. But golf isn’t Watson’s top priority. What he considers most important can be gleaned from the description on his Twitter account:”@bubbawatson: Christian. Husband. Daddy. Pro Golfer. Owner of General Lee 1.”
The Background: In an interview with Trevor Freeze of the Billy Graham Evangelistic Association, Watson tells how he uses his Twitter account—-along with his PGA platform—- to share about his faith in Christ.
“For me, it’s just showing the Light,” said Watson. “There’s people who want to put down Christians. I try to tell them Jesus loves you. It’s just a way to be strong in my faith.”
Last month Watson’s Tweeted before his third round: “The most important thing in my life? Answer after I golf 18 holes with @JustinRose99. #Godisgood”
Later that day he posted on his account, “Most important things in my life- 1. God 2. Wife 3. Family 4. Helping others 5. Golf”
“Lecrae said it the best,” Watson said of the Christian rapper he listens to on his iPod. “He doesn’t want to be a celebrity. He doesn’t want to be a superstar. He just wants to be the middle man for you to see God through him.”
Why It Matters: Christians have always been involved in professional sports, so why is the faith of superstars like Tim Tebow, Jeremy Lin, and Bubba Watson suddenly getting the public’s attention? Perhaps it’s because these athletes are open and unapologetic about their willingness to share the Gospel. They also keep their priorities in order, winsomely admitting that their life’s callings are secondary to serving the Creator who has called them. To a culture that is both obsessed and disillusioned with fame and fortune, the centered perspective of these superstars provides a refreshingly countercultural witness. |
This invention relates to medical devices and procedures used during the repair, replacement, or supplement of a medical patient's natural body organ structures or tissues. In particular, this invention relates to catheters with at least one integrated lumen for use in connection with such medical procedures and to methods of their manufacture.
Revascularization of the human heart is a good example of a medical procedure that involves the repair and supplement of a patient's body organ. Early procedures were known for revascularizing the human heart, but there were several disadvantages to these procedures. The earliest procedures involved exposing the heart by means of a midline sternotomy and stopping the beating of the heart to facilitate performance of the procedure. A graft is used to create a new, uninterrupted channel between a blood source, such as the aorta, and the occluded coronary artery or arteries downstream from the arterial occlusion or occlusions. Such a procedure has significant disadvantages, however, because it is highly invasive and requires general anesthesia. In fact, these disadvantages preclude the use of sternotomy procedures on many patients.
Less invasive procedures were later developed for revascularizing the heart, but these have disadvantages as well. For example, a thoracostomy involves surgical creation of ports in the patient's chest to obtain access to the thoracic cavity. Specially designed instruments are then inserted through the ports. Thoracostomy bypass procedures are less traumatic than sternotomy bypass procedures, but they are still too traumatic for some patients and may be inadequate when the number of surgical bypasses is large. Another procedure, which is known as a thoracotomy, revascularizes the human heart by gaining access to the thoracic cavity with incisions between the patient's ribs, but this procedure may still be too traumatic for some patients.
Goldsteen et al. U.S. patent application Ser. No. 08/745,618, filed Nov. 7, 1996, which is hereby incorporated by reference herein, discloses a less traumatic surgical technique for revascularizing the human heart. A key aspect of that invention involves the use of catheters that are inserted into a patient's body through relatively remote entry ports, such as a femoral (leg) artery of the patient, a brachial artery of the patient, or any other suitable entry point. Control of these instruments throughout their use is from a proximal portion that is outside the patient at all times. In order to minimize the number of entry ports or to perform any of the specialized surgical techniques disclosed therein, a single catheter instrument may include two or more lumens. However, as the number of lumens increases, conventional manufacturing methods may yield catheters that have outer diameters that are undesirably large, which may irritate sensitive vessels and preclude their use in narrow vessels. Furthermore, such catheters may be difficult to position and secure in a patient's body.
In view of the foregoing, it is an object of this invention to provide less traumatic methods and apparatus for revascularizing a patient.
It is another object of the invention to provide methods of manufacturing catheters with integrated lumens without substantially increasing the thickness of catheter walls.
It is still another object of the invention to provide a catheter that can create a hemodynamic seal when positioned across vessel walls.
It is yet another object of the invention to provide a catheter that can be positioned in a vessel and used to selectively secure one or more medical devices therein. |
[Comparative study of antibiotic consumption in Hungarian hospitals during 1989-1991].
The data of case histories of the year of 1989 of seven Hungarian hospitals and also the 1990 and 1991 data of two hospitals were collected and integrated in a database. Consumption of antibiotics were represented in DDD (Defined Daily Dose)/1000 hospital day. These data were compared with the data of the drugs delivered by the hospital pharmacy in one of the mentioned hospitals between 1989 and 1992. It was concluded that data based on case histories represent better the real antibiotic consumption than those of the hospital pharmacy. Reason of this phenomenon is the fact, that considerable amount of drug supply gets out of institutions. Great differences were observed between the seven hospitals in the total amount of antibiotic consumption and between the different antibiotic groups as well. Drugs most frequently used were tetracyclines, broad spectrum penicillins, sulfonamides and aminoglycosides. Consumption of penicillins was decreasing. Regarding new drugs only utilisation of quinolones was increasing. It was concluded, that structure of antibiotic selection did not follow the recommendations of the medical literature. The authors suggest that "antibiotic policy" should be introduced in Hungarian hospitals so as to imporve antibiotic utilisation. |
//---------------------------------------------------------------------------
#ifndef RightsH
#define RightsH
//---------------------------------------------------------------------------
#include <Classes.hpp>
#include <Controls.hpp>
#include <StdCtrls.hpp>
#include <Forms.hpp>
#include <Buttons.hpp>
#include <ActnList.hpp>
#include <ImgList.hpp>
#include <Menus.hpp>
#include "GrayedCheckBox.hpp"
#include "PngImageList.hpp"
#include <System.Actions.hpp>
//---------------------------------------------------------------------------
#include <RemoteFiles.h>
#include <GUITools.h>
//---------------------------------------------------------------------------
class TRightsFrame : public TFrame
{
__published:
TLabel *GroupLabel;
TLabel *OthersLabel;
TLabel *OwnerLabel;
TGrayedCheckBox *OwnerReadCheck;
TGrayedCheckBox *OwnerWriteCheck;
TGrayedCheckBox *OwnerExecuteCheck;
TGrayedCheckBox *GroupReadCheck;
TGrayedCheckBox *GroupWriteCheck;
TGrayedCheckBox *GroupExecuteCheck;
TGrayedCheckBox *OthersReadCheck;
TGrayedCheckBox *OthersWriteCheck;
TGrayedCheckBox *OthersExecuteCheck;
TCheckBox *DirectoriesXCheck;
TSpeedButton *OwnerButton;
TSpeedButton *GroupButton;
TSpeedButton *OthersButton;
TPopupMenu *RightsPopup;
TMenuItem *Norights1;
TMenuItem *Defaultrights1;
TMenuItem *Allrights1;
TMenuItem *Leaveasis1;
TActionList *RightsActions;
TAction *NoRightsAction;
TAction *DefaultRightsAction;
TAction *AllRightsAction;
TAction *LeaveRightsAsIsAction;
TPngImageList *RightsImages;
TMenuItem *N1;
TAction *CopyTextAction;
TAction *CopyOctalAction;
TAction *PasteAction;
TMenuItem *CopyAsText1;
TMenuItem *CopyAsOctal1;
TMenuItem *Paste1;
TPngImageList *RightsImages120;
TPngImageList *RightsImages144;
TPngImageList *RightsImages192;
TLabel *OctalLabel;
TEdit *OctalEdit;
TGrayedCheckBox *SetUidCheck;
TGrayedCheckBox *SetGIDCheck;
TGrayedCheckBox *StickyBitCheck;
TButton *CloseButton;
void __fastcall ControlChange(TObject *Sender);
void __fastcall RightsButtonsClick(TObject *Sender);
void __fastcall RightsActionsExecute(TBasicAction *Action,
bool &Handled);
void __fastcall RightsActionsUpdate(TBasicAction *Action, bool &Handled);
void __fastcall RightsPopupPopup(TObject *Sender);
void __fastcall FrameContextPopup(TObject *Sender, TPoint &MousePos,
bool &Handled);
void __fastcall OctalEditChange(TObject * Sender);
void __fastcall OctalEditExit(TObject * Sender);
void __fastcall CloseButtonClick(TObject * Sender);
private:
bool FAllowAddXToDirectories;
TNotifyEvent FOnChange;
bool FPopup;
TWinControl * FPopupParent;
TButton * FDefaultButton;
TButton * FCancelButton;
bool FPopingContextMenu;
UnicodeString FAddXToDirectoriesSuffix;
bool FInitialized;
void __fastcall CycleRights(int Group);
bool __fastcall GetAddXToDirectories();
bool __fastcall GetAllowUndef();
TCheckBox * __fastcall GetChecks(TRights::TRight Right);
TRights __fastcall GetRights();
TRights::TState __fastcall GetStates(TRights::TRight Right);
void __fastcall SetAddXToDirectories(bool value);
void __fastcall SetAllowAddXToDirectories(bool value);
void __fastcall SetAllowUndef(bool value);
void __fastcall SetRights(const TRights & value);
void __fastcall SetStates(TRights::TRight Right, TRights::TState value);
UnicodeString __fastcall GetText();
void __fastcall SetText(UnicodeString value);
public:
virtual __fastcall ~TRightsFrame();
__fastcall TRightsFrame(TComponent* Owner);
void __fastcall DropDown();
void __fastcall CloseUp();
__property bool AddXToDirectories = { read = GetAddXToDirectories, write = SetAddXToDirectories };
__property bool AllowAddXToDirectories = { read = FAllowAddXToDirectories, write = SetAllowAddXToDirectories };
__property bool AllowUndef = { read = GetAllowUndef, write = SetAllowUndef };
__property TCheckBox * Checks[TRights::TRight Right] = { read = GetChecks };
__property TNotifyEvent OnChange = { read = FOnChange, write = FOnChange };
__property TRights Rights = { read = GetRights, write = SetRights };
__property UnicodeString Text = { read = GetText, write = SetText };
__property bool Popup = { read = FPopup, write = SetPopup };
__property TWinControl * PopupParent = { read = FPopupParent, write = FPopupParent };
protected:
void __fastcall DoChange();
void __fastcall UpdateControls();
virtual void __fastcall SetEnabled(bool Value);
void __fastcall ForceUpdate();
virtual void __fastcall CreateParams(TCreateParams & Params);
virtual void __fastcall CreateWnd();
virtual void __fastcall Dispatch(void * Message);
void __fastcall CMCancelMode(TCMCancelMode & Message);
void __fastcall CMDialogKey(TCMDialogKey & Message);
void __fastcall WMContextMenu(TWMContextMenu & Message);
bool __fastcall IsAncestor(TControl * Control, TControl * Ancestor);
DYNAMIC void __fastcall DoExit();
void __fastcall SetPopup(bool value);
void __fastcall DoCloseUp();
bool __fastcall HasFocus();
bool __fastcall DirectoriesXEffective();
void __fastcall UpdateOctalEdit();
void __fastcall UpdateByOctal();
INTERFACE_HOOK_CUSTOM(TFrame);
__property TRights::TState States[TRights::TRight Right] = { read = GetStates, write = SetStates };
};
//---------------------------------------------------------------------------
#endif
|
"I don't think Mary had a choice in her decision relative to closing some of these plants and the platforms", he said during an interview on "Countdown to the Closing Bell Thursday".
Next month, Governor-elect Mike DeWine is going to the Detroit Auto Show to meet with GM CEOMary Barra.
General Motors Chief Executive Mary Barra came under harsh criticism from members of Congress from MI on Thursday for building a new vehicle in Mexico while ending production at five North American assembly plants and cutting almost 15,000 jobs. GM says that since 2009 it has invested $22 billion in US facilities.
Barra came under pressure from Ohio's two USA senators and other lawmakers who want GM to shift production of a vehicle from Mexico or build electric vehicles at the Lordstown Assembly plant in their state that the automaker has said it intends to close. She also noted GM is launching a number of new vehicles in MI next year.
Representative Brenda Lawrence, who represents Detroit, said lawmakers were putting GM was on notice about future production decisions, noting the company is making strong profits and got a massive taxpayer bailout a decade ago.
Dingell and her colleagues are asking Trump to visit MI and Ohio.
Portman said he and Brown urged Barra to speed up talks.
The CEO said GM planned to add other products at USA plants next year and that the automaker would have some jobs to fill at other OH facilities in 2019.
Tesla CEO Elon Musk recently made headlines saying he would be willing to buy some of the GM plants if they don't want them anymore.
MI lawmakers, including Democratic Sen. After GM announced its plans, Trump threatened to eliminate subsidies for GM in retaliation. Obviously, we're working with General Motors, but should General Motors choose to sell those facilities or do something with them with someone like Elon Musk, that would obviously be a great opportunity for our community. "I also informed them that all salaried GM workers impacted by these actions are being offered outplacement services to help them transition to new jobs".
Musk's announcement could put some pressure on Barra and GM to keep the Lordstown plant open. The union has asked GM to rescind the decision and resolve the fate of the plants in talks for a new labor contract next year. |
Since my December 2, 2005 Non-Hodgkin Lymphoma diagnosis, I've been on a slow-motion journey of survivorship. Chemo wiped out my aggressive disease in May, 2006, but an indolent variety is still lurking. I had my thyroid removed due to papillary thyroid cancer in 2011, and was diagnosed with recurrent thyroid cancer in 2017. Join me for a survivor's reflections on life, death, faith, politics, the Bible and everything else. DISCLAIMER: I’m not a doctor, so don't look here for medical advice.
Wednesday, November 14, 2007
November 14, 2007 - Here's a Way You Can Help Me
I know how a lot of you friends and family members have been wondering what you can do to help me, as I prepare for whatever further cancer treatment may be in my future. Ever since my chemotherapy ordeal ended and my family and I no longer needed you to bring food over to the house, there hasn’t been much I could suggest by way of concrete action – except, of course, for supporting me with your prayers and good wishes (for which I’m always grateful).
Now, here’s a little something you can do, and it will only take a couple of minutes. It’s along the lines of political action. I know some of you may be more comfortable than others with the idea of writing your U.S. Senators and Representative, but this is one case where your letter could have a very real impact on whether I will eventually have access to medicine that could save my life.
Take a look at this article by Jonathan Alter (a mantle-cell lymphoma survivor, himself), from the recent Newsweek. It’s called “How Washington Is Nixing a Cancer Cure.”
I’ve written, before, about the radio- immuno- therapy drugs, Bexxar and Zevalin (see my June 23, July 14 and July 20 blog entries). Now, it seems that changes in Medicare reimbursement guidelines could make these promising drugs disappear altogether – despite the fact that they’ve been proven highly effective against follicular non-Hodgkin lymphoma (the type I have).
Why should Medicare reimbursement policies make any difference to someone like me, who’s too young for Medicare? The answer is that Medicare is such a large player in the multi-billion-dollar world of pharmaceuticals that their refusal to pay a fair price for a drug will start a domino effect. First, hospitals and clinics will stop offering it. Then, its manufacturers will have no choice but to cut their losses and pull it from the market. Jonathan Alter explains the machinations of the system far more clearly than I could: so, you’ll have to click on the link and read his article, if you truly want to understand all the ins and outs.
For me, this is more than merely a matter of casual interest. It’s personal. Bexxar and Zevalin are high up on the list of possible future treatments for me. I’ve already responded well to rituximab (Rituxan) – the medicine that serves as the targeting mechanism for Bexxar and Zevalin, enabling them to deliver tiny particles of radioactive material directly to malignant NHL cells.
It’s to my advantage that my doctors keep as many treatment arrows in their quiver as they possibly can. Your e-mail to your U.S. Senators and Representative will help make it so.
Here’s how to contact them. First, highlight and copy the text below into your computer’s clipboard. Then, go to this web page, and click on “U.S. Senators” and “U.S. Representatives” to find the appropriate officials. Then, after completing the preliminaries, simply paste the text into the field for the body of the message. Make whatever modifications you wish, of course.
(The following was sent to me by Betsy de Parry, a fellow NHL survivor who received Bexxar five years ago, and has not had a recurrence since then. She had not been responding well to other treatments, and credits Bexxar with saving her life. I’ve edited her text down quite a bit; the original was even longer.)
CLICK HERE to send an e-mail to your U.S. Senators and Representative.
Now, here’s the text of the sample e-mail. (There's also an alternate approach, which involves sending a brief e-mail to Betsy de Parry, who's gathering these for the use of lobbyists; I've included that additional information at the end.)
On November 13, Newsweek released a story entitled “How Washington Is Nixing A Cancer Cure.” The link to it is: http://www.newsweek.com/id/70301
I am writing to you in the hope that you will intercede on behalf of thousands of patients whose very lives depend on these drugs which will soon become extinct if the ruling is allowed to take effect on January 1, 2008. I am writing as an individual, not as a member of any group or organization.
Bexxar and Zevalin have proven to be highly effective treatments for non-Hodgkin lymphoma. They are examples of a type of treatment known as radioimmunotherapy.
Radioimmunotherapy, categorized as a radiopharmaceutical under the Medicare payment system, has presented challenges for the Centers for Medicare and Medicaid Services (CMS) since it was first approved. This is because it does not fit neatly into existing categories. Low Medicare reimbursement rates have already made it financially difficult for hospitals to offer these treatments. Several publications, including the Journal of the National Cancer Institute (Volume 99, Issue 7, April 4, 2007) and the New York Times (July 14, 2007), have reported that only 5% and 10% of patients who are eligible for radioimmunotherapy have actually received it.
On November 1, CMS published CMS-1392-FC, which covers changes to the hospital outpatient prospective payment system (OPPS) and sets payment rates for 2008. The new rate cuts payment for Bexxar to approximately one-half its cost. Similar issues apply to Zevalin. This will force hospitals to choose between subsidizing or abandoning the treatment. Abandonment is the most likely response, as both the American Society of Hematology (ASH) and the American Society of Clinical Oncologists (ASCO), and others, have warned, in letters they sent to CMS during the comment period prior to the final ruling.
ASH, in fact, states that “It (the ruling) will eliminate one of the few treatment options and perhaps the only treatment option for some patients with non-Hodgkins lymphoma who have failed chemotherapy treatment.”
CMS, in its final ruling, disputes this fear, saying that “given that the Medicare population is such a dominant portion of the population to which these services are targeted, we do not believe that hospitals will cease to provide the service.” With all due respect, how does CMS expect hospitals to provide any service for which they will lose money?
Additionally, CMS warns that “under 42 CFR 489.53(a)(2), CMS may terminate the provider agreement of any hospital that furnishes this or any other service to its patients but fails to also furnish it to Medicare patients who need it.” Surely no hospital will jeopardize its provider agreement. Thus, if these treatments are unavailable to Medicare patients, they will also be unavailable to anyone else.
CMS has based their recommended reimbursement rates on data from previous hospital claims that they themselves have admitted is flawed, due to widespread errors in coding. Using data that was known to be flawed, the new rate could not have been set accurately.
One thing is certain. The new rate will have long-term and devastating consequences. It will undoubtedly condemn these drugs to medical history. Several scientists and organizations fear that it will make it harder for pharmaceutical companies to develop future innovative therapies.
Much worse, this ruling surely condemns some patients to death.
Because time is so limited, I am asking that your office intercede on behalf of patients whose very lives depend on this and future treatments. I deeply appreciate your help with this matter. Lives are depending on it.
Thank you.
***
Here's an alternate way to express your views on the Bexxar/Zevalin issue (see the text of the e-mail from Betsy de Parry, below). If you're so inclined, you could do this instead of, or in addition to, e-mailing your Senators and Representative direclty.
Dear Friends,
As you know from reading the Newsweek story ( http://www.newsweek.com/id/70301 ), a very effective cancer drug is about to disappear. Several of us are mounting vigorous opposition - and to those of you have written your reps about it, many thanks. Following is way we can make our voices heard much faster than going through the traditional route of emailing our reps and hoping someone reads it.
We now have help from two lobbyists in Washington who know how to maneuver far better than we do. They are working diligently to arrange meetings with senators and representatives who may be able to help reverse the ruling. Karl Schwartz, President of Patients Against Lymphoma, or Karl and myself, may attend these meetings with him. Whether we do or not, it will be hugely helpful for them (or them and us, as the case may be), to represent the patients, families and friends whose lives will be affected if this ruling takes effect.
But – we don’t have time to create another online petition as we did before (which many of you signed - thank you!). Instead, I have volunteered to collect comments, and we need as many as we can possibly get – and as quickly as we can get them (like by next Monday morning).
I have set up a separate email account specifically for this purpose. If each of you will send your comments to that account, I will print them and get them to Washington. The more comments we have, the more impact we have – so I beg each of you to voice your concerns and to ask everyone you know to do likewise. If 100 of us gets 10 people, that's 1000 messages that go straight to Washington!
The email account is:
bdeparry@gmail.com
Please write “CMS-1392-FC” in the subject line. Your message does not need to be long. Feel free to write whatever you wish, and if you aren’t certain what to write, simply copy and paste the following:
I respectfully request an immediate reversal of CMS-1392-FC as it relates to Bexxar and Zevalin.
Please sign your name and include your city and state.
Your information will not be shared with anyone else. It will only be used for the purposes of lobbying our politicians to reverse this ruling.
Finally, "thank you" is an understatement for your support. Words can't convey how much it means.
5 comments:
Just last week, my oncologist suggested that I receive Bexxar or Zevalin when my lymphoma returns. I am shocked that such effective treatments might soon disappear. Thank you for letting me know so I can write my senators while there is still time to save them.
Thanks for this very practical post especially the explanation of the ripple effect to non-medicare patients. I makes me wonder about all sorts of medications that might fall victim to this type of restriction and the people with all sort of ailments, young and old being affected. My rep has been contacted.
Thanks for this very practical post especially the explanation of the ripple effect to non-medicare patients. I makes me wonder about all sorts of medications that might fall victim to this type of restriction and the people with all sort of ailments, young and old being affected. My rep has been contacted.
Thanks Carl for posting this on your blog. I hope other bloggers with lymphoma are doing the same and other bloggers with cancer. This ruling has the potential to affect drug development for everybody's cancer! I too have lymphoma and I was able to be treated with Bexxar as part of a clinical trial (I have an aggressive variety). I think this is an important treatment option for those with both indolent and aggressive lymphomas.
About Me
I am Pastor of the Lamington Presbyterian Church in Bedminster, New Jersey. From time to time I teach Presbyterian Polity at Princeton Theological Seminary and Presbyterian Studies at New Brunswick Theological Seminary. I am married to the Rev. Claire Pula, Director of the Bereavement Program, Hackensack Meridian Hospice in Wall, NJ. We have two children: Benjamin, a singer-songwriter, and Ania, an artist. |
Q:
Timecomplexity analysis of function, Big O
What time-complexity will the following code have in respect to the parameter size? Motivate.
// Process(A, N) is O(sqrt(N)).
Function Complex(array[], size){
if(size == 1) return 1;
if(rand() / float(RAND_MAX) < 0.1){
return Process(array, size*size)
+ Complex(array, size/2)
+ Process(array, size*size);
}
}
I think it is O(N), because if Process(A, N) is O(sqrt(N)), then Process(A, N*N) should be O(N), and Complex(array, size/2) is O(log(n)) because it halves the size every time it runs. So on one run it takes O(N) + O(log(N)) + O(N) = O(N).
Please correct me and give me some hints on how I should think / proceed an assignment like this.
I appreciate all help and thanks in advance.
A:
The time complexity of the algorithm is O(N) indeed, but for a different reason.
The complexity of the function can be denoted as T(n) where:
T(n) = T(n/2) + 2*n
^ ^
recursive 2 calls to
invokation Process(arr,n*n),
each is O(n(
This recursion is well known to be O(n):
T(n) = T(n/2) + 2*n =
= T(n/4) + 2*n/2 + 2*n =
= T(n/8) + 2*n/4 + 2*n/2 + 2*n
= ....
= 2*n / (2^logN) + ... + 2*n/2 + 2*n
< 4n
in O(n)
Let's formally prove it, we will use mathematical induction for it:
Base: T(1) < 4 (check)
Hypothesis: For n, and for every k<n the claim T(k) < 4k holds true.
For n:
T(n) = T(n/2) + n*2 = (*)
< 2*n + 2*n
= 4n
Conclusion: T(n) is in O(n)
(*) From the induction hypothesis
|
Q:
Alternative of CADisplayLink for Mac OS X
Is iOS there is CADisplayLink, in Mac OS X there is CVDisplayLink, but I can't find a way to use it, all the examples are related to OpenGL.
I created this custom UIView and I want to translate it to a NSView
#import "StarView.h"
#import <QuartzCore/QuartzCore.h>
#define MAX_FPS (100.0)
#define MIN_FPS (MAX_FPS/40.0)
#define FRAME_TIME (1.0 / MAX_FPS)
#define MAX_CPF (MAX_FPS / MIN_FPS)
#define aEPS (0.0001f)
@implementation StarView
@synthesize starImage = _starImage;
@synthesize x, y;
- (void)baseInit {
_starImage = nil;
CADisplayLink *displayLink = [CADisplayLink displayLinkWithTarget:self selector:@selector(runLoop)];
[displayLink addToRunLoop:[NSRunLoop mainRunLoop] forMode:NSDefaultRunLoopMode];
}
- (id)initWithFrame:(CGRect)frame
{
self = [super initWithFrame:frame];
if (self) {
[self baseInit];
}
return self;
}
- (id)initWithCoder:(NSCoder *)aDecoder {
if ((self = [super initWithCoder:aDecoder])) {
[self baseInit];
}
return self;
}
// Only override drawRect: if you perform custom drawing.
// An empty implementation adversely affects performance during animation.
- (void)drawRect:(CGRect)rect
{
[self.starImage drawAtPoint:CGPointMake(self.x, self.y)];
}
-(void) cycle {
self.x += 5;
if (self.x > 230) {
self.x = 0;
}
}
- (void) runLoop {
//NSLog(@"called");
static CFTimeInterval last_time = 0.0f;
static float cycles_left_over = 0.0f;
static float dt2 = 0.0f;
float dropAnimRate = (2.1f/25.0f);
CFTimeInterval current_time = CACurrentMediaTime();
float dt = current_time - last_time + cycles_left_over;
dt2 += dt;
[self setNeedsDisplay];
if (dt > (MAX_CPF * FRAME_TIME)) {
dt = (MAX_CPF * FRAME_TIME);
}
while (dt > FRAME_TIME) {
if (dt2 > (dropAnimRate - aEPS)){
[self cycle];
dt2 = 0.0f;
}
dt -= FRAME_TIME;
}
cycles_left_over = dt;
last_time = current_time;
}
@end
The part that I can't translate is this one
- (void)baseInit {
_starImage = nil;
CADisplayLink *displayLink = [CADisplayLink displayLinkWithTarget:self selector:@selector(runLoop)];
[displayLink addToRunLoop:[NSRunLoop mainRunLoop] forMode:NSDefaultRunLoopMode];
}
I know that I can use a NSTimer, but it doesn't have the same accuracy
A:
You can configure a CVDisplayLink to work independently of OpenGL. The following is code that I've used to set up a CVDisplayLink to trigger regular capture and rendering from an industrial camera:
CGDirectDisplayID displayID = CGMainDisplayID();
CVReturn error = kCVReturnSuccess;
error = CVDisplayLinkCreateWithCGDisplay(displayID, &displayLink);
if (error)
{
NSLog(@"DisplayLink created with error:%d", error);
displayLink = NULL;
}
CVDisplayLinkSetOutputCallback(displayLink, renderCallback, (__bridge void *)self);
my renderCallback function looks something like this:
static CVReturn renderCallback(CVDisplayLinkRef displayLink,
const CVTimeStamp *inNow,
const CVTimeStamp *inOutputTime,
CVOptionFlags flagsIn,
CVOptionFlags *flagsOut,
void *displayLinkContext)
{
return [(__bridge SPVideoView *)displayLinkContext renderTime:inOutputTime];
}
You'll have to replace the above with your own class and callback method, of course. The __bridge in the code is there for ARC support, so you may not need that in a manually reference counted environment.
To start capturing events from the display link, you'd use
CVDisplayLinkStart(displayLink);
and to stop
CVDisplayLinkStop(displayLink);
When done, be sure to clean up after your created CVDisplayLink using CVDisplayLinkRelease().
If I may make one last comment, the reason why you see CVDisplayLink normally paired with OpenGL is that you usually don't want to be doing rapid refreshes on the screen using Core Graphics. If you need to be animating something at a 30-60 FPS rate, you're going to either want to draw directly using OpenGL or use Core Animation and let it handle the animation timing for you. Core Graphics drawing is not the way to fluidly animate things.
|
275 F.2d 377
Paul EGAN, Plaintiff-Appellant,v.CITY OF AURORA, a Municipality under the law of the State ofIllinois, Leo Boucon, William G. Konrad, H. A. Wyeth, Sr.,William B. Robertson, Donald Curran, Hershell Stover, LeRoyStraud, Anthony Rukas, John (Jack) Pfiefer, Ray Schuhow,John Day and Charles Darling, Defendants-Appellees.
No. 12738.
United States Court of Appeals Seventh Circuit.
March 4, 1960.
Sol R. Friedman, Joseph Keig, Sr., I. Stephen Friedman, Edwin R. Armstrong, Chicago, Ill., for appellant.
William C. Murphy, Reid, Ochsenschlager, Murphy & Hupp, Aurora, Ill. (L. M. Ochsenschlager, Aurora, Ill., of counsel), for defendants-appellee.
Before HASTINGS, Chief Judge, DUFFY, Circuit Judge, and STECKLER, District Judge.
DUFFY, Circuit Judge.
1
Plaintiff is the Mayor of the City of Aurora, Illinois. He brought this suit charging violation of the Federal Civil Rights Statutes, Title 42 U.S.C.A. 1983 and 1985, and claims damages in the amount of $5,000,000.00.
2
The complaint alleges that plaintiff, as Mayor, was conducting a public meeting before a group in excess of two hundred people in the Council Chambers in the City of Aurora, when defendant Donald Curran, purporting to be acting as Chief of Police of the City of Aurora, and defendants Stover, Straud, Rukas, Pfiefer, Schuhow and Day, all purporting to be police officers of the City of Aurora, without probable cause, arrested plaintiff under color of an Illinois breach-of-the-peace statute, and incarcerated him in the city jail for a period of more than four hours. Plaintiff alleges such action was the result of a conspiracy between the above-named defendants and defendants Boucon, Konrad, Wyeth and Robertson, acting as individuals and as city commissioners of the City of Aurora, and Charles Darling who purported to be Corporation Counsel of the City of Aurora, to deprive plaintiff of his rights to freedom of speech and assembly.
3
The complaint was later amended by adding the following: 'The defendants in all matters and things herein alleged acted with the design and purpose of depriving the plaintiff of his rights under the Fourteenth Amendment of the Constitution of the United States to freedom of speech and freedom of assembly.'
4
Defendants moved to dismiss the action because the complaint as to each of them failed to state a claim upon which relief can be granted. Another ground of the motion to dismiss was that the District Court had no jurisdiction over the subject matter, and that the alleged claim was frivolous. In the alternative the motion also asked the Court to strike specified paragraphs of the complaint. The motion referred to portions of a 'proclamation' issued by plaintiff on October 13, 1958, the day prior to the date of the meeting.
5
Defendants offered and filed a copy of the proclamation issued by the Mayor, and an affidavit which alleged, in part, that the Mayor was attempting to incite a public disturbance pursuant to a program of obtaining personal publicity for himself. A counter-affidavit was offered and filed by plaintiff Egan. The District Court, without a trial, directed judgment for the defendants.
6
Rule 12(b) provides: 'If, on a motion asserting the defense numbered (6) to dismiss for failure of the pleading to state a claim upon which relief can be granted, matters outside the pleading are presented to and not excluded by the court, the motion shall be treated as one for summary judgment and disposed of as provided in Rule 56 * * *.' We shall so consider the issues before us for decision.
7
There is no dispute that on the day before the meeting, the Mayor did issue a proclamation in manner and form as follows:
8
'Proclamation
9
'Whereas;
10
'A dire emergency exists in Aurora which might spread very conceivably thru out the world, when it is demonstrated openly and conclusively that the power of the people in the choice of their executive can be surmounted and destroyed to the great disadvantage of most of the people including those who did not vote for the head of the government, and such blinking and ignoring the basic and vital things in Aurora, the State of Illinois and the Great formerly idealistic and marvelous Government of the United States of America which from its inception until about 45 years ago was the almost holy light of downtrodden men everywhere, the inspiration for living to those who had faith in us and our ideals, principals and basic pronouncements were the hope of the world, in comparison we are now preserving vicious dictatory, old corrupt and tottering governments and turning all of the wealth created by our ancestors and modern day potentialities in a mad race to keep factories working and a few all powerful against the interests, well being and now the very survival of every man woman and child on this entire earth.
11
'I Therefore Proclaim A State Of Emergency In Aurora Illinois and ask every able bodied citizen of this country, who does not have a criminal, insane or other special unsavory record to come to the city hall council chambers at the city hall at 7 P.M., Tuesday night Oct. 14, 1958 to see if the people can be heard and get a remedy from their ills as some of our predecessors did to thro off the yoke of absolute monarcy and potential slavery in their day. I will welcome all races, all creeds and even legally registered Communists who are welcome if the are sincerely willing to help preserve our laws and bear arms for this purpose and to preserve our great heritage, priceless ideals, principals and purposes or rather and this is important, Come Back To Them, all races creeds and colors are invited to 'bear arms,' as the Constitution provides, the protection and preservation of the law in Aurora and let us once more be the City of 'Lights' which guides the way by attending this most important meeting in the history of Aurora. (s) Paul Egan Mayor of Aurora, Illinois.'
12
The affidavit filed upon behalf of defendants recited a course of conduct by Egan covering three years by which it is claimed he attempted to obtain continuous personal publicity by press, radio and otherwise; that he had discharged or attempted to discharge twelve persons as Chief of Police, most of whom were appointed by him, and that on one occasion he appointed a young lady as Chief of Police who was publicity agent for several Chicago taverns, well knowing she had no experience for the office; that Egan constantly solicited publicity from the press, radio and television for his acts and doings; that he refused release from jail although a person was available and ready to put up bond, and that he demanded his attorney telephone the President of the United States and the Governor of the State of Illinois demanding that he be pardoned or released without bail.
13
The motion of the defendants recited that when Egan asked every able-bodied citizen, even registered Communists, to come to the meeting, bearing arms, for the purpose of throwing off the yoke of potential slavery, the real purpose of Egan was to overthrow by use of force and arms the police department of the City of Aurora. The motion also recited the meeting was attended by a packed and boisterous crowd; that in the event of a disturbance it would have exposed the public to serious threat of injury or loss of life because of inadequate exits from the council chambers; that the offense of disturbing the peace was committed in the presence of the police officers.
14
Egan's affidavit denied he was attempting to incite a public disturbance at the time and place of the events alleged in the complaint; it asserted the crowd was not boisterous; that he never appointed a parrot as Chief of Police of the City of Aurora, and that he did not appoint twelve persons as Chiefs of Police. Egan also recited that he had been acquitted by a jury of the charge he had violated Section 160 of the Criminal Code of the State of Illinois, Ill.Rev.Stat.1959, c. 38, 160, which charge was brought against him after his arrest at the meeting.
15
A summary judgment may not be entered pursuant to Rule 56, Federal Rules of Civil Procedure, 28 U.S.C.A., unless there is no genuine issue as to any material fact, and the moving party is entitled to judgment as a matter of law. We think the questions of fact which are disputed in this case are immaterial, and that the defendants are entitled to judgment as a matter of law.
16
Whether Mayor Egan's course of conduct, including the call to the public meeting in the council chamber, was 'political baffoonery' as characterized by the defendants, or a sincere desire by Egan to discuss issues of the day, is not of great importance under the circumstances under which the citizens of Aurora gathered at the meeting on the evening of October 14, 1958. It is evident that the invitation to attend the meeting extended by the Mayor, suggesting those attending bear arms, was fraught with great danger to all those crowded into the Council Chambers.
17
We are faced with a situation where one state public official seeks to invoke the protection of the Federal Government against other state officials in the preservation of what he conceives to be his official status. Mr. Egan alleges he was conducting the meeting in his 'official capacity as Mayor of the City of Aurora.' Plaintiff's rights, as mayor, arise under and are protected by the laws of the State of Illinois.
18
A local controversy such as this involving a dispute between officials and officers of a municipality is not the kind of case which should be brought in the federal courts. As we stated in People ex rel. Turnbaugh v. Bibb, 7 Cir., 252 F.2d 217, 219: '* * * Federal jurisdiction is to be exerted only in exceptional cases involving such an emergency or great urgency as necessitate action to prevent irreparable injury. The jurisdiction to interfere with the proceedings of state governmental bodies charged with the prosecution and punishment of offenders is an exceedingly delicate one to be exercised with the greatest of care and nicest sense of propriety. * * *'
19
The latest expression of this Court on the subject before us for decision is Monroe v. Page, 7 Cir., 1959, 272 F.2d 365. There an action was brought under the Federal Civil Rights Act because of alleged misconduct of Chicago police officers. The City of Chicago was one of the defendants. We sustained the District Court's dismissal of the cause on the ground the complaint did not state a claim upon which relief could be granted. We relied largely upon our decision in Stift v. Lynch, 7 Cir., 267 F.2d 237.
20
In the Stift case, the defendants were respectively, a sheriff, a deputy sheriff, a justice of the peace, a state's attorney and an assistant state's attorney. We disagreed with the argument that the Federal Civil Rights Act applies to 'every person', pointing to the case of Tenney v. Brandhove, 341 U.S. 367, 71 S.Ct. 783, 95 L.Ed. 1019, where the Supreme Court held members of the California State Legislature acting through a Committee were immune to action under the Federal Civil Rights Act.
21
In Stift, we referred to some decisions in other circuits which were contrary to our holding, but also cited others showing our decision was in accord with the great weight of authority. We distinguished the case of Wakat v. Harlib, 7 Cir., 253 F.2d 59. Relying upon our decisions in Eaton v. Bibb, 7 Cir., 217 F.2d 446, Miles v. Armstrong, 7 Cir., 207 F.2d 284, and United States ex rel. Atterbury v. Ragen, 7 Cir., 237 F.2d 953, we held the complaint did not state a claim under the Federal Civil Rights Act upon which relief could be granted against the sheriff and the deputy sheriff. We held the justice of the peace was a judicial officer to which the common law immunity would apply. We held no claim was stated against the state's attorney and the assistant state's attorney.
22
In Jennings v. Nester, 7 Cir., 217 F.2d 153, 155, we pointed out that the Federal Civil Rights Act was not enacted to discipline local law-enforcement officials. We stated: 'The common law provides adequate actions for damages against errant law enforcement officials.' In Monroe v. Pape,272 F.2d 365, 366, we said: 'Plaintiffs are not without their remedy in the state court.' These comments are applicable to the case at bar.
23
We hold the District Court was correct in entering judgment for all of the defendants.
24
Affirmed.
|
Q:
resignFirstResponder OR - (IBAction) doneButtonOnKeyboardPressed: (id)sender
I was just wondering which approach is better to hide keyboard in iphone application
1> Implement
- (IBAction) doneButtonOnKeyboardPressed: (id)sender
{
}
Method on Textfield 's Did End On Exit Event
OR
In Textfield implement this
-(BOOL)textFieldShouldReturn:(UITextField *)theTextField {
[txtName resignFirstResponder];
return YES;
}
Which Option is better to choose in which situation...? Any one Option has advantage over other...?
A:
I strongly prefer using the target/action pattern by responding to the UIControlEventDidEndOnExit event, usually by wiring it up in Interface Builder by connecting the "Did End On Exit" event shown in IB to the File's Owner, using the method of my choice. This would be the first option you showed.
Here's why I prefer this mechanism:
It many apps (well, at least my apps), it is necessary to distinguish between canceling input, say, by touching outside the text field, and completion of input by the done button on the keyboard (the docs tend to refer to this as the "return" button). Because the -textFieldDidEndEditing delegate method gets called any time -resignFirstResponder is invoked when the field is first responder (regardless of the reason for ending editing), it is necessary to have a variable somewhere to track the path of how you're terminating editing. This introduces a level of complexity that simply isn't necessary.
In my app, I respond to touch events outside the UITextField, invoking -resignFirstResponder to cancel editing without taking further action. If I use the delegate methods, I would need to set state here to record that I'm taking the 'cancel' path through my code, and use the -textFieldShouldReturn delegate method to both set state to indicate I'm going through the 'done' path of my code, and invoke -resignFirstResponder. Messy.
Using the target/action pattern here leads to simpler, cleaner code.
|
My group and I are carrying out an investigation to find out how different concentrations of glucose affect osmosis in potato cells. Osmosis is the passive process of diffusing water which means within this experiment particles of water will move from a lower concentrated area to an area of higher concentration ; in this case it will be a concentration of glucose. Diffusion is when particles move from an area of high concentration to low concentration ; for example when a smell of cooking spreads through the house. Osmosis is a vital mechanism in the transport of fluids in living organisms. This point is clearly proven when you put a plant cell in water.Osmosis causes water to enter the plant cell, which results in swelling. However, the cell will not burst as the plant cell walls are made up of an extremely strong substance called cellulose. The swelling within the cell eventually stops, and at this point, the cell is said to be turgid. This process involving osmosis is important within a plant as it allows the stems to become strong and upright. There are many other cases where osmosis is responsible for the basic survival of living organisms. Here is a diagram which visually shows how osmosis will occur in this situation :
In my group we are going to perform our investigation by firstly taking five equally sized test tubes and a potato which we will take a sample of using a cork borer. Afterwards, we will cut five equal pieces of potato using a knife, so that they are more or less the same weight. Once we have weighed all the five pieces of potato, we will get the five different glucose solutions that will be used throughout the experiment. These solutions will consist of different molars of glucose. For example, there will be a solution of 0 molars, 0.2 molars, 0.4 molars, 0.6 molars and 0.8 molars. Using a measuring cylinder, we will measure accurately 10 ml of each solution and put it in the appropriate test tube which would have been labelled relavant...
YOU MAY ALSO FIND THESE DOCUMENTS HELPFUL
...INTRODUCTION
Osmosis is the movement of water molecules from high concentration to low concentration through semipermeable membranes, caused by the difference in concentrations on the two sides of a membrane (Rbowen, L.). It occurs in both animals and plants cells. In human bodies, the process of osmosis is primarily found in the kidneys, in the glomerulus. In plants, osmosis is carried out...
...
Aim
To investigate the effects of increasing salinity on potatocell mass.
Background Information
This experiment is based upon osmosis. Osmosis can be defined as the net movement of water molecules from a region with high concentration to a region with low concentration. This movement must take place across a partially permeable membrane such as a...
...
Introduction
In this experiment I am going to investigate the effect of varying concentration of a differing glucose solution on the amount of osmotic activity, between the solution and a potato tuber of a given size. The purpose of this experiment is to demonstrate how living cells rely on osmosis, the diffusion of water.
...
...AN INVESTIGATION INTO THE EFFECTS OF OSMOSISPOTATOCELLS
SKILL AREA P: PLANNING
Introduction
The aim of this investigation is to see the effect of varying concentrations of glucose solution on the amount of osmotic activity between the solution and a potato chip. An investigation into Plasmolysis in onion cells was undertaken prior to this...
...EXPERIMENT TO SHOW THE EFFECT OF DIFFERENTCONCENTRATION OF GLUCOSE ON POTATO STRIPS
INTRODUCTION: Molecules of liquid and gas are constantly in motion, they move randomly in all directions and bounce around in all directions and bounce around and into each other. As they move, they tend to spread out moving from areas with many molecules to areas with fewer molecules . This process of spreading out is called...
...glass was filled with cold tap water. A drop of red food coloring was dropped in the cup. A stopwatch was used to measure the time it took for the food coloring to get to the bottom of the cup. The average diffusion rate was .78 cm a second. If a different color was used, I do not think it would have made a difference in the results. The mood of the person experimenting could possibly alter the results. For example, if a person is under stress, they may accidentally squeeze too...
...I am going to investigate osmosis when potato is placed in differentconcentrations of sucrose. I am aiming to witness osmosis in 5
differentconcentrations of sucrose. I will use 5 varying
concentrations so that I have a wider spread to compare the results,
and check that I don’t have any anomalies
Prediction
Osmosis is the process of diffusion of... |
News
Business activity continues to grow
Economic activity across England and Wales continued to grow strongly at the start of the second quarter of the year, according to the latest Lloyds Bank Regional Purchasing Managers’ Index.
Robust increases in both business activity and new work were recorded in April, although growth rates were slightly slower on average than in March. The survey also revealed the steepest monthly drop in prices charged since 2009, highlighting continued deflationary forces.
The fastest overall increase in business activity in England was recorded in the North East where growth reached a nine-month high. Other standout performers were London and the South East which recorded the biggest rise in activity since November 2014. The slowest-growing region was the East Midlands followed by the South West.
Wales continued to see strong growth in April, while new business saw the sharpest increase for 13 months.
The survey also showed further broad-based growth in employment during April although the overall rate of job creation slowed. The East of England was the best-performing region, while Wales, the North East and the North West also recorded improved rates of employment growth.
However, average prices charged for goods and services decreased during April, with the fastest rate of decline since September 2009. The average price of inputs meanwhile rose at a modest rate; slightly higher than the previous month.
Tim Hinton, managing director, mid markets and SME banking, Lloyds Banking Group said: “The strong growth in business activity and job creation seen in April are clear signs that business confidence continues to be high across England and Wales.
“However, companies may need to be careful over coming months. Increasing cost pressures might mean that they have less room to provide discounts for customers as a way of encouraging demand.” |
Pages
October 28, 2011
Canonigo
18 October 2011
Catered by Les Paellas Cafe
On my last entry - the Canon Pixma Exclusive Product Launch - the organizers gave us free meal and it was catered by Las Paellas Cafe. They have 3 dishes captured me and I'll feature this first for I am so guilty about this. Hear (read) my confession.
The first time I heard about Canonigo is in a culinary show which is sadly off the air months ago. The chef who made the demo in TV said Canonigo is just like Leche Flan. To make it more "appetizing" she made is as tall a possible, its really like Leche Flan in a bucket.
For this one, I like its sweetness (not so light and not so heavy either) sadly, I forgot to put the custard sauce on my share. I want to come back for more. *&^% I should had got 3 slices on my first take. |
Hypophagia induced by endogenous or liposome-encapsulated 3,4-dihydroxybutanoic acid.
Hypophagia induced by 3,4-dihydroxybutanoic acid (2-deoxytetronic acid, 2-DTA), an endogenous short-chain polyhydroxymonocarboxylic acid, was investigated in rats. Intraperitoneal injection of 2500 mumol 2-DTA did not suppress feeding, but 2.5 mumol 2-DTA injected into the third cerebroventricle did. To efficiently transport exogenous 2-DTA into the brain, its encapsulation and delivery in specially made sulfatide liposomes was attempted. Feeding was suppressed dose-dependently by intraperitoneally injected 2-DTA in liposomes. Injection of 2500 mumol 2-DTA into the common carotid artery also suppressed feeding. Administration by either route prolonged postprandial intermeal interval with no change in meal size, as was observed after central administration of 2-DTA. Injection of 2.5 mumol 2-DTA into the third cerebroventricle elevated plasma glucose level, leaving insulin and free fatty acids unaffected. These findings, together with previous results, indicate that at least one site for the physiological action of 2-DTA is in the hypothalamic centers for food intake. |
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The rational design of enzyme-like catalysts is an important long range goal. This proposal explores the possibility of using antibodies, which are exquisite receptor molecules, to catalyze chemical reactions. Our strategy involved synthesis of compounds which mimic the transition state structure of a particular reaction, eliciting immune responses against such substances, and characterization of the specific antibodies generated. Two reactions will be examined: the unimolecular Claisen rearrangement of chorismate to prephenate and the bimolecular Diels-Alder process. These reactions are good to model, since they do not require nucleophilic or general acid-base catalysis but should be susceptible to induced strain and proximity effects. The latter mechanisms of catalysis are what distinguish enzymes from most chemical catalysts. It is important to understand how enzymes work, and antibodies that mediate the Claisen and Diels-Alder reactions would be useful tools for investigating how enzymes utilize binding energy to achieve enormous rate enhancements and remarkable regio- and stereoselectivity. Furthermore, the Claisen and Diels-Alder reactions are among the most valuable synthetic reactions available to organic chemists for preparation of important health-related natural products. The ability to catalyze these processes with the high rates and selectivities typical of enzymes would therefore represent a significant scientific advance. We expect, finally, that the knowledge gained in this project will lead to the elaboration of general strategies for design of artificial enzymes for any chemical reaction, capitalizing on the virtually limitless supply of highly specific antibody binding sites. |
Currently, the wireless communication systems such as the mobile telephone systems or the wireless LANs (Local Area Networks) are widely used. In order to further improve the transmission rate and the transmission quality in the wireless communication technologies, next-generation technologies are being actively discussed. Some wireless communication technologies enable communication between two wireless communication devices (e.g., between a wireless base station and a mobile station) by use of multiple carriers. In some cases, the use of multiple carriers is called carrier aggregation, and each carrier in the carrier aggregation is called a component carrier.
In wireless communication, control signals are transmitted from a first wireless communication device to a second wireless communication device. The information transmitted by the control signals may include information which is to be referred to by the second wireless communication device for receiving data transmitted from the first wireless communication device (e.g., information indicating the format used in the data transmission), or information designating a transmission procedure to be used by the second wireless communication device in transmission of data (e.g., information designating a format to be used in the data transmission). For example, information transmitted from a wireless base station to a mobile station may include information to be referred to by the mobile station when the mobile station receives data through a downlink data channel, or information to be referred to by the mobile station when the mobile station transmits data through an uplink data channel.
In the case where wireless communication is performed between two wireless communication devices by use of multiple carriers, a wireless resource used in transmission of a control signal may belong to a carrier different from the carrier to which a wireless resource used in data transmission controlled by the control signal belongs. In this case, the problem is the method for recognition of the carrier to which the control signal is applied.
According to a technique which has been proposed as a recognition method, the carrier to which the control signal is applied is clearly indicated by attaching a bit for carrier recognition (carrier indicator) to the control signal. (See, for example, “Final Report of 3GPP TSG-RAN WG1 #57 v1.0.0”, 3GPP (3rd Generation Partnership Project) TSG RAN WG1 #57bis, R1-092292, July 2009, Section 15.4.)
Another recognition method which has been proposed is applied to a wireless base station scrambling a CRC (Cyclic Redundancy Check) bits attached to a control signal, with a scrambling sequence corresponding to an ID (Identifier) assigned to a mobile station as an opposite party. (See, for example, “Control signaling for carrier aggregation”, 3GPP TSG RAN WG1 #55bis, R1-090375, January 2009.) According to the above recognition method, IDs in the number of carriers are assigned to each mobile station, and the CRC bits are scrambled with a scrambling sequence corresponding to one of the IDs. Therefore, it is possible to concurrently recognize both of the mobile station and the carrier to which the control signal is applied.
However, according to the technique disclosed in 3GPP TSG RAN WG1 #57bis, R1-092292, the wireless resources are excessively spent for attaching the carrier indicator to the control signal, so that the transmission efficiency of the control signal is lowered. In addition, according to the technique disclosed in 3GPP TSG RAN WG1 #55bis, R1-090375, the IDs in the number of carriers are assigned to the opposite party. Therefore, the ID exhaustion becomes a problem. In the case where the length of the bit sequence of the ID is fixed, the number of opposite parties with which communication can be performed in parallel is limited. For example, in the case where five carriers are available in total, the upper limit of the number of opposite parties with which communication can be performed in parallel can become one fifth of the number of opposite parties with which communication can be performed in parallel when only one carrier is used in communication with each opposite party.
Further, in some cases, information indicating a border between the control signal and a data channel is transmitted as a separate control signal. In such cases, when the separate control signal is not correctly received, the border between the control signal and the data channel cannot be correctly detected, so that data cannot be correctly received through the data channel. |
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EMBO Mol Med (2019) 11: e10378
Introduction {#emmm201910378-sec-0001}
============
The efficient propagation of electrical nerve impulses requires the establishment of myelin sheaths around axons (Baumann & Pham‐Dinh, [2001](#emmm201910378-bib-0003){ref-type="ref"}). Multiple sclerosis (MS) is a disease affecting nerve conduction as a consequence of myelin damages. Current treatments of MS are not curative but only fighting the associated inflammation without repairing myelin. The myelination process is orchestrated by integrated cellular and molecular interactions unravelling potential therapeutic strategies for remyelination (Mi *et al*, [2007](#emmm201910378-bib-0014){ref-type="ref"}; Abu‐Rub & Miller, [2018](#emmm201910378-bib-0001){ref-type="ref"}). During development, precursors of oligodendrocytes (OL), the myelinating cells of the central nervous system (CNS), use guidance cues to migrate towards their target neuronal cells. Strikingly, this process is not restricted to developmental phases but also occurs to a certain extent in the adult brain. This is also the case in CNS‐demyelinating diseases in which partial spontaneous remyelination closely mimicking developmental myelination is observed. Combinations of soluble factors and membrane‐bound cues are considered to create a specific molecular environment dictating the precise recognition process leading to the myelination of axons (Piaton *et al*, [2010](#emmm201910378-bib-0022){ref-type="ref"}). Among these factors regulating the early phase of myelination, members of the Semaphorin family have been shown to regulate OL precursor cell migration in the optic nerve (Tsai & Miller, [2002](#emmm201910378-bib-0031){ref-type="ref"}) and inhibit adult OL process outgrowth (Ricard *et al*, [2001](#emmm201910378-bib-0026){ref-type="ref"}). Strikingly, an analysis of human multiple sclerosis sample tissues and the use of an experimental model of demyelination revealed a clear spatio‐temporal regulation of Sema3A expression, thereby strengthening the idea of a role of Semaphorins in myelination (Williams *et al*, [2007](#emmm201910378-bib-0034){ref-type="ref"}). However, little is known about the expression of Semaphorin receptors in this context. This is indeed a crucial issue because the biological functions of Semaphorins are intimately linked to the composition of a receptor complex associating various partners in order to trigger appropriate growth‐promoting or growth‐inhibiting effects of Semaphorins (Derijck *et al*, [2010](#emmm201910378-bib-0008){ref-type="ref"}). Thus, we decided to investigate the expression of Plexin‐A1, one of the major Sema3A‐transducing receptors so far essentially described as a transducer of Sema3A inhibiting signal in neurons (Püschel, [2002](#emmm201910378-bib-0023){ref-type="ref"}) or for its role in the immune system (O\'Connor & Ting, [2008](#emmm201910378-bib-0018){ref-type="ref"}). We found that Plexin‐A1 is overexpressed in the white matter of MS patients. Moreover, in mouse, only few CNP‐positive OL expressed Plexin‐A1. However, we observed a sevenfold increase in CNP‐positive OL in animals exhibiting cuprizone‐induced lesions. This was concomitant to local deposition of Sema3A in the demyelinated regions. Moreover, *in vitro* studies showed that blocking Plexin‐A1 counteracted the anti‐migratory and anti‐differentiation effect of Sema3A in oligodendrocytes. Hence, we showed that the administration of the Plexin‐A1 antagonist peptide MTP‐PlexA1 improved myelin content and locomotor activity in mice fed with cuprizone to induce demyelinated lesions or in the context of experimental autoimmune encephalomyelitis (EAE). Altogether, our results suggest a therapeutic potential of inhibiting Plexin‐A1 in myelin diseases such as multiple sclerosis in which we found overexpression of Plexin‐A1.
Results {#emmm201910378-sec-0002}
=======
Plexin‐A1 is expressed in human oligodendrocytes {#emmm201910378-sec-0003}
------------------------------------------------
Previous studies showed the expression of Sema3A in MS lesions (Williams *et al*, [2007](#emmm201910378-bib-0034){ref-type="ref"}) where it is supposed to contribute to the lack of remyelination. We now examined whether OL express the Sema3A signalling receptor Plexin‐A1. To this end, we performed immunocytochemical staining for Plexin‐A1 on a human brain tissue array. The results confirm previous data (Jacob *et al*, [2016](#emmm201910378-bib-0010){ref-type="ref"}), showing neuronal expression of Plexin‐A1 in several CNS locations, but also demonstrate the expression in oligodendrocyte cells in the white matter (Fig [1](#emmm201910378-fig-0001){ref-type="fig"}A). The global analysis of the tissue array revealed a wide expression in several regions of the central nervous system including the cortex, striatum or the spinal cord (data not shown). To confirm the identity of Plexin‐A1‐expressing cells in the white matter, we performed a CNP (3′,5′‐cyclic nucleotide phosphodiesterase) staining (pan‐marker of oligodendrocytes) on the adjacent section of the tissue array. False colour coding of the microphotographs allowed overlay of the two sections to exemplify the co‐expression of Plexin‐A1 and CNP (Fig [1](#emmm201910378-fig-0001){ref-type="fig"}B).
{#emmm201910378-fig-0001}
Plexin‐A1 is overexpressed in MS patients {#emmm201910378-sec-0004}
-----------------------------------------
In order to evaluate the Plexin‐A1 level of expression in the context of multiple sclerosis, we first performed data mining from published gene array profiles using the GEO platform. The analysis of four chronic plaques and two healthy controls (Han *et al*, [2012](#emmm201910378-bib-0009){ref-type="ref"}) revealed an averaged 4.2‐fold increase in *Plexin‐A1* mean expression (4.7‐fold increase in the median) and 3.2‐fold increase in *SEMA3A* mean expression (2.8‐fold increase in the median) in the disease condition ([Appendix Fig S1](#emmm201910378-sup-0001){ref-type="supplementary-material"}). We next collected and analysed white matter samples of 11 MS patients and nine healthy controls from the Netherlands Brain Bank (see [Appendix Table S1](#emmm201910378-sup-0001){ref-type="supplementary-material"} for details). We performed a Western blot analysis to evaluate Plexin‐A1 content and found a 2.3‐fold increased expression in MS patients (Fig [2](#emmm201910378-fig-0002){ref-type="fig"}A and B). The proportion of MS patients exhibiting such a twofold increase in Plexin‐A1 expression above the averaged expression measured in healthy controls reached 45% of the patients (Fig [2](#emmm201910378-fig-0002){ref-type="fig"}C). To further characterize this overexpression of Plexin‐A1, we also determined the number of CNP‐Plexin‐A1‐positive cells by immunocytochemistry conducted on fresh‐frozen sections of the white matter samples. As seen in Fig [2](#emmm201910378-fig-0002){ref-type="fig"}D and E, we found a threefold increase in Plexin‐A1‐positive CNP‐expressing cells in MS patients compared to healthy controls. This suggested that Plexin‐A1 may represent an interesting target in the context of MS.
{#emmm201910378-fig-0002}
Plexin‐A1 is overexpressed in OL in experimental demyelination conditions {#emmm201910378-sec-0005}
-------------------------------------------------------------------------
To address whether Plexin‐A1 may be involved in demyelination/remyelination conditions in the adult, we used the cuprizone model. In this model, a demyelination/remyelination process is obtained by feeding mice with 0.3% cuprizone (*bis*‐cyclohexanone oxal‐dihydrazone) for 4 weeks (acute demyelination phase) and normal diet for 2 weeks (initiation of remyelination) or 4 weeks (induction of total remyelination; see Fig [3](#emmm201910378-fig-0003){ref-type="fig"}A for representative examples). Administration of 8‐week cuprizone diet induces permanent demyelination as described previously (Matsushima & Morell, [2001](#emmm201910378-bib-0013){ref-type="ref"}). We performed double immunostaining for CNP and Plexin‐A1 and determined at the corpus callosum level the number of double‐positive cells in the different demyelination/remyelination status (Fig [3](#emmm201910378-fig-0003){ref-type="fig"}B). Only few OL expressed Plexin‐A1 in control conditions (normal diet, 5.3% of CNP‐positive OL). However, we found that Plexin‐A1 was significantly overexpressed in OL after 4‐week cuprizone and 2‐week normal diet administration (37.8% of CNP‐positive OL, ANOVA, *P* = 0.0015; Fig [3](#emmm201910378-fig-0003){ref-type="fig"}C). Strikingly, Plexin‐A1 expression was back to control level after total recovery (4‐week cuprizone + 4‐week normal diet), while it was not overexpressed in the 8‐week cuprizone group (Fig [3](#emmm201910378-fig-0003){ref-type="fig"}C). Moreover, similar to what has been previously described in human samples of multiple sclerosis (Williams *et al*, [2007](#emmm201910378-bib-0034){ref-type="ref"}), we also found that Sema3A expression transiently increased in acute phases of cuprizone‐induced demyelination (Fig [3](#emmm201910378-fig-0003){ref-type="fig"}D). The overexpression of Sema3A was not uniform throughout the brain but rather matched with cuprizone‐induced lesion sites. This suggested that a Sema3A/Plexin‐A1 signalling is reactivated in the adult in case of demyelination/remyelination process.
{#emmm201910378-fig-0003}
MTP‐PlexA1 cancels Sema3A repulsive effect on oligodendrocyte migration {#emmm201910378-sec-0006}
-----------------------------------------------------------------------
Sema3A deposit inhibits OPC recruitment into MS lesions, explained in part by Sema3A repulsive effect (Boyd *et al*, [2013](#emmm201910378-bib-0004){ref-type="ref"}). We first addressed the involvement of Plexin‐A1 in Sema3A signalling by RNA interference. We obtained a significant 50% knockdown of Plexin‐A1 in Oli‐neu cells as seen by immunocytochemistry and RT--qPCR (Fig [4](#emmm201910378-fig-0004){ref-type="fig"}A). We used XCELLigence transwell chambers to monitor 2% serum‐induced chemotactic migration of the OPC cell line Oli‐neu during 8 h. Compared to control migration without Sema3A, addition of 20 ng/ml Sema3A in the lower chamber decreased migration of 37% of Oli‐neu cells transfected with siRNA control. Oli‐neu cells transfected with siRNA targeting Plexin‐A1 reached 95% of control migration (Fig [4](#emmm201910378-fig-0004){ref-type="fig"}B). This result confirmed the requirement of Plexin‐A1 to drive the inhibitory effect of Sema3A. This lower expression was indeed sufficient to significantly decrease the number of NRP1/Plexin‐A1 dimers (a key step to trigger semaphoring signalling) as determined by proximity ligation assay (Fig [4](#emmm201910378-fig-0004){ref-type="fig"}C). We next used the recently developed peptidic antagonist MTP‐PlexA1. This peptide blocks receptor dimerization and signalling by interfering with the transmembrane domain of Plexin‐A1. It has been successfully used *in vitro* to antagonize Plexin‐A1 signalling and cell migration, while it showed anti‐tumour effect *in vivo* (Jacob *et al*, [2016](#emmm201910378-bib-0010){ref-type="ref"}). Strikingly, the addition of MTP‐PlexA1 induced a similar disruption of NRP1/Plexin‐A1 dimers, thereby confirming the inhibitory effect of the peptide (Fig [4](#emmm201910378-fig-0004){ref-type="fig"}D). Pre‐incubation of the Oli‐neu cells with MTP‐PlexA1 (10^−7^ M) cancelled Sema3A inhibitory effect by bringing migration up to the control condition without Sema3A (Fig [4](#emmm201910378-fig-0004){ref-type="fig"}E). This effect of MTP‐PlexA1 was dose‐dependent with a loss of efficacy from 10^−9^ M. Because of the toxicity of the vehicle (LDS) alone on Oli‐neu cells (data not shown), we were not able to correctly evaluate higher concentrations of the peptide. However, because a maximal effect was obtained with 10^−7^ M we chose this concentration to define the dose for *in vivo* evaluation consistently with previous studies (Jacob *et al*, [2016](#emmm201910378-bib-0010){ref-type="ref"}).
{#emmm201910378-fig-0004}
MTP‐PlexA1 favours oligodendrocyte differentiation in the presence of Sema3A {#emmm201910378-sec-0007}
----------------------------------------------------------------------------
Remyelination failure in MS lesions can result from the lack of OPC recruitment as well as inhibition of OPC differentiation into mature myelinating oligodendrocytes. Our second *in vitro* functional test evaluated by RT--qPCR MTP‐PlexA1 ability to increase a late oligodendroglial marker (*MBP*) expression during neural stem cell (NSC) differentiation. Plating of multipotent NSC onto PLO (poly‐L‐ornithine) with low growth factor concentration induces their differentiation into oligodendroglial, astrocytic glial and neural lineage. By adding triiodothyronine hormone and ascorbic acid into differentiation medium, we favoured oligodendroglial lineage. 100 ng/ml of Sema3A reduced by twofold the expression of *MBP* mRNA after 4 days of differentiation, whereas concomitant treatment with 10^−7^ M of MTP‐PlexA1 brings back *MBP* mRNA expression to 1.2‐fold of control condition without Sema3A (Fig [4](#emmm201910378-fig-0004){ref-type="fig"}F).
MTP‐PlexA1 exhibits no toxicity *in vivo* {#emmm201910378-sec-0008}
-----------------------------------------
Because of the expression of Plexin‐A1 in adult neurons (Jacob *et al*, [2016](#emmm201910378-bib-0010){ref-type="ref"}), we had to check whether a chronic treatment with MTP‐PlexA1 could induce cognitive disabilities. We first evaluated the locomotion capability of vehicle (LDS) and MTP‐PlexA1‐treated animals that had received 1 μg/kg MTP‐PlexA1 three times a week for 4 weeks in an open‐field task (Fig [5](#emmm201910378-fig-0005){ref-type="fig"}A). No difference was found between the two groups, indicating that mice had the same exploration capacity. We then investigated mouse anxiety with an elevated plus maze (EPM) test. After quantification, groups exhibited no significant difference in open‐arm exploration, demonstrating that MTP‐PlexA1 has no measurable effect on plus maze‐evaluated anxiety compared to vehicle (Fig [5](#emmm201910378-fig-0005){ref-type="fig"}B). Hence, we assessed the hippocampal function integrity with a spatial recognition task. Here again, the object discrimination capacity (determined through the recognition index) was identical in the two groups, thereby demonstrating no impact of MTP‐PlexA1 on mouse cognitive functions (RI vehicle 0.36, RI MTP‐PlexA1 0.35; Fig [5](#emmm201910378-fig-0005){ref-type="fig"}C and D).
{#emmm201910378-fig-0005}
To further address the innocuity of MTP‐PlexA1, we also performed a blood sample analysis on four vehicle and four MTP‐PlexA1‐treated animals. White and red cell numeration showed no difference. Kidney and hepatic functions were also equivalent in the two groups ([Appendix Table S2](#emmm201910378-sup-0001){ref-type="supplementary-material"}). This lack of toxicity offered the possibility to test this administration schedule and dosing similar to what was previously performed to treat tumours (Jacob *et al*, [2016](#emmm201910378-bib-0010){ref-type="ref"}) in a model of demyelination to demonstrate the therapeutic potential of MTP‐PlexA1.
MTP‐PlexA1 rescues corpus callosum myelination in demyelinating cuprizone murine model {#emmm201910378-sec-0009}
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To investigate the therapeutic potential of MTP‐PlexA1, we followed by MRI (DTI and T2WI) and histology the evolution of the white matter using the cuprizone‐induced demyelination--remyelination mouse model (Fig [6](#emmm201910378-fig-0006){ref-type="fig"}A). Averaged food intake monitoring confirmed equal consumption of the cuprizone diets in all experimental groups (Fig [6](#emmm201910378-fig-0006){ref-type="fig"}B). The analysis of T2WI signal intensity was used to evaluate the level of inflammation (signing the existence of a toxic effect of cuprizone), whereas DRAD (radial diffusion) signal increase was used as a readout of demyelination (Klawiter *et al*, [2011](#emmm201910378-bib-0012){ref-type="ref"}). We found that the T2WI signal intensity was identical in the two groups and the highest at W4 (123% of the baseline in control and 124% MTP‐PlexA1). The T2WI signal progressively decreased in an equivalent manner for both groups at W6 and W8, thereby confirming an equal cuprizone‐induced demyelination process (Fig [6](#emmm201910378-fig-0006){ref-type="fig"}C and D). To evaluate myelin damages, the t0 session was used as a normalization value for each mouse. DRAD was averaged to localize the demyelination areas in the corpus callosum (red to yellow pixels; Fig [6](#emmm201910378-fig-0006){ref-type="fig"}E). From t0 to W4, DRAD does not differ significantly from the baseline between vehicle (98%) and MTP‐PlexA1 (97%). At W6, perturbation in the DRAD is observed for the vehicle group (110%) but not for MTP‐PlexA1 group (103%), which almost stays at the baseline (Fig [6](#emmm201910378-fig-0006){ref-type="fig"}F). Strikingly, at W8, DRAD signals are significantly higher (two‐way ANOVA, *P* = 0.0196) in the vehicle group (113%) than in the MTP‐PlexA1 group (101%; Fig [6](#emmm201910378-fig-0006){ref-type="fig"}F). To confirm the apparent gain in myelination in MTP‐PlexA1‐treated mice, brains were collected at the end of the protocol to perform myelin histological staining. We performed Luxol fast blue staining on 6‐μm‐thick sagittal brain slices (Fig [7](#emmm201910378-fig-0007){ref-type="fig"}A). We analysed Luxol staining in the splenium and adjacent half of the corpus callosum body, being the structures the most severely demyelinated by cuprizone. We expressed staining intensity as a percentage of healthy control. Mice fed 4 weeks with cuprizone and injected with vehicle during the whole protocol exhibited 67% of healthy control staining, whereas averaged staining of mice fed 4 weeks with cuprizone and treated with MTP‐PlexA1 during the whole protocol reaches 100% of healthy control staining (Fig [7](#emmm201910378-fig-0007){ref-type="fig"}A). Histological pictures show a strong demyelination in the whole analysed area of demyelinated control. In mice treated with vehicle, intermediate demyelination appears and there was a clear lack of myelin compaction inside the splenium, which is not observed in healthy control and MTP‐PlexA1‐treated mice. This is further illustrated by immunostaining of PLP or staining with Fluoromyelin to visualize myelin content at the different stages (Fig [7](#emmm201910378-fig-0007){ref-type="fig"}A). Hence, we performed a proximity ligation assay on histological slices at the level of the lesions in the corpus callosum and splenium (Fig [7](#emmm201910378-fig-0007){ref-type="fig"}B). This analysis showed a dramatic loss of the number of NRP1/Plexin‐A1 dimers (−50%, *P* = 0.0027) in treated animals compared to controls. While demonstrating that a sufficient amount of the peptide crossed the blood--brain barrier and reached the lesion sites, this analysis provides a demonstration of the mechanism of action of the peptide to exert its protective effects. Therefore, MTP‐PlexA1 appears to rescue myelin in corpus callosum to a normal level despite cuprizone treatment by disrupting the signalling capability of Plexin‐A1.
{#emmm201910378-fig-0006}
{#emmm201910378-fig-0007}
Analysis of functional recovery with CatWalk Assay {#emmm201910378-sec-0010}
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MTP‐PlexA1 allowing myelin recovery at the histological level, we looked for a positive effect *in vivo* on functional recovery. Locomotion disorders represent a functional disability found in MS patients and observed in demyelination models. We used CatWalk assay to measure cuprizone feeding effect on mice gait and to evaluate any curative potential of MTP‐PlexA1. We established a 10‐week experiment where mice were fed 6 weeks with cuprizone, then treated 4 weeks with MTP‐PlexA1 or vehicle for recovery. Cuprizone treatments induced alteration of temporal and kinetic parameters. Because the results obtained with forelimbs (Fig [8](#emmm201910378-fig-0008){ref-type="fig"}) and hindlimbs (see [Appendix Fig S2](#emmm201910378-sup-0001){ref-type="supplementary-material"}) are identical at the same time points, they are described here without distinction. Stand Time, Swing and Step Cycle duration increased transiently after 2 weeks of curative treatment in mice receiving vehicle injections, whereas mice receiving MTP‐PlexA1 presented no alteration of these parameters (Fig [8](#emmm201910378-fig-0008){ref-type="fig"}A). There was a correlated significant Swing Speed decrease in mice receiving vehicle at 2 weeks of recovery (Fig [8](#emmm201910378-fig-0008){ref-type="fig"}B). No significant alteration occurred in Stride Length (Fig [8](#emmm201910378-fig-0008){ref-type="fig"}B). Therefore, 6 weeks of cuprizone feeding induced a transitory locomotor disorder, which appearance is prevented by MTP‐PlexA1. Increased Stand Time indicated a longer postural phase, whereas increased Swing Duration without longer Stride Length indicated a slower and less effective propulsion phase. This altered propulsion could have different origins such as ataxia, muscular asthenia or spasticity, reflecting a large impact of cuprizone counteracted by a protective effect of MTP‐PlexA1 treatment.
{#emmm201910378-fig-0008}
MTP‐PlexA1 exhibits protective effect in demyelinating EAE murine model {#emmm201910378-sec-0011}
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We extended our therapeutic approach to the EAE mouse model. This remitting--relapsing model is characterized by the appearance of an ascending paralysis. First, we validated expression of our therapeutic target at the peak of clinical score (reached 13 days after immunization). Indeed, Plexin‐A1 mRNA is significantly more expressed in EAE mice compared to sham mice (Fig [9](#emmm201910378-fig-0009){ref-type="fig"}A). We next investigated the therapeutic potential of MTP‐PlexA1 on the evolution of clinical scores and at the histological level with spinal cord myelin staining. Mice treated with vehicle only displayed first clinical signs 10 days after immunization. Then mean clinical score described a bell shape evolution with a maximum at day 13. Non‐linear regression showed that mean clinical score of mice treated from day 1 with 1 μg/kg MTP‐PlexA1 was significantly lower compared to mice treated with vehicle (Fig [9](#emmm201910378-fig-0009){ref-type="fig"}B). This effect translated into a marked improvement of the myelin content at the lumbar spinal cord level in treated animals (Fig [9](#emmm201910378-fig-0009){ref-type="fig"}C). The Fluoromyelin analysis of the dorsal region showed +48% signal (*P* = 0.0124) and +41% in the lateroventral region (*P* = 0.0448; vehicle group *n* = 3; MTP‐PlexA1 *n* = 3 for a total of 21 microscopy fields; Fig [9](#emmm201910378-fig-0009){ref-type="fig"}D). Moreover, the clinical scores of mice treated with 10 μg/kg MTP‐PlexA1 were significantly improved compared to the 1 μg/kg condition. Hence, we challenged in a second experiment the therapeutic potential of MTP‐PlexA1 when the treatment was administrated after first clinical symptoms appeared (in this case at day 10) and prolonged up to relapse phase. Strikingly, MTP‐PlexA1‐treated mice display no relapse in the next 28 days, whereas 28.6% of vehicle‐treated mice display relapse with score ≥ 2 (see [Appendix Fig S3](#emmm201910378-sup-0001){ref-type="supplementary-material"}). Importantly, the administration of MTP‐PlexA1 had no impact on the inflammatory level as seen by the similar levels of circulating TNF‐α measured by ELISA in all conditions at day 11 (Fig [9](#emmm201910378-fig-0009){ref-type="fig"}E).
{#emmm201910378-fig-0009}
Discussion {#emmm201910378-sec-0012}
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Our expression data showed the expression of Plexin‐A1 in mouse and human oligodendrocytes. This expression is higher in patients with MS compared to healthy controls. The *in vitro* studies demonstrated the capacity of MTP‐PlexA1 to counteract Sema3A inhibitory effects on oligodendrocytes migration and differentiation. *In vivo* studies showed that administrations of MTP‐PlexA1 every 3 days at the dose of 1 μg/kg induced beneficial therapeutic effects exemplified by myelin integrity signal recovery in DTI‐MRI longitudinal follow‐up of animals presenting cuprizone‐induced demyelination. Histological examination of the brains at the end of the protocol showed complete restoration of myelin content in MTP‐PlexA1‐treated animals compared to only partial recovery in vehicle‐treated animals. This myelin recovery is consistent with the functional recovery observed for MTP‐PlexA1‐treated animals for which five independent parameters of gait efficiency were measured. Such a protective effect was also observed in the EAE model in which we found that MTP‐PlexA1 had a positive impact on the myelin content of lumbar spinal cord. Hence, behavioural studies combined with blood analysis demonstrated the innocuity of the MTP‐PlexA1 peptides at the cognitive and biological levels.
Several studies indicate that Semaphorins play a role in OPC migration and OL process outgrowth (Piaton *et al*, [2009](#emmm201910378-bib-0021){ref-type="ref"}). Our results demonstrate that Plexin‐A1, one of the major signalling receptors of Sema3A (Tamagnone *et al*, [1999](#emmm201910378-bib-0028){ref-type="ref"}; Tamagnone & Comoglio, [2000](#emmm201910378-bib-0029){ref-type="ref"}), is expressed in CNP‐positive OL and overexpressed in case of induced demyelination. We also show that a Sema3A/Plexin‐A1 signalling event triggers the inhibition of OL migration and differentiation.
Neuropilins and CRMPs are key components of class 3 Semaphorin signalling in OPC and OL (Ricard *et al*, [2000](#emmm201910378-bib-0025){ref-type="ref"}, [2001](#emmm201910378-bib-0026){ref-type="ref"}; Cohen, [2005](#emmm201910378-bib-0005){ref-type="ref"}). Other members of the Plexin family have also been suggested to act as regulators of Semaphorin signalling in OL. This is the case of Plexin B1 for the transduction of Sema4D (Moreau‐Fauvarque *et al*, [2003](#emmm201910378-bib-0015){ref-type="ref"}) and that of Plexin A4 in OPC (Okada *et al*, [2007](#emmm201910378-bib-0019){ref-type="ref"}). Moreover, Sema4D‐plexin‐B1 interactions are critical for the pathogenesis of EAE (Okuno *et al*, [2010](#emmm201910378-bib-0020){ref-type="ref"}). Our data now show that Plexin‐A1 expression spatially and temporally fits with a role for Sema3A signal transduction in OL. Plexin‐A1 expression significantly increased when provoking cuprizone‐induced demyelination--remyelination process. In this situation, Plexin‐A1 expression was only detected during the phase of acute demyelination during which we also found transient expression of Sema3A in the lesion sites mirroring what was previously shown in MS human samples (Williams *et al*, [2007](#emmm201910378-bib-0034){ref-type="ref"}), predominantly in the posterior third of the corpus callosum. This strongly supported a role of Plexin‐A1 in the transduction of the Sema3A‐induced migratory inhibition preventing remyelination. Indeed, we found *in vitro* that the silencing of Plexin‐A1 using RNAi or the blockade with an antagonist peptide abolished the Sema3A‐induced inhibition of OL migration. Interestingly, the inhibitory effect of Sema3A on OPC differentiation was also counteracted when blocking Plexin‐A1. This suggests that the beneficial effect seen *in vivo* with MTP‐PlexA1 is related both on a pro‐migratory effect and on a differentiation effect.
To block Plexin‐A1, we have used a synthetic peptide mimicking its transmembrane domain. This type of antagonist previously described for other targets such as NRP1 (Nasarre *et al*, [2010](#emmm201910378-bib-0017){ref-type="ref"}; Arpel *et al*, [2016](#emmm201910378-bib-0002){ref-type="ref"}), HER2 (Arpel *et al*, [2016](#emmm201910378-bib-0002){ref-type="ref"}) or Eph‐A2 (Sharonov *et al*, [2014](#emmm201910378-bib-0027){ref-type="ref"}) has been shown to act by the direct modulation of Plexin‐A1 homodimerization or heterodimerization with Neuropilin‐1. Long‐lasting effects were described on brain tumour growth when the peptide was administrated every 3 days *in vivo* (Jacob *et al*, [2016](#emmm201910378-bib-0010){ref-type="ref"}). We now observed therapeutic effects of MTP‐PlexA1 in the demyelination models when administrated with the same schedule and same dosage. While the exact fraction of peptide reaching the lesions remains to be determined, the efficient protective effects observed in two different models of induced demyelination suggest that the required therapeutic dose is in the range of μg/kg. A proximity ligation assay indeed demonstrated a significant decrease in NRP1/Plexin‐A1 dimers in brain slices prepared from treated animals. This assay confirmed a direct impact of MTP‐PlexA1 on the signalling of Plexin‐A1 in the lesion sites. However, membrane targeting peptides exhibit no specificity towards OL cells and Plexin‐A1 is also expressed in adult neurons. While previous studies in cancer settings showed no obvious deleterious effects after 1‐month treatments, we performed here series of behavioural experiments to challenge whether the peptide would or would not impair cognitive functions in a context of chronic administration. We found no effect on cognitive performance after 4‐week treatments in three different tests. This was also correlated with no sign of hematologic or metabolic alteration. Thus, this antagonist peptide could be seen as an interesting drug candidate in the MS context. A translation to humans should be facilitated by the 100% homology between the mouse and human Plexin‐A1 transmembrane domain sequences. Moreover, our comparative analysis of white matter from nine healthy controls or 11 MS patients revealed a marked increase in Plexin‐A1 expression in MS. Future investigations will explore the dynamic of Plexin‐A1 expression along the disease evolution to better identify the right therapeutic window while treating patients with anti‐inflammatory medications. Hence, because current treatments of MS are essentially combating inflammation but are not repairing lesions, future studies will address whether a combination treatment associating anti‐inflammatory molecules with Plexin‐A1 antagonist creates all conditions for repairing MS lesions.
Materials and Methods {#emmm201910378-sec-0013}
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Cell culture {#emmm201910378-sec-0014}
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Oli‐neu cells (Jung *et al*, [1995](#emmm201910378-bib-0011){ref-type="ref"}) kindly provided by Dr Trotter\'s laboratory were cultured at 37°C, with 5% CO~2~, and expanded in Sato medium containing 1% horse serum (DMEM, with 0.2% (w/v) sodium bicarbonate, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 220 nM sodium selenite, 100 μM putrescine, 500 nM triiodothyronine, 520 nM thyroxine and 200 nM progesterone). Migration experiments were performed in Sato medium without progesterone, triiodothyronine and thyroxine on surfaces coated with poly‐[l]{.smallcaps}‐lysine.
E15 embryos were collected from pregnant C57BL/6 mice for brain dissection. Cells from cerebral hemispheres were mechanically dissociated and grown in suspension as neurospheres in Neurobasal‐A medium supplemented with 2 mM [l]{.smallcaps}‐glutamine, 2% B27, penicillin (100 U/ml), streptomycin (100 μg/ml), EGF (20 ng/ml) and bFGF (20 ng/ml). Differentiation was induced by plating neurospheres on poly‐[l]{.smallcaps}‐ornithine surface in Neurobasal‐A medium supplemented with 2% B27, penicillin (100 U/ml), streptomycin (100 μg/ml), EGF (2 ng/ml), FGF (2 ng/ml), triiodothyronine (12.5 ng/ml) and ascorbic acid (50 μM).
Peptide {#emmm201910378-sec-0015}
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We used as previously described (Jacob *et al*, [2016](#emmm201910378-bib-0010){ref-type="ref"}) the specific Plexin‐A1 antagonist peptide corresponding to the transmembrane domain of Plexin‐A1 (TLPAIVGIGGGGGLLLLVIVAVLIAYKRK, amino acid T1240 to K1268 according to UniProt entry P70206). The peptide (MTP‐PlexA1) was synthetized and purified by Peptide Specialty (Heidelberg, Germany) by automatic peptide synthesis (Fmoc chemistry). Peptide purity estimated by RP‐HPLC was more than 92% according to the manufacturer\'s indication. The peptide was solubilized in 72 mM LDS (lithium dodecyl sulphate) at a concentration of 1 mg/ml for stock solution and used at 10^−7^ M *in vitro* and administrated *in vivo* every 3 days at 1 μg/kg or 10 μg/kg.
RNA interference {#emmm201910378-sec-0016}
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siRNA oligo control (AllStars Negative Control) and siRNA targeting mouse Plexin‐A1 (Mm_Plxna1_5) were purchased from Qiagen and transfected into Oli‐neu cells with INTERFERin (Polyplus‐transfection).
Cell migration assay {#emmm201910378-sec-0017}
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Migration of Oli‐neu cells was performed in Transwell CIM‐Plate 16 (8 μm pore size filter ACEA Biosciences, Inc.) with xCELLigence RTCA DP Instrument (ACEA Biosciences Inc.). Cells were pre‐incubated 1 h with vehicle alone or MTP‐PlexA1 (10^−7^ M). The 1 × 10^5^ cells were seeded in the upper chamber with 150 μl of medium. The bottom well contained 160 μl of medium supplemented with 2% foetal bovine serum for chemoattraction and 20 ng/ml Sema3A purified as previously described (Treinys *et al*, [2014](#emmm201910378-bib-0030){ref-type="ref"}) for repulsion. Analysis was performed after 6 h of migration according to the manufacturer\'s instructions. Data are expressed as a percentage of positive control migration, i.e. the migration of Oli‐neu with 2% serum and without Sema3A.
RNA analysis {#emmm201910378-sec-0018}
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Total EAE mouse brain RNA was extracted with TRIzol (Sigma) and analysed by reverse transcription--PCR. cDNA was amplified using the CFX Connect Real‐Time PCR Detection System (Bio‐Rad). A mix was done using the TaqMan™ Universal PCR Master Mix (Applied Biosystems) and the specific probe for Plexin‐A1 (probe ID: Mm00501110_m1, Thermo Fisher). For normalization of gene expression, GAPDH (probe ID: Mm99999915_g1 Thermo Fisher) was used as housekeeping gene. MBP expression was analysed with SYBR Green Supermix (Bio‐Rad) using following primers: GCCTGTCCCTCAGCAGAT and GCCTCCGTAGCCAAATCC. The following programme was used: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, and polymerization at 60°C for 1 min. MBP expression was monitored after 4 days of differentiation in the presence of Sema3A with MTP‐PlexA1 treatment or vehicle.
Animal experimentation {#emmm201910378-sec-0019}
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All experiments were performed in accordance with the French animal protection laws and were approved by the Animal Care Committee of the University of Strasbourg (APAFIS number \#8755‐2017011817246097 and \#9374‐201605111128746v2). We used C57BL/6 female mice (Charles River Laboratories) for Cuprizone study. SJL/J female mice used for EAE were purchased from Janvier and Charles River. All mice were 8 weeks of age and fed in a controlled environment (25°C) with free access to food and water and housed on a 12‐h/12‐h day/night cycle. Before any behavioural test, mice were put in experimentation room 1 h before every session to acclimatize to environmental conditions. After each trial, experimental set‐ups were cleaned with 70% ethanol to eliminate odorant cues. All experiments were conducted in blind conditions with regard to mice treatments. At the end of protocols, mice were euthanized, and brains were collected for histological examination.
Cuprizone‐induced demyelination {#emmm201910378-sec-0020}
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Mice were housed in plastic cages pre‐bedded corn (Innovive reference M‐BTM‐C8) with pre‐filled acidified water bottle (Innovive reference M‐WB‐300A). Mice were paired‐housed (one control with one treated), and cages were changed weekly. After 1 week of acclimation to environment, mice were fed *ad libitum* with a 0.3% cuprizone‐containing diet (Safe Nutrition, E82220, Version 0373 A04) changed three times per week. Cuprizone diet consumption (three times a week) and body weight (once a week) were monitored during the whole protocol. Intraperitoneal administration of vehicle (LDS, 0.072 mM) or MTP‐PlexA1 (1 μg/kg) was done three times per week.
Animals (five mice per condition: vehicle vs. MTP‐PlexA1) subjected to DTI‐MRI (Diffusion Tensor Imaging Magnetic Resonance Imaging), toxicity tests and histology analysis received 4 weeks of cuprizone diet, and those evaluated for locomotion recovery using the CatWalk system received 6 weeks of cuprizone diet followed by 4 weeks of normal diet *ad libitum*. For DTI‐MRI experiments, animals received treatments (vehicle, MTP‐PlexA1) from day 0 (start of cuprizone diet) and were imaged every 2 or 4 weeks (note that one mouse died during acquisition in the treated group at week 4). For CatWalk analysis (one session at t0, W4, W6, W8 and W10), animals received treatments (vehicle, MTP‐PlexA1) for 4 weeks from the end of cuprizone diet (6 weeks).
Preparation of brain sections {#emmm201910378-sec-0021}
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Animals were deeply anaesthetized before intracardiac perfusion of 4% formaldehyde at 4°C. Brains were then removed and postfixed in 4% (v/v) formaldehyde (FA) at 4°C for 2 h. After 24 h washing in TBS, frontal vibratome sections (70 μm thick) were performed and collected in TBS for immediate use or in Watson medium for −20°C conservation.
Induction and assessment of active experimental autoimmune encephalomyelitis {#emmm201910378-sec-0022}
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SJL/J female mice (9 weeks old) were immunized with the kit developed by Hooke laboratories (EK‐2120). Emulsion of PLP139‐151 fragment (HSLGKWLGHPDKF) in CFA (complete Freund\'s adjuvant) was administered as four subcutaneous injections of 50 μl in the flank according to the manufacturer\'s protocol. Mice received 0.4 μg of pertussis toxin intraperitoneally on the day of immunization. Sham mice received only pertussis toxin and CFA without PLP. Clinical score was performed on 21 mice (vehicle *n* = 8; MTP‐PlexA1 *n* = 8 for 1 μg/kg and *n* = 5 for 10 μg/kg). EAE was assessed clinically in blind conditions on a daily basis according to the following criteria: 0, no disease; 1, decreased tail tone; 2, impaired righting reflex and partial hind limb paresis; 3, complete hind limb paralysis; 4, hind limb paralysis with partial forelimb paralysis; and 5, moribund or dead. To evaluate myelin integrity, we collected the spinal cord of three vehicle and three MTP‐PlexA1 (1 μg/kg)‐treated animals 11 days postimmunization. We also collected sera to determine TNF‐α concentration with an ELISA kit (RD System MTA00B).
Immunostaining {#emmm201910378-sec-0023}
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For immunohistochemistry on the US Biomax tissue array (BNC17011b SF40/41), we used adjacent tissue sections of 6 μm thickness. Sections were deparaffinized with toluene, then boiled with the antigen retrieval sodium citrate buffer (pH = 6) for 10 min. Sections were incubated overnight at 4°C with primary antibodies Plexin‐A1 (ab23391, Abcam, 1:200), CNP (ab6319, Abcam, 1:500) or PLP (homemade, 1:500). Slides were then incubated with biotinylated secondary antibodies (Vector Laboratories, 1:200), amplified with the ABC Elite Vectastain kit and developed with the DAB kit from Vector Laboratories. Images of the two sections were false‐colour‐coded and overlaid in order to exemplify double‐stained cells using ImageJ software.
Human cryosections of control and MS patients were prepared from frozen tissue blocks of white matter. All samples were obtained from the Netherlands Brain Bank, the Netherlands Institute for Neuroscience, Amsterdam (open access: [www.brainbank.nl](http://www.brainbank.nl)). All material has been collected from donors for or from whom a written informed consent for a brain autopsy and the use of the material and clinical information for research purposes had been obtained by the NBB in accordance with the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Cryosection were co‐stained with the same Plexin‐A1 (1:200) and CNP (1:250) antibodies, before detection with Cy3 anti‐rabbit and A488 anti‐mouse secondary antibodies (Jackson ImmunoResearch, 1:300).
For Plexin‐A1 expression study, brain sections were incubated overnight at 4°C under agitation with anti‐Plexin‐A1 antibodies and mouse anti‐CNP antibodies before detection using Alexa488‐conjugated anti‐rabbit IgG and Cy3‐conjugated anti‐mouse IgG. The brain sections (prepared from three animals) were then mounted in Vectashield medium with DAPI. The percentage of Plexin‐A1‐positive oligodendrocytes was determined on at least five frontal sections at the level of the corpus callosum, the cortex and the striatum. Figure [3](#emmm201910378-fig-0003){ref-type="fig"}A shows Plexin‐A1 expression in the corpus callosum, while quantification reflects averaged expression in the three brain regions.
Western blot {#emmm201910378-sec-0024}
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Proteins were extracted in a PBS/Triton 1% buffer with protease inhibitors (Sigma) using the Minilys system (Bertin). Proteins were resolved in a 4--20% SDS--PAGE gel and transferred onto a nitrocellulose membrane (Trans‐Blot Turbo System, Bio‐Rad). The blots were soaked in blocking solution (TBS/0.1% Tween/5% milk) for 1 h at RT. Primary antibody (Plexin‐A1 PA5‐77697; Invitrogen, 1:1,000) was incubated overnight at 4°C. After several washes (three times for 5 min, TBS/Tween 0.1%), secondary antibody (anti‐rabbit HRP; Bio‐Rad, 1:3,000) was incubated for 1 h at RT in TBS/1% Tween/5% BSA. The revelation step was performed using Clarity ECL Blotting Substrates (Bio‐Rad) according to the manufacturer\'s instructions. Images of the immunoblots were acquired and analysed thanks to Chemidoc Touch Imaging System (Bio‐Rad) and normalized with the stain‐free method.
Proximity ligation assay {#emmm201910378-sec-0025}
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Cells were seeded on Lab‐Tek Permanox slides overnight, and then treated with appropriate peptide 10^−7^ M for 1 h or transfected with the appropriate siRNA for 48 h. After fixation with 1% PFA for 10 min, slices were permeabilized with PBS/0.1% Triton X‐100. Primary antibodies (NRP1 from Evitria, 1:500; and Plexin‐A1 from Abcam, ab23391, 1:200) were incubated overnight at 4°C in PBS. The proximity ligation assay was then performed according to the manufacturer\'s recommendations with the "detection orange" kit (Sigma). Quantification of the dots was performed using ImageJ software.
Myelin staining {#emmm201910378-sec-0026}
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Plaques of demyelination (Matsushima & Morell, [2001](#emmm201910378-bib-0013){ref-type="ref"}) were observed at the histological level by the mean of osmium tetroxide (OsO~4~) impregnation.
For histological analysis, brains were fixed with 10% FA, dehydrated and embedded in paraffin. Sagittal slices of 6 μm were cut with microtome. Luxol fast blue staining (Abcam) was performed according to the manufacturer\'s instructions. Images were collected with Leica microscope (LEITZ DMRB; equipped with AxioCam Zeiss) using ×1.6 objective. Images were processed with ImageJ software (National Institutes of Health) as follows. Red channel was extracted from RGB pictures and grey values inverted to get myelin signal. For each slice, the relative Luxol fast blue staining intensity was calculated by dividing the mean intensity measured in splenium and adjacent half of corpus callosum body by the mean intensity measured in cortex. Mean intensity of each brain was calculated from at least three slices.
EAE spinal cord was stored in a 4% PFA solution overnight at 4°C and then placed in 15% sucrose--phosphate‐buffered saline solution for 24 h before embedding in OCT (optimal cutting temperature compound). Fluoromyelin staining (Invitrogen ref F34651 diluted at 1:200) and DAPI staining were performed on 10 μm coronal cryosections of the lumbar region of the spinal cord. Slice images were collected on Hamamatsu Nanozoomer S60 (FITC 80 ms, DAPI 8 ms). Fluoromyelin intensity was measured using ImageJ software. Regions of interest were drawn manually, D region corresponds to the dorsal part of the spinal cord and VL to ventrolateral (vehicle *n* = 3; MTP‐PlexA1 1 μg/kg *n* = 3; the analysis was performed on a total of 24 microscopic fields).
For PLP detection, 6‐μm paraffin sections were dewaxed, unmasked with citrate buffer (Sigma) and incubated overnight with anti‐PLP antibody (PLP Lp6, 1:500), followed by incubation with a biotinylated goat anti‐rabbit antibody diluted at 1:200 for 1 h at room temperature. After washing, slides were incubated with ABC Vectastain amplification system (Vector Laboratories) and revealed with DAB (Vector Laboratories). Counterstaining was performed using haematoxylin (Sigma).
MRI recording {#emmm201910378-sec-0027}
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MRI examinations were performed on 7/30 Biospec System (Bruker BioSpin, Ettlingen, Germany). Transmission was achieved with a quadrature volume resonator (inner diameter 86 mm), and a standard mice brain quadrature surface coil (\~ 19 × 19 mm^2^) was used for signal reception (Bruker BioSpin, Ettlingen, Germany). MRI experiments were executed with ParaVision 6.0.1 software. T2‐weighted axial anatomical dataset was acquired using RARE sequence employed using 39 contiguous slices with 0.3 mm thickness and an in‐plane resolution of 0.078 × 0.078 mm^2^. Remaining parameters were as follows: matrix 256 × 256, TE = 26.5 ms, TR = 5 s, *N* avg = 6, RARE factor = 8 and acquisition time: 16 min. DTI was performed using a diffusion tensor spin‐echo echo‐planar imaging (DTI‐SE‐EPI) sequence with the following parameters: four shots, *N* avg = 2, TR = 3 s, TE = 31 ms, 30 directions, 6 *b*‐values, ∆/δ = 12/6 ms and 23 consecutive slices with resolution = 0.1 × 0.1 × 0.5 mm^3^, acquisition time: 60 min. For MRI experiments, anaesthesia was induced with 2.0--2.5% isoflurane. Then, animals were fixed in an animal cradle (Minerve, France) using a tooth bar and ear bars for stable positioning of the head. During MRI experiments, the anaesthesia was maintained with isoflurane to stabilize the breath frequency around 80--90 bpm. Respiration was monitored using a pressure pad under the thorax. MRI acquisitions were done at t0, W4, W6 and W8.
Image processing and analysis {#emmm201910378-sec-0028}
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The signal bias of T2WI induced by the surface coil was corrected with N4 bias correction (Advanced Normalization Tools, ANTs). Brains were automatically extracted and co‐registered to a reference using FSL tools: BET and FLIRT (FMRIB Software Library, Oxford, UK). A set of regions of interest corresponding to corpus callosum and ventricles was designed manually and applied T2WI to extract the signal of the corpus callosum normalized by the signal of the ventricles using ImageJ (NHI, USA).
After interscan drift correction, we used FSL (FMRIB Software Library, Oxford, UK) to reconstruct DRAD maps as a readout of myelin integrity. Then, individuals' T2WI, volume without diffusion gradient, was co‐registered to a reference using FLIRT (FMRIB Software Library, Oxford, UK) and the transformation matrix was applied to the corresponding maps. A set of regions of interest corresponding to corpus callosum was designed manually and applied on all maps to extract values using ImageJ (NHI, USA).
To illustrate the myelin loss, we calculated the mean ratio of DRAD signal at W8/DRAD signal at t0 for each group. Pixels exhibiting ratio values \> 1 (corresponding to demyelination) were selected using a mask before false colour coding and overlaid on the raw DRAD images (ImageJ FIJI).
CatWalk assay {#emmm201910378-sec-0029}
-------------
Gait analysis was performed using the CatWalk XT Automated Gait Analysis System (Noldus Technology Company) according to the manufacturer\'s instructions. Here are definitions of the parameter studied:
\(i\) Stand (s) or Stance Phase is the duration in seconds of contact of a paw with the glass plate. (ii) Swing (s) or Swing Phase is the duration in seconds of no contact of a paw with the glass plate. (iii) Swing Speed is the speed (distance unit/second) of the paw during Swing. Swing Speed = Stride Length/Swing. (iv) Stride Length is the distance (in distance units) between successive placements of the same paw. Calculation of Stride Length is based on the *x*‐coordinates of the centre of the paw print of two consecutive placements of the same paw during Max contact and taking into account Pythagoras' theorem. (v) Step Cycle is the time in seconds between two consecutive Initial Contacts of the same paw.
Values are expressed relatively to week 6 of the protocol corresponding to the last time without measurable deficit. Statistical significance is calculated by comparison with starting time of the experiment (t0).
Elevated plus maze {#emmm201910378-sec-0030}
------------------
The EPM has been used to assess anxiety in animals as proposed for pharmacological agents (Walf & Frye, [2007](#emmm201910378-bib-0033){ref-type="ref"}). In brief, we used a plus‐cross‐shaped apparatus, with two open arms and two arms closed by walls linked by a central platform 50 cm above the floor. Mice were individually put in the centre of the maze facing an open arm to explore the maze for a duration of 10 min. The time spent in the open arm, number of rearing, heading and stretch attend posture were used as an anxiety index. All parameters were measured using a Video Tracker software (Anymaze).
Open field {#emmm201910378-sec-0031}
----------
The locomotion of animals treated for 4 weeks with MTP‐PlexA1 or vehicle was determined in an open‐field test where mice are placed individually in a polyvinyl plastic square open field. Mice that were freely moving and the travelled distance were measured using a Video Tracker software (Anymaze).
Spatial recognition task {#emmm201910378-sec-0032}
------------------------
Mice were subjected to 10‐min exploratory sessions in an open field enriched with three different objects. Five minutes later, one object was placed to the opposite side and the same mouse was introduced in the open field for a second exploratory session. The discrimination index was determined as the ratio of the time spent to explore the displaced object over the time spent to explore not displaced objects.
Blood analysis {#emmm201910378-sec-0033}
--------------
Blood analysis of eight mice (four vehicles and four treated) treated three times per week during 4 weeks with MTP‐PlexA1 (1 μg/kg) or vehicle (0.072 mM) was conducted at the Clinical Chemistry and Hematology Platform of Institut Clinique de la Souris (ICS, Strasbourg, France) according to their laboratory routines.
Statistical analysis {#emmm201910378-sec-0034}
--------------------
Graphs were produced in Prism 5 or 7 (GraphPad), and data are presented as mean ± SEM. Comparisons of one factor between two groups were analysed by unpaired *t*‐test for normal distribution or Mann--Whitney test otherwise. Multiple comparisons were analysed with ANOVA and appropriate postanalysis. Significance was accepted for values where \**P* \< 0.05, \*\**P* \< 0.01 and \*\*\**P* \< 0.001. The number of animals per group for *in vivo* experiments was determined using the LaMorte (Boston University Medical Center) sample size calculation method with an anticipated effect of −30% with α = 0.05 and a power of 0.95. Grubb test analysis was performed to exclude significant outliers in an experimental group. Animals were randomly assigned to experimental groups and assessed in blind conditions with regard to treatments. For animal studies, only one animal dying during the procedure (e.g. anaesthesia accident in the group MTP‐PlexA1 in DTI‐MRI experiment described in Fig [3](#emmm201910378-fig-0003){ref-type="fig"}) was excluded from the analysis. With the exception of Fig [2](#emmm201910378-fig-0002){ref-type="fig"}, variance is similar between groups statistically compared. In this particular Fig [2](#emmm201910378-fig-0002){ref-type="fig"}, we applied Welch\'s correction.
Author contributions {#emmm201910378-sec-0036}
====================
FB performed functional *in vitro* assays. FB & LDP‐V performed *in vivo* experiments and analysed data. GR, CS, LAM & LJ performed immunocytochemical experiments and analysed data. CS performed and analysed Western blot analysis. LM & AGM‐N designed, supervised and analysed behavioural experiments conducted by LPV. CP designed and analysed MRI study performed together with LPV & FB. DBi performed qPCR experiments. VJ produced and scored in blind conditions EAE mice. MVH solubilized and validated peptide solutions. DBa designed and supervised the study, analysed data and wrote the article. All authors provided critical reading of the article.
Conflict of interest {#emmm201910378-sec-0037}
====================
The authors declare that they have no conflict of interest.
{#emmm201910378-sec-0038}
###### The paper explained
Problem {#emmm201910378-sec-0039}
-------
There is currently no treatment to repair the myelin sheaths lost in multiple sclerosis. This crucial step is, however, mandatory to expect full recovery of patients benefitting from anti‐inflammatory treatments used to jugulate the progression of the disease. One strategy to achieve remyelination is to counteract the inhibitory molecules accumulated in the lesion sites, thereby preventing spontaneous remyelination by blocking the arrival of oligodendrocytes for repair.
Results {#emmm201910378-sec-0040}
-------
In this study, we show that counteracting the Sema3A inhibitory molecular barrier by targeting its receptor Plexin‐A1 favours remyelination. The use of a specific peptidic Plexin‐A1 antagonist cancelled the anti‐migratory and anti‐differentiation effects of Sema3A by disrupting the heterodimer NRP1/Plexin‐A1 required to trigger signalling. *In vivo*, the chronic administration of the peptide was remarkably tolerated and showed marked improvement in the myelin content and locomotor functions in two different animal models of MS.
Impact {#emmm201910378-sec-0041}
------
These results demonstrate that the disruption of the Sema3A repulsive functions allows normal myelinating cells to exert their spontaneous remyelinating capacity. This opens unprecedented therapeutic opportunity by combining this novel approach with anti‐inflammatory drugs to envision a possible curative therapeutic option.
Supporting information
======================
######
Appendix
######
Click here for additional data file.
######
Source Data for Figure 2
######
Click here for additional data file.
######
Review Process File
######
Click here for additional data file.
This work was supported by INSERM, ACI JC (\#5327), Labex Medalis (MEDALIS‐DBA‐2016) and SATT Conectus (PLEXREMYEL) to Dominique Bagnard. The authors wish to thank Aurore Loeuillet for help with ELISA and Erwan Grandgirard for help with microphotograph acquisition at the IGBMC platform.
[^1]: These authors contributed equally to this work
|
Introduction {#Sec1}
============
Following the successful application of graphene, great attention has been paid to other layered inorganic graphene analogues due to their peculiar and fascinating physical properties that are correlated with their 2D ultrathin atomic layer structure. Transition metal dichalcogenides (TMDs) have attracted increasing attention in recent years due to their unique optical and electronic properties and have found many applications in catalysts, optoelectronics, and bio-imaging^[@CR1]--[@CR4]^. As the electronic band structure of semiconductor materials is relatively sensitive to the quantum size effect, layered TMDs exhibit excellent fluorescence properties when they are tailored into quantum dots (QDs)^[@CR5]--[@CR8]^. Compared with traditional semiconductor QDs (such as CdS and CdSe), TMDs QDs have been proven to be good candidates for bio-imaging and bio-sensing areas due to their intrinsic low toxicity and good dispersibility^[@CR2],[@CR9]--[@CR11]^.
Similar to the fabrication of well-known carbon QDs or carbon nanodots (C-dots), the synthetic strategies of TMDs can be divided into two groups: top-down and bottom-up methods. Top-down methods mainly use physical or chemical methods to weaken the van der Waals forces between the layers and tailor them into QDs. Although monolayered TMDs can be made by ultrasonication^[@CR12]^, intercalation reaction^[@CR11],[@CR13]^ and CVD methods^[@CR14]^, further reduction of the lateral size of TMDs film to form QDs has remained a significant challenge. For example, P. Wu *et al*. fabricated MoS~2~ and WS~2~ QDs with controllable size using the sonication-assisted liquid exfoliation technique followed by a solvothermal process that was carried out at 140 °C for 9 h^[@CR7]^. Although TMDs QDs have been successfully synthesized by top-down methods, the top-down preparation processes of TMDs QDs is generally time-consuming. In contrast with the top-down methods, the bottom-up methods involve the oxidative condensation of different elements, which is typically used to produce C-dots on the basis of dehydrogenation and carbonization^[@CR15]--[@CR17]^. Due to the difficulties in selecting proper precursors, much less attention has been devoted to TMDs QDs synthesized by the bottom-up methods. W. Song *et al*. obtained MoS~2~ QDs by hydrothermal treatment of a mixture of ammonium molybdate and thiourea. However, their further application was largely hindered because the ammonia solution was harmful to human tissue^[@CR18]^. Based on the above reasons, it is necessary to develop a new fast, green and facile method for preparing TMDs QDs.
Femtosecond laser ablation has attracted much attention due to its outstanding features, such as being fast, clean and efficient^[@CR19],[@CR20]^. When the femtosecond pulses are injected into the targets, multiphoton-absorption ionization occurs, and a plasma plume is formed in a high temperature and high pressure environment^[@CR21],[@CR22]^. Under these extreme conditions, the nanoparticles can be produced through Coulombic explosion, and surface functionalization of the nanoparticles occurs simultaneously^[@CR23]^. Hence, femtosecond laser ablation is a convenient method for preparing different nanoparticles, including iron oxide magnetic nanoparticles^[@CR24],[@CR25]^, alloy nanoparticles^[@CR26],[@CR27]^ and C-dots^[@CR28]--[@CR30]^. Compared with the bottom-up synthetic strategies, the laser ablation method is more environmentally friendly, benefiting from a decrease in the usage of chemical ligands and the residues of reducing agents. In addition, as the size reduction of the particles into nanostructures can be completed in a short time through laser ablation (tens of minutes typically), the femtosecond laser ablation method for TMDs nanoparticles preparation seems to be timesaving compared with top-down methods such as solvothermal approaches^[@CR7],[@CR10]^.
Herein, we designed a facile route to synthesize the TMDs QDs through femtosecond laser ablation combined with sonication-assisted liquid exfoliation. Using this method, bulk TMDs were first tailored into small nanoparticles using femtosecond laser ablation and then exfoliated into few-layered QDs by ultrasonic processing in liquid. The optical properties and chemical structures were characterized using a transmission electron microscope (TEM), atomic force microscope (AFM), UV-Vis absorption spectroscopy, photoluminescence (PL), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy. Meanwhile, the carrier dynamics of the TMDs QDs were investigated using picosecond time-resolved spectroscopy, which showed that the abundance of surface functional groups lead to the TMDs QDs PL. The TMDs QDs fabricated by femtosecond laser ablation exhibited good dispersibility, high purity, bright fluorescence, and low toxicity. In brief, our method is a good candidate for the fabrication of high-quality TMDs QDs as well as boron nitride quantum dots (BNQDs).
Results and Discussion {#Sec2}
======================
The TMDs QDs were prepared by femtosecond laser ablation combined with sonication-assisted liquid exfoliation of bulk TMDs in NMP, a schematic diagram of the process is shown in Fig. [1](#Fig1){ref-type="fig"}, where M represents Mo and W elements. There are two critical steps during the process. First, large bulk MoS~2~ and WS~2~ powders were cut into small multilayer MoS~2~ and WS~2~ nanoparticles by femtosecond laser ablation. Second, the produced multilayer MoS~2~ and WS~2~ nanoparticles were exfoliated into QDs through an ultrasonic exfoliation process. When the two steps were completed, faint yellow solutions containing MoS~2~ and WS~2~ QDs were obtained. Here, NMP was selected as the solvent because its surface energy matched the van der Waals forces of the MoS~2~ and WS~2~ layers^[@CR16],[@CR31]^, which benefits the exfoliation of the MoS~2~ and WS~2~ nanoparticles from the multilayer to the monolayer.Figure 1Schematic illustration of the synthetic procedure of TMDs QDs based on femtosecond laser ablation and sonication-assisted liquid exfoliation.
TEM images were used to characterize the microstructure and size distribution of the MoS~2~ and WS~2~ QDs. The TEM samples were prepared by depositing a small droplet of the TMDs QDs solution onto a microscopic copper grid coated with a thin transparent carbon film. As shown in Fig. [2(a,b)](#Fig2){ref-type="fig"}, the average lateral sizes of the MoS~2~ and WS~2~ QDs were approximately 3.7 nm and 2.1 nm, respectively. The HRTEM images in the inset of Fig. [2(a,b)](#Fig2){ref-type="fig"} indicate that both QDs were well-crystallized. The d-spacing of the MoS~2~ QDs was 0.19 nm, corresponding to the (105) facet of the MoS~2~ crystal^[@CR1]^. A lattice spacing of approximately 0.2 nm could be indexed to the (006) plane of the WS~2~ crystal^[@CR32]^, indicating that these QDs were MoS~2~ or WS~2~ QDs. To further investigate the morphology and thickness of the as-prepared QDs, AFM measurements of these nanostructures were carried out. The AFM images and the height profile (Fig. [2(c,d)](#Fig2){ref-type="fig"}) exhibit typical topographic heights for MoS~2~ and WS~2~ ranging from 1 to 2 nm, corresponding to 1--2 layers of MoS~2~ and WS~2~^[@CR14],[@CR33]^. Due to equipment limitations, the AFM testing was restricted to the current height resolution. These morphological investigations indicated that MoS~2~ and WS~2~ nanoparticles were formed during the laser ablation process and were exfoliated into few-layered QDs after ultrasonic processing in NMP.Figure 2(**a**,**b**) TEM images of the MoS~2~ QDs and WS~2~ QDs. The inset are the size distributions and the HRTEM images of the MoS~2~ QDs and WS~2~ QDs. (**c**,**d**) AFM images of the MoS~2~ QDs and WS~2~ QDs. The inset is the height distribution of MoS~2~ QDs and WS~2~ QDs, marked with a solid red line in (**c**,**d**).
To explore the chemical structure of the prepared TMDs QDs, XPS measurements of the MoS~2~ QDs and WS~2~ QDs were obtained. The high-resolution spectra of Mo (Fig. [3(a)](#Fig3){ref-type="fig"}) showed three peaks at 231, 233 and 234.5 eV, which belonged to Mo^4+^ 3d~5/2~ and Mo^4+^ 3d~3/2~ of 2H-MoS~2~, respectively. Moreover, the existence of Mo^6+^ demonstrated that the Mo edges in the MoS~2~ QDs are oxidized during the preparation process^[@CR1],[@CR34]^. S peaks at 166.5 and 168.3 eV were assigned to S^2−^ 2p~3/2~ and S^2−^ 2p~1/2~ in 2H-MoS~2~ (Fig. [3(b)](#Fig3){ref-type="fig"})^[@CR3]^. As shown in Fig. [3(c,d)](#Fig3){ref-type="fig"}, the XPS spectra of WS~2~ QDs revealed that the structure of S-W-S was maintained through all of the preparation processes. The S peaks (2p~3/2~ at \~166.5 eV and 2p~1/2~ at \~168.3 eV) in Fig. [3(c)](#Fig3){ref-type="fig"} were attributed to the −2 valence state of the S atoms. The peaks for the 4 f level of W atoms that correspond to a bound +4 valence state (WS~2~) are presented in Fig. [3(d)](#Fig3){ref-type="fig"}. The bands at 33.7, 35.2 and 37.3 eV were assigned to W 4f~7/2~, W 4f~5/2~ and W 5p~3/2~, respectively^[@CR35]^. The XPS results indicate that functional groups were attached to the surfaces of the MoS~2~ and WS~2~ QDs during the fabrication process. As reported, ionization of the raw materials and solution occurred in the laser ablation process, and a plasma with a high temperature and high pressure was formed^[@CR23]^. Under these extreme conditions, MoS~2~ and WS~2~ nanoparticles with a size of several nanometres could be produced, and surface functionalization of the nanoparticles occurred simultaneously.Figure 3XPS spectra of (**a**) Mo 3d and (**b**) S 2p regions for MoS~2~ QDs and the (**c**) S 2p and (**d**) W 5p and W 4 f regions for WS~2~ QDs.
The chemical structures of MoS~2~ and WS~2~ QDs were investigated by Raman and FTIR spectroscopy. In Fig. [4(a)](#Fig4){ref-type="fig"}, the Raman spectrum of the bulk MoS~2~ powder had two main modes, the A~1g~ (the out-of-plane vibration of the S atoms) and the E~2g~ (the in-plane vibration of the Mo--S bonds) located at 402 and 377 cm^−1^, respectively^[@CR5]^. The Raman spectrum of the MoS~2~ QDs showed that the A~1g~ peak had blue shifted on the order of 3 cm^−1^, and the peak position of the E~2g~ had also decreased 5 cm^−1^, which was attributed to the A~1g~ softening and E~2g~ stiffening with decreasing layer thickness^[@CR3]^. In the Raman spectra of WS~2~, the bulk WS~2~ also showed two peaks at approximately 415 (A~1g~) and 348 cm^−1^ (E~2g~) (Fig. [4(b)](#Fig4){ref-type="fig"}). For the WS~2~ QDs, the E~2g~ peak blue shifted to 344 cm^−1^, and the A~1g~ peak redshifted to 417 cm^−1^. The blue shift of the E~2g~ was attributed to the reduced long-range Coulomb interactions between the effective charges caused by an increase in the dielectric screening of stacking-induced changes in the interlayer bonding^[@CR36]^. The shift of the A~1g~ may be caused by a decrease in the interlayer Van der Waals interactions, which results in a weaker restoring force in the vibration as WS~2~ QDs form^[@CR32]^. The Raman spectra of the MoS~2~ and WS~2~ QDs confirmed that the bulk TMDs were exfoliated into few-layered QDs during the fabrication process.Figure 4(**a**) Raman spectra of MoS~2~ powder and MoS~2~ QDs. (**b**) Raman spectra of WS~2~ powder and WS~2~ QDs. (**c**) FTIR spectrum of MoS~2~ QDs. (**d**) FTIR spectrum of WS~2~ QDs.
The FTIR measurements were used to study the surface functional groups of the QDs. The FTIR spectrum of the MoS~2~ QDs (Fig. [4(c)](#Fig4){ref-type="fig"}) showed one weak absorption peak at 474 cm^−1^, which could be ascribed to the Mo-S stretching vibration mode of MoS~2~^[@CR37]^. Figure [4(d)](#Fig4){ref-type="fig"} exhibited characteristic absorptions at approximately 821--985 cm^−1^ and 608 cm^−1^, which corresponded to the S-S bond and W-S bond, respectively^[@CR36],[@CR38]^. Apart from the above characteristic peaks, the MoS~2~ QDs and WS~2~ QDs had almost the same FTIR peaks. The appearance of peaks at 3359 cm^−1^ (OH bond stretching), 2924 cm^−1^ (CH~2~ asymmetric stretching), 1673 cm^−1^ (C=O vibration), 1401 cm^−1^ (C-NH-C or C=N-C stretching vibration), 1285 cm^−1^ (C--N stretching frequencies) and 1121 cm^−1^ (C-NH-C or C-N stretching) indicated the attachment of NMP to the QD surface during the femtosecond laser ablation process^[@CR5],[@CR18],[@CR39]--[@CR41]^. In addition, the presence of carboxyl and hydroxyl groups were deemed to be responsible for the good water solubility of the prepared MoS~2~ QDs and WS~2~ QDs.
UV−vis absorbance, PL excitation (PLE) and PL spectra were obtained to study the optical properties of the MoS~2~ QDs and WS~2~ QDs. The as-prepared MoS~2~ QDs and WS~2~ QDs under visible light were yellowish in colour (as shown by the left inset of Fig. [5(a,c)](#Fig5){ref-type="fig"}), while blue-green photoluminescent emission could be observed under UV (395 nm) irradiation (the right inset in Fig. [5(a,c)](#Fig5){ref-type="fig"}). As shown in Fig. [5(a)](#Fig5){ref-type="fig"}, MoS~2~ QDs showed an optical absorption peak at 275 nm with the edge extending to approximately 450 nm, which may be attributed to the functional groups on its surface\[3; 4\]. Similarly, the WS~2~ QDs had almost the same absorption spectrum. Meanwhile, the strongest emission of the MoS~2~ QDs and WS~2~ QDs occurred at 480 nm under 400 nm light excitation with a Stokes shift of 80 nm. Figure [5(b,d)](#Fig5){ref-type="fig"} show that the as-prepared MoS~2~ QDs and WS~2~ QDs all exhibited excitation-dependent PL behaviour, which may be caused by the abundance of surface functional groups of the QDs.Figure 5(**a**,**c**) UV--vis ABS (black line), PLE (red line) and PL (blue line) of the MoS~2~ QDs and WS~2~ QDs, respectively. (**b**,**d**) Excitation-dependent PL emission behaviour of the MoS~2~ QDs and WS~2~ QDs, respectively, excited at wavelengths from 300 to 480 nm. The inserts of (**a**,**c**) show photographs of the bulk materials and the corresponding QDs taken under visible (left) and 395 nm UV (right) lights.
To study the origin and mechanism of the PL process in MoS~2~ QDs, the NMP solvent was replaced with distilled water for the laser ablation process. As shown in Supplementary Fig. [S1](#MOESM1){ref-type="media"}, no photoluminescence appeared in the PL spectrum of the prepared MoS~2~ QDs. Because there were no carbon atoms in water, carbon functional groups were not able to form on the MoS~2~ QDs. Therefore, we could infer that the PL of MoS~2~ QDs prepared in NMP originated from its surface functional groups rather than its intrinsic luminescence^[@CR42]^. Picosecond time-resolved spectroscopy was further used to study the PL mechanism of the prepared MoS~2~ QDs in NMP. The PL emissions were excited using a 404 nm laser, and the temporal behaviour of the emissions at wavelengths of 420, 450, and 480 nm was measured. As shown in Fig. [6](#Fig6){ref-type="fig"}, each of the decay curves of these emissions could be well fitted using a double-exponential function, indicating both a fast decay (0.65\~0.95 ns) and a slow decay (4.90\~7.95 ns). The fitting results are given in Supplementary Table [S1](#MOESM1){ref-type="media"}. Generally, with increasing emission wavelength, the slow time component in the PL dynamics increased, and the average lifetime of the PL was prolonged. Similar to the PL mechanism in C-dots prepared using laser ablation methods, when the MoS~2~ QDs were excited, there were two pathways for electron-hole recombination in the prepared MoS~2~ QDs: direct radiative recombination of the surface states (a fast decay), and a relaxation of carriers from the intrinsic states of MoS~2~ QDs to the surface states followed by radiative recombination of the surface states (a slow decay)^[@CR43]^. When the emission wavelength was increased, the lower electron energy levels of the surface states were corresponded, and relaxation from the intrinsic states to the excited surface states was prolonged, causing an increase in the slow time components of the PL lifetime.Figure 6Time-resolved PL spectra of the prepared MoS~2~ QDs at detection wavelengths of 420, 450, and 480 nm under 404 nm excitation.
Similar to the TMDs QDs, the newly emerged BNQDs have also attracted great attention^[@CR44],[@CR45]^. Unfortunately, the synthesis of BNQDs has also been limited to time-consuming top-down methods due to the difficulty in selecting proper precursors for the bottom-up synthetic strategies^[@CR46]^. The proposed method in this report, based on femtosecond laser ablation and sonication-assisted liquid exfoliation, was also successfully used to fabricate BNQDs. The experimental details and results are given in the supporting information. TEM images (Supplementary Fig. [S2](#MOESM1){ref-type="media"}) and XPS (Supplementary Fig. [S3](#MOESM1){ref-type="media"}) demonstrate the formation of the BNQDs. The PL spectra and the Raman survey (Supplementary Fig. [S4](#MOESM1){ref-type="media"}) indicate the excellent fluorescence properties of the products.
Conclusion {#Sec3}
==========
In summary, a fast and simple method for the synthesis of high-quality TMDs QDs based on femtosecond laser ablation and sonication-assisted liquid exfoliation is proposed. The bulk MoS~2~ and WS~2~ were cut into small nanoparticles by femtosecond laser ablation, and the ultrasonic process exfoliated these nanoparticles into MoS~2~ QDs and WS~2~ QDs. By analysing the results of TEM, AFM, XPS, FTIR and PL, we found that the prepared MoS~2~ QDs and WS~2~ QDs were few layered and exhibited good optical properties. To study the origin and mechanism of the PL process in MoS~2~ QDs, time-resolved PL was also investigated. In addition, our work also provides a fast, low-cost, and simple synthetic strategy for the synthesis of transitional metal dichalcogenides QDs and other 2D nanomaterials.
Methods {#Sec4}
=======
Materials {#Sec5}
---------
Bulk hexagonal boron nitride (hBN), bulk MoS~2~ powders, bulk WS~2~ powders and N-methyl-2-pyrrolidone (NMP, 99.5%) were commercially purchased from Aladdin Industrial Co. Ltd. (Shanghai, China). All materials were of analytical grade and used without further purification.
Preparation of TMDs QDs {#Sec6}
-----------------------
Using the MoS~2~ QDs as an example, the MoS~2~ QDs were prepared using the following procedures. A total of 3.2 mg of MoS~2~ powder was dispersed into 40 mL of NMP solution, and the mixture was sonicated for 2--3 minutes to obtain a uniform distribution. Next, 10 mL of the above solution was placed into a glass beaker (outside diameter × height: 25 mm × 35 mm) for ablation. By focusing with a lens (focal length 100 mm), a femtosecond laser with a wavelength of 800 nm that was produced by a Ti:sapphire laser (with 80 fs pulse duration, 400 mW laser power, and 1 kHz repetition rate) was directed into the solution for approximately 0.5 h. During laser irradiation, a magnetic stirrer was used to prevent gravitational settling of the initial powder. After laser ablation, the solution was centrifuged for 20 min at 12000 rpm to remove large MoS~2~ particles. The supernatant was collected and processed by ultrasound for 2 h with 500 mW power. During the sonication process, an ice/water system was used to maintain a temperature of 10 °C. After sonication, the prepared MoS~2~ QDs contained in the supernatant were collected for use. The fabrication process of the WS~2~ QDs was similar to that of the MoS~2~ QDs. The yield of QDs from the powder suspensions was seriously affected by the ablation conditions, such as the pulse energy, irradiation time and spot size^[@CR23],[@CR47]^. In our experiments, the yield of the QDs for 30-min laser ablation was estimated to be approximately 11%, which is comparable with that reported in previous studies^[@CR7]^.
Instrumentation {#Sec7}
---------------
The PL spectrum measurements were conducted with a spectrometer (Omni-λ, China). The UV-vis absorption spectra were obtained from a spectrophotometer (UV-2600, China). TEM and high resolution TEM (HRTEM) images of the BNQDs were obtained using a high-resolution transmission electron microscope (JEM-ARM200F, Japan). XPS experiments were carried out with an X-ray photoelectron spectrometer (ESCALAB Xi+, USA). The AFM images were obtained using an atomic force microscope (DIMENSION IOON, Germany). The Raman spectra were acquired from a Raman System (HR800, France) with a 532 nm laser excitation. FTIR spectroscopy was performed with a time-resolution infrared spectrometer (Vetex70, Germany) using the KBr pellet method. The time-resolved PL spectra of the BNQDs were monitored with a time-correlated single-photon counting system (FLSPP20, UK) (excited by picosecond pulsed LDs, a time resolution of 100 ps, pulse duration: \<850 ps, repetition rate: 10 MHz).
"Ethics" {#Sec8}
--------
We were not required to complete an ethical assessment prior to conducting our research, and no permissions were required prior to conducting our research.
Supplementary information
=========================
{#Sec9}
Dataset 1
**Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
=================================
**Supplementary information** accompanies this paper at 10.1038/s41598-019-38929-5.
This work was supported by the National R&D Program of China (2017YFA0207400), the National Natural Science Foundation of China (Grant No. 61690221, 11674260 and 11474078), the Fundamental Research Funds for the Central Universities, and the collaborative Innovation Center of Suzhou Nano Science and Technology. The TEM work was performed at the International Center for Dielectric Research (ICDR), Xi'an Jiaotong University, Xi'an, China. The AFM work was measured by the Electronic Materials Research Laboratory, Key Laboratory of the Ministry of Education &International Center for Dielectric Research, School of Electronic and Information Engineering, Xi'an Jiaotong University. The authors also thank Mr. Ma and Ms. Lu for their help in collecting the TEM images.
Yanmin Xu carried out the experimental work, data analysis and writing of the manuscript. Lihe Yan helped design the experiments. Xiaoyu Li and Huanhuan Xu contributed to the discussion of the results.
All of the data generated or analysed during this study are included in this published article and its Supplementary Information files.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
|
Dipole antennas have long been used in various communications systems, including radio, television, and radiotelephone systems. It is well known that the lengths of the dipole arms on the antenna should be adapted to the wavelengths (.lambda.) of the signals transmitted and received. Typically, a plurality of arms having different lengths are used, in order to cover a predetermined range of frequencies. The sequence and spacing of these arms, and of any reflector behind them, determines various characteristics of the resulting beam or radiation field. These characteristics include vertical beam width, horizontal beam width, and front-to-back (F/B) ratio, i.e. the ratio of signal strength in front of the antenna to signal strength in back of the antenna. When a number of different arms are used, each arm makes its own contribution to the resulting field, and the overall expected result rapidly becomes difficult to calculate mathematically in advance. Therefore, considerable experimentation is often needed to achieve desired beam characteristics.
A well-known log periodic dipole antenna (LPDA) design is the "tree" configuration, in which parallel arms extend sideways from a central "trunk" or "standoff," the bottom arm near the base is the longest, and each successive arm is shorter toward the top of the antenna. Such LPDA designs typically result in a front-to-back (F/B) ratio less than 40 dB. This F/B ratio is considered insufficient for use in current PCS (Personal Communication System) cellular telephone sites, since radiation emanating out the back of the antenna tends to cause interference among adjacent sites. A horizontal beam width of 90 degrees is typical. However, in highly congested urban environments, it is preferable to have horizontal beamwidth of 65 degrees, which is obtained by using two parallel columns of dipoles, spaced 0.25 .lambda. to 0.30 .lambda. apart. The wavelength lambda (.lambda.) is the inverse of the frequency. The frequency band allotted for PCS use in the United States is between 1.85 GigaHertz and 1.99 GigaHertz, with a center frequency 1.92 GHz. The PCS band allotted in Europe has a center frequency 1.78 GHz, meaning that the wavelength is about 8% greater. Accordingly, antenna dimension examples stated for the U.S. should be scaled up about 8% for use in Europe.
My earlier LPDA design work has included an "hourglass" dipole strip configuration, in which top and bottom arms are longer than one or more middle arms. This design works well for generating a 90 degree beamwidth, but when used for generating a 65 degree beamwidth, typically results in F/B ratios in the range between 37 dB and 42 dB, better than provided by the "tree" configuration, but still insufficient. Reference is made to pending application U.S. Ser. No. 08/807,560 by myself and a colleague. |
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Exploring Highland Glen, the New GVLT Trail
Submitted by Anonymous on June 18, 2013 - 11:43am
“Come on Mango, come on boy, you can make it!” I yelled at my aging golden retriever plodding along behind me. Mango is 12, which in dog years puts him somewhere in his late seventies. Even the tamest trails are a struggle these days. Today, we’re on the new Gallatin Valley Land Trust (GVLT) trail by the hospital: several miles of fresh singletrack installed a few weeks ago on National Trails Day.
GVLT participated in National Trails Day on June 1, attracting nearly 200 volunteers from the Bozeman area that worked from 9am to noon and put in over two miles of trail on donated hospital land. Other volunteers worked spreading 20 tons of gravel on a half-mile of trail. With so many volunteers, GVLT says that this was their biggest and most successful Trails Day ever.
I had returned to my family home to find this brand-new trail in the familiar field across from my house, and my dog wheezing to get up the neighborhood hill. After a year cooped up on the East Coast, spent daydreaming of forest trails and the Big Sky State's blue skies, getting outdoors and spending time with my dog were top priorities—but I had to find a trail that Mango could get around on.
GVLT has been working on their Main Street to the Mountains program for four years now, and the results have finally reached the eastern part of town. Thanks to this, Mango’s new walking ground can be reached by slipping through a barbed-wire fence after crossing Kagy.
There are two routes on the new trail, and we chose the upper path to survey Bozeman from a new angle. The path follows the wheat field along Highland, which ends right across from a llama farm. We tromped through lupines and lilies and bugs and bees as we followed the virgin trail. The llamas stood at the fence as we neared; at our distance it was unclear if they were on our side of the barrier, preparing to charge, or simply curious and behind barbed wire. Mango's hackles raised and thoughts of newspaper clippings blaring, "Girl mauled by pack of furious llamas" filled my brain. After the llamas, the trail dips into a gully filled with trees and a little stream. Mango splashed around in the creek, ending up covered with mud—now a brown retriever—but he was quick to roll it off in the tall grass.
A short switchback through the trees and we were headed back towards Kagy. We chose to head home, but those with more stamina can continue on the trail towards Main Street or connect to Peets Hill by turning up the hill. As we walked along, we noticed flags denoting other trails that were not in place yet, indicating the huge potential this network has to offer.
The casual expedition only took an hour, but runners and more intrepid hikers passed us frequently as Mango and I made our way home.
According to GVLT's website, the official name for this trail system is the Highland Glen Nature Preserve, and it has been made possible by over $35,000 in grants from local groups. More than two miles of the new trails are complete, with just under three miles to go.
The website also states that the trail is not yet open to the public... oops.
Mango and I will take another foray into the Highland Glen Nature Preserve once it's officially open, but until then, the public can help the project reach completion and enjoy other GVLT projects.
GVLT still needs people to help complete the remaining three miles of trails. For volunteer opportunities, email the trail boss, Josh Olsen, at [email protected]. And be sure to hit up the Longest Day of Trails this Friday, June 21.
Did you know GVLT has an updated Main Street to the Mountains map? Check out our video below, then grab your own map and hit the trail. Trail maps available at GVLT headquarters and at local retailers. |
Sunday, March 13, 2016
I have a right to be here.
Walking/bussing around today on a lovely Spring day in Austin, was reminded of Sylvia Plath's mother's late-1950s words to her from "Desiderata" when her daughter had obviously been feeling low:
You are a child of the universe, no less than the trees and the stars;
you have a right to be here.
And whether or not it is clear to you, no doubt the universe is unfolding as it should.
I cannot stress how much negativity has surrounded me, beginning with my parents. The core of ME trumpeted out: "Wow! I see more than others! I must be special." But my parents both treated me like shit, although I was lucky enough to be recognized in grade school and high school for being smart. When I got to college, I was suddenly being judged not for my insight but because I read "Time" magazine (i.e., I hadn't been reading the correct things). In personal relations, among men, I was too smart; when I came out in 1987 --- among butches, I was too smart.
In other words, my natural enthusiasm and intellect has been completely tamped down. Except by myself, of course. :)
Anyway, today I was doing stuff on my own on a very pretty day in Austin... And I thought of Aurelia Plath's words to Sylvia Plath, that I was indeed a "child of the universe," that I had a right to be here... It didn't matter that I was unloved --- I had a right to be here, enjoying the pretty day, just like anyone else. |
Domex Philippines joined World Toilet Day on November 13
According to a study conducted by the World Health Organization (WHO), children around the world miss an estimated 443 million school days each year because of diseases caused by poor sanitation and hygiene practices, including using unsanitary toilets. Simply put, this number of sick days is equivalent to all grade school and high school classrooms in the Philippines being empty for one month. This sanitation-related concern hinders the children’s learning and significantly reduces their quality of life.
Illnesses due to poor toilet sanitation include diarrhoeal disease and parasitic worm infections which can lead to nutritional deficiencies, physical and mental stunting, and death.
Every day, millions of children in rural communities and urban households nationwide are exposed to these health problems by using dirty toilets populated by disease-carrying germs.
According to Dr. Luisa Efren of the Philippine Public Health Association (PPHA), said that the simple act of proper toilet sanitation can help prevent the spread of these germs.Children continue to be at risk from these deadly diseases because many households still use ordinary laundry bleach to clean their toilet bowls, which is not enough to kill all toilet germs.
“A toilet that looks clean to the naked eye may not necessarily be free from germs and bacteria. So, it is very important to properly sanitize all surfaces using a germ-kill expert with proven efficacy in eliminating bacteria, and not just any ordinary laundry bleach,” Dr. Efren warned.
For the past three years, Unilever Philippines through its germ-kill expert brand Domex, has been working with UNICEF and PPHA to champion the One Million Clean Toilets Movement. This advocacy program aims to educate Filipinos on proper toilet hygiene and the need for sanitized household toilets to keep their families safe against disease-causing germs.
On November 13, World Toilet Day, join the One Million Clean Toilets Movement in the fight against disease causing germs to save lives, one clean toilet at a time. To learn more about Domex and how you can stay safe from disease with a germ-free home, visit Domex Philippines on Facebook. |
Sensitive optomechanical transduction of electric and magnetic signals to the optical domain.
We report a radio-frequency-to-optical converter based on an electro-optomechanical transduction scheme where the electrical, optical, and mechanical interface was integrated on a chip and operated with a fiber-coupled optical setup. The device was designed for field tests in a magnetic resonance scanner where its small form-factor and simple operation is paramount. For the appurtenant magnetic resonance detection circuit at 32 MHz, we demonstrate transduction with an intrinsic magnetic field sensitivity of 8 fT/Hz, noise figure 2.3 dB, noise temperature 210 K, voltage noise 99 pV/Hz, and current noise 113 pA/Hz, all in a 3 dB-bandwidth of 12 kHz. Such sensitivity and bandwidth make the transducer a valuable alternative to conventional electronic preamplifiers that additionally is directly compatible with fiber communication networks. |
Ogre Tubeholic Overdrive Pedal
The Ogre Tubeholic pedal gives you that lovely rich tube driven overdrive tone with a long sustain. Back off a bit and the pedal acts as a clean boost.
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Ogre Tubeholic Overdrive Pedal
Designed by musician and studio engineer Kim Sangkil, Ogre pedals really deliver sonic excellence. All pedals are made from aluminium to withstand years of use and abuse. Controls are neatly protected by a slide back cover so thear are no embarrassing setting changes mid solo! Oh - and the designs are also pretty unique!
We've tried them and really like them - so now we stock them. Come on in and give them a try.
The Ogre Tubeholic pedal gives you that lovely rich tube driven overdrive tone with a long sustain. Back off a bit and the pedal acts as a clean boost.
Specifications:
Controls
Level / Tone / Gain
Connections
Input / Output / DC input
Effect On
Press the pedal switch to turn on the effect(the LED indicator lights up) |
1. Field of the Invention
The present invention relates to the field of torque measurement; and more particularly, to means and method for determining the amount of torque that occurs in a stationary or rotating shaft.
2. Description of the Prior Art
When torque is applied to a shaft, two principal lines of stress are induced along helical lines which are orthogonal to each other on the surface of the shaft. Various different methods of torque measurement have been available, but no method has been totally satisfactory. Two common methods of measuring torque, strain gage and optical, are well described in the literature. See "Sensor and Analyzer Handbook," by Harry Norton, Prentice Hall, 1982, pp 131-142.
Torque is most accurately measured by bonding strain gages in a cross arrangement along the helical lines of compression and tension. The strain gages are electrically configured in a balance-bridge and coupled to measuring electronics via slip rings or noncontacting rotary transformers. Generally, these cross arrangements are difficult to implement and usually require custom installation. In another variation, disclosed by Gurenko and Krutkis in Soviet patent 2,493,268, a light source is used to couple the gage signal to a stationary photodiode.
In optical torque transducers, light beams, code patterns and light sensors convert the differential angular displacement between two positions on a shaft, due to applied torque, into an output signal. Specifically, identical patterns made of light reflecting strips are arranged circumferentially around the shaft at two locations. The patterns are illuminated by laser diodes and the reflected light is sensed by photocell. The output of each photocell is a pulse train and the phase difference between them is a measure of the torque. In a similar device, by Kawamoto, U.S. Pat. No. 4,767,925, a pair of light emitting and receiving elements produce an output depending on the amount of light transmitted due to the relative rotation of two slotted disks. Levine in U.S. Pat. No. 4,433,585 discloses passing a beam of light through two diffraction gratings placed at different locations along a shaft and sensing the phase of the two resulting beams. These are not robust devices as they requires exact alignment. U.S. Pat. No. 5,001,937 to Bechtel et al. discloses an optically based torsion sensor that measures the phase displacement between two bands of alternating high and low reflectivity regions. A major drawback of this device is its dependence on the initial alignment of the two bands. In addition, minor differences in the rise time of detecting electronics will cause serious errors in measurement. U.S. Pat. No. 4,525,068 to Mannava et al. discloses a torque sensor utilizing optical Doppler measurements. Since Doppler measures velocity only, this device suffers from a very serious shortcoming in that it must infer torque from changes in instantaneous rotational velocity of two different sections of a shaft.
Two optical methods for measuring strain of an object are noteworthy. U.S. Pat. No. 4,939,368 to Brown discloses an optical grating to measure strain in a stationary object. The device is complicated in that it requires two frequencies of light and has no provision for measuring a moving object such as a rotating shaft U.S. Pat. No. 4,432,239 to Bykov discloses an apparatus for measuring the deformation of an object. The device utilizes an electrooptical frequency modulator to produce two components from an incident laser beam. A polarization splitter further splits the light into two different frequencies which illuminate a diffraction grating on a stationary object. This device is complicated and expensive, and has no provision for measurement of a moving object such as a rotating shaft.
The literature discloses a capacitive torque sensor consisting of two encoders either mounted perpendicular to the shaft at each end, or mounted circumferentially at two closely placed points along the shaft. See "Interest in Misfire Detection Technology Grows", Automotive Electronics Journal, Nov. 6, 1989, pg 12. Each encoder has two parts: a stator that consists of up to 256 radial fingers that are alternately charged; and a rotor that is mounted on the shaft. As the shaft turns, the rotor's potential switches between positive and negative at a frequency proportional to speed. A disk, in the center of the stator, electrically isolated from the charged fingers collects the signal. Like the optical torque sensor, the twist of the shaft is determined by measuring the phase difference between the two encoders. Also like the optical sensor this device requires exact alignment.
Finally, magnetic torque sensors comprise much of the prior art. The magnetic properties of most ferromagnetic materials change with the application of stress to such an extent that stress may be ranked with field strength and temperature as one of the primary factors affecting magnetic change. Magnetostriction is a measure of the stress sensitivity of a material's magnetic properties. Magnetic based torque sensors take advantage of the magnetostrictive properties of ferromagnetic metals, such as carbon steel. See "Noncontact Magnetic Torque Transducer," Sensors, 11/90, pp. 37-40. These sensors make a contactless measurement of changes of magnetic permeability in shaft materials, which are caused by torsional stress. In place of strain gages, magnetic flux is directed into the shaft and along the helical lines of compression and tension. A positive magnetostriction shaft experiencing torsion will exhibit increased permeability along the line of tension and decreased permeability along the line of compression. At low stress levels the permeability is nearly linear with stress, but varies dramatically at high stress. Another drawback of a magnetostrictive torque sensor is in the need for calibrating it individually with each shaft This requirement is obvious because the torque measurement is made by means of the magnetostrictive properties of the shaft material and cannot be predetermined in the manufacture of the sensor by itself. The variability in magnetostrictive properties is usually correlated with the variability of the mechanical hardness of the material. Hardness variability of shaft materials typically ranges from +10 percent to +40 percent. The shaft-to-shaft variability problem has been addressed in recent research by adding either a sleeve or coating of a well defined and magnetically soft material, such as nickel, permalloy, or ferromagnetic amorphous alloys. While this approach shows promise, installation can not be made in situ, and all magnetic materials, even the softest, can retain some magnetism leading to nonlinearities and drift.
There is a need for an accurate, simple, noncontact sensor for measuring torque in a stationary or rotating shaft. |
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/* C_Axial_L5.1mm_D3.1mm_P10.00mm_Horizontal.step 3D STEP model for use in ECAD systems
* Copyright (C) 2017, kicad StepUp
*
* This program is free software: you can redistribute it and/or modify
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* any later version.
*
* This program is distributed in the hope that it will be useful,
* but WITHOUT ANY WARRANTY; without even the implied warranty of
* MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
* GNU General Public License for more details.
*
* You should have received a copy of the GNU General Public License
* along with this program. If not, see http://www.gnu.org/licenses/.
*
* As a special exception, if you create a design which uses this symbol,
* and embed this symbol or unaltered portions of this symbol into the design,
* this symbol does not by itself cause the resulting design to be covered by
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* This exception does not however invalidate any other reasons why the design
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* If you modify this symbol, you may extend this exception to your version of the symbol,
* but you are not obligated to do so.
* If you do not wish to do so, delete this exception statement from your version
* Risk disclaimer
* *USE 3D CAD DATA AT YOUR OWN RISK*
* *DO NOT RELY UPON ANY INFORMATION FOUND HERE WITHOUT INDEPENDENT VERIFICATION.*
*
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I'm all about ugly food, or, as they say in Portugal, fruta feia, which actually means ugly fruit. It may not be sweeping Europe, but it's growing in Lisbon. It's good because, in a very small way, it helps ensure that less food is wasted. In Europe, according to a Swedish and Dutch report, the amount of wasted food comes to 89 million tons a year.
I can't find an equivalent figure for the United States, but the USDA says that 31 percent of our food goes uneaten.
What the ugly-food people do is comb markets for provisions that other people might reject - tomatoes with torn skin, yellowing spinach, zucchinis so large and misshapen that getting them home would be a challenge.
They like apples with bruises and discolored plums. Appearance doesn't matter; taste matters. By using the ugly food that people won't buy, they're thinking globally and acting locally. Hey, it may be a cliche, but it's still a good strategy. A drop in the bucket, maybe, but if we have enough buckets ...
I'm sure you waste food; I think we all do. We buy something we end up not cooking, and finally it becomes too ancient to be edible. We have cans of bean dip and garbanzo beans and (a representative from my own larder) chipotle peppers in adobo sauce that are years, sometimes decades old.
We have been trained to consume, and we do. We can consume more frugally, and I am attempting to do that, but, you know, guilty as charged.
One thing that's out of my control, though, the subject of rants to my spouse and others: portions at restaurants. When we drove across the country a few years ago, I noticed that as we proceeded eastward, the portions got large and larger.
I was served a breakfast in Wisconsin that must have been 10,000 calories, carbs and fats by the long ton, even some real whipped cream upon demand. (Wisconsin is dairy country.) There was toast and waffles. It had many things the menu didn't mention. Home fries? I didn't finish the damn thing, of course; I barely started it. So I became a food waster, except really it was the restaurant's fault.
Breakfast in restaurants is often a source of waste. In the Bay Area, things can get awfully cute at brunch: orange slices and radish flowers and an organic bagel on the side. If restaurants would cut down on portions, gosh darn it, then we could stop wasting so much food. Fifty million people in this country are "food insecure"; we can at least eat a brown banana.
In other news: We note the growing influence of right-wing nationalist parties in Europe. They are against immigration, many of them believing that their native country is being polluted by foreigners - even if the foreigners have been there for several generations.
The rebels also dislike the European Union. Down south, they blame it for mandatory austerity packages imposed after their nations essentially went bankrupt. The countries in Northern Europe feel that the EU stifles their economic interests, with all its rules and regulations.
And the EU coddles those foreigners. Movement within EU borders is virtually unhindered. Those people can go anywhere. The campaign rhetoric was fiery, and in many low-turnout elections, the nativist parties showed surprising gains.
Well, swell. Anyone remember why the EU was started in the first place? One big reason was that European countries had made it a habit to go to war with each other. Nationalist sentiment created tensions between nations. Armies crossed borders in an effort to right some ancient wrong. Empire-building leaders came into power.
The Hundred Years War, the Thirty Years War, Napoleon and all that, the Kaiser, a couple of world wars - it kind of wasn't working out for Europe. So the nations decided to come together peacefully so that it wouldn't happen again.
And now it's happening again. A vocal group in each country wants to erect borders and quarrel with neighbors. Such a bad idea.
Not only that: I have been reading about the latest nuttiness in Texas. Very, very right-wing Republicans, some righter than the Tea Party that supports them, are claiming or are about to claim local offices around the state.
Some of these guys are buffoons, but no one in the state seems to be calling them on their amazing nonsense. Lord, how I wish Molly Ivins were still alive. She died in 2007, much too soon, and she loved nothing better than skewering politicians. She once said of a Texas congressman: "If his IQ slips any lower, we'll have to water him twice a day."
She also said this: "Practice, practice, practice, that's what Texas provides when it comes to sleaze and stink. Who can forget such great explanations as 'Well, I'll just make a little bit of money, I won't make a whole lot'? And 'There was never a Bible in the room'?" She was funny, and she told the truth.
Enough with the garnishes, already. Save yourself money and us time.
"Oh, don't bother me," said the Duchess; "I never could abide figures!" And with that she began nursing her child again, singing a sort of jcarroll@sfchronicle.com. |
Study and Research
Eight fields of research structure the INHA’s academic programmes: history of ancient art; history of archaeology; history of mediaeval art; history of taste; history of architecture; art beyond the fine and decorative arts design, material culture; history of contemporary art of the 20th – 21st centuries; practices in the history of art; and the arts in globalisation. Programmes are piloted by the Department of Studies and Research, which welcomes current and future scholars for limited periods, under the guidance of academic advisors: resident scholars (young doctors or curators), research assistants (doctoral candidates), student monitors (masters candidates) and foreign scholarship students. These teams are trained in the development and dissemmation of scholarship, in the handling of the documentary dimension of research and in the digital humanities.
The research programmes are led in partnership with French or foreign institutions, either academic institutions or museums, thus bringing together art historians with mixed horizons and making possible a number of ambitious initiatives. These programmes lead to the production of documentary resources, available online for the academic community and the general public via the platform AGORHA (agorha.inha.fr), as well as to the organisation of a multitude of events – symposiums, exhibitions, seminars, conferences – open to all in the spaces of the Galerie Colbert and the publication of books, either copublished or available online (inha.revues.org).
The Department of Studies and Research includes approximately fifty permanent researchers and welcomes each year, for periods of one month to two years, some forty other French and foreign scholars. Other than those who are invited during a stay in Paris, eight traveling scholar programmes organised by the INHA provide financing for trips to Paris or abroad for art historians of all nationalities. |
Gov. Phil Murphy will soon embark on another international trip to help boost business in New Jersey — this time to India, a country with strong ties to the Garden State.
Murphy announced Monday he will be the first sitting New Jersey governor to visit India on official business, taking a seven-day, six-city economic tour in September of a country he called one of the state’s largest trade and investment partners.
The Democratic governor said he will meet with Indian government officials and industry leaders to “chart a course" to bring more business and investment to New Jersey, as well as to help the country with “their priorities.” Murphy said he also hopes to “strengthen the cultural, educational, and political bonds” between the state and the country.
“This can be a true partnership — the two largest democracies in the world working together at both the federal and state levels on issues that span the entire economic spectrum,” Murphy said while making the announcement in a speech at Spice Culture restaurant in South Plainfield.
The trip will last from Sept. 13-22, including travel time. Murphy will visit Delhi, Agra, Hyderabad, Mumbai, Ahmedabad, and Gandhinagar.
It’ll be similar to the eight-day trip Murphy took last October to Germany and Israel.
Like that trip, the bulk of the India visit will be paid for by nonprofit Choose New Jersey. Taxpayers will still foot the bill for the governor’s security detail.
Indian-Americans represent the largest group of foreign-born residents in New Jersey, Murphy’s office said.
Of the state’s nine million residents, about 420,000 are Indian-American — “one of the fastest growing communities in our state," Murphy said.
India is also New Jersey’s second largest foreign direct investor, Murphy’s office said. More than 50 percent of India’s foreign direct investment in the Northeast comes to the state, the office said.
Murphy, a frequent critic of Republican President Donald Trump, painted the India visit as another contrast between his and Trump’s administrations.
“Unlike some in Washington, I have made it clear that international trade and partnership is key to our economic future,” Murphy said. “We have great advantages here in New Jersey. But we cannot ignore that we cannot grow on our own.”
“We are a stronger state and a stronger economy because you are here,” the governor added. “My goal is to ensure you continue to call New Jersey home and continue to contribute in making our state an example of how inclusive leadership can lift millions of people as one.”
Murphy’s office said will the governor meet with leaders in Indian’s life sciences, technology, clean energy, film and media, and manufacturing industries.
Joining Murphy will be his wife, First Lady Tammy Murphy; Tim Sullivan, CEO of the state Economic Development Authority; and Jose Lozano, president of Choose New Jersey.
It’s not uncommon for New Jersey governors to take out-of-country business trips. Murphy’s predecessor, Chris Christie, visited Canada, England, and Mexico on business missions during his eight years as governor.
Brent Johnson may be reached at bjohnson@njadvancemedia.com. Follow him on Twitter @johnsb01.
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Get the latest updates right in your inbox. Subscribe to NJ.com’s newsletters. |
Q:
futures.forEach(CompletableFuture::join) runs all tasks parallel?
I have multiple completable futures created as:
CompletableFuture<Void> future1 = CompletableFuture.runAsync(() -> xxx);
CompletableFuture<Void> future2 = CompletableFuture.runAsync(() -> xxx);
List<CompletableFuture<Void>> futures = Lists.newArrayList(future1, future2);
When I run below, are these two future tasks running in parallel? Will exception throw by one future blocks the other?
futures.forEach(CompletableFuture::join);
A:
By calling CompletableFuture.runAsync(...) the tasks are submitted to the common ForkJoinPool. This pool is managed by the JVM.
Since it is a common pool, no guarantees are made/can be made as to whether the tasks actually run concurrent or in parallel (other tasks may be submitted to the pool, or the pool may have only one thread at its disposal, resulting in a sequential exeuction of the tasks). But in a best-case scenario, they will be processed in parallel.
An Exception in one task will not have any effects on another task (unless explictly implemented in the tasks). In fact, without further configuration, the Exception will be silently ignored (to catch Exceptions, an explicit UncaughtExceptionHandler can be set when constructing a ForkJoinPool). One can also figure out if a ComletableFuture has completed with an Exception by calling isDone() (to know if the task has been executed) and isCompletedExceptionally() (to know whether the task has completed with an Exception). The Exception itself can be obtained by calling get(), but one should beware that the Exception is thrown, not returned when calling get().
|
Prior research suggests that at least two processes are involved in recognition judgments: A fast similarity-based process and a slower recall-like process. However, very little research has explored the specific operation of the recall process. Many researchers have proposed that the recall process operates in a recall-to-reject fashion. In a recall-to-reject process, a test foil that is similar to a studied stimulus is rejected (i.e., called "new") because the studied stimulus is recalled and the mismatch with the similar foil is noticed. For a variety of associative recognition tasks, such as recognizing whether two words were studied together or whether a test word was studied on a particular stimulus list, there is evidence for the use of such a recall-to-reject process. However, item recognition tasks have found no support for the use of recall-to-reject processing. Instead, the data from item recognition tasks are consistent with a recall-to-accept process in which a memory trace that matches the test probe must be retrieved in order for a positive ("old") response to be generated. The proposed research will provide more direct evidence on the operation of the recall-based process in item and associative recognition. New research will: 1) seek converging and unique evidence on the nature of the recall process using receiver- operating characteristic (ROC) curves, 2) estimate the contribution of the underlying processing using conjoint recognition theory, and 3) demonstrate a consequence of the recall process, namely, the inhibition of studied items that are similar to a studied test item. Two pilot studies provide evidence for this inhibitory phenomenon in a recognition task, and the proposed inhibition experiments will a) evaluate the development of that inhibition over processing time; b) determine the extent of the inhibition as a function of the similarity and number of studied items; and c) explore the relationship between the presence or absence of inhibition and the specific operation of the recall process (i.e., recall-to-reject or recall-to- accept). |
- 1/123. Let x be -1*(3/1 + -2). Sort x, -1/6, t in increasing order.
x, -1/6, t
Let a = -7 - -12. Let s = -0.039 + 4.939. Let u = a - s. Put -1/2, 2/9, u in descending order.
2/9, u, -1/2
Let s = -17/27 - 1/27. Let b(a) = -a**3 + 6*a**2 - 2*a + 9. Let n be b(6). Sort s, n, 0.4 in descending order.
0.4, s, n
Let x = 4.29 - 0.29. Sort x, 1/4, -0.3 in increasing order.
-0.3, 1/4, x
Suppose 0 = -5*a - 2*x + 30, 0 = -3*x + 11 + 4. Suppose a*d - 3*d + 2 = 0. Let b be d/7*(-5)/10. Sort b, 5, 3 in decreasing order.
5, 3, b
Let p = 11 - 11. Let g = -39 + 15. Let w be g/(-35) - (-2)/(-7). Sort p, w, 1 in descending order.
1, w, p
Let x = 3 - 5. Let t = 8 + -5. Sort -5, x, t in decreasing order.
t, x, -5
Let k = -1915/7 + 274. Put 2, -3/7, k in ascending order.
-3/7, k, 2
Let v be 2 - (2/(-1) - 0). Let s = 2 + -2. Sort s, -2/5, v in ascending order.
-2/5, s, v
Let t be 16/20 - (-57)/(-15). Sort -5, 5, t in decreasing order.
5, t, -5
Let h = -174 + 179. Let o = -8 + 6. Sort h, o, -4 in decreasing order.
h, o, -4
Let b = 21 + -21.9. Let i = 9.5 + -10. Let r = b - i. Put -1/2, r, 5 in decreasing order.
5, r, -1/2
Let w = 66 - 66.27. Let o = 2.73 - w. Sort 5, -2, o in ascending order.
-2, o, 5
Let u(j) = 23*j**3 - j**2. Let z be u(-1). Let i = 26 + z. Sort i, -1, 4 in descending order.
4, i, -1
Let w = -8 - -11. Let v = -1 + w. Put 3, v, 0.4 in decreasing order.
3, v, 0.4
Suppose -4*u + 4*j - 13 = 11, u + 2*j = 0. Sort -0.5, -0.23, u in decreasing order.
-0.23, -0.5, u
Suppose -5*z - 3*r + 55 = 0, 11 = -4*z - r + 55. Suppose -1 - z = 4*s. Sort 4, 5, s in decreasing order.
5, 4, s
Suppose -s - 3 = 16. Let z = -15 - s. Sort 0, z, 5 in descending order.
5, z, 0
Let h = -7 - -10. Let s be 3/(-5) - (-17)/20. Put s, -0.1, h in decreasing order.
h, s, -0.1
Let v = 32 - 25. Let j(d) = -3*d + 20. Let x be j(v). Put 4, 1, x in descending order.
4, 1, x
Let y = -1.9 + -0.1. Let s = -47/33 + 1/11. Let r = -0.096 - 0.304. Put r, s, y in increasing order.
y, s, r
Let u(j) = j**3 - 10*j**2 - 3. Let r be u(10). Put -14, r, -2 in ascending order.
-14, r, -2
Let k = 4 - 4.5. Let w = -0.39 + 0.09. Sort w, -4/7, k in ascending order.
-4/7, k, w
Let m be 6/6*(-4 + 1). Sort 1, -5, m in increasing order.
-5, m, 1
Let a be 0/(2 + -1) - -2. Suppose -a*s = 2*s - 8. Put s, 3, 1 in descending order.
3, s, 1
Suppose -13 = -3*q - 16. Suppose -25 = -4*w - w. Put w, q, 4 in descending order.
w, 4, q
Let b be 25/10*6/(-5). Let a(s) = -s**2 - s - 5. Let u be a(0). Sort u, b, -2.
u, b, -2
Let b(d) = 2*d - 1. Let s be b(-1). Let a = -17 - -28. Let z = -15 + a. Sort -5, z, s in ascending order.
-5, z, s
Let t = -0.12 + 0.32. Let x = t - 3.2. Put x, -0.2, -1/3 in descending order.
-0.2, -1/3, x
Suppose 0 = 2*q + 2*l, q + 2 = -4*l + 17. Let y be (-13)/(-4) + (-1)/4. Suppose 5 - 2 = y*z, -2*z = 2*g - 8. Put g, q, -1 in descending order.
g, -1, q
Let g be 2*2*(-2)/8. Let q be 2/(-4)*(-32)/88. Let l = 0 - 0.2. Sort q, g, l in increasing order.
g, l, q
Suppose -12*g = -14*g + 2. Sort -1, g, -9 in increasing order.
-9, -1, g
Let w = 0.01 + 5.99. Let u = 6.4 - w. Let a = -68/3 - -206/9. Put -1/3, a, u in descending order.
u, a, -1/3
Let a(n) = n + 9. Let p be a(-6). Let u be -6*((-4)/(-3) + -2). Put u, p, -1 in decreasing order.
u, p, -1
Let d = -68 - -141/2. Sort 23, d, 3 in decreasing order.
23, 3, d
Let b(c) = c**2 + 6*c + 5. Let j be b(-6). Let o(i) = i + 3. Let a be o(-6). Put 0, j, a in ascending order.
a, 0, j
Let x = 155 - 160. Sort 0.2, x, 3, 1 in ascending order.
x, 0.2, 1, 3
Let l(s) = 3*s**2 - 14*s - 3. Let o be l(5). Sort 1, o, 5.
1, o, 5
Let q = 1 + 3. Suppose -4 = -4*o - 0. Let l = o + -3. Sort 0, l, q in decreasing order.
q, 0, l
Suppose -g = -3 + 4. Let a(r) = -6*r - 1. Let z be a(g). Sort -5, 2, z in descending order.
z, 2, -5
Let i = 3 + -2. Sort 5, -5, i in decreasing order.
5, i, -5
Let d = -2 + 5. Let f be ((0 - -1) + 0)/(-1). Sort f, d, 2 in decreasing order.
d, 2, f
Suppose 0*a + 4*o = a, -3*a + o + 11 = 0. Sort -8, -4, a in descending order.
a, -4, -8
Let r be ((-1)/3 - (-275)/105) + -2. Let v be (-2)/3 + (-6)/(-9). Sort 1/6, r, v.
v, 1/6, r
Let b = 81 + -78. Let q be -3*-5*2/(-6). Put 2, q, b in ascending order.
q, 2, b
Let c = -2 + 0. Let a = 2 + 0. Suppose 5*z = a*z - 12. Put z, c, -5 in decreasing order.
c, z, -5
Let l(d) = d**2 + 6*d + 3. Suppose -6*v - 20 = -2*v. Let u be l(v). Sort u, -1/3, 2 in increasing order.
u, -1/3, 2
Suppose -d = 3*f - 9, 0 = -3*f + 3*d - d. Let y(p) = -4*p**2 - 3 + 6*p**2 - p**2 - 3*p. Let n be y(3). Sort f, 0, n in descending order.
f, 0, n
Let g be (-1)/1 - 0/1. Let s be (g/(-9))/(3/6). Sort -5, s, 5 in decreasing order.
5, s, -5
Let r = -23 + 16. Let s = r + 4. Sort -5/3, 1, s in decreasing order.
1, -5/3, s
Let g(a) = 2*a**3 - 18*a**2 + 5. Let t be g(9). Put 4, 21, t in increasing order.
4, t, 21
Let m = -0.019 - -4.019. Sort 3, m, 0.
0, 3, m
Let r(y) = 1 + y**2 - 2*y - 5*y + y - 5. Let o be r(7). Put o, 1, -0.4 in descending order.
o, 1, -0.4
Let f = 18 - 19. Sort -2, f, 2.
-2, f, 2
Let d = -0.06 + -0.24. Let o = 0.01 + 1.99. Put 0, o, d in descending order.
o, 0, d
Let a be -9*4*(-2)/6. Let k be (-2 - 1)/(a/16). Put k, 5, -8 in descending order.
5, k, -8
Let b = -21.7 + 22. Let v be 0 + 2 + (-3)/6. Sort 3, v, b in descending order.
3, v, b
Let t(a) = -a**3 - a**2 - 2. Let n(q) = q**2 + 8*q - 11. Let o be n(-9). Let u be t(o). Let d be (u/7)/(-2)*2. Put 0.5, d, 2/9 in decreasing order.
0.5, 2/9, d
Let v be (-51)/27 - (-3)/(-27). Sort 2, -5, v, 0 in ascending order.
-5, v, 0, 2
Let p = 55 + -59. Sort 2, 1, 4, p in increasing order.
p, 1, 2, 4
Suppose -3*i + 17 = 2. Let o = -20 - -19. Put o, i, 2 in descending order.
i, 2, o
Let v(z) = -z**2 + z - 1. Let l be v(2). Let k = 0.7 + -4.7. Let c = -6 - k. Sort l, c, -0.2.
l, c, -0.2
Let p(y) = y**2 - 3. Let d be p(-3). Let t = d - 4. Put t, -1, 0 in ascending order.
-1, 0, t
Let v be (3 - 2) + 12/3. Let n(u) = -3*u - 3. Let l(x) = -4*x - 3. Let t(b) = v*n(b) - 4*l(b). Let j be t(7). Sort 0, j, 1 in increasing order.
0, 1, j
Suppose -1 = -u + m, -4*m - 7 = -2*u - 15. Sort 1, 3, -3, u in decreasing order.
u, 3, 1, -3
Let z be 2/(3/18 + (-14)/(-60)). Sort -4, -3, z in decreasing order.
z, -3, -4
Let h(j) = j**2 - 6*j. Let g be h(5). Let d be 1/(-3) - 14/(-6). Suppose 0 = -2*n + 10 - d. Put g, -1, n in ascending order.
g, -1, n
Let s(k) = k**2 - 1. Let i be s(-2). Suppose -4*w + 8 = -i*p - 5, 2*p + 2*w = 10. Put 1/3, p, 4 in ascending order.
1/3, p, 4
Let v = -1 + 6/5. Put -5, v, 6/11 in descending order.
6/11, v, -5
Let u = 0.228 + 1.712. Let m = u + -2. Let b = -0.24 + m. Sort b, 3, 2.
b, 2, 3
Let x = -1.9 + 2. Let h = 39 - 28. Suppose f - 4*f - 4*z - h = 0, 5*f + 10 = -5*z. Put -3, f, x in ascending order.
-3, x, f
Let h(g) = g - 4. Let m be h(2). Put 1, 0, m in increasing order.
m, 0, 1
Let t = 17 + -16.65. Let c = t - -0.05. Let u = 11 + -8. Put u, c, -0.2 in ascending order.
-0.2, c, u
Let d(s) = -2*s + 6. Let q be d(0). Put 0.1, q, -2/5 in ascending order.
-2/5, 0.1, q
Let k = -1.5 + 2. Sort k, -2/3, 0 in increasing order.
-2/3, 0, k
Suppose -3*r + 8*r = 20. Put -31/3, 3/4, r in descending order.
r, 3/4, -31/3
Suppose -4*v + 12 = s, 4 = 5*s - 3*v + 13. Let y be (-2 + s)/(8 + 2). Let g = 0.4 - -0.1. Put 3, y, g in descending order.
3, g, y
Let u = 7.3 - 7. Put u, 2/7, 1 in increasing order.
2/7, u, 1
Suppose -2*y + 13 - 3 = 0. Suppose z + 2 = -2*z - 4*q, 4*z + 13 = y*q. Sort -4, z, 1/4.
-4, z, 1/4
Let o be 3/(-1) - -7*1. Suppose 0*t + 19 = 2*t + 5*g, 5 = o*t - g. Put -4, t, -5 in decreasing order.
t, -4, -5
Suppose -10*q - 59 = 41. Sort q, -5, 4 in decreasing order.
4, -5, q
Let c(t) = -t + 8. Let w be c(8). Suppose f = -w + 2. Let x(h) = -h**2 - 4*h + 1. Let m be x(-3). Put f, m, -0.4 in decreasing order.
m, f, -0.4
Suppose 12*z + 5 = 11*z. Sort z, -3, -11 in decreasing order.
-3, z, -11
Let n be (-2)/3 + (-1)/(-2). Sort n, 1/6, 4 in decreasing order.
4, 1/6, n
Let n = 94 + -97. Put -0.01, 1/2, n in descending order.
1/2, -0.01, n
Let b(u) = -u**2 - 8*u + 3. Let d be b(-7). Suppose 3*g + d = 8*g. Sort -2, 5, g in decreasing order.
5, g, -2
Let j(q) = q + 5. Let i(a) = 1. Let c(t) = 3*i(t) - j(t). Let x be c(-6). Sort -4, x, 1 in decreasing order.
x, 1, -4
Let p = 0.7 + -0.4. Let u = 0.131 |
Q:
How customer offsets are maintained in mirrored cluster in Kafka?
Lets say I have two Kafka clusters and I am using mirror maker to mirror the topics from one cluster to another. I understand consumer has an embedded producer to commit offsets to __consumer-offset topic in Kafka cluster. I need to know what will happen if primary Kafka cluster goes down? Do we sync the __consumer-offset topic as well? Because secondary cluster could have different number of brokers and other settings, I think.
Please tell how Kafka mirrored cluster takes care of consumer offset?
Does auto.offset.reset setting play a role here?
A:
Mirror maker does not replicate offsets.
Furthermore, auto.offset.reset is completely unrelated to this, because it's a consumer setting that defines where a consumer should start reading for the case, that no valid committed offset is found at startup.
The reason for not mirroring offsets is basically, that they can be meaningless on the mirror cluster because it is not guaranteed, that the messages will have the same offsets in both cluster.
Thus, in fail over case, you need to figure out something "smart" by yourself. One way would be to remember the metadata timestamp of you last processed record. This allows you to "seek" based on timestamp on the mirror cluster to find an approximate offset there. (You will need Kafka 0.10 for this.)
|
Evaluation of MIDITECH automated colorimetric MIC reading for antimicrobial susceptibility testing.
The MIDITECH colorimetric susceptibility test with automated reading is a modification of the standard broth microdilution method that uses a 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye for detecting viable bacteria. The method can be applied to non-fastidious aerobic Gram-negative bacteria, staphylococci and Enterococcus faecalis. To assess the reliability of this method, we compared susceptibility data obtained by this test with standard NCCLS microdilution assay results. For this purpose, 15 antibiotics and a well characterized set of 527 Gram-negative and Gram-positive bacterial isolates collected and stored at the Division of Infectious Diseases and Chemotherapy (Vienna General Hospital, Austria), yielding 5751 organism-antibiotic combinations, were analysed in duplicate. The overall essential agreement (+/-1 log(2) dilution) between the MIDITECH and NCCLS methods was 96.18 +/- 0.67%. The colorimetric assay compared with the reference method produced MICs < or = 2 log(2) dilutions and > or = 2 log(2) dilutions in 2.34% and 1.48% comparisons, respectively. For 326 Gram-negative bacteria, the absolute interpretative agreement of both methods ranged from 87.12% for ampicillin-sulbactam to 99.85% for meropenem (mean 94.86%); 417 (4.92%) minor, three (0.05%) major and 15 (0.63%) very major errors were found. For 127 staphylococci and 74 E. faecalis isolates, the absolute interpretative agreement ranged from 90.80% for ciprofloxacin to 100% for vancomycin and linezolid (mean 96.96%); 81 (2.77%) minor, three (0.15%) major and eight (0.83%) very major errors were found. For most of the clinically important aerobically growing pathogens, the MIDITECH colorimetric test provided reliable quantitative susceptibility data. The main advantage of this method is simple performance, automated reading and data processing without expensive investments. |
Deepak Bharadwaz is a Market Data Analyst for IHB's Bitcoin Trading Intelligence platform.
Japan Is Over Its Bitcoin Woes
Eighteen months have passed since Mt Gox, the infamous bitcoin exchange based in Tokyo, filed for Bankruptcy. This is overwhelmingly considered to be the prime factor to herald the onset of a bearish trend in Japanese bitcoin sentiment for over a year.
Recently, this sentiment only got worse for Bitcoin in Japan when Mark Karpeles, former CEO of Mt Gox was arrested for allegedly stealing funds and adding them to his personal account from one of the many Mt Gox exchange bitcoin wallets.
This was followed by another painful stroke as the Tokyo District court ruled against a bitcoin compensation plea, stating that "Bitcoin is not subject to ownership" and thus all outstanding claims are moot. However, amidst all the turmoil within the Japanese bitcoin ecosystem, another Japanese bitcoin exchange, bitFlyer, has managed to overshadow the disgraced Mt Gox, by announcing they had raised $4 million in a recent round of funding, potentially setting bitcoin in a positive course in Japan.
Initially founded as the Japanese bitcoin exchange that was going to recapture the vacuum created by Mt. Gox, bitFlyer was launched by Yuzo Kano, a derivatives and convertible bonds trader at Goldman Sachs Group Inc. Right from the start, Mr. Kano was well aware of the fact that bitFlyer's success would depend on bitcoin's reputation improving in Japan. He created a brand that was fun, playful and honest.
Even with stiff competition from other popular bitcoin exchanges like BitOcean, Kraken and ANX, bitFlyer was able to turn the tables in its favor.
How Did BitFlyer Do It?
The firm started with $1.6 million of funding, which happened to be the country’s biggest investment in bitcoin to date. After operating for about six months, bitFlyer launched Japan’s first Bitcoin crowd funding project called ‘fundFlyer’, with the intent of bringing bitcoins to the mainstream in Japan. fundFlyer is a platform for projects to raise money by accepting bitcoin donations.
Similar to kickstarter, it operates by the "Purchase Model" of crowd funding where non-financial returns or "gifts" are given to early backers. fundFlyer places a strong emphasis on the development of long term relationships and communites.
Then on the 24th of September, bitFlyer teamed up with GMO Payment Gateway and was now in the position to offer 48,000 online merchants the option to accept bitcoin. With this move bitFlyer established itself as a leading force in Japan’s still hurting bitcoin ecosystem.
More Money More Success
On 16th October 2014, they took another leap by raising $236,000 from Barry Silbert's New York based Bitcoin Opportunity Corp., citing Singapore as their next target for expansion and then in January 2015 bitFlyer raised an addtional $1.1 million to improve their bitcoin platform.
On 7th April 2015 they launched ‘chainFlyer’ a block explorer with a unique anime style to explore the bitcoin public ledger. On July 25th they launched a sophisticated professional trading platform called ‘bitFlyer Lightning’ targeting professional traders and with them came heavy chunk of market share.
Amidst all the negative bitcoin sentiment surrounding Mt. Gox during the first week of August, they still managed to raise $4 million in this latest round of funding. For now, bitFlyer has strategically grown to upend Mt Gox as the most famous bitcoin company from Japan.
How bitFlyer's influence towards the growing use of bitcoins in Japan is to be seen. |
1. Introduction {#sec1-molecules-22-01720}
===============
Certain metabolites derived from polyunsaturated fatty acids (PUFAs) play a key role in mammalian physiology, where they orchestrate both inflammatory response as well as the return to homeostasis \[[@B1-molecules-22-01720],[@B2-molecules-22-01720],[@B3-molecules-22-01720],[@B4-molecules-22-01720]\]. By combining total synthesis with chemical biology and molecular pharmacology, a number of distinct eicosanoids and docosanoids have been identified, which are active in the cascade elicited by noxious stimuli \[[@B5-molecules-22-01720],[@B6-molecules-22-01720],[@B7-molecules-22-01720],[@B8-molecules-22-01720],[@B9-molecules-22-01720],[@B10-molecules-22-01720],[@B11-molecules-22-01720],[@B12-molecules-22-01720],[@B13-molecules-22-01720],[@B14-molecules-22-01720],[@B15-molecules-22-01720],[@B16-molecules-22-01720],[@B17-molecules-22-01720],[@B18-molecules-22-01720],[@B19-molecules-22-01720],[@B20-molecules-22-01720],[@B21-molecules-22-01720],[@B22-molecules-22-01720],[@B23-molecules-22-01720]\]. As a result, natural products with an underlying PUFA motif are of great interest as potential immunomodulators.
Since antiquity, sea dwelling organisms have proven to be a particularly abundant source of new chemical entities, set apart from those found in the terrestrial environment \[[@B24-molecules-22-01720],[@B25-molecules-22-01720]\]. Thus, the ancient Phoenicians were renowned for their trading with Tyrian purple from the *Murex* sea snail \[[@B26-molecules-22-01720],[@B27-molecules-22-01720]\]. Rising above mere prospecting, modern-day discovery, enabled by the advent of powerful analytical instruments and methods, has found a wealth of bioactive compounds in the marine environment \[[@B28-molecules-22-01720],[@B29-molecules-22-01720],[@B30-molecules-22-01720],[@B31-molecules-22-01720],[@B32-molecules-22-01720],[@B33-molecules-22-01720],[@B34-molecules-22-01720]\].
Ostensibly, mucosin (**1**) is a natural product that was isolated from the Mediterranean sponge *Reniera mucosa* as methyl ester **2** \[[@B35-molecules-22-01720]\]. Formally classified as an eicosanoid, it has been conjectured to originate from arachidonic acid (**3**), based on the C~20~-architechture ([Figure 1](#molecules-22-01720-f001){ref-type="fig"}). While sharing some noticeable features with the prostane scaffold, the compound differs by having an unusual bicyclic core. Clearly, in the structure proposed for mucosin (**1**), the characteristic cyclopentane ring is integrated in a *cis*-fused bicyclo\[4.3.0\]non-3-ene system. However, turning to the elucidation, the assignment of topology poses a challenge. Though only a small molecule, the structure is compact in terms of the four contiguous stereocentres. In NMR experiments on the isolated methyl ester, pertaining to both 1D- and 2D-techniques, the distinguishing resonances/correlations are ensconced in a crowded aliphatic region. Consequently, with the absence of any coupling pattern to corroborate the configuration of the carbocycle, the assignment published by Casapullo et al. does not convince on its own \[[@B35-molecules-22-01720]\].
Fascinated by the structure and the prostane-like motif, we devised a practical, divergent and synthetically unambiguous strategy to establish the proposed stereochemistry. At the end of the campaign, capitalizing on X-ray crystallography to pinpoint the relative arrangement, it was concluded that mucosin (**1**) does not represent the portrayed compound \[[@B36-molecules-22-01720]\]. In a pursuit to identify the natural product isolated from *Reniera mucosa*, our intent is to achieve the goal by manipulation of the bicyclo\[4.3.0\]non-3-ene system. We herein detail synthesis of the mucosin diastereomer **1\***, demonstrating aspects of the chosen route with regard to stereochemical control ([Figure 2](#molecules-22-01720-f002){ref-type="fig"}). From the point of potential biological activity, the putative structure of mucosin shares some apparent structural similarities with bicyclic prostaglandins. Thus, providing that sufficient amounts could be made available, material could be screened in similar assays. Currently, it is the bearer of unknown properties.
2. Results and Discussion {#sec2-molecules-22-01720}
=========================
In 2012, Whitby and co-workers reported that they had completed the first total synthesis of antipodal mucosin (*ent*-**1**) \[[@B37-molecules-22-01720]\]. Using zirconium induced co-cyclisation as the pivotal feature, the preliminary experimental work led them to conclude that thermodynamic control would favour the relative stereochemistry assigned by Casapullo et al. \[[@B35-molecules-22-01720]\]. Applied to the actual sequence, elaboration of the key zirconacycle afforded a \~3:1 mixture of diastereomers \[[@B37-molecules-22-01720]\]. While it was conjectured that the major component could be processed to *ent*-**1**, the minor component would in turn yield *ent*-**1\***. However, although Whitby and co-workers provided data that aligned with the natural product, the authors of the present paper demonstrated irrefutably, that mucosin is not represented by the relative topological connectivity featured in structure **1** \[[@B36-molecules-22-01720]\]. This therefore raised the question as to which diastereomer had been taken on by Whitby and co-workers, and whether mucosin in fact corresponds to structure **1\***. In order to resolve this pressing issue, we designed a strategy to access structure **1**\*.
A central feature in our divergent strategy ([Scheme 1](#molecules-22-01720-sch001){ref-type="scheme"}) was to take advantage of the efficient desymmetrization of *meso*-ketone **4** \[[@B36-molecules-22-01720]\]. ([Supplementary Materials pp. S3, S4](#app1-molecules-22-01720){ref-type="app"} provides the synthetic sequence to obtain *meso*-ketone **4**) After a chiral foothold had been established, it would then be a matter of introducing a functional pattern amenable for subsequent stereoiteration. Ideally, in order to uncover the topologically deviant point(s), the prerequisite *cis*-fused keto ester **5** should also be interconvertible with the *trans*-fused system if need be. However, we first chose to examine the configuration at the appended positions. Thus, along these lines and having previously established a diastereochemical bias, addition of some suitable nucleophile to conjugate ester **7** was judged to follow the precognised trend. By completing the sequence, a new compound **1\*** with the topology inverted at C8 and C16 would result. Aptly, this could then be named *exo*-mucosin **1\***, since the bulky group added during the stereodifferentiating step was projected to occupy the *exo* face of the bicycle ([Figure 3](#molecules-22-01720-f003){ref-type="fig"}). Once *exo*-mucosin **1\*** had been made, the physical data recorded could be compared against those published for the natural product.
By the developed protocol, our synthesis commenced with desymmetrization of *meso*-ketone **4** \[[@B36-molecules-22-01720]\], using Mander's reagent in combination with the lithium amide of (+)-*bis*\[(*R*)-phenyethyl\]amine ([Scheme 2](#molecules-22-01720-sch002){ref-type="scheme"}). This chiral amide is sometimes also referred to as Simpkins' base \[[@B38-molecules-22-01720],[@B39-molecules-22-01720],[@B40-molecules-22-01720]\]. Then, with asymmetric keto ester **9** in hand, conjugated ester **10** was prepared by a three-step procedure, involving sequential manipulation of the keto moiety. Accordingly, the ketone in **9** was reduced, whereupon the corresponding alcohol was turned into a mesylate. Finally, the intermediate mesylate was subjected to base-induced elimination, whereby the Michael acceptor motif was produced.
Having carried out the delineated transformation, the key stereoiterative concept could be tested in the elaboration of conjugated ester **10**. While addition to the less hindered *exo*-face seemed inevitable, the resulting stereochemistry at the ester-appended chiral centre was somewhat uncertain a priori. Simplistically, depending on whether the supervening ester enolate is intercepted by H^+^ at the equatorial or axial position of C8, the protonated species will correspond to the kinetic and the thermodynamic product, respectively. Reflecting the ambivalent stereochemical nature of the C8-carbanion, and based on our previous experience \[[@B36-molecules-22-01720]\], conjugate addition to **10** could consequently lead to a mixture of epimers.
In reality, with Cu(I)-catalysed conjugate addition, using BuMgCl as nucleophile in the presence of TMSCl, the reaction gave ester **11** as the sole compound ([Scheme 3](#molecules-22-01720-sch003){ref-type="scheme"}). Presumably, the Lewis acid takes on dual roles: Not only does TMSCl lower the LUMO of the Michael acceptor, but also stabilizes the ester enolate \[[@B41-molecules-22-01720],[@B42-molecules-22-01720],[@B43-molecules-22-01720],[@B44-molecules-22-01720],[@B45-molecules-22-01720],[@B46-molecules-22-01720]\]. Hence, ester **11** ought to be the conjectured thermodynamic product. Subsequent reduction provided the corresponding carbinol **12**, which could also be readily derivatized for the purpose of X-ray analysis. By obtaining suitable crystals of the dinitrobenzoate **12-DNB**, the relative configuration of the four contiguous stereocentres could be established ([Figure 4](#molecules-22-01720-f004){ref-type="fig"}). This also confirmed the *exo*-facial and thermodynamic preference in the reaction of **10**, using the specified conditions. ([Supplementary Figure S-74](#app1-molecules-22-01720){ref-type="app"} provides a side perspective of the single crystal X-ray structure **12-DNB**).
With the intended topological pattern confirmed, carbinol **12** was taken through a course of four steps to install an alkyne handle by the Ohira-Bestmann protocol \[[@B47-molecules-22-01720],[@B48-molecules-22-01720],[@B49-molecules-22-01720],[@B50-molecules-22-01720]\]. For the last step, it may be noted that Taber et al. have provided an interesting alternative to the rather pricy reagent \[[@B50-molecules-22-01720]\]. Although ^1^H-NMR of the natural product clearly indicates the presence of an *E*-alkene \[[@B35-molecules-22-01720]\], the *en route* aldehyde **13** could also serve as a relay point for *Z*-selective olefination. However, with the cited observation in mind, alkyne **14** was transformed accordingly to provide the featured *E*-configured alkenyl ester motif. This was achieved by performing three consecutive reactions in one-pot. Thus, by means of stereospecific hydrometallation \[[@B51-molecules-22-01720],[@B52-molecules-22-01720],[@B53-molecules-22-01720],[@B54-molecules-22-01720],[@B55-molecules-22-01720]\] and halodemetallation \[[@B56-molecules-22-01720]\], alkyne **14** rendered the corresponding *E*-vinyl halide as substrate for Pd-catalysed cross-coupling with a commercial zinc reagent \[[@B57-molecules-22-01720],[@B58-molecules-22-01720]\]. The target molecule, *exo*-mucosin **1\***, was then obtained after hydrolysis of ester **15**. Finally, re-esterification gave methyl ester **2\***, to be compared with the data published by Casapullo et al. \[[@B35-molecules-22-01720]\].
The *cis*-fused bicyclo\[4.3.0\]non-3-ene system is not often encountered in nature. Adhering to the supposition that arachidonic acid (**3**) is the biogenetic origin of mucosin \[[@B59-molecules-22-01720],[@B60-molecules-22-01720],[@B61-molecules-22-01720]\], the geometry proposed for the core structure invokes a formal disrotatory ring-closure \[[@B62-molecules-22-01720]\]. At a more profound level, the machinery leading to the natural product may traverse any number of pericyclic pathways \[[@B36-molecules-22-01720]\]. Of particular interest, though, is the ongoing discussion regarding whether or not enzyme-catalysed Diels-Alder reactions are implicated in biological systems \[[@B63-molecules-22-01720]\]. The preceding biosynthetic transformation of **3**, into a suitable conjugated precursor for cycloaddition, is known to take place in several marine species \[[@B59-molecules-22-01720],[@B60-molecules-22-01720],[@B61-molecules-22-01720],[@B64-molecules-22-01720],[@B65-molecules-22-01720],[@B66-molecules-22-01720],[@B67-molecules-22-01720],[@B68-molecules-22-01720],[@B69-molecules-22-01720],[@B70-molecules-22-01720],[@B71-molecules-22-01720],[@B72-molecules-22-01720],[@B73-molecules-22-01720],[@B74-molecules-22-01720],[@B75-molecules-22-01720],[@B76-molecules-22-01720],[@B77-molecules-22-01720]\]. However, in all the cases where a Diels-Alderase could be claimed to provide the transformative impetus \[[@B78-molecules-22-01720],[@B79-molecules-22-01720],[@B80-molecules-22-01720],[@B81-molecules-22-01720],[@B82-molecules-22-01720],[@B83-molecules-22-01720],[@B84-molecules-22-01720],[@B85-molecules-22-01720]\], the authors of this paper have found no example of *cis*-fusion.
Cycloaddition via a non-enzymatic pathway is also possible. Thus, Gerwick has proposed allylic carbocations as conceptual intermediates in the biogenesis of marine carbocyclic oxylipins, such as prostaglandin A2 (PGA~2~) \[[@B86-molecules-22-01720],[@B87-molecules-22-01720],[@B88-molecules-22-01720]\]. In this sense, arachidonic acid (**3**) provides a link between mucosin and the prostanoid scaffold, pointing towards a possible mechanism. Yet, for the majority of examples found, the annulation produces a *trans*-1,2-disubstituted cyclopentane ring \[[@B89-molecules-22-01720],[@B90-molecules-22-01720],[@B91-molecules-22-01720],[@B92-molecules-22-01720],[@B93-molecules-22-01720],[@B94-molecules-22-01720]\].
Although being an uncommon structural feature, it would be premature to conclude that the *cis*-fused bicyclo\[4.3.0\]non-3-ene system was incongruous. Nevertheless, when recordings were made on methyl ester **2\***, the data did not match those reported for the compound isolated from *Reniera mucosa*. This was most convincingly demonstrated by comparing the ^13^C-NMR spectra ([Table 1](#molecules-22-01720-t001){ref-type="table"}): Out of the 20 resonances that are observable for the carbon framework, excluding the methoxy group, 16 display deviating shifts (see also [Supplementary Figure S-38](#app1-molecules-22-01720){ref-type="app"}). Furthermore, the optical rotation of **2\*** did not only differ in magnitude, but also in sign: While the naturally occurring material and its purported structure **2** have values of $\left\lbrack \alpha \right\rbrack_{D}^{26} = - 35.5{^\circ}$ and $- 9.8{^\circ}$ ($c = 0.8$, hexane), respectively \[[@B35-molecules-22-01720],[@B36-molecules-22-01720]\] the diastereomer **2\*** had an $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 64.0{^\circ}$ ($c = 0.8$, hexane). Whitby and co-workers have reported $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 38.2{^\circ}$ ($c = 0.8$, hexane) for the material obtained via zirconium induced co-cyclisation \[[@B37-molecules-22-01720]\].
By achieving a rational synthesis of *exo*-mucosin **2\***, the target selection has been narrowed down. Yet, in terms of the *cis*-fused bicycle, there are permutants still unaccounted for. However, given the obvious sterical encumbrance of the two remaining *syn*-diastereomers, they seemed unlikely candidates considering the biogenesis of marine carbocyclic oxylipins \[[@B86-molecules-22-01720],[@B87-molecules-22-01720],[@B88-molecules-22-01720]\]. Additionally, it is worth noticing that the *anti*-relationship of the appended groups seems to rest on a sounder foundation: Diagnostic correlations between the C7-methylene group and the C16-proton have been observed by NOESY and ROESY \[[@B35-molecules-22-01720]\]. Furthermore, DFT calculations by Whitby and co-workers on the relative stability of zirconacycles \[[@B37-molecules-22-01720]\], indicate that the *anti*-geometries are favoured over the *syn*-geometries. Hence, based on our synthetic endeavours, it was inferred that the natural product named mucosin has a *trans*-fused bicyclo\[4.3.0\]non-3-ene ring system and the featured substituents are *anti*-related.
Albeit that the outlined synthesis did not yield the ultimate target, the sequence provided an answer to a central question: namely, the question about the geometry of the fused bicycle. Moreover, taken together with what we have detailed before \[[@B36-molecules-22-01720]\], the current findings have demonstrated a fascinating chemical aspect of the *cis*-fused bicyclo\[4.3.0\]non-3-ene scaffold, that unfolds when a Michael acceptor motif is incorporated. Thus, swapping the functional group at the β-position of the Michael acceptor with the functional group of the Michael donor, a complete inversion of diastereoselectivity was observed. The transformation proved to be doubly orthogonal, as even the stereochemistry at the α-position was inverted in the process. Bearing in mind the chosen protocol, it must be assumed that the favoured epimer is not obtained via spontaneous equilibration of the incipient ester enolate anion. Rather, the reactive constellation between BuMgCl and TMSCl intercepts the Michael adduct as a silyl ketene acetal; it is therefore in the succeeding protonation of the trapped ester enolate anion that the observed epimeric configuration is established ([Scheme 4](#molecules-22-01720-sch004){ref-type="scheme"}). The stereoselective protonation of enolates is a concept, which has received a great deal of attention and in its purest form constitutes a biomimetic approach to establish α-chirality \[[@B95-molecules-22-01720],[@B96-molecules-22-01720],[@B97-molecules-22-01720],[@B98-molecules-22-01720]\]. In our example, the pre-existing topology works in consonance to dictate which face is being protonated.
In summary, the conjugate system shown in [Scheme 3](#molecules-22-01720-sch003){ref-type="scheme"} (vide supra) displays a remarkable diastereotopic preference, enabling excellent control over the reactive manifold.
3. Experimental Section {#sec3-molecules-22-01720}
=======================
3.1. General Information {#sec3dot1-molecules-22-01720}
------------------------
All commercially available reagents and solvents were used in the form they were supplied without any further purification. (+)-Bis\[(R)-1-phenylethyl\]amine hydrochloride (optical purity $\geq$ 99% ee by GLC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The stated yields are based on isolated material. The melting points are uncorrected. Thin layer chromatography was performed on silica gel 60 F~254~ aluminum-backed plates fabricated by Merck (Kenilworth, NJ, USA). Flash column chromatography was performed on silica gel 60 (40--63 µm) fabricated by Merck. NMR spectra were recorded on a Bruker Ascend^TM^ 400 (Bruker, Billerica, MA, USA) at 400 MHz for ^1^H-NMR and at 100 MHz for ^13^C-NMR. Coupling constants (*J*) are reported in hertz (Hz) and chemical shifts are reported in parts per million (δ) relative to the central residual protium solvent resonance in ^1^H-NMR (CDCl~3~ = δ 7.27) and the central carbon solvent resonance in ^13^C-NMR (CDCl~3~ = δ 77.00 ppm). The following abbreviation, appt, has been used to designate an apparent triplet. Mass spectra were recorded at 70 eV on Waters Prospec Q spectrometer (Waters Corporation, Milford, MA, USA) using EI as the method of ionization. IR spectra (4000--600 cm^−1^) were recorded on a Perkin-Elmer Spectrum BX series FT-IR spectrophotometer (Waltham, MA, USA) using a reflectance cell (HATR). Optical rotations were measured using a 1 mL cell with a 1.0 dm path length on a Perkin Elmer 341 polarimeter using the stated solvents. Determination of enantiomeric excess was performed by GLC on an Agilent Technologies 7820A GC instrument (Agilent Technologies, Santa Clara, CA, USA) with split (1:30) injection, FID detector and equipped with a chiral stationary phase (Agilent J&W GC columns, CP-Chirasil-DEX CB, 25 m, 0.25 mm, 0.25 μm) applying the conditions stated. X-ray crystallography was performed on a Bruker D8 Venture diffractometer with InCoatec ImuS Microfocus radiation source and Photon 100 CMOS detector. Data collection with Apex2 \[[@B99-molecules-22-01720]\], data integration and cell refinement with SAINT,1 absorption correction by SADABS \[[@B99-molecules-22-01720]\], structure solution with SHELXT \[[@B100-molecules-22-01720]\], structure refinement with SHELXL \[[@B101-molecules-22-01720]\]. Molecular graphics from Mercury \[[@B102-molecules-22-01720]\].
3.2. Synthesis of Keto Ester ***9*** {#sec3dot2-molecules-22-01720}
------------------------------------
(+)-Bis\[(R)-1-phenylethyl\]amine hydrochloride (2.5 g, 9.60 mmol, 1.58 equiv.) was added in one portion to dry THF (10 mL) at ambient temperature and stirred for 5 min. The stirring suspension was then cooled to −78 °C and BuLi (2.5M in hexane) (7.67 mL, 19.18 mmol, 3.16 equiv.) was added dropwise. The suspension changed colour from cloudy white to pale orange. After stirring at −78 °C for 15 min the suspension was warmed to ambient temperature whereby a transparent yellow solution was formed. This recooled to −78 °C and *meso*-(1*S*,6*R*)-bicyclo\[4.3.0\]non-3-ene-8-one **4** (0.826 g, 6.07 mmol, 1.0 equiv.) was added dropwise over 10 min in dry THF (10 mL). This mixture was then stirred for 45 min whereby a purple colour evolved. Methyl cyanoformate (0.96 mL, 12.14 mmol, 2.0 equiv.) was then added dropwise over 5 min. and the mixture immediately turned bright yellow in colour. This mixture was left stirring for 2.5 h and then quenched by addition of H~2~O (2 mL) at −78 °C. The mixture was then warmed to r.t. and extracted with EtOAc (2 × 50 mL). The resulting organic layer was then washed with H~2~O (2 × 100 mL), 0.5 M HCl (1 × 100 mL) and brine (1 × 100 mL). The organic layer was then dried over MgSO~4~, filtered and concentrated in vacuo. The resulting crude keto ester was purified by column chromatography (hexane/EtOAc 5:1) to form a colourless oil. This oil was then recrystallised from hexane at 0 °C, filtered and air dried to obtain the compound **9** as white crystals. All spectroscopic and physical data were in full agreement with those reported in the literature \[[@B103-molecules-22-01720]\]. Yield: 0.812 g (69%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = - 161{^\circ}$ ($c = 0.1$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~): δ 5.73--5.66 (m, 2H), 3.76 (s, 3H), 3.04 (d, *J* = 11.1 Hz, 1H), 2.88--2.83 (m, 1H), 2.52--2.38 (m, 3H), 2.33--2.21 (m, 2H), 2.04 (dd, *J* = 1.9, 18.2 Hz, 1H), 1.67--1.61 (m, 1H); ^13^C-NMR (100 MHz, CDCl~3~): δ 211.6, 169.7, 124.9, 123.9, 57.7, 52.4, 46.6, 37.3, 29.7, 26.8, 25.3; IR (neat, cm^−1^) 3034 (w), 2945 (m), 2908 (m), 2837 (w), 1751 (s), 1718 (s) 1656 (w) 1433 (s) 1404 (m); HRMS (EI+): Exact mass calculated for C~11~H~14~O~3~ \[M\]^+^: 194.0943 found 194.0933; m.p.: 59--61 °C; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.42.
3.3. Synthesis of Michael Acceptor ***10*** {#sec3dot3-molecules-22-01720}
-------------------------------------------
### 3.3.1. Synthesis of α-Hydroxy Ester ***pre*-10a** {#sec3dot3dot1-molecules-22-01720}
To stirring solution of **9** (1.40 g, 7.21 mmol, 1.0 equiv.) in MeOH (50 mL) at 0 °C was added NaBH~4~ (0,410 g, 10.8 mmol, 1.5 equiv.). The reaction was monitored by TLC and was deemed complete after 1 h. Then, dilute aq. HCl (10 mL, 1 M) was added drowise at 0 °C. The quenched reaction was concentrated in vacuo to afford a crude mixture. This was poured over Et~2~O (50 mL), whereupon water (50 mL) was added. The organic layer was separated and the aqueous layer was extracted with Et~2~O (2 × 50 mL). The organic layers were combined, washed with brine (1 × 100 mL), dried over MgSO~4~, filtered and concentrated in vacuo to afford a colourless, oily, residue. The crude was purified by column chromatography on silica (hexane/EtOAc 7:3) to afford the C8-epimeric compound ***pre*-10a** as colourless oil. Yield: 1.07 g (76%); ^1^H-NMR (400 MHz, CDCl~3~): δ 5.81--5.71 (m, 2H), 4.57--4.39 (m, 1H), 3.73 (s, 3H), 2.65--2.50 (m, 1H), 2.38--2.10 (m, 6H), 2.10--1.84 (m, 2H), 1.52--1.42 (m, 1H); ^13^C-NMR (100 MHz, CDCl~3~): δ 175.5 (major), 174.8 (minor), 127.5 (minor), 126.7 (minor), 126.6 (major), 125.6 (major), 75.3 (major), 73.0 (minor), 57.3 (major), 53.9 (minor), 51.8 (major), 51.7 (minor), 42.1 (minor), 40.9 (major), 39.0 (major), 38.0 (minor), 34.0 (minor), 33.6 (major), 27.9 (minor), 27.7 (major), 26.3 (major), 26.2 (minor); IR (neat, cm^−1^) 3439 (br), 3026 (w), 2914 (w), 2841 (w), 1712 (s), 1438 (m); HRMS (EI+): Exact mass calculated for C~11~H~16~O~3~ \[M\]^+^: 196.1099, found 196.1087; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.30.
### 3.3.2. Synthesis of Mesylate ***pre*-10b** {#sec3dot3dot2-molecules-22-01720}
To a stirring solution of C8-epimer ***pre***-**10a** (1.07 g, 5.45 mmol, 1.0 equiv.) in dry CH~2~Cl~2~ (50 mL) at ambient temperature was added Et~3~N (1.14 mL, 8.18 mmol, 1.5 equiv.) in a dropwise manner. The resulting mixture was left stirring for 5 min. and then cooled to 0 °C. Subsequently, methanesulfonyl chloride (0.51 mL, 6.59 mmol, 1.2 equiv.) was added in a dropwise manner and the reaction mixture was left stirring for 10 min. with continued cooling. Then, the cooling was discontinued and the reaction mixture was allowed to attain ambient temperature overnight. At this point the reaction mixture had turned from colourless to yellow. Brine (20 mL) was added in a dropwise manner and the volatiles were removed in vacuo. The resulting yellow liquid was poured over EtOAc (50 mL) and satd. aq. NaHCO~3~ (50 mL) was added. The organic layer was separated and the aqueous layer was extracted with EtOAc (2 × 50 mL). The organic layers were combined and washed with brine (1 × 100 mL), dried over MgSO~4~, filtered and concentrated in vacuo to afford a yellow, oily, residue. The crude was purified by column chromatography on silica (hexane/EtOAc 4:1) to afford the C8-epimeric compound ***pre*-10b** as a yellow oil. Yield: 1.16 g (77%); ^1^H-NMR (400 MHz, CDCl~3~) δ 5.81--5.77 (m, 0.3H, minor), (m, 1.7H, major), 5.37--5.31 (m, 1H), 3.74 (s, 2.6H, major), 3.72 (s, 0.4H, minor), 3.01 (s, 2.6H, major), 2.96 (s, 0.4H, minor), 2.88--2.82 (m, 1H), 2.4.06 (m, 6H), 2.02--1.87 (m, 1H), 1.86--1.78 (m, 1H); ^13^C-NMR (100 MHz, CDCl~3~) δ 173.9, 127.3 (minor), 126.8 (minor), 125.6 (major), 124.4 (major), 83.9 (major), 83.8 (minor), 54.6 (major), 53.6 (minor), 52.2 (major), 51.9 (minor), 40.9 (minor), 39.8 (major), 39.1 (major), 38.3 (minor), 37.9 (major), 36.4 (minor), 33.9 (major), 33.5 (minor), 27.6 (minor), 26.6 (major), 26.0 (minor), 25.4 (major); IR (neat, cm^−1^) 3031 (w), 2942 (w), 2847 (w), 1734 (s), 1438 (w), 1354 (s); HRMS (EI+): Exact mass calculated for C~12~H~18~O~5~S \[M\]^+^: 274.0875, found 274.0865; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.45.
### 3.3.3. Synthesis of Michael Acceptor **10** {#sec3dot3dot3-molecules-22-01720}
To a stirring solution of C8-epimer ***pre*-10b** (1.40 g, 5.10 mmol, 1.0 equiv.) in dry toluene (30 mL) at ambient temperature was added DBU (1.73 mL, 11.6 mmol, 2.3 equiv.) in a dropwise manner over 5 min. The reaction mixture was stirred overnight at the stated conditions. Having deemed the reaction complete by TLC, water (10 mL) and dilute aq. HCl (10 mL, 0.5 M) was added. The resulting mixture was poured over Et~2~O (20 mL) and the organic layer was separated. The aqueous layer was extracted with Et~2~O (2 × 30 mL). The organic layers were combined, washed in succession with water (1 × 50 mL) and brine (1 × 50 mL), dried over MgSO~4~, filtered and concentrated in vacuo to obtain a oily residue. The crude was purified by column chromatography on silica (hexane/EtOAc 9:1) to afford the compound **10** as colourless oil. Yield: 0.866 g, (95%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 180{^\circ}$ ($c = 0.8$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 6.80--6.69 (m, 1H), 5.95--5.84 (m, 1H), 5.84--5.74 (m, 1H), 3.74 (s, 3H), 3.04--3.92 (m, 1H), 2.67--2.51 (m, 2H), 2.50--2.36 (m, 1H), 2.34--2.12 (m, 2H), 2.00--1.82 (m, 2H); ^13^C-NMR (100 MHz, CDCl~3~) δ 165.6, 143.5, 140.8, 128.3, 127.0, 51.2, 41.2, 39.4, 36.0, 27.8, 26.6; IR (neat, cm^−1^) 3031 (w), 2931 (w), 2841 (w), 1712 (s), 1628 (w), 1438 (m); HRMS (EI+): Exact mass calculated for C~11~H~14~O~2~ \[M\]^+^: 178.0994, found 178.1000; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.60.
3.4. Synthesis of Michael Adduct ***11*** {#sec3dot4-molecules-22-01720}
-----------------------------------------
To a solution of Michael acceptor **10** (0.421 g, 2.36 mmol, 1.0 equiv.) in dry THF (20 mL) at −35 °C was added in succession CuI (0.045 g, 0.24 mmol, 0.1 equiv.) and TMSCl (0.641 g, 0.75 mL, 5.90 mmol, 2.5 equiv.). The resulting slightly heterogenous mixture caused by suspended CuI was stirred for 5 min, whereupon BuMgCl (2.0 M in THF) (2.36 mL, 4.72 mmol, 2.0 equiv.) was added in a dropwise manner during the course of 2 h, maintaining the temperature between at −35 °C. Initial colours cycled between clear and yellow, but gradually took a transient purple hue while reverting to clear. Upon completing the addition, the purple colour persisted (cloudy amethyst). At this point, TLC revealed that the starting material had been consumed. The reaction was treated with aq. satd. NH~4~Cl (5 mL) and diluted with Et~2~O/water (30 mL, 2:1). The phases were separated and the aq. phase was extracted with Et~2~O (3 × 20 mL). The combined org. phases were washed with brine (15 mL), dried over MgSO~4~, filtered and the solvent was evaporated *in vacuo*. The residue was purified by column chromatography on silica (hexane/EtOAc 90:10) to afford compound **11** as a colourless oil. Yield: 0.496 g (81%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 63{^\circ}$ ($c = 0.8,$ CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 5.66--5.56 (m, 2H), 3.67 (s, 3H), 2.58--2.46 (m, 2H), 2.36--2.16 (m, 3H), 1,95--1.68 (m, 4H), 1.45--1.35 (m, 2H), 1.35--1.15 (m, 5H), 0.87 (t, *J* = 7.0 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ 174.8, 124.7, 124.6, 56.0, 51.3, 38.9, 37.4, 36.8, 36.0, 35.3, 30.4, 26.5, 22.8, 22.6, 14.1; IR (neat, cm^−1^) 3020 (w), 2925 (m), 1734 (s); HRMS (EI+): Exact mass calculated for C~15~H~24~O~2~ \[*M*\]^+^: 236.1776, found 236.1763; TLC (hexanes/EtOAc 80:20, KMnO~4~ stain): R~f~ = 0.70.
3.5. Synthesis of Carbinol ***12*** {#sec3dot5-molecules-22-01720}
-----------------------------------
Michael adduct 11 (0.496 g, 2.10 mmol, 1.0 equiv.) was dissolved in hexane (10 mL) at ambient temperature and stirred for 5 min. The solution was then cooled to 0 °C and DIBAL-H (1M in hexane) (4.2 mL, 4.20 mmol, 2.0 equiv.) was added dropwise over 5 min. The reaction was then left to warm to r.t. After 1 h the reaction was cooled back to 0 °C and quenched with sat. aq. NH~4~Cl (5 mL). The reaction mixture was allowed to warm to ambient temperature whereby a cloudy suspension occurred. This suspension was poured over sat. aq. NH~4~Cl (20 mL) and the organic layer separated. The aqueous layer was extracted with EtOAc (2 × 50 mL) and the organic layers combined, washed with H~2~O (1 × 100 mL), brine (1 × 100 mL), dried over MgSO~4~, filtered and concentrated in vacuo to give a crude cloudy oil. This was then purified by column chromatography on silica (hexane/EtOAc 95:5) to afford compound **12** as a colourless oil. Yield: 0.400 g, (92%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 104{^\circ}$ ($c = 0.8$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) 5.77--5.51 (m, 2H), 3.77--3.58 (m, 2H), 2.38--2.22 (m, 1H), 2.22--2.05 (m, 2H), 2.05--1.71 (m, 4H) 1.71--1.52 (m, 2H), 1.52--1.35 (2H) 1.35--1.14 (m, 6H), 0.88 (t, *J* = 7.1 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ 125.3, 124.9, 63.3, 53.7, 38.1, 36.8, 36.3, 35.5, 35.4, 30.8, 26.6, 22.9, 21.6, 14.1; IR (neat, cm^−1^) 3328 (br.), 3020 (w), 2925 (s); HRMS (EI+): Exact mass calculated for C~14~H~24~O \[*M*\]^+^: 208.1827, found 208.1832; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.40. The enantiomeric excess was determined by chiral GLC analysis (CP-Chirasil-DEX CB, using the following program: 60 °C (45 min)---1 degrees/min to 160 °C---160 °C (5 min)): *t~r~*(*e~1~*, major) = 65.08 min and *t~r~*(*e~2~*, minor) = 65.67 min; *ee*: \> 99% \[[@B104-molecules-22-01720]\].
3.6. Synthesis of 3,5-Dinitrobenzoate ***12-DNB*** {#sec3dot6-molecules-22-01720}
--------------------------------------------------
To a stirring solution of carbinol **12** (0.129 g, 0.546 mmol, 1.0 equiv.) in dry DCM (20 mL) was added Et~3~N (0.23 mL, 1.64 mmol, 3.0 equiv.) dropwise. The solution was then cooled to 0 °C and 3,5-dinitrobenzoyl chloride (0.215 g, 0.933 mmol, 1.7 equiv.) was added in one portion. The reaction was slowly warmed to ambient temperature and monitored by TLC until completion. After 2 h, the reaction mixture was poured over H~2~O (10 mL) and the organic layer separated. The aqueous layer was then extracted with DCM (2 × 10 mL) and the organic layers combined. The organic layers were then washed with H~2~O (1 × 30 mL), brine (1 × 30 mL), dried with MgSO~4~, filtered and concentrated in vacuo to form a crude orange oil. This was purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound **12-DNB** as a white powder. Yield: 0.193 g (88%), $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 42{^\circ}$ ($c = 0.8$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 9.23 (t, $J = 2.2\ {Hz}$, 1H), 9.14 (d, $J = 2.2\ {Hz}$, 2H), 5.70--5.61 (m, 2H), 4.49 (s, 1H), 4.47 (d, $J = 1.9\ {Hz}$, 1H), 2.37--2.10 (m, 4H), 2.02--1.88 (m, 2H), 1.88--1.68 (m, 3H), 1.58--1.43 (m, 2H), 1.40--1.23 (m, 5H), 0.89 (t, $J = 6.7\ {Hz}$, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ 162.5, 148.7, 134.1, 129.3, 125.4, 124.3, 122.3, 67.7, 49.5, 38.6, 36.9, 36.7, 35.5, 35.4, 30.8, 26.5, 22.9, 21.8, 14.1; IR (neat, cm^−1^) 3098 (w), 3020 (w), 2931 (m), 1723 (s), 1538 (s); HRMS (EI+): Exact mass calculated for C~21~H~26~N~2~O~6~ \[*M*\]^+^: 402.1791, found 402.1797; m.p.: 117 °C; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.75. \[[@B105-molecules-22-01720]\]
3.7. Synthesis of Aldehyde ***13*** {#sec3dot7-molecules-22-01720}
-----------------------------------
### 3.7.1. Synthesis of Mesylate ***pre*-13a** {#sec3dot7dot1-molecules-22-01720}
To a stirring solution of carbinol **12** (0.400 g, 1.92 mmol, 1.0 equiv.) in dry CH~2~Cl~2~ (5 mL) at ambient temperature, was added Et~3~N (0.54 mL, 3.84 mmol, 2.0 equiv.) dropwise. This solution was left stirring for 5 min then cooled to 0 °C. Then methanesulfonyl chloride (0.45 mL, 5.76 mmol, 3.0 equiv.) was added dropwise and the reaction was left at 0 °C for 10 min then warmed to ambient temperature and left overnight. The reaction mixture turned colourless to yellow. Then, brine (10 mL) was added dropwise and the volatiles concentrated in vacuo to afford a yellow liquid. This was poured over EtOAc (50 mL) and sat. aq. NaHCO~3~ (50 mL) was added. The organic layer was separated and the aqueous layer extracted with EtOAc (2 × 50 mL). The organic layers were combined and washed with brine (1 × 50 mL), dried over MgSO~4~, filtered and concentrated in vacuo to afford a crude yellow oil. This was then purified by column chromatography on silica (hexane/EtOAc 95:5) to afford the compound ***pre*-13a** as a colourless oil. Yield: 0.497 g, (90%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 79{^\circ}$ ($c = 0.8$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 5.60--5.50 (m, 2H), 4.21--4.12 (m, 2H), 2.94 (s, 3H), 2.25--2.11 (m, 1H), 2.11--1.99 (m, 2H), 1.98--1.83 (m, 2H), 1.83--1.76 (m, 1H), 1.71--1.55 (m, 3H), 1.40--1.30 (m, 2H), 1.28--1.10 (m, 5H), 0.82 (t, *J* = 7.0 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ 125.3, 124.4, 70.3, 50.0, 38.1, 37.4, 36.6, 36.4, 35.4, 35.3, 30.7, 26.4, 22.8, 21.4, 14.1; IR (neat, cm^−1^) 3020 (w), 2925 (m), 1354 (s); HRMS (EI+): Exact mass calculated for C~15~H~26~O~3~S~2~ \[*M*\]^+^: 286.1603, found 286.1627; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.50.
### 3.7.2. Synthesis of Nitrile ***pre*-13b** {#sec3dot7dot2-molecules-22-01720}
To a stirring solution of mesylate *pre*-13a (0.497 g, 1.74 mmol, 1.0 equiv.) in dry DMSO (30 mL) was added solid KCN (0.675 g, 10.4 mmol, 6.0 equiv.) in one portion. The reaction mixture was then heated to 70 °C for 2 h. The reaction mixture changed from colourless to yellow. Then, the reaction was cooled to r.t. and H~2~O (5 mL) was added dropwise. The reaction mixture turned from yellow to colourless. This was then poured over EtOAc (20 mL) and the organic layer separated. The aqueous layer was extracted with EtOAc (2 × 20 mL) and the organic layers combined. They were then washed with brine (1 × 50 mL), dried over MgSO~4~, filtered and concentrated in vacuo to afford a crude brown oil. This was then purified by column chromatography on silica (hexane/EtOAc 98:2) to give the compound ***pre*-13b** as a colourless oil. Yield: 0.325, (86%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 111{^\circ}$ ($c = 0.8$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 5.82--5.47 (m, 2H), 2.42--2.28 (m, 2H), 2.18--2.14 (m, 2H), 2.04--1.85 (m, 3H), 1.74--1.65 (m, 3H), 1.51--1.35 (m, 2H), 1.33--1.19 (m, 5H), 0.89 (t, *J* = 7.0 Hz, 3H) ^13^C-NMR (100 MHz, CDCl~3~) δ 125.4, 124.2, 119.6, 47.3, 41.4, 37.9, 35.8, 35.7, 35.1, 30.6, 26.5, 22.8, 21.5, 17.7, 14.1; IR (neat, cm^−1^) 3026 (w), 2919 (s), 2248 (w), 1465 (w) 1436 (w); HRMS (EI+): Exact mass calculated for C~15~H~23~N \[*M*\]^+^: 217.1830, found 217.1845; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.80.
### 3.7.3. Synthesis of Aldehyde **13** {#sec3dot7dot3-molecules-22-01720}
A stirring solution of nitrile ***pre*-13b** (0.322 g, 1.48 mmol, 1.0 equiv.) in hexane (10 mL) was cooled to −78 °C. Then DIBAL-H (1M in hexane) (2.20 mL, 2.22 mmol, 1.5 equiv.) was added dropwise over 5 min and the reaction left to stir for 20 min. Then sat. aq. Rochelle salt (5 mL) was added dropwise to the reaction mixture and then left to warm to ambient temperature. The resulting cloudy suspension was poured over EtOAc (20 mL) and sat. aq. Rochelle salt (20 mL). The organic layer was separated and the aqueous phase extracted with EtOAc (2 × 20 mL). The organic phases were combined and washed with brine (1 × 50 mL), dried over MgSO~4~, filtered and concentrated in vacuo to afford a crude cloudy oil. This was then purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound **13** as a colourless oil. Yield: 0.253 mg, (78%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 101{^\circ}$ ($c = 0.8$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 9.79 (t, *J* = 2.3 Hz, 1H), 5.68--5.51 (m, 2H), 2.49--2.44 (m, 2H), 2.35--2.23 (m, 1H), 2.22--2.12 (m, 1H), 2.10--1.98 (m, 2H), 1.92--1.78 (m, 2H), 1.74--1.60 (m, 3H), 1.47--1.35 (m, 2H), 1.35--1.15 (m, 5H), 0.88 (t, *J* = 7.0 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ 202.9, 125.3, 124.7, 45.2, 44.7, 41.4, 37.5, 35.7, 35.6, 34.9, 30.8, 26.7, 22.9, 22.1, 14.1; IR (neat, cm^−1^) 3020 (w), 2919 (m), 2712 (w), 1723 (s); HRMS (EI+): Exact mass calculated for C~15~H~24~O \[*M*\]^+^: 220.1827, found 220.1824; TLC (hexane/EtOAc 4:1, KMnO~4~ stain): R~f~ = 0.80.
3.8. Synthesis of Alkyne ***14*** {#sec3dot8-molecules-22-01720}
---------------------------------
To a stirring solution of aldehyde **13** (0.253 g, 1.15 mmol, 1.0 equiv.) in dry MeOH (6 mL) at 0 °C was added solid K~2~CO~3~ (0.381 mg, 2.76 mmol, 2.4 equiv.) in one portion and Ohira-Bestmann reagent (10% *w*/*w* in MeCN) (3.9 mL, 3.32 g, 1.73 mmol, 1.5 equiv.). The suspension was then warmed to ambient temperature and left stirring 1 h. After analysis by TLC the mixture was treated with sat. aq. NaHCO~3~ (20 mL), and the resulting mixture poured over CH~2~Cl~2~ (20 mL). The organic phase was separated and the aqueous phase was washed with CH~2~Cl~2~ (3 × 10 mL). The organic phases were then combined, dried over Na~2~SO~4~, filtered and concentrated in vacuo to afford a crude oil. This was purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound **14** as a colourless oil. Yield: 0.193 g, (78%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 119{^\circ}$ ($c = 0.8$, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 5.72--5.58 (m, 2H), 2.35--2.24 (m, 2H), 2.18--2.09 (m, 3H), 2.07--1.98 (m, 1H), 1.91 (t, *J* = 2.7 Hz, 1H), 1.89--1.83 (m, 1H), 1.83--1.75 (m, 1H), 1.75--1.55 (m, 3H), 1.55--1.45 (m, 1H), 1.45--1.36 (m, 1H), 1.36--1.13 (m, 5H), 0.89 (t, *J* = 7.1 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ; 125.2, 125.0, 84.4, 67.9, 50.2, 41.2, 37.7, 36.1, 35.9, 35.2, 30.8, 26.8, 22.9, 21.4, 18.9, 14.1; IR (neat, cm^−1^) 3311 (m), 3020 (w), 2919 (s), 1432 (m); HRMS (EI+): Exact mass calculated for C~16~H~24~ \[*M*\]^+^: 216.1878, found 216.1867; TLC (hexane, KMnO~4~ stain and anisaldehyde dip): R~f~ = 0.25.
3.9. Synthesis of exo-Mucosin ***1\**** {#sec3dot9-molecules-22-01720}
---------------------------------------
### 3.9.1. Synthesis of Ethyl Ester **15** {#sec3dot9dot1-molecules-22-01720}
To a stirring solution of Cp~2~ZrCl~2~ (0.400 g, 1.37 mmol, 2.0 equiv.) in dry THF (8 mL) at 0 °C was added DIBAL-H (1M in hexane) (1.37 mL, 1.37 mmol, 2.0 equiv.) via dropwise addition. The resulting homogenous mixture was then protected from light and stirred at 0 °C for 1 h after which time a colourless heterogeneous mixture formed. Then alkyne **14** (0.148 g, 0.684 mmol, 1.0 equiv.) dissolved in dry THF (4 mL) was added dropwise to the reaction mixture at 0 °C. After 1 h at 0 °C iodine (0.260 g, 1.02 mmol, 1.5 equiv.) was added in one portion to the homogeneous yellow reaction mixture. The reaction mixture was then warmed to ambient temperature and stirred for 1 h. To the preformed vinyl iodide was successively added 4-ethoxy-4-oxobutylzinc bromide solution (0.5M in THF) (2.7 mL, 1.37 mmol, 2.0 equiv.) dropwise and (Ph~3~P)~4~Pd (0.079 g, 0.068 mmol, 0.1 equiv.) in one portion. The resulting light brown mixture was stirred at ambient temperature for 1 h and monitored by TLC. Once the reaction had gone to completion 1 M HCl (10 mL) was added dropwise and the reaction poured over Et~2~O (15 mL). The aqueous phase was extracted with Et~2~O (3 × 50 mL) and the organic phases combined, dried over MgSO~4~, filtered and concentrated in vacuo to form a crude brown oily mixture. This oily mixture was purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound **15** as a colourless oil. Yield: 0.196 g, (86%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 67{^\circ}$ (*c* = 0.8, CHCl~3~); ^1^H-NMR (400 MHz, CDCl~3~) δ 5.69--5.56 (m, 2H), 5.48--5.33 (m, 2H), 4.12 (q, *J* = 7.1 Hz, 2 H), 2.31--2.21 (appt, *J* = 7.5 Hz, 3H), 2.16--1.82 (m, 8H), 1.75--1.50 (m, 6H), 1.48--1.41 (m, 1H), 1.39--1.22 (m, 9H), 0.88 (t, *J* = 7.1 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ 173.8, 131.2, 129.1, 125.3, 125.1, 60.2, 51.6, 41.3, 37.3, 36.2, 35.6, 35.5, 33.7, 33.0, 31.9 31.0, 26.9, 24.8, 23.0, 21.7, 14.2, 14.1; IR (neat, cm^−1^) 3020 (w), 2925 (m), 1734 (s); HRMS (EI+): Exact mass calculated for C~22~H~36~O~2~ \[*M*\]^+^: 332.2715, found 332.2709; TLC (hexane/EtOAc 95:5, KMnO~4~ stain): R~f~ = 0.65.
### 3.9.2. Synthesis of *exo*-Mucosin **1\*** {#sec3dot9dot2-molecules-22-01720}
To a stirring solution of ethyl ester **15** (0.177 g, 0.533 mmol, 1.0 equiv.) in THF/MeOH/H~2~O (2:2:1) (5 mL) at ambient temperature was added lithium hydroxide monohydrate (0.783 mg, 18.7 mmol, 35.0 equiv.) in one portion. The reaction mixture was left stirring and monitored by TLC. Left overnight, the reaction had gone to completion and was acidified to pH 2 by 1M HCl (5 mL). The reaction mixture was then poured over EtOAc (5 mL) and the aqueous phase extracted with EtOAc (3 × 5 mL). The organic phases were combined and washed with brine (1 × 20 mL), dried over MgSO~4~, filtered and concentrated in vacuo to provide a colourless oil. This was purified by column chromatography on silica (hexane/EtOAc, 3:2) to afford the compound **1\*** as a colourless oil. Yield: 0.154 g, (95%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 77{^\circ}$ ($c = 0.8$, hexane); ^1^H-NMR (400 MHz, CDCl~3~) δ 11.63 (br, 1H), 5.67--5.56 (m, 2H), 5.50--5.34 (m, 2H), 2.34 (t, *J* = 7.5 Hz, 2H), 2.33--2.22 (m, 1H), 2.15--2.12 (m, 8H), 1.77--1.49 (m, 6H), 1.48--1.40 (m,1H), 1.40--1.08 (m, 6H), 0.88 (t, *J* = 6.7 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ 180.3, 131.4, 128.9, 125.3, 125.1, 51.6, 41.3, 37.3, 36.2, 35.6, 35.5, 33.4, 33.0, 31.8, 31.0, 26.9, 24.5, 23.0, 21.7, 14.2; IR (neat, cm^−1^) 3020 (w), 2925 (m), 1712 (s); HRMS (EI+): Exact mass calculated for C~20~H~32~O~2~ \[*M*\]^+^: 304.2402, found 304.2400; TLC (hexane/EtOAc 3:2, KMnO~4~ stain): R~f~ = 0.40.
### 3.9.3. Synthesis of Methyl Ester **2\*** {#sec3dot9dot3-molecules-22-01720}
To a stirring solution of *exo*-mucosin **1\*** (0.023 g, 0.076 mmol, 1.0 equiv.) in toluene/MeOH (3:2) (5 mL) at ambient temperature was added TMS diazomethane solution (2M in hexane) (0.06 mL, 0.113 mmol, 1.5 equiv.) dropwise over 2 min. The reaction mixture bubbled and turned transparent yellow. The reaction was monitored by TLC and after 1 h had gone to completion. The reaction mixture was then concentrated in vacuo and directly purified by column chromatography on silica (hexane/EtOAc, 95:5) to afford the compound **2\*** as a colourless oil. Yield: 23 mg, (96%); $\left\lbrack \alpha \right\rbrack_{D}^{26} = + 64{^\circ}$ (*c* = 0.8, hexane); ^1^H-NMR (400 MHz, CDCl~3~) δ 5.69--5.56 (m, 2H), 5.49--5.32 (m, 2H), 3.66 (s, 3H), 2.29 (t, *J* = 7.5 Hz, 2H), 2.26--2.21 (m, 1H), 2.13--1.82 (m, 8H), 1.75--1.63 (m, 3H), 1.63--1.50 (m, 3H), 1.40--1.08 (m, 7H), 0.88 (t, *J* = 7.1 Hz, 3H); ^13^C-NMR (100 MHz, CDCl~3~) δ; 174.2, 131.2, 129.0, 125.3, 125.1, 51.6, 51.4, 41.3, 37.2, 36.2, 35.5, 35.4, 33.4, 33.0, 31.9, 31.0, 26.9, 24.7, 23.0, 21.7, 14.1; IR (neat, cm^−1^) 3020 (w), 2925 (s), 1745 (s); HRMS (EI+): Exact mass calculated for C~21~H~34~O~2~ \[*M*\]^+^: 318.2559, found 318.2552; TLC (hexane/EtOAc 95:5, KMnO~4~ stain): R~f~ = 0.65.
4. Conclusions {#sec4-molecules-22-01720}
==============
To recapitulate our findings, we have investigated the *anti*-diastereomer **2\*** of the proposed structure **2**. Through the execution of 13 discrete linear steps, the target molecule was obtained in an overall yield of 11% and in multi-milligram quantities. By incorporating a Michael acceptor motif, we have revealed an innate topological bias of the *cis*-fused bicyclo\[4.3.0\]non-3-ene system. Combined with our previously developed three step one-pot alkyne iteration, we have shown that *exo*-mucosin **1**\* is not identical to the natural product. In terms of the synthetic sequence detailed by Whitby and co-workers \[[@B37-molecules-22-01720]\], our findings must necessarily have some mechanistic implications. Thus, we conclude that zirconium induced co-cyclisation did not deliver any of the two *anti*-diastereomers, **1** and **1\*** respectively, by the published procedure. Achieving a synthesis that establishes the true structure of mucosin will therefore also provide mechanistic insight into this matter. Based on the present work and what has previously been published \[[@B36-molecules-22-01720],[@B37-molecules-22-01720]\], the fusion geometry of the hydrindane core embedded within mucosin is most likely *trans*.
The authors are much indebted to Dag Ekeberg for the performance of mass spectrometric analysis. Scholarships for S.G.A. and H.G.-S. from the Department of Chemistry, the Norwegian University of Life Sciences, as well as funding from the Research Council of Norway for a research scholarship to J.M.J.N. and grants to Y.H.S. (NFR 209335 and NFR 244351) are gratefully acknowledged.
**Sample Availability:** Samples of *exo*-mucosin (**1\***) and methyl ester **2\*** are available from the authors.
Electronic supplementary information is available online with full experimental procedures and characterisation data for all new compounds; crystal data and refinement details are included to give evidence of the relative stereochemistry of the late stage intermediate **12**.
######
Click here for additional data file.
S.G.A. and H.G.-S. contributed equally to the practical work. C.H.G. performed the crystallographic acquisition and analysis. T.V.H. and Y.H.S. supervised and oversaw the project. Y.H.S. prepared the crystals for X-ray analysis. J.M.J.N. executed the diastereoselective addition and the alkyne iteration, developed the key stereopermutation concept and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare no conflict of interest.
Figures, Schemes and Table
==========================
{#molecules-22-01720-f001}
![Stereopermutation on the *cis*-bicyclo\[4.3.0\]non-3-ene scaffold.](molecules-22-01720-g002){#molecules-22-01720-f002}
{#molecules-22-01720-sch001}
{#molecules-22-01720-f003}
{#molecules-22-01720-sch002}
{#molecules-22-01720-sch003}
{#molecules-22-01720-f004}
{#molecules-22-01720-sch004}
molecules-22-01720-t001_Table 1
######
Comparative ^13^C-NMR of methyl ester **2\*** (δ-values). ^†,‡^
Entry Casapullo et al. \[[@B35-molecules-22-01720]\] Whitby et al. \[[@B37-molecules-22-01720]\] Previous Work \[[@B36-molecules-22-01720]\] This Work
------- ------------------------------------------------ --------------------------------------------- --------------------------------------------- -------------
1 174.2 174.2 174.2 174.2
2 130.0 130.3 130.4 ***131.2***
3 129.8 129.8 129.9 ***129.0***
4 127.0 127.3 ***126.3*** ***125.3***
5 127.0 127.1 ***126.1*** ***125.1***
6 52.1 52.2 ***51.4*** ***51.6***
7 51.4 51.4 51.0 51.4
8 47.1 47.2 ***44.0*** ***41.3***
9 42.1 42.3 ***40.3*** ***37.2***
10 39.9 40.1 ***38.1*** ***36.2***
11 36.7 37.0 ***37.7*** ***35.5***
12 36.5 36.74 ***37.1*** ***35.4***
13 36.4 36.68 ***34.9*** ***33.4***
14 33.2 33.4 33.4 33.0
15 32.0 32.4 31.9 31.9
16 31.7 31.9 ***31.0*** ***31.0***
17 31.5 31.6 ***27.8*** ***26.9***
18 30.7 ^§^ 30.7 ***27.7*** ***24.7***
19 24.5 24.7 24.8 ***23.0***
20 22.6 22.9 22.9 ***21.7***
21 13.8 14.1 14.1 14.1
^†^ The italic bold numbers indicate deviating δ-values compared to Ref. \[[@B35-molecules-22-01720]\]. ^‡^ Upon request, we have not been able to procure the original ^13^C-spectrum from the authors quoted in Ref. \[[@B35-molecules-22-01720]\] for comparison. ^§^ According to communication rendered in the supporting information accompanying Ref. \[[@B37-molecules-22-01720]\], the resonance at δ 30.7 had been omitted in Ref. \[[@B35-molecules-22-01720]\], while an additional signal at δ 36.3 was observed. The data were subsequently revised and this fact is not touched upon in the main paper.
[^1]: Dedication: Dedicated to Lars Skattebøl on the occasion of his 90th birthday.
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Desperate parents of nail-biting kids and furniture-chewing dogs often turn to foul-tasting compounds to deter their loved ones’ gnawing. In the future, we might be trying something similar with mosquitoes, as scientists now say that malaria-causing mosquitoes use scent and taste to decide who to bite. They published their research in the journal Nature Communications.
The mosquito, that scourge of summer, is more than just a nuisance. Mosquito-borne viruses like Zika and dengue are on the rise. Rates of malaria are down, but still high; around 214 million people were affected in 2015 alone. As drug developers race to develop vaccines, other scientists are hoping to find ways to keep disease-carrying mosquitoes from biting in the first place. To do that, they’ve got to know mosquitoes inside and out.
A mosquito lives by its noses. Yes, noses, plural. Every mosquito has three sets of scent-detecting parts: two antennae, two fuzzy mouthparts called maxillary palps, and two little spots called labella at the end of its proboscis. The antennae and palps are scent-only, but the labella contain neurons for sensing both smell and taste.
That’s a lot of olfactory information for a teeny tiny brain to take in. To find out how the mosquitoes do it, researchers tinkered with the genes of the malaria-carrying mosquito Anopheles gambiae. They tricked out the mosquitoes with a gene that would cause cells called odorant receptors (ORs) to glow bright green, which would make them easier to spot under a microscope. Building fluorescent proteins into bug parts is not a new technique, but it’s never been done before in mosquitoes.
This is a female Anopheles gambiae mosquito with olfactory neurons on the antennae, maxillary palp, and labella labeled in green. Image Credit: Olena Riabinina and Courtney Akitake, Johns Hopkins Medicine
By looking at the mosquitoes’ glowing ORs, the team was able to trace the paths from the pests’ sense organs to their brains. They found that information taken in by the antennae and maxillary palps was sent to brain regions called antennal lobes (this process is the same in flies). But signals from the labella went over to an area called the subesophageal zone—an area that had previously only been associated with taste.
The researchers say this likely means that a mosquito not only sniffs us but tastes us, too, poking with the end of its proboscis to confirm we’re edible before unsheathing its gross, syringe-like feeding needles.
It’s an unsettling concept, to be sure, but it might just help us out down the road. Co-author Christopher Potter, a neuroscientist at Johns Hopkins University, says we could use An. gambiae's brain cells against it—all we have to do is convince it that we taste terrible. “Our goal is to let the mosquitoes tell us what smells they find repulsive and use those to keep them from biting us,” he said in a statement.
Lead author Olena Riabinina, now at Imperial College London, noted that their success with the glowing protein has created new possibilities for mosquito research. “We were pleasantly surprised by how well our genetic technique worked and how easy it is now to see the smell-detecting neurons,” she said. “The ease of identification will definitely simplify our task of studying these neurons in the future."
Know of something you think we should cover? Email us at tips@mentalfloss.com. |
Corrections & clarifications: This story has been updated to reflect that AXON is not the manufacturer of the Greenville County Sheriff’s Office’s body cameras.
GREENVILLE, S.C. – Footage captured by a South Carolina deputy's body camera shows the moment he shot and injured a homeowner last month, and the events that led up to and after the shooting.
The events in the video differ from the original account of the June 14 shooting that was reported by the Greenville County Sheriff's Office. In the video, the deputy shoots the man through the window of the Simpsonville house. Initially, the agency said the man was shot after he opened the door and pointed his gun at the deputy.
There was no audio for the first 30 seconds of the video, including when the deputy fired his gun.
AXON, a leading manufacturer of police body cameras, explains on its website that while in "buffer mode" body cameras are recording video, but do not capture audio, and only create 30-second clips that are not saved to permanent memory until cameras are fully activated.
AXON does not produce the Greenville County Sheriff's Office's body cameras.
After he is shot, the man shouts for someone to "call the cops." The deputy responds, "I am the cops."
Florida body cam video: Former Florida officer allegedly planted drugs during traffic stops, 'tailored' body cam video
The man who was shot was not charged, according to a critical incident report released by the Sheriff's Office.
A deputy responded to the house after a cellphone emergency alarm was reported at 11:49 p.m. to Greenville County Communications. The deputy went to the home after failed attempts to contact the cellphone from which the alarm originated.
Capt. Tim Brown, of the Sheriff's Office's Office of Professional Standards, said in the critical incident briefing video that was posted to YouTube that the deputy walked away from the porch, but approached it again when he saw movement inside the house. Brown said the deputy saw a man holding a gun and pointed his flashlight at him.
Story continues
The Sheriff's Office said there would be no further comment about the video. The agency declined to share a copy of the edited video file since the primary public information officer was out of town, said Lt. Jimmy Bolt, a spokesman for the Sheriff's Office.
Brown said the man turned and pointed his gun at the deputy, at which point the deputy fired multiple shots through the window as he left the porch.
The body camera footage shows the deputy approach the front door, and a man can be seen through the front window. The deputy is then seen pointing his flashlight through the window and firing, but the light prevents the camera from capturing what's going on inside the house.
Officer shot: 'Officer down! Officer down!' Chilling footage reveals deadly shootout in Sacramento
“The Sheriff’s Office’s statement for weeks after the shooting (was) that my client opened his front door and aimed at deputy and you can look at that bodycam – that ends that version,” attorney Beattie Ashmore said Monday morning after the clips of body camera footage was posted to YouTube by the Sheriff’s Office. “It’s difficult to explain how something like this could have happened.”
For about 40 seconds after firing shots through the window, the deputy communicates with the man inside while standing in the front yard. The man can be heard yelling in pain during the exchange.
The deputy then goes inside and finds the man on the ground near the front door.
The man, whose face is pixelated in the video, tells the deputy that he's been shot in the groin and the chest.
After entering the house, the deputy proceeds to give the man first aid as they wait for the ambulance to arrive.
As the deputy gets ready to treat him, the man yells, "I saw lights and I heard the door bell ring, so I got my gun. I'm a concealed weapons guy."
Several seconds later, the man asks, "Why did you do that?" and the deputy responds, "You pointed a gun at me, man." The man replies, "Dude, you came to my house at 12 o'clock at night, I'm sleeping. (Expletive), I've got to protect my house."
Ashmore is representing the homeowner, Dick Tench, who he described as a “fiercely patriotic” citizen and concealed weapons permit holder.
Tench saw reflections from the deputy’s flashlight and came to see who was at his home, wondering whether an intruder had even broken into his house, Ashmore said.
Arrest caught on camera: Video shows Sacramento police placing 'spit mask' on 12-year-old boy as he shouts 'I can't breathe!'
“He carries a concealed weapons permit. That’s a four-hour class where they teach you to first know what you’re shooting at when you first pull the trigger. Apparently, the officer didn’t attend that class.”
Ashmore said the video is evidence to show the deputy lied about the justification behind his actions.
"Somebody was," he said, after being asked whether the deputy was lying about the nature of the confrontation.
Tench is recovering but spent the first 30 days after the shooting with two bullets still lodged into his body, Ashmore said. He suffered one gunshot wound to his aortic artery and one to his pelvis. Two other bullets grazed his side, Ashmore said.
“This has shaken him to the core,” Ashmore said. “They are super fine people. There’s just more to the story here.”
The alarm that Greenville County Communications received was a medical emergency alarm, but there was no emergency at the home, Brown said in the video.
The State Law Enforcement Division is investigating the shooting. The Sheriff's Office also is conducting an internal investigation to determine whether the agency's use of force policy was violated during the incident. The deputy was placed on paid administrative leave after the shooting.
The Sheriff's Office has declined to release the name of the deputy.
Follow Conor Hughes and Daniel J. Gross on Twitter: @ConorJHughes and @DanieljGross
This article originally appeared on The Greenville News: Deputy-involved shooting: Video shows deputy shoot man through window |
<?php
/**
* amadeus-ws-client
*
* Copyright 2015 Amadeus Benelux NV
*
* Licensed under the Apache License, Version 2.0 (the "License");
* you may not use this file except in compliance with the License.
* You may obtain a copy of the License at
*
* http://www.apache.org/licenses/LICENSE-2.0
*
* Unless required by applicable law or agreed to in writing, software
* distributed under the License is distributed on an "AS IS" BASIS,
* WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
* See the License for the specific language governing permissions and
* limitations under the License.
*
* @package Amadeus
* @license https://opensource.org/licenses/Apache-2.0 Apache 2.0
*/
namespace Amadeus\Client\Struct\Offer\Create;
/**
* TotalPrice
*
* @package Amadeus\Client\Struct\Offer\Create
* @author Dieter Devlieghere <dermikagh@gmail.com>
*/
class TotalPrice
{
/**
* @var MonetaryDetails
*/
public $monetaryDetails;
/**
* @var MonetaryDetails
*/
public $otherMonetaryDetails;
/**
* TotalPrice constructor.
*
* @param int $amount
* @param string $currency
*/
public function __construct($amount, $currency)
{
$this->monetaryDetails = new MonetaryDetails(
$amount,
$currency
);
}
}
|
Sunday, July 31, 2016
In my last post about mining ethereum, I explained why I preferred Genoil's fork of the Ethereum Foundation's ethminer. After that post, I started having stability problems with one of the newer releases of Genoil's miner. I suspected the problem was likely deadlocks with mutexes that had been added to the code. They had been added to reduce the chance of the miner submitting stale or invalid shares, but in this case the solution was worse than the problem, since there is no harm in submitting a small number of invalid shares to a pool. After taking some time to review the code and discuss my ideas with the author, I decided to make some improvements. The result is ethminer-nr.
A description of some of the changes can be found on the issues tracker for Genoil's miner, since I expect most of my changes to be merged upstream. The first thing I did was remove the mutexes. This does open the possibility of a rare race condition that could cause an invalid share submit when one thread processes a share from a GPU while another thread processes a new job from the pool. On Linux the threads can be serialized using the taskset command to pin the process to a single CPU. On a multi-CPU system, use "taskset 1 ./ethminer ..." to pin the process to the first CPU.
As described in the issues tracker, I added per-GPU reporting of hash rate. I also reduced the stats output to accepted (A) and rejected (R), including stales, since I have never seen a pool submit fail, and only some pools will report a rejected share. The more compact output helps the stats still fit on a single line, even with hashrate reporting from multiple GPUs: m 13:28:46|ethminer 15099 24326 15099 =54525Khs A807+6:R0+0
To help detect when a pool connection has failed, instead of trying to manage timeouts in the code, I decided to rely on the TCP stack. The first thing I did was enable TCP keepalives on the stratum connection to the pool. If the pool server is alive but just didn't have any new jobs for a while, the socket connection will remain open. If the network connection to the pool fails, there will be no keepalive response and the socket will be closed. Since the default timeouts are rather long, I reduced them to make network failure detection faster:
sudo sysctl -w net.ipv4.tcp_keepalive_time=30
sudo sysctl -w net.ipv4.tcp_keepalive_intvl=5
sudo sysctl -w net.ipv4.tcp_keepalive_intvl=3
I wasn't certain if packets sent to the server will reset the keepalive timer, even if there is no response (even an ACK) from the server. Therefore I also reduced the default TCP retransmission count to 5, so the pool connection will close after a packet is sent (i.e. share submit) 5 times without an acknowledgement.
sudo sysctl -w net.ipv4.tcp_retries2=5
I was also able to make a stand-alone linux binary. Until now the Linux builds had made extensive use of shared libraries, so the binary could not be used without first installing several shared library dependencies like boost and json. I had to do some of the build manually, so to make your own static linked binary you'll have to wait a few days for some updates to the cmake build scripts. If you want to try it now anyway, you can add "-DETH_STATIC=1" to the cmake command line.
As for future improvements, since I've started learning OpenCL, I'm hoping to optimize the ethminer OpenCL kernel to improve hashing performance. Look for something in late August or early September.
Sunday, July 17, 2016
Programs for mining on GPUs are usually written in OpenCL. It's based on C, which I know well, so a few weeks ago I decided to try to improve some mining OpenCL code. My intention was to both learn OpenCL and better understand mining algorithims.
The first thing I tried was replacing the rotate code in the ror64 function to use amd_bitalign. The bitalign instruction (v_alignbit_b32) can do a 32-bit rotate in a single cycle, much like the ARM barrel shifter. I was surprised that the speed did not improve, which suggests the AMD OpenCL drivers are optimized to use the alignbit instruction. What was worse was that the kernel would calculate incorrect hash values. After double and triple-checking my code, I found a post indicating a bug with amd_bitalign when using values divisible by 8. I then tried amd_bytealign, and that didn't work either. I was able to confirm the bug when I found that a bitalign of 21 followed by 3 worked (albeit slower), while a single bitalign of 24 did not.
It would seem there is no reason to use the amd_bitalign any more. Relying on the driver to optimize the code makes it portable to other platforms. I couldn't find any documentation from AMD saying the bitalign and other media ops are deprected, but I did verify that the pragmas make no difference in kernel:#pragma OPENCL EXTENSION cl_amd_media_ops : enable
Mining on Windows is relatively easy, with nanopool posting a binary build of siamining's gominer fork. For Ubuntu, you need to build it from the source. For that, you'll need to install go first. If you type 'go' in Ubuntu 14.04, you'll get the following message:The program 'go' is currently not installed. You can install it by typing:apt-get install gccgo-go
I tried the similar package 'gccgo', which turned out to be a rabbit hole. The version 1.4.2 referred to in the gominer readme is a version of the package 'golang'. Neither gccgo-go or gccgo have the latest libraries needed my gominer. And the most recent version of golang in the standard Ubuntu repositories is 1.3.3. However the Ethereum foundation publishes a 1.5.1 build of golang in their ppa.
Even with the golang 1.5.1, building gominer wasn't as simple as "go get github.com/SiaMining/gominer". The reason is that the gominer modifications to support pooled mining are in the "poolmod3" branch, and there is no option to install directly from a branch. So I made my own fork of the poolmod3 branch, and added detailed install instructions for Ubuntu:
Once I got it running on a single GPU, I wanted to find out if it was worthwhile to switch my eth mining rigs to sia. I couldn't find a good sia mining calculator, so I pieced together some information about mining rewards and used the Sia Pulse calculator. I wanted to compare a single R9 290 clocked at 1050/1125, which gets about 29Mh/s mining eth, earning $2.17/day. For Sia, the R9 290 gets about 1100Mh, which if you put that into the Sia Pulse calculator along with the current difficulty of 4740Th, it will calculate daily earnings of 6015 SC/day. Multiplying by the 62c/1000SC shown on sia.nanopool.org will give you a total of $3.73/d, but that will be wrong. The Sia Pulse calculator defaults to a block reward of 300,000, but that goes down by 1 for each block. So at block 59,900, the block reward is 240,100. and the actual earnings would be $2.99/d.
Since the earnings are almost 40% better than eth, I decided to switch my mining rigs from eth to sia. I had to adjust the overclocking settings, as sia is a compute-intensive algorithm instead of a memory-intensive algorithm like ethereum. After reducing the core clock of a couple cards from 1050 to 1025, the rigs were stable. When trying out nanopool, I was getting a lot of "ERROR fetching work;" and "Error submitting solution - Share rejected" messages. I think their servers may have been getting overloaded, as it worked fine when I switched to siamining.com. I also find siamining.com has more detailed stats, in particular % of rejected shares (all below 0.5% for me).
I may end up switching back to eth in the near future, since a doubling in network hashare for sia will eventually mean a doubling of the difficulty, cutting the amount of sia mined in half. In the process I'll at least have learned a bit about golang, and I can easily switch between eth and sia when one is more profitable than the other.
About Me
I have four children, two step-children, and a grand-daughter. Wife 1.0 was a failure, but as with many nerd projects, version 2.0 is a big improvement. :-) As an adult I was diagnosed with ADHD and Asperger's. gmail: ralphdoncaster |
Q:
Need help parsing php date using moment.js
My PHP script is giving me following date:
"Friday, 31-Mar-17 07:45:47 UTC"
I want to parse this date using moment.js and I want to store each parsed token into separate JavaScript variable. How can I do this in moment.js?
A:
Simply use moment(String, String) to parse it. Then you can use format() or getters to get each parsed token:
var m = moment.utc("Friday, 31-Mar-17 07:45:47 UTC", "dddd, DD-MMM-YY HH:mm:ss Z");
console.log(m.format());
var dayOfWeek = m.format('dddd'); // Friday
var dayOfMonth = m.date(); // 31
var shortMonthName = m.format('MMM'); // Mar
var shortYear = m.format('YY'); // 17
var minutes = m.minutes(); // 45
console.log(dayOfWeek, dayOfMonth, shortMonthName, shortYear, minutes);
<script src="https://cdnjs.cloudflare.com/ajax/libs/moment.js/2.18.1/moment.min.js"></script>
|
Folate levels in human liver from autopsies in Canada.
Liver folate is considered to be a direct measure of body folate stores. Specimens of 560 livers were therefore collected during autopsies at hospitals in seven cities across Canada, and analyzed for "total" folate. The values obtained ranged from 2.7 to 15.6 micrograms/g. Stillborn infants had the lowest mean liver folate levels (5.9 microgram/g). Mean liver folate levels tended to increase from birth to a peak between 11 to 30 years (8.8 micrograms/g), and then decreased with increasing age. This trend was reflected in an increased proportion (6 to 15%) of folate levels in the 3.1 to 5.0 microgram/g range in older subjects. Liver folate levels of male subjects did not differ from those of females. Mean liver levels tended to be slightly higher, and the number of liver levels between 3.1 to 5.0 micrograms/g was lower, in subjects dying accidentally (7.8%) and from heart and coronary disease (8.7%), than in those dying from cancer, respiratory diseases or other causes (11.7 to 15.3%). Only two subjects had liver folate levels below 3 microgram/g. In this survey, there was thus little evidence of severe folate deficiency. |
Influence of self-reported work conditions and health on full, partial and no return to work after long-term sickness absence.
This study aimed at describing the frequency of full, partial, and no return to work after long-term sick leave and at ascertaining the influence of psychosocial work conditions, work ability and health, reported before the onset of sick leave, on full and partial return to work. Altogether 853 public-sector employees in Sweden, mainly women, with at least one sick leave lasting > or = 28 days, were studied. The outcome was the level of sick leave 2 years after the sick leave began. Potential predictors were self-rated health, work ability, and psychosocial work conditions assessed by questionnaire before the sick leave. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated by multinomial regression analyses. Altogether 41% of the participants went directly from full sick leave to full return to work; 21% had periods of partial return to work, but, at the 2-year follow-up, were fully back to work; 15% had partial return to work; and 23% were still not working. A relaxed work situation, a combination of low demands and high decision latitude, increased the odds for full (OR 2.72, 95% CI 1.60-4.62) and partial (OR 2.42, 95% CI 1.21-4.85) return to work. Negative consequences of organizational changes were associated with decreased odds for full return to work (OR 0.54, 95% CI 0.38-0.77). Good self-rated health and work ability were associated with full return to work. Partial return to work often precedes full return to work, but also operates as a long-term solution for remaining occupationally active. Promoting relatively low demands and high decision latitude at work may support both full and partial return to work after long sick leaves. |
**What was known?**
Leishmaniasis recidiva cutis is an unusual form of cutaneous leishmaniasis.
Introduction {#sec1-1}
============
Cutaneous leishmaniasis (CL) is a protozoan disease caused by the protozoa Leishmania subtypes and transmitted by the bite of an infected female sand-fly. Leishmaniasis recidiva cutis (LRC) is an unusual form of CL. It appears after a variable period of time (from months to years) in the same area as, or very close to the old scar of, a previous acute lesion healed apparently.\[[@ref1]\]
Herein, we present a case of a large and horrifying example of LRC of the lips mimicking granulomatous cheilitis.
Case Report {#sec1-2}
===========
An 8-year-old, healthy, Syrian boy was admitted to our outpatient clinic of dermatology with a swelling and disfigurement of his lips existing for the last 4 years. The initial lesion had started as asymptomatic papules, 0.5-1 cm in diameter on cheek and lip when he was two years old. After the treatment with intralesional meglumine antimoniate (Glucantime^(R)^) in Syria at that time, the lesions disappeared; however, cribriform scarring on the lip and cheek remained. Two years later, new lesions began as papules, 1 cm in diameter around the cribriform scars on the lips and cheeks. Dermatologic examination showed the presence of a severe swelling and purulent draining, crusty infiltrated granulomatous plaques on the lower and upper lips. A 5 cm cribriform scarring and 1 cm papule on this scar were seen on the left cheek \[Figures [1a](#F1){ref-type="fig"} and [b](#F1){ref-type="fig"}\]. General physical examination did not show any pathological findings. Regional lymph nodes were not involved. Laboratory tests revealed Hb 10.3 g/dl (12.2-18.1 g/dl), Hct 30.5% (37.7-53.7%), sedimentation rate 70 mm/h (0-12 mm/h), C reactive protein 42.4 mg/dl (0-5 mg/dl). Abundant intra and extracellular Leishmania amastigotes were determined in the smear prepared from the lesion with Giemsa stain \[[Figure 2](#F2){ref-type="fig"}\]. Histopathology showed foamy histiocytes and leishmania parasites within the cytoplasm of macrophages in the epidermis and a dense dermal mixed type inflammatory cell infiltrate composed of lymphocytes and foamy histiocytes with multinucleated giant cells \[[Figure 3](#F3){ref-type="fig"}\]. On the basis of these findings, LRC was diagnosed. The patient was treated with 20 mg/kg/day systemic meglumine antimoniate intramuscularly, for 20 days. Because the healing was slow, he was also prescribed oral fluconazole 5 mg/kg/day for 4 weeks. Cryotherapy was applied to the residual papular lesions. The lesion improved markedly at the first month of the treatment \[Figures [1c](#F1){ref-type="fig"} and [d](#F1){ref-type="fig"}\].
{#F1}
{#F2}
{#F3}
Discussion {#sec1-3}
==========
In LRC, the actual cause of reactivation of the disease is unclear; insufficient treatment or the use of non-effective drugs may be possible causes of recurrence, but the most identified theory is a defect in the T-lymphocyte activation by the protozoa and the inability of the macrophages to kill all amastigotes.\[[@ref1][@ref2]\] Clinically, differential diagnosis includes lupus vulgaris, bacterial infections, pseudolymphoma and squamous cell carcinoma and when the lesions found on the lips syphilitic chancre, granulomatous cheilitis, Melkersson-Rosenthal syndrome and foreign body granuloma should be considered.\[[@ref1][@ref3]\] The treatment for LRC includes systemic therapy with pentavalent antimony, alone or in combination with allopurinol, and amphotericin B, and local therapy with intralesional antimonials, cryosurgery, or excision.\[[@ref2]\] In children, fluconazole may represent an effective and well-tolerated therapy.\[[@ref4]\]
Few cases of LRC have been reported previously.\[[@ref1][@ref5][@ref6][@ref7][@ref8][@ref9]\] These lesions can have variable clinical appearance. To our knowledge, lips of LRC mimicking granulomatous cheilitis have not been reported so far. The difference of our case from other cases is that our patient is a child, he had a very giant lesion and lesion was located on the lip. We consider there is unusually abundant amastigotes because of unavailability of a sufficient therapy due to existing social situations.
In summary, we want to report a case with a large and horrifying example of LRC of the lips mimicking granulomatous cheilitis. The absence of PCR examination is a limitation of our study. Considering the different forms of the disease nature, any unusual skin lesion located on the lips in an endemic area should always be investigated for CL and thus, clinicians should have a high level of suspicion to make a diagnosis of LRC and an appropriate treatment.
**What is new?**
Considering the different forms of the disease nature, any unusual skin lesion in an endemic area should always be investigated for cutaneous leishmaniasis.Clinicians should have a high level of suspicion to make a diagnosis of leishmaniasis recidiva cutis and an appropriate treatment.
**Source of support:** Nil
**Conflict of Interest:** Nil.
|
Q:
Invoking native code with hand-written assembly
I'm trying to call a native function from a managed assembly. I've done this on pre-compiled libraries and everything has went well. At this moment I'm building my own library, and I can't get this work.
The native DLL source is the following:
#define DERM_SIMD_EXPORT __declspec(dllexport)
#define DERM_SIMD_API __cdecl
extern "C" {
DERM_SIMD_EXPORT void DERM_SIMD_API Matrix4x4_Multiply_SSE(float *result, float *left, float *right);
}
void DERM_SIMD_API Matrix4x4_Multiply_SSE(float *result, float *left, float *right) {
__asm {
....
}
}
Hereafter we have the managed code which loads the library and create a delegate from a function pointer.
public unsafe class Simd
{
[UnmanagedFunctionPointer(CallingConvention.Cdecl)]
public delegate void MatrixMultiplyDelegate(float* result, float* left, float* right);
public static MatrixMultiplyDelegate MatrixMultiply;
public static void LoadSimdExtensions()
{
string assemblyPath = "Derm.Simd.dll";
IntPtr address = GetProcAddress.GetAddress(assemblyPath, "Matrix4x4_Multiply_SSE");
if (address != IntPtr.Zero) {
MatrixMultiply = (MatrixMultiplyDelegate)Marshal.GetDelegateForFunctionPointer(address, typeof(MatrixMultiplyDelegate));
}
}
}
Using the sources above the code runs without errors (the function pointer is obtained, and the delegate is actually created.
The problem raises when I call the delegate: it is executed (and I can debug it also!), but at function exit the managed application raises a System.ExecutionEngineException (when it doesn't exit without exceptions).
The actual problem is the function implementation: it contains a asm block with SSE instructions; if I remove the asm block, the code works perfectly.
I suspect I am missing some registry save/restore assembly, but I'm completly ignorant on this side.
The strange thing is that if I change the calling convention to __stdcall, the debug version "seems" to work, while the release version behave as if __cdecl calling convetion was used.
(And just because here we are, can you clarify if the calling convetion matters?)
Ok, thank to the David Heffernan comment I find out that the bad instructions causing the problem are the following:
movups result[ 0], xmm4;
movups result[16], xmm5;
movups instructions moves 16 bytes into (unaligned) memory.
The function is called by the following code:
unsafe {
float* prodFix = (float*)prod.MatrixBuffer.AlignedBuffer.ToPointer();
float* m1Fix = (float*)m2.MatrixBuffer.AlignedBuffer.ToPointer();
float* m2Fix = (float*)m1.MatrixBuffer.AlignedBuffer.ToPointer();
if (Simd.Simd.MatrixMultiply == null) {
// ... unsafe C# code
} else {
Simd.Simd.MatrixMultiply(prodFix, m1Fix, m2Fix);
}
}
Where MatrixBuffer is a class of mine; its member AlignedBuffer is allocated in the following way:
// Allocate unmanaged buffer
mUnmanagedBuffer = Marshal.AllocHGlobal(new IntPtr((long)(size + alignment - 1)));
// Align buffer pointer
long misalignment = mUnmanagedBuffer.ToInt64() % alignment;
if (misalignment != 0)
mAlignedBuffer = new IntPtr(mUnmanagedBuffer.ToInt64() + misalignment);
else
mAlignedBuffer = mUnmanagedBuffer;
Maybe the error is caused by Marshal.AllocHGlobal or IntPtr black magic?
This is the minimal source to spot the error:
void Matrix4x4_Multiply_SSE(float *result, float *left, float *right)
{
__asm {
movups xmm0, right[ 0];
movups result, xmm0;
}
}
int main(int argc, char *argv[])
{
float r0[16];
float m1[16], m2[16];
m1[ 0] = 1.0f; m1[ 4] = 0.0f; m1[ 8] = 0.0f; m1[12] = 0.0f;
m1[ 1] = 0.0f; m1[ 5] = 1.0f; m1[ 9] = 0.0f; m1[13] = 0.0f;
m1[ 2] = 0.0f; m1[ 6] = 0.0f; m1[10] = 1.0f; m1[14] = 0.0f;
m1[ 3] = 0.0f; m1[ 7] = 0.0f; m1[11] = 0.0f; m1[15] = 1.0f;
m2[ 0] = 1.0f; m2[ 4] = 0.0f; m2[ 8] = 0.0f; m2[12] = 0.0f;
m2[ 1] = 0.0f; m2[ 5] = 1.0f; m2[ 9] = 0.0f; m2[13] = 0.0f;
m2[ 2] = 0.0f; m2[ 6] = 0.0f; m2[10] = 1.0f; m2[14] = 0.0f;
m2[ 3] = 0.0f; m2[ 7] = 0.0f; m2[11] = 0.0f; m2[15] = 1.0f;
r0[ 0] = 0.0f; r0[ 4] = 0.0f; r0[ 8] = 0.0f; r0[12] = 0.0f;
r0[ 1] = 0.0f; r0[ 5] = 0.0f; r0[ 9] = 0.0f; r0[13] = 0.0f;
r0[ 2] = 0.0f; r0[ 6] = 0.0f; r0[10] = 0.0f; r0[14] = 0.0f;
r0[ 3] = 0.0f; r0[ 7] = 0.0f; r0[11] = 0.0f; r0[15] = 0.0f;
Matrix4x4_Multiply_SSE(r0, m1, m2);
Matrix4x4_Multiply_SSE(r0, m1, m2);
return (0);
}
Pratically after the second movups, the stack changes the result value (stored on the stack), and stores the values of xmm0 on the modified (and wrong) address stored in result.
After having stepped out from *Matrix4x4_Multiply_SSE*, the original memory isn't modified.
What am I missing?
A:
You assembly was flawed. There is a difference between
void DoSomething(int *x)
{
__asm
{
mov x[0], 10 // wrong
mov [x], 10 // also wrong
mov esi,x // first get address
mov [esi],500 // then assign - correct
}
}
The first two examples did not write to the memory location pointed to the pointer but to the storage location of the pointer itself. Since the parameter comes from the stack you did overwrite with the movups instruction your stack. You can see this in the debugger window when you call e.g.
int x=0;
DoSomething(&x);
With mov [x],10 you do not set x to 10 but you write into your stack.
|
Media playback is unsupported on your device Media caption Mark Little pays tribute to Vivean Gray, who played his mother Mrs Mangel in Neighbours.
Vivean Gray, best known for playing the gossipy Mrs Mangel in Australian soap opera Neighbours, has died aged 92.
Born Jean Vivra Gray in 1924, the Cleethorpes native moved to Australia in 1952 to pursue an acting career.
She went on to appear in shows such as The Sullivans and Prisoner Cell Block H, before landing her best-known role as Ramsay Street's resident busybody.
Mark Little, who played her on-screen son Joe, said he had been "privileged to know and work with her".
"We laughed a lot creating The Mangel Dynasty," the comedian and actor said on Twitter.
Image copyright @themarklittle/Twitter
Speaking on BBC Radio 5 live, Little said Gray had played a "nosy parker" on Neighbours "so, so well".
"She was only in the show for two years but she made such an impact."
Gray played Nell Mangel in more than 250 episodes of Neighbours from 1986 to 1988.
She gave up acting after the show, complaining of the verbal attacks she received from people who could not distinguish her from her on-screen character.
"I could barely set foot outside my own door without someone screaming abuse at horrid old Mrs Mangel," she was quoted as saying in 1989.
Image copyright Rex Features Image caption The actress, pictured in 1980, was born in Cleethorpes in Lincolnshire
"People didn't seem to appreciate it was acting. So I decided to take a break."
"One of the sadnesses of her life was that she was basically hounded out of existence," said Little.
"She came back to England and hid, basically."
'Soap legend'
Gray's other roles included a schoolteacher in Peter Weir's 1975 film Picnic at Hanging Rock.
She worked with the director again on his 1977 film The Last Wave, in which she played an expert in Aboriginal history.
"Vivean's contribution to Australian drama will never be forgotten. It is a very sad day for the Neighbours family," said Rick Maier of Australia's Network Ten, which broadcasts the soap.
"Mrs Mangel was the ultimate busybody, remembered for her conservatism and her caustic wit," said Neighbours' executive producer Jason Herbison, describing Gray as "a true soap legend".
Follow us on Twitter @BBCNewsEnts, on Instagram at bbcnewsents, or email entertainment.news@bbc.co.uk. |
"Oh, Rick had cleaned up." "He was back on track." "A photo like this gets into the hands of the authorities, that's strike three." "You write up a new contract and allocate it all to me." "I found an old stripper well on miss Henderson's land." "If I deepen it a little, I'll hit another oil reservoir." "I know you had feelings for John Ross." "What do you say you and I make a new deal?" "$20,000?" "I can't risk you coming back in a few years after I've built a company and trying to claim ownership." "It's about time we had a little talk..." "About that e-mail you sent Elena." "There's something I need to tell you..." "About..." "The e-mail Elena got two years ago." "The one telling her..." "You weren't showing up for the wedding." "How do you even know about that?" "I love you, Christopher, more than anything." "The last thing I want to do is hurt you." "But John Ross is blackmailing me." "Three years ago, Tommy moved to Dallas." "His roommate knew you." "Knew about you and Elena." "All I knew was that you were gonna be on the train!" "That you had just broken up with someone." "But I didn't know." "I swear I didn't know what Tommy had done." "And what did Tommy do?" "He sent the e-mail to Elena and made it look like it had come from your computer." "I swear I didn't know!" "I didn't know he did it." "I didn't know until yesterday when John Ross confronted me." "You need to believe that." "You need to believe I love you." "Wait." "♪ Theme music plays ♪" "Hey, Tommy!" "No!" "No!" "Christopher, stop!" "Stop!" "No, no, no." "Stop!" "Stop!" "Please." "Stop." "What the hell did I do to you?" "!" "I had to tell him, Tommy." "He knows you sent the e-mail to Elena breaking them up." "You did what?" "!" "It was a scam!" "The both of them!" "For our money." "No, don't blame it on Becca." "It was all me." "I'm sorry, man." "Why?" "!" "You son..." "no, no, no!" "How could you do that to him?" "!" "Use us all like this." "I just did." "You son of a bitch!" "Chad!" "Get this punk off my ranch." "Christopher!" "I love you." "I never would've taken your money." "You've got to believe that." "You've got to believe me!" "Just believe me." "Make sure I never see you again." "I'm sorry I didn't believe you." "I shouldn't have accused you of sending that e-mail." "I just need to know you're not gonna chase after him." "He offered to pay me off." "He said I had no integrity." "Christopher and I are done." "All right." "Morning." "Bueno." "Now I can do something." "Mr. Bobby won't let me help him." "I think it's rude." "Just trying to stay busy." "No word from Christopher?" "No." "I'm sure he's okay." "Can you imagine?" "Ugh." "And that brother." "I never liked him." "I hate myself for trusting him." "And her too." "Yes." "Of course." "She just looked so devastated when she left." "I mean, you think it's possible that the whole thing was an act?" "Everything?" "Honey, it doesn't matter." "I left word for Mitch Lobell." "See if there's anything we can do legally about Rebecca and her brother." "I just hope Christopher's okay." "Hey, there." "Can I help ya?" "Robert James ewing?" "Yeah, that's me." "What do you got?" "Thank you." "What is it?" "It's from Lobell." "It's probably about the sale of the ranch." "Mm." "What's wrong?" "J.R." "Southfork's deed is in your name." "Is this some kind of joke?" "No, Bobby." "It's not a joke." "Say, bum, give Bobby and me a couple of minutes, will you?" "Now, Bobby, let me explain, before you get your boxers in a knot." "Oh, I am gonna want to hear this story." "You know, I always felt a little funny about that Marta del sol girl, ever since I saw her at the cattle baron's ball." "Daddy always said beautiful women were the most dangerous." "I know all the things daddy used to say." "Well, a couple of weeks ago," "I heard she was planning on selling Southfork to cliff Barnes." "I was surprised she could put it right back on the market after buying it from you." "But, hell, I figured that was just the deal you and Lobell made with her." "That wasn't the deal, J.R., and you know it." "Well, Bobby kept me out of the deal." "So, no, I don't know it, darlin'." "When I heard that vulture Barnes was trying to steal Southfork, get his hands on the ranch and all the oil my boy found under it," "I got a group of investors together and swooped in and bought it from del sol." "Well, I don't think cliff Barnes knew what hit him." "And I guess maybe neither did you." "Now she's mine." "I'm gonna start sinking more Wells as soon as I can." "Ewing oil is back in business, Bobby." "That's not gonna happen, J.R." "This is not gonna happen." "That's why I wanted to sell Southfork in the first place... to stop all this feuding and leave a legacy to mama." "You want to carry on mama's legacy?" "Well, I want to carry on daddy's." "I'm taking back what should have been mine in the first place." "That's how you justify this?" "How twisted can you get?" "Now, why don't you settle down and accept what's what?" "The deed is real, and this place is mine." "But you can stay here as long as you like, Bobby." "We're family." "I'm gonna make this right." "And I'm gonna take you down..." "Brother." "Always did get a little hot under the collar when he didn't get his way." "But he'll come around." "You'll see." "They warned me, my whole marriage, they told me about you." "But in my wildest imagination," "I never thought you could stoop to this." "Well, Annie, you're just gonna have to work on your imagination." "Bobby!" "Where are you going?" "Lobell's." "Dad?" "Dad!" "What happened?" "J.R. Stole Southfork." "He's gonna drill." "He'd what?" "You heard me." "I can only imagine John Ross is deep in this, too." "Dad, wait!" "Wait!" "Wait." "I'm coming with you." "Oh, my God." "Son of a bitch!" "Lew, thanks for helping." "Yeah, I'm sorry about all this, Bobby." "It's devastating what Lobell has done." "I've known him for a long time, and I pegged him as a good guy." "Well, I worked with him for 30 years." "I want to break his neck." "I would, too, son." "So, how bad is it?" "Well, my team and I have gone through the files you brought." "I spoke with Carlos del sol and Dallas p.D." "Here's where things stand right now." "The del sol conservancy was never really in this deal..." "Bobby." "Lobell lied to you about all of it." "All his due diligence was a fraud, as was the woman pretending to be Marta del sol." "Dallas p.D. Has an apb out for her arrest, but they're pessimistic without her real identity." "Her cellphone number and her address were bogus." "Even the moneys that were exchanged were done through untraceable accounts offshore." "Police think it's likely she's left the country, and the same is true for Lobell and his family." "Then you can prove it was a fraud." "Just undo the deal and put the deed back in my name." "It's not that simple, Bobby." "How is it not that simple?" "Son, just a minute." "No jury is gonna doubt what went on here." "The sale of Southfork to Marta del sol was a fraud." "But it was also a bona fide sale." "Your father was paid the contract price and cashed the check." "Then I'll give the money back." "You can't, Bobby, because of the second sale to J.R." "He was a third-party purchaser for value, which means the sale of Southfork to him was valid..." "And cannot be undone unless we can prove he was involved in or knew about the fraud of the first deal." "And how the hell am I supposed to do that, lew?" "Well, first things first." "We'll need all the private records you have on the deal." "And berman here will sit you down and take you through an oral timeline." "You're just lucky I was smart." "Getting all this stuff before you blew us up." "I told you, John Ross was gonna tell Christopher." "If there was a chance of Chris believing me," "I had to tell him myself." "Well, that worked real well." "Do you really think I wanted him to hate me?" "Exactly the opposite." "At least I got us access to Christopher's computer." "So we can track his progress with the methane extraction." "And when that works, there's gonna be a lot of people we can sell those plans to." "And all these files that I copied... there's got to be something in them we can monetize." "Don't you think?" "Yeah." "Hey." "I put two years into this." "I plan on coming out a millionaire." "So you better pull yourself together and start helping me find something." "You're hurting me." "Don't forget we're in this together..." "For the long haul." "If I'd have known you were coming I'd have got up earlier." "What have you done, John Ross?" "Southfork!" "Your daddy told Bobby this morning that it's his, that he's drilling section 18." "My daddy did what?" "Don't play games, John Ross." "The deed's in J.R.'S name." "That's impossible." "I thought you let it go." "That we were moving forward with drilling the Henderson well." "J.R. And Bobby are at war!" "They think you're a part of this, too." "I got to talk to my father." "That's all you're gonna say, really?" "You're not even gonna deny it?" "I don't know what's going on!" "All right?" "!" "I was just starting to trust you again." "This is J.R. Ewing." "After the beep, tell me what you know." "P" "J.R.?" "Open this door!" "J.R.!" "He left, Bobby." "An hour ago." "Oh, damn it." "He's already got Ryland tankers headed to section 18." "Well, what did the lawyer say?" "Ha!" "Not only did Lobell hang me out to dry, but if I want to get Southfork back," "I have to prove J.R. Was in on the fraud, or at least he knew about it." "So they're interviewing everybody they can think of." "They're gonna subpoena his bank records and his phone records and even the visitor's log at the nursing home." "But I know my brother pretty well, and he's covered his tracks." "Well, what about Marta del sol?" "Oh, well, actually, she wasn't Marta del Sol, and the police are looking for her." "We need to start fighting fire with fire." "Dad, we need to start dealing with the same type of people they do to find what we need to bring them down." "We are not breaking the law to fix this, son." "J.R. And my cousin are gonna be pumping by the end of the week!" "If not sooner!" "We need to do more, and we need to do it now!" "I'm going to fight this with everything I've got," "Christopher, but I'm not doing business with crooks." "That's not who we are." "Maybe not you." "Son..." "The whole point of selling Southfork in the first place was to stop this family's descent." "I'm not going to let us lower ourself any further." "Look what it's already cost us." "Look at what Rebecca and Tommy's lies have done to you." "I am done sitting around while people walk all over me in this family!" "So am I!" "I have fought J.R. Before, son, and I beat him." "And I'll beat him here..." "my way." "No." "We are doing this the right way." "Honey, you can have everybody just start unpacking, because we are not going anywhere." "Okay." "Your father isn't here." "I don't know where he is." "Tell me it isn't true." "You tell me you did not cut me out of this deal and put Southfork into my father's name!" "You need to discuss that with J.R." "No, I brought you into this deal." "How could you turn around and give all of Southfork to J.R.?" "!" "I warned you not to mix business with pleasure." "I gave you chance after chance." "What the hell are you talking about?" "It was taken yesterday." "You had me followed." "Your father did." "I guess he couldn't trust you, either." "You said you and Elena were broken up." "You lied to me." "You lied to me about where you were the other day, and you blew me off to be with her yesterday." "You hurt me." "I warned you." "I warned you not to toy with me." "I warned you not to confuse our business dealings by getting involved." "You let me fall for you." "I could have explained... with another lie." "That picture's pretty obvious." "You undo what you did." "You put my name back on that deed." "It's too late." "Besides, it was a much better business deal anyway." "I should tell the cops where you are." "Who you are." "And lose any claim you have to Southfork by blowing up your father's deal?" "You stay the hell away from me." "If you know what's good for you." "I need you to be certain..." "Because there's no turning back." "I've never met anyone like you." "We're two of a kind." "Are you sure you won't mix business with pleasure?" "What the hell did we do last night?" "Oh, I remember you like it rough, huh?" "John Ross." "I'm your daddy's friend, bum." "You're the son of a bitch who took those pictures." "I ought to kill you." "J.R. Asked me to come get you." "May you have as strong a second half as you had the first, Jerry." "Good to have you back in form, J.R." "And I appreciate you making sure everybody knows it." "I'll let you be alone with your boy." "Good to see you, John Ross." "You've got quite a father there." "I'll say." "Marta called." "Trying to know where your head is at." "A little word of advice... you ought to be a lot nicer to that girl." "She's a bit of a loose Cannon." "I should call the police." "Have you arrested for what you did." "There's no reason for that." "The deed to Southfork is still yours for the having, John Ross." "It's just a matter of time." "You and I were supposed to be partners... now." "That was the deal." "The deal was to teach you the oil business." "And I am." "I'm leaving town in a little while." "I'm putting you in charge." "I'm throwing you into the deep end just like my daddy did to me." "I'm gonna see if you can swim." "Or sink." "I don't need to be tested." "I was ready to do this all by myself." "Well, now if that were true, you wouldn't have had the deal stolen out from under you." "Here's my power of attorney so you can run things." "And bum's staying behind." "If you get into trouble, he can be resourceful, which I think you know." "Just start drilling." "By the time I'm back," "Bobby will have gotten used to oil being pumped on Southfork and the damage will be done." "Just don't screw it up." "Jerry Jones and the cowboys organization is proud to welcome J.R. Ewing to the cowboys stadium." "Smile for the camera." "Let's all give him a big Texas-size welcome." "Oh, I'm so glad you picked up." "I was terrified you wouldn't." "What do you want, Rebecca?" "I'm so sorry, Ann." "You were so nice to me." "You made me feel like family." "I didn't mean to... once you started the lie, there was only one way out." "Can I see you?" "Explain it all to you in person?" "So you can look at me in the eye and see that I am not as horrible as you think I am." "Please, Ann." "Please." "I really appreciate the money, Mr. ewing." "I barely get tips around here, even at Christmas." "Hey, I just appreciate you doing my cousin a solid." "John Ross would be freaking out if I wasn't able to pick up this oil lease report that he left here." "Well, there she is." "I'll wait here for you." "You know what?" "Uh, it might take me a while to find it." "I wouldn't want you to get in trouble for..." "Not Manning the front door." "I'll lock up on my way out." "Yeah, I-I should really wait, I think." "You know, I've already put you out enough already." "It's fine." "Trust me." "Marta oh, I remember you like it rough, huh?" "You like it rough..." "Veronica." "I'm sorry it had to be done like this." "But I'm counting on you." "Make your daddy proud." "Where are you goin'?" "Well, what fun would I get out of telling you that?" "Besides, what you don't know, you can't tell." "When are you coming back?" "That depends on you, son." "There you go." "My father said you weren't moving out." "I'm glad." "Don't you come in this house and be smart with me, young man." "I'll toss you right out that door." "I'm in the mood." "Besides..." "Your dad is not here." "I know." "Will you give my Uncle and I a minute, please?" "I don't want any trouble with you, Uncle Bobby." "Now, I know we've had our differences in the past, but what's gone on here is between you and my father." "I ain't got nothing to do with it." "You really expect me to believe that." "All you've ever wanted is to drill on this ranch." "You see my name anywhere on my father's deed?" "If you ask me, though, you should have let me drill when Elena and I asked." "If you did, I don't think my father would have been pushed to this." "You should've known J.R. Wouldn't let a billion barrels of oil slip through his hands." "Yeah, well, I am gonna fight him on that." "And I think that hurt him." "One of the reasons why he left Dallas this afternoon." "J.R. Left?" "I just saw him go." "He's put me in a real uncomfortable situation." "J.R. Gave you his power of attorney." "Well, the two of you must really be enjoying this little joke." "Oh, I assure you it is no joke." "At my father's request, I'm moving in to Southfork to keep an eye on things in his absence." "Into his suite, of course." "I'm gonna start pumping section 18 as soon as I can." "Now, I know you're gonna have a problem with that." "But I'm hoping you and I can stay out of each other's way." "The last thing I'd want to do is have to kick you off this ranch." "Like you did to me." "♪ Music plays ♪" "I keep thinking if I could go back, make better choices..." "Meet Christopher under different circumstances." "The funny thing is..." "I'd probably have never met him if things had been different." "You're young, Rebecca." "You make mistakes when you're young." "It doesn't mean you can't change." "Move beyond this in time." "You really believe that?" "I've had to." "I keep letting myself fall into bad patterns." "Letting myself be swayed by my brother." "Your choices are yours, not his." "I know." "The thing is..." "I don't know what he would do..." "If he felt that I didn't have his back." "Where's Tommy now?" "Trying to figure out his next move." "And you?" "Trying to figure out a way to tell him to go to hell." "Maybe the first step to that is coming clean with Christopher." "Do you think Christopher could ever forgive me?" "It's more than forgiveness." "It's trust." "And somehow proving to him that you're worthy of a second chance." "No..." "I heard you this morning." "Now, hear me out." "I spoke to my father." "He did what you said." "Southfork, the oil, it's all in his name." "He left town." "He wants me to do his bidding until he gets back." "Till Uncle Bobby settles down." "He thinks it will be better this way." "Well, I guess you got what you wanted." "I wanted it." "But not this way." "He's put my head in the cross hairs doing things his way." "And he has left me..." "alone." "I never wanted to do it alone." "I wanted to do it with you." "Trust me." "I feel the same way about J.R." "I've spent my whole life trying not to be his son." "But this place..." "That oil..." "It's my birthright." "And now that it's all on me," "I got to do whatever I can to make it work." "But Bobby doesn't deserve... no, Bobby was hurt by my father, not me." "Not you." "Bobby also wanted to give away Southfork." "All of it." "I cannot say that I'm sorry that my father stopped him from doing that." "Uh..." "You have accused me of awful things that I did not do." "And yet I'm still here, at your door..." "Asking you to take a chance..." "On me." "Asking you to do what we always said we would do..." "Together." "I can't be a part of that." "You can't be a part of that..." "Or a part of me?" "I don't know." "J.R. Thought he could get out of all of this by leaving town, so he put John Ross in charge." "I'm just afraid he's gonna tear up this whole ranch digging for oil before he's done." "I did this, Annie, by rushing it." "I tried to make things better, and I made them worse." "What the hell was I thinking?" "You were shouldering everything yourself." "Especially the cancer." "And you didn't make this bad." "J.R. Did." "You are the strongest man I know." "You will fix this." "Yeah, but they're gonna start pumping before I figure this out." "And I promised mama that wouldn't happen." "I've got to find a way to slow him down." "You will figure this out." "I just wish I was as certain as you." "Elena!" "Open the door!" "Elena!" "Please?" "You're drunk, Christopher." "You need to go home." "I'm sorry." "I'm sorry for what I said the other day." "I'm just..." "I'm sorry." "Oh, God." "Okay, come on." "Come in." "You helped me last night?" "You could barely make it to the couch, let alone the house." "Here." "Drink this." "Then get out." "I-I thought that... you thought because I let you sleep on my couch that I forgive you?" "I let you sleep there because it was the decent thing to do." "You could learn something about decency, Christopher." "I'm sorry." "You know exactly what I'm insecure about, what hurts me, and still you went there." "I'm sorry about Rebecca, but don't come here looking to make peace." "To think that I let you put in my head that it was John Ross this whole time when that's what you think." "That's exactly how you think of me." "John Ross?" "!" "Do you have any idea what he's up to?" "Whatever you think, more than anyone he has always had my back." "And he has never, ever..." "Made me feel bad..." "The way you made me feel the other day." "Mr. Ryland?" "Your ex-wife is here to see you." "Thank you for seeing me, Harris." "Why don't you come over here and give me a hug?" "I don't think that's necessary." "Oh, well, I do." "You know, I expected you to sit up on your high horse for at least a couple more minutes before you caved." "Especially since, as I remember," "I make your skin crawl." "Of course you always were prone to hyperbole." "I don't want to fight with you." "That's pretty obvious, Annie." "You're here for a favor." "Why would you want to start a fight?" "You have a contract with J.R. Ewing to transport 2,000 barrels a day from his well on Southfork." "I want you to cancel the contract." "I'm the only one in Dallas with enough trucks to move that kind of oil." "If J.R. Can't move his oil, then he's got to stop drilling." "Is that the endgame you're looking after?" "You can just nod your head if I'm hitting the mark." "After all these years, I can still read your mind." "Forget it." "After all we've been through," "I thought you might have changed." "Oh, people don't." "Now, maybe the clothes we wear, the size of one's heels." "It's nice to see you're not afraid to be wearing them anymore, by the way." "But the soul?" "I think that stays pretty constant." "Otherwise you wouldn't be here." "I'll do it." "I like your husband." "And I always thought his brother was a prick." "So I'll pull my trucks." "It's gonna cost you a hug." "Saw the completion rig outside." "Crew already gone for the day?" "I thought you were in a hurry." "Well, they finished the casing faster than expected." "It still needs to dry, so I broke them early." "I'm a good boss, remember?" "Never met a crew that didn't like you." "A tycoon who gets his hands dirty... who can resist that?" "So, you in this with me?" "I thought Bobby was wrong to not let us drill." "We were never gonna ruin the land." "But I can't drill on Southfork." "I can't be in the middle of that war." "I'll work my leases on the Henderson land." "Maybe give you a run for your money." "You're an independent woman." "I like that." "I understand why you're doing this." "It's your birthright." "You have to prove yourself to your father." "But promise me that you had nothing to do with stealing Southfork." "That it was all J.R." "Promise me you're the man I think you are." "I swear..." "None of it was me." "Oh, you pulled yourself together." "Yeah." "I'm feeling better about things." "Good." "Find anything yet?" "Hmm." "Doorman let me in." "He's under the impression the two of us are close." "What are you doing here?" "Got something to show you." "Come on, John Ross." "You know what I like." "Don't you say?" "I remember you liked it rough." "Where'd you get this?" "Where do you think?" "She left it for you." "Here." "I especially like the part where you call Marta by her real name..." "Veronica." "Proves that you knew who she was before the deal was made." "That you were conspiring against my father." "So what?" "The deed's not in my name." "Show it to the police, your father..." "It's not gonna help you get Southfork back." "Or are you just gonna hold it over my head with Elena?" "You'd do anything to get her to hate me." "I wouldn't show her that." "I wouldn't do that to her." "Might not undo the deal, but fraud and conspiracy..." "Are still a crime in the state of Texas." "I want proof..." "From you that J.R. Was in on the fraud." "Enough proof to undo the deal." "Or you're going to jail." |
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XBTfreelancer: Employers currently posing include coinbase with other hosts of blogging, development, marketing etc. Employers pay freelancers using bitcoin as a means of payment.
Cryptocurrencies are uniquely designated for its online security and anonymity which physical currencies lack in most cases. The word ‘cryptocurrency’ was borne out of the word term cryptography, a term that describes the code used by the internet to track purchases and transfers.
The history of cryptography itself began during the Second World War as there was a desire to create security of communication. In this digital era, it has taken its own different shape, making use of computer science and maths theory to become a means of securing information, communication and money on the internet. While the preconception of cryptocurrency began in 2008 by Satoshi Nakamoto and was finally implemented in 2009 as bitcoin, it continued to encounter a constant rise and fall in value as compared to the US Dollars. Although it didn’t gain an initial credit or a wide base investors but it went into a gradual but steady climb into gaining popularity and was harnessed by investors and its customer base skyrocketed in early 2011.
The first ever transaction with bitcoin was valued at 10,000 bitcoin which was used to make a purchase of two pizzas. It was after a frequent rise and fall in the value of bitcoin that it rose gradually to over $1000 in august, and it continued to rise up till November 2017 and has settled at a value of over $7000, causing a stir in the currency market.
The cryptocurrency is one very huge aspect of digitization and in fact a breakthrough in the world’s economy. The major advantages of the cryptocurrencies apart from primarily being a medium of exchange, its other secondary uses are to secure transactions, control the creation of other units and verification of assets transfer.
Cryptocurrencies can be traded as well on various platforms such as coinbase, and volumes can be offered through bank transfers or credit cards.
Bitcoin hadn’t achieved much interest in the wonderful world of business and financing prior to the 12 months during 2009. This rose to fameduring the 2011-2012 period when it obtained over 300%. Consequently, capital raising companies and traders all over the world always have kept high respect for cryptocurrency.
During the first-half of 2014, capital raising companies invested $57 million in Bitcoin, accompanied by an additional $73 million in the next quarter which had amounted to a complete of $130 million. This was 50% higher than previous year’s total of $88 million.
These types of figures show certainly that Bitcoin will probably be worth the expense, which usually begs the question, how will you purchase and spend money on Bitcoin?
A recommendation for beginner traders in Bitcoin.
The simplest and least difficult method to spend money on Bitcoin is through acquiring more bitcoins. There are a great number of businesses, primarily in the whole of the US and also overseas, who welcome the trading ofBitcoins (BTC).
Coinbase
Coinbase gives it can customers with BTC at 1% more than the prevailing selling price. Citizens of America have the choice to synchronize their particular Coinbase purses with their bank account. Consequently, potential repayment exchanges are created hassle-free. The corporation likewise provides you the choice of automated bitcoin buying every once in a while. For occasion, if you are interested to buy 50 dollars in bitcoins at the start of every month, Coinbase enables you to auto purchase intended for that quantity.
BitStamp
BitStamp fits certain requirements of a typical bitcoin exchange. Bitcoin functions while an intermediary that allows one to operate with various other users and not the business itself. Right here the exchange is usually larger and you will have an excellent prospect to discover and find someone who is ready to trade along. There can be an initial charge of 5% which may be reduced to 0. 2% in the event that you transact $150 in an interval of 30 days.
Local Bitcoins
Exchanging isn’t the only way of doing business in Bitcoins. Local Bitcoins is usually frequently utilized to get BTC offline. The web site is made to connectwith potential audience and retailers. The bitcoins will be encrypted from the merchant via an escrow and may only be released to purchasers.
Shopping for bitcoins off-line isn’t usually very dependable or protected. Therefore it can more suitable to meet up with the retailers during daytime and allow a buddy tag along in the event if things do not turn out as intended.
Bitcoin is usually not only a modern pattern. Capital raising companies consider Bitcoin to become a good alternative to standard foreign currency over time. You will find lots of methods that you can use in the world of Bitcoin expenditure. As stated just before, BitStamp, Coinbase and Local Bitcoins will be the majority of well-known stations foracquiring bitcoin in USA. After you’ve done the research and figured out through which means you can get the best results, start trading!
It was reported in the Bitcoin Magazine that Bitcoin Cash could be encountering a freeze in their blockchain over a short period of time in the coming future. The report was the outcome of an assumption where they considered miners to act according to their self-interest while having a vision only for the short run. In the following weekend of the report, the Bitcoin Cash situation intensified to another level when miners called for a partial emergency difficulty adjustment which led to few significant shift in hash power, block time periods which were not reliable and also caused inflation to rise.
The story unfolds with another twist in the tale. In present times, Bitcoin Cash is reported to be making less profit than BItcoin in order to undertake to mine. Based on the statement of one of the operators of a mining pool, this situation was created intentionally. Some miners are reportedly working in order to maintain the Bitcoin Cash difficulty as it is now, which is relative to Bitcoins itself. This was done in order to keep the prices of the two coins relatively similar to each other. We can conclude that Bitcoin Cash is being kept less profitable to mine than Bitcoin intentionally.
As explained in the article before, miners are likely to shift to the chain that is most profitable to be mine if they are driven by their short-term incentives to achieve higher finance but they are not influenced by the activities of other miners. Irrespective of the fact that Bitcoin Cash is experiencing lower profits, miners are still continuing to mine and that is taking place at a constant rate as before. Blocks are found to be in the state that it was operating at, the rate of inflation seems to be within the expected range. These facts help to support the fact that the situation Bitcoin Cash is experiencing is a stable situation.
This means miners are leaving money for the sake of Bitcoin Cash to keep it usable but the main question is why is it happening.
A simplified reasoning for the particular situation is mainly due to the fact that miners believe and has claimed that they are likely to see a massive increase in the exchange rate for BCH. On the basis of their predictions, we can figure out that the present miners are willingly taking the risk and are continuing to mine just so that they are able to enjoy the fruits of their labor which they had put in despite knowing the fact that the gamble was too risky for them. Moreover, more miners could be employed for Bash’s sake in order to keep their investment and Bitcoin Cash itself running smoothly.
Recently you might have noticed the looks of a new and incredibly nasty kind of computer threat known as Ransomware. Kaspersky confirmed that a computer gets contaminated with a ransomware every 10 seconds! During 2017, a lot more than 150 countries got afflicted from adifferent variantof ransomware known as WannaCry. It really did make a whole lot of individuals want to cry, because the harm it inflicted was approximated to be over $1 Billion!
Ransomware will usually attempt to infect your personal computer via two ways. The first one is through spreading infection by E-mail attachments. Utilizing a technique called phishing, hackers can find out about you through your LinkedIn or Facebook profiles, then they would send you an e-mail making it look like it originated from your colleague or friend. This E-mail would then encompass a name highly relevant to something you’ll receive from them. By studying you as well as your practices, hackers make deceptive email messages more credible, and boosts the chances that you’ll go through thisinfected attachment.
Yet another way ransomware infects your personal computer is through infected or compromised web pages. In cases like this, you can receive a contact, text on your telephone, or even a Facebook or Linkedin post with a web link. This sort of message or post is usually crafted to make it look genuine and would entice you to select it, getting your credentials with an infected webpage. From then on, the ransomware on the web page scans your personal computer for vulnerabilities. If it discovers one, then the ransomware straightaway uses it to infect your personal computer. The hacker will then ask for payment to decrypt your computer. This is mostly done via Bitcoins, as it cannot be traced back. If his demands are not met, you will end up losing all of your data.
Just what exactly do you do? How can you stop this nightmare from happening? There are many things you might do to decrease the chance of infection:
Keep your operating-system updated.
It is widely confirmed that the majority of the ransomware uses vulnerabilities within operating program such as Home windows 7, 8 and 10. By regularly updating your operating-system, you repair those vulnerabilities, therefore when ransomware attempts to infect your personal computer the loopholes are shut! In Windows operating-system, you can arrange it so it updates instantly and all you need to do is usually restart the computer once in a while when the improvements are applied.
Correctly choose and install your anti-malware solution.
Your antivirus takes on a huge part in defending your personal computer from a variety of malicious viral programs (malware) which also includes ransomware. It could identify malicious behavior and prevent it in its tracks before it could do significant damage. Keeping appropriate and updated antivirus is completely necessary to keep your computer safe and secure.
The ultimate frontier of protection: Backup.
You might be surprised to listen to that the very best protection against WannaCry Ransomware is when you are proactive. As an alternative of attempting to recover your personal computer after it’s been contaminated (which proves to become increasingly more difficult lately) you just restore it to the prior uninfected state! You retain backups of all of your computer on exterior and protected drives. If your personal computer gets inflicted by a ransomware assault, instead of spending money on hackers and praying that they can decrypt your files, just restore your personal computer from the prior backup! There are numerous back-up solutions out there in the marketplace, which can only help you with backing up your computer, nevertheless the current leading one is named Acronis. It could make a thorough backup of your personal computer and very easily bring back it to the prior condition when disaster strikes. |
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Liberty Tax
More than 140 million people file taxes in the U.S. each year, and more than half use a professional tax preparer. These consumers are served each year at Liberty Tax locations across the country – and our numbers are growing. Opening a tax franchise doesn’t require tax expertise. Liberty’s proven system provides the tools and makes the tax business a great business opportunity. Since 1997, Liberty Tax has grown to more than 4,300 offices in the United States and Canada. Find out more about the Liberty Tax opportunity at LibertyTaxFranchise.com.
For many entrepreneurs, travel and entertainment (T&E) tax deductions can be a minefield. Certain expenses for business travel are deductible as long as they meet two criteria: They must be “ordinary and necessary” in the course of doing business, and they must be documented.
Back taxes can have serious consequences for a small business. They accrue interest and penalties, so your tax debt can quickly snowball. The IRS will hold any tax refund your business is entitled to until the past-due taxes are paid.
If your business is an activity that many people enjoy as a hobby – for example, making jewelry, painting oil portraits or playing in a cover band — and isn't consistently profitable, there can be a fine line between "hobby" and "business." |
Breaking News
Britney Spears took a huge amount of amphetamines the night she was strapped to a stretcher and placed on a 5150 hold -- so claims Sam Lutfi's lawyer in the opening statements of his defamation case.
Lutfi's lawyer, Joseph Schleimer, told the jury, on January 28, 2008, Britney had an amphetamine script filled. Schleimer says she took 6 to 8 pills early in the day, and several more later and went off the rails.
Lutfi's lawyer also said he tried to get Britney to meet with a psychiatrist 2 days before she was 5150'd, but Brit refused. |
The present technology relates to implantable compositions that exhibit desired release profiles of bioactive agents.
Various approaches have been employed in attempting to provide implantable compositions that provide systemic or local delivery of drug actives. Some active technologies, e.g., insulin pumps, have been developed to provide variable rate drug delivery. However, these require specialized equipment and are desirable for use in only chronic conditions. Alternatively, passive drug releasing implants have been developed to provide a post-implantation burst or spike of the active agent after in vivo biodegradation of a long-term coating. Many such approaches simply provide long-term release at a constant rate. However, among other issues, these materials can exhibit premature release of the bioactive agent through the layer of long-term biodegradable coating. Therefore, it would be advantageous to provide implants capable of releasing bioactive agents in a manner that is more selective, with decreased risk of premature release, and that is adaptable to provide any of various pre-selected drug release profiles. |
Q:
Web testing frameworks for ASP.NET web application
I'm trying to implement some front end testing in a ASP.NET web application, and I would like to know how do the several web testing frameworks compare. Especially MSTest's "Web Tests", because I haven't seen a lot of info about them, and since it seems to have a nice integration with Visual Studio.
Related posts:
WatiN or Selenium?
Watir vs Selenium vs Sahi
Selenium
WatiN
Visual Studio Team System "Web Tests"
Thanks in advance.
A:
One important thing to note about the Microsoft Web Tests (with VSTS 2008) is that they are at the HTTP level, not the UI level. They'll work great to test navigation, form submission, and other HTTP calls, but will not test the UI components on a page. This is something important to keep in mind when comparing with other apps such as QuickTest Professional (QTP) which does test at the UI component level.
The much-improved test features of VSTS 2010 will include testing at the UI level.
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Canada Post unveils new community boxes
canada post new boxes
As the post office starts phasing out door-to-door home delivery, it is unveiling an updated community mailbox, with flatter slots and bigger parcel boxes.
“These boxes are meant for the realities of today and the future,” said Canada Post spokesman Jon Hamilton. “The future is less mail in the box, and more boxes in the mail.”
The new design, with a larger aluminum base, can easily handle online purchases such as small electronics or clothing, but any item that requires a signature it will still be delivered to the door, he said.
Oakville is among the first 11 cities to switch entirely to community mailboxes with home delivery slated to be eliminated by this fall. In all, 25,300 residential addresses and 1,100 business addresses in Oakville will be affected.
Rural routes and delivery to apartment and condo buildings will remain unchanged.
Canada Post is targeting suburban areas, since some of those neighbourhoods already use community mailboxes.
Dense neighbourhoods in big cities like Toronto and Montreal will be among the last to change over in the five-year plan, in part because the post office still needs to figure out how best to adapt to small lots and narrow streets.
First introduced in the 1980s, these super mailboxes were placed in all new subdivisions or communities as a cost-saving measure. Canada Post says it will consult with those impacted with a mail-in survey as well as a more extensive online survey.
“We are going to work neighbourhood by neighbourhood,” Hamilton said. “Based on that we will get a good idea as to whether people are looking for, fewer boxes closer to the home, or a cluster of boxes nearby.”
Each box serves 16 addresses along with two boxes to hold parcels and an outgoing mail slot.
Residents will be asked whether they prefer two to three community mailboxes, often on the sides of corner lots, spread throughout a neighbourhood. Or the other option is one larger site, serving more households, up to 200, but it will be located farther away from their homes.
The online survey also asks people about different colour schemes including grey and beige, as well as different decal coating, from Canadian icons to landscapes to the Canada Post logo, to prevent graffiti. As well, it asks about other concerns including lighting, safety and traffic.
“We hope to find what works best, understanding that we might not be able to make everyone happy,” Hamilton said.
The final decision on community mailboxes in urban neighbourhoods has not been made, but Hamilton said possibilities include affixing mailboxes to the side of a building or even boxes of smaller sizes or shapes.
“They could also be in convenience stores, pharmacies or grocery stores where people can combine their shopping and errands,” he said.
But the union representing postal carriers says it still gearing up to fight Canada Post’s plan that calls for the elimination of 6,000 to 8,000 jobs over the next five years.
“It’s really a political fight. It’s rallying people,” said Denis Lemelin, national president of the Canadian Union of Postal Workers. “We need to make it clear to the government that they have to stop it.”
While the federal government has made clear it supports Canada Post’s plan, Lemelin said some municipalities as well as the union are studying whether they can mount a legal challenge to block the changes. |
This post originally appeared at In These Times.
A striking feature of the current political moment is that many activists on the left are flocking to the Democratic Party. At first glance, this makes sense simply as a reaction to the narrow and disputed electoral victory of the bizarre and dangerous Donald Trump.
But the Democrats are not merely gaining voters. They are gaining activists, people who are committing not only to pull the party lever in the voting booth, but who are determined to rejuvenate and transform the party, beginning at the local level. This development is encouraging, and not only because it could make a difference in the 2018 midterms and the next presidential election.
Until the shock and fear of a Trump-led government took center stage, some on the left viewed elections and movement building as separate, even irreconcilable, paths to reform. While their skepticism about the Democratic Party was not misplaced, we argue that movements also depend on electoral politics. The growth, morale and effectiveness of today’s movements will depend on the success of the current surge of enthusiasm for Democratic Party activism.
Why Some on the Left are Turning to the Democratic Party
Compared to the Obama years and the noisy 2016 election itself, the enthusiasm for Democratic Party activism welling up on the broad left today is startling. It already overshadows the usual Democratic Party electoral ground game of enlisting labor and other grass-roots constituencies to knock on doors, distribute literature and make phone calls to prime voters. Hundreds of groups at the national and local levels have organized to recruit new Democratic candidates and work on campaigns. Long-standing organizations that support Democratic candidates, such as Emily’s List, are seeing unprecedented growth, and movement organizations, from the Democratic Socialists of America to the Movement for Black Lives, are getting involved in local and state races.
While fear of Trump has galvanized even centrist liberals, much of the new energy and organizing know-how is coming from left-leaning activists. Though surprising, it is not hard to explain the sudden enthusiasm for electoral action. The dangers of Donald Trump in the White House and a right-wing Republican Party in control of Congress, the Supreme Court and more than half of the states are glaring.
The path toward this new electoral activism was paved by the Bernie Sanders campaign, which made credible the prospect of engaging in a fight within the Democratic Party for a more radical and democratic economic program, for racial and social justice and for peace. As historian Max Elbaum has observed, the polarization of the country grew during the 2016 election. But the sectors of the left that grew the most were those energized by the Sanders campaign. Our Revolution, an organization inspired by that campaign, now claims some 400 local chapters that are trying to shift the Democratic Party to the left, in part by backing progressive local and state candidates.
The path to victory will be difficult. In the House, if the Democrats can hold on to the seats they already have, they still need to win an additional 24 seats. In the Senate, the prospects are more daunting: The Democrats must defend three times as many seats as the Republicans, 10 in states won by Trump, half of those by double digits.
It will take time, then, to oust the Republicans from their commanding position at the national level. That is why so much left energy has been focused on down-ballot elections. Victories on the local level can matter. Not only do they boost the morale of electoral and movement activists alike, but in our federal system, localities often have significant policy authority.
An astonishing number of state and local elected offices go uncontested. One study found that, between 1992 and 2010, a third of all state legislative incumbents did not face a challenger in the primary and general elections. Another study found that, in six states, half of all mayoral candidates ran unopposed.
In Virginia, for example, where all statewide offices are held by Democrats and Clinton defeated Trump by 5 points, the lower House of Delegates has long been dominated by Republicans. Forty of the Republicans’ 66 seats were uncontested by Democratic challengers in 2015. While it still might be a long shot, with the election of Trump and the new energy for electoral politics on the left, in 2017 the GOP could lose the 17 Republican House of Delegates seats that voted for Clinton in 2016.
An astonishing number of state and local elected offices go uncontested. One study found that, between 1992 and 2010, a third of all state legislative incumbents did not face a challenger in the primary and general elections. Another study found that, in six states, half of all mayoral candidates ran unopposed.
Such a victory would be unprecedented, but the challenge is being embraced by a new grass-roots political action committee, Activate Virginia, founded by Josh Stanfield, a 30-year-old Sanders delegate to the Democratic National Convention, and two other activists.
Virginia’s example, which points to a key weakness of the Democratic Party, also offers an opportunity to strengthen the influence of the left. The two major political parties are not parties in the sense of disciplined, unified, hierarchical membership organizations. Rather, they are loose and conflict-ridden confederations of separate leadership groups whose overall structure reflects the complex constitutional and institutional arrangements of the US federal system.
The point, however, is not to belabor the weakness of fractious and institutionally hamstrung political parties, but rather to note that the institutional fragmentation of the Democratic Party makes it susceptible to takeover. As an example of how centrists have exploited this political reality, consider the creation of the Democratic Leadership Council (DLC) in 1985. The so-called Third Way was designed to stymie the progressive, pro-labor party activism stimulated by Jesse Jackson’s presidential campaigns in 1984 and 1988, and other efforts to move the party to the left. And it succeeded — until the recent Sanders challenge loosened the grip that centrists had on the party for the past 30 years.
This kind of synergy between electoral and movement politics may be emerging in the area of health care. On the one side, Trump and the right-wing majority in Congress have put forward a series of Draconian legislative proposals to dismantle the Affordable Care Act, and especially the provisions that underwrite health care for the poor. On the other side, the political furor over these efforts has given a big bump to the Sanders-backed Medicare for All Act, with 16 Democratic senators now signed on. The legislative drama, in turn, is likely to boost the morale and increase the energy of the longer-term movement for a publicly funded health care system.
Why Movements Need Electoral Politics
The two major parties also matter because they play a very large role in shaping the life course of movements. This dynamic is often overlooked because the fundamental dynamics of movements and electoral campaigns are different. Movement activists work to raise the issues that divide and anger constituencies, while electoral operatives tend to smooth over the divisions that inhibit the building of the winning majority that elections require. In these respects, movement and electoral dynamics are antagonistic. But that is by no means the whole of it.
Movements also depend on elected leaders who are susceptible to or embrace the challenges that movements generate. They thrive when they get the rhetorical support of the elected leaders who worry about defections from movement-influenced constituencies. Moreover, the policy victories that movements score are ultimately fashioned by elected politicians.
Refusal isn’t easy, and that is an important reason why movements depend on electoral politics.
As an example, consider the recent fortunes of the environmental movement. The same year Barack Obama was first elected president, a Canadian firm, TransCanada, had applied for a permit to build a 1,200-mile pipeline across the American Midwest to connect Canadian tar sands oil with Gulf Coast oil refineries. The company and the oil lobby misleadingly claimed that the project would create 140,000 jobs and billions in economic benefits; the Canadian government pressured a newly elected President Obama to approve the project. In April 2010, the US State Department concluded the pipeline would have a limited effect on the environment. Political strategists inside the White House convinced the president to stop using the term “climate change” and to focus on “clean energy jobs” and a “clean energy economy” to avoid drawing fire from the fossil-fuel industry and conservatives. Meanwhile, oil lobbyists used propaganda to successfully shift public opinion on climate change. In 2008, acceptance that its causes were human-made was 72 percent. Two years later, only 52 percent agreed.
Environmental activists rejected their insider tactics and began to build a coalition of grassroots groups that went well beyond normal lobbying and interest group politics, bringing together ranchers and land rights advocates in red states like Nebraska and Native tribes whose land would be violated and water threatened by the project.
At the time, Bill McKibben, a leader of the movement, wrote, “Now we know what we didn’t before. Making nice doesn’t work … we may need to get arrested.” In late August 2011, protestors mounted a two-week campaign in front of the White House, joined by some of the large environmental groups that are not usually associated with civil disobedience and more than 1,200 people got arrested. On Nov. 6, 2011, thousands of protesters surrounded the White House in what they called a “solidarity hug” to urge Obama to veto the pipeline. Under intense pressure from the Republican-controlled Congress to move the project forward, in 2015, Obama exercised his veto power for only the third time.
The re-energized environmental movement did its work in the streets. But the crucial point is that friendly Democrats ultimately conceded to the demand. The delays won by a broad and inclusive coalition of opposition groups to the pipeline using direct action, civil disobedience and mass arrests exerted political pressure on a wobbly president.
The great and transformational movements of the past — the radical Democrats of the Revolutionary War era, or the abolitionists of the 19th century, or the 20th-century labor movement, or the black freedom movement, or the women’s movement, or the movements for personal rights included under the LBGTQ acronym — all scored their successes because they activated the elementary and fundamental power of ordinary people. The essence of that power is the refusal to cooperate in the basic institutional arrangements of a society. That is what movement power is: The power of the strike writ large, encompassing not only refusal in the workplace but in civil society itself.
Refusal isn’t easy, and that is an important reason why movements depend on electoral politics. All of the influences of the institutions that mold daily life collaborate to make the exercise of movement power difficult, as do the immediate threats and punishments that the dominant society imposes on movements. Most of the time, electoral politics legitimizes those threats and punishments, giving the authority of tradition and legal procedure to the threat of force that usually suppresses rebellion. But sometimes, when electoral calculations lead politicians to recognize that they need voter support from among emerging movement constituencies, mass discontent is sufficient to lead at least some political leaders to rhetorically side with the discontented. By doing so, they of course give courage and moral support to emerging movements, as Roosevelt’s campaign rhetoric gave courage to an emerging labor movement, or Kennedy’s rhetoric nourished the black freedom movement, or Obama’s sympathy for Trayvon Martin encouraged the Movement for Black Lives. The importance of this encouragement cannot be overstated.
Electoral context matters for another reason. The disruption that ensues from movement leverage can cleave the electoral base of a governing party, compelling political elites to respond with ameliorating reform. When that happens, it is elected politicians who fashion the policy measures that respond to movement demands and disorder. We need politicians in charge of that process who lean toward the left and its movements. Even when movement leverage succeeds in forcing action on policy reform, the movement itself is only one of the influences in crafting the policy change. We want the thumbs of legislators like Elizabeth Warren and Bernie Sanders on the scale in legislative deliberations. And that means movements need a rejuvenated Democratic Party.
An earlier version incorrectly identified Josh Stanfield as a founder of Progressive House VA. |
Q:
Android Custom Switch
I'm trying to make a custom switch like this:
with these properites:
text on both sides always shown.
different colors for on and off.
and these are two problems I faced since the switch only shows the text on the chosen side , and I can't seem to find a place where I can specify two different colors?
can I achieve this using the regular switch in android studio or must I use some library?
Thank you.
A:
After researching I found a way that gives me exactly what I needed, this is what I got:
in case of anyone looking for a way to do it, this is how:
based on this post answer , which worked great for me.
this is what I did, I created two drawables one for On and another for Off :
switch_on.xml
<?xml version="1.0" encoding="utf-8"?>
<selector xmlns:android="http://schemas.android.com/apk/res/android">
<item android:state_enabled="false" android:state_checked="true" android:drawable="@color/colorGray"/>
<item android:drawable="@color/colorPrimary" android:state_checked="true" />
<item android:drawable="@color/colorPrimaryDark" android:state_pressed="true" />
<item android:drawable="@color/transparent" />
</selector>
switch_off.xml
<?xml version="1.0" encoding="utf-8"?>
<selector xmlns:android="http://schemas.android.com/apk/res/android">
<item android:state_enabled="false" android:state_checked="true" android:drawable="@color/colorGray"/>
<item android:drawable="@color/gray_light" android:state_checked="true" />
<item android:drawable="@color/black_overlay" android:state_pressed="true" />
<item android:drawable="@color/transparent" />
</selector>
Next , created a drawable for the outline of the switch.
outline.xml
<?xml version="1.0" encoding="utf-8"?>
<shape xmlns:android="http://schemas.android.com/apk/res/android"
android:shape="rectangle">
<corners android:radius="2dp" />
<solid android:color="#80ffffff" />
<stroke
android:width="1dp"
android:color="@color/gray_light" />
</shape>
one thing that I added is a drawable for the text color, because the color of the text changes depending on whether it's checked or not, this is it :
switch_text.xml
<?xml version="1.0" encoding="utf-8"?>
<selector xmlns:android="http://schemas.android.com/apk/res/android">
<item android:state_pressed="true" android:color="@color/colorWhite"/>
<item android:state_checked="true" android:color="@color/colorWhite"/>
<item android:color="@color/gray_light"/>
</selector>
and finally, created RadioGroup in my layout this way:
<RadioGroup
android:id="@+id/toggle"
android:layout_width="wrap_content"
android:layout_height="50dp"
android:background="@drawable/outline"
android:checkedButton="@+id/off"
android:orientation="horizontal">
<RadioButton
android:id="@+id/off"
android:layout_width="wrap_content"
android:layout_height="match_parent"
android:layout_marginBottom="1dp"
android:layout_marginStart="1dp"
android:layout_marginTop="1dp"
android:layout_weight="1"
android:background="@drawable/switch_off"
android:button="@null"
android:gravity="center"
android:padding="@dimen/fab_margin"
android:text="@string/off"
android:textColor="@drawable/switch_text" />
<RadioButton
android:id="@+id/on"
android:layout_width="wrap_content"
android:layout_height="match_parent"
android:layout_marginBottom="1dp"
android:layout_marginEnd="1dp"
android:layout_marginTop="1dp"
android:layout_weight="1"
android:background="@drawable/switch_on"
android:button="@null"
android:gravity="center"
android:padding="@dimen/fab_margin"
android:text="@string/on"
android:textColor="@drawable/switch_text" />
</RadioGroup>
Notice the usage of each drawable in the right place:
android:background="@drawable/outline" for the RadioGroup
android:background="@drawable/switch_off" for the first RadioButton
android:background="@drawable/switch_on" for the second RadioButton
android:textColor="@drawable/switch_text" for both Radio Buttons
And that's all.
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James Gunn’s Tour of Walt Disney’s Restored Private Office
During a visit to Walt Disney Studios, James Gunn had the opportunity to tour Walt Disney’s office, which has very recently been restored to the exact state it was in at the time of the filmmaker’s death. Disney, the mastermind behind the brand that bears his name, died of lung cancer in 1966 at the age of 65.
Luckily for us, Gunn is a social media maven and often lets us into his world via Facebook.
James Gunn: “This is Walt’s working office where most of his actual work got done.”
James Gunn: “This is the actual page on which Walt wrote down ‘Kirt Russell’ – one of the last things he ever wrote – something Kurt and I talked about on one of our first dinners in Atlanta as we were about to start Vol. 2.”
James Gunn: “I took the tour with Robert D Cabana, the director of NASA’s John F Kennedy Space Center and the first man ever on a space station – he turned on the power and computers! Disney’s Katie Reese was also there.”
James Gunn: “A few of Walt’s favorite food items are kept stocked in the cupboard in a kitchenette behind an electric door in the side of the room (some of the few things in the room that aren’t the originals).”
James Gunn: “Nearby one of the original animation tables was set up.”
James Gunn: “This is the desk in the front, more formal office, used less often for work.”
Gunn has recently wrapped filming on Guardians of the Galaxy Vol. 2 for Marvel, a subsidiary of Walt Disney Studios. Clearly there are advantages to creating masterpieces for the House of Mouse whilst being an all-around awesome person. Tours like this are not common. According to The Nerdist:
“Tours of Walt Disney’s office suite begin in 2016 as part of the D23: The Official Disney Fan Club offer to their Gold Members.”
So even if you’re not in the unique position of bringing Star Lord to the big screen, you can still have a shot of seeing the interior space of one of Hollywood’s most iconic and visionary minds. If you snag a D23 Gold Membership, that is. It’s surprisingly not a total bank breaker at $79.99 per year for individuals.
Guardians of the Galaxy Vol. 2 opens in US theaters May 5, 2017 and in the UK April 28, 2017.
Images via James Gunn’s Facebook page
Posted By
Denise Heard-Bashur
Managing editor for GeekFeed.com living in the desolation of the west Texas desert. Proud mom, photo editor by day, former film major, MCU maven. Founder of the Free Snowpiercer & Free Loki campaigns. Also hung up on LOTR, Star Wars, Game of Thrones, Outlander, Friends, and cinema in general. Good, bad...it's all awesome. I'm a firm believer that if you haven't read the book yet, see the movie first. Saves on tons of disappointment and spoilers. SAN DIMAS HIGH SCHOOL FOOTBALL RULES! |
from .render import GerberContext
from ..excellon import DrillSlot
from ..excellon_statements import *
class ExcellonContext(GerberContext):
MODE_DRILL = 1
MODE_SLOT =2
def __init__(self, settings):
GerberContext.__init__(self)
# Statements that we write
self.comments = []
self.header = []
self.tool_def = []
self.body_start = [RewindStopStmt()]
self.body = []
self.start = [HeaderBeginStmt()]
# Current tool and position
self.handled_tools = set()
self.cur_tool = None
self.drill_mode = ExcellonContext.MODE_DRILL
self.drill_down = False
self._pos = (None, None)
self.settings = settings
self._start_header()
self._start_comments()
def _start_header(self):
"""Create the header from the settings"""
self.header.append(UnitStmt.from_settings(self.settings))
if self.settings.notation == 'incremental':
raise NotImplementedError('Incremental mode is not implemented')
else:
self.body.append(AbsoluteModeStmt())
def _start_comments(self):
# Write the digits used - this isn't valid Excellon statement, so we write as a comment
self.comments.append(CommentStmt('FILE_FORMAT=%d:%d' % (self.settings.format[0], self.settings.format[1])))
def _get_end(self):
"""How we end depends on our mode"""
end = []
if self.drill_down:
end.append(RetractWithClampingStmt())
end.append(RetractWithoutClampingStmt())
end.append(EndOfProgramStmt())
return end
@property
def statements(self):
return self.start + self.comments + self.header + self.body_start + self.body + self._get_end()
def set_bounds(self, bounds, *args, **kwargs):
pass
def paint_background(self):
pass
def _render_line(self, line, color):
raise ValueError('Invalid Excellon object')
def _render_arc(self, arc, color):
raise ValueError('Invalid Excellon object')
def _render_region(self, region, color):
raise ValueError('Invalid Excellon object')
def _render_level_polarity(self, region):
raise ValueError('Invalid Excellon object')
def _render_circle(self, circle, color):
raise ValueError('Invalid Excellon object')
def _render_rectangle(self, rectangle, color):
raise ValueError('Invalid Excellon object')
def _render_obround(self, obround, color):
raise ValueError('Invalid Excellon object')
def _render_polygon(self, polygon, color):
raise ValueError('Invalid Excellon object')
def _simplify_point(self, point):
return (point[0] if point[0] != self._pos[0] else None, point[1] if point[1] != self._pos[1] else None)
def _render_drill(self, drill, color):
if self.drill_mode != ExcellonContext.MODE_DRILL:
self._start_drill_mode()
tool = drill.hit.tool
if not tool in self.handled_tools:
self.handled_tools.add(tool)
self.header.append(ExcellonTool.from_tool(tool))
if tool != self.cur_tool:
self.body.append(ToolSelectionStmt(tool.number))
self.cur_tool = tool
point = self._simplify_point(drill.position)
self._pos = drill.position
self.body.append(CoordinateStmt.from_point(point))
def _start_drill_mode(self):
"""
If we are not in drill mode, then end the ROUT so we can do basic drilling
"""
if self.drill_mode == ExcellonContext.MODE_SLOT:
# Make sure we are retracted before changing modes
last_cmd = self.body[-1]
if self.drill_down:
self.body.append(RetractWithClampingStmt())
self.body.append(RetractWithoutClampingStmt())
self.drill_down = False
# Switch to drill mode
self.body.append(DrillModeStmt())
self.drill_mode = ExcellonContext.MODE_DRILL
else:
raise ValueError('Should be in slot mode')
def _render_slot(self, slot, color):
# Set the tool first, before we might go into drill mode
tool = slot.hit.tool
if not tool in self.handled_tools:
self.handled_tools.add(tool)
self.header.append(ExcellonTool.from_tool(tool))
if tool != self.cur_tool:
self.body.append(ToolSelectionStmt(tool.number))
self.cur_tool = tool
# Two types of drilling - normal drill and slots
if slot.hit.slot_type == DrillSlot.TYPE_ROUT:
# For ROUT, setting the mode is part of the actual command.
# Are we in the right position?
if slot.start != self._pos:
if self.drill_down:
# We need to move into the right position, so retract
self.body.append(RetractWithClampingStmt())
self.drill_down = False
# Move to the right spot
point = self._simplify_point(slot.start)
self._pos = slot.start
self.body.append(CoordinateStmt.from_point(point, mode="ROUT"))
# Now we are in the right spot, so drill down
if not self.drill_down:
self.body.append(ZAxisRoutPositionStmt())
self.drill_down = True
# Do a linear move from our current position to the end position
point = self._simplify_point(slot.end)
self._pos = slot.end
self.body.append(CoordinateStmt.from_point(point, mode="LINEAR"))
self.drill_mode = ExcellonContext.MODE_SLOT
else:
# This is a G85 slot, so do this in normally drilling mode
if self.drill_mode != ExcellonContext.MODE_DRILL:
self._start_drill_mode()
# Slots don't use simplified points
self._pos = slot.end
self.body.append(SlotStmt.from_points(slot.start, slot.end))
def _render_inverted_layer(self):
pass
|
Here is a sad story about the way the Philadelphia Jewish community is using its energy and resources: battling any criticism of Israel in the local press.
Last Thursday, Philly.com, the website for two Philadelphia newspapers, published an op-ed piece by Susan Abulhawa, the Palestinian novelist, protesting the fact that the Philadelphia Orchestra was planning a trip to Israel with patrons to celebrate the country’s 70th anniversary, even as Israel has been slaughtering unarmed protesters in Gaza.
Abulhawa said that more than 100 musicians and activists along with 35 social justice organizations had sent a letter to the orchestra urging it to cancel the trip, but the orchestra’s co-presidents, Ryan Fleur and Matthew Loden, dismissed the appeal in a short letter.
One of the things mentioned in our letters was a scheduled dinner with [Culture Minister] Miri Regev, a politician who once referred to African asylum-seekers as “a cancer” and later apologized to cancer patients for the association. She said, “Heaven forbid … I did not compare them [Africans] to human beings.” The itinerary (open to orchestra members and patrons joining the trip) boasts multiple visits with military personnel, including a “VIP visit to an IDF [military] base.” This is the same military that has killed 40 Palestinians and wounded 5,000 more in the span of just four weeks…. Fleur and Loden claim neutrality, invoking lofty verbiage like “cultural diplomacy,” “brotherly and sisterly love,” and “peace and tolerance.”
When Abulhawa’s op-ed appeared, the Israel lobby in Philadelphia flew into action. “The Jewish Federation, our JCRC [Jewish Community Relations Council], the Anti-Defamation League (ADL) and the Philadelphia Orchestra immediately began communicating on a strategy for a strong and best possible response,” Naomi Adler, the CEO of the Jewish Federations, wrote in a letter to the Board of Rabbis in Philadelphia.
Please bear in mind that the Jewish Federations is a sponsor of the orchestra’s trip, and that its ceo, Adler, is close to David Cohen, a donor to many institutions and a top exec at the largest cable company in the world, Comcast, which owns MSNBC; and that David Cohen and his wife Rhonda have run fundraisers for the Jewish Federations, and Cohen is a former vice chair of the Federations.
Adler characterized Abulhawa’s piece as a “very disturbing and upsetting op-ed from an anti-Israel activist” and said her groups had sought an assurance from the Philadelphia Inquirer that it would not publish Abulhawa’s piece in print. They failed in that effort; the op-ed was printed in the paper last Sunday.
But “we were able to reach a compromise with the Opinion editor,” Adler said. A rebuttal piece was published alongside Abulhawa, by Nancy Baron-Baer, the regional director of the Anti-Defamation League. So, a lot of executives were involved in this pushback! Baron-Baer said the trip was allowing the orchestra to engage with the “complex realities” of Israel. Not demonize Israel:
Let us be clear: Anyone has a right to criticize the State of Israel for its policies, just as it is fair to criticize the policies of the United States, France, or India. However, we strongly disagree with these activists’ demonization of all things Israel, and their spurious, one-sided presentation of a complicated situation. An honest accounting reveals that the Israeli-Palestinian conflict is extremely complex, challenging, and heartbreaking on all sides. To claim that Israel is the sole cause of the conflict belies this fact, and to advocate for an international boycott of the Jewish state actively undermines any productive efforts to end the conflict.
Baron-Baer also claimed that in an April 6 protest of the orchestra at the Kimmel Center for the Performing Arts — Sidney Kimmel is also a big supporter of the Jewish Federations — pro-Palestinian groups had voiced hate speech and bigotry. “People at the protest reported to the Anti-Defamation League (ADL) that they heard phrases that the ADL would clearly classify as anti-Semitic.”
Abulhawa says there was no such thing.
Most of our speakers on April 6th were African Americans, including Rev Graylan Hagler, Dr Tony Montiero, Pam Africa, and more. Not to mention the fact that many of us were physically and verbally attacked on that day and every other day we’ve gone out to protest. One man walked through the line of protesters spitting on at least three people. Another man, former Israeli soldier, aggressively ripped a poster from my hands. Three different women tried to wrestle the microphone from our speakers, and one of them was so aggressive that a police officer threatened to arrest her if she didn’t leave.
Since that first op-ed countering Abulhawa, philly.com has run two more pieces attacking the protesters. First, there was this very high-minded article by Ryan Fleur of the Philadelphia Orchestra Association saying the trip was supported by the “private sector” in Philadelphia and that it was going to serve the peace process.
The U.S. State Department, with which the orchestra works closely, has told us our appearances help bring people together on the path to a long-term peace process… we hope to help heal by sharing our musical gifts with a part of the world that has known too much conflict.
Then Philly.com ran a column by Stu Bykofsky, saying that Boycott, Divestment and Sanctions (BDS) campaign is anti-semitic.
There are some Israeli practices that many American Jews don’t like, and some that Israelis don’t like. By holding Israel, the only majority Jewish state, to a standard it imposes on no other nation, BDS invites suspicion it is animated by anti-Semitism.
Bykofsky also said that Palestinians inside Israel have full rights (which is simply not the case), and that there are some separate roads for Palestinians and Jews in the West Bank “to thwart attacks.”
Naomi Adler indicated in her letter to the rabbis last week that she’s not done. She’s sending in a letter to the editor of the newspaper. So is Dan Segal, chair of the Jewish Community Relations Council.
In addition to all of this advocacy, we had already planned a BDS training session for members of the media for Thursday, May 3rd, and in response to this situation, we will insist that reporters and staff at the Inquirer be in attendance. We will also plan on requesting an Inquirer editorial board meeting sometime in the future. As you know, we have dealt with anti-Israel media coverage in the past and certainly will continue to do so in the future. The Jewish Federation will always be prepared and ready to join with our partner organizations to stand in support of Israel and Jewish communities across the world.
But who is listening. It’s clear that many readers of the Philadelphia papers are appalled by Israel’s actions and want to see Americans taking action.
Philly residents are upset at their orchestra. They want @philorch to cancel its June tour in Israel, according to letters to the editor of the @PhillyInquirer #PhillyDontOrchestrateApartheid pic.twitter.com/Z4bTfJZ2nV — Adalah-NY (@AdalahNY) May 4, 2018
But Naomi Adler surely has a lot of clout in Philadelphia. Her org is supported by Sidney Kimmel. And her friend David Cohen– who says, “Israel needs financial support from the United States” — was once the political guru of former mayor and governor Ed Rendell, a regular on MSNBC and defender of Israel. Cohen and Jonathan Lavine are co-chairs of a big charity based in Boston. Lavine joined the board of the Democratic Party’s thinktank after it hosted Benjamin Netanyahu in 2015, just weeks after Netanyahu had tried to submarine the Iran deal.
And the Israel tour by the Philadelphia Orchestra, which includes a lot of restaurants and sightseeing, is aimed at engaging “new members of the Philadelphia community” associated with the Jewish Federations.
These donors are the new Jewish establishment: civic leaders in liberal states who are also big supporters of the Democratic Party, and Israel.
Just imagine if one drop of this clout was spent urging Israel not to shoot unarmed protesters! |
Q:
How to open multiple Visual Studio windows on Windows 8.1?
I was using Windows 7 and I was able to launch more than one Visual Studio and work at the same time. Now I have windows 8.1 OS and whenever I try to launch second Visual Studio it brings me the one currently launched. Is there a solution for that or should I settle to this new thing?
A:
If you have one instance open of Visual Studio, locate it in the taskbar.
Rightclick on the Visual Studio icon and click the line "Visual Studio[version]".
A:
Middle click your Visual Studio icon in the task bar.
A:
Press shift and click the visual studio icon
|
Q:
How to read config from string or list?
Is it possible to read the configuration for ConfigParser from a string or list?
Without any kind of temporary file on a filesystem
OR
Is there any similar solution for this?
A:
You could use a buffer which behaves like a file:
Python 3 solution
import configparser
import io
s_config = """
[example]
is_real: False
"""
buf = io.StringIO(s_config)
config = configparser.ConfigParser()
config.read_file(buf)
print(config.getboolean('example', 'is_real'))
In Python 2.7, this implementation was correct:
import ConfigParser
import StringIO
s_config = """
[example]
is_real: False
"""
buf = StringIO.StringIO(s_config)
config = ConfigParser.ConfigParser()
config.readfp(buf)
print config.getboolean('example', 'is_real')
A:
The question was tagged as python-2.7 but just for the sake of completeness: Since 3.2 you can use the ConfigParser function read_string() so you don't need the StringIO method anymore.
import configparser
s_config = """
[example]
is_real: False
"""
config = configparser.ConfigParser()
config.read_string(s_config)
print(config.getboolean('example', 'is_real'))
|
Title: Dopamine Neuron Regulation and its Implications for the Treatment and Prevention of Schizophrenia
========================================================================================================
**Abstract**
Substantial evidence shows a role for the dopamine system in schizophrenia. However, studies indicate that it is not the dopamine neurons themselves that are responsible for these pathological states, but instead the disorder appears to arise due to a disruption of dopamine neuron regulation by afferent inputs. Dopamine neurons recorded in vivo are known to exhibit multiple functional activity states, including baseline tonic firing and phasic activation in response to salient stimuli. Phasic burst firing is believed to be the behaviorally relevant "signal" of the dopamine neuron, whereas the level of tonic discharge represents the "gain" or the level of amplification of this signal. This tonic gain is differentially regulated by multiple brain regions, with the ventral hippocampus playing a major role. Using the MAM developmental disruption model of schizophrenia in rats, we found that parvalbumin interneuron loss in the hippocampus leads to abnormally high tonic dopamine neuron firing, causing the system to be hyper-responsive to phasic stimuli. This would lead to over- or misinterpretation of external events. Restoring GABAergic balance in the hippocampus by a selective GABA A alpha 5 positive allosteric modulator restores baseline dopamine neuron firing and behavioral responses to amphetamine. In contrast, this drug is not effective if the rats are pre-treated with a D2 blocking antipsychotic drug, which has implications for the failure of novel target compounds in clinical trials.
This disruption in hippocampal interneurons appears to be driven by abnormal responses to stress occurring in the peripubertal time period. Thus, in MAM rats there is hyperactivity peripubertally both in the amygdala and in its target area, the ventral hippocampus. Such hyperactivity is proposed to lead to the interneuron loss, thereby producing abnormal hippocampal function in the adult. We found that administration of an anti-anxiety drug during the peripubertal period at doses that relieve the hyperanxious state prevents the development of dopamine system hyper-responsivity in the adult rat. Therefore, examining the circuitry underlying dopamine system disruption in this developmental model of psychosis can contribute both to a better understanding of the pathophysiology of major psychiatric disorders, as well as glean insights into novel avenues of treatment and potentially in preventing the emergence of schizophrenia in susceptible individuals.
|
Q:
JavaScript prototype inside "Immediately-Invoked Function Expressions"
I am writing functions inside Immediately-Invoked Function Expressions (IIFE).
(function(){
"use strict";
function EventService(){
//MASTER CLASS
}
EventService.prototype.eventObj = function(){
var ES = getEvwentMapping();
return EventService;
};
EventService.prototype.popup = function(){
eventService.eventObj.createPopup();
};
EventService.prototype.drilldown = function(){
eventService.eventObj.createDrilldown();
};
})();
eventService();
Here I am getting error as eventService() is undefined.
A:
Yes, your function is enclosed in your IIFE and doesn't become part of any external scope. If you need it outside your function, then try returning it and assigning it to a variable. For example:
var eventService = (function(){
"use strict";
function eventService(){
//MASTER CLASS
}
eventService.prototype.eventObj = function(){
var ES = getEvwentMapping();
return EventService;
};
eventService.prototype.popup = function(){
eventService.eventObj.createPopup();
};
eventService.prototype.drilldown = function(){
eventService.eventObj.createDrilldown();
};
return eventService;
})();
Or the module patern suggested by @ppoliani is also good if you have several functions and/or properties you need to expose.
Another question, however, is why you think you need an IIFE here anyway? This would work the same by just removing the IIFE altogether.
Here's how you'd use the thing:
var myES = new eventService();
myES.popup();
http://jsfiddle.net/k7ZJY/
EDIT: As I said before, there is no reason to actually use an IIFE here because you aren't hiding anything and you code doesn't actual execute anything anyway. But, one way you might use this is to create a singleton and I'm wondering if that was your actual objective here. What you could do is this:
var eventServiceSingleton = (function(){
"use strict";
function eventService(){
//MASTER CLASS
}
eventService.prototype.eventObj = function(){
var ES = getEvwentMapping();
return EventService;
};
eventService.prototype.popup = function(){
eventService.eventObj.createPopup();
};
eventService.prototype.drilldown = function(){
eventService.eventObj.createDrilldown();
};
return new eventService();
})();
Now you have a fully constructed eventService assigned to the variable eventServiceSingleton and there is no way to create another eventService object. So now:
eventServiceSingleton.popup(); // works
but:
var anotherEventService = new eventService(); // eventService is undefined
A:
You need to apply the module pattern which returns a constructor.
var EventService = (function(){
"use strict";
function EventService(){
//MASTER CLASS
}
EventService.prototype.eventObj = function(){
var ES = getEvwentMapping();
return ES;
};
EventService.prototype.popup = function(){
this.eventObj().createPopup();
};
EventService.prototype.drilldown = function(){
this.eventObj().createDrilldown();
};
return EventService;
})();
and then you can simply invoke the method by doing
var eventService = new EventService();
eventService.popup();
The issue is that you are trying to apply a sort of encapsulation with the aid of JavaScript closures; that is anything defined within the closure is not accessible from the outside unless you explicitly export them.
However, you're definitely doing something wrong here; As @Matt Burland mentioned, you don't need to use an IIFE here. You can benefit from a IIFE when you want to conceal some variables so that you don't pollute the global scope or you simply don't want to make them accessible. I cannot see that intention in your code.
|
Nightmare Moon's Manifestation inside Princess Luna
The Lunar Eclipse Briefing
Princess Luna still however continue to give Maya nightmares Lady Maya became sentient in her own dreams
and confronted Luna once again during that dream.
This confrontation led Maya and Luna to set up a duel against each other.
This duel made both Maya and Princess Luna's darker half to spike so high both of them became Berserk at some point of the duel.
Unfortunately the once the duel was over, Princess Luna's emotions became so out of rack that Nightmare Moon once again took over Luna's body
and begin fighting Maya and injuring her severally.
Nightmare Moon continue to torture Maya's injured should blaming her that she should of never came to see her that day. Maya argued that Nightmare Moon was the one that took Luna's happiness away from her. She was going to be "The Goddess of Passion" in Equestria but Nightmare Moon put all of that doubt in her mind and made her fall in to despair.
Maya persist that the only one who deserves to end her life is her Sister Princess Celestia, That's when Nightmare Moon got angry enough to release her sword from Maya's shoulder to decapitate her, and give Maya an opportunity to use her magic concealing bullets to knock her out and try to reach for Excalibur. Before Maya can reach for Luna's sword Nightmare Moon engulfed her into her Nightmare realm telling her she will sent her to Hell before she dies. And that's where I left off in this story.
have every attention for this Nightmare Moon to live up to her name.
EIGHT
Hit me up on Twitter:
and Tumblr:
for updates or you want to chat. I'm trying to more active there you know.
Software used: Daz Studio 4.9 Pro, Face Den Artist Pro, Adobe Photoshop CC 2017, Iray, Poser Pro 2014 |
Posts tagged "localized"
When you use the localized version of LiveCycle ES 8.2.1 Workspace in a web browser, clicking the link to the Help files may return a 404 error. The link works correctly when you use the standard English locale. This issue has been reported particularly on French and German locales.
Reason
This was a bug in LC ES 8.2.1.2 and previous versions.
Solution
This issue has been fixed in LC ES2 9.0.0.0 and later versions. There is a patch available for LiveCycle ES 8.2.1.x, so you should contact enterprise support if you require this patch.
Installation on a localized OS is supported as long as it is one of the supported OS versions, however, localized operating systems have not been tested and you may encounter issues.
Solution
Install on an English operating system if possible. If you only have a localized OS, then proceed with the installation and report any issues to Adobe support. We can provide patches as necessary to get LiveCycle running on the localized OS you are using. |
Vince,
A reminder that I'm gone on Tuesday to Spearman.
Status of projects:
1. Compound option for power structuring (Bernie, Edith Cross, Nick ?)
Alex has finished the model. Paulo has done some validation so we can
probably release it.
This is will be used for an exotic book, so we may have to help with VAR
interface, etc.
2. Followed up with Laine Borgman on DG contract. We still need legal's
sign off.
3. Left a message and email about Tom Barkley with Molly Magee and told Tom
that an offer would be coming by next week. |
LONDON -- As the women's final at Wimbledon approached, most experts predicted that Serena Williams would overcome Simona Halep to win the title and equal Margaret Court's record Grand Slam singles title count of 24.
But Halep, much like Angelique Kerber last year in the Wimbledon final against Williams, seized her moment. She took advantage of an extremely slow start by Williams and never faltered or relented as she locked down the second Grand Slam title of her career, rolling to a 6-2, 6-2 victory in 56 minutes. Here's how Halep accomplished it:
The relentless running of Simona Halep proved too much for Serena Williams in the Wimbledon women's singles final. Nic Bothma/EPA
One more ball
It's one of the oldest commandments in the tennis lexicon: Try to get that one more ball back. You never know when your opponent will take her eye off the ball or make an error. Halep has been doing that for her entire career, which is how she has been able to play three finals on the most rally-friendly surface of them all. But she never felt quite comfortable with her footing on grass.
Halep finally cracked the grass-court code here this year. Her ability to track and return shots -- often in ways that posed awkward questions for Williams -- were a major component in her win. The greatest example: Halep recorded the critical go-ahead break in the fifth game of the second set when, after chasing down a few heavy Williams blasts, she hooked a desperation crosscourt shot that left Williams with an easy backhand down-the-line putaway. But Williams cracked the shot just beyond the baseline to provide Halep with the lead, 3-2.
Avoiding errors
Halep made a grand total of just three unforced errors in the match, which left her with a +10 ratio of winners to unforced errors. It wasn't like Halep unfurled a stream of winners. She had just 13 in the match. But combine that with just three unforced errors and it's easy to see why Williams had so little room to operate.
Williams smacked 17 winners, but her differential was -9 because she littered the court with 26 unforced errors. Each time she hit a signature placement or teed off on a Halep serve, an error soon followed to wipe out her advantage. Williams was equally erratic off wings, making 11 forehand errors and 14 with her backhand.
When you can play error-free tennis while keeping the ball in play and weathering the best a powerful opponent can throw at you, you're in great shape.
First serve
Before the match, most observers felt that Williams would be able to tee off on Halep's serve, especially her second serve. But while Halep is short of power (her fastest in the final was 108 mph, 10 miles slower than Williams'), she's very good at protecting her serve by getting the first ball into play. She ranks No. 3 on the WTA Tour with a first-serve conversion rate of 69.7%. She did even better in Saturday's final, putting in 76% of her first serves.
Halep won 83% of her first-serve points, an excellent percentage at any time. That allowed her to, if not exactly take control of the points, at least meet most of Williams' returns with her feet planted and ready to rally. Granted, Williams did not have a great day in any dimension, including returning. But in the last game of the match, when Halep might have been vulnerable to nerves with the match on the line, she delivered two serves that Williams wasn't able to return, including the one that brought her to match point.
play 1:13 Serena: Halep played 'out of her mind' Serena Williams says Simona Halep played amazing and there wasn't much she could have done. She also thanks her team for all the support.
Taking it down the line
One of the keys to Halep's success in general is her ability to go down the line off either forehand or backhand wing. It's more difficult than going crosscourt (both technique-wise, and because the net is higher at either side of the court) but it's also the greatest weapon in a baseliner's arsenal. It takes time away from an opponent, opens up the court, and ends or completely redirects a rally.
After breaking Williams for a 1-0 lead, Halep built a 40-love lead and secured the game with a down-the-line blast that declared her intentions. She broke Williams again in the next game -- again with a down-the-line backhand winner. She would continue to do damage with the shot throughout the match.
Break chances
Nobody would have predicted that Williams would fail to break Halep even once, and see no more than one break point -- or that Halep would convert four of five break points against the greatest of all women servers. Williams squandered her only break point with a crosscourt forehand error. She also gifted Halep with break-point errors twice, while Halep converted her other two break points with those down-the-line backhand winners. |
Subsets and Splits
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