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// license:BSD-3-Clause
// copyright-holders:James Wallace
/*************************************************************************************
AWP Hardware video simulation system
*************************************************************************************/
#ifndef AWP_VIDEO
#define AWP_VIDEO
#include "machine/steppers.h"
void awp_draw_reel(running_machine &machine, const char* reeltag, stepper_device &reel);
#endif
| 2024-06-01T01:26:19.665474 | https://example.com/article/3332 |
Meta
RedState
8 Things To Understand About Panic Attacks & How To Deal With Them
place. Thats a bad place. Yet, its a deeply-ingrained reaction to feeling physically out of control. (Google flight or fight response.) All youre feeling while youre having one is that you would give anything to feel normal again panic attacks are so scary that I used to have panic attacks because I was worried about having panic attacks! The reality is that after the person calms down and starts breathing normally again, he or see will feel better soon. Better, of course, is relative, but what I mean is that when she calms down, shell be able to see that shes not immediately at risk. In the moment, though, her focus is on her inability to breathe, tense muscles, dizziness, trembling and her wildly-beating heart. For the original version, visit http://www.thefrisky.com/2014-06-25/8-things-to-understand-about-panic-attacks-how-to-deal-with-them/ | 2023-09-17T01:26:19.665474 | https://example.com/article/5797 |
Q:
How can DejaDup backup folders requiring root permissions?
I'm using etckeeper to manage /etc and Deja-Dup for /home. I wanted to back up /etc/.git/ as well, using Deja-Dup, but this folder has owner:group set to root:root. Obviously, running Deja-Dup as my user fails to touch it.
I configured Deja-Dup from system settings, and I found no option that would help me tackle this there. Any ideas? Or do I go to duplicity and run it from cron? If so, where can I see what exact calls to duplicity are made from Deja-Dup (is there anything clearer than setting a debug flag as in DEJA_DUP_DEBUG=1 deja-dup --backup and hunting for calls in wall-of-text that follows)?
I would like to avoid changing the permissions to my user, if possible (I believe etckeeper folks did this under root:root for a reason).
A:
It's fine to include such directories in a backup as long as your user can READ the files (users can often, but not always, read root-owned files).
But if your user can't read the files, I recommend running duplicity directly via cron. Deja Dup is designed for users, not system administrators.
| 2023-10-26T01:26:19.665474 | https://example.com/article/7218 |
Association between the use of selective serotonin reuptake inhibitors and multiple sclerosis disability progression.
Benefits of selective serotonin reuptake inhibitors (SSRIs) in modifying the multiple sclerosis (MS) disease course have been suggested, but their ability to delay disability progression remains unknown. We examined the association between SSRI exposure and MS disability progression. A nested case-control study was conducted using the British Columbia (Canada) Multiple Sclerosis clinical data linked to health administrative data. The primary outcome was a sustained score of 6 (requires a cane to walk) on the Expanded Disability Status Scale (EDSS), and the secondary outcome was the onset of secondary progressive MS (SPMS, an advanced stage of MS). The cases were those who reached a study outcome and were matched with up to four randomly selected controls by sex, age, EDSS and calendar year at study entry using incidence density sampling. The associations between disability worsening and SSRI exposure were assessed with conditional logistic regression models, adjusted for confounders. A total of 3920 patients were included in the main analyses, of which 272 reached sustained EDSS 6 and 187 reached SPMS. SSRI exposure was significantly different between patients who reached sustained EDSS 6 and controls [adjusted odds ratio (adjOR):1.44; 95% confidence interval (CI):1.03-2.01]. However, SSRI exposure was not significantly different between those who reached SPMS and their controls (adjOR:1.35; 95%CI:0.89-2.04). We found no evidence to suggest that SSRI exposure was associated with a delay in MS disability accumulation or progression. Copyright © 2016 John Wiley & Sons, Ltd. | 2023-12-20T01:26:19.665474 | https://example.com/article/8169 |
unbalance voltages
Unbalance phase voltages on a three (3) power system produce negative sequence components of the voltage. Even a small voltage unbalance will result in large current unbalance during the running of motor by a factor… | 2024-04-13T01:26:19.665474 | https://example.com/article/1971 |
Locals react to inauguration, new president
PLATTSBURGH - Not only was it a big day in Washington D.C., but it was in Plattsburgh as well.
An estimated 200 peopled gathered at the State University of New York at Plattsburgh's Yokum Hall to celebrate the inauguration of President Barack Obama Jan. 20.
Organizer Marti Martin, an alumnus of SUNY Plattsburgh, felt it was necessary for the community to come together to watch the historic event.
"I just felt like it was such an occasion to bring the community together, to celebrate this great day," she said. "I was just wondering, and I started asking around, 'Is there anything special planned?' and I couldn't imagine that there wasn't."
With no other public viewings of the inauguration happening in the area, Martin took it upon herself to make contacts with people she knew at the college and said it "snowballed" from there.
"The people here at the college just took up with the idea and made it happen," she said.
Fellow organizer Lauren Kiefer, a professor at the college, said the whole idea was "very last minute," with six people meeting about a week earlier to get the celebration afoot.
With so little time, there was still a great turnout, said Kiefer.
"I was gratified by how many people came," she said. "People really did want to see it. People really did think it was historic."
A standing ovation was seen throughout the room as Obama took the oath of office, and cheers were heard as he gave what some called an "amazing" speech.
"That we are in the midst of crisis is now well understood," Obama said. "Our economy is badly weakened ... Homes have been lost; jobs shed; business shuttered. Our health care is too costly; our schools fail too many; and each day brings further evidence that the ways we use energy strengthen our adversaries and threaten our planet." | 2023-08-18T01:26:19.665474 | https://example.com/article/8779 |
Run Away or Get Married
Run Away or Get Married () is an Armenian comedy written by Ashot Abrahamyan and directed by Hayk Kbeyan, starring Iveta Mukuchyan and Mkrtich Arzumanyan. The film was completed in 2016 and was released on March 3, 2016.
Plot
Aghasi's (Mkrtich Arzumanyan) father Arshavir Xojoyan, is the person who made the first Armenian car called "EpA3 Rocket" during the years of the USSR. Even though Moscow discontinued the production, one of the cars remains with Aghasi, which he uses as a taxi. One day Aghasi's life immediately changes when Satenik (Iveta Mukuchyan) comes in, who has just escaped from her wedding.
Cast
Iveta Mukuchyan as Sathenik
Mkrtich Arzumanyan as Aghasi
External links
Official Facebook page
References
Category:Armenian comedy films
Category:Armenian films
Category:2010s comedy films
Category:Films shot in Armenia | 2023-11-23T01:26:19.665474 | https://example.com/article/2675 |
Q:
Dimension of polynomially parameterized set of points
I'm seeking a general method for determining the dimension of a set of points that have polynomial parameterizations. Any information about what this type of problem would be called would be helpful, any resources, or a clear method for the problem below. I have very little background in this area of mathematics.
Example Problem:
Suppose we have the parameterized functions
\begin{equation} x(a,b,c,d) = ac\end{equation}
\begin{equation} y(a,b,c,d) = bc + ad \end{equation}
\begin{equation} z(a,b,c,d) = bd \end{equation}
What is the intrinsic dimension of the set \begin{equation} M = \{ (x,y,z) \in \mathbb{R}^3 : (a,b,c,d) \in \mathbb{R}^4\}? \end{equation}
Clearly it is not all of $\mathbb{R}^3$ since points are missing from $M$, such as $(1,0,1)$. But could it be a volume, surface or just a curve?
Possible method of solution:
It was suggested be a friend that maximizing the rank of the Jacobian matrix where the parameterization is smooth gives the dimension of $M$.
The Jacobian is
\begin{equation}
J = \begin{pmatrix} c&0&a&0 \\ d&c&b&a \\ 0&d&0&b \end{pmatrix}.
\end{equation}
Since the three rows here are linearly independent in general, one can conclude that $\dim(M) = 3$. Is this a valid method to determine the dimension of $M$? If so, I could imagine that this becomes increasingly difficult for larger and more complicated systems. References on general methods would be greatly appreciated.
A:
The algebraic-geometry perspective on this is that you have a morphism of varieties $X\to Y$ and you're trying to find the equations of (the closure of) the image. The general way to do this is to write $x=ac$, $y=bc+ad$, and $z=bd$ and then eliminate $a,b,c,d$ from these equations using elimination theory.
If you're instead focusing on more of the real-valued aspect of the problem, you can analyze this via semialgebraic geometry. A good starting point to become familiar with this (depending on your level) is Coste's notes.
In this case, there's a no-tech way to approach the problem: the equations are simple enough that we can just solve for $a,b,c,d$ on a suitable open set. When $x\neq 0$, we can set $a=1$, $b=\frac{y\pm\sqrt{y^2-4xz}}{2x}$, $c=x$, and $d=\frac{y\mp\sqrt{y^2-4xz}}{2}$ which shows that image of our map contains the set $\{(x,y,z)\in \Bbb R^3\mid x\neq 0, y^2-4xz>0\}$, which is open in the usual topology. So the dimension of the image is three (we're glossing over a bunch of different ways to define definition and their possible equivalence here, hope you don't mind).
I wrote the above answer before your edit discussing the Jacobian loaded. If you want to use the Jacobian to answer this problem, you may refer to the answers here which discuss how locally in the standard topology, a map which has Jacobian of rank $k$ in a neighborhood of a point locally looks like a coordinate projection, and the image has dimension $k$. So this is indeed a valid way to solve your problem, as we again speed past the correct notion of dimension to apply here.
| 2023-11-10T01:26:19.665474 | https://example.com/article/8152 |
Behind a black door just steps from the golden domes of Novodevichy monastery, a group of young men and women sit huddled at computers. They are surrounded by racks of the red and white jumpers and scarves that mark the devoted fans of FC Spartak, Moscow's leading football club.
This is the headquarters of Fratria, the unofficial Spartak fan club that lost one of its members when he was killed during a brawl with a gang from the Caucasus, the restive mainly Muslim region on Russia's southernmost flank.
The shooting of Yegor Sviridov on 6 December has sparked the worst race riots Moscow has seen since the fall of the Soviet Union. The killing and beating of immigrants has increased, racist and anti-Semitic graffiti has proliferated, and the atmosphere is tense.
For the past week, groups of roaming youth have taken to the streets daily, shouting "Russia for Russians! Moscow for Muscovites!" The youths manning the shop at Fratria insist they are not involved. "We only support legal, peaceful forms of protest," says Lena Sekhina, the club's press secretary.
Russia has a growing racism problem, with nationalist feelings increasingly stoked by the government since the collapse of the Soviet Union left an ideological vacuum. Yet many are beginning to wonder why tensions have finally boiled over. There are the conspiracy theories that say the unrest was stoked by the government of Vladimir Putin to prove the need for his authoritarian rule.
Others wonder whether nationalist groups are taking advantage of the weakened political climate following the dramatic firing of Moscow's veteran mayor, Yury Luzhkov, two months ago.
For Yevgeny Valyaev the cause is clear. "It's not one death. It's a pressure that's been building for several years," said the shaven-headed leader of Russky Obraz, an ultra-nationalist group that helped gather some of the 5,000 men who descended upon the Kremlin on 11 December, launching the current unrest. "There was no way we could not gather – because he was a football fan and because he was Russian," he said, referring to Sviridov. "It was a protest against ethnic banditry."
The link between ultra-nationalism and football has flourished in Russia, which boasts some of the rowdiest supporters in Europe. On Friday, UEFA fined Spartak €75,000 (£63,600) after its supporters invaded the pitch during a Champions League match away to Zilina in Slovakia. Several Russian clubs have been fined over the years after supporters waved racist banners referring to African players as monkeys.
It is a problem that has been years in the making. Russia has eight years to prepare for its role as host of the 2018 World Cup. Some are concerned that while eight years may be enough time to build stadiums and travel infrastructure, it may not be enough to transform a national mind-set.
"I have always been a patriot," said Valyaev, a stocky 23-year-old who also helps organise the annual neo-Nazi rally in Moscow. "We think the interests of the race should be above the interests of the government – the government must work for the race. To stop this wave now, people must see that their protests have been heard."
Four days after the Kremlin riot, youths gathered in sites around Moscow, shouting racist slogans and standing off with the hundreds of riot police called in to control them. More than 1,000 people were arrested.
Skinheads were few, outnumbered by adolescent boys and girls who had latched on to the nationalist wave. On Thursday, police arrested three teenagers, including a 14-year-old boy, on suspicion of involvement in the stabbing to death of a Kyrgyz man last weekend.
The unrest has already spread to other cities – St Petersburg, Rostov-on-Don, Samara. All are due to host stadiums for the 2018 World Cup. "For a long time, this hasn't just been a problem for just Spartak fans and football fans," said Ivan Kuznetsov, a spokesman for the All-Russian Union of Fans, referring to the influx of migrants to the Russian capital. "This is a problem for all the youth of Moscow and other cities."
He was referring mainly to men from the Caucasus – Chechnya, Dagestan and the other dozen republics that, while a part of the Russian Federation, have never been culturally integrated.
"Kids who are at school, university, who have girlfriends – they're constantly running up against aggressive Caucasian youth," he said.
The problem is further complicated by the fact that much of Moscow's police force fought in Chechnya to put down the republic's separatist rebellion. The conflict, marked by widespread human rights abuses, left lasting scars. In dealing with the riots, police have avoided harsh tactics and some have even appeared sympathetic.
The football fans and skinheads do not fear them. Still, Valyaev has, for the past three years, carried an air pistol whenever he leaves the house. "I've never had to use it, but I want to make sure if a situation arises where it needs to be used, it is close." | 2024-01-12T01:26:19.665474 | https://example.com/article/6705 |
Background {#Sec1}
==========
Lung cancer is a leading cause of cancer-related death, with a 5-year overall survival rate less than 15% \[[@CR1]\], a significantly lower survival rate than that of most epithelial malignancies. Lung cancer is divided into small cell lung cancer and non-small cell lung cancer (NSCLC). The proportion of the NSCLC type is more than 85%, mainly including lung adenocarcinoma and lung squamous cell carcinoma \[[@CR2], [@CR3]\]. Lung squamous cell carcinoma (LSCC) accounts for approximately 30% of all lung cancers, with a extremely high mortality rate \[[@CR4]\]. Because most lung cancer patients are already in the advanced stage of disease at the time of diagnosis, they have lost the opportunity for surgical treatment, and the prognosis of LSCC has not obviously improved, although the finding of high-frequency mutations in epidermal growth factor receptor (*EGFR*) kinase has led to a dramatic change in the treatment of patients with lung adenocarcinoma \[[@CR5], [@CR6]\], and recent data have indicated that targeting mutations in *BRAF, AKT1*, *ERBB2,* and *PIK3CA* as well as fusions that involve receptor tyrosine kinase genes *ALK*, *ROS1,* and *RET* may also be successful \[[@CR7], [@CR8]\]. Unfortunately, the activating mutations in *EGFR* and *ALK* fusions are limited in lung adenocarcinoma and are not present in LSCC \[[@CR9]\], and targeted agents developed for these activating mutations are largely ineffective in LSCC.
Recent researches have accumulated approximately 29 possible pathogenic genes for LSCC and are widely accepted \[[@CR10]--[@CR12]\]. However, therapeutic drugs targeting these driver genes are lacking. Interestingly, a search of the TCGA database revealed that approximately 30% of LSCCs undergo recurrent mutations in *KEAP1* and *NFE2L2(also named as NRF2)* \[[@CR11], [@CR12]\]. In our previous study, we identified that *KEAP1* and *NRF2* mutations are recurrent in Chinese patients with LSCC, with a 5.8% frequency for *KEAP1* and a 27.9% frequency for *KEAP1/NRF2* mutations. However, mutations in *KEAP1/NRF2* in Chinese patients with lung adenocarcinoma are rarely found, which is consistent with reports from Takahashi T \[[@CR13]\]. Interestingly, *KEAP1* and *NRF2* mutations show mutual exclusive in Chinese patients with LSCC \[[@CR12]\]. *KEAP1* and *NRF2* are the two key genes that regulate the oxidative stress pathway. At physiological homeostasis, NRF2 is bound by the adapter protein KEAP1, which recruits the CUL3 ubiquitin ligase, leading to the proteasomal degradation of NRF2 \[[@CR14]\]. Oxidative stress acts on KEAP1, causing its conformation change and dissociation from NRF2, thereby losing the ability to mediate NRF2 degradation \[[@CR15], [@CR16]\] and leading to NRF2 activation and subsequent antioxidative properties, which is important in maintaining physiological homeostasis. However, it has been reported that NRF2 activation involves in chemotherapy drugs inactivation through rapid metabolism of these drugs in cells, significantly reducing their anti-tumor efficacy \[[@CR17]--[@CR19]\]. More recently, the data have also shown that loss of function of *KEAP1* promotes *KRAS*-driven lung cancer and results in the dependence on glutaminolysis \[[@CR20]\].
Therefore, we aimed to test whether mutations in *KEAP1*, identified in our previous study, accelerate the development of lung cancer, and whether a NRF2 inhibitor can be used as a targeted therapeutic drug in patients with lung cancer carrying *KEAP1/NRF2* mutations.
Materials and methods {#Sec2}
=====================
Cell culture, reagents, and nude mice {#Sec3}
-------------------------------------
The NCI-H1299,A549, H838, H460,H1299, 95D, and SPCA1 human lung cancer cell lines and HEK293T cells were obtained from American Type Culture Collection (Manassas, VA, USA). H1299, H838, H460, H292, 95D, and SPCA1 cells were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA). A549 cells were cultured in F-12 K(Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. Twelve 4--6-week-old male BALB/c nude mice were purchased and reared from the Shanghai Ninth People's Hospital Central Laboratory Animal Law.
Plasmids, site-directed mutagenesis, and stable transfection {#Sec4}
------------------------------------------------------------
Mutations were conducted using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and were validated by sequencing; the primer sequences for mutagenesis are shown in (Supplementary Table [1](#MOESM2){ref-type="media"}). A retrovirus-mediated infection system was used to construct A549 and H460 cells stably over-expressing 3FLAG-tagged KEAP1(WT or mutant). For PMSCV production, DNA encoding 3FLAG-tagged KEAP1 was inserted into the multi-cloning site of the pMSCV vector. Each PMSCV vector was co-transfected with gag-pol and VSVG using Lipofectamine 2000(Invitrogen,Waltham, MA, USA) in 293 T cells. The virus was collected 2 days later and was transfected into A549 and H460 cells. The infected cells were selected with 1 μg/mL (A549) or 0.5 μg/mL (H460) of puromycin for 3--4 weeks.
Gene editing using CRISPR/Cas9 system {#Sec5}
-------------------------------------
Target-specific guide RNA within NRF2 gene locus was designed on CRISPRDESIGN (<http://crispr.mit.edu/>). The following target sgRNA sequences were used in this study:sgRNA-F 5′-TGCCTGTAAGTCCTGGTCAT-3′, sgRNA-R 5′-TCTCTGGTGTGTTCTCACAT-3′. Igonucleotides for guide RNA were inserted into CRISPR Nuclease vector and then the vector was transfected into A549 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Specific lentivirus transfecting method was the same as above. Finally, 3 homozygous knockout cell lines were selected (Supplementary Fig. [1](#MOESM2){ref-type="media"}a).
Western blot and immunoprecipitation analyses {#Sec6}
---------------------------------------------
The antibodies used in our study were as follows: anti-FLAG M2 monoclonal (Sigma, St. Louis, MO, USA), anti-NRF2 (Abcam, Cambridge, UnitedKingdom), anti-β-actin (Cell Signaling,Danvers, MA, USA), anti-HO-1 (Cell Signaling), anti-LaminB1 (Cell Signaling), anti-Lamin A/C (Cell Signaling). The Nuclear and Cytoplasmic Protein Extraction kit was obtained from (Thermo Fisher Scientific, Waltham, MA, USA). For immunoprecipitation, Whole cell lysate (WCL) was used respectively as the negative control. Cell lysates were cleared by centrifugation and were incubated with FLAG resin (Sigma) before washing with lysis buffer, followed by overnight incubation at 4 °C. After washing three times by 1× phosphate-buffered saline (PBS), the precipitates were analyzed by immunoblotting.
Real-time quantitative PCR {#Sec7}
--------------------------
Total RNA was prepared from cells using Trizol reagent (Invitrogen), and tumors using Animal Total RNA Isolation kit (Sangon Biotech,Shanghai, China) and reverse transcription were performed using the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio,Shiga, Japan). The sequences for each primer are listed in Supplementary Table [2](#MOESM2){ref-type="media"}.
ROS measurement {#Sec8}
---------------
After the cells were washed with PBS twice, the cells were incubated with 1 μM CM-H2DCF-DA (Nanjing Jiancheng, Nanjing, China) in culture conditions for 30 min in the dark and then were trypsinized and collected. The ROS levels were examined by flow cytometry.
Colony-formation assay {#Sec9}
----------------------
Exponentially growing cells were counted,diluted, and seeded in triplicate at 800--1000 cells/well in 6-well plates. To assess clonogenic survival following drug exposure, the cell cultures were incubated in complete growth medium at 37 °C for 11--14 days. Colonies were fixed with precooled methanol and then were stained with 0.5% (w/v) crystal violet for 30 min at room temperature, followed by washing with PBS, photographing and counting. Only colonies with more than 50 cells were counted \[[@CR21]\].
Wound-healing assay {#Sec10}
-------------------
Cell motility was determined by measuring the movement of cells close to an artificial wound. Cells were wounded with a 200-μL pipette tip, washed with PBS, and incubated in F12-K or RPMI 1640 medium without FBS. The distances removed by cells were monitored by microscopy at the indicated time points. The scratched area was analyzed using ImageJ.
Tumor xenograft model {#Sec11}
---------------------
Twelve BALB/c nude mice (4--6 weeks old, male) were randomly assigned to two groups (WT or mutant). We infected A549-*KEAP1*-WT or A549-*KEAP1*-R320W cells (1 × 10^8^) subcutaneously into the right flank of BALB/c nude mice and measured the tumor dimensions by calipers every 3--4 days. The tumor volumes were calculated using the formulalength \[(mm) × width (mm)^2^\]/2 \[[@CR22]\]. All experimental protocols conducted on mice were performed in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Shanghai Jiaotong University Animal Care and Use Committee.
Statistical analysis {#Sec12}
--------------------
All experiments were performed in quadruplicate and were repeated at least three times with similar results unless otherwise indicated. All statistical analyses were performed using unpaired two-tailed Student's t-test and the mean ± standard error of the mean. A *P*-value of 0.05 or less was considered statistically significant. These analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) or GraphpadPrism 7 (GraphpadSoftware, San Diego, CA, USA).
Results {#Sec13}
=======
The KEAP1/NRF2 pathway was activated in lung cancer cell lines with *KEAP1* mutations {#Sec14}
-------------------------------------------------------------------------------------
To explore the effect of *KEAP1* loss on activation of the KEAP1/NRF2 pathway, we collected three NSCLC cell lines with *KEAP1* mutations (A549, NCI-H460, and NCI-H838) and four NSCLC cell lines without *KEAP1/NRF2* mutations (NCI-H1299, NCI-H292, 95D, and SPCA1). In the present study, we have found the frequency of *KEAP1/NRF2* mutation was higher in human LSCC than that in LUAD patients, thus we hope find the LSCC cell lines with *KEAP1* mutation to investigate the effect of *KEAP1* mutation on the function of *KEAP1/NRF2* pathaway. However, after serching the literatures and cell line database, we found none LSCC cell lines carried *KEAP1/NRF2* mutation. We further collected all available 7 lung cancer cell lines and found three of them carried the *KEAP1* mutation (A549, NCI-H460, NCI-H838), thus, in the present study, all the seven lung cancer cell lines were used to study the influence of *KEAP1* mutation on the function of *KEAP1/NRF2* pathway. As expected, Sanger sequencing revealed that the A549, H460, and H838 cell lines carried homozygous point mutations at D236H, G333C, and E444\* in *KEAP1*, respectively; however, H1299, NCI-H292, 95D, and SPCA1 cell lines did not carry mutations in neither *KEAP1* nor *NRF2***(**Fig. [1](#Fig1){ref-type="fig"}a). Next, we found that the mRNA expression of downstream target genes of the KEAP1/NRF2 pathway, such as *GCLC*, *GCLM*, *TXN*, *TXNRD*, *HO1*, *NQO1*, *GSR,* and *G6PD*, which encode detoxifying enzymes and antioxidant proteins, were significantly higher in *KEAP1* mutant cell lines than in wild-type (WT) lung cancer cells by real-time polymerase chain reaction (PCR), but the mRNA expression of *NRF2* showed no significant difference between lung cancer cell lines with and without *KEAP1* mutation **(**Fig. [1](#Fig1){ref-type="fig"}b**)**. Given that NRF2 protein translocates into the nucleus to activate the transcription of downstream target genesin the KEAP1/NRF2 pathway, we further examined the protein expression of nuclear NRF2 and the downstream target HO-1 in these tumor cells by western blot analysis. As expected, function loss of *KEAP1* significantly increased nuclear NRF2 levels and HO-1 levels incytoplasm (Fig. [1](#Fig1){ref-type="fig"}c). Thus, *KEAP1* loss decreased the degradation of NRF2 to activate the KEAP1/NRF2 pathway. Fig. 1The KEAP1/NRF2 pathway was activated in lung cancer cell lines with loss-of-function mutations in *KEAP1*. **a** The mutations of *KEAP1* in A549, H460, and H838 cells were detected by Sanger sequencing. The red rectangle indicates the mutation sites of *KEAP1*. **b** Compared with lung cancer cells with neither *KEAP1* nor *NRF2* mutation, the expression levels of *NRF2* target genes were significantly increased in lung cancer cells with *KEAP1* mutation. **c** The protein expression levels of NRF2 and its target protein HO-1 were increased in lung cancer cells with *KEAP1* mutation compared with those in lung cancer cells with neither *KEAP1* nor *NRF2* mutation. **d***KEAP1* mutation suppressed ROS production in lung cancer cells. The fluorescence intensity in cells was detected by flow cytometry. Quantitative analysis of the mean fluorescence intensity using unpaired two-tailed Student's t-test. The results are expressed as mean ± standard error of the mean. \* *P* \< 0.05, \*\* *P* \< 0.01, \*\*\* *P* \< 0.001, and \*\*\*\* *P* \< 0.0001
Activation of the KEAP1/NRF2 pathway decreases intracellular reactive oxygen species (ROS) levels through upregulating the expression of detoxifying enzymes and antioxidant genes. Thus, we checked whether *KEAP1* loss could decrease ROS production in lung cancer cell lines. As shown in Fig. [1](#Fig1){ref-type="fig"}d, ROS levels were significantly decreased in lung cancer cells with *KEAP1* mutation. Collectively, these findings demonstrate that *KEAP1* loss can enhance the ability of lung cancer cells to resist oxidative stress.
Mutations in *KEAP1* identified in Chinese patients with lung cancer promoted tumorigenesis via activation of the KEAP1/NRF2 pathway in lung cancer cells {#Sec15}
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In our previous study, we identified five nonsynonymous mutations in *KEAP1* from five patients with LSCC. However, the role of these *KEAP1* mutations in tumorigenesis is unclear. The function these five nonsynonymous mutations in *KEAP1* were predicted by *Polyhen2_HDIV* and *SIFT*. The results showed that these five nonsynonymous mutations in *KEAP1* were harmful, affecting protein function (Table 1).
To verify whether these five somatic mutations in *KEAP1* influence the function of *KEAP1*, WT and five *KEAP1* mutants were stably transfected with retroviral vectors into A549/H460 lung cancer cell lines that carry loss-of-function mutations in *KEAP1*. As expected, nuclear NRF2 protein levels and expression of the NRF2 target gene *HO-1* were significantly decreased after A549 or H460 lung cancer cell lines were stably transfected with WT *KEAP1* (Fig. [2](#Fig2){ref-type="fig"}a). However, the levels of nuclear NRF2 protein and expression of NRF2 target gene *HO-1* showed no significant difference after A549 or H460 lung cancer cell lines were stably transfected with these five *KEAP1* mutants (Fig. [2](#Fig2){ref-type="fig"}a). These lung cancer cell lines that carry loss-of-function mutations in *KEAP1*, were losing the ability to mediate NRF2 degradation. Activated NRF2 will be degradated when WT *KEAP1* overexpression. Fig. 2*KEAP1* mutation promoted the activity of lung cancer cell lines in vitro. **a** Compared with A549 or H460 lung cancer cell lines transfected with empty vector, expression levels of nuclear NRF2 and its target protein HO-1 were significantly decreased in A549 or H460 cells transfected with wild-type (WT) *KEAP1* by western blot analysis. The levels of nuclear NRF2 and its target gene HO-1 showed no significant difference after A549 or H460 cells were transfected with the five *KEAP1* mutants. **b** The mRNA expression levels of *NRF2* and its target genes were significantly decreased after the cell lines were stably transfected with WT *KEAP1*. However, the mRNA levels of *NRF2* and its target genes *HO-1*, *GCLC,* and *FTH1* were significantly increased in A549 or H460 lung cancer cell lines stably transfected with the five *KEAP1* mutants. **c** Position of the mutation in the KEAP1 protein. Some mutants (R174L, R234W, and R320Q) enhanced NRF2 binding, while others (R413L and D749H) weakened NRF2 binding. Flag-tagged KEAP1 was co-expressed in A549 cells. Whole cell lysate (WCL) was used respectively as the negative control. Immunoprecipitation experiments were performedusing anti-flag M2 beads, and immunoblot analysis was performed using anti-flag and anti-NRF2 antibodies. **d** Colony-formation assay showed that the proliferation of A549/H460 lung cancer cell lines stably transfected with WT *KEAP1* was significantly decreased, while that of cells transfected with mutant *KEAP1* was significant increased. **e** The scratch wound-healing assay showed that the migration of A549/H460 cells stably transfected with mutant *KEAP1* was faster at 0 h, 24 h, 48 h, and 72 h than that of A549/H460 cells transfected with WT *KEAP1*. Scratch area quantitative analysis by Image J software. Each assay was repeated at least three times. Mean ± standard error of the mean (SEM) are reported (\* *P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001)
Compared with A549 or H460 lung cancer cell lines stably transfected with empty vector*,* mRNA levels of *NRF2* and its target genes *HO-1*, *GCLC,* and *FTH1* were significantly decreased after the cell lines were stably transfected with WT *KEAP1* (Fig. [2](#Fig2){ref-type="fig"}b). However, mRNA levels of *NRF2* and its target genes *HO-1*, *GCLC,* and *FTH1* were significantly increased in the A549 or H460 lung cancer cell lines stably transfected with these five *KEAP1* mutants (Fig. [2](#Fig2){ref-type="fig"}b). Together, these data suggested that these five nonsynonymous mutations in *KEAP1,* derived from Chinese patients with LSCC, were loss-of-function mutations that upregulated the expression of detoxifying enzymes and antioxidant genes.
*KEAP1* is located at 19p13.2 and its protein has three major domains: an N-terminal broad complex, tramtrack, and the bric-a-brac (BTB) domain; a central intervening region (IVR); and a series of six C-terminal Kelch repeats \[[@CR23]\]. The Kelch repeats of KEAP1 bind the NEH2 domain of NRF2 \[[@CR24]\], whereas the IVR and BTB domains are required for the redox-sensitive regulation of NRF2 through a series of reactive cysteines present throughout this region \[[@CR14], [@CR25]\]. In our present study, we found somatic mutations at R320Q, R413L, and D479H in the Kelch repeat domains of KEAP1, a somatic mutation at R234W in the IVR domain, and a somatic mutation at F174L in the BTB domain of KEAP1 (Fig. [2](#Fig2){ref-type="fig"}c). The binding of KEAP1 mutants to NRF2 was detected in coimmunoprecipitation experiments. Interestingly, mutants at R413L and D479H in the Kelch repeat domain of *KEAP1* did not bind to NRF2 (Fig. [2](#Fig2){ref-type="fig"}c). However, compared with WT KEAP1, binding of the mutants at F174L in the BTB domain and at R234W in the IVR domain of NRF2 was significantly increased (Fig. [2](#Fig2){ref-type="fig"}c). Unexpectedly, binding of NRF2 to the *KEAP1* mutants at R320Q in the Kelch repeat domain was not affected (Fig. [2](#Fig2){ref-type="fig"}c).
To uncover whether these five somatic mutations in KEAP1 influence the biological behavior of lung cancer cells, cell proliferation and migration were detected by colony-formation and scratch experiments. Compared with A549/H460 lung cancer cell lines stably transfected with retroviral empty vector, the colony formation and migration of A549/H460 lung cancer cell lines stably transfected with WT KEAP1 were significantly decreased (Fig. [2](#Fig2){ref-type="fig"}d, e). However, after being stably transfected with KEAP1 mutants, the colony formation and migration of A549/H460 lung cancer cell lines significantly increased (Fig. [2](#Fig2){ref-type="fig"}d, e). To further validate that their mechanism were really passed through the NRF2 pathway, we knockdown NRF2 using double sgRNAs and detected the expression of well established downstream genes of NRF2. As shown in (supplementary Fig. [1](#MOESM2){ref-type="media"}b, c), the expression of NRF2 target gene HO-1protein level was significantly decreased and the mRNA level of HMOX1,HO-1, GCLC, NQO1, FTH1, NRF2 when NRF2 was knockdown. As expected (supplementary Fig. [1](#MOESM2){ref-type="media"}d, e), the reduced number of colonies and migration in cells knockouted with NRF2.These data suggest that the newly found somatic mutations in KEAP1 promote tumor cell activity through activating NRF2 antioxidant stress signaling pathways.
The somatic mutation at R320Q in *KEAP1* accelerated tumor growth in vivo {#Sec16}
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Due to we have considered that R320Q mutant has a considerable effection to KEAP1'S function, the ability of the antioxidative stress or proliferation and migration was significantly increased. Therefore, we choosed the R320Q mutant for our next experiments. To further examine the effect of the somatic mutations in *KEAP1* on the growth of lung cancer cell lines in vivo, the A549 cell lines stably transfected with WT *KEAP1* or the R320Q mutant of *KEAP1* were grafted subcutaneously into 4 to 5-week-old nude mice. After the cancer cell lines were grafted subcutaneously into nude mice, tumor sizes were measured using Vernier caliperseach day for 4 days. After 45 days of subcutaneous engraftment, tumors were peeled from the subcutis of the nude mice. The tumor sizes of the A549 cell line stably transfected with the R320Q *KEAP1*mutant were significantly larger than those of the A549 cell line stably transfected with WT *KEAP1***(**Fig. [3](#Fig3){ref-type="fig"}a). Additionally, the tumor growth of the A549 cell line stably transfected with the R320Q *KEAP1* mutant was strongly accelerated compared with that of the A549 cell line stably transfected with WT *KEAP1*, as measured by the change in tumor volume **(**Fig. [3](#Fig3){ref-type="fig"}b**).** Consistent with the in vitro results, the *KEAP1* mutant showed significantly accelerated tumor growth in vivo. These results indicate that *KEAP1* likely is a novel tumor driver gene for LSCC. Fig. 3The somatic mutation R320Q in *KEAP1* accelerates tumor growth in vivo. **a** Tumor volumesof nude mice subcutaneously injected with A549 cells stably transfected with the *KEAP1* mutant (R320Q) were significantly larger than those injected with A549 cells transfected with wild-type (WT) *KEAP1*. Twelve 4--6-week-old male BALB/c nude mice were separated into two groups and injected with A549 cells transfected with WT *KEAP1* or mutant *KEAP1* (R320Q). Solid tumors were peeled from mouse subcutaneous tissues 7 weeks after injection. **b** Tumor growth was significantly faster in nude mice subcutaneously injected with A549 cells stably transfected with mutant *KEAP1* (R320Q) than those with A549 cell transfected with WT *KEAP1*. **c** The expression of *NRF2* and its target genes in xenograft tumors from A549 cells stably transfected with mutant *KEAP1* (R320Q) were higher than that from A549 cells transfected with WT *KEAP1*. Total RNA of xenograft tumors was extracted, and the indicated mRNA levels were determined by real-time PCR. **d** The nuclear protein levels of NRF2 and its downstream target protein HO-1 were increased in *KEAP1*-mutant (R320Q) xenograft tumors. Mean ± SEM are reported (\* *P* \< 0.05, \*\* *P* \< 0.01, \*\*\* *P* \< 0.001)
Next, we examined the expression of oxidative stress-related genes in the grafted tumor tissues from the nude mice. Compared with the expression of NRF2 in the nucleus and its target protein HO-1 in the cytoplasm in tumor tissues from the A549 cell line transfected with WT *KEAP1,* the expression levels of NRF2 and its target protein HO-1 were significantly increased in the tumor tissues from the A549 cell line transfected with the R320Q *KEAP1* mutant **(**Fig. [3](#Fig3){ref-type="fig"}d**).** The mRNA levels of *NRF2* and its target genes *HMOX1*, *HO-1*, *GCLC,* and *NQO1* in the grafted tumor tissues from the A549 cell line transfected with the R320Q *KEAP1* mutant were remarkably increased compared with that in grafted tumor tissues from the A549 cell line transfected with WT *KEAP1*(Fig. [3](#Fig3){ref-type="fig"}c**)**.
The NRF2 inhibitor ML385 inhibited proliferation of lung cancer cells carrying *KEAP1* mutations {#Sec17}
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Increased cellular oxidative stress levels by small-molecule compounds to enhance cytotoxicity have been identified as a viable cancer treatment strategy \[[@CR26]\]. High levels of ROS not only inhibit cancer cell proliferation but also trigger apoptosis. A subset of NRF2 inhibitors has been reported to inhibit the proliferation of cancer cells by down-regulating the expression of *NRF2*, resulting in elevated levels of intracellular ROS and increased cytotoxicity. However, it is unknown whether NRF2 inhibitors have different effects on these LSCCs with or without *KEAP1* somatic mutations. Thus, we selected an effective NRF2 inhibitor, ML385, which specifically and directly interacts with NRF2 protein, blocks *NRF2* transcriptional activity, and enhances the efficacy of carboplatin and other chemotherapeutic drugs in lung cancer cells \[[@CR27]\]. The lung cancer cell line A549 transfected with the R320Q *KEAP1* mutant (*KEAP1-*R320Q mutant) or with WT *KEAP1* (*KEAP1-WT*) and H1299 lung cancer cells, which carry both WT *KEAP1* and *NRF2*, were selected and treated with ML385. The number of formed colonies was decreased in all three groups in a dose-dependent manner with increased ML385 treatment **(**Fig. [4](#Fig4){ref-type="fig"}a). Interestingly, when the lung cancer cell lines were treated with ML385 at a low dose, such as 0.25 or 0.5 μM/L, the proliferation of theA549 lung cancer cell line transfected with the R320Q *KEAP1* mutant showed more significant inhibition than that of A549 cells transfected with WT *KEAP1* or that of H1299 lung cancer cells **(**Fig. [4](#Fig4){ref-type="fig"}a, b). The proliferation of lung cancer cell lines showed no significant difference between A549 cells transfected with WT *KEAP1* and H1299 lung cancer cells without *KEAP1* and *NFR2* mutation **(**Fig. [4](#Fig4){ref-type="fig"}a, b). These results suggest that lung cancer cell lines with *KEAP1* mutations may have higher sensitivity to ML385 treatment. Fig. 4NRF2 inhibitor ML385 effectively inhibited proliferation of lung cancer cells carrying *KEAP1* mutations. **a** The number of formed colonies was decreased in the three groups as the dose of ML385 increased. When the lung cancer cell lines were treated with ML385 at a low dose (0.5 or 1.0 μM/L) the proliferation of A549 lung cancer cells transfected with the R320Q *KEAP1* mutant was more significantly inhibited than that of A549 cells transfected with wild-type (WT)*KEAP1* or that of H1299 lung cancer cells, which carried neither *KEAP1* nor *NRF2* mutation. Three groups of cells were treated with different doses of ML385 for 72 h. At the end of treatment, the medium was added and cells were further incubated for 8--10 days and stained with crystal violet. **b** The number of formed coloniesrapidly deceased in A549 cells transfected with the R320Q *KEAP1* mutant after treatment with ML385 compared with that in A549 cells transfected with WT *KEAP1* or H1299 lung cancer cells. Clonal formation rate = effective clones/plating cell numbers × 100% (\**P* \< 0.05). **c** Protein levels of NRF2 and HO-1 were decreased in *KEAP1* mutant cells after drug intervention. Total protein was extracted after 3 days of treatment with 1 μM ML385. **d** The mRNA levels of NRF2 and HO-1 were decreased in *KEAP1* mutant cells. Total RNA was extracted after 3 days of treatment with 1 μMML385. Mean ± SEM are reported (\* *P* \< 0.05, \*\* *P* \< 0.01, \*\*\* *P* \< 0.001)
To further detect whether the effect of ML385 on lung cancer cell proliferation is mediated by inhibiting the KEAP1/NRF2 pathway, the expression of *NRF2* and its target genes *HO-1, GCLC* and *NQO1* in lung cancer cell lines treated with ML385 were investigated by western blot and real-time PCR. As shown in Fig. [4](#Fig4){ref-type="fig"}c, the expression levels of HO-1 and NRF2 protein in A549 lung cancer cells transfected with F174L, R234W, R320Q, and R413L *KEAP1* mutants were significantly inhibited by ML385. However, the expression levels of HO-1 and NRF2 protein showed no significant difference between A549 transfected with WT *KEAP1* treated with and without ML385 (Fig. [4](#Fig4){ref-type="fig"}c). Although mRNA expression levels of *NRF2* and its target gene *NQO1* in A549 cells transfected with WT *KEAP1* were significantly inhibited by ML385, the mRNA expression levels of the *NRF2* target genes *GCLC* and *HO-1* were not influenced by ML385 (Fig. [4](#Fig4){ref-type="fig"}d). Notably, the mRNA expression levels of *NRF2* and its target genes *HO-1*, *GCLC,* and *NQO1* in A549 cells transfected with F174L, R234W, R320Q, and R413L *KEAP1* mutants were dramatically decreased after treatment with ML385 (Fig. [4](#Fig4){ref-type="fig"}d).
Discussion {#Sec18}
==========
Recently, high-frequency somatic mutations of *KEAP1* and *NRF2* in the oxidative stress response pathway have been identified in patients with NSCLC by large-scale genomic studies \[[@CR28]\]. Previous studies have revealed that *NRF2* is involved in cancer development, especially lung cancer \[[@CR29]--[@CR32]\]. In addition, recent evidence suggests that in mouse models of lung cancers, activated Nrf2 inhibits the Fbxo22-dependent degradation of Bach1 via induction of Ho-1 expression, and high levels of Bach1 promoting metastasis \[[@CR33]\]. The loss-of-function mutation of *KEAP1* promoted the tumorigenesis of *Kras*- and *Pten*-driven lung cancer cell lines in mice \[[@CR20], [@CR34]\]. Moreover, the KEAP1/NRF2 pathway synergized with *TP53* deletion mutation could induce LSCC and radiation resistance \[[@CR22]\]. However, no evidence that *KEAP1* or *NRF2* mutations identified in lung cancer patients are involved in tumorgenesis has been reported. In the present study, we found that the ability of the antioxidative stress response mediated by activation of the KEAP1/NRF2 pathway is higher in lung cancer cell lines with *KEAP1* mutation (A549, NCI-H460, NCI-H838) than in lung cancer cell lines without *KEAP1* mutation (NCI-H1299, NCI-H292, 95D, and SPCA1). Interestingly, compared with the lung cancer cell lines A549 and H460, which carry *KEAP1* mutation, after stable transfection with WT *KEAP1*, the mRNA levels of *NRF2* and its target genes are significantly increased in A549 and H460 lung cancer cell lines transfected with *KEAP1* mutants. Moreover, colony formation and migration were increased in A549 and H460 lung cancer cell lines transfected with *KEAP1* mutants*.* Similarly, the grafted subcutaneous tumor sizes in nude mice were significantly larger in A549 cells transfected with the R320Q *KEAP1* mutant than those inA549 cells transfected with WT *KEAP1*. These data suggest that the somatic mutations of *KEAP1* identified in Chinese patients with LSCC likely promote the development of lung cancer through activation of the antioxidative stress response in the KEAP1/NRF2 pathway.
Although genomic analysis identified some high-frequency gene mutations from LSCC, such as *TP53*, *PI3KCA*, and *SOX2*, no clear operationable targets for the treatment of LSCC have been found thus far \[[@CR11], [@CR12], [@CR35]\]. Practically, molecular targeted therapy improves the survival of patients with lung adenocarcinoma, but no effective targeted drugs have been identifiedin clinical trials for LSCC \[[@CR36]\]. Convenient treatment of LSCC remains to be platinum-based chemotherapy, added with immune checkpoint inhibitors that emerged recently and has brought certain benefits for the treatment of LSCC \[[@CR37]\]. Additionally, cancer cells acquire novel nutrient dependencies to support oncogenic growth by changing metabolic pathways, Sarah E first points that dietary restriction or enzymatic depletion of asparagine can lead to suppression of Keap1 mutant tumor growth \[[@CR38]\]. However, the overall effects of therapy for LSCC remain grim, revealing the immediate need for an effective treatment. In the present study, we found that the loss-of-function mutation in*KEAP1* promotes the development of lung cancer mediated by activation of the KEAP1/NRF2 pathway. Thus, it is tempting to presume that inhibitors of the KEAP1/NRF2 pathway will likely treat Lung cancer patients carrying *KEAP1/NRF2* mutations. To date, some inhibitors of the KEAP1/NRF2 pathway have been identified, such as NRF2 inhibitors that inhibit this pathway: ML385 \[[@CR27]\], Brucea chinensis \[[@CR39]\], clonazepa propionate \[[@CR40]\], luteolin, all-trans retinoic acid, and flavonoid molecular compounds \[[@CR41], [@CR42]\]. The NRF2 inhibitor ML385 blocks activation of the pathway by inhibiting *NRF2* expression. We further explored the effect of ML385 on the development of lung cancer cell lines with or without *KEAP1* mutations. Compared with A549 lung cancer cell lines trasfected with WT *KEAP1*, proliferation of the A549 lung cancer cell line trasfected with the R320Q *KEAP1* mutant was dramatically inhibited by ML385. These preliminary datas suggest that ML385 inhibits the proliferation of lung cancer cells with *KEAP1* mutations by blocking the KEAP1/NRF2 antioxidant stress response pathway. It will provide a new option for therapy targeted to the KEAP1/NRF2 pathway in patients with LSCC carrying the *KEAP1* mutation.
Conclusion {#Sec19}
==========
In summary, high-frequency mutation in *KEAP1* has been identified in Chinese patients with LSCC. The somatic nonsynonymous mutations in *KEAP1* derived from patients with lung cancer likely promote tumorigenesis via activation of the KEAP1/NRF2 antioxidant stress response pathway. Notably, NRF2 inhibitor ML385 may inhibit the proliferation of tumor cells with *KEAP1* mutation by enhancing the oxidative stress level of lung cancer cells.
Supplementary information
=========================
{#Sec20}
**Additional file 1: Supplementary Fig. 1**. (A). Gene editing methods in A549 lung cancer cell lines with NRF2 homozygous knockout (The yellow part represents target sequences; the dotted line represents the base sequence of the knockout) (B). Compared with A549, expression NRF2 and its target protein HO-1 were significantly decreased in A549 with NRF2 knockout by western blot analysis. (C). The mRNA expression levels of *NRF2* and its target genes were significantly decreased after the cell lines were knockout NRF2.(D) Colony-formation assay showed that the proliferation of A549 lung cancer cell lines depleted with NRF2 was significantly decreased.(E) The scratch wound-healing assay showed that the migration of A549 lung cancer cell lines depleted with NRF2 was slower at 0 h,24 h, and 48 h than that of A549 cell lines. Mean ± standard error of the mean (SEM) are reported (\* *P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001).
NRF2/ *NFE2L2*
: A nuclear factor erythroid 2-related factor 2
NSCLC
: Non-smallcell lung cancer
LSCC
: Lung squamous cell carcinoma
EGFR
: Epidermal growth factor receptor
WT
: Wild-type
*Polyhen2_HDIV*
: <http://genetics.bwh.harvard.edu/pph2/>
*SIFT*
: <http://sift.jcvi.org/>
ROS
: Reactive oxygen species
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Meiling Gong and Yan Li contributed equally to this work.
Supplementary information
=========================
**Supplementary information** accompanies this paper at 10.1186/s12964-020-00568-z.
Not applicable.
ML-G, Y-L and LL-Z were major contributor in writing the manuscript. CX-Z was Corresponding Author. The authors read and approved the final manuscript.
This work was supported by grants from the National Natural ScienceFoundation of China (No. 81772460 and 81472177).
Not applicable.
Experiments with Nude mouse tumor formation experiment were carried out under the ethical approval of Research Ethics Committee of the Shanghai Ninth People's Hospital Central Laboratory Animal Law.
Not applicable.
The authors declare that they have no competing interests.
| 2023-08-12T01:26:19.665474 | https://example.com/article/8211 |
Man charged in death tied to heroin use
A 29-year-old Powell man has been charged with negligent homicide on allegations that he provided heroin that led to the death of his neighbor.
Christopher J. Nichols was arrested Friday night as part of a Powell Police Department investigation into the Dec. 4 death of Jeremy W. Ernest, 30.
Ernest was initially taken to Powell Valley Hospital, unconscious and vomiting, around 1:44 a.m. and a roommate overheard a doctor saying he had opiates, a group of drugs that includes heroin, in his system, according to an affidavit from Powell Police Investigator Mike Hall submitted in the case.
Ernest was released from the hospital at 5:10 p.m., but less than an hour and a half later, another 911 call was placed from Ernest’s East Sixth Street apartment complex reporting he was struggling to breathe, Hall wrote.
Hall’s summary of the investigation suggests that after leaving the hospital the first time, Ernest may have ingested black tar heroin — an illegal and highly addictive opiate and depressant.
Ernest was pronounced dead at the hospital at 7:22 p.m.
No obvious cause of death was found in an autopsy and toxicology results will likely not be available for three or four weeks, said Park County Coroner Tim Power. Hall says hospital records indicate Ernest had opiates, benzodiazepines and THC — the active ingredient in marijuana — in his system.
Nichols has been charged with delivering the heroin to Ernest, a felony, and negligent homicide, a misdemeanor charge that alleges Nichols caused Ernest’s death “by conduct amounting to criminal negligence.” Hall quotes Ernest’s roommate, Morgan Wiles, as saying Nichols was slow to call 911 the two times Ernest collapsed and quotes two unnamed informants as saying Nichols admitted he’d provided Ernest with heroin.
Nichols claimed to have warned Ernest that the heroin was strong and he shouldn’t use it all at once, Hall wrote of the informants’ accounts. They recalled Nichols saying “‘the stupid son of a (expletive) wouldn’t listen to me’ or words to that effect,” Hall wrote.
Nichols also faces a third charge of illegally possessing a felony-level amount of anabolic steroids; police allegedly found more than .3 grams of the substance in a safe in Nichols’ bedroom, making the count a felony.
At an initial Monday afternoon appearance in Circuit Court, Nichols acknowledged having the steroids, but said the negligent homicide and heroin delivery charges “were out of the ballpark” and later added “I just think that this is ridiculous.”
In arguing for a lower bond than the $40,000 figure requested by a prosecutor, Nichols said he works as a server, goes to Northwest College as a full-time nursing student and attends church in Clark.
Nichols said unspecified attorneys have told him the individuals referenced in the affidavit are drug addicts or dealers.
“I came out here and I turned my life around, sir, like, been doing good and they (Ernest and Wiles) come over always trying to party and when all this started happening, like, I actually called 911 both times,” Nichols said in a lengthy statement. “Morgan (Wiles) never did anything.”
When Ernest became sick in the early morning hours of Dec. 4, Nichols wanted Wiles to drag Ernest back to his own apartment next door before calling police, saying his place was “dirty” and that he couldn’t have cops there, Hall wrote of Wiles’ account.
Nichols did call 911, though when officers arrived, he “took an abnormally long time answering the door for an emergency call,” Hall wrote.
Wiles said while at the hospital, he found a small black cellophane-wrapped ball in Ernest’s pocket; Wiles put it back, but told Hall he later argued with Ernest about not using the drug anymore. A cellophane wrapper would be found by police later that night — empty — in Ernest’s bathroom, Hall said.
Wiles told Hall he went to his girlfriend’s residence after Ernest’s initial release from the hospital. Shortly after that, Wiles said his girlfriend got a call from Nichols saying they needed to come back to the apartment for Ernest. Wiles told police he found Ernest unconscious and again insisted Nichols call 911.
Police searched Nichols’ apartment on the morning of Dec. 5 and found steroids but no heroin, according to court records; Hall quotes one of the unnamed informants as saying Nichols had confided that he’d cleaned out his apartment after Ernest’s death.
After arresting Nichols at his workplace at around 9 p.m. Friday, “I started asking Nichols why it took him so long to call 911 for Ernest and why he called Wiles’ girlfriend instead,” Hall wrote. “Nichols didn’t have an answer, but said if we were going to talk about Ernest, he needed an attorney.”
Hall said when he later searched Nichols’ phone, he found all the call and text records from Dec. 4 and the days before were gone. Hall said he did find photos showing marijuana, what looked like crystal methamphetamine, a computer screen referencing heroin and morphine and an image of Nichols holding a piece of foil with a black substance.
Speaking generally of heroin in a Tuesday interview, Powell Police Chief Roy Eckerdt said “we hear of it, but we don’t see it very often.”
“Heroin is making a comeback nationwide,” he added. “It was kind of replaced or off the radar for awhile with meth and then that became harder to get and we saw the upsurge in prescription (medication abuse), and now with the focus on prescription meds and the lack of availability of other drugs, heroin’s making a comeback.”
In court on Monday, Deputy Park County Attorney Tim Blatt cited Nichols’ limited ties to the area (he’s been here a year), some past misdemeanor criminal charges and a risk to the community in asking Circuit Court Judge Bruce Waters for a $40,000 bond.
“Anytime you have a delivery of a controlled substance, that’s a community danger and I think in this particular case, it shows exactly what that danger is to the community,” Blatt said.
Nichols said he was not a flight risk, wanted to complete his Northwest College finals this week and said he’d been “nothing but cooperative” with law enforcement.
“I willingly gave them information, my cell phone, all this stuff,” he said.
Waters went with the prosecutor’s recommendation.
“All these sort of add up to a pretty serious situation, a very concerning situation,” the judge said.
A preliminary hearing, where Waters will weigh if there’s enough evidence for the case to proceed to a trial, has tentatively been set for Monday, Dec. 16.
17 comments
posted by TIP
December 30, 2013 2:29 am
My little brother came back to Powell to help me take care of my little boys. He came here sober. He enrolled in college and was trying to better himself. As time went by he started going out and meeting people. After a couple months of hanging out with the ( locals) he wasn't even the same person. He hadnt known Morgan or his nieghbor when he got here only a few months before. He came here to help and died because no one cared to help him. I'm so sorry I ever asked him to come here. He made some bad choices, but he didn't have to die. Indifference , ignorance , selfishness and plain stupidity! Are just as much to blame.
Pretty obvious Powell has a massive drug problem,and has had for years.There are a few locals who have been in the dope business for years around Powell,and the cops have look the other way,making one wonder who gets what in Powell.So glad I left that town.
I think the bigger picture that needs to be seen is what a HUGE problem drugs are in the Powell community. As former resident during my college days, I know first hand how rampant drug abuse is in town. Doesn't the fact that this was the second drug overdose resulting in death within a week raise huge flags about the state of matters in Powell? Drug abuse is getting more and more prevalent, but I don't see much being done to reverse the spread.
Chris Nichols did NOT force Ernest to take the heroin. many times i stayed the night with Nichols at his house and Ernest would come wake us up at 2,4 am trying to sell chris drugs. if any of u knew chris like i do u would see he is a good man not a murderer or druggie. he was sober and happy and has a bigger heart than most people.And he was an nwc stdent not a cna yet so if u think he should be drug tested then test all nwc studens. Get and know the facts first not media facts..real facts
Stop pointing fingers!!! The guy that died was old enough to know right from wrong. Everyone makes there own outcome in life and you can't blame a situation like this on one person. Remember it all starts at home so if your going to point fingers start from the begining not at the end .
Jeremy died because he invested herion. He was not forced or held against his will to do drugs.but jermey made a willing choice to do them his self. You want to talk about friends if Morgan was such a friend why did he put the herion back in Jeremy's pocket and tell no one. You don't just become a drug addict over night and let,s look at the bigger picture powell valley health care they knew he has herion in his system and don't call the police. To end this Jeremy had more then herion in his system and until the toxicallogy report comes back the cause of death is still unknown .when you play with fire you get burned and now people just want someone to blame for an accidental over dose which happens all the time
it was Jeremys choice to take the drugs. No one put a knife to his throat and said DO IT!!! Thats what i dont get, its the addicts choice to do the drugs. Dealers just sale it but they do know the risk. as it states he was warned how bad it was!!. I do not support dealers NOR addicts. but i dont think he should get man slaughter, due to the fact Jeremy KNEW he might die a second time around!!! and it was Jeremys choice to start drugs again if he was a recovery addict!! he choice the wrong crowd.. but still sorry for your loss to his family!! May he Rest In Peace
Well I'm sure whoever actually had the heroine didn't just "give" the stuff away, and its unfortunate that a person died but they were both breaking the law regardless. Ernest had to go to the hospital for having a bunch of drugs in his system then barely an hour after getting out of the hospital went home and did the rest? I don't think charging Nichols with negligible homicide is necessarily fair but either way he's going to end up behind bars for a long time. And what about Morgan Wiles and these two "informants"?? I hardly think they were just standing around while heroine was being eaten by people, why aren't they being charged with criminal negligence?? My point is the drug problem in park county doesn't come down to one or two people, who knows how many other people bought some from whoever sold it to Ernest. And unfortunately "having a good heart" or not "meaning to hurt anyone" or being a good student trying to have a good life or being sober or whatever else doesn't matter within the confines of the law its about evidence. Elected officials are being convicted of drug related crimes for crying out loud, valedictorians from right here in park county, etc etc etc. There's a reason Justice wears a blindfold.
my little brother was a great man and nickels should get at least five years for giving someone in recovery drugs like that this loss has left a family without a father and brother I hope they throw the book at him
Addiction is a very sad thing and has taken many people in my life. Now it has takin my only daughters father I do not feel like this situation is being I did live in WY for some time and swore never to bring my girls up there it was bad even when I was there and now this. Do not let this go unknown please show this town,state how bad it really is please make a statement outta this KID before it happens to ur own.
Chris Nichols is a good person and moved to Wyoming to get away from bad habits, seeing him during thanksgiving I can absolutely say that Chris is clean and happy about it. This is one giant misunderstanding.
A full-time nursing student? Aren't random drug tests completed? Maybe not in college I guess but sure should be random tests while employed as a nurse. Frightening to think how many at Powell Valley Healthcare(staff-not just nurses) couldn't pass a drug test. I know quite a few that use Mary Jane-at the minimum-random drug tests must not occur. Why not?
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Fields marked (*) are required. | 2023-11-03T01:26:19.665474 | https://example.com/article/9641 |
Effect of wearing personal protective clothing and self-contained breathing apparatus on heart rate, temperature and oxygen consumption during stepping exercise and live fire training exercises.
Fire fighter breathing apparatus instructors (BAIs) must possess the ability to respond to both the extrinsic stress of a high temperature environment and the intrinsic stress from wearing personal protective equipment (PPE) and self-contained breathing apparatus (SCBA), repeatedly and regularly, whilst training recruits in live fire training exercises (LFTEs). There are few previous investigations on BAIs in hot environments such as LFTEs, since the main research focus has been on regular fire fighters undertaking exercises in temperate or fire conditions at a moderate to high exercise intensity. In this study, the intrinsic cardiovascular stress effects of wearing PPE + SCBA were first investigated using a step test whilst wearing gym kit (control), weighted gym kit (a rucksack weighted to the equivalent of PPE + SCBA) and full PPE + SCBA (weight plus the effects of protective clothing). The extrinsic effects of the very hot environment were investigated in BIAs in LFTEs compared to mock fire training exercises (MFTEs), where the fire was not ignited. There was an increase in heart rate due to the modest workload imposed on the BAIs through carrying out the MFTEs (25.0 (18.7)%) compared to resting. However, when exposed to fire during the LFTEs, heat storage appears to be significant as the heart rate increased by up to 39.8 (+/-20.1)% over that of the mock LFTEs at temperate conditions. Thus, being able to dissipate heat from the PPE is particularly important in reducing the cardiovascular responses for BAIs during LFTEs. | 2023-12-01T01:26:19.665474 | https://example.com/article/4454 |
7, -984, -2083, -3164, -4221, -5248?
n**3 - 1118*n + 1244
What is the s'th term of -5589, -11106, -16623, -22140, -27657, -33174?
-5517*s - 72
What is the c'th term of -776, -776, -768, -746, -704?
c**3 - 2*c**2 - c - 774
What is the p'th term of -612, -711, -820, -945, -1092, -1267?
-p**3 + p**2 - 95*p - 517
What is the q'th term of 45, 462, 1595, 3804, 7449, 12890, 20487, 30600?
60*q**3 - 2*q**2 + 3*q - 16
What is the b'th term of 665544, 665542, 665540, 665538, 665536?
-2*b + 665546
What is the l'th term of 1965, 15728, 53083, 125826, 245753, 424660, 674343?
1966*l**3 + l - 2
What is the r'th term of 425, 1546, 3385, 5942, 9217, 13210, 17921?
359*r**2 + 44*r + 22
What is the r'th term of 44497, 177984, 400461, 711928, 1112385, 1601832?
44495*r**2 + 2*r
What is the k'th term of 2571, 1934, 1297?
-637*k + 3208
What is the j'th term of -851407, -851405, -851401, -851395, -851387, -851377?
j**2 - j - 851407
What is the m'th term of 10852, 21707, 32562?
10855*m - 3
What is the r'th term of -3653, -7241, -10755, -14189, -17537, -20793, -23951?
r**3 + 31*r**2 - 3688*r + 3
What is the y'th term of -1741, -3300, -4837, -6340, -7797?
2*y**3 - y**2 - 1570*y - 172
What is the s'th term of -19746, -39511, -59288, -79083, -98902, -118751?
-s**3 - 19758*s + 13
What is the y'th term of 1183211, 2366416, 3549621, 4732826?
1183205*y + 6
What is the f'th term of -31616, -31618, -31620, -31622, -31624, -31626?
-2*f - 31614
What is the d'th term of 1351, 1328, 1305?
-23*d + 1374
What is the a'th term of -36, -60, -128, -264, -492, -836, -1320, -1968?
-4*a**3 + 2*a**2 - 2*a - 32
What is the p'th term of 1174, 1196, 1206, 1198, 1166, 1104, 1006?
-p**3 + 29*p + 1146
What is the r'th term of -902, -901, -898, -893?
r**2 - 2*r - 901
What is the v'th term of -9994, -10007, -10020, -10033, -10046?
-13*v - 9981
What is the m'th term of 111, 34, -227, -672, -1301, -2114, -3111?
-92*m**2 + 199*m + 4
What is the t'th term of -1534, -1531, -1516, -1483, -1426, -1339, -1216, -1051?
t**3 - 4*t - 1531
What is the p'th term of 668331, 1336670, 2005007, 2673342, 3341675, 4010006, 4678335?
-p**2 + 668342*p - 10
What is the h'th term of 230194, 230191, 230188?
-3*h + 230197
What is the l'th term of 15149, 15138, 15113, 15068, 14997, 14894, 14753?
-l**3 - l**2 - l + 15152
What is the d'th term of -40, -172, -400, -742, -1216, -1840, -2632?
-3*d**3 - 30*d**2 - 21*d + 14
What is the f'th term of 89300, 89303, 89306, 89309, 89312?
3*f + 89297
What is the r'th term of 80, 190, 300, 410, 520?
110*r - 30
What is the a'th term of 901, 1783, 2643, 3475, 4273?
-a**3 - 5*a**2 + 904*a + 3
What is the w'th term of -612, -985, -1358, -1731, -2104, -2477?
-373*w - 239
What is the s'th term of 11690, 23376, 35062?
11686*s + 4
What is the q'th term of 11405, 11597, 11917, 12365, 12941, 13645?
64*q**2 + 11341
What is the p'th term of -181, -558, -1179, -2038, -3129, -4446?
p**3 - 128*p**2 - 54
What is the r'th term of -6676, -6676, -6684, -6706, -6748, -6816?
-r**3 + 2*r**2 + r - 6678
What is the u'th term of -212657, -212659, -212661, -212663, -212665?
-2*u - 212655
What is the g'th term of -1006, -928, -776, -532, -178, 304, 932, 1724?
3*g**3 + 19*g**2 - 1028
What is the z'th term of 68, -599, -1718, -3295, -5336, -7847, -10834?
-z**3 - 220*z**2 + 289
What is the y'th term of -14, 239, 682, 1321, 2162, 3211, 4474, 5957?
y**3 + 89*y**2 - 21*y - 83
What is the u'th term of 184, 379, 574?
195*u - 11
What is the n'th term of -394, -1548, -3470, -6160?
-384*n**2 - 2*n - 8
What is the a'th term of -22, -4, -10, -58, -166, -352?
-3*a**3 + 6*a**2 + 21*a - 46
What is the r'th term of -39755, -79494, -119211, -158900, -198555?
r**3 + 5*r**2 - 39761*r
What is the x'th term of -45847, -45668, -45489, -45310, -45131, -44952?
179*x - 46026
What is the v'th term of 157, 402, 777, 1282, 1917, 2682, 3577?
65*v**2 + 50*v + 42
What is the t'th term of 151, 211, 271, 331, 391, 451?
60*t + 91
What is the v'th term of -1090874, -1090875, -1090876, -1090877, -1090878?
-v - 1090873
What is the z'th term of -1045, -1751, -2461, -3175, -3893?
-2*z**2 - 700*z - 343
What is the l'th term of -35178, -140787, -316802, -563223, -880050?
-35203*l**2 + 25
What is the f'th term of -55057, -55058, -55059, -55060?
-f - 55056
What is the b'th term of 6607, 6791, 6997, 7237, 7523, 7867?
2*b**3 - b**2 + 173*b + 6433
What is the i'th term of 530, 1056, 1548, 1988, 2358, 2640, 2816?
-3*i**3 + i**2 + 544*i - 12
What is the c'th term of -5026874, -10053751, -15080630, -20107511?
-c**2 - 5026874*c + 1
What is the c'th term of -374, -774, -1174?
-400*c + 26
What is the h'th term of -3892, -7643, -11392, -15139?
h**2 - 3754*h - 139
What is the s'th term of 27685, 27692, 27699?
7*s + 27678
What is the d'th term of 3225, 6422, 9589, 12726?
-15*d**2 + 3242*d - 2
What is the l'th term of -157, -939, -2257, -4123, -6549?
-2*l**3 - 256*l**2 + 101
What is the a'th term of 3039, 3009, 2979, 2949, 2919?
-30*a + 3069
What is the m'th term of 21929, 43865, 65801?
21936*m - 7
What is the d'th term of 11839, 11859, 11879, 11899, 11919?
20*d + 11819
What is the u'th term of -81, 7, 137, 291, 451, 599, 717, 787?
-3*u**3 + 39*u**2 - 8*u - 109
What is the t'th term of 4846, 4947, 5050, 5155, 5262, 5371?
t**2 + 98*t + 4747
What is the f'th term of 224, 264, 332, 428, 552, 704, 884?
14*f**2 - 2*f + 212
What is the m'th term of 3824, 3374, 2924, 2474, 2024, 1574?
-450*m + 4274
What is the o'th term of -248, -509, -790, -1091, -1412, -1753?
-10*o**2 - 231*o - 7
What is the u'th term of -368, -358, -336, -296, -232, -138, -8?
u**3 + 3*u - 372
What is the m'th term of -36, -14, 16, 60, 124, 214, 336, 496?
m**3 - 2*m**2 + 21*m - 56
What is the b'th term of -3007, -2791, -2573, -2353, -2131, -1907, -1681?
b**2 + 213*b - 3221
What is the u'th term of -463, -946, -1453, -1984, -2539, -3118, -3721?
-12*u**2 - 447*u - 4
What is the s'th term of -1644, -1646, -1640, -1620, -1580, -1514, -1416, -1280?
s**3 - 2*s**2 - 3*s - 1640
What is the r'th term of -125, -126, -135, -158, -201, -270, -371?
-r**3 + 2*r**2 - 126
What is the m'th term of -6198, -24751, -55680, -98991, -154690?
-m**3 - 6182*m**2 - 15
What is the y'th term of -38579, -77142, -115705, -154268?
-38563*y - 16
What is the l'th term of 124, 499, 1120, 1987, 3100, 4459?
123*l**2 + 6*l - 5
What is the o'th term of 444, 1979, 4518, 8061, 12608, 18159?
502*o**2 + 29*o - 87
What is the b'th term of -342, -1525, -3508, -6303, -9922, -14377?
-2*b**3 - 388*b**2 - 5*b + 53
What is the i'th term of 553, 4354, 14673, 34768, 67897, 117318?
543*i**3 + i**2 - 3*i + 12
What is the h'th term of 6053, 24269, 54625, 97115, 151733, 218473, 297329, 388295?
-h**3 + 6076*h**2 - 5*h - 17
What is the i'th term of 267, 1073, 2405, 4239, 6551?
-4*i**3 + 287*i**2 - 27*i + 11
What is the q'th term of 168, 367, 614, 915, 1276?
q**3 + 18*q**2 + 138*q + 11
What is the j'th term of -267, -1100, -2519, -4548, -7211, -10532?
-4*j**3 - 269*j**2 + 2*j + 4
What is the g'th term of 1613, 3057, 4491, 5909, 7305, 8673, 10007?
-g**3 + g**2 + 1448*g + 165
What is the x'th term of 23929, 47861, 71793, 95725, 119657?
23932*x - 3
What is the o'th term of 2509, 2431, 2353, 2275, 2197, 2119?
-78*o + 2587
What is the v'th term of -124, -151, -182, -217, -256?
-2*v**2 - 21*v - 101
What is the c'th term of 1728, 3460, 5198, 6942, 8692, 10448, 12210?
3*c**2 + 1723*c + 2
What is the b'th term of -975, -3680, -8105, -14244, -22091?
b**3 - 866*b**2 - 114*b + 4
What is the j'th term of 28470, 56982, 85494, 114006, 142518?
28512*j - 42
What is the s'th term of -336, -1718, -4024, -7254, -11408, -16486?
-462*s**2 + 4*s + 122
What is the j'th term of -588, -1188, -1788, -2388, -2988, -3588?
-600*j + 12
What is the v'th term of 21431, 42864, 64297, 85730, 107163?
21433*v - 2
What is the c'th term of -320611, -641224, -961837, -1282450, -1603063?
-320613*c + 2
What is the p'th term of 11, 27, 119, 341, 747?
9*p**3 - 16*p**2 + p + 17
What is the h'th term of 6403, 25618, 57643, 102478, 160123, 230578?
6405*h**2 - 2
What is the n'th term of 4324, 4291, 4258, 4225?
-33*n + 4357
What is the u'th term of -333, -2068, -5205, -9738, -15661, -22968, -31653?
u**3 - 707*u**2 + 379*u - 6
What is the q'th term of 53345, 106702, 160059, 213416, 266773?
53357*q - 12
What is the | 2024-02-07T01:26:19.665474 | https://example.com/article/8894 |
As from 1st September 2012 racing and drinking shirts will be priced at $35 (for members and non-members)
And from shirts to shorts: if your new racing top makes your appearance at races look spiffy then don’t short-change your nether regions. You certainly don’t want your undercarriage attire getting short-shrift and below-the-belt comments from team mates as your ageing velcro is rent asunder at the meekest attempt at a Sumo during warm-ups…
British Dragons Shorts - $30
Gill has procured some Men’s beach-style shorts with mesh pant lining – navy blue with union jack logo (the same as Paul has been modelling recently).
Only 10 pairs are available at just $30 each – so first come first serve.
Here are the available sizes:
Small – waist up to 81cm / 32″
Medium – waist 84-89cm / 33-35″
Large – waist 91-96cm / 36-38″
(note that the sizes come up slightly on the large side).
We now also have waterproof British Dragons stickers at $3 each.
British Dragons stickers - White - $3
Coat of arms on clear background (does not show up well on dark colours): 4 3/4″ by 3 3/4″ | 2024-01-17T01:26:19.665474 | https://example.com/article/8737 |
Srila Prabhupada’s Original Krsna Book Now On-Line
O Lord Krsna, please protect Me and maintain Me. O Lord Rama, descendant of King Raghu, please protect Me. O Krsna, O Kesava, killer of the Kesi demon, please maintain Me.
We have the great pleasure of announcing Srila Prabhupada’s original Krsna Book is now available for reading on the internet at http://www.krsnabook.com
Reading Krsna Book is very important and we simply present a few quotes from His Divine Grace A.C. Bhaktivedanta Swami Prabhupada on the necessity and great spiritual benefits of reading Krsna Book.
Prabhupada: Yes. If you read Krsna book and if you believe, then you see Krsna. He’s not different from the book. (N98:760427CC.AUC)
Prabhupada: Krsna book is written in such a way that everyone can read. But the scheme is like that. To understand Krsna, one has to go through this… The idea is that Krsna book should be read. (721104SB.VRN Lectures)
Read Krsna book regularly. Why these books are written? Only for selling? Taking statistics, “How many books you have sold?” You learn, read. Always read, twenty-four hours. As soon as you get time, read. I do that. I do that. Reading, writing, or chanting. But when there is no other way, you sleep little. Not to enjoy sleep, but because it is not possible to continue, all right, sleep one hour, two hours, three hours, four hours, five hours. Not more than that. Not that I am sleeping, enjoying life, up to eight o’clock, twelve o’clock. (720423SB.TOK Lectures)
Even if you hear yourself, you read books, you will save your life. If you simply read Krsna book or Bhagavad-gita or Teachings of Lord Caitanya, then you know the… As long as you are reading, the sun is unable to take your life. It is not possible for the sun to take your life. (720612SB.LA Lectures)
Another process is, save time, if you chant Hare Krsna mantra, then the time is not lost. Time is not lost. Hare Krsna mantra, read book, Krsna book. Any moment, any time you spend for thinking of Krsna, working for Krsna, chanting for Krsna, eating for Krsna, dancing for Krsna, that time is saved. That time is saved. Therefore it is recommended, harer nama harer nama harer namaiva kevalam, kalau nasty eva nasty eva… Otherwise, by yoga process, it is not… How many men can actually practice yoga? It is all farce. This is possible. Even a child can take part, Hare Krsna mantra. (740120SB.HAW Lectures)
If you actually want to become free from the contamination of this material world, then you should always be engaged in chanting, sankirtanam. Not only the holy name, but reading the Krsna book, The Nectar of Devotion, Teachings of Lord Caitanya. If you feel tired chanting, you read these books. Sometimes there is psychology, transfer. You are reading some book, you want to read another book. So we have got so many books. If you feel tired this book, then transfer your attention to another book, or another book, or chant. Find out. Simply, not to waste a single moment. Kirtaniyah sada harih. Caitanya Mahaprabhu says sada. Sada means always. So we have got materials for engaging ourself always in Krsna consciousness. Not that we have got only this one item. No. We have got so many items. That is also accepted. (710214SB.GOR Lectures)
It is so nice. Because you will understand Krsna, what is Krsna, and here it is said, janma karma ca me divyam ye janati tattvatah. It is explained. The people who are reading Krsna book very seriously, and tries to understand Krsna, he will understand. Krsna is very kind. As soon as he begins reading Krsna book with a little faith and adherence, Krsna will be very much pleased. Srnvatam sva-kathah krsnah punya-sravana-kirtanah. As soon as Krsna is within your heart so when you read Bhagavad-gita or Krsna book with little seriousness, then Krsna understands, “Now he is serious to understand me.” He’s with the…. You haven’t got to search out Krsna, He’s already within you. Simply you have to become little serious. “Krsna, kindly give me knowledge so that I can understand.” Buddhi-yogam dadami tam. If you are serious devotee, if you are in love with Krsna, tesam satata-yuktanam bhajatam priti-purvakam. If you are engaged in His worshiping, in chanting, man-mana bhava mad-bhakto mad-yaji mam namaskuru. If you simply follow these four principles, simply thinking of Krsna, simply, always, sa vai manah krsna-padaravindayoh. Simply meditate. This is meditation. (740329BG.BOM Lectures)
Our Krsna consciousness movement is the same: it is simply questioning about Krsna and hearing the answer. It is loka-mangalam. Anywhere this vibration will go on, there will be all auspicity. Loka-mangalam. Krsna-samprasno yenatma suprasidati. Another feature is Krsna is all-attractive; therefore, talks about Him is also attractive. In our Krsna book there are so many topics about Krsna, janma karma me divyam, about His birth, about His transference from real father’s house to another foster father, then the attack by the demons upon Krsna, Kamsa. All these activities, if we simply study and hear the krsna-samprasnah, then we are liberated. Without any doubt, our liberation is guaranteed. Simply by hearing about Krsna. (720717SB.EDI Lectures)
Krsna is killing so many demons, Krsna is dancing with gopis, Krsna is playing with His cowherd boys, Krsna is going there, this is lila, smaranam. The Krsna book reading means remembering all these activities of Krsna. Simply go on reading Krsna book, repeat it, you are in the perfect stage of transcendental position. Simply read our Nectar of Devotion. Lila, atulyan. Nisamya karmani gunan atulyan, viryani lila-tanubhih krtani, yadatiharsotpulakasru-gadgadam. Pulakasru, in ecstasy. Pulaka is trembling and faltering voice and tears coming down, perspiring, these are (called) asta-sattvika-vikara. Protkantha udgayati rauti nrtyati. (710911sb.mom Lectures)
Even a respectable person, ordinary person, a big man or a rich man or a king comes, we become so much respectful. And what about Krsna when He’s present as arca-murti or as the holy name? These are the realization point of view: Krsna, His name, His form, His quality, His pastimes. When you hear about Krsna, that is also Krsna. Actually, when you are reading Krsna book, Krsna is fighting with demons, that is also His pastime. So Krsna is present. So you should be very attentive and worship this hearing. Unless we come to this point, there is lack of realization what is Krsna. (710216LE.GOR Lectures)
His Divine Grace A.C. Bhaktivedanta Swami Prabhupada | 2023-10-23T01:26:19.665474 | https://example.com/article/4424 |
Israel's top court ruled Monday that Prime Minister Benjamin Netanyahu must divulge information on his phone conversations with Sheldon Adelson and the editors of the Israel Hayom (Israel Today) daily, published by Adelson and considered supportive of Netanyahu.
>> Explained: How long can Netanyahu remain PM?
>> Clearer, sharper, restrained: Netanyahu's heiress
The Supreme Court accepted the appeal submitted by investigative reporter Raviv Drucker, deciding that Netanyahu must release call logs detailing the times of his discussions with top Israel Hayom representatives, including Adelson and the former editor-in-chief Amos Regev. This decision overturned last year's district court ruling regarding Druker's freedom of information request.
"It is in the public's interest to expose this important information," wrote Judge Menachem Mazuz in the decision, responding to Adelson and Regev who insisted that releasing the call logs would violate their privacy. Mazuz continued, "The public's interest, in this situation, supersedes Adelson, Regev, and the prime minister's right to privacy."
Meanwhile, the State Attorney's Office supported the position of the Prime Minister's Office that Regev and Adelson's call logs need not be released, stating that "the two involved parties are personal friends of the prime minister, and their conversations are private and unrelated to his ministerial work."
Drucker originally submitted a freedom of information request regarding the call logs in February of 2015, which was rejected in September of that year. In appealing the decision, Drucker's told the court that "the relationship between the prime minister, Adelson, and Regev is not a matter of gossip, it's a matter of understanding the relationship between the government, the newspaper, and its funder."
Drucker further insisted that call log could reveal if a correlation exists between the dates of their communications and a variety of items published in the newspaper, and on whether or not the prime minister was the de facto chief editor of Israel Hayom.
Last week, Adelson told Israel Police investigators that Netanyahu asked him to consider canceling the possibility of publishing weekend supplements, Channel 2 reported on Saturday. This information was reportedly shared with investigators during Adelson's second questioning, about a month ago, and referred to a discussion that took place when the paper had no weekend supplements.
The so-called "Case 2000" involves suspicions that Netanyahu promised to rein in the Israel Hayom, which is heavily pro-Netanyahu, in exchange for getting more favorable coverage in Noni Mozes's Yedioth Ahronoth newspaper. | 2024-02-04T01:26:19.665474 | https://example.com/article/5852 |
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Estimated Delivery 5 business day(s) - Wed 12/20 - The products will be shipped directly from the Manufacturer and will require additional time for delivery.5 business days - Wed 12/20 - The products will be shipped directly from the Manufacturer and will require additional time for delivery.
When our offices recently moved into a new office, I was responsible for organizing and distributing all keys for doors, desks and file cabinets.These key tags were the easiest way to mark all the keys. We then kept the Master Key for each to put in our key box.
How many flashdrives do you own? I am sure your answer is too many! I use these tags to organize my flashdrives. On one side of the tag I write my name and phone number. On the other side I label the drive. I have not lost a flashdrive since I started using the tags!
All my employees use these on their file cabnet keys. When they are hung up on the wall, they look very professional. They do not fall apart and are easy to slip the key on. I would highly recommend this product to anyone looking for an easy, professional, realible key ring!
I purchased this product to label a basket of scissors that are used by all the teachers in my school. They are great! I haven't lost a pair of scissors yet! The tags last a long time and don't fall off of the items you place them on. I personally like the assorted package colors.
Until I had these Key Tags it took forever to find the right key. These made it so much easier to find the key I needed very quickly. I would recommend these to anyone with a ton of keys! They work great and last a long time!
I have a billion keys and I can't stand when its raining and I have to go through at least 4 keys till I get the right one to open my door! These Key tags made it so much easier for me! I looked down read which key was what and I was good to go! So easy!
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experience with the same product for their open and honest feedback. | 2024-06-03T01:26:19.665474 | https://example.com/article/8797 |
Q:
Android: Provide a resource if width is smaller than specific DP
How do I use the resource qualifiers system in Android to specify that I want a resource to apply only if the width is smaller than a particular value?
To elaborate, suppose I want to provide one layout whenever the current available width is up to - say 320dp - and another layout for all other cases. This is what comes to mind:
layout-w320dp/mylayout.xml
layout/mylayout.xml
However, as per my understanding of the resource matching algorithm, even a large device (say, a tablet in landscape) qualifies as w320dp - because, well, the available width would be greater than 320dp.
As such, the resource from layout-w320dp would always be picked - even for larger phones and tablets. The only time the default resource from the layout folder is picked is if the available width is less than 320dp.
So, how do I express a "smaller than" relationship using the Android resource matching system?
A:
Ugh! Just after posting the question I realized that one way to do this would be to negate the equation - i.e.:
layout-w320dp/mylayout.xml --> layout for screens larger than 320dp
layout/mylayout.xml --> layout for up to 320dp
However, I'm still interested in knowing if the original "smaller than" relation can be achieved since it makes the intent much clearer.
| 2024-02-16T01:26:19.665474 | https://example.com/article/7745 |
on •
LEXIE CANNES STATE OF TRANS — An unidentified trans woman waiting at a bus stop in the Newham area of London was violently assaulted by a man who got out of his car and walked up to her. The attacker was urged on by the car’s other occupants who were shouting “kill him, kill him,” the victim told police.
The attack happened late at night May 16th and was reported in the UK news media today. Police still do not have suspect in custody and are now asking for help from the public.
Detective Constable Ben Rouse according to London 24: “This was an unprovoked attack. This suspect is clearly willing to use violence against innocent members of the community.”
I was unable to gather further details on the incident, including information of the victim’s condition other than a broken nose.
—–
Just when you think maybe, just maybe, things are on the upswing, this kind of thing happens. Be safe.
Trigger Warning: http://www.london24.com/news/crime/transvestite_attacked_as_onlookers_screamed_kill_him_kill_him_1_4113547
Watch LEXIE CANNES right now: http://www.amazon.com/Lexie-Cannes-CourtneyODonnell/dp/B00KEYH3LQ Or get the DVD: http://www.amazon.com/gp/product/0963781332
Read Lexie Cannes in The Huffington Post: http://www.huffingtonpost.com/courtney-odonnell/
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Categories: Transgender, Transsexual, Trans, Transphobia, exploitation, dehumanizing, violence, hate | 2024-06-16T01:26:19.665474 | https://example.com/article/3058 |
On the economic performance of the waste sector. A literature review.
In a context of increasing international concern about public cost savings, research interest in the waste sector has gradually emerged. The literature on waste cost and inefficiency, particularly the use of parametric and non-parametric methods has increased exponentially in the last years. This paper reviews the developments, themes, objectives, concerns and characteristics of this kind of research, by reviewing a comprehensive database consisting of more than 100 relevant papers on economic performance of the waste services were published since 1965. Based on the econometric and mathematical programming methods (cost and product ion frontiers) used so far, the paper identifies characteristics of the waste research community (such as the authors' case-studies, aims of research, methods adopted, among others). Finally, it also identifies the main targets in this research field, such as market structure, ownership, incentives (through regulation and legal mechanism) and performance assessment. | 2024-04-25T01:26:19.665474 | https://example.com/article/9695 |
Plant neighborhood influences colonization of Brassicaceae by specialist and generalist aphids.
A plant's own characteristics, but also those of its neighbors, might have an impact on its probability of being colonized by herbivorous insects. A plant might be less colonized and experience associational resistance when it grows near repellent neighbors. In contrast, it might be more colonized and experience associational susceptibility near attractive neighbors. To date, mechanisms that drive associational defense are not really understood. In order to gain insights into the occurrence of associational resistance versus associational susceptibility under field conditions, we conducted an experiment to determine the influence of neighboring plants on the colonization of a focal plant by aphids. The focal plant was always Brassica oleracea. The neighbors were B. oleracea (control), B. napus, B. nigra, or Solanum lycopersicum, which represent contrasting levels of physical and chemical defenses. The focal plant, B. oleracea, was more colonized by the specialist aphid Brevicoryne brassicae, and experienced associational susceptibility when it was surrounded by B. nigra or B. napus. In contrast, B. oleracea was less colonized by the generalist aphid Myzus persicae, and experienced associational resistance when it was surrounded by S. lycopersicum, B. nigra or B. napus. Neighboring plants had no significant impact on host plant choice by the generalist aphid Macrosiphum euphorbiae. In conclusion, attraction or repulsion of the specialist aphid B. brassicae and the generalist aphid M. persicae by B. nigra, B. napus, and S. lycopersicum resulted in associational susceptibility or associational resistance for B. oleracea. | 2023-12-12T01:26:19.665474 | https://example.com/article/1224 |
laces?
9
What is -1119680 rounded to the nearest one thousand?
-1120000
What is -0.000062911 rounded to 5 dps?
-0.00006
Round 0.09151 to one decimal place.
0.1
What is 0.000912 rounded to four dps?
0.0009
What is -0.00000773 rounded to 6 decimal places?
-0.000008
Round -28640000 to the nearest 100000.
-28600000
Round 6680000 to the nearest 1000000.
7000000
What is -1339.13 rounded to the nearest one hundred?
-1300
Round 4.505 to zero dps.
5
Round 0.0003094 to 4 dps.
0.0003
Round 0.05246 to 2 decimal places.
0.05
Round -5900.1 to the nearest 1000.
-6000
What is -0.000037063 rounded to 7 decimal places?
-0.0000371
Round 247.9 to the nearest 10.
250
What is -71.93 rounded to the nearest ten?
-70
Round 0.0000481 to six decimal places.
0.000048
Round 54270000 to the nearest 1000000.
54000000
What is -2470.1 rounded to the nearest 100?
-2500
Round -58166000 to the nearest one hundred thousand.
-58200000
Round -2161.2 to the nearest ten.
-2160
Round -0.13996 to 2 dps.
-0.14
Round 4734200 to the nearest ten thousand.
4730000
Round 50614000 to the nearest one million.
51000000
Round -1.1282 to 3 dps.
-1.128
What is -390.9 rounded to the nearest 100?
-400
What is 0.000280018 rounded to five decimal places?
0.00028
Round -0.00436 to four dps.
-0.0044
Round 39082 to the nearest 100.
39100
Round -0.02232 to 3 decimal places.
-0.022
Round 226.79 to 0 decimal places.
227
What is 0.02559 rounded to 3 dps?
0.026
What is -3192900 rounded to the nearest 1000000?
-3000000
Round 0.00058862 to four dps.
0.0006
Round -0.3694 to 2 decimal places.
-0.37
Round -61013 to the nearest 1000.
-61000
Round 1.46755 to 2 dps.
1.47
Round 0.000000337 to 7 decimal places.
0.0000003
Round 0.5056 to two dps.
0.51
Round 9989 to the nearest 1000.
10000
What is 15.756 rounded to one decimal place?
15.8
Round -0.0029539 to 4 decimal places.
-0.003
What is 0.00256 rounded to three dps?
0.003
Round 3776000 to the nearest one hundred thousand.
3800000
What is 0.0000706 rounded to four decimal places?
0.0001
What is 0.0011547 rounded to four decimal places?
0.0012
Round -5253500 to the nearest one million.
-5000000
Round 0.5796 to 2 dps.
0.58
What is -3476 rounded to the nearest one hundred?
-3500
What is -0.0145161 rounded to three decimal places?
-0.015
What is 0.000012111 rounded to 6 decimal places?
0.000012
What is -2.674 rounded to one decimal place?
-2.7
What is -0.0032928 rounded to 4 decimal places?
-0.0033
Round 0.012837 to three dps.
0.013
What is 1223.3 rounded to the nearest 10?
1220
What is -6212.8 rounded to the nearest one thousand?
-6000
Round -0.00483 to 2 dps.
0
What is -1.284 rounded to one decimal place?
-1.3
Round 0.986 to 1 decimal place.
1
Round -35750 to the nearest 10000.
-40000
What is -26916 rounded to the nearest 100?
-26900
What is 0.004205 rounded to three decimal places?
0.004
What is -521200 rounded to the nearest 10000?
-520000
What is -290.33 rounded to the nearest one hundred?
-300
What is -4.896 rounded to the nearest integer?
-5
Round -0.00001518 to six dps.
-0.000015
Round 79.245 to the nearest ten.
80
What is -0.196 rounded to the nearest integer?
0
Round 6003 to the nearest 1000.
6000
What is -7927800 rounded to the nearest 1000000?
-8000000
What is 0.003507 rounded to 4 decimal places?
0.0035
Round 0.0030775 to 5 decimal places.
0.00308
Round 0.0002219 to 5 dps.
0.00022
Round -0.0022447 to three dps.
-0.002
Round 96910000 to the nearest 1000000.
97000000
What is -0.0012 rounded to 3 decimal places?
-0.001
Round -0.00023101 to 5 decimal places.
-0.00023
Round 5.227 to the nearest integer.
5
Round -6.694 to one dp.
-6.7
Round 0.05809 to 1 decimal place.
0.1
Round -0.0004906 to four decimal places.
-0.0005
Round 0.000022318 to seven decimal places.
0.0000223
Round -0.00243 to three decimal places.
-0.002
What is -41215.7 rounded to the nearest ten thousand?
-40000
Round -980.18 to the nearest one hundred.
-1000
Round -15.827 to the nearest integer.
-16
Round 0.006753 to four dps.
0.0068
What is -13.039 rounded to 0 dps?
-13
What is 0.00051032 rounded to 4 dps?
0.0005
Round 755160 to the nearest ten thousand.
760000
Round 7.627 to zero decimal places.
8
Round 4210 to the nearest one hundred.
4200
Round -0.0000050403 to seven decimal places.
-0.000005
What is -78492000 rounded to the nearest 1000000?
-78000000
What is 30000 rounded to the nearest 100000?
0
Round 0.0004125 to four decimal places.
0.0004
What is 0.00001514 rounded to 7 dps?
0.0000151
Round -1859040 to the nearest ten thousand.
-1860000
What is -0.04409 rounded to three decimal places?
-0.044
Round -21913 to the nearest one thousand.
-22000
Round -0.019078 to 4 dps.
-0.0191
Round -0.00006012 to 6 decimal places.
-0.00006
Round 27338000 to the nearest 1000000.
27000000
What is 31763 rounded to the nearest ten thousand?
30000
Round -54.656 to 1 decimal place.
-54.7
What is 6378 rounded to the nearest 1000?
6000
What is 0.0047096 rounded to 4 decimal places?
0.0047
Round -0.000009601 to six decimal places.
-0.00001
What is -558780 rounded to the nearest 10000?
-560000
What is 1.2397 rounded to 1 decimal place?
1.2
What is 163.4 rounded to the nearest 10?
160
What is -5550 rounded to the nearest one thousand?
-6000
What is -6177.7 rounded to the nearest ten?
-6180
Round 959.53 to the nearest 100.
1000
What is 0.000077579 rounded to five decimal places?
0.00008
What is -6684000 rounded to the nearest 100000?
-6700000
What is 0.0001852 rounded to 6 dps?
0.000185
What is -1421.5 rounded to the nearest 10?
-1420
Round 17390.9 to the nearest 100.
17400
What is -8300 rounded to the nearest 10000?
-10000
What is 0.0000557 rounded to five decimal places?
0.00006
What is -0.29533 rounded to 1 decimal place?
-0.3
Round 41.9338 to 1 decimal place.
41.9
What is -0.000078276 rounded to six dps?
-0.000078
What is -32440000 rounded to the nearest 1000000?
-32000000
Round -0.10161 to two dps.
-0.1
Round -0.06706 to three decimal places.
-0.067
What is 45.124 rounded to the nearest integer?
45
What is -0.00637 rounded to three decimal places?
-0.006
Round -521000 to the nearest one hundred thousand.
-500000
Round 0.00090765 to four dps.
0.0009
Round -3987.93 to the nearest 10.
-3990
Round -39.93 to 0 decimal places.
-40
What is 15950 rounded to the nearest one thousand?
16000
Round 0.000001672 to 6 decimal places.
0.000002
What is -39416 rounded to the nearest one thousand?
-39000
Round -0.1058 to two dps.
-0.11
What is 979 rounded to the nearest one hundred?
1000
Round 0.000559 to 5 dps.
0.00056
Round 1.293 to 1 dp.
1.3
What is 0.02573 rounded to three dps?
0.026
What is 4229200 rounded to the nearest one hundred thousand?
4200000
Round 19810 to the nearest one thousand.
20000
Round -20000000 to the nearest 1000000.
-20000000
What is -0.0718 rounded to 2 dps?
-0.07
What is -6149.1 rounded to the nearest one hundred?
-6100
Round 247.081 to the nearest ten.
250
What is 99791 rounded to the nearest 100?
99800
Round -0.00000109823 to 7 decimal places.
-0.0000011
What is 4.672 rounded to one dp?
4.7
Round -0.49951 to one dp.
-0.5
Round 0.00002397 to 6 decimal places.
0.000024
What is 0.0000000455 rounded to 7 dps?
0
What is -0.000032694 rounded to six dps?
-0.000033
Round -26 to the nearest ten.
-30
Round -0.00018857 to six decimal places.
-0.000189
Round -2526540 to the nearest 10000.
-2530000
What is 41430 rounded to the nearest one thousand?
41000
Round -0.03216 to 2 decimal places.
-0.03
What is -0.60405 rounded to two dps?
-0.6
What is -0.0001715 rounded to 4 dps?
-0.0002
What is 797.6 rounded to the nearest one hundred?
800
What is 1053.6 rounded to the nearest ten?
1050
What is -8.49 rounded to zero decimal places?
-8
What is -0.04112 rounded to four decimal places?
-0.0411
What is -0.00025551 rounded to four decimal places?
-0.0003
Round 0.0000227 to 5 dps.
0.00002
What is -228250 rounded to the nearest one thousand?
-228000
What is -17510 rounded to the nearest 1000?
-18000
Round -0.00000548 to 7 dps.
-0.0000055
What is -0.0000113 rounded to 7 decimal places?
-0.0000113
What is 330000 rounded to the nearest one hundred thousand?
300000
What is -0.00000075 rounded to 6 dps?
-0.000001
Round -100270 to the nearest 10000.
-100000
Round 4216000 to the nearest one hundred thousand.
4200000
Round 7.8354 to | 2024-06-06T01:26:19.665474 | https://example.com/article/3004 |
Q:
Did the financial sector make up 40% of the US economy before the 2008 financial crisis?
I'm watching a video that claims that 40% of the US economy is made up by the financial sector. It then goes on to talk about the Financial crisis of 2008, so I would assume it's referring to pre-crisis levels. Is that true?
A:
The most standard way of looking at how much of the economy any given entity comprises is to compare the value of its production to the Gross Domestic Product (GDP) of the country for the same time period. From this perspective saying that the financial sector accounts for 40% of the economy would seem to be far overblown. This NYU study includes a comparison of the size of the financial sector to GDP right up to the finiancial crisis, and the financial sector maxes out at about 8% of GDP.
The video by postcarboninstitute doesn't give any hint as to what metric they are using, but the most likely one I could come up with with is that companies in the financial sector made up 40% of all corporate profits in the years before 2008. The problem is that corporate profits are not "the economy" -- in fact all corporate profits last year amounted to about $1.6 trillion (I couldn't find numbers that were Q4, but it probably won't change much), while the total annual GDP was close to $14 trillion.
None of this is to make a judgement on whether or not this is a good state of affairs, but postcarboninstitute should have probably phrased that statistic differently for the sake of clarity.
A:
No, the 40% figure is not true.
These figures from the Bureau of Economic Analysis in the US Department of Commerce suggest that in each year over the last decade the finance and insurance industry made up about 7.2%-8.4% of GDP ("value added" is the technical term) and 4.5% of employment.
Widening this to finance, insurance, real estate, rental, and leasing gives between 19%-22% of GDP and about 6% of employment.
More tables here if anyone is interested.
| 2024-06-26T01:26:19.665474 | https://example.com/article/5258 |
Sandy Pierre, editor of the wonderful online news magazine Shire Liberty News, sent the following essay to her subscribers last week. It is such an important message, The Freecoast requested permission to reprint it here. If you fancy yourself an activist, don’t do anything until you read this.
Are You an Activist?
by Sandy Pierre
I have long considered myself to be a liberty activist. And as someone who lives in New Hampshire and interacts with other Free State Project early movers on a regular basis, many of the people in my social circle consider themselves to be activists as well. But are they really? Am I? What constitutes “activism”?
Activism – the doctrine or practice of vigorous action or involvement as a means of achieving political or other goals, sometimes by demonstrations, protests, etc.
Activism
Implementing legislative change on the local level is activism. I say this because, while philosophically I consider myself an anarchist, in the real world we find ourselves in right now, anything that makes it less likely to get fined or arrested for a victimless crime is a good thing.
Writing a pro-liberty bill that gets passed (or that has a good shot at passing) counts. So does shooting down an anti-liberty bill. Working to get such a bill passed by speaking to a committee, writing to/calling local legislators, writing effective Letters to the Editor that get published and read… these all count as activism. Running for local office, winning, and then actually showing up, counts too.
Changing someone’s mind in a pro-liberty direction is activism. This can be done via face-to-face conversation, writing a book, blogging, podcasting, writing music, handing out flyers. However, the number of books, blogs, podcasts, songs and conversations that actually achieve this (i.e. change someone’s mind in a pro-liberty direction) is a small fraction of the total. In activism, unlike gift-giving, it’s NOT the thought that counts! It’s the effective communication of an idea that counts.
Not Activism
In my opinion, trying to reform the federal government is not activism. Such endeavors are worse than useless, because they consume time, energy and money on things that might actually make a difference! The same goes for voting in federal elections (note: I actually still do this myself… but I’m not sure why. Tradition?).
Running for office when there’s not a snowball’s chance in Hell that you’ll win – That’s just stroking your own ego and/or trying to prove a point which eludes me. Now, some people do seem to run for offices they know they have no chance of winning on the grounds that it gives them a platform from which to promote the principles of liberty. If a campaign actually does give them an opportunity to participate in a debate with the other candidates, get significant media coverage, etc., then perhaps there is some value there.
Starting a blog, a podcast, a cable access TV show, etc. – These activities, in and of themselves, do not count as activism. They may count, if they successfully perform one of the activities in the Activism section above. I don’t consider my personal blog to be activism; it’s just an outlet for creative expression and a way to amuse myself and my friends.
Publicity – Some claim “any publicity is good publicity”. I respectfully disagree, and will stoop to using a distasteful recent example to make my point. A liberty activist (not in New Hampshire, thankfully) recently admitted to molesting a young girl on his Facebook wall. His post has garnered over 100 shares, over 2000 comments, and presumably thousands of views. That is a lot of publicity! This man is (or was) associated with pro-liberty organizations such as CopBlock and C4SS. Do you think that either of these organizations are grateful for the spotlight this self-confessed child molester has shone on them this week via his association with them? Um, yeah….
Running an “agorist” business – As far as I can tell, the definition of an agorist business seems to be refusing to comply with laws regarding business licensure and taxes. While it may be laudable to refuse to “feed the beast” by refusing to pay taxes, this is not promoting or expanding liberty any more than simply being unemployed would be. Please don’t misunderstand: I’m not saying that there is no value in running an agorist business! I’ve made use of several myself over the past few years and am grateful for their existence. But it’s not “activism”.
Anti-Activism
A minority of self-described activists do things that actually drive potential converts away from the ideas of liberty. If you engage in any of these pastimes, please, stop!
Insulting/namecalling/engaging in flame wars – You may be right. You may be smarter than the other person. But if you call him names, or put him down, or act condescending, you are not doing positive activism. On the contrary, you’re doing negative activism. Calling people names, or cursing at them, will not convince them that you are right and they are wrong. It will anger them and put them on the defensive, which makes them much less likely to actually hear any valid points you might have been trying to make.
Being a deadbeat/mooch – Self-ownership is one of the foundational principles of libertarianism. Owning yourself includes being responsible for yourself, taking care of yourself. It boggles my mind how many self-described activists are seemingly unable to feed and house themselves and their children without relying upon the charity of others. Some even go so far as to renege on contracts they’ve voluntarily entered into for housing or basic services. Please, if you haven’t got the bare minimum self-supporting aspects of being an adult down yet, stop calling yourself an activist.
Metrics
Action that doesn’t achieve the desired outcome is a waste of your precious limited time (and money, if you’re spending any on your project). It’s not enough to just “do something”; you need to do something that achieves your goal, or at least gets you part of the way there. Ideally, you need to do it efficiently. As an example, spending a million dollars on a futile political campaign may result in turning X number of people on to the ideas of liberty. But was that the most effective use of that million dollars? If the money had been spent another way, might X have wound up being a larger number?
Good feedback – People writing or speaking to you, saying they got something out of your article/video/podcast/etc. They may write to say they agree with you. They may disagree. But if you can maintain a respectful dialog with those who disagree, you may accomplish something. Even if you don’t succeed in changing their mind, if you can end the debate in a civil manner, with mutual respect, then at least you’ve left that reader with positive thoughts about you and your position.
Indeterminate feedback – Number of social media friends/followers. People who follow you on social media and/or read your articles because you’re so outrageous they just want to see what you’ll say or do next are not a valid measure of how well you’re promoting liberty. I’m not going to name names here, but I have been told by a number of friends that certain high-profile “celebritarians” are followed/read for just such a reason. They are the human equivalent of a gory highway accident. Not all of your followers may be fans or supporters; some of them are simply rubber-neckers. Others are just people who, for whatever reason, will follow/link to anyone who follows/links to them first.
I’ve noticed this myself on Twitter, which I only started making use of in the last several months for purposes of promoting Shire Liberty News. I’ve been systematically following people who identify themselves as “libertarian”, and many of them have followed me back. But I can tell by some of their tweets that, well… to paraphrase the immortal words of Inigo Montoya “I do not think that word means what they think it means”. If many of them actually took the time to read what we publish in this newsletter, they’d probably disagree, some of them angrily. This is not to say that linking to them on Twitter serves no positive purpose at all. Due to Twitter’s rules, you need to have X number of followers in order to be allowed to follow Y users yourself. So in that sense, every single follower does help, albeit indirectly. But I have no illusions that, because SLN has over a thousand followers on Twitter, that equates to a thousand Twitter FANS, or even readers.
Negative feedback – If you’re so abrasive or obnoxious that even your allies unfriend/unfollow you, block you, ban you from their events… you might want to take a moment of quiet reflection and assess the efficacy of your activism. | 2024-03-27T01:26:19.665474 | https://example.com/article/9967 |
Yesterday, Swami Aseemanand and three others were acquitted by the Special NIA Court at Panchkula in the 2007 Samjhauta Express Blast Case. The others acquitted were Lokesh Sharma, Kamal Chauhan, and Rajinder Chaudhary.
Along with expected lines, liberals have risen up in arms against the Court verdict. Senior Congress leader Kapil Sibal said sarcastically that it was a “proud day” for the Judiciary in India.
2007 : Samjhauta Express Bomb Blast 68 killed
NIA charged 8 accused Verdict : No one knows who killed the 68 victims Must be a proud day for our criminal justice system ! — Kapil Sibal (@KapilSibal) March 21, 2019
However, unlike what Sibal would have us believe, we do have a fair idea about who is responsible for the terror attack that killed 68. The United Nations holds Arif Qasmani, a Pakistani national, responsible for the terror attack. He was sanctioned by the global body on the 29th of June, 2009.
The UN says, “Arif Qasmani was listed on 29 June 2009 pursuant to paragraphs 1 and 2 of resolution 1822 (2008) as being associated with Al-Qaida, Usama bin Laden or the Taliban for “participating in the financing, planning, facilitating, preparing or perpetrating of acts or activities by, in conjunction with, under the name of, on behalf or in support of”; “supplying, selling or transferring arms and related materiel to” or “otherwise supporting acts or activities of” Lashkar-e-Tayyiba (QDe.118) and Al-Qaida (QDe.004).”
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It adds further, “Arif Qasmani is the chief coordinator of Lashkar-e-Tayyiba’s (LeT) (QDe.118) dealings with other organizations and has provided significant support for LeT terrorist operations. Qasmani has worked with LeT to facilitate terrorist attacks, including the July 2006 train bombing in Mumbai, India, and the February 2007 Samjota Express bombing in Panipat, India. Qasmani utilized money that he received from Dawood Ibrahim Kaskar (QDi.135), an Indian crime figure and listed terrorism supporter, to facilitate the July 2006 train bombing in Mumbai, India. Qasmani also conducted fundraising activities on behalf of LeT in late 2005.”
Qasmani was known to provide financial assistance to terror organizations. The UN remarks, “Arif Qasmani has also provided financial and other support to Al-Qaida (QDe.004). As of late 2006, Qasmani provided funding to Al-Qaida members and facilitated the return of foreign fighters to their respective countries. Between 2004 and 2005, Qasmani provided Al-Qaida with supplies and weapons and facilitated the movement of Al-Qaida leaders in and out of Afghanistan. In return for Qasmani’s support, Al-Qaida provided him with operatives to support the July 2006 train bombing in Mumbai, India, and the February 2007 Samjota Express bombing in Panipat, India. Qasmani also facilitated the movement of Al-Qaida personnel out of Afghanistan in 2001. In 2005, Arif Qasmani provided Taliban leaders with a means to smuggle personnel, equipment and weapons into Afghanistan.”
RVS Mani, who served as undersecretary in the Ministry of Home Affairs from February 2006 to August 2010, too asserted that under the then UPA administration Qasmani had escaped while the government was busy creating the bogey of Hindu terror. He had stated that Qasmani had claimed the responsibility for the blasts. Also, later links to SIMI and other such organisations were exposed. He had remarked that in all the years of his service he has never come across a single actionable input regarding Hindu Terror and the term was deliberately coined.
Mani told ANI last year in June, “I’ve said this earlier too that there was no official information on Hindu terror till 2010. Even after that, there was no such thing. I’ve written a book which clearly states how Digvijay Singh laid the foundation of Hindu terror and spread it.” “In name of Hindu terror, he saved real terrorists using government resources. Arif Qasmani, the accused Samjhauta Express blast had escaped. In Mecca Masjid blast case, Bilal escaped. I don’t know his political agenda but there’s no Hindu terror,” he added.
Thus, it appears, unlike what Sibal would have us believe, we do have a fair idea about who is possibly responsible for the Samjhauta blast. Of course, Congress leaders would have hoped for the conviction of Hindus regardless of their guilt as it would have deflected attention from the despicable efforts it made to perpetuate a mythical narrative on Hindu terror. | 2023-12-21T01:26:19.665474 | https://example.com/article/7426 |
Baltimore’s Arabbers: Simple Solutions to Public Problems?
Recently I was on conference call with a group of community-based food leaders. I asked them for a wish list—a list of any resource imaginable that could help them create a more equitable food system in their community. I told them: “the sky is the limit—just tell me what you need.” The usual suspects were rattled off…more time, money, staff…and then a quiet voice said, “I wish Baltimore would bring back the horses.”
Huh? I quickly looked at my Union of Concerned Scientists’ colleagues—I mouthed the word “horse?” —and they all nodded “yes”. That voice, now becoming more confident, went on: “Yes, I would like to see the arabbers on the streets again.” I had no idea what an arabber was and was too embarrassed to ask. Fortunately, Clayton Williams, farm manager of Strength to Love II, a job training program to support residents returning from incarceration, saved me and asked, “Do you all know what I’m talking about?—Baltimore used to have arabbers traveling through neighborhoods with horses and carts selling fresh produce. They traveled right to the neighborhood—right into the heart of the community.”
Arabber on 36th Street in Baltimore. (Photo by Jason Hoffman)
Following the Civil War, horse-cart vending was one of a only few jobs available to African American entrepreneurs. Arabbers sold produce on colorful horse-drawn carts—and Baltimore’s arabbers are the last horse-cart vendors in the United States. However, over the past several years city health officials and animal control have shut down many operations due to sanitary concerns and poor living conditions for the horses. While sanitation and animal welfare are legitimate concerns, cities should try and work with arabbers more closely to address these issues.
I followed-up with Clayton to learn more about Baltimore’s arabbers. I asked him to tell me about his experiences with them and he said: “When I was a kid, my siblings and I spent a lot of time with my grandmother. We would sit on her front porch and watch the arabbers coming down the street handing out peaches, cherries, and watermelons. It was just like the ice cream man—you were always happy to see him. They were part of the community—you would see the different horses and carts. Each horse had their own style.” More importantly, Clayton said, “They got food right into the neighborhood. Right there!”
Baltimore’s arabbers were a simple solution to a public problem: a broken food system. Clayton’s grandmother didn’t drive, and she cared for Clayton and his two siblings after school. If she wanted to purchase food for their family, they would have to walk to the grocery store. Clayton remembers carrying grocery bags with his siblings all the way home—“It was pretty inconvenient.” So when the arabbers came through the neighborhood calling out the different types of produce they had for sale, Clayton said his grandmother would always buy from them. “The arabbers were a morale booster. Their carts were colorful and they made people feel good.”
A new study released this month by the Baltimore Food Policy Initiative and the Johns Hopkins Center for a Livable Future found that 1 in 4 Baltimore residents lack access to healthy food. The study also found that African Americans and children are disproportionally affected by this lack of access. In terms of finding solutions to this problem, Clayton says: “Bringing Baltimore’s arabbers back would get fresh produce right into the community—it’s good for the entrepreneur, it’s good for the people. It may sound small and old-fashioned, but if they could find a way to get the horses back it would be a beautiful solution.”
Solutions to public problems don’t always have to be complex. Local governments, non-profit organizations, and entrepreneurs across the country are working to address food access in lower-income communities. New York City’s Green Carts program consists of mobile food carts that offer fresh produce in neighborhoods with limited food access. Arcadia Center for Sustainable Food and Agriculture operates farm-stands-on-wheels that travel directly to underserved communities in the Washington, DC area selling local, sustainably produced food to residents. Breaking Bread Cafe in North Minneapolis is a restaurant serving healthy, flavorful food while trying to improve the health of its community through food and economic development opportunities.
Small-scale, community-inspired solutions can serve as the building blocks to creating a more equitable food system.
Support from UCS members make work like this possible. Will you join us? Help UCS advance independent science for a healthy environment and a safer world.
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Wow! I so remember those horse carts when I was young growing up in Bmore. It is funny how we can watch horses run at the Preakness and yet can’t have them walk around bring freshly grown produce to the people. I would love to support the building of a clean area for horses and carts to reside. The Penn / North area was where they were housed and it was not the greatest for sure. Good is possible. I would love to see that again.
Lindsey Haynes-Maslow
Thank you for your comment Coach! I know there is a Baltimore Arabber’s Preservation Society, but I am unaware of any current movement to get a different area for the horses’ stables. I will be keeping a close watch on any policy movement. However, if you’re still in the Baltimore area, I do think you could ask a city official to consider your suggestion.
My grandfather sold produce from a horse drawn cart in the Detroit area when he was a young man. It was about 1915 or so. He did that successfully for a few years. Then with his savings he and his wife bought a small retail store in a residential neighborhood. They changed it into a hardware store in a few years when they thought they could do better. That became the family business on which their four sons were raised. It also provided us 11 grandchildren support for our college educations! He finally sold the business almost 60 years later.
Lindsey Haynes-Maslow
Richard, thank you for your comment. That is such an amazing story. I think it is so important to share these stories so they are not lost or forgotten for future generations. Do you have any pictures of your grandfather’s cart or store in Detroit?
Richard Solomon
Thanks for asking! Below is a copy of the one photo that I have of the store. It was taken around 1925….maybe 7-8 years after the store had been in operation. He called it Red Front Hardware because of the red brick wall in the front of the building. He is the one standing behind the counter.
Lindsey Haynes-Maslow
That is such a great photo! I love the store and all the items hanging from the ceiling! How lucky of you to have this photo in your possession! | 2023-10-06T01:26:19.665474 | https://example.com/article/8335 |
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abstract:
- |
The energetics of ionic selectivity in the neuronal sodium channels is studied. A simple model constructed for the selectivity filter of the channel is used. The selectivity filter of this channel type contains aspartate (D), glutamate (E), lysine (K), and alanine (A) residues (the DEKA locus). We use Grand Canonical Monte Carlo simulations to compute equilibrium binding selectivity in the selectivity filter and to obtain various terms of the excess chemical potential from a particle insertion procedure based on Widom’s method. We show that K$^{+}$ ions in competition with Na$^{+}$ are efficiently excluded from the selectivity filter due to entropic hard sphere exclusion. The dielectric constant of protein has no effect on this selectivity. Ca$^{2+}$ ions, on the other hand, are excluded from the filter due to a free energetic penalty which is enhanced by the low dielectric constant of protein.
Monte Carlo, primitive model electrolytes, ion channel, selectivity 61.20.Qg, 68.03.-g, 81.05.Rm, 61.20.Ja, 07.05.Tp
- |
Проведено дослідження енергетики іонної селективності в нейронних каналах натрію. Використано просту модель, сконструйовану спеціально для селективного фільтру. Селективний фільтр канального типу містить залишки аспарату (D), глютамату (E), лізину K) та аланіну (A) (область DEKA). Використано моделювання методом Монте Карло у великому канонічному ансамблі для обчислення селективності рівноважного зв’язування у селективному фільтрі і для отримання різних членів надлишкового хімічного потенціалу в результаті процедури вставляння частинок на основі методу Відома. Показано, що іони K$^{+}$ у суперництві з Na$^{+}$ ефективно вилучаються з селективного фільтра за рахунок ентропійного виключення твердих сфер. Діелектрична проникність протеїну не має жодного впливу на дану селективність. З іншого боку, іони Ca$^{2+}$ вилучаються з фільтра за рахунок вільного енергетичного “пенальті”, що підсилюється низькою діелектричною проникністю протеїну.
Монте Карло, примітивна модель електролітів, іонний канал, селективність
author:
- 'D. Boda[^1], G. Leaf, J. Fonseca, B. Eisenberg'
- 'Д. Бода, Г. Ліф, Дж. Фонсека, Б. Айзенберг'
date: 'Received September 2, 2014'
title: Енергетика іонної конкуренції у селективному фільтрі DEKA нейронних натрієвих каналів
---
Introduction {#sec:intro}
============
Sodium (Na) channels can be categorized on the basis of their function, the cell in which they are found, structure of the protein (both secondary and tertiary), and the structure of the selectivity filter (SF). The SF is a narrow region of the permeation pathway, where the channel discriminates between different ions. The selectivity properties of different channels primarily depend on the type of amino acid motifs present in their SF.
The two most widely studied classes of Na channels are the neuronal (this is the one studied here) and bacterial Na channels. The SF of neuronal Na channels has a DEKA locus made of aspartate (D), glutamate (E), lysine (K), and alanine (A) residues. On the basis of their homology with L-type calcium (Ca) channels [@2000_koch_jbc_34493], these amino acids seem to face the permeation pathway. The accurate structure of the DEKA Na channels is still unknown, so theoretical studies are restricted to using models based on homologies of the known structures or on reduced models based on minimal structural information available. This is the approach used in this work, while the minimal structural information is that the SF has the DEKA locus.
The bacterial Na channels, on the other hand, have X-ray structures measured recently [@2012_mccusker_nc_1102; @2011_payandeh_n_353; @2013_stock_jpcb_3782; @2013_ulmschneider_pnasusa_6364]. These channels include NavMs [@2012_mccusker_nc_1102; @2013_ulmschneider_pnasusa_6364], NavAb [@2011_payandeh_n_353; @2013_stock_jpcb_3782], and NaChBac [@2001_ren_s_2372]. The structure of a Ca$^{2+}$-selective mutant of NavAb is also available [@2014_tang_n_56]. These channels have a lot of aspartates and glutamates in their SF. Therefore, at a first glance, they look like a Ca channel. Hydration plays an important role in the selectivity mechanisms of these channels [@2014_finnerty_jpcl_subm], but this is not the subject of the present study.
Simulation studies for Na channels have been based on models of different resolutions. All-atom, explicit-water models are usually used when X-ray structures are available. They are generally studied using molecular dynamics (MD) simulations [@2013_ulmschneider_pnasusa_6364; @2013_stock_jpcb_3782; @2014_finol-urdaneta_jgp_157]. In the case of the DEKA channel, Lipkind and Fozzard [@2008_lipkind_jgp_523] performed MD simulations to explore the Na$^{+}$ vs. K$^{+}$ selectivity for various mutants of DEKA based on extreme homology modelling.
Boda et al. [@boda-pccp-4-5154-2002; @boda-bj-93-1960-2007; @csanyi-bba-1818-592-2012] and Vora et al. [@2008_vora_bj_1600] used reduced models of Na channels in the implicit solvent framework. In these models, only the SF amino acids were represented in an explicit way, while other parts of the channel protein were reduced into a dielectric body. This is the modelling level that we use in this work. An intermediate approach is that of Finnerty et al. [@2013_finnerty_jctc_766], who proposed a localization method, where SF aminoacid terminal groups are localized into certain positions inspired by structural information.
The advantage of reduced models is that they permit the design of simulation setups in time and length scales that mimic experimental setups and are capable of studying a wide range of concentrations and voltage. Also, they make it possible to focus on the essential features of the system (SF structure, pore geometry, bath concentrations, voltage, etc.) and to take the effect of the remaining degrees of freedom into account in an averaged, but physically well-based manner (dielectric response as well as external constraints such as the walls of the channel and the membrane).
Simulations can also be distinguished on the basis of the fact whether they were performed in or out of equilibrium. Equilibrium simulations can study the selective binding of various ions in the SF. Monte Carlo (MC) simulations, especially in the grand canonical (GC) ensemble (Grand Canonical Monte Carlo, GCMC) are ideal tools for this purpose [@boda-pccp-4-5154-2002; @boda-bj-93-1960-2007; @csanyi-bba-1818-592-2012; @2013_finnerty_jctc_766]. MD simulations can also be used to study selective binding [@2008_lipkind_jgp_523; @roux_2010; @roux_2005]. Some properties of transport, however, can be extrapolated even from equilibrium simulations on the basis of the integrated Nernst-Planck equation as suggested by Gillespie et al. [@gillespie-bj-95-2658-2008; @boda-jgp-133-497-2009]. Simulating transport requires a dynamical simulation method. These can be MD simulations [@2013_ulmschneider_pnasusa_6364; @2013_stock_jpcb_3782; @2014_finol-urdaneta_jgp_157], Brownian Dynamics simulations [@2008_vora_bj_1600; @chung-bj-77-2517-1999; @corry-bj-2001; @im_bj_2000], and Dynamical Monte Carlo (DMC) simulations [@boda-jpcc-118-700-2014; @csanyi-bba-1818-592-2012; @hato-jcp-137-054109-2012]. Transport can also be studied with theoretical methods such as the Energy Variational approach of Eisenberg et al. [@eisenberg_envara; @hyon_cms_2011; @liu_jpcb_2013; @liu_jcp_2014].
To extend equilibrium binding-selectivity simulations to non-equilibrium situations of steady-state ionic transport is of crucial importance because experimental data are available for ionic currents from electrophysiological measurements [@1965_Chandler_jpl_788; @1969_binstock_jgp_342; @1971_hille_jgp_599; @1972_feldman_ab_317; @1972_hille_jgp_637; @1973_meves_jpl_225; @1976_binstock_jgp_551; @1976_campbell_jgp_295; @1976_ebert_jgp_327; @1992_heinemann_n_441; @1994_canessa_n_463; @1995_tomaselli_bj_1814; @2000_sheng_jbc_8572; @2001_kellenberger_jgp_679; @2003_li_jbc_13867; @2007_anantharam_jgp_55]. The relation of the fluxes carried by the competing ions (flux ratio) defines dynamical selectivity. How binding selectivity is related to dynamical selectivity is, however, a non-trivial issue as shown by Rutkai et al. [@rutkai-jpcl-1-2179-2010]. In particular, the flux is determined not only by the occupancy of a given ionic species in the channel, but also by its mobility.
Measurements show permeability ratios 0.06 and 0.13 for K$^{+}$/Na$^{+}$ and Ca$^{2+}$/Na$^{+}$, respectively [@1965_Chandler_jpl_788; @1969_binstock_jgp_342; @1971_hille_jgp_599; @1972_feldman_ab_317; @1972_hille_jgp_637; @1973_meves_jpl_225; @1976_binstock_jgp_551; @1976_campbell_jgp_295; @1976_ebert_jgp_327; @1992_heinemann_n_441], while $<0.01$ flux ratio for K$^{+}$/Na$^{+}$ [@1994_canessa_n_463; @1995_tomaselli_bj_1814; @2000_sheng_jbc_8572; @2001_kellenberger_jgp_679; @2003_li_jbc_13867; @2007_anantharam_jgp_55]. To a first approximation, we can assume that binding selectivity agrees well with the above selectivity values measured in terms of flux. To what degree this assumption is valid can be studied by dynamical simulation methods. Our first attempt in this direction is the DMC study of Csányi et al. [@csanyi-bba-1818-592-2012].
In this paper, we focus on equilibrium binding, so it is a direct continuation of our previous papers [@boda-pccp-4-5154-2002; @boda-bj-93-1960-2007], where the binding selectivity of the DEKA locus was studied with GCMC simulations using a reduced model of the SF. These studies used the charge-space competition (CSC) mechanism of Nonner and Eisenberg [@2000_nonner_bj_1976; @2001_nonner_jpcb_6427; @eisenberg_cpl_2011; @crowded_charges; @eisenberg_fnl_2012; @eisenberg_p_2013; @eisenberg_bj_2013] extended later to inhomogeneous models of the channels studied by GCMC simulations [@boda-jpcb-105-11574-2001; @boda-pccp-4-5154-2002; @boda-mp-100-2361-2002; @boda-jcp-125-034901-2006; @boda-prl-98-168102-2007; @boda-bj-93-1960-2007; @gillespie-bj-95-2658-2008; @boda-bj-94-3486-2008; @boda-jgp-133-497-2009; @malasics-bba-1798-2013-2010].
The main conclusions of those papers [@boda-pccp-4-5154-2002; @boda-bj-93-1960-2007] were that K$^{+}$ ions are excluded from the SF by steric repulsion, while Ca$^{2+}$ ions are excluded by an electrostatic penalty. The new aspect of this study is that we provide an energetic analysis for the phenomena described in our 2007 paper [@boda-bj-93-1960-2007]. The energetic analysis is performed by separating the free energy (more exactly, the chemical potential) into various terms corresponding to various interactions such as volume exclusion, ion-ion, ion-dielectrics, self-energy, etc., interactions. This approach was introduced by Gillespie [@gillespie-bj-2008; @dirk-janhavi-mike] in his density functional studies for the Ryanodine Receptor Ca channel and was extended to three-dimensional models including inhomogeneous dielectrics using a GCMC methodology [@boda-jcp-134-055102-2011] on the basis of Widom’s particle insertion method [@widom63; @widom78].
In our previous work, we analyzed the energetics of the selectivity of the L-type Ca channel [@boda-jcp-134-055102-2011]. In that paper, the dielectric constant of the protein ($\epsilon_{\mathrm{pr}}$) was allowed to be different from that of the baths ($\epsilon_{\mathrm{w}}$). It was shown that the low dielectric protein surrounding the pore focusing the electric field, and thus enhancing the electrostatics, is necessary to reproduce the micromolar selectivity observed for the L-type Ca channels [@AlmersMcCleskey1984; @Almersetal1984]. We also extended that work for the case of a dielectric constant different inside the channel ($\epsilon_{\mathrm{ch}}$) from that of the bath [@boda-jcp-139-055103-2013]. This model is a simple representation of solvation. Our results showed that solvation plays a minor role in the selectivity mechanism of the L-type Ca channel. The explanation is that the solvation penalty for Ca$^{2+}$ is balanced by stronger interactions of Ca$^{2+}$ with the SF charges. Our simulations extending this work to the DEKA locus are in progress and will be published in a subsequent paper.
In this work, however, we restrict ourselves to the case, where the dielectric constants inside and outside the channel are the same ($\epsilon_{\mathrm{ch}}=\epsilon_{\mathrm{w}}$). This is the model that was considered in our work [@boda-bj-93-1960-2007] in 2007. The SF of the Ca channel is highly charged (EEEE locus, four glutamate residues providing $-4e$ charge). The DEKA filter, on the other hand, is weakly charged ($-1e$ altogether). Therefore, it does not favor divalent ions (Ca$^{2+}$). Additionally, the bulky terminal group of the lysine is present, which, according to our hypothesis, is there to exclude large ions such as K$^{+}$. This paper examines how these mechanisms work and their energetic basis.
Model {#sec:model}
=====
In our model, most of the atomic structure of the Na channel is reduced to a coarse-grained geometry (figure \[Fig1\]). The channel protein is represented as a continuum solid with dielectric coefficient $\epsilon_{\mathrm{pr}}$. The three dimensional body of the protein is obtained by rotating the thick line in figure \[Fig1\] about the $r=0$ axis. The protein thus forms an aqueous pore that connects the two baths. Water in the baths and pore is described as an implicit solvent that is a continuum dielectric having uniform dielectric coefficient $\epsilon_{\mathrm{w}}=80$. The central cylindrical part of the pore (with radius $R=3.5-4.5$ [Å]{} and length 10 [Å]{}) forms the selectivity filter that includes the only atoms of the protein that are treated explicitly. These atoms are four half-charged ‘oxygen ions’ O$^{1/2-}$ \[figure \[Fig1\] (B), *red spheres*\] representing the charged terminal groups of the D and E residues, while a positive ‘ammonium ion’ NH$_{4}^{+}$ \[figure \[Fig1\] (B), *blue sphere*\] represents the terminal group of the K residue. The alanine is ignored. The structural oxygen ions are confined to the selectivity filter (their centers are in the region $r \leqslant R-R_{i}$, $|z| \leqslant 5~\text{\AA}-R_{i}$, where $R_{i}$ is the ionic radius), but they can move freely inside the filter.
The ions are modelled as charged hard spheres with crystal radii (see caption of figure \[Fig1\]). The computation of the intermolecular energy terms due to screened Coulomb potentials and interactions with polarization charges induced on the dielectric boundaries \[the boundary of the protein and the electrolyte; thick line in figure \[Fig1\] (A)\] are described in our previous works [@boda-pre-69-046702-2004; @boda-jcp-125-034901-2006; @boda-jcp-134-055102-2011]. Ions are restricted to the aqueous space of the model and cannot overlap with hard walls in the system. Figure \[Fig1\] (A) shows only the small central region of the simulation cell. The entire simulation cell is a cylinder having typical dimensions of radius 40 [Å]{} and length 180 [Å]{}. The channel is embedded in a membrane region that excludes ions by hard walls as described before [@boda-jcp-125-034901-2006].
[![(Color online) Model of ion channel, membrane, and electrolyte. The three-dimensional geometry (B) is obtained by rotating the two-dimensional shape shown in panel A around the $z$-axis. The simulation cell is much larger than shown in the figure. The blue lines represent the grid over which the excess chemical potential profiles are computed. The grid is finer inside the channel (width 0.5 [Å]{}), while it is coarser outside the channel (width 2 [Å]{}). The selectivity filter ($|z|<5$ [Å]{}) contains 4 half charged oxygen ions O$^{1/2-}$ (*red spheres* in panel B) and an ammonium ion NH$_{4}^{+}$ (*blue sphere* in panel B). For the radii of the ions, the Pauling radii are used: 0.6, 0.95, 1.33, 1.52, 1.7, 0.99, 1.81, 1.4, and 1.5 [Å]{} for Li$^{+}$, Na$^{+}$, K$^{+}$, Rb$^{+}$, Cs$^{+}$, Ca$^{2+}$, Cl$^{-}$, O$^{1/2-}$, and NH$_{4}^{+}$ respectively.[]{data-label="Fig1"}](Fig1a "fig:"){width="47.00000%"}]{} [![(Color online) Model of ion channel, membrane, and electrolyte. The three-dimensional geometry (B) is obtained by rotating the two-dimensional shape shown in panel A around the $z$-axis. The simulation cell is much larger than shown in the figure. The blue lines represent the grid over which the excess chemical potential profiles are computed. The grid is finer inside the channel (width 0.5 [Å]{}), while it is coarser outside the channel (width 2 [Å]{}). The selectivity filter ($|z|<5$ [Å]{}) contains 4 half charged oxygen ions O$^{1/2-}$ (*red spheres* in panel B) and an ammonium ion NH$_{4}^{+}$ (*blue sphere* in panel B). For the radii of the ions, the Pauling radii are used: 0.6, 0.95, 1.33, 1.52, 1.7, 0.99, 1.81, 1.4, and 1.5 [Å]{} for Li$^{+}$, Na$^{+}$, K$^{+}$, Rb$^{+}$, Cs$^{+}$, Ca$^{2+}$, Cl$^{-}$, O$^{1/2-}$, and NH$_{4}^{+}$ respectively.[]{data-label="Fig1"}](Fig1b "fig:"){width="36.00000%"}]{}
Method of energetic analysis {#sec:energetic}
============================
In an equilibrium GCMC simulation, the acceptance of ion insertion/deletions of ions is governed by the configurational chemical potential of the respective ionic species $i$ defined as $$\mu_{i} = kT \ln c_{i} (\mathbf{r}) + \mu^{\mathrm{EX}}_{i} (\mathbf{r}) = kT\ln c_{i}(\mathrm{B}) + \mu_{i}^{\mathrm{EX}}(\mathrm{B}),
\label{eq:chempot}$$ where $k$ is Boltzmann’s constant, $T$ is the temperature, $c_{i}(\mathbf{r})$ is the concentration profile, $\mu_{i}^{\mathrm{EX}}(\mathbf{r})$ is the excess chemical potential profile, $c_{i}(\mathrm{B})$ is the bulk concentration, and $\mu_{i}^{\mathrm{EX}}(\mathrm{B})$ is the bulk excess chemical potential. Although $kTc_{i}(\mathbf{r})$ and $\mu_{i}^{\mathrm{EX}}(\mathbf{r})$ can be different in different regions (they are position dependent), their sum is constant due to equilibrium. The bulk excess chemical potentials $\mu_{i}^{\mathrm{EX}}(\mathrm{B})$ corresponding to the prescribed bulk concentrations $c_{i}(\mathrm{B})$ are calculated using the Adaptive GCMC method [@malasics-jcp-132-244103-2010]. By rewriting equation (\[eq:chempot\]), the excess chemical potential difference is defined as $$\Delta \mu_{i}^{\mathrm{EX}}(\mathbf{r}) = \mu_{i}^{\mathrm{EX}}(\mathbf{r})-\mu_{i}^{\mathrm{EX}}(\mathrm{B}) = -kT \ln \left(\dfrac{c_{i}(\mathbf{r})}{c_{i}(\mathrm{B})} \right) .
\label{eq:deltamu_ex}$$ It can be identified with the binding free energy of an ion moved from a bath (B) to position $\mathbf{r}$ of the channel [@boda-jcp-134-055102-2011]. If we write up equation (\[eq:deltamu\_ex\]) for Na$^{+}$ and K$^{+}$ and take the difference, we can derive that $$\ln \left( \dfrac{c_{\mathrm{Na}^{+}}(\mathbf{r})}{c_{\mathrm{K}^{+}}(\mathbf{r})} \right) =
\ln \left( \dfrac{c_{\mathrm{Na}^{+}}(\mathrm{B})}{c_{\mathrm{K}^{+}}(\mathrm{B})} \right) +
\dfrac{\Delta\Delta \mu^{\mathrm{EX}}(\mathbf{r})}{kT} \,,
\label{eq:finaladv}$$ where $$\Delta\Delta\mu^{\mathrm{EX}} (\mathbf{r}) = \Delta \mu^{\mathrm{EX}}_{\mathrm{Na}^{+}} (\mathbf{r}) - \Delta \mu^{\mathrm{EX}}_{\mathrm{K}^{+}} (\mathbf{r}) .$$ Similar equations can be given for other pairs of ions.
In equation (\[eq:finaladv\]), the left-hand side is called ‘binding selectivity’ because it expresses the degree to which Na$^{+}$ is favored over K$^{+}$ at location $\mathbf{r}$ (binding selectivity is positive if location $\mathbf{r}$ is selective for Na$^{+}$ over K$^{+}$). The corresponding term on the right-hand side containing the bulk concentrations is called ‘number advantage’ [@gillespie-bj-2008] because it expresses the advantage that an ionic species gets from outnumbering the other species in the bulk. The channel can become selective for a given ionic species for two reasons: either from the number advantage or the energetic advantage expressed by $\Delta \Delta \mu^{\mathrm{EX}} (\mathbf{r})$.
The energetic advantage, however, contains terms due to different interactions present in the system as described in appendix \[sec:wid\]. The EX term can be divided in various ways. Here, we use the division used in our latest work [@boda-jcp-139-055103-2013]: $$\Delta\mu^{\mathrm{EX}}_{i}(\mathbf{r}) = \Delta\mu^{\mathrm{HS}}_{i}(\mathbf{r}) + \Delta\mu^{\mathrm{II}}_{i}(\mathbf{r}) + \Delta\mu^{\mathrm{ID}}_{i}(\mathbf{r}) + \Delta\mu^{\mathrm{SELF}}_{i}(\mathbf{r})$$ or briefly $\mathrm{EX}=\mathrm{HS}+\mathrm{II}+\mathrm{ID}+\mathrm{SELF}$, where HS means hard sphere exclusion, II means interaction with the ions, ID means interactions with the dielectrics (polarization charges induced by other ions), and SELF means interactions with the polarization charges induced by the ion itself. (In the division of our earlier work [@boda-jcp-134-055102-2011], we used the DIEL term that contained the SELF term, namely, $\mathrm{DIEL}=\mathrm{ID}+\mathrm{SELF}$.) We can also use the division $\mathrm{EX}=\mathrm{HS}+\mathrm{MF}+\mathrm{SC}+\mathrm{SELF}$, where MF means the interaction with the mean (average) electric field of all the existing charges in the system (ionic and induced). SC expresses correlations beyond the mean field level (SC refers to ‘screening’) [@gillespie-bj-2008]. The SELF term is a one-particle term (mean-field in nature) and corresponds to the average electrostatic interaction energy of the inserted ion with its self-induced charge. It is not included in the ID or the MF term. The SELF term corresponds to the dielectric boundary force or energy of reference [@nadler_2003].
The computation of all these terms can be found in our original paper [@boda-jcp-134-055102-2011] and in appendix \[sec:wid\]. Briefly, the total EX chemical potential can unambiguously be obtained by inserting charged hard spheres (representing the ions) in the Widom particle insertion method. Different terms of EX are computed by inserting particles interacting only through short-ranged (HS) or more direct (II) interactions and obtaining the rest as residuals. For example, it is reasonable to compute the HS term by inserting uncharged hard spheres with the same radius as the respective ion in the Widom procedure. All the remaining terms (II, ID, SELF) are electrostatic in nature and obtained by deducting the HS term from the EX term. (The separation of HS and electrostatic terms and their effect on selectivity can already be found in the work of Nonner et al. [@2000_nonner_bj_1976] in the context of the mean spherical approximation.) Similar procedures are used to separate the II and ID, as well as the MF and SC terms, as described in appendix \[sec:wid\].
The $\mathbf{r}$-dependence of various terms is computed by ion insertions into grid cells shown in figure \[Fig1\]. Note that the concentration profile can be computed in two different ways. First, sampling the number of ions in a volume element, computing the average ion number and dividing by the volume of the element. This is advantageous when the concentration and/or the volume element is large so there is a large enough sample of ions. The concentration, on the other hand, can also be computed from equation (\[eq:chempot\]) by computing the EX term from the Widom method and deducting it from the chemical potential. This approach is useful where the concentration is low. This method was used in our simulations for the DEKA channel.
Our grid is two-dimensional because we have rotational symmetry. Our profiles, therefore, are expressed in terms of the $(z,r)$ cylindrical coordinates. In this work, however, we show the results that are averaged over the $r$-coordinate $$\Delta \mu_{i}^{\mathrm{EX}}(z) = \dfrac{2}{R_{\mathrm{min}}^{2}(z)} \int_{0}^{R_{\mathrm{min}}(z)} r\, \Delta \mu_{i}^{\mathrm{EX}} (z,r) \,\rd r ,
\label{r-average}$$ where $R_{\mathrm{min}}(z)=R(z)-R_{\mathrm{larger \, \, ion}}(z)$ is the cross-section that is accessible to the center of the larger of the competing ions, \[$R(z)$ denotes the radius of the simulation domain at $z$\].
Results and discussion {#sec:results}
======================
We start our discussion with competition of ions of the same charge. Specifically, we study the selectivity of Na$^{+}$ over various monovalent ions. In the classical mole fraction experiment, the mole fraction of one ion (Na$^{+}$, for example) is changed while keeping the total cation concentration constant (when divalent ion is present, the total ionic strength is kept constant in some studies). These results are seen in figure 5 of reference [@boda-bj-93-1960-2007]. In this work, the concentration of the two competing cations in the baths is the same (50 mM), so the number advantage is zero.
Figure \[Fig2\] shows the various terms of the $\Delta \mu ^{\mathrm{EX}}_{i}(z)$-profiles for Na$^{+}$ and K$^{+}$ for protein dielectric constant $\epsilon_{\mathrm{pr}}=10$ and filter radius $R=3.5$ [Å]{}. The value $\epsilon_{\mathrm{pr}}=10$ is the value fixed in our studies for the L-type Ca channel [@boda-jcp-125-034901-2006; @boda-prl-98-168102-2007; @gillespie-bj-95-2658-2008; @boda-jgp-133-497-2009; @malasics-bba-1788-2471-2009; @malasics-bba-1798-2013-2010; @boda-jcp-134-055102-2011; @giri-pb-8-026004-2011]. The value $R=3.5$ [Å]{} was used in our DMC study for the DEKA Na channel to reproduce experimental data [@csanyi-bba-1818-592-2012].
The EX terms are related to the concentration ratios through $-\ln [c_{i}(\mathbf{r})/c_{i}(\mathrm{B})]$ \[see equation (\[eq:deltamu\_ex\])\]. Therefore, where the EX term (or any component) is negative, it energetically favors the ionic species, so it increases the concentration of that ionic species. As also seen in figure 6 of our previous paper [@boda-bj-93-1960-2007], there are peaks at the entrances of the SF and the vestibules ($|z|\sim 5$ [Å]{}). In the center of the SF, on the other hand, the concentrations are low. This region forms a depletion zone for both ions, where ions have difficulty to enter. The question, therefore, is which ion is excluded less from this region. The answer is that there are more Na$^{+}$ than K$^{+}$ in the SF (the EX term is lower for Na$^{+}$), so the SF is Na$^{+}$-selective.
All the electrostatic terms (II, ID, MF, SC) are negative except the SELF term. The SELF term is repulsive because the ions are in the $\epsilon_{\mathrm{w}}=80$ region, so the sign of the induced charge on the $\epsilon_{\mathrm{pr}}|\epsilon_{\mathrm{w}}$ boundary is the same as the sign of the inserted ion itself. This practically corresponds to the dielectric penalty an ion must pay when it passes the low dielectric membrane region as described in classical works [@neumcke_bj_1969; @parsegian_n_1969; @levitt_bj_1978; @jordan_bj_1982]. The SELF term is slightly larger for Na$^{+}$ because the smaller Na$^{+}$ can get closer to the channel wall and can induce a larger polarization charge.
The other term that is positive is the HS term describing the volume exclusion. This is the term that is very different in the case of Na$^{+}$ and K$^{+}$; it is larger in the case of K$^{+}$. Since the size of K$^{+}$ ions (we talk about the dehydrated (Pauling) radius) is larger, it is more difficult to insert such an ion in the SF. Therefore, K$^{+}$ has a larger entropic penalty than Na$^{+}$ does. This difference is especially apparent in the center of the SF, where the NH$_{4}^{+}$ (the structural ion representing the large terminal group of the lysine) profile has a peak (see figure 6 of reference [@boda-bj-93-1960-2007]). Without the HS term (ions of finite size) we could not get a Na$^{+}$-selective filter (against K$^{+}$) in this model.
The MF term is negative, because the SF is negatively charged. There is no space for the cations to fully neutralize the SF charge. The SC term is similar to the MF term in order of magnitude indicating that mean field theories are not sufficient to study ionic systems in crowded confined spaces such as the SF of ion channels.
The dominant term that drives Na$^{+}$ vs. K$^{+}$ selectivity is the HS term. In figures \[Fig3\] and \[Fig4\], therefore, only the differences of the EX and HS terms are shown for various cases. In this special case, where the number advantage is zero, the EX difference is equal to the binding affinity \[see equation (\[eq:finaladv\])\], while the HS term is the dominant term of $\Delta\Delta\mu^{\mathrm{EX}}(\mathbf{r})$. Since the differences are obtained by deducting the K$^{+}$ terms from the Na$^{+}$ terms, positive values favor Na$^{+}$.
[![ (Color online) The binding affinity and HS advantage curves for Na$^{+}$ vs. K$^{+}$ competition for $\epsilon_{\mathrm{pr}}=10$ (top panels) and 80 (bottom panels) for filter radii $R=3.5$, 4, and 4.5 [Å]{} (50–50 mM bath concentrations).[]{data-label="Fig3"}](Fig3 "fig:"){width="85.00000%"}]{}
[![(Color online) The binding affinity and HS advantage curves for the competition of Na$^{+}$ against various monovalent ions (Li$^{+}$, K$^{+}$, Rb$^{+}$, Cs$^{+}$) for $\epsilon_{\mathrm{pr}}=10$ (top panels) and 80 (bottom panels) (50–50 mM bath concentrations, $R=3.5$ [Å]{}). []{data-label="Fig4"}](Fig4 "fig:"){width="85.00000%"}]{}
Figure \[Fig3\] shows the profiles for various pore radii for $\epsilon_{\mathrm{pr}}=10$ (top panels) and $\epsilon_{\mathrm{pr}}=80$ (bottom panels). Narrower channels favor Na$^{+}$ even more, as expected, because it is even more difficult to find space for the large K$^{+}$ ions in the small SF compared to Na$^{+}$. Putting it in another way, Na$^{+}$ vs. K$^{+}$ selectivity is better for narrow channels, where stronger competition is forced by the confinement and lack of space, so the smaller size of Na$^{+}$ has the advantage. The binding affinity curves (left-hand panels) and the HS advantages (right-hand panels) behave similarly with small differences due to other energetic terms (see figure \[Fig2\]).
Another conclusion of the figure is that Na$^{+}$ vs. K$^{+}$ selectivity does not depend on the dielectric constant of the protein; the curves for $\epsilon_{\mathrm{pr}}=10$ (top panels) and $\epsilon_{\mathrm{pr}}=80$ (bottom panels) behave practically the same.
Figure \[Fig4\] shows the same curves but now for a fixed pore radius ($R=3.5$ [Å]{}) and different monovalent cations (Li$^{+}$, K$^{+}$, Rb$^{+}$, Cs$^{+}$) competing with Na$^{+}$. The main conclusion is similar to those drawn in figure \[Fig3\]; the crowded SF favors the smaller ion. The pore is selective for Li$^{+}$ against Na$^{+}$, while it is selective for Na$^{+}$ against the larger ions.
The protein dielectric constant does not have an effect on these profiles. Of course, the value of $\epsilon_{\mathrm{pr}}$ has a large effect on the individual ionic profiles and the occupancies (see figure 8 of reference [@boda-bj-93-1960-2007]), but not on the relative ones that we study here.
In the second half of this section, we analyze the competition of Na$^{+}$ against Ca$^{2+}$. The other usual way to study the behavior of the channel having varying electrolyte composition is to keep the concentration of one species fixed (Na$^{+}$, for example) and to add another species (Ca$^{2+}$, for example) gradually. This added salt experiment was done by Almers and McCleskey in their experiment for the L-type Ca channel [@Almersetal1984; @AlmersMcCleskey1984]. We performed this kind of experiment in our previous simulations for the DEKA locus and its DEEA mutant, see figure 2 of reference [@boda-bj-93-1960-2007].
[![(Color online) Ca$^{2+}$ and Na$^{+}$ concentration profiles for two different Ca$^{2+}$ concentrations (10 and 40 mM in top and bottom panels, respectively) with a 50 mM Na$^{+}$ background ($R=3.5$ [Å]{}). The profiles are shown for protein dielectric constants $\epsilon_{\mathrm{pr}}=80$ (left-hand panels) and 10 (right-hand panels).[]{data-label="Fig5"}](Fig5 "fig:"){width="90.00000%"}]{}
Those simulations qualitatively reproduced the experiment of Heinemann et al. [@1992_heinemann_n_441]. Heinemann et al. found that mutating the DEKA locus into a DEEA locus, the selectivity behavior of the channel is reminiscent to Ca channels rather than Na channels. In experiment, the current drops to half (IC50) at Ca$^{2+}$ concentration $10^{-4}$ M, while in our simulations, the number of Na$^{+}$ ions drops to half at the same concentration. The explanation is that the DEEA mutation has $-3e$ charge producing a Ca channel, but with weaker selectivity than in the case of the $-4e$ charge (EEEE locus). The DEKA locus, on the other hand, shows Na$^{+}$ over Ca$^{2+}$ selectivity. This selectivity is stronger for smaller $\epsilon_{\mathrm{pr}}$ (see figure 10 (A) of reference [@boda-bj-93-1960-2007]). The dielectric constant of the protein, therefore, has a strong effect in the case of monovalent vs. divalent competition.
In figure \[Fig5\], we show the results only for two chosen Ca$^{2+}$ concentrations, 10 mM (top panels) and 40 mM (bottom panels) — both are well above the physiological values ($\sim$1–2 mM).
The background Na$^{+}$ concentration is 50 mM. The Na$^{+}$ and Ca$^{2+}$ concentration profiles are shown for $\epsilon_{\mathrm{pr}}=80$ (left-hand panels) and 10 (right-hand panels).
There are more Na$^{+}$ than Ca$^{2+}$ ions in the filter in the case of $\epsilon_{\mathrm{pr}}=10$ for both concentrations. A single Na$^{+}$ ion efficiently counterbalances the filter charge. Ca$^{2+}$ ions, on the other hand, overcharge the filter, which is electrostatically unfavorable. To counterbalance this overcharge, a Cl$^{-}$ would be needed, but there is no space left for it in the filter.
In the case $\epsilon_{\mathrm{pr}}=80$, on the other hand, there are more Ca$^{2+}$ ions at \[Ca$^{2+}]=40$ mM. The explanation is that Ca$^{2+}$ is still double charged so the SF attracts it more strongly. The overcharged filter is balanced by Cl$^{-}$ ions from outside the filter. In this case, this is possible because the Coulomb forces are more long-ranged and more screened than in the case of $\epsilon_{\mathrm{pr}}=10$, where the low-dielectric protein focuses the electric field. This means that the low dielectric protein is needed to exclude Ca$^{2+}$.
The energetics of this phenomenon is analyzed in figures \[Fig6\] and \[Fig7\]. The difference in Na$^{+}$ vs. Ca$^{2+}$ selectivity is more clearly seen by plotting the binding selectivity curves. When this is positive, the pore is Na$^{+}$-selective, while it is Ca$^{2+}$-selective in the opposite case. The number advantages are also indicated with dashed horizontal lines. As bath Ca$^{2+}$ concentration is increased, this line and the binding selectivity curve with it are shifted downwards. The shape of the binding selectivity curves does not change much with the bath Ca$^{2+}$ concentration. We can conclude, therefore, that Na$^{+}$ vs. Ca$^{2+}$ selectivity does not depend on the bath Ca$^{2+}$ concentration. This is because the DEKA locus is a singly occupied SF; only one cation occupies the SF at one time (or none).
This was not true for the L-type Ca channel. That channel could be multiply occupied, so selectivity behavior was a function of Ca$^{2+}$ concentration due to correlations of cations in the filter. Furthermore, the SF of the EEEE locus became more charge neutral as Ca$^{2+}$ concentration was increased. Because of that, the MF terms decreased (see figure 7 of Boda et al. [@boda-jcp-134-055102-2011]). That effect is absent here; the probability that a channel becomes charge neutral does not depend on ionic concentrations, but it rather depends on entropic effects (available space in the channel given by filter radius and ion sizes).
The difference of binding selectivity and number advantage defines the free energy advantage, $\Delta\Delta\mu^{\mathrm{EX}} (\mathbf{r})$, \[see equation (\[eq:finaladv\])\]. The terms of that advantage are analyzed for $\epsilon_{\mathrm{pr}}=10$ and 80 for a given Ca$^{2+}$ concentration (10 mM) in figure \[Fig7\].
The top panels show the $\epsilon_{\mathrm{pr}}=10$ data. The left-hand panel shows the II and ID terms ($\mathrm{EX} = \mathrm{HS} + \mathrm{II} + \mathrm{ID} + \mathrm{SELF}$), while the right-hand panel shows the MF and SC terms ($\mathrm{EX} = \mathrm{HS} + \mathrm{MF} + \mathrm{SC} + \mathrm{SELF}$). The EX and SELF terms are shown both in the left-hand and right-hand sides. The HS term (not shown) is close to zero because the ions are of similar sizes. The EX term is also close to zero in this case, but this is the effect of the balance of the different free energy advantage terms. The SELF term is very positive, so it favors Na$^{+}$. This term is about four times larger for Ca$^{2+}$ than for Na$^{+}$ so it plays the role of solvation penalty in this model. Without the SELF term, we could not get a Na$^{+}$ selective filter (against Ca$^{2+}$) in this model. Both the II and ID terms (as well as the MF and SC terms, see right-hand panel) favor Ca$^{2+}$ because Ca$^{2+}$ is attracted twice as strongly by the SF charges (ionic and induced) as Na$^{+}$.
The bottom panels show the $\epsilon_{\mathrm{pr}}=80$ data. Here, the ID and SELF terms are absent, because there is no dielectric boundary present. The ID term favors Ca$^{2+}$, while the SELF term favors Na$^{+}$. Since the SELF term is larger in absolute value, these two terms together ($\mathrm{ID}+\mathrm{SELF}$) still favor Na$^{+}$, so the channel becomes less Na$^{+}$ selective in their absence.
The SC term is small for $\epsilon_{\mathrm{pr}}=80$, which means that Na$^{+}$ vs. Ca$^{2+}$ selectivity is chiefly a mean-field effect in this case; the O$^{1/2-}$ ions attract Ca$^{2+}$ twice as strongly as they attract Na$^{+}$. In the case of $\epsilon_{\mathrm{pr}}=10$, on the other hand, SC is quite large indicating a SF of higher density and correlations beyond the mean-field level (mainly, with induced charges).
Summarizing, the EX term is negative for $\epsilon_{\mathrm{pr}}=80$, so it is rather a Ca channel. The EX term is close to zero for $\epsilon_{\mathrm{pr}}=10$, which means that neither ions are favored energetically. Binding selectivity is driven by the number advantage, which results in a Na$^{+}$ selective channel (against Ca$^{2+}$) at physiological Ca$^{2+}$ concentrations (1–2 mM).
Conclusions {#sec:concl}
===========
We analyzed the energetics of ion selectivity in the SF of the DEKA Na channels. The reduced model studied before [@boda-bj-93-1960-2007] was capable of reproducing the basic characteristics of this channel. We showed that K$^{+}$ ions are excluded from the SF due to entropic hard sphere exclusion. The dielectric constant of the protein has no effect on this selectivity. In general, this filter favors smaller ions over larger ones.
Ca$^{2+}$ ions, on the other hand, are excluded from the filter due to a free-energetic penalty which is enhanced by the low dielectric constant of the protein. The DEKA locus works as a Na channel in the Na$^{+}$ vs. Ca$^{2+}$ competition by *not favoring* Ca$^{2+}$. The dominant term is the number advantage in the bulk solutions. In physiological situations this mechanism suffices.
We showed that the dominant term of the energetic penalty is the SELF term, which is a dielectric penalty — the interaction of the ion with the polarization charges induced by itself. This dielectric penalty is a simple, implicit representation of solvation penalty in the framework of this model, where $\epsilon_{\mathrm{ch}}=\epsilon_{\mathrm{w}}$. Simulations, where a different dielectric constant inside the channel is used, take solvation into account explicitly.
Acknowledgements {#acknowledgements .unnumbered}
================
We gratefully acknowledge the computing resources provided on Blues and/or Fusion, high-performance computing cluster operated by the Laboratory Computing Resource Center at Argonne National Laboratory. We acknowledge the support of the Hungarian National Research Fund (OTKA NN113527) in the framework of ERA Chemistry. Present publication was realized with the support of the projects TÁMOP-4.2.2/A-11/1/KONV-2012-0071 and TÁMOP-4.1.1/C-12/1/KONV-2012-0017.
Widom particle insertion method to compute the components of the excess chemical potential {#sec:wid}
==========================================================================================
The excess chemical potential profile can be computed using Widom’s particle insertion method [@widom63; @widom78]. We divide the simulation cell into small volume elements as described in reference [@boda-jcp-134-055102-2011] and insert “ghost” particles into uniformly generated positions in these volume elements. We compute the interaction energy $U(\mathbf{r})$ of the “ghost” ion inserted at position $\mathbf{r}$ with the whole system and use it in the operation $$\mathcal{W}\left[ U(\mathbf{r})\right] = -kT \ln \left\langle \re^{-U(\mathbf{r})/kT}\right\rangle ,
\label{eq:w-operation}$$ where the brackets denote GC ensemble average. If the interaction energy $U(\mathbf{r})$ contains all the terms (no matter how it is divided into terms), operator $\mathcal{W}$ provides the full excess chemical potential $$\mu_{i}^{\mathrm{EX}}(\mathbf{r}) = \mathcal{W}\left[ U_{i}^{\mathrm{HS}}(\mathbf{r})+ U_{i}^{\mathrm{II}}(\mathbf{r})+ U_{i}^{\mathrm{ID}}(\mathbf{r}) + U_{i}^{\mathrm{SELF}}(\mathbf{r}) \right].$$ A diverging term $U_{i}^{\mathrm{WALL}}(\mathbf{r})$ corresponding to overlap with protein and membrane walls is omitted in this equation, because we evaluate the excess chemical potential only at the allowed positions.
The II term of the energy is obtained as $U_{i}^{\mathrm{II}}(\mathbf{r})=\sum_{j\neq i} z_{i}z_{j}e^{2}\psi_{ij}^{\mathrm{II}}(\mathbf{r},\mathbf{r}_{j})$, where $$\psi_{ij}^{\mathrm{II}}(\mathbf{r}_{i},\mathbf{r}_{j})= \dfrac{1}{8\pi \epsilon_{0} \epsilon_{\mathrm{w}} |\mathbf{r}_{i}-\mathbf{r}_{j}|}
\label{eq:psi_II}$$ describes the Coulomb interaction between two unit charges at positions $\mathbf{r}_{i}$ and $\mathbf{r}_{j}$. The ID term is obtained as $U_{i}^{\mathrm{ID}}(\mathbf{r})=\sum_{j\ne i} z_{i}z_{j}e\psi_{ij}^{\mathrm{ID}}(\mathbf{r},\mathbf{r}_{j})$, where $$\psi_{ij}^{\mathrm{ID}} (\mathbf{r}_{i}, \mathbf{r}_{j} ) = \dfrac{1}{8\pi \epsilon_{0}} \left[
\int_{\mathcal{B}} \dfrac{h_{j}(\mathbf{r}_{j},\mathbf{s})}{|\mathbf{r}_{i}-\mathbf{s}|} \rd\mathbf{s} +
\int_{\mathcal{B}} \dfrac{h_{i}(\mathbf{r}_{i},\mathbf{s})}{|\mathbf{r}_{j}-\mathbf{s}|} \rd\mathbf{s} \right]
\label{eq:psi_ID}$$ describes the interaction of a unit charge at $\mathbf{r}_{i}$ with the polarization charge, $h_{j}(\mathbf{r}_{j},\mathbf{s})$, induced by another unit charge at $\mathbf{r}_{j}$ (or vice versa). Vector $\mathbf{s}$ runs over the dielectric boundary $\mathcal{B}$. The polarization charge is determined using our Induced Charge Computation method [@boda-pre-69-046702-2004; @boda-jcp-125-034901-2006].
We define the terms in the excess chemical potential that correspond to different interactions as suggested by Gillespie [@gillespie-bj-2008]. The definition of these terms is not unique. In our previous work [@boda-jcp-134-055102-2011], we suggested a possible and physically well-based procedure. The HS term in the excess chemical potential is computed by inserting uncharged hard spheres into the system with the same size as the corresponding ion, but without the charge: $$\mu_{i}^{\mathrm{HS}} (\mathbf{r})= \mathcal{W}\left[ U_{i}^{\mathrm{HS}} (\mathbf{r})\right] .$$ The $\mathrm{II}+\mathrm{ID}+\mathrm{SELF}$ part is the difference $\mathrm{EX}-\mathrm{HS}$. If we insert charged hard spheres into the system, but ignore their interactions with the polarization charges, we can compute an excess chemical potential term describing the ion-ion interactions including the HS interactions: $\mathcal{W}\left[ U_{i}^{\mathrm{HS}} (\mathbf{r}) +U_{i}^{\mathrm{II}} (\mathbf{r}) \right]$. The II term (that corresponds solely to the interaction with the ionic charges) is obtained by subtracting the HS term: $$\mu_{i}^{\mathrm{II}} (\mathbf{r}) = \mathcal{W}\left[ U_{i}^{\mathrm{HS}} (\mathbf{r}) +U_{i}^{\mathrm{II}} (\mathbf{r}) \right] - \mathcal{W}\left[ U_{i}^{\mathrm{HS}} (\mathbf{r})\right].$$ The ID term (that corresponds to the interactions with polarization charges induced by other ions) is what remains: $$\mu_{i}^{\mathrm{ID}}(\mathbf{r}) = \mu_{i}^{\mathrm{EX}} (\mathbf{r}) - \mu_{i}^{\mathrm{HS}} (\mathbf{r}) - \mu_{i}^{\mathrm{II}} (\mathbf{r}) -\mu^{\mathrm{SELF}}_{i}(\mathbf{r}).$$ The SELF term is a one-particle term that corresponds to the $i=j$ term of the ID energy in equation (\[eq:psi\_ID\]). The MF term is simply the interaction with the mean electric field computed by sampling with a unit point charge as described in reference [@boda-jcp-134-055102-2011]. The SC term, again, is what remains: $\mathrm{SC} = \mathrm{EX} - \mathrm{HS} - \mathrm{MF} - \mathrm{SELF}$.
[99]{} Koch S.E., Bodi I., Schwartz A., Varadi G., J. Biol. Chem., 2000, **275**, No. 44, 34493; .
McCusker E.C., Bagn[é]{}ris C., Naylor C.E., Cole A.R., D’Avanzo N., Nichols C.G., Wallace B.A., Nat. Commun., 2012, **3**, 1102; .
Payandeh J., Scheuer T., Zheng N., Catterall W.A., Nature, 2011, **475**, 353; .
Stock L., Delemotte L., Carnevale V., Treptow W., Klein M.L., J. Phys. Chem. B, 2013, **117**, No. 14, 3782; .
Ulmschneider M.B., Bagn[é]{}ris C., McCusker E.C., DeCaen P.G., Delling M., Clapham D.E., Ulmschneider J.P., Wallace B.A., Proc. Natl. Acad. Sci. USA, 2013, **110**, No. 16, 6364; .
Ren D.J., Navarro B., Xu H.X., Yue L.X., Shi Q., Clapham D.E., Science, 2001, **294**, No. 5550, 2372;\
.
Tang L., [Gamal El-Din]{} T.M., Payandeh J., Martinez G.Q., Heard T.M., Scheuer T., Zheng N., Catterall W.A., Nature, 2014, **505**, No. 7481, 56; .
Finol-Urdaneta R.K., Wang Y., Al-Sabi A., Zhao C., Noskov S.Y., French R.J., J. Gen. Physiol., 2014, **143**, No. 2, 157; .
Lipkind G.M., Fozzard H.A., J. Gen. Physiol., 2008, **131**, No. 6, 523; .
Boda D., Busath D.D., Eisenberg B., Henderson D., Nonner W., Phys. Chem. Chem. Phys., 2002, **4**, No. 20, 5154; .
Boda D., Nonner W., Valisk[ó]{} M., Henderson D., Eisenberg B., Gillespie D., Biophys. J., 2007, **93**, No. 6, 1960; .
Cs[á]{}nyi [É]{}., Boda D., Gillespie D., Krist[ó]{}f T., Biochim. Biophys. Acta-Biomembr., 2012, **1818**, No. 3, 592;\
.
Vora T., Bisset D., Chung S.H., Biophys. J., 2008, **95**, No. 4, 1600; .
Finnerty J.J., Eisenberg R., Carloni P., J. Chem. Theory Comp., 2013, **9**, No. 1, 766; .
Roux B., Biophys. J., 2010, **98**, No. 12, 2877; .
Roux B., Annu. Rev. Bioph. Biom., 2005, **34**, 153; .
Gillespie D., Boda D., Biophys. J., 2008, **95**, No. 6, 2658; .
Boda D., Valisk[ó]{} M., Henderson D., Eisenberg B., Gillespie D., Nonner W., J. Gen. Physiol., 2009, **133**, No. 5, 497; .
Chung S.H., Allen T.W., Hoyles M., Kuyucak S., Biophys. J., 1999, **77**, No. 5, 2517;\
.
Corry B., Allen T.W., Kuyucak S., Chung S.H., Biophys. J., 2001, **80**, No. 1, 195; .
Im W., Seefeld S., Roux B., Biophys. J., 2000, **79**, No. 2, 788; .
Boda D., Csányi E., Gillespie D., Kristóf T., J. Phys. Chem. C, 2014, **118**, No. 1, 700; .
Hat[ó]{} Z., Boda D., Krist[ó]{}f T., J. Chem. Phys., 2012, **137**, No. 5, 054109; .
Eisenberg B., Hyon Y.K., Liu C., J. Chem. Phys., 2010, **133**, No. 10, 104104; .
Hyon Y., Eisenberg B., Liu C., Commun. Math. Sci., 2011, **9**, No. 2, 459; .
Liu J.L., Eisenberg R., J. Phys. Chem. B, 2013, **117**, No. 40, 12051; .
Liu J.L., Eisenberg B., J. Chem. Phys., 2014, **141**, No. 7, 075102; .
Chandler W.K., Meves H., J. Physiol.-London, 1965, **180**, No. 4, 788; .
Binstock L., Lecar H., J. Gen. Physiol., 1969, **53**, No. 3, 342; .
Hille B., J. Gen. Physiol., 1971, **58**, No. 6, 599; .
Feldman H.A., Anal. Biochem., 1972, **48**, No. 2, 317; .
Hille B., J. Gen. Physiol., 1972, **59**, No. 6, 637; .
Meves H., Vogel W., J. Physiol.-London, 1973, **235**, No. 1, 225; .
Binstock L., J. Gen. Physiol., 1976, **68**, No. 5, 551; .
Campbell D.T., J. Gen. Physiol., 1976, **67**, No. 3, 295; .
Ebert G.A., Goldman L., J. Gen. Physiol., 1976, **68**, No. 3, 327; .
Heinemann S.H., Teriau H., Stuhmer W., Imoto K., Numa S., Nature, 1992, **356**, No. 6368, 441;\
.
Canessa C.M., Schild L., Buell G., Thorens B., Gautschi I., Horisberger J.D., Rossier B.C., Nature, 1994, **367**, No. 6462, 463; .
Tomaselli G.F., Chiamvimonvat N., Nuss H.B., Balser J.R., P[é]{}rez-Garc[í]{}a M.T., Xu R.H., Orias D.W., Backx P.H., Marban E., Biophys. J., 1995, **68**, No. 5, 1814; .
Sheng S., Li J., McNulty K.A., Avery D., Kleyman T.R., J. Biol. Chem., 2000, **275**, No. 12, 8572;\
.
Kellenberger S., Auberson M., Gautschi I., Schneeberger E., Schild L., J. Gen. Physiol., 2001, **118**, No. 6, 679; .
Li J., Sheng S., Perry C.J., Kleyman T.R., J. Biol. Chem., 2003, **278**, No. 16, 13867; .
Anantharam A., Palmer L.G., J. Gen. Physiol., 2007, **130**, No. 1, 55; .
Rutkai G., Boda D., Krist[ó]{}f T., J. Phys. Chem. Lett., 2010, **1**, No. 14, 2179; .
Nonner W., Catacuzzeno L., Eisenberg B., Biophys. J., 2000, **79**, No. 4, 1976; .
Nonner W., Gillespie D., Henderson D., Eisenberg B., J. Phys. Chem. B, 2001, **105**, No. 27, 6427;\
.
Eisenberg B., Chem. Phys. Lett., 2011, **511**, No. 1–3, 1; .
Eisenberg B., In: [Advances in Chemical Physics]{}, vol. [148]{}, Rice S.A., Dinner A.R. (Eds.), John Wiley & Sons, Hoboken, [2011]{}, [77–223]{}; .
Eisenberg B., Fluct. Noise Lett., 2012, **11**, No. 1, 1240001; .
Eisenberg B., Physiology, 2013, **28**, No. 1, 28; .
Eisenberg B., Biophys. J., 2013, **104**, No. 9, 1849; .
Boda D., Henderson D., Busath D.D., J. Phys. Chem. B, 2001, **105**, No. 47, 11574; .
Boda D., Henderson D., Busath D.D., Mol. Phys., 2002, **100**, No. 14, 2361; .
Boda D., Valisk[ó]{} M., Eisenberg B., Nonner W., Henderson D., Gillespie D., J. Chem. Phys., 2006, **125**, No. 3, 034901; .
Boda D., Valisk[ó]{} M., Eisenberg B., Nonner W., Henderson D., Gillespie D., Phys. Rev. Lett., 2007, **98**, No. 16, 168102; .
Boda D., Nonner W., Henderson D., Eisenberg B., [D. Gillespie]{} D., Biophys. J., 2008, **94**, No. 9, 3486;\
.
Malasics M., Boda D., Valisk[ó]{} M., Henderson D., Gillespie D., Biochim. Biophys. Acta-Biomembr., 2010, **1798**, No. 11, 2013; .
Gillespie D., Biophys. J., 2008, **94**, No. 4, 1169; .
Gillespie D., Giri J., Fill M., Biophys. J., 2009, **97**, No. 8, 2212; .
Boda D., Giri J., Henderson D., Eisenberg B., Gillespie D., J. Chem. Phys., 2011, **134**, No. 5, 055102;\
.
Widom B., J. Chem. Phys., 1963, **39**, No. 11, 2808; .
Widom B., J. Stat. Phys., 1978, **19**, No. 6, 563; .
Almers W., McCleskey E.W., J. Physiol., 1984, **353**, 585; .
Almers W., McCleskey E.W., Palade P.T., J. Physiol., 1984, **353**, 565; .
Boda D., Henderson D., Gillespie D., J. Chem. Phys., 2013, **139**, No. 5, 055103; .
Boda D., Gillespie D., Nonner W., Henderson D., Eisenberg B., Phys. Rev. E, 2004, **69**, No. 4, 046702;\
.
Malasics A., Boda D., J. Chem. Phys., 2010, **132**, No. 24, 244103; .
Nadler B., Hollerbach U., Eisenberg R., Phys. Rev. E, 2003, **68**, No. 2, 021905; .
Malasics A., Gillespie D., Nonner W., Henderson D., Eisenberg B., Boda D., Biochim. Biophys. Acta-Biomembr., 2009, **1788**, No. 12, 2471; .
Giri J., Fonseca J., Boda D., Henderson D., Eisenberg B., Phys. Biol., 2011, **8**, No. 2, 026004; .
Neumcke B., L[ä]{}uger P., Biophys. J., 1969, **9**, No. 9, 1160; .
Parsegian A., Nature, 1969, **221**, No. 5183, 844; .
Levitt D.G., Biophys. J., 1978, **22**, No. 2, 209; .
Jordan P.C., Biophys. J., 1982, **39**, No. 2, 157; .
[^1]: Author for correspondence: boda@almos.vein.hu
| 2024-04-28T01:26:19.665474 | https://example.com/article/9563 |
Hypochondriacal beliefs and attitudes in family practice and psychiatric patients.
Beliefs and attitudes which can be responsible for hypochondriacal behavior were explored by administering the Illness Attitude Scales and two distress scales to patients attending a family practice clinic, nonpsychotic psychiatric outpatients and a random group of employees. Family practice patients were more distressed, had more hypochondriacal concerns and had more bodily preoccupations than employees and took more precautions about their health. Psychiatric patients were more distressed and had more fears about illness and death than family practice patients, yet took fewer precautions about their health. The findings appear to have implications for treatment. | 2023-09-24T01:26:19.665474 | https://example.com/article/6285 |
1. FIELD OF INVENTION
This invention relates to compressed ethylene and systems involving the piping of compressed ethylene along relatively great distances. In its broader aspects, it relates to reaction boundary suppressors in facilities in which a reaction boundary such as a flame front of an undesired reaction (initiated by an accident) has a propensity to migrate through a gas pipeline. The similar minimizing of the spreading of flames through a gas pipeline system is sometimes discussed in relation to the strategy of placement of flame arrestors. | 2024-04-13T01:26:19.665474 | https://example.com/article/4062 |
528 So.2d 896 (1988)
Robert Anthony PRESTON, Appellant,
v.
STATE of Florida, Appellee.
No. 70835.
Supreme Court of Florida.
May 26, 1988.
Rehearing Denied August 19, 1988.
*897 Larry Helm Spalding, Capital Collateral Representative and Billy H. Nolas, Staff Atty., Office of the Capital Collateral Representative, Tallahassee, for appellant.
Robert A. Butterworth, Atty. Gen. and Sean Daly, Asst. Atty. Gen., Daytona Beach, for appellee.
PER CURIAM.
Appellant's conviction of first-degree murder and sentence of death were affirmed in Preston v. State, 444 So.2d 939 (Fla. 1984). Five days before his scheduled execution, appellant filed a motion to vacate the judgment and sentence pursuant to Florida Rule of Criminal Procedure 3.850. The trial judge stayed the scheduled execution. The judge then held an evidentiary hearing, after which he denied appellant's motion. Appellant appeals that order. Inasmuch as we hear appeals from final judgments imposing the death penalty, we have jurisdiction of postconviction attacks on such judgments. Art. V, § 3(b)(1), Fla. Const.
At the outset it should be noted that after the stay of execution was entered, the judge permitted appellant to amend substantially his motion for postconviction relief. Thereafter, the hearing was postponed at the request of the Capital Collateral Representative so as to permit further time for investigation. The hearing was *898 not held until almost one year after the original motion had been filed. At the beginning of the hearing, a new lawyer from the office of the Capital Collateral Representative asked to be permitted to take over the case, even though he was not admitted to the Florida Bar. The judge granted the request upon the understanding that he was prepared to go forward with the hearing. Following the close of testimony, appellant's counsel requested to file a memorandum to "supplement the 3.850 proceeding." The court granted the motion after receiving assurances that this was a legal memorandum directed to the issues addressed at the evidentiary hearing. Three weeks later, appellant's counsel moved for a continuance and asked for supplemental relief. He also filed a supplemental memorandum in which he sought to raise new substantive issues based on affidavits which had been signed after the evidentiary hearing. In addition, appellant filed another motion seeking to have a witness produced for testimony "essential to the proper disposition of the instant motion." Finally, a "consolidated addendum" to the motion to vacate as well as a "request for further fact-finding proceedings" was filed along with an "addendum to the proposed order previously submitted." The record does not reflect any attempt to call these motions up for hearing. When the trial judge ultimately denied the motion to vacate, its order addressed only the issues raised in the original amended motion that had been considered at the evidentiary hearing.
In this appeal, appellant raises a myriad of issues, some of which are predicated upon the motions which were filed after the evidentiary hearing and which sought to inject new issues into the case. Under the circumstances, the judge properly declined to rule on these issues, and they will not be further addressed in this opinion. To the extent, if any, that the content of such motions reflects newly discovered evidence tending to exonerate appellant, this may be presented through the filing of a motion for writ of error coram nobis. We note, however, that at least two of the affidavits upon which appellant relies were given by persons who had already testified at the evidentiary hearing.
Appellant contends that the state violated the dictates of Brady v. Maryland, 373 U.S. 83, 83 S.Ct. 1194, 10 L.Ed.2d 215 (1963), by failing to notify appellant's counsel that the police had discovered keys bearing the name "Marcus A. Morales" in the victim's automobile. The existence of the keys came to light during the original trial. Appellant asserted the Brady violation in his motion for new trial, which the court denied on the premise that appellant had failed to demonstrate the materiality of the keys. This issue could have been raised on direct appeal, and appellant is procedurally barred from now raising the claim. Even if there were no procedural bar, the court did not abuse its discretion in excluding evidence of the keys.
Appellant also asserts that the state committed a Brady violation by failing to disclose to the defense an unfavorable personnel evaluation of a hair analysis expert who testified at appellant's trial. In rejecting this contention, the trial court stated:
The court finds as a matter of fact that Diana Bass' testimony was not misleading or based upon improper technique. The record at best shows only that Diana Bass was the subject of a critical employee evaluation and was being retrained. Robert Kopec, the author of the critical evaluation, indicated that he had no knowledge of her work on this case. James Halligan, the Defendant's expert, could not disagree with Diana Bass' conclusion, could not state that her conclusion was misleading, and could not state that she had not used proper techniques.
We find no error in this conclusion. We do not believe that the state's responsibility under Brady extends to examining in depth the personnel files of proposed expert witnesses and divulging possible adverse comments to the defense.
Appellant also contends that appellant's conviction and sentence should be reversed on the theory of a conflict of interests with respect to his former attorney. Several years before the murder involved *899 in this case, appellant was represented on a misdemeanor charge by Don Marblestone, who subsequently became an assistant state attorney. Marblestone played no substantive role in the prosecution of appellant in this case. He testified that he had not discussed any privileged communications or other matters with reference to appellant with any members of the state attorney's office. Marblestone did appear at a 1980 continuance hearing at the request of the prosecuting attorney who was unable to attend but did so only to communicate that attorney's objection to any motion to continue. In the order rejecting this claim, the court stated:
A. FINDINGS OF FACT.
The court finds that Donald Marblestone represented the Defendant in an unrelated misdemeanor case in the early 1970's and later became employed by the State Attorney's Office. The court further finds that Mr. Marblestone did not participate in the prosecution of the Defendant, nor did he provide any prejudicial information relating to the charges against the Defendant, and that his presence at a motion to continue on October 23, 1980, was at the request of prosecuting attorney, Alan Robinson, to state an objection to motion to continue, which was done without any vigorous objection. The appearance of Mr. Marblestone at that hearing is assistance; however, it is not the character of assistance in the prosecution as contemplated by State v. Fitzpatrick, 464 So.2d 1185 (Fla. 1985), justifying disqualification of the entire State Attorney's Office.
We agree with this analysis.
Appellant contends that in imposing sentence upon him, the trial judge did not properly consider all of the nonstatutory mitigating evidence and points to the recent decision of Hitchcock v. Dugger, 481 U.S. 393, 107 S.Ct. 1821, 95 L.Ed.2d 347 (1987), in an effort to avoid procedural default. Appellant's position is not well founded. The jury was properly instructed concerning nonstatutory mitigating evidence, and there is no indication that the judge did not properly consider the nonstatutory mitigating evidence in his sentencing decision. See Johnson v. Dugger, 520 So.2d 565 (Fla. 1988).
Relying upon the rationale of Caldwell v. Mississippi, 472 U.S. 320, 105 S.Ct. 2633, 86 L.Ed.2d 231 (1985), appellant further asserts that the judge's instructions to the jurors misled them with respect to the significance to be attached to their sentencing verdict. Appellant cannot now raise this claim, not only because there was no objection interposed at the trial but because the issue was not raised in his direct appeal. Moreover, even if the claim were not procedurally barred, it could not be sustained on the merits. See Combs v. State, 525 So.2d 853 (Fla. 1988); Grossman v. State, 525 So.2d 833 (Fla. 1988).
Appellant's claims based upon the rationale of Estelle v. Smith, 451 U.S. 454, 101 S.Ct. 1866, 68 L.Ed.2d 359 (1981), are procedurally barred because that decision had been issued by the time of appellant's trial, and there was no objection raised at the trial and no argument of the issue on appeal. Even if the point were not procedurally barred, we agree with the trial court that under the facts of this case, he was not entitled to relief. Unlike the circumstances in Estelle, appellant underwent a court-ordered psychiatric examination only after placing his sanity in issue and after notice to his counsel. Moreover, the psychiatrist's testimony of which he complains was presented after he had opened the door through the introduction of psychiatric testimony of his own on the subject. See Hargrave v. State, 427 So.2d 713 (Fla. 1983); Parkin v. State, 238 So.2d 817 (Fla. 1970), cert. denied, 401 U.S. 974, 91 S.Ct. 1189, 28 L.Ed.2d 322 (1971).
The record supports the trial court's conclusion that appellant was not denied the effective assistance of trial counsel. The balance of appellant's claims should have been argued on direct appeal and are not timely raised in these proceedings.
We affirm the order denying the motion for postconviction relief.
It is so ordered.
*900 McDONALD, C.J., and OVERTON, EHRLICH, SHAW, GRIMES and KOGAN, JJ., concur.
BARKETT, J., concurs in result only.
| 2024-05-20T01:26:19.665474 | https://example.com/article/5319 |
Calendar
After the Kansas City Chiefs finished their three day rookie mini-camp, Todd Haley and CB Donald Washington met with the media to discuss the weekend. Haley on the new guys picking up the system on day three: “Yeah, I would say today was the best day. Again, the conditioning aspect of it was pretty obvious […] | 2024-05-29T01:26:19.665474 | https://example.com/article/5051 |
Structural characterization of myo-inositol monophosphatase from bovine brain by secondary structure prediction, fluorescence, circular dichroism and Raman spectroscopy.
Structural aspects of myo-inositol monophosphatase were examined by spectroscopic techniques and empirical prediction methods. The enzyme belongs to the alpha/beta class of proteins, with approx. 33% alpha-helix and 29% beta-sheet, as shown by circular dichroism (CD), Raman spectroscopy and prediction based on the amino-acid sequence. The Raman spectrum also suggests that the three tryptophan residues in myo-inositol monophosphatase are not exposed to solvent. This was confirmed by a blue shift of 25 nm in the fluorescence emission spectrum, as compared to tryptophan in water, and by quenching studies with acrylamide. The enzyme shows a transition temperature of 87 degrees C for the CD signal at 222 nm. This remarkable heat stability is not due to the presence of disulfide bonds, since both the Raman spectrum and chemical modification studies clearly indicate that all six cysteine residues are in the reduced state. | 2024-05-29T01:26:19.665474 | https://example.com/article/4391 |
+ 7*s - 21*s)*(s**3 - 3*s**3 + s**3)*(-s - 5 + 5).
-17*s**5
Expand (0 - 1 - 1)*(8*t - 3*t - t) + t + t - 3*t.
-9*t
Expand (-g - g - 2*g)*(-5 + 3 - 3).
20*g
Expand (2*z**2 + 2*z**2 + 0*z**2)*(2*z**2 - 6 + 3 - 3*z**2).
-4*z**4 - 12*z**2
Expand (6 - w - 6)*(-2 - 4 + 1)*(-w**3 - 2*w**3 + w**3).
-10*w**4
Expand (-2*k - k - 2*k)*(-4*k - 4*k + 3*k + (4*k - 5*k - k)*(2 + 4 - 4) - 3*k + 3*k + 2*k).
35*k**2
Expand 2*r**4 - r**4 - 3*r**4 + (17 + 6*r**3 - 17 + (-1 + 3 + 0)*(-2*r**3 + 5*r**3 - 2*r**3))*((0 - 1 + 2)*(0*r + 5*r - 3*r) + 15*r - 4*r + 4*r).
134*r**4
Expand -2*s - s**2 + 2*s + (2*s + 2*s - 3*s)*(-2*s - 1 + 1) - 3*s**2 + 4*s**2 - 3*s**2 + 3 + 3 + 3 - 2*s**2.
-7*s**2 + 9
Expand -2*d**4 - 5*d**4 + 3*d**4 + 4*d**4 + d**4 - 3*d**4 + 6*d**4 - 5*d**4 - 2*d**4 + (0*d**4 + d**4 + 0*d**4)*(0 - 1 - 1).
-5*d**4
Expand 2*n**4 - 5*n**4 + 0*n**4 + (-1 - 2*n**4 + 1)*(2 - 7 + 3) + n**4 + n**4 + 4*n**4.
7*n**4
Expand (-6 + 1 + 2)*(2*l**3 + 4*l**3 - 4*l**3) + (8 - 8 + 3*l)*(-42*l + 42*l - 5*l**2).
-21*l**3
Expand 0*b**3 - b**3 + 2*b**3 + (-2*b**3 + 4*b**2 - 4*b**2)*(2 + 0 + 0) - 1 + 2*b**3 + 1 - 4*b**3 + b**3 - 6*b**3.
-10*b**3
Expand -i**3 - 2 + 2 + 2*i**3 + 10*i**3 + 3*i**3 + 2*i**3 + 0 + 0 - i**3 + 2*i**3 + i**3 + (3 - 3 + 2*i**2)*(-2*i - 3*i + 3*i) + 4 + i**3 - 4.
15*i**3
Expand -2*z**2 + 1 - 1 + (37*z - 19*z + 20 - 16*z)*(-1 + 1 - z).
-4*z**2 - 20*z
Expand (-4*k + 3*k - k)*(-k - k + k + (-71*k + 28*k + 32*k)*(-2 + 0 + 3)).
24*k**2
Expand (-2*t + t + 3*t)*(-2 + 2 - t) - t**2 - 4*t**2 + 3*t**2 + (5 - 5 - 3*t**2)*(5 - 5 + 6).
-22*t**2
Expand (w + 0*w - 2*w)*(w**3 + w - w) + 2*w**4 - 2*w**4 - w**4 - 3*w**4 + 2*w**4 + 7*w**4 + 1 + w**4 - 1 + (w + 2*w - 2*w)*(2*w**3 - w**2 + w**2).
7*w**4
Expand (4 - 1 - 1)*(-7*l - 3*l**2 + 7*l + (2*l + l - l)*(l - 4*l + 2*l) - 2*l**2 + 3*l**2 - 4*l**2).
-16*l**2
Expand (-19 - 9 + 10)*(4*p + 0*p - 2*p)*(-5 - 1 + 4).
72*p
Expand (-2*c**4 + c**4 + 3*c**4)*(3 - 4 - 4) - c + 2*c**4 - c - 4*c**4.
-12*c**4 - 2*c
Expand (4*k**2 + 3*k**2 - 4*k**2)*(-2 + 2 - 8*k)*(2 - k - 2).
24*k**4
Expand (2 - 3*a - 2 + 4*a + 0*a - 5*a + (1 - 1 + 1)*(-2*a + 7*a - 2*a))*(-a**3 - 3 + 3).
a**4
Expand -s**5 + 2*s**2 - 2*s**2 + (-3 + 0 + 4)*(-4*s**3 + 0*s**3 + 5*s**3)*(3*s - 2*s**2 - 3*s).
-3*s**5
Expand (5 - 2*b**4 + 4*b**2 - 5)*(3*b - b - b).
-2*b**5 + 4*b**3
Expand (-3 + 0 - 1)*(0*a - a + 0*a)*(-3 - 1 + 3).
-4*a
Expand (-5*i**3 - i**3 + 5*i**3)*(i**2 + 0 + 0 + (5*i - 20*i - 2*i)*(-3 + 3 - 3*i)).
-52*i**5
Expand (c - 2*c**3 - c)*(-2*c - 2 + 2) - 4*c**2 - c**4 + 4*c**2 + (4 + 7*c**3 - 4)*(-5*c + 2*c + 2*c).
-4*c**4
Expand (-9 + 9 + 9*g + (-1 + 1 - 1)*(-2*g + g - g))*(4*g**3 - 2*g**3 + 0*g**3).
22*g**4
Expand ((s + 4 - 4)*(0 + 5 - 3) - 2*s + 5*s - 4*s)*(4*s**2 + 5*s**2 - 2*s**2).
7*s**3
Expand -63*l + 63*l - 21*l**4 + (-l**4 - l**3 + l**3)*(-5 + 1 + 2).
-19*l**4
Expand 15*w**3 - 10*w**3 + 11*w**3 + 3*w**3 + 0*w**3 - 2*w**3 + (-6*w**2 + 4*w**2 + 0*w**2)*(-w + 1 - 1).
19*w**3
Expand (-2*n + 2*n + 2*n)*(-9*n - 4*n + 4*n).
-18*n**2
Expand (-6 + 4 + 3)*(1 + 2 + 0)*(4*n - 3*n - 4*n)*(4*n - 2*n - n).
-9*n**2
Expand ((4*u - 4*u - u)*(-3*u + 0*u + 5*u) + 7*u**2 + 3*u - 2*u**2 + 4*u**2)*(-2 + 2*u + 2).
14*u**3 + 6*u**2
Expand (3 - 6 + 1)*(2*b - 2 + 2) + (1 + 0 + 1)*(0*b - 5*b + 0*b) + (0*b - 2*b + 4*b)*(-2 - 1 + 4).
-12*b
Expand u + 2*u - 2*u + (-3 + 3 - 2)*(5 + 2 - 3)*(-u - u + 0*u).
17*u
Expand -12*i + 12 - 21 + 9 + (2 - 1 - 4)*(i + 0 + 0).
-15*i
Expand (-2*p**3 - 1 + 1)*(-4*p + 2*p + 0*p) + 0*p**4 - p**4 + 0*p**4 - 3*p**4 + 2*p**4 + 0*p**4.
2*p**4
Expand (-378 + 378 - 28*z)*(2 + 3 - 2)*(-2*z + z - z).
168*z**2
Expand -112 + 112 + 21*s - 3*s + 6*s - 5*s + (-3 + 3 - 2*s)*(5 - 5 + 1) + 2*s - 2 + 2.
19*s
Expand (-2*x + 0*x + 0*x)*(-6 + 2 + 3) - 27*x + 6 - 32*x + 61*x.
4*x + 6
Expand (8*q - 4*q**2 - 48*q + 2*q**2 + (-2*q - 2*q + 5*q)*(q + q - 3*q))*(2 + 1 - 2).
-3*q**2 - 40*q
Expand (t**2 + 2*t**2 + 6*t**2)*(-3 - 1 + 3 + (4 + 1 - 6)*(-2 + 0 + 4)).
-27*t**2
Expand (-2*s**2 - s**2 + 2*s**2)*(0 + 9 - 5)*(-s + 1 - 1).
4*s**3
Expand -25 + 25 + 20*a**3 + (-a**2 - 3*a**2 + 6*a**2)*(-1 + 1 + 2*a) + (-1 + 1 - 1)*(a**3 + 0*a**2 + 0*a**2) + 4*a**3 - 2*a**3 + 0*a**3.
25*a**3
Expand (0*j**3 + 0*j**3 + j**3)*(-54*j + 49 - 49)*(6 + 0 - 5).
-54*j**4
Expand (-4 + 4 - 2*k)*(-2*k + 2*k + 2*k) + 3*k**2 + 0*k**2 - 2*k**2 + (0*k + 3*k - 6*k)*(-4*k + 2*k + 4*k).
-9*k**2
Expand -16*x**2 - 39*x**2 + 14*x**2 - 18*x**2 + 0*x + 0*x + x**2 + (-2*x + 2*x - 2*x)*(2 - 2 + 2*x) + 3*x**2 - 3*x**2 + 2*x**2.
-60*x**2
Expand -2*h**2 + 3*h**2 - 3*h**2 + (0*h + 0*h - 2*h)*(0 + h + 0) - 9*h**2 + 34*h**2 + 0*h**2.
21*h**2
Expand (-1 - 2*g + 1)*((-3*g**2 + 3*g**2 + g**2)*(22 - 13 - 22) - 6*g**2 + 5*g**2 + 3*g**2).
22*g**3
Expand (4 + 0 - 3)*(2 + 1 - 2)*(-2 - 1 + 6)*(0*g + 0*g - g).
-3*g
Expand (2*s**2 - 4*s**2 + 11*s**2)*(-2 - s + 2) + 2*s**2 + s**3 - 2*s**2.
-8*s**3
Expand ((-2 - 3 + 4)*(2 + 3 - 2) - 5 + 1 + 2)*(-3 + 2 + 2)*(-4*w + 0*w + 3*w).
5*w
Expand (-5 + 5 - 2*n)*(2*n - n - 2*n + (5 - 1 - 2)*(-3*n + 4*n - 2*n)).
6*n**2
Expand 0*o**2 + 0*o**2 - 5*o**3 + (-1 + 1 - 2)*(-2*o - 2*o**3 + 2*o) + (-5*o**2 + 7*o**2 + 3*o**2)*(-4*o + 3*o + 4*o).
14*o**3
Expand (w - w + w**2)*(36 - 15 - 15).
6*w**2
Expand (7 - 7 + 6*s)*(2 + 1 - 2)*(-2 + 2*s**4 + 4 + 0*s**4).
12*s**5 + 12*s
Expand -2*x**3 + 4*x**3 - 3*x**3 + (8*x - 20*x - 27*x)*(x**2 - x**2 - x**2).
38*x**3
Expand (-u - u**3 + u)*((-5 + 5 - 2)*(0 - 1 + 3) + 1 + 0 - 3) + (-2 + 0 + 1)*(u**3 + 0*u**3 + u**3).
4*u**3
Expand 4*k**3 - 2*k**3 - k**3 + (0 + 0 - 2*k)*(3*k**2 - 3*k**2 - k**2) + 2*k**3 - 2*k**2 + 2*k**2 + 2.
5*k**3 + 2
Expand 7*f**2 + 3*f**5 - 7*f**2 + 4*f**3 + f**5 - 4*f**3 + (f**2 - f**2 + 2*f**4)*(2*f - 4*f + 3*f).
6*f**5
Expand -9*p + 3 - 1 - 7*p + (1 + 1 - 1)*(-3*p + 2*p + 0*p).
-17*p + 2
Expand (k**4 + 4 - 4 + (2*k**2 + 4*k - 4*k)*(-2 + k**2 + 2) + k**4 + 0*k**2 + 0*k**2 + 4*k + k**4 - 4*k)*(-9 - 6 + 10).
-25*k**4
Expand (0*v + 0*v + 2*v)*(-2*v - v + 5*v + 3*v + 0 + 0 + (-5*v + v - v)*(3 + 2 - 3)).
-10*v**2
Expand (-w + w - w)*(2*w - 3*w - 3*w)*((2 + 1 - 4)*(-2*w + 2*w - 2*w) + 0 + w + 0).
12*w**3
Expand (-k**2 + k**2 - k**2 + (-k - 3 + 3)*(0*k - 2*k + 0*k))*(-k**3 - 7*k**3 - k**3).
-9*k**5
Expand (o**2 - 5*o**2 + 3*o**2)*(-9 + 1 - 3).
11*o**2
Expand (-3*p + 0*p + 5*p)*((-1 - 3 + 3)*(7 - 7*p**2 - 7) + 5*p**2 + p**2 - 4*p**2).
18*p**3
Expand 19*y**2 - 65*y + 65*y + (-3*y**2 + 3*y**2 + y**2)*(-1 + 1 - 1) - y**2 - 2 + 2 + 3*y**2 - 2*y**2 + 0*y**2 + 2*y**2 - 3*y**2 - y**2.
16*y**2
Expand (4 - 4 + 1)*(1 + 3*v - 1) + 0 + 0 - v + (5 + v - 5)*(-2 + 3 + 2) - v - 1 + 1 + (-2 - 1 + 5)*(v + 0 + 0).
6*v
Expand -h**2 - 2*h**2 + 4*h**2 - 1 + 1 + h**2 + (2 - h - 2)*(3 - 3 - h) + 3*h**2 - 4*h**2 + 2*h**2.
4*h**2
Expand (-1 - h + 1)*(-7 + 8 - 37) + (0 + 5 - 3)*(-1 - h + 1).
34*h
Expand (4 + 2*d + 2*d**2 - 4)*(d + 2*d - d).
4*d**3 + 4*d**2
Expand (1 - 1 + z)*((-102 + 102 - 15*z**4)*(0 - 2 + 1) - 5*z**4 + 0*z**4 + 3*z**4).
13*z**5
Expand (9*g**4 - g**5 + 41*g**3 + 2 - 41*g**3)*(-3 + 2 + 5).
-4*g**5 + 36*g**4 + 8
Expand (-3 - 2*f + 3)*(1 + 3 - 2) + f - 3*f - 3*f.
-9*f
Expand -6*y + 0*y - 2*y + y + 0 + 0 - 2*y + 4*y + 0*y - 2*y + 5*y - y + (1 + 0 + 1)*(4*y - y - y) + 0*y + 0*y - 2*y + (2*y - 2*y + 2*y)*(1 - 6 + 3).
-5*y
Expand (-3*c**3 + 4*c - 4*c + c)*(5*c**2 - 2*c**2 - 5*c**2).
6*c**5 - 2*c**3
Expand (-4 + 1 + 5)*(-p + 0*p + 0*p) - 4 + 4 + 6 - p.
-3*p + 6
Expand (0 - 6 + 0)*(1 - 3*g - 1)*(g**4 - 6*g**4 + 4*g**4).
-18*g**5
Expand (-p**3 + 5*p**3 - 2*p**3)*(0 - 2*p**2 + 0) + (0*p**4 + p**4 - 3*p**4)*(5*p - p - 5*p) + (-3*p**3 - 2*p**3 + 7*p**3)*(2*p - 3*p**2 - 2*p).
-8*p**5
Expand (2 - 3 + 3)*(-4*p + 2*p - p) + 2 + 2*p - 2 - 4*p + 0*p + 6*p - p + 0*p + 3*p + (2*p - p + 0*p)*(0 + 3 - 1) + p - 1 + 1 + p - 5*p + 2*p.
p
Expand (5*p - 3*p - 3*p - p**2)*(8*p + 0*p - p).
-7*p**3 - 7*p**2
Expand (4 + 3*i - 4)*(1 + 2*i**3 - 1).
6*i**4
Expand (c**2 - 10*c - 3 + 8*c - 7)*(2*c**3 + 3*c**3 - 2*c**3).
3*c**5 - 6*c**4 - 30*c**3
Expand m**2 - m**2 - 2*m**2 + (-3*m + 0*m + 5*m)*(-m + 0 + 0) + 5*m**2 - 2*m**2 - 4*m**2.
-5*m**2
Expand 2 - 2 - 2*r**3 + (0*r**2 + r**2 - 2*r**2)*(2*r + 5*r - 5*r) + (1 + 2 - 2 + (3 + 2 - 4)*(1 - 1 + 1))*(9*r**3 + 8*r**3 - 10*r**3).
10*r**3
Expand 8*t**2 - 3*t**2 + 0*t**2 + (-2*t**2 + 3 - 3)*(-3 + 2 + 0) + t**2 - 3*t**2 + 3*t**2 - t**2 + t**2 + t**2.
9*t**2
Exp | 2023-11-16T01:26:19.665474 | https://example.com/article/8346 |
The BBC's Rupert Wingfield-Hayes"The Chinese are adamantly opposed to any form of missile defence" real 56k
Friday, 15 June, 2001, 08:57 GMT 09:57 UK
Shanghai summit backs ABM Treaty
Russia and China want to increase co-operation
Russia, China and four central Asian countries meeting in Shanghai have signed a joint statement in support of the Anti-Ballistic Missile (ABM) Treaty, which the United States wants to scrap to make way for a missile defence shield.
Attending the summit
Russia
China
Kyrgyzstan
Tajikistan
Kazakhstan
Uzbekistan
The newly named Shanghai Co-operation Organisation (SCO) agreed that the 1972 treaty between the US and the former Soviet Union was the "cornerstone of global stability and disarmament", a Russian official said.
The construction of the US anti-missile shield would effectively lead to the collapse of the ABM treaty, which Mr Bush says is no longer relevant to US-Russian ties and global non-proliferation.
Participants at the Shanghai summit have also signed an agreement aimed at stepping up efforts to defeat Islamic militants in Central Asia.
Arms race
Correspondents say that despite US assurances that the proposed shield is aimed at containing rogue states such as Iraq and North Korea, there is strong opposition from China and Russia who have warned that it could trigger a new global arms race.
The summit is against US plans for a missile defence system
Analysts say China could try to combat the shield by building as many new missiles as possible.
Russian President Vladimir Putin met Chinese President Jiang Zemin for talks, in the first of several meetings planned between the two leaders this year.
President Putin is next due to hold his first meeting with Mr Bush in Slovenia on Saturday with the missile shield and the ABM Treaty top of the agenda.
Russian Foreign Minister Igor Ivanov told reporters in Shanghai that Russia and China regularly consulted about US missile defence plans and that their views "fully coincide".
Fighting Islamic extremism
At its meeting, the SCO signed another agreement, dubbed the Shanghai Convention, aimed at curbing "extremism, terrorism and separatism".
The organisation, originally called the Shanghai Five, was set up in 1996 to sort out lingering Sino-Russian border disputes.
It has evolved into a security mechanism aimed at combating the rise of Islamic militancy, mainly from Taleban-ruled Afghanistan.
On Thursday, Uzbekistan became the sixth member of the group, which Moscow and Beijing hope will counterbalance growing US influence.
A BBC correspondent in Moscow says the addition of Uzbekistan - where authorities are fighting one of the region's strongest rebel groups - is a logical progression.
China, with a large and restive Muslim population in its far west, is keen to stem the growth of Islamic militancy in Central Asia and prevent groups there linking up with Muslim separatists inside China.
All the countries at the summit are dealing with Muslim guerrillas to some extent, many of whom are believed to receive support from Afghanistan's militant Muslim Taleban movement.
But China and Russia also have broader ambitions to build the Shanghai group into a bulwark against American influence in Central Asia. | 2023-08-01T01:26:19.665474 | https://example.com/article/5894 |
Already tracking to be one of our GOATs. Funny to think how cooked we'd have been without him hoping that Petracca, Brayshaw, Viney, Tyson, Stretch and Salem pulled something together (before Harmes looked decent), given he was drafted after all of them. Would take one Oliver over the rest of that group together.
Norm Smith Medallist
Already tracking to be one of our GOATs. Funny to think how cooked we'd have been without him hoping that Petracca, Brayshaw, Viney, Tyson, Stretch and Salem pulled something together (before Harmes looked decent). Would take one Oliver over the rest of that group together.
Premium Platinum
Im not, high profile Victorian players dont ever leave to other Victorian clubs in their prime. He will be an RFA.
Think back over the years an tell me one high profile Vic kid to move to another Vic club | 2023-12-26T01:26:19.665474 | https://example.com/article/9009 |
Theresa May to Brexiteers: back me on compromise plan or risk full customs union
Theresa May has warned warring ministers to fall in line behind her favoured plan for close customs ties with the EU or risk being pushed into a full customs union by MPs, it has emerged.
The Prime Minister's so-called Brexit war cabinet is at odds over Downing Street’s favoured ‘customs partnership’ plan, which eurosceptics fear will hobble future trade deals with countries outside of the EU and reduce the UK to acting as the “taxman of Europe”.
Despite opposition from figures including Home Secretary Sajid Javid, however, Mrs May is expected to table a tweaked version of the proposal at a fresh meeting this week.
Amid intense efforts by Downing Street to convince ministers to back the proposal, the Financial Times reports that the Prime Minister has told them to accept the deal or see MPs impose a full customs union on the Government.
An ally of Mrs May told the FT: “If we don’t make this decision, parliament could do it for us."
The prospect of Commons trouble over the customs union has been raised since ministers suffered a major defeat in the House of Lords on the subject last month.
Several rebel Tory MPs have meanwhile put their names to amendments on the Trade Bill backing close customs ties with the EU.
"DISGRACEFUL"
With tensions running high over the row, Mrs May’s office has been accused of trying to gag Brexit-backing ministers.
The Telegraph cites senior pro-Brexit figures who reacted furiously to what they saw as an attempt by Business Secretary Greg Clark – known to be in favour of Number 10’s plan – to reignite ‘Project Fear’ during a weekend interview where he raised the threat of thousands of job losses.
A Cabinet source accused Downing Street of “silencing Brexiteers while unsubtly putting forward the Business Secretary to make the case for staying in the customs partnership”.
Former Brexit minister David Jones told the paper that there was “no doubt” Mr Clark’s appearance had been “licenced” by Number 10.
He fumed: “They ought to understand that the customs partnership is dead and finished and they should give up...
"If a eurosceptic minister had spoken out, in the same way, they would have been hauled in front of Number 10. It's disgraceful."
But a Downing Street spokesperson denied that Mr Clark’s appearance had been part of a concerted effort to bang the drum for the partnership plan.
They said: "The idea that Downing Street deliberately put Greg Clark up to fly the flag for a customs partnership is absolute nonsense."
Elsewhere Environment Secretary Michael Gove last night endorsed a Twitter thread written by one of his former aides calling for the favoured partnership plan to be shelved.
Eurosceptics instead want to see the UK go for a ‘maximum facilitation’ option, which would use technology and so-called ‘trusted trader’ schemes to try and keep trade friction to a minimum.
Brexit Secretary David Davis admitted last week that Brussels had reacted coolly to both of the proposals being thrashed out by the Cabinet. | 2024-03-18T01:26:19.665474 | https://example.com/article/6347 |
Q:
Need helping combining data from different rows and columns
I am having trouble getting my query to do what I would like. This is my first time trying something like this. Here is part of the table I am working with:
So the DLSEQUENCE column is grouped together by a series of questions asked, this would make one record. What I am trying to find out is the number of restraints by organization. The two organizations I'll call them ABC and DEF. For the RECODED_RESPONSE column, the 1 would be Yes and 2 would be No for the Restraint?. The hardest part for me is putting the data together that is on separate rows. I would like two columns, ABC and DEF. Then under each column, the total of restraints for organization.
Here is what I have so far:
SELECT COUNT(CASE WHEN Field_Name = 'Organization' AND Recoded_Response = 'ABC'
THEN Recoded_Response
END) AS ABC
, COUNT(CASE WHEN Field_Name = 'Organization' AND Recoded_Response = 'DEF'
THEN Recoded_Response
END) AS DEF
FROM DAILY_LOG_CUSTOM_DATA
WHERE Field_Name = 'Restraint?'
AND Recoded_Response = 1
GROUP BY DLSEQUENCE
I was looking for something like this for an output:
A:
What I am trying to find out is the number of restraints by organization.
Hmmm . . . Does a join and group by work?
select o.recoded_response as organization,
sum(case when r.recoded_response = '1' then 1 else 0 end) as restraints
from DAILY_LOG_CUSTOM_DATA o left join
DAILY_LOG_CUSTOM_DATA r
on o.DLSEQUENCE = r.DLSEQUENCE and
r.Field_Name = 'Restraint?'
where o.Field_Name = 'Organization'
group by o.recoded_response;
| 2023-11-02T01:26:19.665474 | https://example.com/article/7501 |
Brentwood Cafe and Tavern Map
Recent Brentwood Cafe and Tavern Reviews
A diner
from Las Vegas, NV
gave an overall rating of
on June 4, 2012 @ 3:21 PM
The food, drinks and service were fantastic. The Saturday night music sucks. Where did you get that wore out old hillbilly from? He dose not know how to sing. My date and I had to leave because he would not stop laughing at the old man on the mic. Other than his attempt at singing, the restaurant was great. Looking forward to some real music, from some real musicians in the future.
A diner
from Las Vegas, NV
gave an overall rating of
on July 23, 2010 @ 4:38 AM
Brentwood Cafe and Tavern is by far the best neighborhood bar in Las Vegas. The food is magnificent and everyone that works there is really friendly. I have to say that I was very comfortable with my visit. If you haven't been to Brentwood Cafe yet then I think it's time you should head on over and bring your family. I will have to admit that the last couple of times I hate their I have added a couple more meals to my favorites. The burgers and fries are to die for and the pizza as well. If you like firewood pizza then you will love it. Even if you don't like firewood pizza you will. The wings in my opinion have to be better than Buffalo Wild Wings. As anyone would know they have some good wings but I really do think they Brentwood Cafe has them beat hands down. So if you like great food and a great atmosphere with friendly people then you should really head to Brentwood Cafe!!!! | 2024-01-21T01:26:19.665474 | https://example.com/article/9443 |
Introduction
CheckInstall keeps track of all files installed by a "make install" or equivalent, creates a Slackware, RPM, or Debian package with those files, and adds it to the installed packages database, allowing for easy package removal or distribution.
Use CheckInstall instead of just running "sudo make install", as that will likely put files all over the filesystem, with no easy way of removing them if things go wrong. If in the future you try to install a package that contains the same file as the software you are compiling, you will receive errors and the software you compiled may stop working.
(In fact, checkinstall can keep track of files modified by any command line, not just a "make install", so you can use it for any type of installation task outside of apt, and it will keep track of the installation in the package manager.)
CheckInstall is not designed to produce packages suitable for distribution. Do not use it to produce packages intended for the Ubuntu archive or PPAs. Instead, follow the Packaging Guide.
From the checkinstall README: "The Debian support in CheckInstall is still new, so handle it with care. It has been reported to work OK in some Debian systems and it certainly works OK in my Slackware development system with dpkg installed. Your mileage may vary."
Installation
Install the package checkinstall from the Repositories.
For help on installing software in Ubuntu, see InstallingSoftware.
A quick method via the terminal for those who like to copy and paste:
sudo apt-get update && sudo apt-get install checkinstall
Usage
Instead of
sudo make install
you will use
sudo checkinstall
When called with no arguments, checkinstall will call "make install". If you need other arguments, they can be supplied:
sudo checkinstall make install_package
The installed package can then also easily be removed via Synaptic or via the terminal:
sudo dpkg -r packagename
Example:
sudo dpkg -r pidgin
Note that the .deb package it creates can also be used elsewhere, which simplifies installation of the same program on many machines.
Use CheckInstall with auto-apt
You can use auto-apt when you want to build a simple package from source with checkinstall. You need to have auto-apt installed!
Instead of
./configure
you use:
auto-apt run ./configure
If the dependencies are available, a dialog box opens and ask you to install them.
The rest remains the same
make sudo checkinstall
CategorySoftware CategoryCommandLine | 2024-05-29T01:26:19.665474 | https://example.com/article/7133 |
#!/usr/bin/env python
import glob
import sys
from setuptools import setup, Extension
from distutils.command.build_ext import build_ext
sources = ['pycrfsuite/_pycrfsuite.cpp', 'pycrfsuite/trainer_wrapper.cpp']
# crfsuite
sources += glob.glob('crfsuite/lib/crf/src/*.c')
sources += glob.glob('crfsuite/swig/*.cpp')
sources += ['crfsuite/lib/cqdb/src/cqdb.c']
sources += ['crfsuite/lib/cqdb/src/lookup3.c']
# lbfgs
sources += glob.glob('liblbfgs/lib/*.c')
includes = [
'crfsuite/include/',
'crfsuite/lib/cqdb/include',
'liblbfgs/include',
'pycrfsuite',
]
class build_ext_check_gcc(build_ext):
def build_extensions(self):
c = self.compiler
_compile = c._compile
def c_compile(obj, src, ext, cc_args, extra_postargs, pp_opts):
cc_args = cc_args + ['-std=c99'] if src.endswith('.c') else cc_args
return _compile(obj, src, ext, cc_args, extra_postargs, pp_opts)
if c.compiler_type == 'unix' and 'gcc' in c.compiler:
c._compile = c_compile
elif self.compiler.compiler_type == "msvc":
if sys.version_info[:2] < (3, 5):
c.include_dirs.extend(['crfsuite/win32'])
build_ext.build_extensions(self)
ext_modules = [Extension('pycrfsuite._pycrfsuite',
include_dirs=includes,
language='c++',
sources=sorted(sources)
)]
setup(
name='python-crfsuite',
version="0.9.7",
description="Python binding for CRFsuite",
long_description=open('README.rst').read(),
author="Terry Peng, Mikhail Korobov",
author_email="pengtaoo@gmail.com, kmike84@gmail.com",
license="MIT",
url='https://github.com/scrapinghub/python-crfsuite',
classifiers=[
"Development Status :: 4 - Beta",
"Intended Audience :: Developers",
"Intended Audience :: Science/Research",
"License :: OSI Approved :: MIT License",
"Programming Language :: Cython",
"Programming Language :: Python",
"Programming Language :: Python :: 2",
"Programming Language :: Python :: 2.7",
"Programming Language :: Python :: 3",
"Programming Language :: Python :: 3.5",
"Programming Language :: Python :: 3.6",
"Programming Language :: Python :: 3.7",
"Programming Language :: Python :: 3.8",
"Topic :: Software Development",
"Topic :: Software Development :: Libraries :: Python Modules",
"Topic :: Scientific/Engineering",
"Topic :: Scientific/Engineering :: Information Analysis",
"Topic :: Text Processing :: Linguistic",
],
zip_safe=False,
packages=['pycrfsuite'],
ext_modules=ext_modules,
cmdclass={'build_ext': build_ext_check_gcc}
)
| 2024-07-09T01:26:19.665474 | https://example.com/article/5729 |
Q:
HTML Email : adjust vertical line height according to the content in another td
I want to adjust vertical line height as per the content of <td> tag.
<table class="bg-color1" style="background-color:#ffffff;border:none;border-collapse: collapse;mso-table-lspace: 0pt;mso-table-rspace: 0pt; " align="center" border="0" cellpadding="0" cellspacing="0" width="600" >
<tbody>
<tr>
<td width="28%" valign="top" style=" border: none;mso-table-rspace: 0pt; mso-table-lspace:0pt; border-collapse: collapse; text-transform: uppercase; font-family: 'open sans', arial, sans-serif; font-weight: 500; font-size: 18px; letter-spacing: 0px; padding-top: 20px; padding-left:70px; text-align: center; ">
<span style="text-align: center">8:30 - 9:30</span>
</td>
<td width="7%" valign="top" style=";mso-table-rspace: 0pt; mso-table-lspace:0pt; border-collapse: collapse; text-transform: uppercase; font-family: 'open sans', arial, sans-serif; font-weight: 500; font-size: 18px; letter-spacing: 0px; padding-left: 70px; padding-top: 10px; ">
<hr style="height: 20px;">
</td>
<td width="65%" style="mso-table-rspace: 0pt; mso-table-lspace:0pt; border-collapse: collapse; text-transform: uppercase; font-family: 'open sans', arial, sans-serif; font-weight: 500; font-size: 15px; letter-spacing: 0px; padding-left: 10px; padding-top: 10px; text-transform: uppercase; text-align: left; ">
Tea, Registration, Networking
</td>
</tr>
<tr style="border: thin black solid;">
<td width="28%" valign="top" style=" border: none;mso-table-rspace: 0pt; mso-table-lspace:0pt; border-collapse: collapse; text-transform: uppercase; font-family: 'open sans', arial, sans-serif; font-weight: 500; font-size: 18px; letter-spacing: 0px; padding-top: 20px; padding-left:70px; text-align: center; ">
<span style="text-align: center">8:30 - 9:30</span>
</td>
<td width="7%" valign="top" style=";mso-table-rspace: 0pt; mso-table-lspace:0pt; border-collapse: collapse; text-transform: uppercase; font-family: 'open sans', arial, sans-serif; font-weight: 500; font-size: 18px; letter-spacing: 0px; padding-left: 70px; padding-top: 10px; ">
<hr style="height: 20px;">
</td>
<td width="65%" valign="top" style="mso-table-rspace: 0pt; mso-table-lspace:0pt; border-collapse: collapse; text-transform: uppercase; font-family: 'open sans', arial, sans-serif; font-weight: 500; font-size: 15px; letter-spacing: 0px; padding-left: 10px; padding-top: 10px; text-transform: uppercase; text-align: left; ">
Tea, Registration, Networking
Tea, Registration, Networking
Tea, Registration, Networking
</td>
</tr>
</tbody>
<tr>
<td height="40"></td>
</tr>
</table>
Now What I want is when the content of third <td> changes then as per the changes the height of vertical line and the time of 1st <td> should be adjusted to center of the <td> tag.
I want output as per the image.
PS : As I am making HTML Email , I can't use <div> , position property. I have to stick to table and inline css only.
Thank You.
A:
Replacing padding-top: xx; in the first TD's with vertical-align: middle; seems to work.
fiddle
https://jsfiddle.net/Hastig/j3qy132b/1/
I played around a bit further, if it's of use to anyone..
https://jsfiddle.net/Hastig/j3qy132b/3/
A faint border-left instead of boxshadow
https://jsfiddle.net/Hastig/j3qy132b/4/
| 2023-12-15T01:26:19.665474 | https://example.com/article/8196 |
id: dsq-1726873180
replyToId: dsq-1725648097
date: 2014-12-05T01:38:22.0000000-08:00
name: Michael
avatar: https://disqus.com/api/users/avatars/Michael.jpg
message: <p>Hi Phil!<br>Yeah, I did look at the stuff in VSConsole and even tried to debug the VS Extension but got the impression that you really need to know about working with this kind of VS-Addin, the Powershell execution and error handling model, and possibly some other stuff. And to be honest, that looked like a whole bunch of stuff you'd have to first become afluent in before you could figure out the right spot where the error-output is redirected. Plus, start-with-debugger didn't work for me, either because I missed something or my setup is somehow not compatible so I benched my own exploration until a time when I actually have more than an hour or two to investigate deeper.</p><p>Many thanks for pinging this over!</p><p>Best regards, Michael</p>
| 2023-11-28T01:26:19.665474 | https://example.com/article/9232 |
[MRSA-related empyema as thoracic surgical site infection].
The incidence of empyema as a thoracic surgical site infection (SSI) is relating low, but empyema related to MRSA poses an unenviable therapeutic challenge. We review 3 cases of MRSA-related empyema as SSI seem in the last 10 years, and evaluate therapeutic measures. All 3 subjects began being administered vancomycin (VCM) systemically once the diagnosis was established. Subject 1 developed MRSA-related empyema following pulmonary segmentectomy for small-cell lung cancer. The subject was treated following a diagnosis of incisional SSI, with delayed adequate pleural drainage, resulting in treatment difficulties, but was cured without becoming MRSA-negative. Subject 2 developed MRSA-related empyema following pulmonary lobectomy for advanced lung cancer associated with pneumoconiosis. Following bronchoplasty, a chest tube was placed for long-term drainage. The subject did not become MRSA-negative after VCM administration, but became so after linezolid treatment, facilitating a cure. Subject 3, who had secondary pneumothorax, underwent thoracoscopic partial hepatic resection. Intraoperative findings suggested pleural cavity infection, necessitating a prophylactic drain, but MRSA-related pyothorax developed. Fibrinolysis with urokinase effectively cleared up the poor drainage and the subject was cured without becoming MRSA-negative. In conclusion, in controlling MRSA-related empyema as SSI noted that: (1) long-term postperative thoracic drain retention may lead to retrograde infection; (2) surgical procedures reducing the extent of pulmonary resection may effectively prevent pyothorax progression; (3) for poor drainage in advanced pyothorax, fibrinolytic therapy is worth attempting before thoracoscopic surgery; and (4) the timing for discontinuing anti-MRSA drugs should be determined based on the clinical course rather than negative conversion of bacteria. | 2024-03-24T01:26:19.665474 | https://example.com/article/2008 |
This invention relates to methods and apparatus for curling lips about the open mouths of flexible-walled stacked containers, particularly those formed from difficult-to-process thermoplastic, synthetic plastic, material such as polypropylene. A number of systems for curling or beading the rims of thermoplastic containers have been proposed and employed over the years and are exemplified in the following patents:
U.S. Pat. No. 3,096,546 PA1 U.S. Pat. No. 3,337,919 PA1 U.S. Pat. No. 3,339,005 PA1 U.S. Pat. No. 3,358,331 PA1 U.S. Pat. No. 3,487,139 PA1 U.S. Pat. No. 3,355,536 PA1 U.S. Pat. No. 3,579,737 PA1 U.S. Pat. No. 3,676,543 PA1 U.S. Pat. No. 3,861,847 PA1 U.S. Pat. No. 3,910,744 PA1 U.S. Pat. No. 3,949,045 PA1 U.S. Pat. No. 4,048,781
While such systems have proven useful in curling the rims or brims of some plastic containers, they have not, to our knowledge, been sufficiently versatile to handle also containers formed from difficult-to-process materials such as polypropylene. While a suitable brim curling temperature for polypropylene containers may be stated to be generally in the range of 300.degree. to 320.degree. F., the actual range for specific polypropylene materials is normally in the area of plus or minus 5.degree. F. within that general range. For example, with a certain batch of polypropylene which was tested, satisfactory results could be achieved if the brims, at the time of forming were held between 300.degree. and 305.degree. F.
With systems such as disclosed in U.S. Pat. Nos. 3,339,005 3,355,536 and 3,337,919, which employ radiant heat in advance of the forming rolls, the usual result with material such as polypropylene was that the containers were rendered too pliable to properly enter the passageway between the rolls. In other words, sufficient control of the temperature of the container side wall sections, and the temperature of the axial return (brim) sections of the containers, simply could not be achieved to permit the proficient forming of turned-in lips, when the material was a temperature-critical thermoplastic material which requires more heat input relative to surface area such as polypropylene.
Similarly, systems such as disclosed in Edwards U.S. Pat. No. 3,096,546, which utilize only internal roller heating to conductively heat container rims, could not process polypropylene containers efficiently and required rollers of undue length, which were expensive and cumbersome. In contradistinction to the methods previously suggested and commercially employed, the present system utilizes a combination of radiative and conductive heating to bring the container brims quickly, and yet in a controlled manner, to the critical forming temperature, while the rims are engaged in and mechanically supported by, the roller helical grooves. Prior to forming, the radial distance between the brim and the peripheral body wall portion at the mouth of each container is fixed and then, after forming by subjecting the brim to axially opposed pressures, heat is removed conductively by the rollers in a rim cooling zone and while the fixed radial distance mentioned continues to be maintained, until the temperature of the container and brim is lowered sufficiently so that the plastic is set in mechanically stable configuration.
One of the prime objects of the present invention is to provide a versatile apparatus which will operate on difficult-to-form materials such as polypropylene, as well as on other materials, to automatically roll in the edges on plastic containers in controlled, fast, and repetitively trouble-free manner.
Another object of the invention is to accomplish the objects of the invention with an efficient low cost machine having thermally conductive rollers which can be heated efficiently to a critical temperature and which then will form and cool the brims of stacked containers in a manner such that rollers of relatively short length can be employed, even with difficult-to-process thermoplastic materials.
Still another object of the invention is to provide a progressive curling machine of the character described which utilizes a plurality of continuous helical grooves which are capable of processing containers such as beverage cups and cottage cheese containers at higher rates of speed than previously. | 2024-01-06T01:26:19.665474 | https://example.com/article/4613 |
utf8 cpp library
Release 2.3.4
A minor bug fix release. Thanks to all who reported bugs.
Note: Version 2.3.3 contained a regression, and therefore was removed.
Changes from version 2.3.2
- Bug fix [39]: checked.h Line 273 and unchecked.h Line 182 have an extra ';'
- Bug fix [36]: replace_invalid() only works with back_inserter
Files included in the release: utf8.h, core.h, checked.h, unchecked.h, utf8cpp.html, ReleaseNotes
| 2024-06-23T01:26:19.665474 | https://example.com/article/4571 |
/*
* Copyright 2011 Nikhil Marathe <nsm.nikhil@gmail.com>
*
* Permission is hereby granted, free of charge, to any person obtaining a copy
* of this software and associated documentation files (the "Software"), to
* deal in the Software without restriction, including without limitation the
* rights to use, copy, modify, merge, publish, distribute, sublicense, and/or
* sell copies of the Software, and to permit persons to whom the Software is
* furnished to do so, subject to the following conditions:
*
* The above copyright notice and this permission notice shall be included in
* all copies or substantial portions of the Software.
*
* THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
* IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
* FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
* AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
* LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING
* FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS
* IN THE SOFTWARE.
*/
#include "qhttprequest.h"
#include "qhttpconnection.h"
QHttpRequest::QHttpRequest(QHttpConnection *connection, QObject *parent)
: QObject(parent)
, m_connection(connection)
, m_url("http://localhost/")
, m_success(false)
{
}
QHttpRequest::~QHttpRequest()
{
}
| 2024-04-24T01:26:19.665474 | https://example.com/article/2540 |
---
-api-id: M:Windows.UI.Xaml.UIElement.RegisterAsScrollPort(Windows.UI.Xaml.UIElement)
-api-type: winrt method
---
<!-- Method syntax.
public void UIElement.RegisterAsScrollPort(UIElement element)
-->
# Windows.UI.Xaml.UIElement.RegisterAsScrollPort
## -description
Registers an element as representing a scrollable viewport.
## -parameters
### -param element
The element to register as a scrollable viewport.
## -remarks
This method informs the framework that the element's applied clip (either due to layout or an explicitly assigned [Clip](uielement_clip.md)) is acting as a viewport and should receive special consideration.
This method is intended for use by custom controls that do not use the platform's native controls to display a scrollable area. For example, a custom scrolling control could be built using [InteractionTracker](../windows.ui.composition.interactions/interactiontracker.md).
### Effective Viewport
The [Clip](uielement_clip.md) of the registered element is recognized by the framework as the bounds of a *viewport*, which will be used in calculating the property values for the [EffectiveViewportChanged](frameworkelement_effectiveviewportchanged.md) event.
### System Focus Visuals
By default, the focus visual is fully rendered around the outside of the focused element taking into account all clips. When an element is only partially visible within a viewport the framework needs to disambiguate which clip in the element's ancestor chain represents the viewport. The framework uses this knowledge to correctly apply the viewport clip to the rendered focus visual.
## -see-also
[UseSystemFocusVisuals](../windows.ui.xaml.controls/control_usesystemfocusvisuals.md), [InteractionTracker](../windows.ui.composition.interactions/interactiontracker.md), [EffectiveViewportChanged](frameworkelement_effectiveviewportchanged.md), [InvalidateViewport](frameworkelement_invalidateviewport_528063221.md)
## -examples
| 2024-04-10T01:26:19.665474 | https://example.com/article/1855 |
A newborn baby is reportedly the youngest person to test positive for the novel coronavirus.
The baby's mother, unaware that she had the virus and thought to have had pneumonia, was rushed to a hospital in London a few days ago, The Sun reported.
It was unclear whether the baby contracted the virus in the womb or during labor.
The mother and the baby were being treated in separate hospitals, The Sun said.
Visit Business Insider's homepage for more stories.
A newborn baby is reportedly the youngest person to test positive for the novel coronavirus.
The baby's mother was rushed to a hospital in London a few days ago, thought to have had pneumonia, The Sun reported.
Once doctors knew that both the mother and the baby had the coronavirus, they were placed in separate hospitals to receive treatment, The Sun said.
The mother, who did not know she had the virus until after giving birth, was tested at North Middlesex University Hospital, according to The Sun.
The baby was tested within minutes and is still receiving treatment at that hospital. The mother, meanwhile, was transferred to "a specialist infections hospital," The Sun said.
A source told The Sun that hospital staff members who had contact with the mother and the newborn were instructed to self-isolate.
It's unclear whether the baby contracted the virus in the womb or during labor, The Sun said.
"Two patients at North Middlesex University hospital have tested positive for coronavirus. One has been transferred to a specialist centre and one is being treated in an isolation room," North Middlesex University Hospital NHS Trust said in a statement.
"The safety of our patients and staff is our top priority," the statement continued, "so in following guidance from Public Health England, we are regularly deep cleaning the areas where the patients are cared for and staff who were in close contact with these patients were advised to self-isolate." | 2023-11-28T01:26:19.665474 | https://example.com/article/4954 |
1. Field of the Invention
The present invention relates to a multi-band radio terminal and a band switching method used therefor and a program thereof and, more particularly, a band switching method at a radio portable terminal adaptable to multi-band.
2. Description of the Related Art
In recent years, more and more multi-band-adaptable radio portable terminals have been produced. In Japan, dual-band terminals adapted to 800 MHz band (PDC: Personal Digital Cellular) and 1.9 GHz band (PHS: Personal Handyphone System) have been already put on the domestic market and other multi-band terminals having more varieties of frequency bands are expected to be produced hereafter.
Example of a structure of the above-described dual-band terminal is shown in FIG. 5. In FIG. 5, the dual-band terminal includes an antenna (ANT#1) 110 and a Band (#1) adaptation unit 150 adaptable to a band #1 as a first band, an antenna (ANT#2) 120 and a Band (#2) adaptation unit 160 adaptable to a band #2 as a second band, a band switching unit 170 and a host sequence 130.
The Band (#1) adaptation unit 150 includes a radio transmitter and receiver unit 151, a three wire serial signal generation unit 152, a register conversion unit 153, a frequency control unit 154 and frequency setting data 155.
The Band (#2) adaptation unit 160 includes a radio transmitter and receiver unit 161, a three wire serial signal generation unit 162, a register conversion unit 163, a frequency control unit 164 and frequency setting data 165.
The band switching unit 170 controls the radio transmitter and receiver units 151 and 161 and the frequency control units 154 and 164 in response to an instruction from the host sequence 130 to operate either the antenna (ANT#1) 110 and the Band (#1) adaptation unit 150 or the antenna (ANT#2) 120 and the Band (#2) adaptation unit 160, thereby switching a band (see Japanese Patent Laying-Open (Kokai) No. Heisei 11-251951, for example).
In the above-described conventional dual-band terminal, since all control for band switching and setting of a frequency is performed by a control unit (CPU: central processing unit), operation of a control unit (CPU) becomes very complicated. | 2024-01-09T01:26:19.665474 | https://example.com/article/2961 |
// Targeted by JavaCPP version 1.5.4: DO NOT EDIT THIS FILE
package org.bytedeco.systems.macosx;
import java.nio.*;
import org.bytedeco.javacpp.*;
import org.bytedeco.javacpp.annotation.*;
import static org.bytedeco.javacpp.presets.javacpp.*;
import static org.bytedeco.systems.global.macosx.*;
/* special day to force password
* change at next login */
// #endif
@Properties(inherit = org.bytedeco.systems.presets.macosx.class)
public class passwd extends Pointer {
static { Loader.load(); }
/** Default native constructor. */
public passwd() { super((Pointer)null); allocate(); }
/** Native array allocator. Access with {@link Pointer#position(long)}. */
public passwd(long size) { super((Pointer)null); allocateArray(size); }
/** Pointer cast constructor. Invokes {@link Pointer#Pointer(Pointer)}. */
public passwd(Pointer p) { super(p); }
private native void allocate();
private native void allocateArray(long size);
@Override public passwd position(long position) {
return (passwd)super.position(position);
}
@Override public passwd getPointer(long i) {
return new passwd(this).position(position + i);
}
public native @Cast("char*") BytePointer pw_name(); public native passwd pw_name(BytePointer setter); /* user name */
public native @Cast("char*") BytePointer pw_passwd(); public native passwd pw_passwd(BytePointer setter); /* encrypted password */
public native @Cast("uid_t") int pw_uid(); public native passwd pw_uid(int setter); /* user uid */
public native @Cast("gid_t") int pw_gid(); public native passwd pw_gid(int setter); /* user gid */
public native @Cast("__darwin_time_t") long pw_change(); public native passwd pw_change(long setter); /* password change time */
public native @Cast("char*") BytePointer pw_class(); public native passwd pw_class(BytePointer setter); /* user access class */
public native @Cast("char*") BytePointer pw_gecos(); public native passwd pw_gecos(BytePointer setter); /* Honeywell login info */
public native @Cast("char*") BytePointer pw_dir(); public native passwd pw_dir(BytePointer setter); /* home directory */
public native @Cast("char*") BytePointer pw_shell(); public native passwd pw_shell(BytePointer setter); /* default shell */
public native @Cast("__darwin_time_t") long pw_expire(); public native passwd pw_expire(long setter); /* account expiration */
}
| 2024-06-30T01:26:19.665474 | https://example.com/article/6264 |
Every business has their daily process that needs to be fulfilled as part of making the revenue, making sure you’re equipped is what makes that process much more efficient and less complicated. It might be expensive at first to buy all of the necessary equipment, but there are some tools you just can’t go without! Making sure you get the right amount of use out of them is also an essential. Otherwise, you’ll end up purchasing equipment that you really don’t need. While it doesn’t hurt to be prepared for everything, some items don’t fit into your version of “everything”.
Your business
Every business will operate differently, each of which needs different kinds of equipment to help see their job done. It’s possible that you can overcomplicate things when you don’t invest in the right tech, so looking into ways to make your current operations easier can be a great solution to a struggle. For example, if your operation is weather dependent, having to search online is not the most efficient way for you. You’ll find general results for your general area, but you need to know what it’s like at this moment at your location. In this case, you would invest in a commercial windsock to help determine your working conditions. Other situations might need you to invest in an automation system, something that can make calculations or changes in the system more convenient. For example, supermarkets use barcode scanners to quickly calculate and register the products of each customer.
Without these simple adjustments made in the business, each working day can be much more of a struggle, even if you do have a backup method. Making sure you have the fastest option possible is much more beneficial in the long run, even if the costs are high. If your business is popular and you have to run on a schedule, you could also increase your workflow and revenue, knowing and increasing your limits as you go.
Safety and security
Being equipped doesn’t only help your workflow, but it also comes down to creating a secure work environment. If you work with multiple employees, making sure they’re not endangered at any point is vital to your daily practice. In some cases, laws are requiring you to keep certain equipment in the workplace; like fire extinguishers and alarms. You could also invest in making sure you have the best security possible too, like cameras and secure door locks. It’s not guaranteed that people will try to break in. However, it can help prevent such situations if you make it known that there are security measures put in place.
Being unequipped is a surefire way to lose money in your daily workings. If you do end up a victim of theft, then you’ve lost out on whatever they’ve stolen, and also your property will have been damaged where they broke in. If you run a small business, this is where you become most vulnerable, as there’s no such thing as acceptable losses. You can’t survive as a business if your assets are always at risk. | 2023-09-08T01:26:19.665474 | https://example.com/article/7986 |
1. Field of the Invention
The present invention relates to an ophthalmologic examination apparatus having a plurality of ophthalmologic examination functions.
2. Description of the Prior Art
A variety of ophthalmologic examination apparatuses such as fundus cameras and perimeters are conventionally known. A cameras as disclosed in Japanese Patent No. 3359126 is, for example, provided with functions whereby an illuminated eye fundus is once imaged, then magnified and imaged again in the imaging region of a CCD in order to create images of the eye fundus having different magnification ratios. Similarly, a fundus cameras is also known from Japanese Laid-open Patent Application 1979-62691 wherein an illuminated eye fundus can be imaged, the resulting image can be magnified, and the image of the eye fundus can subsequently be divided in a position conjugate with the pupil for stereographical observation.
A fundus camera is also known from Japanese Laid-open Patent Application 1998-155743 wherein a photography unit for photographing an eye fundus is provided separately from a main unit that houses an illuminating optical system and a photographing optical system and an ocular lens are provided on the photography unit side.
However, several problems arise in conventional ophthalmologic examination apparatuses. For example, the apparatuses are primarily intended for a single ophthalmologic examination wherein an eye fundus is observed or imaged, or a visual field is examined. This does not allow various ophthalmologic examinations to be performed. Furthermore, the optical systems of apparatuses that perform a variety of ophthalmologic examinations are complex.
Therefore, an object of the present invention is to provide an ophthalmologic examination apparatus that is capable of performing a variety of ophthalmologic examinations with an inexpensive arrangement. | 2024-02-16T01:26:19.665474 | https://example.com/article/6216 |
Symptom distress, spirituality, and quality of life in African American breast cancer survivors.
This study examined the relationships among the demographic characteristics, symptom distress, spirituality, and quality of life (QOL) of African American breast cancer survivors. A convenience sample of 30 survivors with a mean age of 56 years and a mean survival of 6 years was recruited from African American breast cancer support groups and churches in the Southeastern United States. Data were collected through face-to-face interviews using a demographic questionnaire, the Quality of Life Index-Cancer Version, the Symptom Distress Scale, and the Spiritual Perspective Scale. Statistically significant relationships were found between symptoms and QOL (r = -0.62, P < .05) and between spirituality and QOL (r = 0.70, P < .05). No statistically significant relationships were found between age at diagnosis, income, or education and QOL. This research suggests that symptoms and spirituality are associated with QOL. Culturally appropriate care should be provided to these women to reduce health disparities and to improve their QOL. | 2024-01-04T01:26:19.665474 | https://example.com/article/5271 |
Blog
San Jose ranked #2 “Best Small Business Growth City in America for 2nd year
/ May 7, 2019 by Elisabeth Handler
Retaining its position as the second best metro area for small businesses in the US for the second year, San Jose has shown continued strength in its small business community’s annual revenue, credit score, longevity and other factors. The annual ranking is by Biz2Credit, an online company specializing in small business financing. To determine the rankings, Biz2Credit examined the financials of nearly 30,000 companies that applied for small business financing in 2018.
The top-rated city for small business this year is Miami, which scored #3 in 2018. The #1 city last year was New York, which is fell to#4 in 2019. In the 2019 study, San Jose was ranked #1 in terms of small business credit scores, and #3 in annual revenue, after New York and Miami.
For this analysis, Biz2Credit defined “small businesses” as companies having fewer than 250 employees or less than $10 million in annual revenues. The Top 25 Cities for Small Business in 2019 (with 2018 ranking in parenthesis) are:
Miami (No. 3)
San Jose, CA (No. 2)
San Francisco (No. 4)
New York (No. 1)
San Diego (No. 9)
Los Angeles (No. 5)
Boston (unranked)
Sacramento, CA (No. 10)
Baltimore (No. 16)
Washington, DC (No. 7)
Philadelphia (No. 22)
Chicago (No. 12)
Seattle (No. 14)
Riverside, CA (No. 6)
Minneapolis (unranked)
Phoenix (No. 11)
Las Vegas (No. 18)
Charlotte, NC (No. 24)
Nashville, TN (unranked)
Raleigh, NC (unranked)
Detroit (No. 20)
Oklahoma City (unranked)
Houston (No. 21)
Cleveland (unranked)
Pittsburgh (unranked)
When sorted by credit score, San Jose was the leader, followed by San Francisco, New York, and Miami.
About the Biz2Credit Best Small Business Cities in America Study
Biz2Credit analyzed nearly 30,000 businesses with less than 250 employees and less than $10 million in annual revenues from across the country that have been in operation for more than one year. Founded in 2007, Biz2Credit has arranged more than $2 billion in small business financing and has several times been named to Crain’s New York’s Fast 50 and was recently ranked among the top 200 fast-growing companies on Deloitte’s 2018 Technology Fast 500. Biz2Credit is expanding its industry-leading technology in custom digital platform solutions for leading banks and other financial institutions, investors and service providers in the U.S. Visit www.biz2credit.com | 2023-12-16T01:26:19.665474 | https://example.com/article/9866 |
Embark on a once-in-lifetime cruise holiday on P&O Cruises Adonia; the brand new little sister in the P&O fleet.
Why choose P and O Adonia?
The name speaks for itself - P&O, Britain’s award-winning cruise line can trace its roots back over 175 years to the formation of the Peninsular Steam Navigation Company in 1837, assuring an unquestionable level of expertise when it comes to cruises. Built in 2001 and re-named in 2011, The Adonia is the smallest of P&O’s seven ships and offers a delightfully intimate cruising experience on a ship that radiates charm. Exclusively for adults, the elegant Adonia is a ship that goes back to P&O’s roots and offers a wide range of itineraries departing from Southampton - perfect for older British passengers looking for a cruise reminiscent of classic liner days and for luxury with the added comfort of familiar surroundings.
Destinations and itineraries
The P&O Adonia offers an un-rivalled array of breathtaking, off-the-beaten-track itineraries, setting sail on a range of Arctic, Northern European, Mediterranean, Far Eastern and Atlantic Island cruises, and calling at out-of-the-way ports and offering a wide range of shore excursions.
The beginning of September sees Adonia set sail from her home port of Southampton on a 17-day Baltic cruise, stopping in Denmark, Sweden, Russia, Estonia, Poland and Germany. Passengers can expect some magical excursions, from the Lummelunda Caves of Stockholm and the breath-taking palaces of St Petersburg, to the medieval cities of Eastern Europe and the chalk cliffs of Sassnitz, Germany.
For the true adventurer, Adonia offers a number of once-in-a-lifetime world cruises. Come January, she sets sail on a 113-day Asian Grand Adventure cruise, passing through North Africa, The Middle East, UAE and Asia before returning to the UK back through Europe. From the Pyramids of Egypt and the Souks of Dubai, to the bustling markets of Malaysia and the Great Wall of China, passengers will get a rare chance to see some of the most beautiful corners of the Earth.
With her modest capacity, The Adonia is perfect for adults wanting to experience the cruising camaraderie that comes with being surrounded by friendly, familiar faces. Where waiters learn your name and bar tenders make time to chat, the intimate Adonia with her country-house charm is an ideal choice for a far-flung adventure combined with a personal touch and stylish finish.
With wood-style panelling, a grand staircase and traditional artworks on display, Adonia’s ambience and décor are in keeping with the traditions of cruising. Yet being a 21st century ship, there is certainly no lack of modernity to her detailing, all of which is beautifully crafted to the highest standard. The Adonia offers a wide range of adult-orientated activities and dining options to suit all tastes, and of course being a smaller ship, everything on The Adonia is just a short walk away. The teak-surrounded crystal pool, a focal point of the Adonia’s spacious sun deck, is the perfect place to cool off after a drink in the sun. For those in need of some more intense relaxation, The Oasis Spa area is an ideal choice that offers a range of sensual treatments that will revive and rejuvenate. In fitting with their impeccable standards of service, P&O request that guests aboard The Adonia observe a dress code of smart casual. Longer cruises will also feature optional formal nights, when gents will be expected to sport a black tie, and ladies to wear elegant dresses.
Even at double occupancy, the ship provides a generous crew to passenger ratio of one for every 1.8 passengers, ensuring the highest level of service at all times with crew that will go out of their way at all times to offer service beyond the call of duty. There are a total of 335 extremely well-equipped cabins on board, over 60% of which have balconies that offer stunning views of the ocean. All cabins have very good storage space and all the amenities one expects on a luxury liner including TV, radio, hairdryer, sitting area, telephone, fridge and safe, with suites offering double wardrobes, a dining area and large balconies.
Dining options range from the traditional Pacific Ocean Dining Room with its six course silver service, to a number of speciality restaurants including the fine dining Italian eatery, Sorrento, and the Ocean Grill that serves up burgers, steaks, and an excellent selection of fresh seafood. After dinner guests can enjoy a drink in the many public lounges including the Crow’s Nest Observation Lounge, which offers stunning views of the ocean. The Grand Lounge features a dance floor for later in the evening, and a stage to feature after-dinner entertainment's and guest speakers.
P & O Cruise Liners
P&O Adonia Balcony Stateroom
FEATURES✔ The Balcony Stateroom has two lower beds that are convertible to a king sized bed✔ Bathroom with a shower over small bath and WC✔ Floor to ceiling sliding glass doors that lead to the balcony✔ The room is approximately 214 square feet including the balcony.
Inside Cabin
FEATURES✔ Inside Cabin has two lower beds that are convertible into a king size bed✔ Bathroom with shower over small bath and WC.
Outside Cabin
FEATURES✔ The Outside Cabin has two lower beds that are convertible into a king size bed✔ Bathroom with shower over small bath and WC✔ Approximately 165 square feet including the balcony
Also included: Additionally there is a useful Walk-in dressing area* with iron & ironing board and a hairdryer. The lounge has a sofa & armchairs plus dining table and chairs, with a mini stereo and two flat panel , interactive TVs plus radio and telephone, writing desk, refrigerator and tea/coffee making faculties. There is also a safe. Floor to ceiling sliding glass doors lead to your balcony with loungers*, chairs and table
Balcony with Shower
Also includes: There is a sitting area with chair and table, a TV, plus radio and telephone, writing desk, refrigerator and tea/coffee making facilities. There is also a safe. Floor to ceiling sliding glass doors lead to your balcony with recliner chairs and table.
Balcony (Part obstucted view) with shower
Also includes: There is a sitting area with chair and table, a TV, plus radio and telephone, writing desk, refrigerator and tea/coffee making facilities. There is also a safe. Floor to ceiling sliding glass doors lead to your balcony with recliner chairs and table.
Larger Outside with Shower
FEATURES✔ Two lower beds convertible to king-size bed✔ Bathroom with shower only and WC✔ Outside cabins will have either windows or portholes✔ Size: Approx between 137-181 Square Feet
Special Touches✔ Mineral water on arrival✔ Pamper pack
** When three/four passengers share a cabin with upper berths, for safety reasons the two lower beds cannot be pushed together
Outside with Shower
FEATURES✔ Two lower beds convertible to king-size bed✔ Bathroom with shower only and WC✔ Outside cabins will have either windows or portholes✔ Size: Approx between 137-181 Square Feet
Special Touches✔ Mineral water on arrival✔ Pamper pack
** When three/four passengers share a cabin with upper berths, for safety reasons the two lower beds cannot be pushed together
Outside (obstructed view) with Shower
FEATURES✔ Two lower beds convertible to king-size bed✔ Bathroom with shower only and WC✔ Outside cabins will have either windows or portholes✔ Size: Approx between 137-181 Square Feet
Special Touches✔ Mineral water on arrival✔ Pamper pack
** When three/four passengers share a cabin with upper berths, for safety reasons the two lower beds cannot be pushed together
Inside with Shower
FEATURES✔ Two lower beds convertible to king-size bed✔ Bathroom with shower only and WC✔ Inside cabins have a mirror✔ Approx between 137-181
Special Touches✔ Mineral water on arrival✔ Pamper pack
When three/four passengers share a cabin with upper berths, for safety reasons the two lower beds cannot be pushed together
Help
Tools
Your financial protection
All flights and flight-inclusive holidays on this website are financially protected by the ATOL scheme but ATOL protection does not apply to the other services offered on this site. We act as an agent for licenced Tour Operators. When you pay you will be supplied with an ATOL Certificate. Please ask for it and check to ensure that everything you booked (flights, cruises, hotels and other services) is listed on it. Please see the tour operators booking conditions for further information or go to www.atol.org.uk for more information about financial protection and the ATOL Certificate. | 2024-02-22T01:26:19.665474 | https://example.com/article/6739 |
## Process this file with automake to produce Makefile.in
SUBDIRS = grabbag
EXTRA_DIST = \
alloc.h \
compat.h \
endswap.h \
getopt.h \
grabbag.h \
macros.h \
private.h \
replaygain_analysis.h \
replaygain_synthesis.h \
safe_str.h \
utf8.h \
win_utf8_io.h \
windows_unicode_filenames.h
| 2023-11-16T01:26:19.665474 | https://example.com/article/6937 |
Airbus secures firm commitment for 100 A320neos from China
Airbus China says it has secured a firm commitment for at least 100 A320neos from Chinese customers, and that discussions for several hundreds more of the re-engined narrowbody are ongoing.
In an interview with journalists at Airbus China's office in Beijing, its president Eric Chen disclosed that the commitment for the 100 Neos comes from flag carrier Air China and state-owned leasing company ICBC Leasing, and are subjected to approvals from the local government.
If approved, this would be one of China's first Neo orders. Publicly, the airframer has so far only announced an order from ICBC Leasing for 20 Neos – Chen says a part of this figure is included in the 100 aircraft commitment.
Meanwhile, Air China filed a stock exchange disclosure in May, saying its board has approved an order for 100 A320s, 40 of which will go to subsidiary Shenzhen Airlines. At that time, the Star Alliance carrier did not say whether the order included the re-engined version of the narrowbody. Chen, however, says that 60 of the 100 aircraft on order are Neos.
"Besides this firm commitment for 100 aircraft, we also have an active sales campaign, and several hundreds more Neos are being discussed," says Chen.
He explains that though Chinese carriers only order aircraft based on five-year plans (the current one runs from 2011 to 2015), they are aware that they may not get early slots should they order the Neo later, because of high demand for the type.
"They realise that if they plan in the same way, they may lose the opportunity for slots in years to come. So in this context, there is a lot of anxiety and thus they are discussing with us their needs for Neos," says Chen. "There are many active negotiations ongoing."
There are currently 807 A320 family aircraft being operated by Chinese customers. | 2023-11-17T01:26:19.665474 | https://example.com/article/2266 |
Q:
Internal graphics not initialized yet: javafx
I'm trying to write a javaFx application whit multiple images inside a window.
The short story is that I have an enum class named Candy and each candy has some properties and a path to the image file representing it.
In the constructor of my javafx.application class (Table) I want to fill an array list with those images, so I wrote this so far:
public class Table extends Application {
ArrayList<Image> images;
@Override
public void start(Stage primaryStage) throws Exception {
primaryStage.setTitle("CandyFx");
primaryStage.show();
}
public Table() {
images = new ArrayList<Image>();
for (Candy candy : Candy.values()) {
File file = new File (candy.getImagePath());
Image image = new Image(file.toURI().toString());
images.add(image);
}
}
}
Now every time I want to create an instance of Table class, the application throws a java.lang.RuntimeException: Internal graphics not initialized yet.
How can I initial graphics which it seems I did not?
A:
First of all if you are using linux ,GTK 2.18 is required to run JavaFX .try to install
libswt-gtk-3-java
This exception will thrown whenever your screen is null .Try to create your images inside start method. Just before the primaryStage.show();.
Take a look at this link too
http://cr.openjdk.java.net/~vadim/RT-33475/webrev.00/modules/graphics/src/main/java/com/sun/glass/ui/Screen.java.html
| 2024-03-03T01:26:19.665474 | https://example.com/article/6510 |
Clinicopathological study of pontine hemorrhage.
This report concerns a clinico-pathological study of 60 patients afflicted with primary pontine hemorrhage. The illness was fatal in 43, 17 patients survived. Ophthalmic signs, autonomic disturbances and transient visual hallucination were observed and discussed. A ruptured microaneurysm within the border of a pontine hematoma was detected in this study, and in the first report of such a finding. | 2024-06-24T01:26:19.665474 | https://example.com/article/2293 |
Heading off to weigh in tonight moving forward again from here on in after taking a mental break by targeting off for a while.
Good luck everyone who is weighing today
Jun 14th, 2016, 22:11 PM
PurpleJ
SoSlim Star
Join Date: Jul 2015
Location: Wales
Posts: 1,797
Likes: 583
Well I weighed tonight and am 14st 5lb so 1.5lb to lose to get back to my 3 stone I was awarded. I am at target at the moment so half lb under that, but would like to try and re-focus and lose some more. Work and family life got in the way recently so need to work on that.
So goal for this week is to lose.. anything!
Jun 15th, 2016, 12:54 PM
sarahc4536
Moderator
Join Date: Jun 2015
Location: Manchester
Posts: 2,754
Likes: 687
Well done for staying around the 3 stone mark PJ - I've heard others says it can be difficult to maintain, but it's good that you have managed
Good luck for the next week!
Jun 15th, 2016, 21:40 PM
PurpleJ
SoSlim Star
Join Date: Jul 2015
Location: Wales
Posts: 1,797
Likes: 583
Thanks Sarah I must admit I'm chuffed to be approaching my 1 year anniversary at 3 stone gone and to have managed to maintain for a little while. I think in the long run I'd like to lose another 2 stone but it still feels huge. I was my happiest 3 stone down but that feels impossible! lol
Jun 16th, 2016, 00:12 AM
Looby
SoSlim Rising Star
Join Date: May 2015
Posts: 185
Likes: 91
I've spent the last 3 months or so bobbing around the same few pounds...
...but I've now got my award...
...and my weight now starts with an 11...
...and I've bitten the bullet and set my target for another half stone (11st 6.5lb).
Bring it on!
Jun 16th, 2016, 00:13 AM
Looby
SoSlim Rising Star
Join Date: May 2015
Posts: 185
Likes: 91
Purple J...you've done amazingly to maintain your fab weight loss. Good to see you back. xx
Jun 16th, 2016, 12:22 PM
sarahc4536
Moderator
Join Date: Jun 2015
Location: Manchester
Posts: 2,754
Likes: 687
Looby that's fantastic!!
I'm so pleased for you
Jun 17th, 2016, 23:16 PM
PurpleJ
SoSlim Star
Join Date: Jul 2015
Location: Wales
Posts: 1,797
Likes: 583
Looby HUGE HUGE congratulations! that is truly inspiring stuf Last time I saw an 11 was 20 years ago!! and that wasn't for long! lol
Thanks for your kind words, I'm chuffed to still be here, and feel stronger again
Jun 21st, 2016, 12:08 PM
Kellielou84
SoSlim Newbie
Join Date: May 2016
Location: Port Talbot
Posts: 11
Likes: 1
Lost 2.5lbs this morning!! happy with that! On way to shifting the huuuuge holiday gain I had a couple of weeks ago!! | 2024-02-24T01:26:19.665474 | https://example.com/article/9300 |
CHILDREN as young as eight years are starting to use drugs as experts warn of a dangerous trend of younger addicts showing signs of increasingly more violent behaviour.
The Matt Talbot Adolescent Service in Cork said trends showed the onset of drug use for young people had dramatically lowered – from the age of 12 in 2006 to the age of eight this year.
Young people referred to the services, which caters for 14-23 year olds, are increasingly smoking heroin, using prescription drugs and carrying weapons to defend themselves against drug dealers to whom they owed money.
Strong evidence gathered by MTAS shows that combined benzodiazepines, such as Valium or Xanax, cocaine, and alcohol use correlated with repeated violent criminal behaviour.
Edel Foley, clinical manager at the centre, said there was also a real worry about the increasing levels of suicidal behaviour.
About half of the young people who attended services in January this year had attempted suicide prior to being referred, she said.
All of these young people were under 18 and had not been able to access immediate emergency psychiatric assessment.
Ms Foley welcomed the ban on head shop products this week, which she said were being used by most of the young people currently attending the services.
She said they were exhibiting extreme symptoms including extreme anxiety, depression, visual and auditory hallucinations and high levels of aggressive and challenging behaviour.
Statistics from 2009 for the free centre – which provides assessment, support and treatment services show:
* Referrals to MTAS increased by 70% in first six months of 2009.
* 92% of young people attending the service were multi-drug (POLYDRUG) users.
* 85% of young people were involved in the juvenile justice system.
* 62% of 18-23 year olds attending the service were heroin users.
* Significant number of referral of young people under 18 years who have smoked heroin.
* 42% increase in young people carrying concealed weapons.
Last year, 282 young people were referred to MTAS and 562 parents and carers, which show a significant overall increase of 30% on the previous year. Ms Foley warned that the onset of heroin use among young people was "very frightening" and needed urgent attention and resources.
She said there are only 28 detox beds for an estimated 14,500 heroin users in Ireland and none of those detox beds are in Cork or the Munster area. Waiting lists of up to one year for treatment exist in the public healthcare system, she said.
INCREASING levels of domestic violence and family breakdown is leading to a surge in homelessness among families and young men barred from their family homes.
And in many cases, men who have no reported addictions, physical or mental health issues are also becoming homeless – simply because of economic circumstances.
The disclosures emerged in an unpublished internal report by the HSE’s homeless person’s unit (HPU). The unit has been the main service provider of frontline supports for the homeless in Dublin city for more than 20 years. However, under the Government’s planned restructuring of services, the unit will essentially be dismantled, with its main functions – placement and assessment of homeless people – transferred to Dublin City Council.
In the report, obtained under the Freedom of Information Act, HPU staff make it clear they do not believe the plans are in the best interests of vulnerable homeless people. They say it was a "matter of lingering regret and resentment" the HPU was not subject to proper evaluation before the decision was taken to restructure. Furthermore, staff remain unconvinced that research undertaken reflects the practical realities of homelessness.
"Accumulated wisdom of frontline staff is now apparently being cast aside as erroneous," it states. Correspondence between the council and the HPU also reveals the apparent lack of knowledge the council has regarding homeless services.
A January 1 hand-over date was fixed last August but the council was not ready for a number of reasons. A new date of February 1 was agreed but changed again at the 11th hour to April 1. That deadline has passed without the hand-over being completed.
Documents show as late as December, council officials submitted pages of basic questions relating to HPU practices, which include:
* what is your policy on placing people who have been barred from other accommodations?
* Do you have any formal links with hospitals regarding discharge policies?
* What do you do if a person presents with a known contagious infection?
* Have you any formal links with psychiatric hospitals?
* Are there any agreements in place with domestic violence refuges?
* Have you any formal links with social work teams regarding child protection issues?
* Why do you operate separate clinics for men, women and children?
According to a union representative for the HPU’s community welfare officers, the unit is seen as "surplus to requirements". Social campaigner Fr Peter McVerry said while the Government’s policy was to be welcomed, he was highly sceptical whether the resources were there to make it work. | 2023-08-28T01:26:19.665474 | https://example.com/article/1117 |
Dr. John R Sutherland
- Reviews
The Vitals website is provided for your informational use only. Nothing contained or offered by, on or through Vitals should be construed as medical advice or relied upon for medical diagnosis or treatment. Vitals does not recommend or endorse any particular healthcare provider whose information or ratings appear on this website. We encourage you to read our full Terms of Service.
Rating Overview
24 Ratings
with 5 Comments
4.4
The overall average patient rating of Dr. John R Sutherland
is Great.
Dr. John R Sutherland has been rated by 24 patients.
From those 24 patients 5
of those left a comment along with their rating. The overall rating for
Dr. John R Sutherland is 4.4 of 5.0 stars.
Board Certified
Dr. John R Sutherland is Board Certified in the following:
Obstetrics & Gynecology
Board certification indicates that a doctor is highly qualified in
the medical field in which he or she practices. A board-certified
doctor is more likely than a non-board-certified doctor to have the
most current skills and knowledge about how to treat your medical condition.
* This information is proprietary data maintained in a copyrighted
database compilation owned by the American Board of Medical Specialties.
Copyright 2017 American Board of Medical Specialties. All rights reserved.
Board Certified
Dr. John R Sutherland is Board Certified in the following:
Obstetrics & Gynecology
Board certification indicates that a doctor is highly qualified in
the medical field in which he or she practices. A board-certified
doctor is more likely than a non-board-certified doctor to have the
most current skills and knowledge about how to treat your medical condition.
* This information is proprietary data maintained in a copyrighted
database compilation owned by the American Board of Medical Specialties.
Copyright 2017 American Board of Medical Specialties. All rights reserved.
A review by a patient that verified they have visited Dr. John R Sutherland
- Posted on February 10th, 2016
...he is the man to see!
He is very knowledgeable, kind, caring and all around great person. Oh, and a wonderful doctor. Since I moved to the area over 20 years ago, he has been my doctor. I am happy that I found him AND North Dover first time around.
Awards
4 Awards
Patients' Choice Award
(2015, 2016, 2017, 2018)
Patients' Choice recognition reflects the difference a particular physician has made in the lives of his/her patients. The honor is bestowed to physicians who have received near perfect scores, as voted by patients.
On-Time Doctor Award
(2018)
Vitals On-Time + Promptness Award recognizes doctors with consistent high ratings for timeliness of appointments. The honor is granted based on a physician's overall and promptness scores.
Compassionate Doctor Recognition
(2016, 2017, 2018)
Compassionate Doctor certification is granted to physicians who treat their patients with the utmost kindness. The honor is granted based on a physician's overall and bedside manner scores.
Top 10 Doctor - City
(2014)
Top 10 Doctors are chosen by the millions of patients who visit Vitals each year to find a new doctor and share their experiences by providing ratings and reviews. In order to differentiate highly-regarded doctors from the rest for patients in search of quality care, Vitals awards Top 10 Doctor honors to those physicians within a certain specialty and geographic area who are consistently given top ratings by their patients.
Hospital Affiliations
Dr. Sutherland is affiliated (can practice and admit patients) with the following hospital(s). | 2023-11-19T01:26:19.665474 | https://example.com/article/5068 |
Lumbar laminectomy for herniated disc: a prospective controlled comparison with internal fixation fusion.
This is a controlled prospective study on a matched set of patients with herniated lumbar discs. Both groups received the same bilateral lumbar laminectomy and disc excision by the same surgeon. One group had the addition of an intertransverse fusion with internal fixation. Both groups were studied by an independent examiner at an average of 3 years postoperatively for success rate as determined by activity level, medication, subjective and objective evaluation. Both groups had similar age, sex, and occupational characteristics. No patient had prior surgical treatment or chemonucleolysis. Patients with associated lumbar spine problems such as stenosis, instability, or spondylolisthesis were excluded. Each patient had a positive clinical picture for a herniated lumbar disc, as well as a positive myelogram, venogram or computerized tomographic scan. Most had positive electromyograms. All patients received at least 3 months of conservative care. The 38 patients with fusion had a significantly longer mean time to return to work after surgery versus the 31 patients without fusion. Although the general success rate of both groups was 87%, the best results were in the nonfusion group. A total of 29% of nonfusions had excellent results whereas only 11% of the fusion group had excellent results. The conclusion is that fusions are not necessary and give less excellent results in simple laminectomy cases for herniated lumbar disc. | 2023-10-13T01:26:19.665474 | https://example.com/article/1451 |
In his latest guest blog for Investment Sense, Martin Tilley, Director of Technical Services at Dentons ponders why some SIPP providers are less forthcoming than others when answering industry surveys.
I read with interest the Money Management special report on SIPPs (Self-Invested Personal Pensions) issued earlier this month, edited by Aimee Steen. The survey is one of the most comprehensive of the market and this year features responses from over 50 SIPP providers covering 68 different SIPP Products.
Being one of the largest surveys of its type I image that independent advisors might look to this document as a useful tool amongst others, and a source of reference when comparing potential suitors for their SIPP clients.
Martin Tilley, Director of Technical Services at Dentons
All the more reason why one would think, for the marketing departments of the SIPP providers to step in to demonstrate their product in comparison to others?
However, despite Money Management issuing the survey questionnaire widely, less than half of the FCA authorised and regulated SIPP providers chose to respond and those that did, did not provide all of the information requested. As a useful source of reference then, it diminishes in value.
For example, 32% of the respondents could not confirm a numerical value for the number of SIPPs they had established in the last twelve months, which is unusual since the Regulator requires this information to be collated and reported in the SIPP Providers quarterly returns. Several have provided remarkably round numbers, for several years in a row. One would expect that at the very least, a well run business would wish to maintain this “management information” and particularly so for SIPP cases lost although 40% of the respondents declined to release this information. There is nothing wrong with losing cases. Clients retire and buy annuities, use flexible drawdown and draw out their pot. Some sell the non standard assets that required them to have a SIPP in the first place and move to a more economic vehicle.
Similarly in the commercial property section of the report, two providers could provide an average property value but not the number of properties within their book whilst other providers could determine the number of properties but not break them down between UK, overseas, hotel rooms or land banking.
Perhaps the real reasons for these non disclosures are commercial and the fact that in direct comparison to competitors, certain providers do not want their data analysed. Sometimes saying nothing, says more than you think.
Investment Sense Ltd is registered in England & Wales no. 07050481. Investment Sense Ltd is an Appointed Representative of the Sense Network FCA No. 465124, which is authorised and regulated by the Financial Conduct Authority. The guidance and/or advice contained in this website is subject to UK regulatory regime and is therefore restricted to consumers based in the UK.
The Financial Ombudsman Service is available to sort out individual complaints that clients and financial services businesses aren't able to resolve themselves. To contact the Financial Ombudsman Service please visit www.financialombudsman.org.uk. | 2023-12-25T01:26:19.665474 | https://example.com/article/8486 |
Progressives' academic incest
Academic Collectivism relies on the claims of “experts” rather than original documents as the standard for truth.
~ David Barton
The fifth and final sophism by the left that undermines realism, truth and historical accuracy, or what David Barton in his new book on Jefferson calls “the five malpractices of modern history,” is Academic Collectivism, “whereby writers and scholars quote each other and those from their peer group rather than consult original sources. This destructive and harmful tendency now dominates the modern academic world, with a heavy reliance on peer review as the almost exclusive standard for historical truth.”
I am presently working on a law review article on Oliver Wendell Holmes’ famous 1918 Harvard Law Review article titled “Natural Law.” This work, along with perhaps his 1886 book, “The Common Law” and a handful of dissenting opinions he wrote in the late 1920s and early 1930s as a justice on the Supreme Court, are what we now view as Holmes’ most significant works. Nevertheless, his “Natural Law” article has zero footnotes even though it was published in the venerated Harvard Law Review.
Why? I theorize that Holmes had such contempt for America’s Judeo-Christian worldview and Natural Law, which integrated legality and morality in addition to being the primary legal philosophy of America’s constitutional framers, that he felt it beneath his vaunted and legendary intellect to deign any respect to this subject by citing original sources. In other words, the entire law review article is Holmes merely ranting and raving against America’s Judeo-Christian origins, making his “Natural Law” a most cynical, unremarkable and despicable treatment of the subject matter.
Barton offers an engaging but chilling case of this historical malpractice, citing a 2005 book called “The Godless Constitution.” In that work, Cornell professors Isaac Kramnick and Laurence Moore present the popular academic sophism that the Founding Fathers were nothing but a bunch of atheists, agnostics and deists who knowingly constructed a secular government, a democracy based on humanism. This phony theme has become a staple in most colleges and universities throughout America; law reviews, law journals, courts and other professors reflexively refer to this work as a trustworthy source to “prove” the Founding Fathers’ deficit of religious principle. After presenting such a radical view of history, one would expect to see numerous footnotes at the end of the book, but like my example of Holmes’ “Nature Law,” Kramnick and Moore arrogantly acknowledge that “we have dispensed with the usual scholarly apparatus of footnotes.”
This is an incredible admission by two so-called legal scholars with Ph.D.s! They offer not the slightest support defending their sweeping generalizations and radical claims regarding an alleged lack of faith among the framers of the Constitution, and like sock puppets their peers in academia seemingly in unison deem this book a grand scholarly triumph.
These are some of the great scams of what I call the Progressive Revolution taking over the public schools founded by the Puritans with William Penn as America’s first superintendent. Since the 1870s, progressives have gradually taken over the Christian academy where 187 of the first 200 colleges in America were Christian, Bible-teaching institutions. Entrance to Harvard, Yale and Princeton required strong knowledge of the Bible. In 2012 the academy is essentially a pseudo-academic progressive propaganda mill.
The effects since the late 19th century? Barton writes: “This type of ‘peer review’ is incestuous, with one scholar quoting another, each recirculating the other’s views, but with none of them consulting sources or ideas outside his or her own academic gene pool. The presence of a Ph.D. after one’s name today somehow suggests academic infallibility – but this view must change if truth, accuracy, and objectivity are ever again to govern the presentation of history and historical figures. Primary source documents and historical evidence are the proper standard for historical truth, not professors’ opinions.”
Academic Collectivism relies on the claims of “experts” rather than original documents as the standard for truth, Barton writes. It promotes an adulterous, complex bureaucratic system of peer review scholarship as the sole measurement for determining whether a historical fact is correct or false. However, think about this question: If a liar with a Ph.D. validates another liar with a PhD, isn’t the entire affair one, giant, colossal lie?
This is the state of Academic Collectivism in 2012!
In conclusion, Barton offers these important words to combat liberal bias in the academy, or what Barton calls Academic Collectivism:
The solution for Academic Collectivism is to personally investigate, study, and search out information rather than just accept what the “experts” claim. Become like a jury member of old; get all the evidence, listen to both sides, and reach an independent conclusion warranted by the facts. By practicing these remedies, the five traps of modern historical malpractice can be avoided. Learning accurate history should be our objective.
By the way, history was defined in America’s original dictionary (1828) as “an account of facts” and “a narration of events in the order in which they happened, with their causes and effect.” All five modern historical devices fail to meet important parts of this definition.
Deconstructionists avoid telling about “events” in the way “they happened,” preferring instead to selectively pick out a few things in order to construct a negative image. Poststructuralists avoid “an account of facts,” believing instead that history is subjective and must therefore be individually interpreted based on the way one feels about what happened. Modernists and Minimalists both sidestep “causes and effects,” one by avoiding context and the other by dismissing because examining them “causes and effects” would make things too complicated. And Academic Collectivists avoid “a narration of events,” preferring instead to narrate only what other so-called “experts” has said about those events.
God, realism, truth and history should always be our guide, not the biased rantings of an agenda-driven gang of liberals, progressives and Marxist academics whose pseudo-scholarly aim is to deconstruct everything righteous, good and exceptional about America while rewriting the history of great but flawed men (like all of us), in their own perverted, Marxist, ahistorical image.
President Obama, the Democratic Socialist Party and the liberal academy have all allowed Academic Collectivism to thrive to the detriment of government, culture and society. | 2023-08-08T01:26:19.665474 | https://example.com/article/4452 |
Q:
Table spanning two of three columns in multicol-environment
I have a three-column document where I would like to place a (floating) table that spans the two left columns of the three. The following minimal example works for the placement but overlaps the text and graphics in the middle column. Any suggestions?
\documentclass[paper=a4,paper=landscape, 10pt]{scrartcl}
\usepackage[
left=1.3cm,
right=2cm,
top=1.5cm,
bottom=1.5cm
]{geometry}
\usepackage{wrapfig}
\usepackage{multicol}
\usepackage{array}
\usepackage{lipsum}
\begin{document}
\begin{multicols}{3}
\lipsum[1-2]
\begin{wrapfigure}{l}{2\columnwidth}
\begin{tabular}{m{0.66\columnwidth}m{0.66\columnwidth}m{0.66\columnwidth}}
\hline
A & B & C \\ \hline
A & B & C \\ \hline
\end{tabular}
\end{wrapfigure}
\lipsum[1-5]
\end{multicols}
\end{document}
A:
This feature is neither supported by LaTeX2e nor package wrapfigure.
A manual (cumbersome) workaround is to add some space (\vspace*{...}) in the second column for the table.
The total width of two columns is
\dimexpr 2\columnwidth + \columnsep\relax
And the calculation for the table column has to take \tabcolsep into account:
\dimexpr (2\columnnwidth + \columnsep)/3 - 2\tabcolsep\relax
Example for the manual workaround:
\documentclass[paper=a4,paper=landscape, 10pt]{scrartcl}
\usepackage[
left=1.3cm,
right=2cm,
top=1.5cm,
bottom=1.5cm
]{geometry}
\usepackage{wrapfig}
\usepackage{multicol}
\usepackage{array}
\usepackage{lipsum}
\begin{document}
\begin{multicols}{3}
\lipsum[1-2]
\sbox0{%
\dimen0=\dimexpr(2\columnwidth+\columnsep)/3-2\tabcolsep\relax
\begin{tabular}{m{\dimen0}m{\dimen0}m{\dimen0}}
\hline
A & B & C \\ \hline
A & B & C \\ \hline
\end{tabular}%
}\copy0 %
\edef\TheTableHeight{\the\dimexpr\ht0 + \dp0\relax}
\lipsum[1-2]
Nulla malesuada porttitor diam. Donec felis
erat, congue non, volutpat at, tincidunt tristique, libero.
\vspace{\TheTableHeight}
Vivamus
viverra fermentum felis. Donec nonummy pellentesque ante. Phasellus
adipiscing semper elit. Proin fermentum massa ac quam. Sed diam
turpis, molestie vitae, placerat a, molestie nec, leo. Maecenas
lacinia. Nam ipsum ligula, eleifend at, accumsan nec, suscipit a,
ipsum. Morbi blandit ligula feugiat magna. Nunc eleifend consequat
lorem. Sed lacinia nulla vitae enim. Pellentesque tincidunt purus
vel magna. Integer non enim. Praesent euismod nunc eu purus. Donec
bibendum quam in tellus. Nullam cursus pulvinar lectus. Donec et mi.
Nam vulputate metus eu enim. Vestibulum pellentesque felis eu
massa.
\lipsum[4-5]
\end{multicols}
\end{document}
| 2024-02-05T01:26:19.665474 | https://example.com/article/7195 |
Maria of Gaeta
Maria of Gaeta (born 1020) was an Italian regent, countess of Aquino by marriage and regent of the Duchy of Gaeta for her son in 1062–65.
She was daughter of Pandulf IV of Capua and Maria, was the wife (from before 1038) of Atenulf, count of Aquino, while her sister Sikelgaita was the wife of Atenulf's brother Lando. According to Amatus of Montecassino, Atenulf was consequently supported by Pandulf in taking the duchy of Gaeta from Asclettin, Count of Aversa, on the death of Ranulf Drengot in 1045.
Her eldest son was betrothed to a daughter of Richard I of Capua in 1058, but died before the marriage could take place. Richard extorted the morgengab anyway and Gaeta became a feudatory of Capua.
As senatrix and ducissa of Gaeta, Maria ruled as regent for her and Atenulf's son Atenulf II after her husband's death on 2 February 1062. On 1 June, a pact was confirmed between Maria and the counts of Traietto, Maranola, and Suio. The allies were excluded from forming any pact with the Normans and sworn to protect the territory of the Gaetan duchy. The treaty was finalised at Traietto and was to last for a year. The league was successful in preventing Richard of Capua from extending his conquests during the year. However, Richard skillfully negotiated to prevent a renewal of the pact and on 28 June 1063, he was in possession of Gaeta.
Maria allied with the counts of Traietto and Aquino, her sons Lando and the aforementioned Atenulf, and with William of Montreuil, who repudiated his wife in order to marry her, in late 1064. In February 1065, the revolted were put down by Richard of Capua and Maria and William were expelled from Gaeta. Richard offered to compensate her by marrying her to his son Jordan.
Notes
Sources
Chalandon, Ferdinand. Histoire de la domination normande en Italie et en Sicilie. Paris, 1907.
Southern Italy.
Category:1020s births
Category:11th-century deaths
Category:Women of medieval Italy
Category:Italian nobility
Category:11th-century women rulers | 2023-10-01T01:26:19.665474 | https://example.com/article/1031 |
As a Club, we understand the frustration that our fans must be feeling due to recent events. We are asking that you stand by us and be resilient in the face of adversity, as we strive for a solution with the EFL to help get Bury Football Club back on the right track.
Your support is vital to us, and we are eternally grateful to all of those who are sticking by us as we look to get our season in EFL Sky Bet League One underway as soon as possible.
In the last six months, we have come a long way together. If we all stand united, then we can get through this difficult spell for our historic Club.
Once we have supplied the EFL with the recently requested additional information, we are confident that our embargo will be lifted. In anticipation of this, we would expect that the EFL will not remove us from the Football League.
A lot of people are working very hard to get this over the line. As a Club, we would like to thank the players who are currently training at Carrington with a view to signing for our brilliant football club. Thank you for standing by us, even though the situation we find ourselves in is very difficult.
There is a lot of uncertainty in the Football League with other Clubs outside of Bury, everyone at the Club wishes them all the best with their own circumstances.
VINCIT OMNIA INDUSTRIA | 2023-08-03T01:26:19.665474 | https://example.com/article/9189 |
3
a
Let s = 1125 + -5627/5. What is the closest to 0.4 in 2, 0.2, s?
0.2
Let b(x) = -x**2 - 6*x - 5. Suppose 0*u + 2 = -u. Let m be b(u). Let f = -653/5 - -130. What is the closest to 0 in 1/2, m, f?
1/2
Let h = 0 - 0.1. Let o = -235 - -237. Which is the closest to o? (a) -4 (b) h (c) -0.3
b
Let p = -0.028 - -0.018. Let m = 0 - 0.01. Let z = p - m. What is the closest to -0.1 in -0.1, 0.2, z?
-0.1
Let x = 7 - 1. Let t = x + -7.6. Let g = -6.6 - t. Which is the closest to 0? (a) 3/7 (b) 2/11 (c) g
b
Let y = 0.2 + 0.8. Let r(s) = -s**3 + 2*s**2 + 2*s - 1. Let d be r(3). Which is the closest to y? (a) d (b) -2/9 (c) 1
c
Let j = -191 - -192. Which is the closest to -1/11? (a) -2/19 (b) j (c) 1/7 (d) -2/9
a
Let t = -0.2824 + 0.2824. Which is the nearest to t? (a) -2 (b) 0 (c) -12 (d) -2/53
b
Let y = 167 - 164. What is the closest to 2/17 in y, -6, -5/4?
-5/4
Let p = -7377652/46635 + -1/9327. Let q = 159 + p. Let x = -2.5 + 2.7. What is the closest to -2/5 in -3, x, q?
x
Let r = 2 - 6. Let g = r + 4.5. Let z = -61.86 + 61.9. Which is the nearest to z? (a) 3/5 (b) g (c) -0.4
c
Let x = -37/16 - -275/112. Which is the closest to 0? (a) 9 (b) 2/5 (c) 1 (d) x
d
Let t = 5.4 - 0.4. Let r = t - 4. Let x = 56 + -60. What is the nearest to 0.1 in -2/7, r, x?
-2/7
Let k = -119.6 - -119.4. Let u be 3*(-4)/(-42) - 0. Which is the nearest to 1? (a) k (b) u (c) 0
b
Let v be 7*1*(35/(-5) + 10). What is the closest to 0 in v, -3/5, 0?
0
Let a be ((-6)/(-4))/((-945)/2940). What is the nearest to 0 in a, 0.2, 0.3?
0.2
Let s = -18 - -26. Let y = 5 - s. Let g be (-21)/(-35)*(4/3)/4. Which is the closest to -1? (a) y (b) -3/2 (c) g
b
Suppose -6*m + 48 = 6*m. What is the nearest to 1/2 in -1/3, -4, m?
-1/3
Suppose -382 = -4*k + 5*t - 97, 2*k - 120 = -2*t. What is the closest to 2/7 in 2, -0.2, k, 5?
-0.2
Let b be 35/105 - (1 + 8/(-18)). What is the closest to b in 0.2, -0.7, 4?
0.2
Let v = 2 - -1. Suppose -4 = 4*s + 33*b - 30*b, 2*s + 4*b = -2. What is the nearest to s in v, 0.5, -0.1?
-0.1
Let s = 0.17 - 0.23. Let i = -1.94 + s. Let n be (4 + (-46)/10)/(2/10). Which is the nearest to n? (a) -0.1 (b) i (c) -3
c
Let n = -1531 - -1531.1. What is the closest to 1 in -0.48, n, -0.3, 0.4?
0.4
Let m = -25/4 - -23/4. Let y = -1.421 + 1.021. What is the closest to m in y, -1, 0.4?
y
Let m = -0.06 + 2.06. Let d be 4 - (-2 - -28)/(60/10). Which is the closest to d? (a) 4 (b) m (c) 5
b
Let l = -4 + 6. Let m = 20 - 16. What is the closest to -1/2 in 1/3, l, m?
1/3
Let y = -1/13 + -1/156. What is the nearest to y in 0, -0.1, 4?
-0.1
Let g = 259 - 259. What is the nearest to 0 in g, -4, -2/17?
g
Suppose -2*h = w + 2*w - 15, -2*h - 3 = -3*w. Let d = -0.0467 + -0.0533. What is the nearest to d in 2/7, h, -0.1?
-0.1
Let s = -51.8 - -51.9. Suppose 12 = -4*w, -2*d + 7 = -5*w - 16. Which is the closest to s? (a) 3 (b) d (c) 0
c
Let z = 5 + 1. Let f be (-2)/(-11) - 5/(-33). Let y = 43/63 + -1/9. What is the nearest to f in y, z, -0.3?
y
Suppose -201*v - 72 = -203*v. Which is the nearest to v? (a) -2/3 (b) 3/8 (c) 2/13
b
Let i = 0.1 - 0.08. Let s = 0.66 - 0.16. What is the closest to i in 3/4, s, -3?
s
Suppose 14*n - 72 = 20*n. Let x be (-18)/n*2/9. What is the closest to -0.1 in 4/5, 3/4, x?
x
Let o = -1493 - -1487. What is the closest to -4/9 in 4/7, o, 2, -1/2?
-1/2
Suppose 640*c + 48 = 656*c. What is the closest to 0.4 in -0.5, c, -2.6?
-0.5
Let u = 1474 - 1480. Which is the closest to u? (a) 4 (b) -1/2 (c) 3 (d) 0.1
b
Let x = 1.3 - 1.7. Let d be ((-132)/8)/(3/(-2)). Which is the nearest to 0? (a) x (b) d (c) -0.2
c
Let p = -5/567 + 587/2268. Which is the nearest to -1? (a) 8 (b) p (c) 2/5 (d) 6
b
Let t be 15/16*(-634)/5. Let r = 119 + t. Let v be (4/(-3))/((-420)/(-36) + -11). Which is the closest to 0.1? (a) v (b) -2/41 (c) r
c
Suppose -5*w + 6 + 4 = 0. Let n(t) = -t**3 + 6*t**2 - 4*t - 2. Let z be n(5). Suppose v - z*v + w = 0. What is the nearest to -1 in v, 1/7, -0.5?
-0.5
Let t(m) = -25*m - 106. Let j be t(-4). What is the closest to -0.09 in -3, -4, j?
-3
Let d be (-1)/(-4) - (-111)/(-12). Let w = d - -16. Suppose 2*v = w*v. What is the nearest to v in -5, 3, -1?
-1
Let t = -56/3 - -18. Let x be (-4)/(-2) + (-140)/65. Which is the closest to t? (a) 0.5 (b) 0.4 (c) x
c
Suppose 3*h - 3*j + 1 + 8 = 0, 4*h + 2*j - 6 = 0. Suppose -20 = -3*t - t. Suppose 19 - 4 = -t*u. What is the closest to h in 0.5, u, 2?
0.5
Let j = -129 + 195. Let p = 66 - j. Which is the closest to p? (a) -7 (b) 1/4 (c) 0.3
b
Let d = 1.415 - 1.815. What is the closest to 0.9 in -1/2, 0.3, d, 5?
0.3
Let n be ((-3)/2 - -1)*0. Suppose n = -3*w + 3*o + 21, -19 = 4*w - 5*w + 4*o. Which is the nearest to 4? (a) -5 (b) w (c) 0.5
b
Let n = 226 - 222. What is the nearest to -6 in -2/13, -4, n?
-4
Let u = 1967 - 1963. What is the closest to 0 in -0.4, u, -0.2, 5?
-0.2
Let z = 19507/13 - 1501. What is the closest to 1 in z, 7, -3?
z
Let h = -58/3 - -748/39. Which is the nearest to 0.2? (a) h (b) -0.6 (c) 1/10
c
Let r = -12.26 + 12.06. What is the closest to 1 in r, -3/4, 1/4, -15?
1/4
Let l = -2.2 - 0.9. Let r = l + 2.4. Let y = r + -0.3. What is the nearest to 0 in 0, y, -5?
0
Let r(d) = -d**3 - 14*d**2 - 14*d - 5. Let u be r(-13). Let v be (-4)/((u/4)/(-2)). Which is the nearest to -1/5? (a) v (b) 1 (c) -2
b
Let d be 42/(-9)*(-18)/12. Let n be 0 + (-138)/(-24) + -6. Which is the nearest to 0? (a) n (b) -0.2 (c) d
b
Let o = -1.954 + -0.046. Let w = 0.86 - -0.14. What is the closest to -17 in o, 5, w?
o
Let j = 6.5 + -6.6. Let t be (-108)/525 + 12/42. Which is the nearest to 0.1? (a) t (b) j (c) 0.5
a
Let f = 2593.7 - 2594. Which is the nearest to 1/4? (a) f (b) 0.3 (c) 0.1 (d) -0.01
b
Let o = 60 - 36. Let x = -24.5 + o. Let c = -0.3 - x. What is the nearest to c in 0, -4, -5?
0
Let i = -1.13 - -16.93. Let r = i - 15.9. Which is the closest to r? (a) -3 (b) -1/7 (c) -0.3
b
Let z = -3.5 - -3. Suppose -23*g + 28*g - 1290 = 0. Let i = g + -2836/11. Which is the closest to -1? (a) i (b) -2 (c) z
c
Let s = -0.7 - 0.3. Suppose 2*v - 33 = -5*y, -4*v + 6*v = -y + 13. Which is the nearest to -2/5? (a) s (b) -6 (c) v
a
Let s = -7.7 - -8. Let k = -0.3 + s. Let n = 23/42 + -3/14. What is the closest to k in -0.3, -1/6, n?
-1/6
Let w = -703/4 + 177. Suppose -1 = -2*r + 3. Which is the closest to -3? (a) w (b) 0.3 (c) r
b
Let j = 6/65 - 77/130. Let h = -30 + 23. Which is the nearest to j? (a) -1/6 (b) -2/17 (c) h
a
Suppose 0 = 14*m - 13*m - 6. Let k = 18 + -19. Which is the closest to k? (a) m (b) 0 (c) 0.4
b
Let k = -1493 - -1500. Which is the closest to k? (a) 1/3 (b) 0.5 (c) -5
b
Let y = 197/320 - 1/64. Let k be 2/8 + 14/(-24). Let o = -2630 + 2626. Which is the nearest to -1? (a) o (b) y (c) k
c
Suppose -2*f + 4 = 12*a - 11*a, 0 = -3*a. What is the nearest to 0.01 in 3/2, 1/2, f?
1/2
Let b = 362 + -358. What is the closest to 1/8 in 4/7, b, 1/5?
1/5
Let v = -203 - -206. What is the nearest to 2 in -5, v, 0.07?
v
Let f be 14223/(-60) - ((-26)/8 - -3). Let p = 236 + f. What is the closest to 15/7 in -5, -0.4, p?
-0.4
Let t(l) = -l**3 - 2*l**2 + 2*l - 7. Let j be t(-3). Let n = -83 + 87. Which is the closest to n? (a) -1 (b) -1/2 (c) j
b
Let y be -1 - (2 + (-7)/4). Let s = -111.8 + 111.5. Let t be (-1)/((1 - 4)/(-6)). Which is the nearest to 0? (a) t (b) s (c) y
b
Let k = -1.8 + 1.7. Let t = 311.1 + -311. Which is the nearest to t? (a) -3 (b) -0.4 (c) k
c
Let t = 58 - 292/5. What is the nearest to 0.4 in 2, 0.5, t?
0.5
Let g = 76.8 + -78. What is the closest to g in -5/3, 2, 3?
-5/3
Let d = 0.01 + 6.19. Let u = d + -6. Let w be -2 + ((-54)/21)/(-1). Which is the closest to u? (a) -2/5 (b) 2/5 (c) w
b
Let u = 16/15 - 2/3. Let i be (4/8 - 3)/5. Which is the closest to u? (a) 1/5 (b) 3 (c) i
a
Let w = -1000194/95 + 10528. Let n = w + 3/19. Let z be 1 - 1*(-5)/(-2). Which is the closest to n? (a) -0.2 (b) 1 (c) z
a
Let o = -1516 + 1516.1. Which is the closest to -2/3? (a) 6/11 (b) o (c) -13
b
Let v be 0 + 19/9 - 2. Let g = -584.1 + 583.8. Which is the nearest to 0.3? (a) g (b) v (c) -2/5
b
Let j(m) = -m**2 + 15*m + 2. Let p be j(8). Let z be (-14)/(-12)*(4 - p/14). Which is the nearest to z? (a) -0.1 (b) -0.2 | 2024-06-16T01:26:19.665474 | https://example.com/article/2064 |
/*
* Copyright 2020 Precog Data
*
* 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 quasar.qsu
import quasar.fp._
import quasar.qscript.ReduceFuncs
import quasar.qsu.{QScriptUniform => QSU}
import matryoshka.BirecursiveT
import scalaz.syntax.equal._
object RecognizeDistinct {
import QSUGraph.Extractors._
// pattern from compileDistinct in compiler.scala
// TODO this is dumb; we shouldn't be replicating patterns like this
def apply[T[_[_]]: BirecursiveT](qgraph: QSUGraph[T]): QSUGraph[T] =
qgraph rewrite {
case qgraph @ LPReduce(GroupBy(orig1, orig2), ReduceFuncs.Arbitrary(()))
if orig1.root === orig2.root =>
qgraph.overwriteAtRoot(QSU.Distinct(orig1.root))
}
}
| 2023-11-05T01:26:19.665474 | https://example.com/article/4818 |
Yep
Thats pretty much how the old owners were. The field was mostly run by the refs. They played on the Ghostriders and were the best refs that I have ever seen. Then these new owners bought it some rich doctor with a bunch of nerdy refs. They ran it like a day care. That rich doctor always talked about buying Paintball USA dont know or think if he ever did. Well anyway if you ever hear about anything going on with Warzone let me know it may have been crappy but It was nice having it close to my house. Also if your ever in the area Paintball Zone on Dixie farm road is really turning out to be kind of a nice field. The owners are great but the refs are pricks. | 2024-01-26T01:26:19.665474 | https://example.com/article/8530 |
Poverty and Education
The link between poverty and educational failure has concerned many both in government and research for many years. If poverty in general does lead to educational failure, does this mean that those children born into poverty are born into a vicious circle - the cycle of poverty - that they cannot get out of? Born into poverty, poorly prepared if prepared at all for pre-school, failure when compared to others at primary school, inability to access the full curriculum at secondary school and failure when exams are taken. With such a background, how can a child born into poverty break out of the cycle?
The Carolina Abecedarian Project was a controlled experiment that was conducted in 1972 in North Carolina, United States, by the Frank Porter Graham Child Development Institute to study the potential benefits of early childhood education for poor children to enhance school readiness. It has been found that in their earliest school years, poor children lag behind others, suggesting the fact that they were ill-prepared for schooling. The Abecedarian Project was inspired by the fact that few other early childhood programs could provide a sufficiently well-controlled environment to determine the effectiveness of early childhood training.
Children from disadvantaged backgrounds need to do more than just attend a good school to boost their educational achievement, a report has claimed. It claimed that just 14% of the difference between an individual's performance was down to the quality of the school. Former Schools Minister, Lord Adonis, said that one of the Labour government’s (1997 to 2010) main priorities while they were in government was helping children from disadvantaged backgrounds. He suggested that one of the former government’s achievements was the provision of more activities outside of school which helped children develop their confidence. With the current large cutback in government spending, the fear is that these projects will be easy targets for cuts.
The Joseph Rowntree Foundation has studied the link between poverty and failure or success at school. JRF found that children in poverty face greatly reduced educational prospects and future life chances. They found that this is the conclusion not just of social policy experts and government statisticians, but of young children themselves. Research published by the Joseph Rowntree Foundation (JRF) shows that children are aware of such outcomes from an early age and that their own stereotyping reinforces these differences. This study summarises the messages from the first eight projects in the JRF’s Education and Poverty programme and looks at the experiences of children from different backgrounds and their attitudes to education.
It concluded that low income is a strong predictor of low educational performance and that children from different backgrounds have contrasting experiences at school. Less advantaged children are more likely to feel a lack of control over their learning, and to become reluctant recipients of the taught curriculum. This influences the development of different attitudes to education at primary school that help shape their future.
Their findings were supported by research done by the BBC in September 2007. The study found that poor children are as much as two years behind their peers in educational achievement by age 14 and heading for a "downward spiral". Research for the ‘Campaign to End Child Poverty’ says children from poor homes are up to nine months behind their peers before they even get to school.
For many children living in poverty stricken areas, private schools are not a likely option. The average cost to attend a private school is £10,000, which for those living in poverty is an impossible dream. The next option for them is good state schools, where they are going to be at a disadvantage again because of the ‘postcode lottery’. This is a system which favours those with perceived better postcodes and critics claim it allows schools to best select their future intake so that the school’s reputation is enhanced or maintained.
UK Government set a target of 2020 to eradicate child poverty. The JRF believe that an extra £4.2bn a year will have to be spent on tax credits if the government is to meet its target of halving child poverty by 2015. In 2010, the JRF claimed that 2.3 million children were living in poverty. The government had set a target for 2010 of 1.7 million – but this was set in 1999 well before the recession kicked in to the UK’s economy. With wholesale cuts in government spending in most areas, child poverty campaigners claim that the figure will actually go up and that the 2020 deadline for child poverty eradication will be missed by a long way. | 2023-11-04T01:26:19.665474 | https://example.com/article/2713 |
The Westchester Knicks came back from a double-digit deficit only to fall just short in a 114-113 loss to Raptors 905. The W-Knicks fell behind by as much as 19 points, came back with a 16-2 run, trailed by 10 points in the fourth quarter, went ahead with a 13-0 run, and ultimately couldn't hold on. Basketball, as you may have heard, is a game of runs.
At the very least, it was a nice game for Cleanthony Early on his road to recovery. The second-year forward played 36 minutes -- by far the most he's played since the Dec. 30 shooting -- scored 20 points on 7-12 from the field (2-3 from three), grabbed 11 rebounds and came up with 3 steals. He looked very bouncy, very Cle-like. That's what we like to see, baby!
Highlights
Cleanthony Early
Jordan Bachynski
Travis Trice
Jimmer Fredette
Meanwhile, the mystery of what happened to Thanasis Antetokounmpo grows. Thanasis is listed as a DNP-coach's decision. Starting in his place was Keith Wright, who ... I dunno. He went to Harvard, so that's something. He's not as good as Thanasis. Hopefully our Greek buddy just has a stomach bug or something. The kid certainly a key member of the W-Knicks.
Only five games remain for Westchester. They lead Iowa by three games in the loss column for the fourth and final playoff spot in the East. Based on my shoddy math skills, their magic number to clinch a playoff berth should be 3. Let's get to it, guys! | 2023-09-15T01:26:19.665474 | https://example.com/article/8648 |
81 So.3d 113 (2011)
Curtis D. LEE, Plaintiff-Appellee
v.
SAFEWAY INSURANCE COMPANY OF LOUISIANA and Henry Lee, Jr., Defendant-Appellant.
No. 46,716-CA.
Court of Appeal of Louisiana, Second Circuit.
December 9, 2011.
*114 Tracy L. Oakley, Ruston, LA, for Appellants.
Edward Larvadain, III, for Appellee.
Before PEATROSS, DREW and LOLLEY, JJ.
DREW, J.
Safeway Insurance Company of Louisiana and Henry Lee, Jr. ("Henry Lee, Jr."), defendants, appeal a judgment awarding plaintiff, Curtis D. Lee ("Lee"), special damages of $1,956.00 and general damages of $7,500.00 for injuries sustained or preexisting injuries aggravated in a collision on November 19, 2008. Because the record contains insufficient evidence to establish the connection between plaintiff's *115 alleged injuries and this wreck, the judgment of the trial court is reversed and the plaintiff's action is dismissed with prejudice.
A little over two months before the collision which is the subject of this litigation, Lee was in another auto accident on September 15, 2008. The first crash occurred when Lee was traveling about 60 mph behind a U-Haul van which suddenly pulled to the right revealing a car stopped in the road. Although he slammed on his brakes, Lee collided with the stopped car and another vehicle then hit him. He could not change lanes because the other lanes were occupied. Lee was treated and released at the emergency room of Schumpert Hospital.
In the second accident (November 19, 2008) from which Lee is seeking damages in this litigation, Lee stated that he was a guest passenger in his own car being driven with his permission by his cousin, Henry Lee, Jr., who failed to yield at a yield sign and collided with a car driven by Nolan S. Taylor. The parties stipulated to liability. Stating his vehicle was totaled in the collision, Lee asserted that in the wreck, he sustained lumbar and cervical strain, lower back pain, and general body aches and bruises.
Lee sought care from two different chiropractors, each of whom treated Lee apparently without being aware Lee had been in another accident for which he was receiving therapy from another chiropractor. The following timeline sets out the accidents and the treatment related to each. The items relevant to the second accident are indicated in italics. Dates on which Lee was treated by both chiropractors are shown in boldfaced type and underlined.
September 15, 2008 First accident
October 10, 2008 First appointment with Dr. Grady Michael Stimits for first accident, at which he received a detailed exam, Hot and Cold Packs, and Mechanical Traction
October 13, 2008 Treatment by Dr. Stimits, including Chiropractic Manipulative Treatment ("CMT") 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
October 20, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
October 27, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
November 3, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
November 5, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
November 10, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
November 17, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
November 19, 2008 Second accident
November 24, 2008 Re-exam by Dr. Stimits and treatment including Mechanical Traction and Hot and Cold Packs
December 1, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
December 8, 2008 Treatment by Dr. Stimits, including CMT 3-4 Region, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
*116 December 8, 2008 First appointment with Dr. John Lawrence for second accident
December 12, 2008 Treatment by Dr. Lawrence, including CMT 3-4 Regions, Hot Pack, Mechanical Traction, Electrical Stimulation, and Hydrotherapy
December 15, 2008 Treatment by Dr. Stimits, including CMT 3-4 Regions, Mechanical Traction, Hot and Cold Packs, and Myofacial Release
December 15, 2008 Treatment by Dr. Lawrence, including CMT 3-4 Regions, Hot Pack, Mechanical Traction, Electrical Stimulation, and Hydrotherapy
December 22, 2008 Treatment by Dr. Lawrence, including CMT 3-4 Regions, Mechanical Traction, Electrical Stimulation, and Hydrotherapy
December 29, 2008 Treatment by Dr. Stimits, including CMT 3 Region, Mechanical Traction, Hot and Cold Packs and Myofacial Release
December 29, 2008 Treatment by Dr. Lawrence, including CMT 3-4 Regions, Mechanical Traction, Electrical Stimulation, and Hydrotherapy
January 5, 2009 Treatment by Dr. Stimits, including re-exam, Mechanical Traction, and Hot and Cold Packs
January 5, 2009 Treatment by Dr. Lawrence, including CMT 3-4 Regions, Mechanical Traction, Electrical Stimulation, and Hydrotherapy
January 12, 2009 Treatment by Dr. Lawrence, including CMT 3-4 Regions, Mechanical Traction, Electrical Stimulation, and Hydrotherapy
January 20, 2009 Last appointment with Dr. Stimits, who did re-exam
January 21, 2009 Treatment by Dr. Lawrence, including CMT 3-4 Regions, Mechanical Traction, and Hydrotherapy
January 26, 2009 Last appointment with Dr. Lawrence with treatment including CMT 3-4 Regions, Mechanical Traction, and Hydrotherapy
TRIAL TESTIMONY
The parties stipulated that Dr. Grady Michael Stimits, a chiropractor called by the defense, was an expert. In brief, the plaintiff stated that the only witnesses at trial were himself and Dr. John Lawrence. Actually, the only trial witnesses were the plaintiff and Dr. Stimits. The exhibits were the accident report, the medical records from both chiropractors, and the insurance policy submitted as a joint exhibit. Although Lee's deposition was referenced in his cross-examination, that deposition was not filed into evidence.
Dr. Stimits testified that:
he treated Lee for injuries sustained in the September 15, 2008, wreck;
the course of treatment ran from October 10, 2008, until January 20, 2009;
he was unaware of the November 19, 2008, accident;
he could not testify as to what Lee may or may not have told his staff, although he thought it likely his staff would have mentioned a second accident;
it was important that he be informed about a second accident because of issues involving injuries, treatment, and litigation;
he did not know that Lee was simultaneously being treated by another doctor;
Lee initially complained of pain in his neck and back, weakness and pain in his right shoulder, and numbness in his right arm;
during a reevaluation examination on November 24, 2008, Lee reported pain on the right side where the pelvis meets *117 the spine, along with neck and back pain,[1] and right arm numbness;
on that date, there was improvement in ranges of motion and decreases in spasms and numbness;
though Lee had reported pain in all ranges of motion in the initial exam on October 10, he had pain in only one range of neck motion on November 24;
the initial evaluation showed spasms and tenderness all the way down his back;
by November 24, the tenderness and spasms were only on the right side of his back;
by January 5, 2009, Lee's range of motion was not quite normal, but pretty close, on his neck and back, with some mild numbness in the right arm and pain in the neck;
Lee reported no pain, and was normal neurologically on January 20, 2009;
when checked for spasms on that date, there was no abnormal tension or tightness and both his neck and back were pain free;
the doctor found no disability and a good prognosis;
too many chiropractic adjustments "can actually hard (sic) the patient";
thus it was absolutely vital that patients be truthful with the chiropractor; and
not knowing another chiropractor is performing adjustments on a patient can create a lot of issues.
On cross-examination, Dr. Stimits stated he was certain a reference to the right knee in the records was an error because all other references were to the left knee. At the November 24 examination (just days following the November 19 wreck), Lee had signs and symptoms of injury. On January 5, he still had mild numbness in the right arm and off-and-on pains in the neck and back, while reporting his left knee was okay and he was sleeping much better. The doctor recommended Lee be treated twice a week but he said his work schedule would allow only once a week.
Lee's final visit was January 20, 2009, at which time Dr. Stimits thought it unlikely that he was pain free as the doctor had observed spasms on January 5. Lee said he needed to go and Dr. Stimits respected that request, which is common with his patients who decide they can no longer continue treatment. The chiropractor suspected that Lee still had some pain and discomfort on January 20.
Regarding the November 19 accident, Lee testified that:
he received a knot on his head;
the right side of his body hit the door; and
his injuries included headache, pain in his right arm, lower right back, knee and lower leg, as well as numbness from his right shoulder to his elbow.
Lee's attorney sent him to a chiropractor, Dr. John Lawrence, whom he first saw on December 8, 2008, and last saw on January 26, 2009. Lee asked the doctor to release him because he had no transportation to continue treatment and work. He still had discomfort at the January 18, 2011, trial.
Lee testified that as a result of the November 19 accident, he was no longer able to play baseball and that his sleep was restless due to arm and leg pain. He also described being nervous with people close by as a result of the November 19 wreck. Because of these injuries, he also said he was not able to continue his job as a caretaker for an elderly couple, which involved lifting the man who was wheelchair *118 bound. Never having been arrested, Lee testified that his only legal trouble was having his license suspended for an unpaid traffic ticket when he was 15.
Lee acknowledged on cross examination that in December 2008 and January 2009 he was working two jobs (caretaker of the elderly and security guard at Villagio) and stated at his deposition that he discontinued medical treatment because he had to go to work. Lee did not mention his transportation problems at the deposition. He agreed that he was being treated by two different chiropractors, Dr. Stimits (for the September accident) and Dr. Lawrence (for the November accident). Additionally, Lee said that the injuries for which he sought to recover in this action were the same injuries for which Dr. Stimits treated him because Lee contended his injuries from the first accident were aggravated by the second collision.
At his deposition, Lee stated, "I don't think I told him (Dr. Stimits about the second accident)." When questioned about whether he informed Dr. Lawrence about his first accident, Lee's response was unclear. When asked to explain why he did not think it was important to tell each doctor he was being treated by the other, Lee stated, "Naw (inaudible) two separate accidents (inaudible)."
On redirect examination, Lee stated in response to an inquiry about whether he informed Dr. Stimits about the second collision, "Yeah. I think I told him. I am not for sure.... I could have told the secretary."
With a referral from his attorney, Lee first saw Dr. Lawrence on December 8, 2008, and received extensive examination and diagnostic testing. Dr. Lawrence's initial report, right after the November accident, noted complaints of neck and low back pain along with right shoulder and arm pain. At the December 8, 2008, office visit, Lee's reported symptoms were:
dull throbbing bilateral headaches;
sharp burning, cramping, stinging bilateral neck pain;
dull spastic, throbbing constricting pain on the right side of his lower back;
sharp, shooting, stinging pain in right shoulder;
sharp, shooting, cramping, stinging pain in upper right arm;
weakness, listlessness, lack of energy; and
inability to sleep due to pain.
Dr. Lawrence's initial report contained the following very enlightening information:
HISTORY:
I have determined that Mr. Lee's history has not contributed to his present condition. Mr. Lee denies any prior symptoms, injuries or accidents similar to those described in this report. Therefore, it is my opinion, based on medical probability, that this patient's condition, as outlined in this report, is the direct result of the injuries sustained on November 19, 2008, as described by the patient.
Social History:
The patient's social history is non-contributory in that it does not contain any information that would have contributed to his present complaints. The patients's family history in non-contributory in that it does not contain any information that would have contributed to his present complaints.
Noting that he expected Lee to experience favorable treatment results, Dr. Lawrence diagnosed Lee with whiplash injury, multiple subluxations (term generally meaning a misalignment) of cervical, thoracic, and lumbosacral spine; headache, *119 myalgia, lumbosacral sprain, and sleep disturbances.
On a December 12, 2008, office visit, Dr. Lawrence reported that Lee was progressing as expected and experienced some improvement. On December 29, 2008, Dr. Lawrence noted that Lee was again progressing as expected and showed moderate improvement from the previous visit. The identical assessment was made on January 5, 2009. On January 12, 2009, Dr. Lawrence observed that Lee's progress was moving along as expected and he had experienced moderate improvement. On January 20, 2009, the chiropractor observed that Lee was progressing as expected and showed marked improvement. In all the foregoing reports, Dr. Lawrence stated that Lee was receiving medically necessary therapeutic care and had not yet reached MMI ("maximum medical improvement").
On January 26, 2009, Dr. Lawrence found somewhat restricted range of motion in the cervical spine along with subluxations and slight pain on palpation of his cervical and lumbar spine. The assessment was that Lee was progressing as expected, had not reached MMI, but showed a marked improvement since his last visit. The doctor recommended chiropractic adjustments once a week for two weeks. That was the last visit Lee made to Dr. Lawrence.
REASONS FOR JUDGMENT
After hearing the testimony, the trial court found Lee to be a "real credible" witness who sustained the injuries alleged. Additionally, the trial court concluded that the second accident aggravated some of the old injuries and caused a couple of new injuries. Other factors cited were plaintiffs inability to play baseball and the pain suffered. The judgment granted plaintiff special damages of $1,956.00 and general damages of $7,500.00.
DISCUSSION
It is axiomatic that a court of appeal may not set aside a trial court's finding of fact in the absence of "manifest error" or unless it is "clearly wrong." See Rosell v. ESCO, 549 So.2d 840 (La.1989). In Welch v. Willia-Knighton Pierremont, 45,554 (La.App.2d Cir.11/17/10), 56 So.3d 242, writs denied, 2011-0075 (La.2/25/11), 58 So.Sd 457, and 2011-0109 (La.2/25/11), 58 So.3d 459, this court explained that to reverse a trial court, the appellate court must find that a reasonable factual basis does not exist in the record for the finding and that the determination is clearly wrong. Where two permissible views of the evidence exist, the factfinder's choice between them cannot be manifestly erroneous or clearly wrong. If the documentary or objective evidence so contradicts the witness's testimony, or the testimony is so internally inconsistent or implausible on its face that a reasonable factfinder would not credit the testimony, the court of appeal may find manifest error even where the finding is purportedly based on a credibility determination. Absent a finding of clear error and if the factfinder's conclusion is based on its decision to credit the testimony of one or more witnesses, that finding can virtually never be manifestly erroneous or clearly wrong. Welch v. Willis-Knighton Pierremont, supra.
In a personal injury action seeking damages, the plaintiff has the burden of proving a causal relationship between the accident and any alleged injuries. The plaintiff must prove causation by a preponderance of the evidence. That burden is satisfied when the plaintiff proves through medical and lay testimony that it was more probable than not that the injury was caused by the accident. Bradshaw v. *120 Brookshire Grocery Co., 38, 960 (La.App.2d Cir.10/27/04), 886 So.2d 623.
Whether the accident caused the plaintiff's injuries is a factual question which should not be reversed on appeal absent manifest error. Reversal is warranted only if the appellate court finds that a reasonable factual basis for the trial court's finding does not exist in the record and that the finding is clearly wrong on the record. Bradshaw, supra.
The trial court found Lee to be a credible witness, and we note that Dr. Lawrence's tests for malingering were negative. However, we respectfully find that a reasonable factual basis does not exist in this record to support the conclusion that Lee's injuries were sustained in or worsened by the November 19, 2008, mishap. The medical evidence either fails to support or simply contradicts Lee's assertion that his injuries were related to the second accident. The trial court's decision was clearly wrong.
Lee was poorly served by the failure to provide the chiropractors with an accurate history of his dual accidents. Without that information, Dr. Lawrence's opinion that Lee's injuries were a direct result of the November 19, 2008, wreck is fatally flawed, since those injuries were substantially present prior to the accident.
Lee's testimony that his injuries were aggravated by the November 19, 2008, accident is belied by the fact that Dr. Stimits noted improvement in Lee's condition on his November 24, 2008, visit. Lee's statements that his injuries were worsened by the second wreck are not supported by the medical evidence in the record. Simply put, Lee failed to prove he suffered new injuries or aggravations of preexisting conditions in the November collision. On this record, a decision to the contrary is manifestly erroneous.
DECREE
The judgment of the trial court is reversed, and plaintiffs action is dismissed with prejudice at his costs.
REVERSED.
NOTES
[1] Upon flexion and extension. The doctor also noted that on November 10, 2008, the patient had a tender right side joint, referring to the right sacroiliac joint.
| 2023-12-16T01:26:19.665474 | https://example.com/article/5148 |
Scott Wartman
swartman@enquirer.com
A streetcar crossing the Ohio River might seem far-fetched, considering the obstacles it faced in Cincinnati.
But some residents and leaders have, at least, started to lay out the framework for a possible streetcar in Northern Kentucky.
In a proposed design revealed Thursday, the streetcar would cross the Taylor-Southgate Bridge into Newport. It would then go through the Ovation development site across the Fourth Street Bridge into Covington.
A group of residents, business leaders and elected officials known as the Northern Kentucky Streetcar Committee came up with the design and will seek $300,000 from the federal government for a study, said Ian Budd, a Newport resident and head of the committee.
"It connects Newport and Covington, which I think is fantastic," Budd said. "At the moment when I travel on public transportation from Newport to Covington, I have to go through Cincinnati. I think we want the cities to be more connected and working together."
Budd and streetcar proponents from Cincinnati spoke about the benefits of a streetcar Thursday in Covington, at a lunch hosted by the Covington Business Council.
Cincinnati City Councilman Chris Seelbach, former Cincinnati Mayor Roxanne Qualls and Cincinnati streetcar advocate John Schneider also spoke at the event touting the streetcar.
Budd and Newport City Commissioner Beth Fennell will travel to Washington, D.C. in February to lobby for federal funds to study a streetcar in Northern Kentucky.
"The opportunities are limitless," Budd said. "Connecting the streetcar to the airport is quite possible, as well."
All the proponents of the streetcar acknowledged the uphill battle faced in Northern Kentucky. After all, this is a place where tea party groups tried to eliminate library funding. Even repairs to a clubhouse in Devou Park faced opposition.
It'll probably be 20 years before a streetcar crosses into Northern Kentucky, said Covington Mayor Sherry Carran. But it needs to be talked about, she said.
"I think through having a discussion we can learn the pros and cons as to whether the streetcar would be a good fit for Northern Kentucky," Carran said. "We won't know that until there's a discussion. It's a shame that you're criticized for even wanting to explore the opportunity."
The groundwork laid by Cincinnati could expedite the process in Northern Kentucky, the Cincinnati officials from Cincinnati told the crowd Thursday. It'll be an easier sale when they see how it works in Cincinnati, Schneider said.
"We made a terrible mistake in Cincinnati: We let our opponents define the project," Schneider said. "For 20 years, we had an idea in Cincinnati. Now we have a product. It's tough to argue with a product."
The idea of streetcars in Northern Kentucky isn't new. The predecessor to Vision 2015, Forward Quest, proposed a monorail in Northern Kentucky in the 1990s. Cincinnati built lower ramps to facilitate streetcars going into Northern Kentucky when it redesigned Fort Washington Way in the late 1990s, Qualls said.
"People have been anticipating this for a long time," Qualls said. "It's a local decision and it's going to require tremendous local support from the constituencies and courage on the part of elected officials to put up with a lot of pushback." | 2023-11-02T01:26:19.665474 | https://example.com/article/5760 |
Archives for April 2017
Some people are afraid of flying but I happen to love it. (Which is great since my family lives in Poland and the only way I get to visit them is to hop on the airplane and fly to Europe.)
What are you afraid of?
Maybe you love flying like I do, but you are afraid of failing in your daycare business. Or maybe you are afraid to confront your daycare parents about an ongoing issue. Perhaps licensing visits give you a lot of anxiety.
We are all afraid of something.
Let me share with you the beginning of a scary adventure called "Living My American Dream."
I remember landing in Chicago and going through customs, which took forever, and finally meeting my friend Marcin who was picking me up at the airport. I was very excited, yet somewhat nervous about starting college here in the US.
We arrived on campus late at night. Since I was not assigned to a dorm room yet, I had to stay in a guest room.
I was alone - laying in bed, dealing with jet lag, and out of the blue I started to cry as I wondered, “What have I done? Was moving here a mistake?”
I couldn’t sleep, so I called my mom just to talk. When she answered, I was sobbing and unable to form sentences. I was terrified, to say the least. I was scared of this new country, new culture, new language and my new unknown adventure.
New beginnings are not always easy, are they? Starting an in-home daycare can feel very similar.
Perhaps you have just opened a daycare and now you are asking yourself “What am I doing?” Or maybe you have been running a daycare for a while, yet still those voices in your head are questioning: “Did I make a mistake by opening daycare? Was I foolish by quitting a full time job and starting my own business?"
Fear tells us to quit, to give up before we even start. Fear moves in on us and causes us to doubt ourselves and the abilities we have within us. And fear would win the day if it had it's way - but we are tough women, and we don’t surrender easily, do we?
The obvious way out for me would have been to never unpack my luggage and move back to Poland before school even started.
But I decided not to give up on my college education here in the US. And - you do not have to give up on building a strong and successful daycare business.
So how did I do it? How did I not allow fear to totally overtake me?
Here’s my secret…
After my first sleepless night at college, the morning finally came. I was exhausted both physically and emotionally, but I needed to get up, meet with my advisor and register for classes. While I was getting ready and fighting tears, I heard a "knock" on my door. It was my friend Marcin, who attended the same college I was about to enroll in.
He offered to walk me around campus, show me classrooms, and introduce me to his friends and his beautiful wife, Bethany.
Suddenly things did not seem so scary anymore!
Instead I was very excited because I realized I did not have to do my new life in America alone. Marcin and Bethany were there for me not only on my first day of college, but they have been there for me ever since. I am forever grateful for their investment in my life - I know I couldn’t have done it without them. They modeled to me what a successful mentorship looks like.
And guess what? YOU don’t have to do life or run a daycare business alone either!
It’s so much less intimidating when you have somebody by your side. We all need mentors and friends who will be there for us when we are scared, walk with us when things are challenging, and believe in us when we have no faith.
Just like Marcin and Bethany were there for me, I would like to be there for you.
And even though we might not be able to avoid every daycare struggle that comes our way, when we approach them together we can overcome the paralyzing fear and become bold and confident daycare owners.
You don't have to do it alone, my friend. Let's do it together!
xoxo,
Sylvie
PS - If you want more guidance and practical tips how to run a successful daycare business without feeling alone and misunderstood, the Fabulous Provider Guide might have the message you need to hear. It is packed with personal stories and inspiring advice. | 2023-10-31T01:26:19.665474 | https://example.com/article/5311 |
Deep-fill hyaluronic acid for the temporary treatment of the naso-jugal groove: a report of 303 consecutive treatments.
To report a 2-year experience of treating the naso-jugal groove with injectable hyaluronic acid gel, using a deep-fill method. This was a consecutive, retrospective, nonrandomized case series of patients presenting with concerns involving dark circles, lower eyelid hollows, or other contour irregularities that make up the naso-jugal groove. One author performed all treatments, consisting of transcutaneous injection of hyaluronic acid gel filler, to address the naso-jugal groove. The filler was placed deep on the anterior lip of the orbital rim and molded to the desired shape. Between December 2003 and December 2005, 164 patients (34 male and 130 female) received hyaluronic acid gel filler in the face. Ninety-eight patients were treated just once and 66 had multiple treatment sessions. The mean dose of filler per session was 1.53 +/- 0.8 ml, with 0.84 +/- 0.38 ml divided between the two lower eyelids. The most common complication was localized swelling, followed by bruising, asymmetry, cellulitis (2 cases), and migraine (1 case). There were no cases of visual loss. Hyaluronic acid gel fillers have had an enormous impact on the practice of cosmetic surgery, and this series demonstrates the usefulness of these fillers for treatment of the lower eyelid and midface. The authors recommend the deep-fill method described as a reliable means of addressing the hollow created by the naso-jugal groove. | 2024-04-27T01:26:19.665474 | https://example.com/article/9429 |
Influence of the load of nylon capsules on their passage through the digestive tract and specific gravity.
The passage and specific gravity of nylon capsules were evaluated in five trials. In individual trials, different lactating cows were fed the same diet consisting of maize silage, alfalfa hay and concentrate. In each trial the different feeds (or no feed) were used to fill the capsules. The capsules were made of nylon cloth (42 microm pore size, 10 mm external diameter). The different weights of the load (L1-L5) were obtained using a combination of 2 and 3 mm stainless steel balls. The highest recovery of the capsules was obtained with the L3 and L4 loads (91.4 and 92.3%, resp.). After 14 hours of incubation in the rumen, the calculated values of functional specific gravity of the capsules ranged from 0.92 to 2.05 g x cm(-3). It was concluded that L3 (one 2 mm and one 3 mm ball) was the suitable weight of the load. | 2024-07-03T01:26:19.665474 | https://example.com/article/7420 |
include/mt_client.h
lib/libmt_client.a
lib/libmt_client.so
lib/libmt_client.so.0
lib/libmt_client.so.0.0.0
| 2024-04-24T01:26:19.665474 | https://example.com/article/8589 |
Q:
How can I change the scale of the legend in highcharter in R?
Here is some open source code from the highcharter package website that details how to load in a map using hcmap.
library(highcharter)
# produces the following map inline
hcmap("countries/us/us-all", data = data_fake, value = "value",
joinBy = c("hc-a2", "code"), name = "Fake data",
dataLabels = list(enabled = TRUE, format = '{point.name}'),
borderColor = "#FAFAFA", borderWidth = 0.1,
tooltip = list(valueDecimals = 2, valuePrefix = "$", valueSuffix = " USD"))
What I would like to know is whether/how I can alter the scale of the legend in the bottom right corner. What if I wanted a logarithmic scale or some custom values? Is there a way to do that with this package?
A:
For a logarithmic scale, try to pipe to hc_colorAxis(type = "logarithmic"):
library(highcharter)
library(dplyr)
data("USArrests", package = "datasets")
USArrests <- mutate(USArrests, `woe-name` = rownames(USArrests))
set.seed(1)
data_fake <- tibble(test = sample(1:300000, 50, replace = TRUE),
`woe-name` = USArrests$`woe-name`)
hcmap(map = "countries/us/us-all", data = data_fake,
joinBy = "woe-name", value = "test", name = "woe-name",
dataLabels = list(enabled = TRUE, format = '{point.name}'),
borderColor = "#FAFAFA", borderWidth = 0.1,
tooltip = list(valueDecimals = 2, valuePrefix = "$", valueSuffix = " USD")
) %>% hc_colorAxis(type = "logarithmic")
Edit: Custom color scales are also possible, see https://www.highcharts.com/forum/viewtopic.php?t=33569.
| 2024-03-30T01:26:19.665474 | https://example.com/article/4178 |
Homozygous familial hypercholesterolaemia in Vietnam: Case series, genetics and cascade testing of families.
Familial hypercholesterolaemia has not been previously described in the Vietnamese population. We aimed to describe the features of patients with homozygous familial hypercholesterolaemia (hoFH) in Vietnam and the outcomes of screening family members using genetic and cholesterol testing. Mutation testing by massively parallel sequencing for genes causative of FH was undertaken in five index cases presenting to a single cardiac center with a presumptive diagnosis of hoFH. Cascade testing of all available family members was subsequently undertaken. The number of new cases of FH detected and commenced on lipid-lowering treatment was evaluated. All five index cases had true homozygous mutations in the LDL receptor gene (LDLR). Cascade screening was undertaken in four families. 107 relatives were screened and FH was identified in 56 relatives (52%), including 3 new cases of hoFH. Only 5 FH relatives (9%) were subsequently treated owing to the adverse perceptions and comparative high cost of drug treatment, and lack of awareness of FH among patients and local doctors. HoFH due to LDLR mutations is a severe disorder in Vietnam that needs early detection and treatment with LDL-cholesterol lowering drugs. Cascade testing of families allows effective detection of new cases of FH that may also benefit from early treatment. However, convincing patients to commence statin treatment is a challenge. Extended education and awareness programs and treatment subsidies are imperative to improve the care of patients and families suffering from FH in Vietnam. | 2024-06-19T01:26:19.665474 | https://example.com/article/5328 |
Pfizer says construction to start on new Saudi plant
Global pharmaceutical giant Pfizer has announced that construction work is set to start on a new manufacturing plant in Saudi Arabia.
The foundation stone has been laid at its new pharmaceutical manufacturing plant in King Abdullah Economic City (KAEC).
Incorporating medicine solid dose manufacturing, packaging, and warehousing facilities, the new plant will be operational in 2015, Pfizer said in a statement.
At a total production capacity of 18 million packs per year, the facility will produce a broad range of Pfizer's medicines currently supplied to Saudi Arabia.
The development will also facilitate the transfer of Pfizer's expertise to the local market in Saudi, ensuring production meets Pfizer's rigorous and globally recognised quality standards, the statement added.
Abdulatif Bin Ahmed Al Othman, governor of the Saudi Arabian General Investment Authority (SAGIA), said: "We are sending a very strong and positive message to the world - that Saudi Arabia is set to transform the economy and its infrastructure to host and support knowledge-based industry leaders and manufactures like Pfizer."
He added: "Pfizer's decision and commitment to set up its first ever manufacturing facility throughout the GCC on the land of Saudi Arabia is a clear signal that SAGIA's vision to make Saudi Arabia a major hub between East and West is attainable."
Pfizer's new manufacturing facility will be instrumental in providing Saudi patients with improved access to Pfizer broad portfolio of medicines. It will also create new employment opportunities.
Keith Dennie, vice president Operations, Pfizer, said: "We intend that the new facility will serve the growing needs of the kingdom now and in the future."
"With a growing population and high prevalence of endemic diseases such as diabetes and cardiovascular disease, Saudi Arabia healthcare need is on rise." added Hussein El Hakim, country manager, Pfizer Saudi Arabia.
"For us, investing in setting up a world-class manufacturing facility in Saudi is not only a rewarding business opportunity, but also a commitment to the health and wellbeing of the people of Saudi Arabia."
Pfizer Pharmaceutical manufacturing plant will be built at the Industrial Valley, King Abdullah Economic City, on the west coast of the Red Sea, north of Jeddah. | 2024-06-24T01:26:19.665474 | https://example.com/article/4006 |
Introduction {#s1}
============
In mammals, reproductive organs are already formed in the embryo, with gametogenesis being completed after birth. By contrast, plant reproductive development is initiated only after the transition from the juvenile to the adult phase, with the production of flowers that harbor the reproductive organs. Despite these differences, both kingdoms tightly control the progression from initiation to maturation of reproductive organs and the acquisition of reproductive competence. The progression relies on successive genetic programs that govern a delicate balance of cell division and differentiation, organ growth and maturation, as well as cell death. Coordination of these events in plants makes use of a host of interacting hormones, including cytokinins, auxin, gibberellins (GAs), jasmonate (JA), ethylene and brassinosteroids. Hormone action is often mediated by dedicated transcription factors (TFs) such as the auxin response factors (ARFs), several of which are microRNA (miRNA) regulated. As an example, expression of the paralogous master regulators *ARF6* and *ARF8* ("*ARF6/8*") of *Arabidopsis thaliana* are fine-tuned in specific floral organs by miR167 [@pgen.1003374-Wu1]. Increased miR167 levels or reduced ARF6/8 function both result in underdeveloped floral organs and impaired male and female fertility [@pgen.1003374-Wu1]--[@pgen.1003374-Nagpal1]. *ARF6/8* activity is also sufficient for driving growth of floral organs, as shown with plants in which miR167 function is blocked [@pgen.1003374-Wu1], [@pgen.1003374-Todesco1]. ARF6/8 regulate the expression of auxin homeostatic genes and help to set the boundaries of cytokinin-dependent meristematic activity, in addition to modulating JA biosynthesis [@pgen.1003374-Tabata1], [@pgen.1003374-Nagpal1], [@pgen.1003374-Reeves1]. Therefore, some of the defects in flowers with reduced *ARF6/8* levels resemble symptoms of auxin, GA and JA deficiency and cytokinin overproduction [@pgen.1003374-Wen1]--[@pgen.1003374-Pautot1]. Further complexity comes from miR167 having several isoforms that differ in their expression patterns and in their ability to downregulate their ARF6/8 targets [@pgen.1003374-Wu1].
Defects seen in *arf6/8* mutant flowers are reminiscent of ones observed when the function of two other miRNAs, miR159 and miR319, is compromised. Such flowers suffer from retarded development of organs in the three outer whorls, the sepals, petals and anthers. The targets of these miRNAs are MYB and TCP transcription factors [@pgen.1003374-Achard1]--[@pgen.1003374-Rhoades1]. miR159-mediated restriction of MYB33 and MYB65 activity to anthers is necessary for normal floral organ growth and fertility [@pgen.1003374-Millar1], [@pgen.1003374-Allen1], [@pgen.1003374-Allen2]. MiR159 levels are positively regulated by GA [@pgen.1003374-Achard1], and at least in rice, this is also true for miR319 expression [@pgen.1003374-Liu1]. MiR319 is encoded by three genomic loci with overlapping expression patterns in *A. thaliana*. Transcription of *MIR319A* coincides with *MIR319C* at the base of all floral organs, in stamen filaments and petals, while *MIR319B* is restricted to stamens and the abscission zone of sepals [@pgen.1003374-Warthmann1], [@pgen.1003374-Nag1]. Reduction of miR319 activity results in smaller flowers with short petals, strap-like petals and underdeveloped stamens; in extreme cases, petals and stamens are lost [@pgen.1003374-Todesco1], [@pgen.1003374-Nag1].
Here, we report how these three evolutionary conserved miRNA-TF pairs (miRNA-TF nodes hereafter) are organized into a regulatory network that is responsible for several checkpoints in floral organ maturation. MiR159 and miR319 regulate directly interacting targets, which in turn control the expression of *MIR167* family members during this process. Restriction of *MIR167A* expression allows the progression from meristematic, cytokinin-dependent programs to auxin-dependent organogenesis, culminating in GA- and JA-dependent maturation. We propose that the convergence of unrelated miRNA targets onto common downstream targets is an important feature of miRNA networks in plant development.
Results {#s2}
=======
Floral defects caused by altered miR159, miR167, and miR319 activities {#s2a}
----------------------------------------------------------------------
Flowers of *Pro35S:MIM159* and *Pro35S:MIM319* transgenic plants, in which miR159 and miR319 activities are impaired by constitutive expression of target mimics ([Figure S1](#pgen.1003374.s001){ref-type="supplementary-material"}), have defects reminiscent of those found in plants with reduced ARF6/8 activity [@pgen.1003374-Todesco1]. We therefore compared in detail the consequences of reducing ARF6/8, miR159 and miR319 function during reproductive development. Similar to plants with reduced ARF6/8 function (*arf6*-2 *arf8*-3 double mutants, *Pro35S:MIR167A* and *Pro35S:MIR167C* plants), the maturation of sepals, petals and anthers was delayed to various degrees in *Pro35S:MIM159* and *Pro35S:MIM319* plants. In addition, the flower stems, or pedicels, were shorter, and the angles of flowers relative to the main stem were more acute ([Figure 1A](#pgen-1003374-g001){ref-type="fig"} and [Figure S2](#pgen.1003374.s002){ref-type="supplementary-material"}). Stamens did not completely elongate in any of the genotypes. The strongest defects were seen in *arf6 arf8*, *Pro35S:MIR167A* and *Pro35S:MIM319* plants, which never shed pollen ([Figure 1A](#pgen-1003374-g001){ref-type="fig"}). Thus, stamen filament elongation seemed to be more sensitive to loss of ARF6/8 activity than anther dehiscence.
{ref-type="supplementary-material"}.](pgen.1003374.g001){#pgen-1003374-g001}
Smaller organ size was likely due to reduced cell size, as seen by scanning electron microscopy of stamen filaments ([Figure 1B](#pgen-1003374-g001){ref-type="fig"}). In addition to epidermal defects, vascular development in stamen filaments appeared to be arrested at the procambium stage, since the expression of the procambial marker Q0990 [@pgen.1003374-Sawchuk1] was expanded in plants with diminished miR159, miR319 and ARF6/8 activities ([Figure 1C](#pgen-1003374-g001){ref-type="fig"}). Petal vasculature was also compromised, lacking secondary loops in *Pro35S*:*MIM319* plants, or having only two instead of four loops in *Pro35S*:*MIM159* and *Pro35S:MIR167c* plants ([Figure 1D](#pgen-1003374-g001){ref-type="fig"}).
Since petals and stamens were particularly sensitive to depletion of miR159 and miR319, we investigated the specific functions of two of their main targets, *MYB33* and *TCP4* [@pgen.1003374-Millar1], [@pgen.1003374-Palatnik1], [@pgen.1003374-Allen1], [@pgen.1003374-Allen2], [@pgen.1003374-Palatnik2], by expressing miRNA-non-targetable versions (*mMYB33* and *mTCP4*) under the control of the petal- and stamen-specific *APETALA3* (*AP3*) promoter [@pgen.1003374-Jack1]. We grew those plants along with plants expressing *MIM159* and *MIM319* decoys under the same *AP3* promoter for comparison ([Figure 1E](#pgen-1003374-g001){ref-type="fig"}). While mis-expression of the unmodified versions had little, if any, phenotypic effects, the non-targetable forms led to underdeveloped petals and anthers in *ProAP3*:*mMYB33* flowers, and elimination of petals and anthers in *ProAP3*:*mTCP4* plants ([Figure 1E](#pgen-1003374-g001){ref-type="fig"}) [@pgen.1003374-Nag1]. Flowers expressing the miRNA insensitive form of *MYB33* faithfully resembled the developmental impairments found in petals and stamens of *ProAP3:MIM159* plants ([Figure 1E](#pgen-1003374-g001){ref-type="fig"}). Nevertheless, flowers from *ProAP3:mTCP4* presented more severe developmental defects than *ProAP3:MIM319* flowers, in line with previous reports [@pgen.1003374-Nag1]. The phenotypic differences might be explained by a crosstalk between different miR319 TCP targets, or, as suggested in [@pgen.1003374-Nag1], by TCP4 movement or feed-forward regulation.
A sequence of hormone-related events regulated by coordinated miRNA action {#s2b}
--------------------------------------------------------------------------
Previous studies that have examined the role of different hormones during flower maturation suggested a sequential pattern for hormone action in the three outer whorls of the flower, especially in petals and stamens. Early in flower development, cytokinin-dependent maintenance of meristematic activity is delimited by the action of auxins [@pgen.1003374-Tabata1]. Auxins subsequently trigger GA- and JA-based programs required for organ elongation and maturation during floral development [@pgen.1003374-Nagpal1], [@pgen.1003374-Wolbang1], [@pgen.1003374-Cheng2].
A major consequence of *ARF6/8* deficiency, and therefore reduced auxin signaling, is the expanded expression of positive meristem regulators involved in cytokinin action, such as the class I *KNOX* genes *SHOOTMERISTEMLES*S (*STM*) and *BREVIPEDICELLUS* (*BP*) [@pgen.1003374-Tabata1]. The phenotypic alterations described above, along with premature flower bud opening, might thus be attributed to the expansion of cytokinin-related meristematic activity and reduced auxin action [@pgen.1003374-Scofield1], [@pgen.1003374-Ori1], [@pgen.1003374-Li1], [@pgen.1003374-Bartrina1]. As similar developmental defects were found in plants deficient in miR159 and miR319 activity, we asked whether their absence was also affecting the sequence of hormone-related events leading to flower maturation. We analyzed *STM* and *BP* activity in *Pro35S*:*MIM159* and *Pro35S:MIM319* inflorescences, and in *MIR167C* overexpressors, which have reduced ARF6/8 expression. In all three backgrounds, *STM* and *BP* expression were increased ([Figure 2A](#pgen-1003374-g002){ref-type="fig"}). Suppression of the *bp*-1 mutant phenotype by *Pro35S:MIM319* indicated that increased expression of other *KNOXI* genes could bypass the *BP* requirement ([Figure 2B, 2C](#pgen-1003374-g002){ref-type="fig"}). Increased *KNOXI* activity might also explain the abnormal angle of petiole growth in mutants affected in ARF6/8, miR159 and miR319 activities. Additional defects caused by increased *KNOXI* activity are in vasculature development and stamen elongation and maturation, which are linked to reduced auxin transport [@pgen.1003374-Tabata1], [@pgen.1003374-Feng1]. Accordingly, *STM* and *BP* upregulation was paralleled by reduced expression of the *PIN1* auxin transporter gene in *Pro35S:MIM159*, *Pro35S:MIR319* and *Pro35S:MIR167C* lines ([Figure 2A](#pgen-1003374-g002){ref-type="fig"}). These results showed that miR159, miR319, just like ARF6/8, were negative regulators of the expression of class I *KNOX* genes, thereby confining cytokinin-dependent meristematic activity.
{#pgen-1003374-g002}
In addition to demarcating cytokinin action, ARF6/8 also contribute to flower development in later stages of maturation. ARF6/8 activate the expression of genes encoding two major JA biosynthetic enzymes, *LOX2* and *DAD1* [@pgen.1003374-Tabata1], [@pgen.1003374-Nagpal1], a role that they share with the miR319 target TCP4 [@pgen.1003374-Schommer1]. JA is important for the latest stages of floral organ development, including final stamen elongation, positioning of the anthers at the stigma at the time of dehiscence, and pollen maturation. Hence, without JA, plants are infertile [@pgen.1003374-Feys1]--[@pgen.1003374-Caldelari1]. In addition, JA modulates petal growth and vascular development [@pgen.1003374-Brioudes1], [@pgen.1003374-Sehr1]. *LOX2* is not essential for fertility [@pgen.1003374-Bell1], but the *LOX2* promoter is active in stamens and sepals, where its expression increases from stages 12 to 15 of flower development [@pgen.1003374-Jensen1], [@pgen.1003374-Schmid1]. Consistent with a role of miR159, miR167 and miR319 in JA regulation, *LOX2* promoter activity was reduced in sepals of *Pro35S:MIM319*, *Pro35S:MIM159* and *Pro35S:MIR167c* plants ([Figure 2C](#pgen-1003374-g002){ref-type="fig"}), with ectopic activation at the base of *Pro35S*:*MIM319* pedicels, where *MIR319B* is normally expressed ([Figure 2C](#pgen-1003374-g002){ref-type="fig"}, [Figure S4C](#pgen.1003374.s004){ref-type="supplementary-material"}).
Taken together, the phenotypic and physiological resemblance of plants with a reduction in *ARF6/8* or miR159/miR319 activities supports links between miR167, miR159 and miR319 in growth and hormone-dependent maturation of sepals, petals and anthers. The coordinated roles of the three miRNAs and their targets initially enable organogenesis by confining cytokinin-dependent meristematic activity. Later on, they contribute to the JA-dependent maturation of floral organs.
Mediation of miR159 and miR319 effects by miR167 {#s2c}
------------------------------------------------
We next assayed *ARF6/8* expression in *Pro35S:MIM159* and *Pro35S:MIM319* plants in order to determine how *ARF6/8* are regulated by miR159 and miR319. In wild type, both genes are expressed in the vasculature of floral organs, including in sepals, in petals, in stamen filaments, and in the precursor of the transmitting tract, the medial ridge of the carpel [@pgen.1003374-Wu1]. Expression of *ARF6/8* was slightly reduced in *Pro35S:MIM159* and *Pro35S:MIM319* flowers, especially in the stamen filament vasculature ([Figure 3A](#pgen-1003374-g003){ref-type="fig"}). In addition, we found that the expression of the *ProARF8:ARF8-GUS* reporter was specifically absent from stamens when miR159 and miR319 activities were reduced ([Figure 3B](#pgen-1003374-g003){ref-type="fig"}).
{#pgen-1003374-g003}
To determine whether miR159 and miR319 targets are likely to regulate *ARF6/8* directly or through miR167, we compared the response of *ProARF8*:*ARF8-GUS* and its miRNA insensitive form, *ProARF8*:*mARF8-GUS*, to changes in miR159 and miR319 activity ([Figure 3B](#pgen-1003374-g003){ref-type="fig"}). While the expression of *ProARF8:ARF8-GUS* was altered in *Pro35S:MIM159* and *Pro35S:MIM319* plants, that of *ProARF8*:*mARF8-GUS* was not, indicating that miR159 and miR319 targets regulate *ARF8* by increasing miR167 activity ([Figure 3B](#pgen-1003374-g003){ref-type="fig"}). This hypothesis was further supported by the finding that petal and stamen development as well as fertility in *Pro35S:MIM159* and *Pro35S:MIM319* flowers were substantially restored when we reduced miR167 activity with the *Pro35S:MIM167* construct ([Figure 3C, 3D](#pgen-1003374-g003){ref-type="fig"}). These results placed miR159 and miR319 upstream of miR167. Plant miRNAs can regulate the expression of their targets through mRNA cleavage and/or translational inhibition [@pgen.1003374-Todesco1], [@pgen.1003374-Brodersen1]. The observed difference between the effects of changes in miR167 expression on *ARF6/8* mRNA and protein indicated that miR167 acts through both mRNA cleavage and translational inhibition.
Simultaneous sequestration of both miR159 and miR319 resulted in additive phenotypic and molecular effects ([Figure 4A, 4B, 4D](#pgen-1003374-g004){ref-type="fig"}), suggesting that the miR159- and miR319-regulated MYB and TCP transcription factors regulate shared target genes. We found that miR159 targets affected anther and petal development even when miR319 targets were suppressed in *jaw*-D plants ([Figure 4C](#pgen-1003374-g004){ref-type="fig"}), indicating that MYBs and TCPs work in parallel to trigger miR167-mediated ARF6/8 regulation. Since TFs that regulate the same gene often form higher-order heteromeric complexes [@pgen.1003374-Smaczniak1], we hypothesized that this might be the case for miR159-targetd MYBs and miR319-targeted TCPs. Consistent with this idea, the expression patterns of *MYB33* and *TCP4* in the affected floral organs overlap ([Figure S3](#pgen.1003374.s003){ref-type="supplementary-material"}) and the proteins can interact in yeast and plant assays ([Figure 4E, 4F](#pgen-1003374-g004){ref-type="fig"}). This implies that the two miRNA pathways converge on common downstream targets by modulating the composition of regulatory TF complexes.
{#pgen-1003374-g004}
Differential regulation of *MIR167* genes by miR159 and miR319 targets {#s2d}
----------------------------------------------------------------------
The divergent promoter activities of *MIR167* family members are paralleled by their different abilities to downregulate ARF6/8, both of which indicate subfunctionalization [@pgen.1003374-Wu1]. It appears likely that the different *MIRNA167* genes fine-tune ARF6/8 expression within different organs. To better understand miR159- and miR319-dependent regulation of *MIR167* genes, we assayed the transcriptional activity of *MIR167A*, *MIR167B* and *MIR167C* in *Pro35S:MIM159* and *Pro35S:MIM319* plants. We also analyzed *Pro35S:MIR167C* plants deficient in ARF6/8 function, to address whether altered reporter expression was a direct consequence of the absence of miR159 and miR319, or a downstream consequence of disrupting development by reducing ARF6/8 levels.
A GUS reporter under control of the *MIR167A* promoter is expressed in anther procambium, sepal vasculature and ovaries; activity of the *MIR167B* promoter has been detected in ovaries and at the base of flower pedicels; and the *MIR167C* promoter is mainly active in stamen filaments and petals [@pgen.1003374-Wu1]. As expected, in *Pro35S:MIM159* and *Pro35S:MIM319* flowers, *ProMIR167A:GUS* was ectopically expressed in tissues with abnormal development, including sepal vasculature, petals and stamen filaments, while *MIR167C* overexpression had no effect ([Figure 5A](#pgen-1003374-g005){ref-type="fig"} and [Figure S4A, S4B](#pgen.1003374.s004){ref-type="supplementary-material"}). In addition, similarly to the *LOX2* promoter in *Pro35S:MIM319* flowers ([Figure 2C](#pgen-1003374-g002){ref-type="fig"}), the *MIR167A* promoter was ectopically activated at the base of pedicels, in contrast to the repression of the *MIR167B* promoter in this region ([Figure 5A](#pgen-1003374-g005){ref-type="fig"} and [Figure S4A, S4C](#pgen.1003374.s004){ref-type="supplementary-material"}). Likewise, reduced ARF6/8 function in *Pro35S:MIM159* and *Pro35S:MIM319* plants or in *MIR167C* overexpressors caused the *MIR167C* promoter to be less active in stamen filaments ([Figure 5A](#pgen-1003374-g005){ref-type="fig"}). These results point to miR159 and miR319 as coordinating the expression pattern of *MIR167* family members in petals, sepals and stamen.
{ref-type="supplementary-material"}.](pgen.1003374.g005){#pgen-1003374-g005}
*MIR167A* promoter activity correlated with mature miRNA levels, as shown by *in situ* hybridization with an LNA probe ([Figure 5B](#pgen-1003374-g005){ref-type="fig"}). *MIR167A* was initially expressed in emerging stamen, becoming confined to the procambium of stamen filaments from stage 7 on, and its levels were increased in *Pro35S:MIM159* and *Pro35S:MIM319* flowers ([Figure 5C](#pgen-1003374-g005){ref-type="fig"}). The expression pattern of miR167a in ovaries, which was unaffected by changes in miR159 and miR319, resembled its promoter activity as well ([Figure 5B, 5C](#pgen-1003374-g005){ref-type="fig"}) [@pgen.1003374-Wu1], [@pgen.1003374-Valoczi1]. Together, these results suggested that the effects of miR159 and miR319 are mainly mediated by *MIR167A*. The *MIR167A* promoter has two predicted TCP binding sites ([Table S1](#pgen.1003374.s006){ref-type="supplementary-material"}). Mutating either of two potential TCP sites rendered the *ProMIR167A:GUS* reporter unresponsive to increased TCP levels in *Pro35S:MIM319* plants ([Figure 6A, 6B](#pgen-1003374-g006){ref-type="fig"}, and [Figure S5A](#pgen.1003374.s005){ref-type="supplementary-material"}). We tested whether *MIR167A* regulation by TCP4 is direct by chromatin immunoprecipitation (ChIP) using anti-GFP antibodies and extracts from *ProTCP4:TCP4-GFP* Pro35S:*MIM31*9 inflorescences. Quantitative PCR revealed specific enrichment overlapping one of the two potential TCP binding motifs ([Figure 6C](#pgen-1003374-g006){ref-type="fig"}, [Table S1](#pgen.1003374.s006){ref-type="supplementary-material"}). The *A. thaliana* genome encodes 24 TCPs, including several in the miR319-regulated TCP4 clade, and many can homo- and heterodimerize ([Figure S5B](#pgen.1003374.s005){ref-type="supplementary-material"}) [@pgen.1003374-Cubas1]--[@pgen.1003374-MartinTrillo1]. It is therefore conceivable that one of the TCP consensus motifs is bound by a TCP other than TCP4.
![Direct regulation of *MIR167A* by TCP4.\
(A) Diagram of *MIR167A* promoter with potential TCP binding sites (inverted triangles) that were mutated (1: 5′-G\[G/t\]TCCC-3′; 2: 5′-GGA\[C/a\]CA-3′; lower case indicates mutant). Transcription start site is from [@pgen.1003374-Xie1]; transcribed region is not to scale. (B) Reporter gene assay with *ProMIR167A:GUS*. In contrast to the wild-type promoter ([Figure 4A](#pgen-1003374-g004){ref-type="fig"}), the mutant promoter variants do not respond to a change in miR319 activity. Scale bar indicates 1 mm. (C) TCP4-GFP ChIP with material from *ProTCP4:TCP4*-GFP *Pro35S:MIM319* inflorescences, normalized against empty vector control (dotted line). Amplicons 1 and 2 are indicated as lines in (A); *LOX2* served as positive, *HSF1* as negative control. Error bars indicate range of two biological and two technical replicates. See also [Tables S1](#pgen.1003374.s006){ref-type="supplementary-material"}, [S2](#pgen.1003374.s007){ref-type="supplementary-material"} and [Figure S5](#pgen.1003374.s005){ref-type="supplementary-material"}.](pgen.1003374.g006){#pgen-1003374-g006}
Discussion {#s3}
==========
We have analyzed how three miRNA-target nodes interact to control consecutive checkpoints during floral organ maturation in the three outer whorls. First, we have discovered that the miR159-MYB and miR319-TCP nodes can independently regulate the miR167-ARF node. Second, we conclude that the convergent downstream effects of miR159 and miR319 are at least partially due to direct interaction of their MYB and TCP transcription factor targets. The finding of a link between these three miRNA-TF nodes reinforces the observation that miRNAs in plants are disproportionately often involved in auxin signaling [@pgen.1003374-RubioSomoza1] and that they can be organized into miRNA networks [@pgen.1003374-RubioSomoza2].
The miR159-miR167-miR319 circuit acts in sepals, petals and anthers to modulate the activity of ARF6/8, which control a large number of floral genes [@pgen.1003374-Nagpal1]. MiR159 and miR319 dampen the expression of their TF targets, which can otherwise lead to miR167 misexpression both individually and cooperatively through engaging in common protein complexes. In addition to regulation by miR159 and miR319 targets, there is cross-regulation among *MIR167* genes, one example being repression of *MIR167C* in stamen filaments by miR167a. All these inputs are required to avoid miR167 misexpression. Moreover, the interactions can be complex, with miR319 contributing to activation of *MIR167B* and repression of *MIR167A* at the base of pedicels.
MiR159 and miR319 regulation enable ARF6/8 to play a central role in setting the cytokinin-auxin differentiation boundary by delimiting the expression of *KNOXI* genes. Inhibition of *KNOXI* expression leads to repression of cytokinin-based programs, which allows cells to exit the undifferentiated state [@pgen.1003374-Tabata1], [@pgen.1003374-Scofield1], [@pgen.1003374-Ori1]. MiR159- and miR319-dependent ARF6/8 function contributes to later aspects of development by promoting JA synthesis in sepals through the induction of *LOX2* expression and in petals and anthers through the regulation of *DAD1* expression [@pgen.1003374-Tabata1]. In addition, *LOX2* is directly controlled by miR319-targeted TCP transcription factors at the base of pedicels independently of ARF6/8 levels. The tissue-specific differences in JA regulation by miR319 deserve further investigation.
Petal and anther development are particularly sensitive to perturbations in the miR159-miR167-miR319 network. Hormone action in the development of these floral organs starts with the maintenance of meristematic activity linked to *KNOXI*-dependent cytokinin biosynthesis and signal transduction ([Figure 7](#pgen-1003374-g007){ref-type="fig"}). Auxin action is a prerequisite for the initiation of flower organ primordia [@pgen.1003374-Cheng1], [@pgen.1003374-Tao1]--[@pgen.1003374-Zhao1]. Later on, miR159- and miR319-dependent ARF6/8 activities control a checkpoint for a transition that requires inhibition of *KNOXI* genes, which in turn regulates auxin transport and GA signaling ([Figure 7](#pgen-1003374-g007){ref-type="fig"}) [@pgen.1003374-Tabata1], [@pgen.1003374-Wen1], [@pgen.1003374-Jasinski1], [@pgen.1003374-Fleishon1]. Both processes are essential for the elongation and maturation of petals and anthers as well as for JA biosynthesis, which contributes to the last steps of the maturation of both organs ([Figure 7](#pgen-1003374-g007){ref-type="fig"}) [@pgen.1003374-Tabata1], [@pgen.1003374-Nagpal1], [@pgen.1003374-Wen1], [@pgen.1003374-Griffiths1], [@pgen.1003374-Feng1], [@pgen.1003374-Cecchetti1], [@pgen.1003374-Feys1], [@pgen.1003374-Stintzi1], [@pgen.1003374-Brioudes1], [@pgen.1003374-Peng1]. Auxin action, mediated by ARF5/MP and ARF6/8, also promotes cambium development [@pgen.1003374-Nagpal1], [@pgen.1003374-Hardtke1], [@pgen.1003374-Donner1], and we propose that progression of vascular development which appears to follow a similar sequence of signaling events as floral organ maturation ([Figure 7](#pgen-1003374-g007){ref-type="fig"}) [@pgen.1003374-Sehr1], [@pgen.1003374-Dettmer1], is mediated by the miR159-miR167-miR319 network as well.
{#pgen-1003374-g007}
Sepals, petals and stamens, which make up the three outer whorls of floral organs, have key roles in evolutionary transitions between different plant mating strategies. The most common change is from obligate cross-pollination (allogamy) to self-fertilization (autogamy), which is found in up to half of all flowering plants, including many domesticated species [@pgen.1003374-Chen1]. The window of time during which the stigma is exserted beyond the outcrossing-protective sepals and petals before the stamens elongate, is a central determinant of outcrossing opportunities [@pgen.1003374-Peng2], [@pgen.1003374-Tantikanjana1]. Since the miR159-167-miR319 network is crucial for the development of sepals, petals and stamens, modulation of its activity might provide a regulatory entry point for different reproductive strategies.
Materials and Methods {#s4}
=====================
Plant material {#s4a}
--------------
Plants were grown on soil in long days (16 h light/8 hours dark) under a mixture of cool and warm white fluorescent light at 23°C and 65% humidity. *ProMIR167A:GUS*, *ProMIR167B:GUS*, *ProMIR167c:GUS ProARF8:ARF8-GUS*, *ProARF8:mARF8-GUS* and *afr6-*2 *arf8-*3 [@pgen.1003374-Wu1], [@pgen.1003374-Nagpal1], artificial miRNA target mimics [@pgen.1003374-Todesco1], *ProLOX2:GUS* and *ProTCP4*:*TCP4-GFP* [@pgen.1003374-Schommer1], the Q0990 marker [@pgen.1003374-Sawchuk1], and *bp*-1 mutants [@pgen.1003374-Venglat1] have been described.
Transgenic plants {#s4b}
-----------------
*MIR167A* and *MIR167C* fragments were PCR amplified from Col-0 genomic DNA, and placed behind the constitutive CaMV 35S promoter (Pro35S) [@pgen.1003374-Odell1]. Unmodified and modified *MYB33* and *TCP4* coding sequences [@pgen.1003374-Palatnik1] were placed behind the *AP3* promoter [@pgen.1003374-Jack1]. The *ProMIR319B:GUS* reporter included a 2,662 bp genomic DNA fragment that begins 182 bp upstream of the first nucleotide of pre-miR319b. The *MIR167A* promoter was according to [@pgen.1003374-Wu1]. See [Table S2](#pgen.1003374.s007){ref-type="supplementary-material"} for oligonucleotide primers. Constructs were introduced into Col-0 plants by *Agrobacterium tumefaciens*-mediated transformation [@pgen.1003374-Weigel1]. Their names can be found in [Table S3](#pgen.1003374.s008){ref-type="supplementary-material"}.
RNA analyses {#s4c}
------------
Total RNA was extracted from 30-day old inflorescences of T1 or F1 plants using TRIzol Reagent (Invitrogen) with two biological replicates using tissue pooled from 10 to 15 plants each. After reverse transcription with the RevertAid First Strand cDNA Synthesis Kit (Fermentas) of 1 µg of total RNA that had been treated with DNase I (Fermentas). PCR was carried out in presence of SYBR Green (Invitrogen) and monitored in real time with the Opticon Continuous Fluorescence Detection System (MJR/BioRad). For in situ hybridization [@pgen.1003374-Wollmann1] to detect *ARF6*/8, we used sections of inflorescences from 30-day old plants, with sense and antisense probes as described [@pgen.1003374-Wu1]. For LNA in situ hybridization [@pgen.1003374-Wang1] to detect miR167, 10% polyvinylalcohol was added to the colorimetric reaction buffer to increase the sensitivity of the assay.
Histology {#s4d}
---------
Inflorescences were fixed in 90% acetone, and GUS activity was assayed as described [@pgen.1003374-Blzquez1]. For *ARF8-GUS* reporters, ten-fold lower concentrations of ferri- and ferrocyanate were used to increase sensitivity. Gus pictures are representative of three different experiments that included at least 10 samples each. Petals were sequentially cleared with chloral hydrate and ethanol/glycerol/lactic acid (3∶1∶1).
Chromatin immunoprecipitation {#s4e}
-----------------------------
About 150 inflorescences were collected and fixed in two biological replicates, and immunoprecipitation was performed as described [@pgen.1003374-GomezMea1], with 3 µl of anti-rabbit GFP antibody (ab290; Abcam). Twenty µl eluate from Minelute columns (Qiagen) was diluted 1∶5 and used as template for two technical replicates of real-time PCR. Enrichment was calculated by comparing amplification in post-binding and input fraction and normalized to enrichment in samples from empty vector plants. We used a DNA fragment located within the 1 Kb upstream region of the *Hsf1* gene (at4g17750) and lacking any putative TCP binding site as negative control.
Protein--protein interaction {#s4f}
----------------------------
Interaction in the yeast two-hybrid system was assayed on selective medium (Leu^−^, Trp^−^, His^−^) supplemented with 60 mM 3-amino 1,2,4-triazole (3-AT). The assay was repeated four times. For the luciferase complementation assay [@pgen.1003374-Chen2], fusions of non-targetable forms of MYB33, TCP2 and TCP4 were expressed from the 35S promoter. *Agrobacterium tumefaciens* cultures with the different constructs at OD~600~ = 0.3 each were mixed in equal ratios with a P19 silencing suppressor culture at OD~600~ = 0.1. Leaves were imaged three days after inoculation. Details for the constructs can be found in [Table S3](#pgen.1003374.s008){ref-type="supplementary-material"}.
Supporting Information {#s5}
======================
######
Expression levels of representative miR159 and miR319 targets in plants with specific miRNA attenuated function. (A) Expression of three representative miR319-TCP targets in *Pro35S:MIM319* inflorescences. (B) Expression of miR159-MYB targets in *Pro35S:MIM159* inflorescences. Expression was monitored by real-time RT-PCR. Error bars indicate range of two biological and two technical replicates. Expression values of the different genes in the mutant plants were normalized to their expression in wild type inflorescences (dashed lines).
(PDF)
######
Click here for additional data file.
######
Flowers and inflorescences of mutant and transgenic plants. (A) Scanning electron micrographs of flowers from transgenic plants. MIMXXX and MIRXXX lines express the target mimics and miRNAs from the 35S promoter. MIM159, MIM319 and MIR167C plants were 30 days, MIM167 plants 38 days old. Scale bar indicates 700 µm. (B) Entire inflorescences. Note that flowers grow more upwards, that is, at a smaller angle relative to the main stem, in MIM159 and MIM319 plants, or in *arf6 arf8* mutants. Scale bar indicates 2 mm.
(PDF)
######
Click here for additional data file.
######
MYB33 and TCP4 expression patterns in flowers. (A) *ProMYB33:GUS* is broadly express in every flower organ. (B) *ProTCP4:GUS* is expressed in the vasculature of sepals and anther filaments (procambium), petals and female reproductive organs.
(PDF)
######
Click here for additional data file.
######
Effect of miR159, miR167 and miR319 on *MIR167A* promoter activity, and comparison of *MIR167A* and *MIR319B* promoter activities. (A) Activity of *ProMIR167A:GUS* in different backgrounds. Note particularly strong ectopic activity in sepals, petals, and at the base of flowers and pedicels of MIM319 expressers. (B) Close-up of mature petals. Note ectopic activity in vasculature. (C) ProMIR319B:*GUS* expression in wild-type inflorescence. Activity is notable at the base of petioles and floral organs, and in anthers. Scale bars indicate 2 mm (A), 500 µm (B). See also [Figure 5](#pgen-1003374-g005){ref-type="fig"}.
(PDF)
######
Click here for additional data file.
######
Regulation of *MIR167A* promoter by TCP transcription factors. (A) Reporter gene assay with *MIR167A* promoter. Mutations in either of the two TCP binding motifs suppress ectopic activity in response to MIM319 overexpression. Scale bar indicates 2 mm. (B) Heterodimerization of TCP2 and TCP4 assayed by firefly luciferase complementation assay in *N. benthamiana* leaves. Luciferase activity is shown in false color, with highest levels red and lowest levels blue. See also [Figure 6](#pgen-1003374-g006){ref-type="fig"}.
(PDF)
######
Click here for additional data file.
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Potential R2R3MYB and TCP binding motifs in *MIR167A* and *LOX2* promoters.
(DOC)
######
Click here for additional data file.
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Oligonucleotide primer sequences.
(DOC)
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Click here for additional data file.
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List of plasmids.
(DOC)
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Click here for additional data file.
We thank Jason Reed and Enrico Scarpella for graciously responding to multiple requests for seeds from different transgenic lines; Jia-Wei Wang for the plasmids used in bimolecular luminiscence complementation assays; Carla Schommer for the unpublished MYB33 and TCP4 reporter lines; Jürgen Berger and Steffen Lau for assistance with microscopy; and Roosa Laitinen, Sascha Laubinger, Miguel Ángel Moreno-Risueño, Rebecca Schwab, and Jia-Wei Wang for comments and discussion.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: IR-S. Performed the experiments: IR-S. Analyzed the data: DW IR-S. Wrote the paper: IR-S DW.
| 2023-09-03T01:26:19.665474 | https://example.com/article/1209 |
Q:
Converting Ant module to Maven, what is the best practices?
Many tools and ways are used to convert from ant to maven but what was the best for this migration ?
Any help is appreciated.
A:
Though, these may not be enough, but I've compiled some common practices while transferring module from ant to maven.
Delete your build.xml completely. You'd have to rewrite it again because maven doesn't use it at all.
Introduce a new pom.xml.
Define main class in <mainClass>src.com.test.Main</mainClass> using maven-compiler-plugin. See this answer.
For .jar that you're using in your build.xml search their dependencies online and add then in pom.xml. You can also add local dependencies in your pom.xml.
Transfer your images, text, audio, video, docs, pdf, xml to resources folder. (see also: What is the purpose for the resource folder?)
For more best practices see this answer also.
Moving onto Maven Anyway...
| 2023-12-30T01:26:19.665474 | https://example.com/article/8937 |
Minimum payment due still applies to 0% offer balances. Any remaining balance due after the 4-month promotional period or any baskets under £99 will be charged interest at 19.9% p.a. (variable). In order to maintain the 0% offer, you need to keep up monthly repayments and stay within your credit limit. Credit subject to status. Terms and conditions apply. UK residents only. PayPal Credit is a trading name of PayPal (Europe) S.à.r.l. et Cie, S.C.A., 22-24 Boulevard Royal L-2449, Luxembourg
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(Posted on 20/02/2019)
Product as expected
Great friendly customer service pre purchase. Place order on a Friday afternoon and delivered Saturday despite confirmation saying delivery Monday. Product as expected and seems decent quality although time will tell.
Delivery
Quality
Price
Review by J Williams
(Posted on 01/02/2019)
SGS Compressors and Tools
Have purchased two compressors and various tools from SGS. Compressors used for stapling, drilling, sanding and sandblasting - also blow/dusting other tools. Quality is very very good for the price. Advice given online on what to purchase is very sound - you need to read it and take heed. Compressors used for stapling, drilling, sanding and sandblasting - also blow/dusting other tools. Very prompt delivery. Only one minor error made in the order of some compressor oil. Dealt with very efficiently and without any fuss.
Price
Delivery
Quality
Review by Dave
(Posted on 25/10/2018)
Great
Brilliant kit for the money. Delivered on time
Quality
Price
Delivery
Review by MR.Alwyn Berry
(Posted on 10/07/2018)
24L AIR COMPRESSOR
I have just used my compressor for the first time and I must say for the price it was very good nd did the job I had bought it for, I would highly recomend this product to anyone wishing to buy a compressor from SGS .
The SGS 24 litre high flow air compressor is ideal for DIY and trade applications thanks to it's impressive 24L capacity and 8 bar maximum working pressure. It offers a fantastic combination of performance, safety and value for money. Also included are are all the hoses, accessories and fittings needed to start using the compressor with the air tool of your choice. The powerful engine ensures a short wait for the tank to pressurise - keeping you working. Fitted with safety valve and pressure regulator with gauges for controlled air pressure.
The powerful 2.5 HP motor is capable of delivering up to 9.6 CFM.
The 24 litre tank is ideal for nailing, stapling, sanding, drilling and cutting.
Boasting twin outlets, this compressor can have two tools connected to it at once.
SGS are a British company; our air compressors are hand built by our expert engineers to the highest standard of quality and are European CE marked and German GS certified for product safety. Parts, spares and consumables such as oil and air filters are available from SGS. The compressor is shipped with oil but may require additional oil before first use.
Please note this compressor is for semi-pro and not commercial use.We would not recommend this compressor for use with most impact wrenches due to the tank size. We would recommend a 50L or greater tank size for use with impact wrenches.
Reviews
I bought from them and fast delivery only thing I could say that they could improve upon is the instructions for the air compressor other than that would happily buy again
Price
Delivery
Quality
Review by Snoweyjoey
(Posted on 20/02/2019)
Product as expected
Great friendly customer service pre purchase. Place order on a Friday afternoon and delivered Saturday despite confirmation saying delivery Monday. Product as expected and seems decent quality although time will tell.
Delivery
Quality
Price
Review by J Williams
(Posted on 01/02/2019)
SGS Compressors and Tools
Have purchased two compressors and various tools from SGS. Compressors used for stapling, drilling, sanding and sandblasting - also blow/dusting other tools. Quality is very very good for the price. Advice given online on what to purchase is very sound - you need to read it and take heed. Compressors used for stapling, drilling, sanding and sandblasting - also blow/dusting other tools. Very prompt delivery. Only one minor error made in the order of some compressor oil. Dealt with very efficiently and without any fuss.
Price
Delivery
Quality
Review by Dave
(Posted on 25/10/2018)
Great
Brilliant kit for the money. Delivered on time
Quality
Price
Delivery
Review by MR.Alwyn Berry
(Posted on 10/07/2018)
24L AIR COMPRESSOR
I have just used my compressor for the first time and I must say for the price it was very good nd did the job I had bought it for, I would highly recomend this product to anyone wishing to buy a compressor from SGS .
Product Questions
All our air compressors use specialist ISO VG 68 oil for effective and reliable lubrication. Our part code is SO2068 and you can purchase the oil from here.
Help Centre
Our semi pro air compressors are great for a wide range of home based work or DIY tasks, such as: drilling, hammering, sanding, grinding, cutting, nailing, stapling, wrenching and more. We've put together a few compressor how-to guides and some safety information to get you started. These will tell you everything you need to know about our electric air compressors. For the best portable and small air compressors UK customers can find, SGS Engineering is the place to go. | 2024-02-01T01:26:19.665474 | https://example.com/article/7357 |
Aging in the Right Place Research Study
NSF-funded researchers at the University of Minnesota investigate how neighborhood environments affect the health and wellbeing of community-dwelling older residents across the Minneapolis metropolitan area.
Project Supervisor: Jessica Finlay, PhD Candidate
Nicollet Mall, Minneapolis, MN
The majority of older Americans live in single-family home communities designed for the able and mobile. These neighborhoods are increasingly unsuitable for aging residents, but a growing number either choose not to or cannot afford to move to the limited supply of accessible supportive housing.
Older people overwhelmingly prefer to "age in place" – to stay in their homes or apartments for as long as possible, rather than moving to new settings. But this is increasingly a challenge given unsupportive infrastructure.
The NSF-funded research project bridges geography and gerontology to focus in-depth on local experiences of later life. It critically considers how neighborhood environments across the Twin Cities area affect the physical, mental, and social welfare of older residents.
3-13-17 John Hines Show 11AMAudio: 32:42Jessica Finlay, an environmental gerontologist at the University of Minnesota talks about her research into making Cities more accessable for the eldery and John takes calls on what people think should happen to Surdyks for opening on Sunday's four months early.
What helps Minnesota seniors age in place? U researcher has some clues
February 24, 2017By Claude Peck, Star Tribune
Jan and Art Larson on the balcony of their downtown Minneapolis condo.
University of Minnesota doctoral candidate Jessica Finlay focuses on how senior citizens interact with their homes and neighborhoods.
"Aging in (the Right?) Place" Research Study Overview
"Aging in (the Right?) Place" Research Study Areas
Case Study Areas
(1) North Minneapolis(2) Downtown Minneapolis(3) Eden Prairie
Phase I:
Interviews and neighbourhood tours with community-dwelling older residents living in North Minneapolis, Downtown Minneapolis, and Eden Prairie.
Interview: Researchers will interview participants in their home or a nearby public place of their choosing. The interview will last approximately 60 minutes, and be audio recorded. Researchers will ask about daily routines, social and community engagements, and experiences in and opinions of the neighborhood.
"Tour" of the Neighbourhood: After the interview concludes, participants will be asked to take researchers for a quick "tour" of the home and neighborhood. This mobile interview will last approximately 15 minutes, though the length of time, route, speed, and mode of transit are completely up to participants. Most often the "tour" consists of a walk around the block where we can chat informally and see the neighborhood together.
Phase II:
Follow-up sessions with select interview participants.
Researchers will spend more time with randomly selected participants to learn more about their daily routines, interactions, and everyday life.
Phase II participants will spend 5 to 10 mornings and/or afternoons with a researcher over an 9 to 12-month period (ideally 3-5 hours once a month, 15-30 hours total). A researcher will follow along a participants' daily routine in ordinary settings of their choosing (e.g. around the home/neighborhood, at the grocery store), and informally chat with participants about your everyday life.
Phase III:
Interviews with municipal staff, community organizations, and local representatives regarding aging demographics in the Twin Cities
Researchers will interview staff members of municipal departments, service providers, and community representatives.
Jessica will offer to present and share findings with staff. Departments/organizations/leaders can benefit from the study by learning new strategies and methods to improve the health and wellbeing of diverse constituents, particularly aging citizens. | 2023-11-02T01:26:19.665474 | https://example.com/article/7684 |
replicaCount: 1
clusterName: eshop-aks
pathBase: /mobileshoppingapigw
image:
repository: envoyproxy/envoy
tag: v1.11.1
service:
type: ClusterIP
port: 80
adminPort: 8001
ingress:
enabled: true
annotations:
nginx.ingress.kubernetes.io/rewrite-target: "/"
ingress.kubernetes.io/rewrite-target: "/"
tls: []
resources: {}
nodeSelector: {}
tolerations: []
affinity: {}
env: {}
envoy:
configPath: /etc/envoy
probes:
liveness:
path: /ready
initialDelaySeconds: 5
periodSeconds: 15
port: 8001
readiness:
path: /ready
initialDelaySeconds: 5
periodSeconds: 60
port: 8001 | 2024-01-29T01:26:19.665474 | https://example.com/article/1938 |
the partitions at my company end about two feet from the ceiling, so when things in my cubicle are quiet i can hear what's going on in the cubes around me. and sometimes, even when things aren't so quiet, i can hear mike. (his real name, and oh i hope someday he finds this and reads it.) mike is loud. mike is from los angeles. mike studied film at ucla, which (he has loudly claimed from his cell next to mine) has one of the best film schools in the nation. i haven't bothered to check this out; i just tacitly accept that he's lying, the loud, self-aggrandizing bastard. mike has worked (as an intern, he admitted to one questioning student) at pixar, mike doesn't like roppongi, mike thinks that very soon the IT industry will give in to the broadcasting industry and media conglomerates will take over the internet.
it is not these facts and opinions that piss me off so much. (although the one about the internet is kind of annoying, especially since his reasoning for this belief is that people don't like to have choices, that people prefer to be told what they can do and when, and that people also like commercials... oh, and that the main interest of everyone in the IT industry is money, which, seriously, hasn't he ever heard of linux?) it isn't even the fact that he's so loud. it's that he speaks in the most fragmented, moronic english i have ever heard coming from a teacher's mouth.
"where... you go... in tokyo?" one of his students asked the other day. inwardly, i cringed, awaiting his response. i tried to focus on the book in my lap, but his voice was like the scent of rotten fish in a bakery. "i go ebisu. don't like... rop-pon-gi." oh god, i thought. oh god oh god oh god please someone turn the music up i don't care that it's def leppard just turn it up please jesus-- "most of time, though, make mov-ie. an-i-ma-tion? you like... an-i-ma-tion?"
mike talks non-stop to his students about this twelve-minute long animation that he made over the past three years. so far, from what i can tell, he's only suckered one of his students into watching it on his laptop, which he brings into school with him every day and uses to listen to music between lessons. i do not care very much about the music or the fact that he brings the laptop in. and i don't care about the fact that he talks all the time about his animated short. what i care about is that he omits nearly every particle-- articles, prepositions, conjunctions, and others (are there others?)-- when he is talking about it. he often uses the present tense to speak about the past or the future, and in many cases his statements or questions will consist of one word, the 'sentence' type denoted only by inflection. he even omits the 's' at the end of plural words and verb conjugations.
i didn't suspect him of such villainy at first. no, i thought he seemed like a decent guy (if a little bit of an egoist/misogynist-- one of the first sentences out of his mouth was "i've almost had enough of these japanese girls, it's time for me to go back to cali and sell my movie." i think i may have hurt him when i didn't ask him anything more about this 'movie'). after three days of working next to him, though, i began to harbor fantasies of unmasking him for the slacking verbalizer he is. i imagined standing on a chair, poking my head over the partition and saying "hey, mike, don't you mean 'most of the time?' 'most of time' is incorrect, and i really think that you should speak english properly if you're going to teach it. oh, and you're talking about computers, not about 'com-pu-ter.'" then i realized that if his students put up with his awful language they probably wouldn't understand my critique.
next i began to dream about getting him fired. i would call my boss and say "hey, how's it going, do you remember hiring mike? yeah, well, his english is absolutely terrible. no, not when he's talking to me, but when he's talking to his students he sounds like a slightly precocious two-year-old who learned the language using flash cards. yeah. yeah, that's right, haha, good one, like a parrot with a speak-n-spell." then i thought that maybe the students like this method. it is entirely possible that for some low-level students, the exaggerated enunciation and omission of important parts of speech makes for easier learning, or at least makes for the illusion that they are understanding and speaking proper english. i would not want to deprive my boss of the income he makes off of these people. (and i wouldn't want to risk getting fired for complaining about it.)
so today i finally made my move. we finished our last lessons at the same time, and i held the elevator open until he was done hitting on our (engaged) receptionist. "thanks," he said as he slid in. he opened his mouth again to speak but i cut him off.
"what you do... on week-end?" i spoke a little louder than i normally do, partly because i was nervous and partly because we were in a tiny japanese elevator and everything just sounds louder in there.
he looked at me a little funny and mumbled something about working on his movie.
"nice!" i said. "i go to... ha-ra-ju-ku. you know ha-ra-ju-ku? people there... wear costume. dress funny." the doors opened on the ground floor and we walked out. he was looking at me strangely again, as though he knew i was getting at something but he couldn't tell what. "i go now, use com-pu-ter. send e-mail. see you to-mor-row!"
i walked away as fast as i could. at the corner i looked back and he was walking slowly down the sidewalk, shaking his head and staring at the ground.
Here I am, writing my first node in I-don't-know-how-long. I could easily look it up, but I'm too lazy for that sort of thing. Facts are for liberals anyway. HateQuest 2006 inspired me. I have no idea what I am going to write, but I don't want to stop typing until I bang out a few paragraphs.
I'm what is known in the local dialect as a fled noder. I used to be fairly active. I used to write a lot. I mean, look at one of my highest rated nodes - Union of Soviet Socialist Republics - and see that I used to spend some time working on serious nodecraft. But then "it" just left me. What was "it"? "It" was a combination of interest, industry, and eagerness to connect with people out there in the nodegel. One day it was just gone. I figured E2 wouldn't miss me. It will take care of itself.
Then came all sorts of management decisions I didn't personally agree with. And then I noticed a lot of old timers had left too. A day would pass and the New Writeups nodelet would hardly move.
Now I use Wikipedia and E2 with almost equal frequency to look up shit. I use e2 for a more subjective slant. For instance if I wanted to look up XYZ widget for my computer, I would look it up here expecting not just a dry description but also some valuable input like "nice piece of gear but a bitch to install and maintain." Likewise, with new music or movies or books, I will turn here to see what people think because I find I have similar tastes to a lot of noders.
Of course, one could attribute my "fledness" to sour grapes. During my active periods, I semi-actively campaigned to become a content editor. I'm certain that the Management's decision not to so empower me had to do with my low writing frequency and my minimal social interaction on the catbox and/or #everything. Or it could have been that everybody thought my writing sucks, which I can accept. I wanted to be more than just another writer. At one time I really wanted to participate and contribute. For a while I maintained some supernodes, like some of the recipies nodes. But, like everything else I mentioned before, "it" just sort of went away.
Now I don't feel any compulsion to participate in the E2 community in any way. Sometimes I throw a non sequitir into the catbox to get a rise out of people or just to be stupid.
See? Already I have lost interest, so I am going to stop writing now and answer my email.
Hatequest 2006 eh? Fled Noders eh? I have a writeup in violence? Since when? Ok, I feel as if that's enough linking. Back in the day someone started messaging me and telling me all my daylog write ups would be erased unless I used more hardlinks in them, so that nodes that didn't have many softlinks could get some meaningless day attatched to them. Oh noes!
One day I thought about going back to e2 and doing random stuff, but when I contacted most people I knew who were doing it back in "yon day" they basically told me they had little to nothing to do with the whole thing anymore, quote unquote, "Eventually you get tired of a literary circle jerk."
That settled things.
Imagine you have an editor, and imagine that your editor is a gigantic pain in your butt, not because his recommendations are entirely inaccurate, but because everything you write is technically his and not yours and he has the ultimate final say on any creative decisions you might make. Now imagine that everything2 is a place where everyone acts like they are or has powerks akin to an editor. Well, there you have the root of more or less every conflict on e2 ever.
Wikipedia has the exact same problems, but it's superior in that there is a percievable direction in which the site is going. When contributing here you run the risk of having material destroyed for really no reason whatsoever save wimsy. That's all fine and good, and certainly one way to do things, but I find myself more interested in my own desires rather than the desires of others.
Of course things may have changed in the interim,
Or maybe not. I'm not saying this just to be a dick, but word spreads through such channels I do visit and it's like, hm, wow, it's exactly the same as it was however many years ago when I was an active contributor. The difference between then and now is of course experience and wisdom. Also i've gotten laid a lot more since then, so i'm considerably calmer.
First of all, nothing good can come of it unless you consider getting personal and offensive a good thing. There's enough huge egos around this place (at times, mine included) to go around and all I can see is people taking offense and being the subject of drive by downvoting and innuendo just because someone else has got it in for them. That's not what I would call an adult way of going about our business.
Look man, in the GOOD OLD DAYS we were more sly than to downvote people we didn't like. Instead we upvoted and cooled the enemies of our enemies, and oh man, it got to people. Actually that wasn't the intention at the time, but it had a tendency to be percieved as such.
Often times here at E2 we find ourselves bemoaning the fact that we're not attracting new talent and a lot of the old talent is falling by the wayside in ever increasing numbers. Oh, maybe they hang around but you hardly ever see their names on the new write-ups list. That's all well and good and we all come here for our own reasons but when I see only 50 or so users on the user list and half of them have been here only a month or two, something is wrong. If anybody thinks that's the way the way to attract new people then in my ever so humble opinion, they're dead wrong. All it does is encourage some of our newer folks to spew out whatever venom it is they have on their mind. My advice is if you want to do that, go get yourself a blog or possibly a good shrink.
Well, maybe the system is broken. In fact, i'm pretty sure that's exactly whats going on here. Instead of letting everything take care of itself it relies on a core group of editors to decide whats in and whats out. Upvotes, downvotes, and cools are at best a weak guideline. With such a high degree of intervention in writing it's hard to feel as if your work is your own or simply the product of someone elses guidelines. (and no, I don't really care whether or not it's legally someone's work or not.)
I grew up with a skewered view of the world based upon a household that at times seemed to be filled with hate. It left me bitter and uncaring for more years than I care to count. I've worked for the last twenty or so to rid myself of that and I'm still working hard on it every day. It's not the lesson I care to impart on those that are much more important to me than I am.
As a content editor, I'm supposed to be unbiased and judge each w/u on its own merits. In good conscience, I can't say I can do that with anything that would be contributed to this cause. Just the though of it leaves a bitter taste in my mouth and I don't want to play loose with the truth.
It's not that I don't sympathize with you, because I do, but that statement also describes the precise reason why I feel as if any time I spend on e2 would be better spent elsewhere. Unless something is unintelligable, pornographic, copyrighted, plagarized, or otherwise unfit by some very clear guideline, well, I just can't see removing it and still saying that e2 contributes something unique or even particularly interesting to the intranets.
As soon as you do, e2 is just another version of The escapist, wired, or as was derisively pointed out in the past, Slashdot, and it is this way because of the vision and biases of editors contrbituing trimmings of w/u's "on their own merit."
I think I like the general outline of e2 as a database, and I can see things that I would like to do with it, but at the same time I honestly have no interest in the current implimentation and direction of e2, and thats fine, but maybe you ought to start asking yourself exactly who and what the audience is and whether or not this is really the way you'd like to use e2.
I believe I would start by pointing out that my statement is directed at those who have the power of life and death over nodes. However, who are those people, why do they have that power, and does what sort of content does that implimentation generate? | 2024-05-22T01:26:19.665474 | https://example.com/article/9820 |
If I asked you to talk about the states of consciousness, how would you answer? Would you say that being awake and asleep are states of consciousness or would you have a more spiritual answer such as astral travel? Is déjà vu a form of consciousness and what about meditation? Well, you can make a [...]
When the 3rd eye awakening happens, there are some remarkable effects on your thinking and feeling. The third eye (also known as the mind's eye situated between your eyebrows) is the sixth chakra with powerful abilities. Usually, this area or chakra becomes active when the person has reached a certain level of awareness and consciousness. [...]
Being aligned with your intuition can reap benefits and guide you through life’s challenges. The ability to follow our intuition – to understand something instinctively without any reasoning – is something we are all naturally equipped with. Einstein himself defined it as ‘the only real valuable thing’. It can guide us in ways that can [...]
Have you ever experienced the higher levels of consciousness and do you know how to maintain the level you have already reached? At the end of the day, life is not about giving or taking, it is about evolving in harmony with the energy-informational field and sharing your knowledge with others. It seems that today [...]
In Part 1 of this series, I explained some of my beliefs pertaining to the way that our world works and what I understand to be our physical perception of reality, as well as the association of this perception with our conscious minds and the convergence of this theory on how people may vary in [...]
If you want to learn to perceive energy, the first step is to get to know the concepts of the astral realm and ESP (extrasensory perception). Some of my earlier articles were pertaining to the ability to see people’s auras or energy that objects and lifeforms give off, how I realized that not everybody could, [...]
According to a new study, anxious people have a huge advantage over others as their brains have a different reaction to danger, which, in fact, could be life-saving. An anxious personality can be viewed as a negative characteristic. I know that’s what I’ve always encountered, being diagnosed, in part, with an anxious disposition. With that [...]
We have a sixth sense. All of us. And it is not the ability we saw in the famous movie with the little guy telling Bruce Willis “I see dead people”. It is the ability to understand and work with numbers. Numerosity is our sixth sense and there is a specific brain region that ‘hosts’ [...] | 2023-12-02T01:26:19.665474 | https://example.com/article/6057 |
“Science is a way of talking about the universe in words that bind it to a common reality.
Magic is a method of talking to the universe in words that it cannot ignore.
The two are rarely compatible.”
― Neil Gaiman, The Books of Magic
“Science is a way of talking about the universe in words that bind it to a common reality.
Magic is a method of talking to the universe in words that it cannot ignore.
The two are rarely compatible.”
― Neil Gaiman, The Books of Magic
Hey all, i got into Magick and the occult a year ago and have found it very interesting. I created a video exploring chaos magick as taught through the Gallery of Magick and had some interesting experiences.
Anyhow this is the video, i look forward to getting to know you and this community
“Science is a way of talking about the universe in words that bind it to a common reality.
Magic is a method of talking to the universe in words that it cannot ignore.
The two are rarely compatible.”
― Neil Gaiman, The Books of Magic
I am a long time Dialectic monist (Zen/Chan oriented mystic) and a returning student of the Occult (I began in the 90s studying whatever I could find at the local bookstores, I never heard of Chaos Magick, but apparently I was doing a great deal of it intuitively. I was also dealing with a chaos form of Evokation, Invokation, and far left hand Demonology.).
I have recently felt the Call to return to the dark arts, and armed with my more mature Monist understanding of reality, I am now much better equipped to handle the mind games of the left-hand path and the "Entities" encountered within.
I have recently felt a calling most strongly to Lovecraftian themed 23 current occult threads, and am about to embark on using S. Ben. Qayins work with the Black Book of Azathoth. We'll see soon if I follow through, and what kind of results I get from it.
I have started a blog which is kind of a mix of creative writing autobiographical and a place to vomit forth ideas. I have also recently rebranded a Youtube Channel that is going to focus mostly on the occult, mysticism and the unexplained. I plan on documenting my experiences with the Black Book of Azathoth, and what results I get on the blog and on the youtube channel.
Hoping to get some feedback and share my content with interested people.
**********************************************************
Come on over and read my blog, it's got Demons, Great old ones, Zen Mysticism, Chaos Magick, and even a few ninjas.
Sounds like some interesting things you got going on there, I look forward to hearing more about them
“Science is a way of talking about the universe in words that bind it to a common reality.
Magic is a method of talking to the universe in words that it cannot ignore.
The two are rarely compatible.”
― Neil Gaiman, The Books of Magic | 2024-02-07T01:26:19.665474 | https://example.com/article/3062 |
I like to figure out how many hours I have left to live if I die at a reasonable age like... say 80. If I die when I'm 80, that means that I only have about 525,600 hours left to live. Take a third of that away for sleeping and its even less. Only 350,800 hours of conscious life. That's what I can reasonably expect if I can live to 80. | 2023-12-25T01:26:19.665474 | https://example.com/article/3331 |
Name Heavy Assault
ShortName kskAssault2
AddItem m60
AddItem usp
AddItem m67 1
AddItem mwKnife
AddItem m60Ammo 2
AddItem uspAmmo 3
Hat juggernautHelmet
Chest juggernautBody
Legs juggernautPants
Shoes juggernautBoots | 2023-11-27T01:26:19.665474 | https://example.com/article/3049 |
The present invention relates to forming semiconductor devices. More specifically, the present invention relates to profile and CD uniformity control of spacers in the formation of semiconductor devices.
During semiconductor wafer processing, spacers, such as nitride spacers, may be used for etch or implantation masks. | 2023-10-12T01:26:19.665474 | https://example.com/article/7970 |
I had a unique pleasure of working together with David Evans on helping a group of testers understand agile software development last week. While talking about the value of quick feedback and short iterations, David used an analogy which I haven’t heard before and I liked it very much, so I decided to share it on this blog. He started the story by going back in history to primitive societies and comparing hunters and gatherers.
Gatherers go around with a basket, looking at what’s available and picking up fruit and veg. When they encounter something new and unknown, they can take a bite and see if it tastes good, then put it in the basked it they like it or throw it away. They pick up small things, one thing at a time, so if something is rotten or not edible, they quickly move on and find something that is fine.
Hunters chase a wild beast. This is a bold and dangerous task so they have to carefully pick a relatively easy target and go after it. Setting up a trap or chasing the animal takes a lot longer than gathering fruit and if the one they targeted gets away they need to do it all over again. If for some reason they can’t eat the animal, the entire effort is wasted.
What’s really important in the story is what happens at the end of the day if things don’t go according to plan:
It’s OK if you don’t quite fill the the basket. It’s not OK if you don’t quite catch the animal.
Short iterations and quick feedback make it much less risky to run software projects. They allow us to do a piece of functionality at a time and see how we can handle that. At the end of the project phase, if not everything is quite there but we have most of it, especially the high priority stuff, everything is fine. Without quick feedback and short iterations, we’re basically hunting the mammoth. Everything depends on that final hit and there is very little room for mistakes.
I think that there is probably room for one more idea in this metaphor, where having the safety net of a half-full basket allows us to go and hunt a large animal every once in a while and put some meat on the table as well. | 2024-04-19T01:26:19.665474 | https://example.com/article/7524 |
Q:
Pre-compiled Closure Templates - "Variable referenced before declaration" Warning in Closure Compiler
java -jar SoyToJsSrcCompiler.jar --shouldGenerateJsdoc --outputPathFormat
simple.js --srcs simple.soy
SoyToJsSrcCompiler generates a js file which looks like this:
if (typeof templates == 'undefined') { var templates = {}; }
if (typeof templates.simple == 'undefined') { templates.simple = {}; }
/**
* @param {Object.<string, *>=} opt_data
* @param {(null|undefined)=} opt_ignored
* @return {string}
* @notypecheck
*/
templates.simple.tinyButton = function(opt_data, opt_ignored) {
.....
};
I am using Closure Compiler with --warning_level=VERBOSE and --compilation_level ADVANCED_OPTIMIZATIONS
and I am getting this warning:
simple.js:1: WARNING - Variable referenced before declaration: templates
if (typeof templates == 'undefined') { var templates = {}; }
How can I clear this warning?
A:
One workaround is to declare the variables in an externs file with:
/** @suppress {duplicate} */
var template;
But the Soy compiler should be fixed. I expect people don't see this because you typically use it with Closure Library and in that mode the Soy compiler should be generating:
goog.provide('template.simple')
| 2023-11-14T01:26:19.665474 | https://example.com/article/7299 |
1. Introduction {#sec1-nutrients-09-00831}
===============
The role of vitamin C in health and disease has been actively studied since its discovery over 80 years ago \[[@B1-nutrients-09-00831]\]. Vitamin C has a number of well-recognized biological functions, all of which depend upon its ability to act as an electron donor \[[@B2-nutrients-09-00831]\]. One of the most significant of these is its cofactor activity for a variety of enzymes with critical functions throughout the body. These include the copper-containing monoxygenases dopamine hydroxylase and peptidyl-glycine α-amidating monooxygenase \[[@B3-nutrients-09-00831]\] and the Fe (II) and 2-oxoglutarate-dependent family of dioxygenases \[[@B4-nutrients-09-00831]\]. The latter is a large and varied family, with a continually expanding membership that includes the collagen prolyl hydroxylases responsible for stabilization of the tertiary structure of collagen, the prolyl and asparaginyl hydroxylases which regulate hypoxia-inducible factors (HIF) activity, and DNA and histone demethylases involved in the epigenetic regulation of gene expression. Vitamin C also functions as a highly effective water-soluble antioxidant, protecting in vivo biomolecules from oxidation \[[@B5-nutrients-09-00831]\], and there is good evidence to suggest it is involved in the regeneration of vitamin E in vivo \[[@B6-nutrients-09-00831],[@B7-nutrients-09-00831]\].
Because humans are unable to synthesize their own vitamin C, it must be obtained from the diet, principally through fruit and vegetable consumption. Inadequate dietary intake results in the potentially fatal deficiency disease, scurvy. As little as 10 mg/day vitamin C is sufficient to prevent overt scurvy \[[@B8-nutrients-09-00831]\] and, although scurvy is considered to be relatively rare in Western populations, vitamin C deficiency is the fourth most prevalent nutrient deficiency reported in the United States \[[@B9-nutrients-09-00831],[@B10-nutrients-09-00831]\]. Hypovitaminosis C (defined as a plasma concentration ≤23 µmol/L) affects a significant proportion of the population, with estimates as large as 15--20% in the United States \[[@B9-nutrients-09-00831]\]. Similar data for the New Zealand population are lacking, although dietary vitamin C intake has been used to estimate the prevalence of inadequate intake, defined as not meeting the estimated average requirement (EAR) \[[@B11-nutrients-09-00831]\].
The classical symptoms of scurvy, such as joint pain, lassitude, bleeding and ulceration are thought to be due to the loss in activity of the vitamin C-cofactor enzymes, particularly the collagen hydroxylases. It is becoming increasingly acknowledged, however, that vitamin C is required at concentrations above those needed for the prevention of scurvy for the maintenance of good health \[[@B12-nutrients-09-00831],[@B13-nutrients-09-00831]\]. For example, individuals with hypovitaminosis C are known to present with fatigue, depression and deficiencies in wound healing \[[@B14-nutrients-09-00831],[@B15-nutrients-09-00831]\], suggesting a requirement for vitamin C status to be above 23 µmol/L in plasma to support these functions. There is also epidemiological evidence to support a role for vitamin C in the prevention of some chronic disease, with intakes \>100 mg/day recommended \[[@B12-nutrients-09-00831]\]; these intakes will provide adequate plasma levels (i.e., \>50 µmol/L) \[[@B14-nutrients-09-00831],[@B16-nutrients-09-00831]\]. Although the Australasian Recommended Dietary Intake (RDI) for vitamin C is only 45 mg/day, the New Zealand Ministry of Health, in accord with other international bodies, has a suggested dietary target of \~200 mg/day vitamin C for the reduction of chronic disease risk \[[@B17-nutrients-09-00831]\]. As the many cofactor functions of vitamin C become more widely understood, epidemiological studies in areas in which its biological activity can be justified are required.
The CHALICE (Canterbury Health, Ageing and Lifecourse) study is a unique New Zealand study comprising a comprehensive database of determinants of health. It has prospectively recruited \~400 fifty-year-olds at random from the electoral roll within the Canterbury region. Participants have undergone extensive health, dietary and social assessments \[[@B18-nutrients-09-00831]\]. Here we report on the plasma vitamin C status and dietary vitamin C intake of the participants, and examine the relationships between these measures and a range of health indicators.
2. Materials and Methods {#sec2-nutrients-09-00831}
========================
2.1. Study Population {#sec2dot1-nutrients-09-00831}
---------------------
Participants were from a random sample drawn from the New Zealand electoral roll, recruited to take part in a prospective longitudinal study of health and wellbeing (2010--2013), called the Canterbury Health, Ageing and Lifecourse (CHALICE) study (detailed in \[[@B18-nutrients-09-00831]\]). Participants had to be aged 49--51 years, intend to reside within the greater Christchurch area for at least 6 of the next 12 months, live in the community (i.e., not in a prison or a rest home) and be able to complete the assessment (e.g., speak English proficiently). Māori, the indigenous people of New Zealand, were over-sampled so that they represented 15% of the CHALICE study sample. Enrolment statistics estimate that, in 2012, 94.9% of the target population were registered to vote in the Christchurch City Council area \[[@B19-nutrients-09-00831]\]. Relative to the rest of New Zealand, the Canterbury area has a slightly higher proportion of people aged ≥40 years and a higher proportion of people living in the least economically deprived national quintile \[[@B20-nutrients-09-00831]\]. Ethical approval was obtained from the Upper South A Regional Ethics Committee (URA/10/03/021) and all participants provided written informed consent.
Data were collected during a 4--6 h interview, via self-completed questionnaires and lifestyle diaries, and from blood and urine tests. The full cohort was 404 participants, and the present analysis is based on the 369 participants for whom fasting plasma vitamin C measurements were obtained and a sample of 250 for whom dietary vitamin C intake was determined.
2.2. Blood Sample Collection {#sec2dot2-nutrients-09-00831}
----------------------------
Fasting blood samples were collected into EDTA anticoagulant tubes and sent to Canterbury Health Laboratories, an International Accreditation New Zealand (IANZ) laboratory, for analysis of biomarkers. Additional fasting samples were centrifuged at 4000 rpm for 10 min at 4 °C to separate plasma, and the plasma stored at −80 °C for vitamin C analysis.
2.3. Sample Preparation for Vitamin C Analysis {#sec2dot3-nutrients-09-00831}
----------------------------------------------
Stored EDTA-plasma was rapidly thawed and a 500 µL aliquot was treated with an equal volume of ice-cold 0.54 M HPLC-grade perchloric acid solution (containing 100 µmol/L of the metal chelator DTPA) to precipitate protein and stabilize the vitamin C. Samples were mixed, incubated on ice for a few minutes, then centrifuged. A 100 µL aliquot of the deproteinated supernatant was treated with 10 µL of the reducing agent TCEP (100 mg/mL stock) for 2 h at 4 °C to recover any oxidized vitamin C \[[@B21-nutrients-09-00831]\]. Samples were further diluted with an equal volume of ice-cold 77 mM perchloric acid/DTPA solution for HPLC analysis.
2.4. Vitamin C HPLC Analysis {#sec2dot4-nutrients-09-00831}
----------------------------
The total vitamin C content (ascorbic acid plus dehydroascorbic acid) of the samples was determined by HPLC with electrochemical detection as described previously \[[@B22-nutrients-09-00831]\]. Samples (20 µL) were separated on a Synergi 4 µ Hydro-RP 80A column 150 mm × 4.6 mm (Phenomenex NZ Ltd, Auckland, New Zealand) using a Dionex Ultimate 3000 HPLC unit (with autosampler chilled to 4 °C and column temperature set at 30 °C) and an ESA coulochem II detector (+200 mV electrode potential and 20 µA sensitivity). The mobile phase comprised 80 mM sodium acetate buffer, pH 4.8, containing DTPA (0.54 mmol/L) and freshly added ion pair reagent n-octylamine (1 µmol/L), delivered at a flow rate of 1.2 mL/min. A standard curve of sodium-L-ascorbate, standardized spectrophotometrically at 245 nm (ε = 9860), was freshly prepared for each HPLC run in 77 mmol/L HPLC-grade perchloric acid containing DTPA (100 µmol/L). Plasma vitamin C content is expressed as µmol/L.
Fasting plasma vitamin C concentrations were classified as follows; deficient \<11 μmol/L, marginal 11--23 μmol/L, inadequate 23--50 μmol/L or adequate \>50 μmol/L \[[@B13-nutrients-09-00831],[@B15-nutrients-09-00831]\].
2.5. Metabolic and Heart Health Assessments {#sec2dot5-nutrients-09-00831}
-------------------------------------------
Metabolic health was assessed by body measurements and fasting blood tests. Participants' height, weight and waist circumference were taken by the study interviewer, and body mass index (BMI) calculated (kg/m^2^). Fasting blood tests comprised triglycerides, high-density lipoprotein (HDL), glucose, HbA1c and insulin (Canterbury Health Laboratories).
Heart health was assessed by blood pressure and participants had their NZ cardiovascular risk score calculated. Blood pressure measurements were taken while seated. Five year cardiovascular risk (%) was derived according to the New Zealand adaptation of the Framingham risk score; the following variables are included in the calculation: age, gender, systolic blood pressure, diabetic status, smoking history, and total cholesterol to HDL ratio \[[@B23-nutrients-09-00831]\].
2.6. Dietary Intake Assessment {#sec2dot6-nutrients-09-00831}
------------------------------
Participants were asked to complete the Four Day Estimated Food Diary (4DEFD) in the week after their interview; on one weekend day and three weekdays. The 4DEFD included detailed instructions on how to record portion sizes, using common household measures. The completed 4DEFD were checked by a trained nutritionist and additional information obtained from participants where necessary before the data were entered into the nutrient analysis program Kai-culator (version 1.08d, Department of Human Nutrition, University of Otago, Dunedin, New Zealand). Dietary analysis was performed on 250 of the CHALICE participants, who had dietary data entered and cleaned at the time of analysis, for whom the mean daily intake of vitamin C was calculated. Data entry was undertaken by experienced nutritionists and all diaries were further checked for accuracy by one person who also made any necessary changes, to ensure consistency of data entry.
2.7. Wellbeing, Depression and Cognition {#sec2dot7-nutrients-09-00831}
----------------------------------------
### 2.7.1. Mental Wellbeing {#sec2dot7dot1-nutrients-09-00831}
The Warwick--Edinburgh Mental Wellbeing Scale (WEMWBS) was used to assess general wellbeing. The 14 item questionnaire aims to measure positive mental health by assessing both aspects of well-being: eudaimonic and hedonic \[[@B24-nutrients-09-00831]\].
### 2.7.2. Depression {#sec2dot7dot2-nutrients-09-00831}
During the assessment, trained interviewers used the Mini-International Neuropsychiatric Interview (MINI) for diagnosis of current and past depressive episodes using DSM IV criteria \[[@B25-nutrients-09-00831]\].
### 2.7.3. Cognition {#sec2dot7dot3-nutrients-09-00831}
Participants completed the Montreal Cognitive Assessment (MoCA) version 7.1 (original version) \[[@B26-nutrients-09-00831]\], a short screening test for mild cognitive impairment. It assesses the cognitive domains of attention and concentration, executive functions, memory, language, visuoconstructional skills, conceptual thinking, calculations, and orientation. A score of 26 or more indicates normal functioning, while a score less than 26 might indicate mild cognitive impairment. MoCA scores were excluded from the analysis if English was the second language or if a previous event (e.g., carbon monoxide poisoning) had affected cognitive ability.
2.8. Socio-Economic Status {#sec2dot8-nutrients-09-00831}
--------------------------
The Economic Living Standard Index Short Form (ELSI~SF~) was used to assess standard of living \[[@B27-nutrients-09-00831]\]. Developed in New Zealand, the ELSI~SF~ assesses a person's consumption and personal possessions, calculating a total score by combining information from all items of the survey. The ELSI~SF~ scores range from 0--31, with those who score 0--16 described as being in hardship, scores of 17--24 as comfortable and scores of 25 or above as socio-economically good or very good. The ELSI~SF~ has excellent internal consistency (coefficient alpha of 0.88).
2.9. Statistical Analyses {#sec2dot9-nutrients-09-00831}
-------------------------
Statistical analyses were performed using R 3.3.1 software (R Foundation for Statistical Computing, Vienna, Austria). Univariate tests on continuous variables were t-tests with Satterthwhaite's adjustment for unequal variances while Wald odds ratios and Fisher exact p-values were calculated for categorical variables. Sample characteristics were compared with census proportions using the chi squared goodness of fit test. All health measures were examined independently for association with vitamin C (plasma vitamin C concentration or dietary vitamin C intake) using linear or logistic regression models. The models fitted the dietary measure, gender (dichotomous), Māori ethnicity (dichotomous) and current smoking (dichotomous). Models were fitted on males and females separately and the whole cohort combined. Modeling assumptions were verified with no material departures observed. For each outcome, the *p* values were adjusted for multiple comparisons using the Benjamini and Yekutieli method. The nominal *p* value for statistical significance is the usual 0.05 or 5% type II error rate. All *p* \< 0.1 are shown in the tables with *p* \> 0.1 shown as NS (not significant). The odds of currently smoking for those in the lowest socio-economic strata was 3.8 times that of the highest strata (95% CI 1.7--9.0), *p* = 0.002. Similarly the odds of current smoking were 3.4 times higher in the least educated strata than the most educated (95% CI 1.75, 6.54), *p* = 0.0006. To prevent over fitting, socio-economic status and education were not fitted, however smoking acts as a reasonable proxy for population modeling.
3. Results {#sec3-nutrients-09-00831}
==========
3.1. Characteristics of the Study Population {#sec3dot1-nutrients-09-00831}
--------------------------------------------
Of the full CHALICE cohort (*N* = 404), 46.8% (189) were male, with 83.7% (338) self-identifying as New Zealand European and 14.9% (60) as Māori ([Table 1](#nutrients-09-00831-t001){ref-type="table"}). The majority of the participants were in the highest ELSI~SF~ category. There were 60 current smokers in the cohort.
[Table 1](#nutrients-09-00831-t001){ref-type="table"} compares the CHALICE participants to the New Zealand Census 2006 data for similar age and region. The CHALICE participants had higher rates of Māori ethnicity and higher qualifications than the Canterbury average ([Table 1](#nutrients-09-00831-t001){ref-type="table"}), whereas socio-economic status and smoking were within stochastic limits. This suggests the sample is reasonably representative of Canterbury 50-year-olds and hence the national cohort allowing for regional bias.
The CHALICE cohort also had typical levels of health for a community sample ([Table 2](#nutrients-09-00831-t002){ref-type="table"}). Anthropometric measures were close to those of the New Zealand population. Average metabolic and cardiac markers for the cohort were generally within the healthy range. However, the high prevalence of chronic conditions in the New Zealand population was also readily apparent.
3.2. Vitamin C Status of the Study Population {#sec3dot2-nutrients-09-00831}
---------------------------------------------
Fasting plasma vitamin C measurements were available for 369 of the CHALICE participants. The mean plasma vitamin C concentration was 44.2 μmol/L (95% CI 42.4, 46.0); 62% of the participants were below the adequate level (i.e., 50 μmol/L), and 93% of the participants were below the optimal saturating level (i.e., 70 μmol/L; [Figure 1](#nutrients-09-00831-f001){ref-type="fig"}). Ten percent of the cohort had marginal vitamin C concentrations (i.e., 11--23 µmol/L), and vitamin C deficiency, defined as a plasma concentration of \<11 µmol/L, was apparent in 2.4% of the cohort ([Table 3](#nutrients-09-00831-t003){ref-type="table"}).
Plasma vitamin C status was substantially lower in men than in women (*p* = 0.005), and it also varied by socio-economic status (*p* = 0.003). For example, 8% of those in the lowest socio-economic category were vitamin C deficient compared to 2.4% of the entire cohort (*n* = 369). Smoking status was also associated with plasma vitamin C status with current smokers having lower vitamin C levels (*p* \< 0.001; [Table 3](#nutrients-09-00831-t003){ref-type="table"}).
Study participants with and without vitamin C measurements do not differ significantly by gender, ethnicity, education, socio-economic status, smoking status, waist, weight or BMI (all *p* \> 0.13), hence are treated as missing at random.
3.3. Associations of Vitamin C Status with Markers of Metabolic and Mental Health {#sec3dot3-nutrients-09-00831}
---------------------------------------------------------------------------------
The results of the statistical modeling with plasma vitamin C are summarized in [Table 4](#nutrients-09-00831-t004){ref-type="table"}. Higher plasma vitamin C status was associated with lower weight, BMI and waist circumference in the CHALICE cohort, even after adjustment for gender, ethnicity and current smoking. Of the other markers of metabolic health, plasma vitamin C was negatively associated with blood triglycerides, HbA1c and insulin, and positively associated with HDL levels. However, after multiple adjustment only triglycerides, HbA1c and insulin levels remained significant. No correlation was found between plasma vitamin C and the two indicators of heart health; blood pressure and cardiovascular risk score.
Mild cognitive impairment was assessed by the MoCA test. Higher plasma vitamin C status was correlated with lower mild cognitive impairment, which was maintained after adjustment for gender, ethnicity and current smoking ([Table 4](#nutrients-09-00831-t004){ref-type="table"}). A 1 μmol/L increase in plasma vitamin C was associated with 3% reduced odds of mild cognitive impairment (OR = 0.97, 95% CI = (0.96, 0.99), *p* = 0.004). Indeed, the odds of mild cognitive impairment were twice as high for those below 23 μmol/L plasma vitamin C (OR = 2.1, 95% CI = (1.2, 3.7), *p* = 0.01). Plasma vitamin C status was not associated with wellbeing or depression.
3.4. Dietary Vitamin C Intake {#sec3dot4-nutrients-09-00831}
-----------------------------
Dietary intake analysis was performed on 250 of the CHALICE participants. The average dietary vitamin C intake was 110 mg/day, with 12% falling below the New Zealand recommended dietary intake (RDI, [Table 5](#nutrients-09-00831-t005){ref-type="table"}). There was little effect of gender, ethnicity or socio-economic status on dietary intake. However, those with the lowest educational qualifications tended to have lower dietary vitamin C intake, although this was not quite significant. Current smokers also had a lower dietary intake of vitamin C (*p* \< 0.001). Dietary vitamin C intake correlated somewhat less than expected with plasma levels of vitamin C, although the correlation was statistically significant (Pearson's correlation coefficient *r* = 0.27, *p* = 0.00002).
3.5. Associations of Dietary Vitamin C Intake with Markers of Metabolic and Mental Health {#sec3dot5-nutrients-09-00831}
-----------------------------------------------------------------------------------------
There was evidence that higher dietary intake of vitamin C was associated with lower waist circumference and insulin levels, after adjustment for gender, ethnicity and current smoking ([Table 6](#nutrients-09-00831-t006){ref-type="table"}). Glucose and HbA1c levels were inversely associated with dietary vitamin C intake in the initial models, however they did not remain so after correction for multiple comparisons. Higher dietary vitamin C intake was also associated with lower blood pressure, although there was no effect on cardiovascular risk score. There was little association between dietary vitamin C intake and mental health measures, although dietary intake was inversely associated with mild cognitive impairment in the unadjusted model.
4. Discussion {#sec4-nutrients-09-00831}
=============
These findings were drawn from the first phase of the CHALICE study, a longitudinal observational study of randomly selected 50-year-olds from the Canterbury region, New Zealand in 2010--2013. The comprehensive range of instruments used in the CHALICE study gives a broad picture of the cohort's health and the agreement between the study data and national demographics provides confidence that the study is representative of the health of 50-year-old New Zealanders in 2010. The cohort has typical levels of metabolic and cardiac markers, with indications of overweight/obesity and hypertension in some individuals. Our study provides new evidence that mid-life adults with higher vitamin C levels exhibited better measures of metabolic health and lower levels of mild cognitive impairment.
In New Zealand, dietary vitamin C intake has been estimated by several comprehensive national dietary surveys, including the 2008/2009 New Zealand Adult Nutrition Survey in which the mean usual adult daily intake was 108 mg based on 24 h dietary recall data \[[@B11-nutrients-09-00831]\]. This is close to the average dietary intake of 110 mg/day found in the current study. However, measuring vitamin C concentrations in the body has a number of advantages over dietary intake. It does not rely on participant's recall of their diet, and takes in all sources of the vitamin, including supplements, and the potential impact of vitamin C losses due to food processing and preparation. More particularly, it accounts for confounders of vitamin C status such as smoking, alcohol consumption, prescription medications and health conditions which may affect turnover of the vitamin \[[@B31-nutrients-09-00831]\]. The CHALICE study is the first representative study of plasma vitamin C status within the New Zealand population. Only smaller studies in specific, non-representative groups have measured plasma vitamin C concentrations within the New Zealand population \[[@B32-nutrients-09-00831],[@B33-nutrients-09-00831]\].
In our study, we found that 2.4% of 50-year-olds were deficient in vitamin C (i.e., \<11 µmol/L), putting them at higher risk of developing scurvy and other health effects that may be associated with very low vitamin C status. Men were at greater risk of being deficient than women, and having lower socio-economic status significantly increased risk. Smoking also increased the risk of deficiency, most likely due to increased oxidative stress causing faster turnover of the vitamin \[[@B31-nutrients-09-00831]\]. In addition, in our cohort, smokers had a lower dietary intake of vitamin C. Numerous studies have previously shown gender, socio-economic status and smoking to be important predictors of vitamin C status \[[@B9-nutrients-09-00831],[@B33-nutrients-09-00831],[@B34-nutrients-09-00831],[@B35-nutrients-09-00831],[@B36-nutrients-09-00831],[@B37-nutrients-09-00831]\]. A recent study suggests the effect of gender on vitamin C status may be due to the differing fat free mass between men and women, meaning vitamin C is distributed throughout a higher volume in men, leading to lower vitamin C concentrations in the plasma \[[@B36-nutrients-09-00831]\].
Data from large international cohorts show similar levels of vitamin C deficiency and hypovitaminosis C to the CHALICE cohort \[[@B37-nutrients-09-00831],[@B38-nutrients-09-00831]\], although the United States and lower socio-economic groups in the United Kingdom stand out as having higher rates of deficiency \[[@B9-nutrients-09-00831],[@B34-nutrients-09-00831]\]. In the current study, hypovitaminosis C (i.e., \<23 µmol/L) was apparent in 13% of participants, and this increased to 36% for those in the lowest socio-economic category. Symptoms such as decreased mood and energy levels may be observed with hypovitaminosis C, and are possibly related to the role of vitamin C as a cofactor in carnitine and catecholamine neurotransmitter synthesis \[[@B3-nutrients-09-00831],[@B14-nutrients-09-00831]\]. A high proportion (63%) of our participants had inadequate plasma vitamin C concentrations (i.e., \<50 µmol/L). Indeed, very few of our participants, only 7%, had saturating plasma vitamin C status (i.e., \>70 µmol/L), implying that current Ministry of Health guidelines recommending consumption of at least five servings of vegetables and fruit per day are ineffective \[[@B39-nutrients-09-00831]\]. Since the vitamin C content of fruit and vegetables is quite variable, we suggest that it is important to highlight the consumption of high vitamin C-content fruit and/or vegetables to provide plasma saturation in this age group.
High vitamin C concentrations in the blood were associated with significantly lower weight, waist circumference and BMI, and the effect of plasma vitamin C status was significant enough to survive the correction for multiple comparisons. The association of low vitamin C with obesity in this study replicates results in the literature \[[@B35-nutrients-09-00831],[@B40-nutrients-09-00831],[@B41-nutrients-09-00831],[@B42-nutrients-09-00831],[@B43-nutrients-09-00831],[@B44-nutrients-09-00831]\], and it is apparent that individuals with higher weight require higher intakes of vitamin C to reach adequate vitamin C status \[[@B45-nutrients-09-00831],[@B46-nutrients-09-00831]\]. We also show that higher plasma vitamin C status is associated with lower circulating levels of blood triglycerides, insulin and HbA1c, associations which survive correction for gender, ethnicity and current smoking. These findings are in agreement with a number of smaller intervention studies that have found inverse relationships of vitamin C with various markers of metabolic health \[[@B47-nutrients-09-00831],[@B48-nutrients-09-00831],[@B49-nutrients-09-00831]\], although others have failed to observe an effect of intervention \[[@B50-nutrients-09-00831]\]. Dakhale and coworkers show a small decrease in HbA1c and fasting blood glucose in individuals with type 2 diabetes after vitamin C supplementation of 1 g/day for 12 weeks \[[@B51-nutrients-09-00831]\]. Observational studies also provide evidence that low vitamin C status is associated with increased risk of metabolic syndrome \[[@B52-nutrients-09-00831],[@B53-nutrients-09-00831],[@B54-nutrients-09-00831]\].
A role for vitamin C in the prevention or management of diabetes and/or metabolic syndrome has been suggested \[[@B47-nutrients-09-00831],[@B51-nutrients-09-00831],[@B53-nutrients-09-00831],[@B54-nutrients-09-00831]\]. Obesity is a major risk factor for diabetes, and it may be that vitamin C has a role in moderating the inflammatory effect of adipose tissue. Vitamin C is thought to have anti-inflammatory activity, decreasing levels of inflammatory markers such as C-reactive protein and pro-inflammatory cytokines, although the exact mechanism(s) responsible for this are unknown \[[@B55-nutrients-09-00831],[@B56-nutrients-09-00831]\]. Disorders of energy balance and metabolism are common worldwide. For example, in New Zealand, around 241,000 individuals have been diagnosed with diabetes, and significant numbers have undiagnosed diabetes, or pre-diabetes \[[@B57-nutrients-09-00831]\]. Further, among people aged over 15 years, 65% of individuals meet the criteria for overweight and obesity \[[@B58-nutrients-09-00831]\]. Diet and lifestyle factors are associated with these disorders and represent key modifiable determinants. Interestingly, in the CHALICE cohort there were no consistent significant effects identified between plasma vitamin C status and blood pressure or cardiovascular disease risk, although higher dietary vitamin C intake was associated with decreased blood pressure, an effect that has been observed previously \[[@B59-nutrients-09-00831]\].
In this study, we also demonstrate lower levels of mild cognitive impairment in those with high vitamin C status, even after adjustment for gender, ethnicity and smoking. Current smoking was a good proxy for socio-economic status and educational achievement in the model; thus, the relationship with vitamin C status survived correction for these important predictors of cognitive impairment. The odds of mild cognitive impairment were twice as high for those below 23 μmol/L plasma vitamin C concentration. Vitamin C is present at very high concentrations in the brain \[[@B60-nutrients-09-00831]\], and animal models have shown that the brain is the last organ to be depleted of the vitamin during prolonged deficiency \[[@B61-nutrients-09-00831]\], suggesting an important requirement for vitamin C in the central nervous system. A recent animal study has shown that moderate vitamin C deficiency may play a role in accelerating amyloid plaque accumulation in Alzheimer's disease, the most common form of dementia \[[@B62-nutrients-09-00831]\]. However, epidemiological studies have been inconclusive in regards to whether vitamin C status may affect cognitive decline \[[@B63-nutrients-09-00831],[@B64-nutrients-09-00831]\] and Alzheimer's disease specifically \[[@B65-nutrients-09-00831],[@B66-nutrients-09-00831]\]. Lu and co-workers investigated the relationship between dietary nutrients and mild cognitive impairment in 2892 elderly Chinese participants using the MoCA test, and found that vitamin C intake exhibited a significant protective effect \[[@B64-nutrients-09-00831]\]. Our study has the advantage over many in that plasma vitamin C concentrations have been measured; we were not reliant on dietary intake, which may be susceptible to problems with recall ability and the other confounders mentioned above.
In later life, dementia and disorders of cognition are highly prevalent. Even in the CHALICE sample of 50-year-olds, 15% of the sample scored below the recommended cut point on the MoCA. There is considerable interest in the effect of diet on maintaining cognitive function and delaying neuro-degenerative disease in old age. A 2015 study with 37 older healthy adults demonstrated reduced rates of cognitive decline following consumption of orange juice \[[@B67-nutrients-09-00831]\]. This was attributed to the high flavanone content of the orange juice, since flavonoids have been associated with reduced rates of cognitive decline \[[@B68-nutrients-09-00831],[@B69-nutrients-09-00831]\]. However, it is possible that the vitamin C content of the orange juice may have contributed to the observed effect. In support of this premise, studies have shown that supplementation of older adults with the antioxidant vitamins C and E was able to preserve cognitive performance \[[@B70-nutrients-09-00831],[@B71-nutrients-09-00831],[@B72-nutrients-09-00831]\]. Another study, however, found no impact of antioxidant vitamin supplementation on cognition, despite improvements in markers of oxidative stress \[[@B73-nutrients-09-00831]\], demonstrating mixed results in the literature. Intervention studies often look for relatively short-term impacts on cognition instruments in response to different nutrient intakes. In contrast, the CHALICE study measured the association of plasma vitamin C status and dietary intake, more likely to be markers of longer-term lifestyle patterns, with a cognitive instrument (MoCA) as an assessment of current mild cognitive impairment.
There are several limitations to our study, notably the observational design, in which associations do not imply causation. Many factors impact on the health status of individuals and groups, including diet, exercise, temperament, behaviors, socio-economic status and genetics. These factors typically interact and correlate with each other, as they do in the CHALICE cohort, with the result that predictors of health outcomes are related (e.g., low blood pressure is associated with low BMI). We have addressed multiple testing issues with the use of corrected *p* values, and multi-collinearity does not affect individual models as each model only has one independent predictor, with the dichotomous covariates having limited capacity to induce collinearity. While we have focused on the associations of vitamin C with health outcomes, these associations could include the effects of unmeasured nutrients associated with vitamin C intake. Dietary vitamin C and plasma vitamin C status did not always correlate with the same health indicators. However, as detailed above, this is likely due to fasting plasma vitamin C concentration being a more accurate indicator of body status.
5. Conclusions {#sec5-nutrients-09-00831}
==============
The CHALICE cohort of 404 individuals aged 50 years had an average vitamin C intake of \~110 mg/day, which should provide adequate plasma concentrations \[[@B14-nutrients-09-00831]\]. Despite this, a significant proportion of the participants had inadequate plasma vitamin C status. This indicates the likely effects of confounding factors, such as chronic disease, on plasma vitamin C status, and suggests that dietary interventions targeting increased consumption of fruit and vegetables, and increased vitamin C intake in particular, are required for this age group. Metabolic health markers were significantly better in participants with higher plasma vitamin C concentrations, even after correction for confounders. The association of high vitamin C concentrations with the reduction in risk of impaired cognition is intriguing and merits further investigation.
We would like to acknowledge the participants of the CHALICE study and the CHALICE study investigators. The CHALICE study was supported by grants awarded from the Department of Internal Affairs' Lotteries Health (grant number: AP265022), Canterbury Community Trust, Otago Thyroid Research Foundation and University of Otago Foundation Trust (grant number: TL1060). Funding for the vitamin C analyses was provided by Zespri International Ltd, Mt Maunganui, New Zealand. A.C. is the recipient of a Health Research Council of New Zealand Sir Charles Hercus Health Research Fellowship.
J.S. coordinated study; A.C., J.M.P. and M.V. measured vitamin C status; R.W. and P.S. calculated dietary intakes; V.C. contributed to design of cardiovascular measures; A.C., P.S. and J.F.P. conceived paper; J.F.P. analyzed data; J.M.P., A.C. and J.F.P. interpreted data and wrote paper; and M.V., P.S., J.W., J.S. and V.C. edited paper. J.F.P., J.M.P. and A.C. contributed to the work equally.
The authors declare no conflict of interest.
{#nutrients-09-00831-f001}
nutrients-09-00831-t001_Table 1
######
CHALICE participants compared with Census 2006 50--54-year-olds from same region.
Chalice (*n*, %) Census 2006 (%) *p*
-------------------------------- ---------------------------- ------------------ ----------------- ------ ----------
Gender Female 215 53.2 50.9 NS
Male 189 46.8 49.1
Ethnicity Māori 60 14.9 4.5 \<0.0001
NZ European 338 83.7 74.2
Socio-Economic Status Low (ELSI~SF~ score 0--16) 30 7.4 8.2 NS
Medium (ELSI~SF~ score 17--24) 122 30.2 29.4
High (ELSI~SF~ score 25--31) 252 62.4 62.5
Education No Qualification 53 13.1 23.9 \<0.0001
Secondary School Qualification 110 27.2 35.2
Post-secondary 168 41.6 25.6
University Degree 73 18.1 15.3
Current Smoker 60 14.9 16.6 NS
*N* = 404; *p* (χ^2^*~n~*~−1~) \> 0.1 shown as not significant, NS.
nutrients-09-00831-t002_Table 2
######
Health of CHALICE participants and normal ranges for the New Zealand population.
Female Male
---------------------------- ---------- --------- --------- -------------------- ---------- --------- --------- -------------------
**Body Measurements** **Mean** **Min** **Max** **NZ Female Mean** **Mean** **Min** **Max** **NZ Male Mean**
Weight kg 78.6 49.1 149.9 74.8 (73.5--76.1) 88.4 50.8 143.8 88.0 (86.9--89.1)
BMI kg/m^2^ 29.1 17.4 63.4 28.1 (27.6--28.6) 28.1 19.2 48.6 28.6 (28.2--28.9)
Waist cm 92.0 63.0 144.0 86.6 (85.5--87.6) 98.3 72.5 148.0 98.4 (97.4--99.3)
**Metabolism** **Mean** **Min** **Max** **Healthy Range** **Mean** **Min** **Max** **Healthy Range**
Triglycerides mmol/L 1.3 0.4 11.7 \<1.7 1.6 0.4 11.7 \<1.7
HDL mmol/L 1.4 0.8 2.7 1.0--2.2 1.2 0.7 1.9 0.9--2.0
Glucose mmol/L 5.1 3.2 10.8 \<6.1 5.4 3.7 17.9 \<6.1
HbA1c mmol/L 38.2 27.0 74.0 \<40 39.9 28.0 102.0 \<40
Insulin pmol/L 60.9 10.0 277.0 10--80 61.2 4.0 480.0 10--80
**Heart Health** **Mean** **Min** **Max** **Healthy Range** **Mean** **Min** **Max** **Healthy Range**
BP (systolic) mmHg 131.1 104.0 183.7 120 134.2 97.7 185.7 120
BP (diastolic) mmHg 82.5 60.3 106.0 80 85.0 61.0 128.3 80
CVD risk score % 2.5--5 \<2.5 20--25 \<2.5 5--10 2.5--5 20--25 \<2.5
**Mental Health** **Mean** **Min** **Max** **Mean** **Min** **Max**
Wellbeing 53.0 16 70 52.7 30 70
Cognition 27.1 19 30 26.6 16 30
Current Depression *n* (%) 17 (7.9) 12 (6.3)
BMI: body mass index, HDL: high-density lipoprotein, BP: blood pressure, CVD: Cardiovascular disease. Body measurements compared with New Zealand mean (95% confidence interval) for 45--55 age range \[[@B28-nutrients-09-00831]\]. Metabolic and heart health compared with normal healthy range \[[@B23-nutrients-09-00831],[@B29-nutrients-09-00831],[@B30-nutrients-09-00831]\]. Wellbeing measured by Warwick--Edinburgh scale, cognition by MoCA. Current depression is those currently clinically depressed excluding those diagnosed bipolar (*N* = 203 female, 179 male). One female has no waist measurement, three females no fasting metabolic measures, one male no fasting metabolic measures, one male glucose assay failed and two males HbA1c assay failed, otherwise data are for 215 females and 189 males.
nutrients-09-00831-t003_Table 3
######
Categories of vitamin C status.
Plasma Vitamin C Deficient Marginal Inadequate Adequate *p*
------------------------ -------------------- ------------------ -------------- ---------- ------------ ---------- ------ ------ ------ ------ ------ ---------
Total 44.2 (42.4, 46.0) 9 2.4 39 10.6 183 49.6 138 37.4
Gender Female 47.4 (44.9, 49.9) 2 1.0 20 10.3 85 43.6 88 45.1 0.005
Male 40.6 (38.2, 43.0) 7 4.0 19 10.9 98 56.3 50 28.7
Ethnicity Non Māori 44.5 (42.6, 46.4) 7 2.2 31 9.8 159 50.3 119 37.7 NS
Māori 42.4 (37.2, 47.6) 2 3.8 8 15.1 24 45.3 19 35.8
Socio- Economic Status Low 36.8 (28.3, 45.3) 2 8.0 7 28.0 9 36.0 7 28.0 0.003
Medium 43.7 (40.3, 47.1) 4 3.5 14 12.3 53 46.5 43 37.7
High 45.3 (43.2, 47.4) 3 1.3 18 7.8 121 52.6 88 38.3
Education None 38.7 (33.6, 43.9) 3 6.1 6 12.2 26 53.1 14 28.6 NS
Secondary School 45.9 (42.1, 49.7) 1 1.0 13 12.6 49 47.6 40 38.8
Post-secondary 43.1 (40.6, 45.7) 5 3.3 16 10.6 75 49.7 55 36.4
University Degree 48.1 (44.4, 51.9) 0 0.0 4 6.1 33 50.0 29 43.9
Tobacco Not Current Smoker 45.9 (44.1, 47.8) 6 1.9 26 8.2 157 49.7 127 40.2 \<0.001
Current Smoker 34.1 (29.2, 38.9) 3 5.7 13 24.5 26 49.1 11 20.8
Plasma vitamin C classified as deficient \<11 μmol/L, marginal 11--23 μmol/L, inadequate 23--50 μmol/L or adequate \>50 μmol/L; *n* = 369.
nutrients-09-00831-t004_Table 4
######
Significant plasma vitamin C effects for body measures, metabolic health and mental health.
Vitamin C \<23 µmol/L (*n* = 47) Vitamin C \>23 µmol/L (*n* = 321) *p* *p* Adjusted
-------------------- ---------------------------------- ----------------------------------- --------- ------------------- ------- ---------
Body measurements
Weight 90.3 (83.3, 97.4) 81.7 (79.8, 83.6) 0.024 0.004
BMI 31.4 (28.7, 34.0) 28.1 (27.5, 28.7) 0.021 \<0.001
Waist 103.3 (97.6, 108.9) 93.3 (91.8, 94.8) 0.001 \<0.001
Metabolism
Triglycerides 1.8 (1.4, 2.3) 1.4 (1.3, 1.5) 0.061 0.029
HDL 1.3 (1.2, 1.3) 1.4 (1.3, 1.4) 0.033 NS
Glucose 5.6 (5.2, 6.0) 5.2 (5.0, 5.3) 0.072 0.073
HbA1c 42.2 (39.6, 44.8) 38.5 (37.7, 39.3) 0.009 0.015
Insulin 91.0 (68.4, 113.6) 56.3 (51.9, 60.8) 0.004 0.000
Heart health
BP (systolic) 132.2 (128.0, 136.4) 132.5 (130.8, 134.2) NS NS
BP (diastolic) 83.6 (81.0, 86.3) 83.5 (82.4, 84.6) NS NS
CVD risk score 5--10% (\<2.5%, 20--25%) 2.5--5% (3.5--5%, 5--10%) 0.057 NS
Mental Health
Wellbeing 50.9 (48.4, 53.4) 53.0 (52.0, 53.9) NS NS
*n* *%* *n* *%*
MCI 17 40.5 66 21.5 0.012 0.02
Current Depression 6 12.5 20 6.2 NS NS
MCI: Mild Cognitive Impairment indicated by MoCA score \<26 for those without excluding conditions. Current depression is for those without Bipolar Disorder. P values less than 0.1 shown otherwise NS: Not Significant. *p* values adjusted for gender, ethnicity and current smoking.
nutrients-09-00831-t005_Table 5
######
Categories of dietary vitamin C intake.
Dietary Vitamin C Below RDI RDI-Average Above Average *p*
----------------------- -------------------- ------------------- ---------------- ------------- --------------- ------ ------ ------ ------ ---------
Total 109.8 (101.5, 118.1) 30 12 126 50.4 94 37.6
Gender Female 107.4 (96.6, 118.2) 13 9.7 73 54.5 48 35.8 NS
Male 112.6 (99.7, 125.6) 17 14.7 53 45.7 46 39.7
Ethnicity Non Māori 112.0 (102.7, 121.2) 22 10.3 111 51.9 81 37.9 NS
Māori 97.2 (79.6, 114.7) 8 22.2 15 41.7 13 36.1
Socio-Economic Status Low 78.8 (54.4, 103.1) 4 26.7 8 53.3 3 20.0 NS
Medium 105.0 (90.7, 119.2) 12 15.2 36 45.6 31 39.2
High 115.3 (104.4, 126.1) 14 9.0 82 52.6 60 38.5
Education None 83.5 (64.2, 102.7) 8 28.6 13 46.4 7 25.0 0.1
Secondary School 117.1 (98.4, 135.7) 6 10.0 32 53.3 22 36.7
Post-secondary 108.6 (97.1, 120.1) 11 9.9 59 53.2 41 36.9
University Degree 118.4 (97.6, 139.2) 5 9.8 22 43.1 24 47.1
Tobacco Not Current Smoker 114.1 (105.3, 122.8) 20 9.0 112 50.7 89 40.3 \<0.001
Current Smoker 77.5 (54.6, 100.5) 10 34.5 14 48.3 5 17.2
The cut-off values for the vitamin C categories are as follows: New Zealand recommended dietary intake is 45 mg/day, the average New Zealand intake is 109 mg/day for men and 106 mg/day for women \[[@B11-nutrients-09-00831]\]; *n* = 250.
nutrients-09-00831-t006_Table 6
######
Significant dietary vitamin C effects based on average intake for body measures, metabolic health and heart health.
Intake \< Average (*n* = 147) Intake \> Average (*n* = 103) *p* *p* Adjusted
-------------------- ------------------------------- ------------------------------- --------- ---------------- ------ -------
Body measurements
Weight 82.2 (79.2, 85.3) 79.8 (76.4, 83.3) NS NS
BMI 28.5 (27.4, 29.6) 27.2 (26.2, 28.1) 0.08 0.063
Waist 94.6 (92.2, 97.0) 91.2 (88.5, 93.8) 0.06 0.047
Metabolism
Triglycerides 1.4 (1.3, 1.5) 1.3 (1.1, 1.6) NS NS
HDL 1.4 (1.3, 1.4) 1.4 (1.3, 1.4) NS NS
Glucose 5.3 (5.1, 5.5) 5.0 (4.9, 5.2) 0.03 0.078
HbA1c 39.6 (38.3, 41.0) 37.8 (36.9, 38.7) 0.03 NS
Insulin 64.6 (55.5, 73.6) 52.3 (44.3, 60.3) 0.05 0.041
Heart health
BP (systolic) 135.0 (132.5, 137.5) 130.6 (127.4, 133.8) 0.03 0.016
BP (diastolic) 85.2 (83.6, 86.7) 82.3 (80.4, 84.1) 0.02 0.007
CVD risk score 2.8 (2.6, 3.0) 2.6 (2.2, 2.9) NS NS
Mental Health
Wellbeing 52.5 (51.1, 53.8) 52.9 (51.3, 54.4) NS NS
***n*** ***%*** ***n*** ***%***
MCI 36 24.5 14 13.6 0.04 NS
Current Depression 13 8.8 4 3.9 NS NS
MCI Mild Cognitive Impairment indicated by MoCA score \<26 for those without excluding conditions. Current depression is for those without Bipolar Disorder. *p* values less than 0.1 shown otherwise NS: Not Significant. *p* values adjusted for gender, ethnicity and current smoking. Average is New Zealand average of 109 mg/day for men, 106 mg/day for women \[[@B11-nutrients-09-00831]\].
| 2024-03-23T01:26:19.665474 | https://example.com/article/6867 |
Greg Thompson
Gregory Francis Thompson, (March 28, 1947 – September 10, 2019) was a Canadian politician who served six terms as a Member of Parliament (MP).
Political career
Thompson, a high school teacher, a businessman and financial planner was first elected to the House of Commons of Canada in the 1988 Canadian federal election as a member of the Progressive Conservative Party of Canada. He was elected in the riding of Carleton—Charlotte. His bid for re-election in the 1993 Canadian federal election was unsuccessful and he was defeated by Harold Culbert of the Liberal Party of Canada by fewer than 1,000 votes.
Thompson however ran again in the next election and was re-elected in the riding of Charlotte, where he defeated Culbert. Thompson was re-elected in the 2000 Canadian federal election in the riding of New Brunswick Southwest and again the 2004 Canadian federal election in the riding of St. Croix—Belleisle. Shortly before the 2004 election, he joined the new Conservative Party of Canada. He was re-elected in the 2006 federal election. In the 2008 federal election he was elected for a sixth term in the riding of New Brunswick Southwest by garnering over 58% of the vote.
During his time in parliament, he has served as the critic of Human Resources Development, the Treasury Board, Regional Development, Health, and Public Accounts, as well as critic of the Atlantic Canada Opportunities Agency. On February 6, 2006, he was appointed Minister of Veterans Affairs in Stephen Harper's Cabinet. In April 2007, he and Harper told the press in Kitchener, Ontario that a Veterans' Bill of Rights would come into effect soon and there would be a new ombudsman for veterans along with it.
He was formerly a high school history teacher at Fundy High School from 1975-1980.
He resigned from his position in Cabinet on January 16, 2010, because years of travel had worn him down and he wasn't looking forward to making a trip to New Zealand due to the length and time he had to invest in the trip. He also announced he would not run in the 2011 federal election.
In 2018 he ran provincially under the Progressive Conservatives in the riding of Saint Croix and won. He served as Minister of Intergovernmental Affairs until his death in September of 2019.
Veterans Affairs privacy issues
In October 2010, Canada's Privacy Commissioner Jennifer Stoddart uncovered evidence that widespread privacy abuses had been occurring at Veterans Affairs Canada.
Among the cases where privacy issues were investigated is that in which highly personal information of an outspoken critic of Veterans Affairs, including confidential medical and financial information, was included in briefing notes prepared for then-minister Greg Thompson.
Electoral record
See also
Veterans Affairs Canada
References
External links
Category:1947 births
Category:2019 deaths
Category:Conservative Party of Canada MPs
Category:Deaths from cancer in New Brunswick
Category:Members of the 28th Canadian Ministry
Category:Members of the Executive Council of New Brunswick
Category:Members of the House of Commons of Canada from New Brunswick
Category:Members of the Queen's Privy Council for Canada
Category:People from St. Stephen, New Brunswick
Category:Progressive Conservative Party of New Brunswick MLAs | 2024-03-17T01:26:19.665474 | https://example.com/article/2061 |
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February 22, 2011 (DURHAM, NC) – Triangle Modernist Houses (TMH), the award-winning non-profit organization dedicated to archiving, preserving and promoting modernist residential design, is featured in the “Advocacy Spotlight” for the month of February in the Docomomo-US Newsletter.
Dcocomomo is an acronym for the DOcumentation and COnservation of buildings, sites and neighborhoods of the MOdern Movement. Founded in 1988 in The Netherlands, Docomomo has national chapters, or “working parties,” in 54 countries and over 2000 individual members. It is an important presence in conservation and in the worldwide architectural culture, working in partnership with other international organizations, national governments, and regional and national associations. Docomomo-US is the national chapter for the United States.
The February newsletter’s Advocacy Spotlight notes that TMH is “one of the largest single archives for residential modern in the United States.” It discusses why the Triangle region of North Carolina has the third largest collection of modernist houses in the nation: “…due in large part to Henry Kamphoefner, the founding Dean of the NC State University School of Design (now College of Design) who recruited top faculty and insisted that they practice as well as teach. Their output was prolific but almost completely undocumented, until the formation of Triangle Modernist in 2007.”
The Spotlight also notes: “TMH maintains a rapidly growing collection of audio, video, and document archives featuring Modernist architects both living and deceased, including over 130 in North Carolina.”
February 23, 2011 (CHARLOTTE, NC) – The Lassiter House, the oldest identified Modernist house in Charlotte, and one of the few designed by architect A.G. Odell still standing, will be torn down if not sold by June.
The house’s only owners put their three-bedroom, three-bath house in Charlotte’s Eastover neighborhood on the market in 2010. Triangle Modernist Houses (TMH), the state’s award-winning non-profit organization for Modernist residential architecture, today issued a National Alert on the Lassiter House. (The group’s last National Alert saved the historic Carr House in Durham designed by architect Kenneth Scott.)
“When most people in the Triangle area think ‘modernist houses,’ they don’t realize that there are some true gems in Rocky Mount and Greenville,” said George Smart, TMH founder and director. “They’ll change their minds after they see these gorgeous homes.”
This series focuses on black design professionals before 1970, those “who followed their hearts into architecture despite great resistance from both society and their own industry,” says George Smart, TMH founder and director. Mechanics and Farmers Bank, The Michael Okoli Agency, and architect Arthur Clement provided financial support for the series.
“Today there are many minority architects in North Carolina, but before 1970 it was another story, and not a nice one,” Smart says. “The field of architecture made choosing the profession nearly impossible for minorities. In North Carolina, there were almost none for decades.” | 2023-11-25T01:26:19.665474 | https://example.com/article/3535 |
Introduction {#s1}
============
Chronic hepatitis B, caused by HBV, globally affects some 400 million people [@pone.0072798-WHO1] and puts them at a high risk of progressing to liver cirrhosis and hepatocellular carcinoma (HCC) [@pone.0072798-Ganem1]. At present, only few and only partially effective therapies are available [@pone.0072798-Ganem1], [@pone.0072798-Zoulim1]. Detailed elucidation of the HBV replication mechanism provides a chance to identify novel antiviral targets for therapeutic intervention [@pone.0072798-Nassal1]. One such potential target is the protein-primed reverse transcription mechanism [@pone.0072798-Wang1] employed by HBV and the related animal hepadnaviruses, such as duck HBV (DHBV), to generate new DNA genomes.
In all hepadnaviruses one of the viral transcripts, the pregenomic (pg) RNA, serves as template for reverse transcription within viral capsids (core particles) [@pone.0072798-Summers1] (for reviews see [@pone.0072798-Beck1], [@pone.0072798-Nassal2]). Co-packaging of pgRNA and the viral reverse transcriptase, called P protein, into newly forming capsids as well as replication are initiated by the specific interaction of P protein with the RNA encapsidation signal ε near the 5′-end of the pgRNA [@pone.0072798-Bartenschlager1]; its overall structure, consisting of a lower and an upper stem, an internal bulge and an apical loop [@pone.0072798-Knaus1], [@pone.0072798-Pollack1], is similar in all hepadnaviruses [@pone.0072798-Beck2]. All P proteins carry, beyond the evolutionarily conserved catalytic reverse transcriptase (RT) and RNase H (RH) domains, a unique Terminal Protein (TP) domain, separated from the RT domain by a dispensable spacer region. TP provides a specific Tyr residue (Y63 in HBV, Y96 in DHBV) as acceptor [@pone.0072798-Weber1], [@pone.0072798-Zoulim2], [@pone.0072798-Lanford1] for the first nucleotide of newly generated DNA (\"protein-priming\"). Binding to the cognate ε activates P for protein-priming, and the 3′ proximal half of the bulge [@pone.0072798-Tavis1], [@pone.0072798-Wang2], for HBV possibly plus the following nucleotide [@pone.0072798-Lanford1], [@pone.0072798-Nassal3], provides the template for a 3- to 4- nt DNA oligo that is covalently linked to the Tyr residue in TP ([Fig. 1](#pone-0072798-g001){ref-type="fig"}). The P-linked DNA oligo is then translocated to a matching acceptor site at the 3′ proximal DR1\* in the terminally redundant pgRNA from where it is extended into full-length (-)-strand DNA. Concomitant degradation of the pgRNA and subsequent (+)-strand DNA synthesis eventually yield the relaxed circular (RC) DNA typically found in virions (for reviews: [@pone.0072798-Beck1], [@pone.0072798-Nassal2]).
{#pone-0072798-g001}
Despite their overall similarity the ε stem-loops from HBV and DHBV are not functionally interchangeable. This and previous mutational studies indicate that the interaction between P protein and its cognate ε is highly specific and the result of both sequence- and structure-specific features in the RNA. Owing to the successful in vitro reconstitution of protein-priming for DHBV in rabbit reticulocyte lysate [@pone.0072798-Wang3] or from purified components [@pone.0072798-Hu1], [@pone.0072798-Beck3], [@pone.0072798-Stahl1], such features are known in much more detail for DHBV than for human HBV where P - ε interaction studies have largely been restricted to mutational analyses of pgRNA encapsidation [@pone.0072798-Knaus1], [@pone.0072798-Pollack1] and DNA formation [@pone.0072798-Nassal3] in transfected cells. Collectively, the DHBV data suggest that the P - ε interaction during priming is a dynamic multi-step process in which the initial RNA binding is followed by conformational changes in both protein [@pone.0072798-Tavis2], [@pone.0072798-Cao1], [@pone.0072798-Stahl2] and RNA [@pone.0072798-Beck4]; these alterations are crucial to reach the priming-active state and require assistance by cellular chaperones [@pone.0072798-Hu2], [@pone.0072798-Hu3], [@pone.0072798-Stahl2], [@pone.0072798-Stahl1], although chaperone-dependence can be circumvented by employing truncated \"miniP\" proteins which lack the RH domain, the spacer and nonessential parts of the TP and RT domains but remain priming-competent [@pone.0072798-Beck5], [@pone.0072798-Boregowda1]. Most important within the DHBV ε RNA for achieving a priming-active complex are the central region comprising the bulge [@pone.0072798-Schaaf1] and the apical loop, whereas in the upper stem various nt exchanges are tolerated [@pone.0072798-Hu4], even in vivo [@pone.0072798-Schmid1]; in fact, part of the right half of the upper stem becomes expelled in priming-active P - ε complexes [@pone.0072798-Beck4].
HBV P protein employed in analogous setups showed specific binding to ε RNA yet no priming activity [@pone.0072798-Hu5]; in addition, binding did not require the upper stem and loop, although these subelements appear crucial for pgRNA packaging and DNA synthesis in cells.
Conversely, RNAs that bind to P protein but do not support virus replication lend themselves as decoys; these are structural mimics of functional RNAs that compete with the original RNA for the natural interaction partner(s) and thus can act as therapeutically useful inhibitors [@pone.0072798-Zhou1]). Particularly strongly binding RNA sequences (aptamers) may be selected from large sequence pools by appropriate SELEX (systematic evolution of ligands by exponential enrichment) procedures (for reviews: [@pone.0072798-Gold1], [@pone.0072798-Taouji1]). To identify potential HBV ε decoys, we have recently performed such an in vitro SELEX screen using ε RNA libraries in which the entire upper stem, or the upper stem but not the 6 nt apical loop sequence, which according to NMR data forms a pseudo-triloop [@pone.0072798-Flodell1], [@pone.0072798-Flodell2], were randomized; selection was for binding to a recombinant HBV miniP protein [@pone.0072798-Feng1]. In line with previous in vitro data [@pone.0072798-Hu5], the selected RNAs displayed no preference for wild-type-like upper stem sequences but rather a general enrichment of A residues at the randomized positions. However, representative aptamers from the pool with maintained loop sequence displayed stronger competition with wild-type ε RNA than those from the completely randomized pool, implying that, at least in an unstructured A-rich upper stem framework, the loop sequence might contribute to P binding even in vitro. No inferences could be drawn on the role of base-pairing or nucleotide identity in the upper stem but according to previous reports [@pone.0072798-Knaus1], [@pone.0072798-Pollack2], [@pone.0072798-Fallows1], [@pone.0072798-Rieger1] numerous mutations in this region interfere with pgRNA packaging and/or replication. However, the near exclusive use of multiple simultaneous mutations hampered more detailed interpretations.
Based on these considerations and the strategic positions of selected residues within or close to non-paired regions (see below) we here focused on mutationally analyzing the functional role of six individual upper stem positions, namely all of the four A residues, i.e. A1 and A2 immediately above the bulge (numbers referring to positions within the upper stem), A9 and A10 close to the apical loop, and U13 and U15 within the pseudo-triloop. To address a potential impact of base-pairing, we also included U28 and U29 which can pair with A1 and A2 to close the bulge.
The corresponding mutations, in the context of a complete HBV genome, were first investigated for their effect on DNA synthesis in transfected human hepatoma cells. Next, in order to trace the underlying mechanism, those mutants displaying substantial defects in DNA accumulation were analyzed for pgRNA packaging. Finally, while this work was in progress, Jones et al. [@pone.0072798-Jones1] reported a new cell-free system to directly address the HBV protein-priming reaction. This provided an opportunity to also look at the effect of selected mutations on the very first steps of HBV replication. As shown below, the combined results revealed very distinct impacts, correlating with the position of the respective mutations within ε, on divergent steps of HBV DNA synthesis, thus confirming the dynamic multi-step nature of the P - ε interaction as well as suggesting clues towards more effective ε decoys.
Results {#s2}
=======
Predicted location of the targeted positions in the HBV ε upper stem and mutant design {#s2a}
--------------------------------------------------------------------------------------
RNA loops and bulges as well as their distorted connections into regular double-helical regions provide a rich repertoire of distinct structural features predestining them as specific recognition elements, often by opening the major groove [@pone.0072798-Weeks1]. Since simple 2D structure representations do not reveal such features, we first combined published NMR 3D structure data for the (isolated) upper stem of HBV ε [@pone.0072798-Flodell2] and structure prediction to model [@pone.0072798-Parisien1] the entire stem-loop and visualize the location of the 6 residues in question. As shown in [Fig. 2A](#pone-0072798-g002){ref-type="fig"}, the A residues are at or close to the junctions between double-helical sections and the single-stranded bulge and the pseudo-triloop which harbors U13 and U15; the A residues face the major groove in the double-helical portion, as do U13 and U15 in the pseudo-triloop. Hence the upper stem A- and apical loop U-residues would be suitably positioned to contribute to the P - ε interaction.
![Predicted spacial location of the targeted upper stem residues and mutational design.\
(A) Model for the 3D structure of the ε stem-loop. 3D structure prediction as implemented in the MC-Fold and MC-Sym program package [@pone.0072798-Parisien1] was combined with NMR-based structural information on the upper stem and apical loop ([@pone.0072798-Flodell2], PDB entry 2IXY) to derive a tentative model for the entire ε element with its lower stem (green), bulge (blue), upper stem (yellow) and apical loop (red). The same model is shown from two different angles to provide a visual impression of the spacial distribution of the targeted residues A1, A2, A9, A10, U13 and U15 (all in cyan). Their major groove location and close juxtaposition to the bulge and loop, respectively, is supported by the NMR data; other features, including the relative orientation of upper stem vs. lower stem, are arbitrary. (B) Specific mutations investigated. Nucleotide exchanges and their positions in a 2D representation of ε together with the designations of the mutants are indicated. A linear representation is shown in [Table 1](#pone-0072798-t001){ref-type="table"}.](pone.0072798.g002){#pone-0072798-g002}
Next we designed a set of 24 mutants, as summarized in [Fig. 2B](#pone-0072798-g002){ref-type="fig"}. A1, A9 and U13 were individually mutated to all other three nucleotides, A10 and U15 in various combinations with mutations at A9 or U13, respectively. To assess the importance of A1 and A2 being base-paired with U28 and U29 we also introduced mutations at the latter positions, thus replacing the original basepairs by others, or preventing basepair formation; mutants in which the first or first plus second base-pair above the bulge is restored were named rb1NN and rb1,2NN, with NN indicating the specific new base-pair. Lastly, we included a mutant (termed \"mut\" below) in which the four consecutive nucleotides C~7~C~8~A~9~A~10~ were replaced by GGUU ([Fig. 2B](#pone-0072798-g002){ref-type="fig"}); while reportedly reducing but not ablating RNA packaging (mutant \"upperL2\" in [@pone.0072798-Pollack2]) the same mutations completely abrogated in vitro P binding (mutant \"U-L-U\" in [@pone.0072798-Hu5]); this variant was therefore used as negative control. All mutations were introduced into the 5′ copy of ε encoded in the 1.1× unit length wild-type HBV genome present in the CMV-IE promoter-driven expression vector pCH-9/3091 [@pone.0072798-Nassal4].
Differential impact of mutations of the upper stem adenines and loop uracils on HBV replication {#s2b}
-----------------------------------------------------------------------------------------------
Formation of HBV replicative DNA intermediates indicates that all prior steps have successfully been passed, including expression of core and P protein, assembly of pgRNA plus P containing nucleocapsids, and protein-primed initiation and extension of minus-strand DNA synthesis. We therefore transfected the HBV expression vectors for the 24 ε upper stem variants, plus the parental wild-type HBV vector as positive control and the P protein binding-deficient ε mut vector as negative control, into HepG2 cells; four days later viral DNAs associated with cytoplasmic nucleocapsids were analyzed by Southern blotting, using a ^32^P-labeled DNA probe covering the whole HBV genome ([Fig. 3](#pone-0072798-g003){ref-type="fig"}). As controls for core protein expression and equal loading, aliquots from the same cytoplasmic lysates were analyzed by Western blotting (panel labeled \"core\") and RT-PCR for β-actin mRNA (panel labeled β-actin mRNA).
{#pone-0072798-g003}
Compared to the replicative DNA intermediates generated by the wild-type HBV vector (lane wt), the mutants displayed distinct classes of phenotypes ([Fig. 3](#pone-0072798-g003){ref-type="fig"}). Some mutants showed wild-type-like patterns and amounts of DNA (A1U; A1,2U; rb1UA; rb1,2GC; A9G; A9C; A9,10U; U13,15G; U13,15C), some others modest reductions (down to ∼30% of the wild-type) in DNA amounts (rb1GC; U29A; rb1,2UA; U28,29A; A1C; U13G; U13C). The remaining variants displayed severe reductions in DNA amounts (\<25% of the wild-type); however some still produced a wild-type-like pattern of apparently full-length DNA (A1 and A1+A2 mutants A1G, A1,2G, A1,2C) while for the others mainly faster migrating but no full-length products were detected (A9, A9+A10, and some U13+U15 mutants). This would be compatible with the former mutations largely affecting efficiency of protein-primed initiation whereas the latter appeared to also impact the quality of minus-strand and possibly plus-strand DNA extension. For the P binding-defective mut variant no signals were detected. The comparable RT-PCR signals for β-actin mRNA indicated that the source lysates were derived from similar numbers of cells. The levels of Western blot detectable core protein varied by less than two-fold and thus could neither account for the massive differences in DNA levels; for instance, the core protein signals for the low DNA samples A1G or A1,2C or the negative control variant mut were not weaker than those for samples showing high DNA levels such as wild-type HBV or mutants rb1UA or rb1,2GC.
To directly confirm a low DNA content of the capsids produced from the mutants giving low Southern blot signals we employed native agarose gel electrophoresis (NAGE) in which intact capsids are analyzed. First, capsids in cytoplasmic lysates from cells transfected with the indicated constructs were separated by NAGE and, after blotting, were subjected to immunodetection of capsids or molecular hybridization with a (+)-polarity probe specific for HBV (−)-DNA (genome positions 837--1284). As shown in [Fig. 4A](#pone-0072798-g004){ref-type="fig"}, all samples contained capsids but only those from the wild-type HBV transfected cells gave a comigrating distinct DNA signal. The lower sensitivity of this assay compared to the Southern blot likely relates, apart from the shorter size of the single-stranded probe, to the fact that in the NAGE assay much smaller fractions of cytoplasmic lysate are employed than were used to isolate capsid-associated DNA for Southern blotting. Clearly, however, the mutant-derived capsids contained much less DNA than wild-type HBV capsids. As a more sensitive test, we next used the endogenous polymerase assay (EPA) in which the capsid-associated P protein extends immature DNAs upon provision of exogenously added dNTPs, one of which is ^32^P labeled. To this end, one aliquot each of the respective cytoplasmic lysates was separated by NAGE for anti-capsid immunoblotting, two other aliquots were prior to NAGE incubated with either α-^32^P-dATP or α-^32^P-dCTP plus the lacking three other dNTPs. A replication-defective P protein mutant in which the protein-priming Tyr63 residue was replaced by Phe (Y63F) was included as further negative control in some experiments. As shown in [Fig. 4B](#pone-0072798-g004){ref-type="fig"}, all samples contained capsids, and both α-^32^P-dCTP and α-^32^P-dATP labeled EPA assays gave comparable results; wild-type capsids yielded strong signals, the mut and Y63F variants no signal, and the signals from the respective mutants, quantitated by phosphorimaging, correlated well with those obtained by Southern blotting ([Fig. 4C](#pone-0072798-g004){ref-type="fig"}).
{#pone-0072798-g004}
Together these data documented that mutations at the A1, A2, A9, A10 positions in the upper stem and at the U13 and U15 positions in the apical loop can, but do not necessarily have to, negatively affect viral DNA accumulation in capsids. While a more detailed interpretation is provided in the Discussion, we note that canonical Watson-Crick base-pairing of the residues at the A1/A2 positions immediately following the bulge is not required for efficient DNA synthesis, as shown by several wild-type-like behaving mutants in which the natural A1+A2 pairing with U28+U29 was absent (e.g. A1U; U28,29A). Similarly, there was no direct correlation between DNA synthesis and the presence of the natural A9+A10 pairing with U19/U20 (e.g. A9C; A9,10U). However, low levels of viral DNA could not only relate to impaired DNA synthesis but also defects in encapsidation of the pgRNA template.
Differential impact of upper stem versus apical loop mutations on pgRNA encapsidation {#s2c}
-------------------------------------------------------------------------------------
Based on the DNA analyses ([Fig. 3](#pone-0072798-g003){ref-type="fig"} and [Fig. 4](#pone-0072798-g004){ref-type="fig"}), we next analyzed variants with strongly reduced viral DNA levels for pgRNA encapsidation; wild-type HBV and the mut variant served as references. To this end, we employed RNase protection assays to compare the levels of pgRNA contained in cytoplasmic nucleocapsids versus in total cytoplasmic RNA preparations. As shown in [Fig. 5](#pone-0072798-g005){ref-type="fig"}, wild-type HBV-like amounts of pgRNA were detected in all total RNA preparations and also in most capsid-associated RNA samples, with two exceptions; the P binding defective mut variant showed no signal and the apical loop double-mutant U13,15A a substantially (by two thirds) weakened signal. This was confirmed by semiquantitatively, via phosphorimaging, relating the signal intensities of capsid RNA versus total RNA in each sample. Accordingly, the apical loop double mutant was the only one for which the low levels of detectable viral DNA ([Fig. 3](#pone-0072798-g003){ref-type="fig"} and [Fig. 4](#pone-0072798-g004){ref-type="fig"}) correlated with substantially reduced pgRNA packaging; however, the defect in DNA synthesis appeared more pronounced. For the A1/A2 and A9/A10 mutants, in contrast, factors other than impaired pgRNA encapsidation must be responsible for the severely reduced DNA accumulation.
{#pone-0072798-g005}
Assessment of upper-stem and apical loop mutants as protein-priming templates {#s2d}
-----------------------------------------------------------------------------
The identification of several ε mutants that did not accumulate viral DNA despite intact pgRNA encapsidation suggested that their defects related to impaired protein-priming, or impaired primer translocation to DR1\*, preventing generation of full-length minus-strand and eventually RC-DNA. Until recently, HBV protein-priming could not be addressed in vitro. Jones et al. [@pone.0072798-Jones1] have now partly overcome this problem by affinity enrichment of FLAG-tagged P protein - ε RNA complexes (plus associated cell factors) from HEK293T cells co-transfected with P protein plus ε RNA expression vectors. The immobilized complexes were capable of in vitro protein-priming when supplied with dNTPs, as detectable by covalent radiolabeling of P protein when α-^32^P-dNTPs are used. We therefore applied an analogous system to address the priming competence of various mutants that had shown severe defects in DNA accumulation. As a first test, we compared P protein expressed in the absence or presence of co-expressed wild-type ε RNA. As shown in [Fig. 6A](#pone-0072798-g006){ref-type="fig"}, clearly detectable P protein labeling upon provision of α-^32^P-dATP required ε RNA co-expression as reported [@pone.0072798-Jones1], and the signal was enhanced by doubling the amount of α-^32^P-dATP (lanes P + ε vs. lane P). The presence of P protein was confirmed by anti-FLAG immuno blotting. Next, analogous assays were performed with complexes from cells co-transfected with the P protein expression vector plus one each of the vectors for the indicated eight mutant ε RNAs. As shown in [Fig. 6B](#pone-0072798-g006){ref-type="fig"}, the wild-type RNA but not the P binding-defective mut variant produced a clear signal whereas for the mutants a wide range of signal intensities was seen. Signals for the double mutants A1,2C and U13,15A were too weak for quantification. The single A1 and A9 mutants A1G and A9U showed weak but distinct bands; band intensity was increased for the U13 mutant U13A and almost reached wild-type levels for the A9/10 double mutants A9,10G and A9,10C; these differences were not due to different amounts of P protein, as shown by anti-FLAG immunoblotting ([Fig. 6B](#pone-0072798-g006){ref-type="fig"}, bottom panel).
![In vitro priming activities of selected ε RNAs showing reduced DNA accumulation in cells.\
(A) Increased priming signals by co-expression of wild-type ε RNA and increased α-^32^P-dATP concentration. FLAG-tagged HBV P protein from cells transfected with only the P protein vector (lane P), or cotransfected with a wild-type ε RNA expression vector (lanes P + ε) was immobilized on anti-FLAG antibody beads. One third each of the immunoprecipitate was incubated with 1 µl or 2 µl of α-^32^P-dATP (3,000 Ci/mmol) as reported [@pone.0072798-Jones1]; subsequently, the beads were boiled in SDS-PAGE sample buffer, and the released material was analyzed by SDS-PAGE and autoradiography. The remaining one third of the immunopellet was analyzed for FLAG-tagged P protein by Western blotting (panel anti-FLAG). (B) In vitro priming activities of selected ε RNA variants. FLAG-tagged P protein complexes with the indicated ε RNAs were expressed, affinity purified and subjected to in vitro priming conditions as in (A), using 2 µl α-^32^P-dATP. The molecular mass marker positions indicated on the left (in kDa) are approximations inferred from the respective marker protein positions on the SDS-PAGE gels used for the anti-FLAG immunoblots which were run in parallel under identical conditions. Numbers below the autoradiogram indicate mean signal intensities ± standard deviation from two independent experiments relative to that produced by the wild-type ε RNA complex which was set to 100.](pone.0072798.g006){#pone-0072798-g006}
Assuming that the quality of the P protein in the nine different preparations was comparable (because ε RNA added after complex isolation from the cells is not accepted as template [@pone.0072798-Jones1], individual preparations are required), and if the results obtained with dATP also apply to the other dNTPs, these data suggest that failure of DNA accumulation for the A1/A2 and U13/U15 mutants results from a general impairment of protein-priming, whereas the A9/A10 mutations and the U13A mutation impede a step following addition of the first nucleotide but preceding primer translocation to the proper acceptor site at DR1\* (see [Fig. 1](#pone-0072798-g001){ref-type="fig"}, and Discussion); as we have not directly determined RNA encapsidation efficiency for the U13A variant, a limited contribution of reduced RNA packaging to lowered DNA accumulation is currently not excluded.
Altogether, these data revealed that seemingly minor changes in the ε sequence can have more differentiated impacts on the different functional aspects of the HBV P protein - ε interaction than suggested by previous studies.
Discussion {#s3}
==========
In this study we have systematically investigated the impact of mutations at six strategically located positions in the upper stem and apical loop of the HBV ε signal with respect to viral DNA accumulation, pgRNA packaging, and in vitro priming activity. The individual mutants and their functional phenotypes are summarized in [Table 1](#pone-0072798-t001){ref-type="table"}. The data revealed that seemingly minor changes in sequence caused several divergent replication phenotypes, ranging from virtually no impact over modest to massive reductions in viral DNA accumulation; as discussed below, these defects in DNA synthesis correlated with distinct defects in the preceding steps that depend on a productive P protein - ε RNA interaction.
10.1371/journal.pone.0072798.t001
###### Sequences of investigated ε mutants and summary of phenotypes.
{#pone-0072798-t001-1}
Designation upper stem (left) loop upper stem (right) [a)](#nt101){ref-type="table-fn"}DNA accumulation [a)](#nt101){ref-type="table-fn"}EPA activity ^b)^Full-length (FL) or Short (S) DNA [a)](#nt101){ref-type="table-fn"}pgRNA packaging [a)](#nt101){ref-type="table-fn"}in vitro priming (dATP)
------------- ------------------- ---------- -------------------- --------------------------------------------------- ----------------------------------------------- --------------------------------------- -------------------------------------------------- ----------------------------------------------------------
wt AAGCCUCCAAG CUGUGC CUUGGGUGGCUU +++ +++ FL +++ +++
A1U U\...\...\.... \...\... \...\...\...\... +++ nd FL nd nd
A1G G\...\...\.... \...\... \...\...\...\... +/− +/− FL +++ +/−
A1C C\...\...\.... \...\... \...\...\...\... ++ nd FL nd nd
A1,2U UU\...\...\... \...\... \...\...\...\... +++ nd FL nd nd
A1,2G GG\...\...\... \...\... \...\...\...\... +/− nd FL nd nd
A1,2C CC\...\...\... \...\... \...\...\...\... +/− +/− FL +++ --
U29A \...\...\..... \...\... \...\...\.....A \+ nd FL nd nd
U28,29A \...\...\..... \...\... \...\...\....AA ++ nd FL nd nd
rb1UA U\...\...\.... \...\... \...\...\.....A +++ nd FL nd nd
rb1GC G\...\...\.... \...\... \...\...\.....C ++ nd FL nd nd
rb1,2UA UU\...\...\... \...\... \...\...\....AA ++ nd FL nd nd
rb1,2GC GG\...\...\... \...\... \...\...\....CC +++ nd FL nd nd
A9U \...\.....U.. \...\... \...\...\...\... \+ \+ S +++ \+
A9G \...\.....G.. \...\... \...\...\...\... +++ nd FL nd nd
A9C \...\.....C.. \...\... \...\...\...\... +++ nd FL nd nd
A9,10U \...\.....UU. \...\... \...\...\...\... +++ nd FL nd nd
A9,10G \...\.....GG. \...\... \...\...\...\... +/− +/− S +++ ++
A9,10C \...\.....CC. \...\... \...\...\...\... +/− +/− S +++ +++
U13A \...\...\..... .A\.... \...\...\...\... +/− nd (FL+) S nd ++
U13G \...\...\..... .G\.... \...\...\...\... ++ nd FL nd nd
U13C \...\...\..... .C\.... \...\...\...\... ++ nd FL nd nd
U13,15A \...\...\..... .A.A.. \...\...\...\... +/− +/− (FL+) S \+ --
U13,15G \...\...\..... .G.G.. \...\...\...\... +++ nd FL nd nd
U13,15C \...\...\..... .C.C.. \...\...\...\... +++ nd FL nd nd
mut \...\...GGUU. \...\... \...\...\...\... -- -- none +/− --
Values are categorized relative to wild-type HBV (100%) as follows: +++, 66--100%; ++, 33--65%; +, 25--32%; +/−, 10--24%; −, not detectable; nd, not determined. ^b)^ Derived by visual inspection of the autoradiograms shown in [Fig. 3](#pone-0072798-g003){ref-type="fig"}.
Tolerated upper stem mutations {#s3a}
------------------------------
In natural HBV isolates, the ε sequence is one of the most highly conserved regions [@pone.0072798-Lok1], [@pone.0072798-Sun1], as confirmed in a recent ultra-deep sequencing study [@pone.0072798-Homs1]. Mutations that affect the ε structure, e.g. stop codons in the overlapping preC ORF which prevent precore translation and HBeAg formation, are almost invariably accompanied by additional compensatory mutations that restore the genuine stem-loop architecture. The HBV ε sequence is also largely conserved in the other mammalian hepadnaviruses; in contrast, especially the upper stem is much more variable between avian hepadnaviruses [@pone.0072798-Hu4], and DHBVs with various mutations in the upper ε stem are viable even in ducks [@pone.0072798-Schmid1]. Hence a reason for the strict conservation of ε amongst the mammalian viruses is not obvious. The ten HBV ε mutants from our study which showed no substantial defect in DNA accumulation indicate that the authentic ε sequence is not a prerequisite for productive interactions with P protein; notably, tolerated mutations were found at all investigated positions, including the structured apical loop [@pone.0072798-Flodell1], [@pone.0072798-Flodell2]. Hence natural sequence conservation may be driven by minor differences in replication efficiency that come to bear only upon continuous genome propagation in vivo but not in single round transfection studies [@pone.0072798-Schmid1]. Candidate factors beyond the immediate P - ε interaction might be the overlapping preC ORF and its translation products, or cis-elements that act after the initial priming step. For instance, base-pairing between the left half-stem of ε and the downstream φ element appears to contribute to replication efficiency [@pone.0072798-Abraham1], [@pone.0072798-Abraham2], [@pone.0072798-Oropeza1]. However, the A1 and U13/U15 positions mutated are not, and the A2 and A9/A10 positions are only peripherally involved in this base-pairing ([Fig. S1](#pone.0072798.s001){ref-type="supplementary-material"}), making a substantial contribution to the poor DNA accumulation of mutants such as A9,10G or A9,10C unlikely.
Impact of the A1/A2 positions and their involvement in base-pairing {#s3b}
-------------------------------------------------------------------
Residues A1 and A2 immediately follow the template region in the ε bulge, and A1 [@pone.0072798-Lanford1], [@pone.0072798-Nassal3] may as well as the preceding C residue [@pone.0072798-Jones1] serve as initiation template for the DNA oligonucleotide primer ([Fig. 1](#pone-0072798-g001){ref-type="fig"}); hence A1 and A2 would be expected to most directly affect protein-priming. Indeed, the single A1G exchange caused a drastic reduction in DNA accumulation ([Fig. 3](#pone-0072798-g003){ref-type="fig"}, [Fig. 4](#pone-0072798-g004){ref-type="fig"}) and gave an extremely weak in vitro priming signal with ^32^P-dATP ([Fig. 6](#pone-0072798-g006){ref-type="fig"}) although it did not negatively impact pgRNA encapsidation ([Fig. 5](#pone-0072798-g005){ref-type="fig"}). In contrast, the analogous A1C exchange had only a modest, and the A1U mutation no detectable influence on DNA accumulation. The corresponding A1/A2 double mutations to GG and UU (A1,2G and A1,2C) showed the same phenotypes as the single G and U mutations, whereas the A1/A2 CC double exchange strongly enhanced the negative impact of the single A1C mutation, both in DNA accumulation ([Fig. 3](#pone-0072798-g003){ref-type="fig"}) and ^32^P-dATP priming ([Fig. 6](#pone-0072798-g006){ref-type="fig"}); however, it also did not interfere with pgRNA encapsidation ([Fig. 5](#pone-0072798-g005){ref-type="fig"}), i.e. a packaging-proficient interaction with P was maintained.
One possibility to explain these different phenotypes was the importance of the base-pairs closing the ε bulge. For instance, preventing A-U pair formation by combining the authentic A1 or A1 plus A2 residues with A substitutions at the opposite U28 and U29 positions (U29A; U28,29A) reduced DNA accumulation similarly as the A1C exchange ([Fig. 3](#pone-0072798-g003){ref-type="fig"}) although the sequence of the template region was preserved; conversely, the strong replication defects caused by the A1G and A1A2\>GG (A1,2G) mutations were partially or even completely rescued by replacing U29 (rb1GC) or U28 and U29 (rb1,2GC) by C residues.
However, the other mutants suggest a more complex correlation. Mutants containing U residues at A1 or A1+A2 (A1U; A1,2U) displayed wild-type-like DNA accumulation although base-pairing with U28/U29 was prevented ([Fig. 3](#pone-0072798-g003){ref-type="fig"}). Replacing the original A1-U29 basepair by a U-A pair (rb1UA) had no negative effect but replacing both the A1-U29 and A2-U28 pair by U-A (rb1,2UA) reduced DNA accumulation.
These different data sets may be reconciled by the negatively acting mutations disturbing, to different extents, formation of a priming-compatible structure of the template region in the bulge by inducing non-productive alternative structures ([Fig. 7A](#pone-0072798-g007){ref-type="fig"}). For instance, a G at the A1 position could pair with the 5′ terminal C of the bulge at the cost of the weak A1G-U29 pair; such an improper cross-bulge base-pairing would be partially counteracted by keeping A1G paired within the upper stem by the U29C mutation (rb1GC), and to an even larger extent in the quadrupel mutant A1A2\>GG plus U28U29\>CC (rb1,2GC). A similar case can be made for improper base-pairing of the A1C and A1,2C mutants to the G residue at the third bulge position. Conversely, with U residues at the A1 and A2 positions no inappropriate cross-bulge base-pairings could occur for lack of A residues in the bulge. Obviously, the current data do not yet prove this model but they provide a conceptual framework for future experiments; particularly revealing should be a direct structural comparison of the corresponding RNAs in their free and P protein-bound state, as previously applied to the DHBV P protein - ε RNA interaction [@pone.0072798-Beck4].
{#pone-0072798-g007}
Impact of the A9/A10 positions at the tip of the upper stem {#s3c}
-----------------------------------------------------------
The A9 and A9+A10 mutants also presented with complex phenotypic DNA patterns. Single replacements of A9 by G (A9G) or C (A9C) were completely tolerated, replacement by U (A9U) was not; in contrast, concomitant substitution of A10 by U (A9,10U) restored function of the A9U mutant whereas two consecutive G (A9,10G) or C (A9,10C) mutations nearly ablated DNA accumulation. Hence the functional impact of individual mutations is highly context-dependent; rather than the mere number of base-pairs that context appears to define whether or not individual nucleotides will be available for productive interactions with P protein or not. Again, elucidating the structures of the free versus P protein-bound RNAs should provide new insights, yet even with the new in vitro priming system this will remain a difficult task because the ε RNA in the P protein - RNA complexes isolated from the transfected cells can not be exchanged in vitro [@pone.0072798-Jones1].
However, already our current data imply that the mechanism by which A9/A10 mutations interfere with DNA accumulation differs from that exerted by the A1/A2 mutations. For the latter mutants, the reduced DNA signals in the Southern blot still extended up to the full-length position ([Fig. 3](#pone-0072798-g003){ref-type="fig"}, A1G, A1,2G, A1U) whereas for the A9/A10 mutants the bulk of products migrated faster, with little or no signal at the full-length position ([Fig. 3](#pone-0072798-g003){ref-type="fig"}, A9U, A9,10G, A9,10C). Furthermore, the corresponding A9/A10 mutants displayed modest (A9U) or even strong (A9,10G; A9,10C) in vitro priming activity ([Fig. 6](#pone-0072798-g006){ref-type="fig"}) whereas very low or no signals were seen for the A1/A2 mutants A1C and A1,2C ([Fig. 6](#pone-0072798-g006){ref-type="fig"}). An interpretation in line with these data ([Fig. 7B](#pone-0072798-g007){ref-type="fig"}) is that mutations at A1/A2 interfere with protein-priming per se, whereas A9/A10 mutations allow priming but negatively impact subsequent primer translocation to DR1\* and/or minus-strand DNA extension. One scenario is that A9/A10 mutations affect initiation site selection, such that the sequence or length of the protein-primed DNA oligonucleotide are incompatible with the genuine DR1\* acceptor; as previously shown, non-DR1\* matching primers can be translocated to alternative acceptor sites [@pone.0072798-Nassal3], [@pone.0072798-Abraham2], preventing full-length minus-strand DNA and subsequent RC-DNA synthesis. Another scenario results from the requirement for minus-strand DNA extension to replace ε as template by the pgRNA sequence preceding the DR1\* acceptor. Hence mutations in ε that enhance binding to P may impede this template replacement. Again the current data do not allow firm mechanistic conclusions but they define a specific set of mutants for future more thorough investigation, in particular definition of the exact DNA sequences that are linked to P protein in the faster migrating products.
Impact of the U residues in the apical loop {#s3d}
-------------------------------------------
Earlier studies indicated that multiple simultaneous loop mutations severely affected DNA accumulation, mostly via impeding pgRNA encapsidation [@pone.0072798-Pollack2], [@pone.0072798-Fallows1], [@pone.0072798-Nassal3]. Single site mutations, such as variants L1 to L6 (named according to the position in the CUGUGC loop sequence [@pone.0072798-Pollack2]; U13 and U15 in our numbering correspond to L2 and L4) produced differing phenotypes, ranging from virtually no impact (L1, L2 and L6) to a substantial reduction (L4 and L5) or complete loss (L3) of encapsidation competence (measured using fusions of the ε sequence to heterologous reporter RNAs). However, only one specific replacement for each loop position was analyzed. In principal accordance, we found an only modest impact on DNA accumulation of replacing U13 by A (U13A, identical to the reported L2 mutation in [@pone.0072798-Pollack2]), and even less so by G or C substitutions (U13G and U13C). The U13A mutant also displayed substantial α-^32^P-dATP in vitro priming activity ([Fig. 6](#pone-0072798-g006){ref-type="fig"}). The strongest negative impact on DNA accumulation was seen for the U13/U15 double A mutation (U13,15A), which also yielded no detectable in vitro priming signal ([Fig. 6](#pone-0072798-g006){ref-type="fig"}). Moreover, this was the only of the tested variants with clearly reduced pgRNA encapsidation capacity ([Fig. 5](#pone-0072798-g005){ref-type="fig"}), suggesting a generally reduced ability to interact with P protein.
More surprising is the only modest or even lacking impact on DNA accumulation exerted by the U13G and U13C variants and the U13,15G and U13,15C double mutants; based on simple Watson-Crick base-pairing one might expect these mutations to abrogate the intricate pseudo-triloop structure which lends itself as a highly specific recognition element. However, as in many triloops, the non-planar arrangement of the loop residues, and often also of the closing base-pair, allows for unconventional pairings between all four bases [@pone.0072798-Lisi1]. In addition, numerous different intraloop interactions can occur, as is the case for the HBV ε apical loop ([@pone.0072798-Flodell2] and PDB: 2IXY) and also in numerous triloop sequences found in other RNA 3D structures [@pone.0072798-Schudoma1]. Conceivably then, formation of a pseudo-triloop structure may not be restricted to the genuine ε loop sequence. In particular our fully functional loop mutants may therefore be attractive candidates for future structural studies by NMR. Notably, ultra-deep sequencing revealed a low frequency of four UGU variants in patient-derived HBV sequences, including one identical to our U13C mutant [@pone.0072798-Homs1] and one similar to U13,15C, except that U13 was preserved (corresponding to U15C in our nomenclature). Although ultra-deep sequencing may pick up nonfunctional sequences the near wild-type-like amounts and patterns of viral DNA seen for the U13C and the U13,15C mutants ([Fig. 3](#pone-0072798-g003){ref-type="fig"}) is in line with in vivo viability of these variants.
Interestingly, the loop mutants U13A and U13,15A displayed an intermediate DNA pattern between A1/A2 and A9/A10 mutants, i.e. some full-length products were formed but the bulk of signals accumulated further down in the gel, though not as low as for the partially defective A9/A10 variants ([Fig. 3](#pone-0072798-g003){ref-type="fig"}). This would be in line with a contribution of the loop to initiation site selection (see above).
Conclusions {#s4}
===========
The interaction between HBV P protein and ε is key to virus replication and thus also an attractive target for intervention. However, in the absence of direct structural data on P protein, let alone active P protein - ε complexes, knowledge of this interaction is still limited. Our mutational analysis of different manifestations of the interaction, i.e. DNA synthesis, pgRNA encapsidation and in vitro protein-priming, substantially extends previous investigations. Given the dependence of RNA structure on sequence and the highly dynamic nature of the P protein - ε RNA interaction (see below), it is not surprising that our study revealed a whole range of phenotypes but no simple correlation allowing to predict how a specific mutant will behave. However, several trends were observed that should be valuable in designing further experiments, including attempts to target the P - ε interaction for therapeutic intervention. First, our identification of numerous mutations that had little impact on replication suggests a substantial sequence space for escape mutants. Second, our data give hints as to which subregions in ε tend have the largest (though not exclusive) impact on general P binding, priming efficiency, and possibly initiation site selection; to our knowledge, this is the first demonstration that these different functional aspects of the P - ε interaction can be genetically uncoupled, which also paves new ways towards more effective ε RNA decoys. Third, our data raise basic structural issues, especially the tolerance of the apical pseudo-triloop to even double mutations although the complex structure of this subelement should make a highly specific recognition element. These variant RNAs therefore lend themselves for further direct structural investigation.
Lastly, the diversity of phenotypes, from no impact at all to interference with distinctly different steps towards DNA synthesis, suggests that each step requires a distinct conformation of the RNA in the P protein complex, or in other words, that there are more conformational states than just non-productive binding (exemplified by in vitro P - ε binding without priming [@pone.0072798-Hu5]) and a priming-competent interaction. Accordingly, individual nucleotides within ε may have dynamically altering contact patterns with P protein, each specific for a specific stage from initial binding over pgRNA encapsidation and priming until the eventual replacement of ε upon primer translocation to DR1\*. Possibly the wild-type ε sequence provides the optimal solution to fully comply with these changing requirements.
Materials and Methods {#s5}
=====================
The sequences of the oligonucleotides used to introduce the indicated mutations into ε and for RT-PCR of β-actin mRNA are given in [Table S1](#pone.0072798.s002){ref-type="supplementary-material"}.
Prediction for the 3D structure of ε {#s5a}
------------------------------------
The 3D structure of ε was modeled using programs MC-Fold and MC-Sym [@pone.0072798-Parisien1], including experimental NMR data (PDB entry: 2IXY) of the apical stem-loop of ε [@pone.0072798-Flodell2].
Plasmid constructs {#s5b}
------------------
The full-length HBV expressing vectors carrying mutant ε sequences were generated by site-directed mutagenesis. First, synthetic oligonucleotides ([Table S1](#pone.0072798.s002){ref-type="supplementary-material"}) carrying the desired nucleotide exchanges ([Fig. 2B](#pone-0072798-g002){ref-type="fig"}) were used as PCR templates, then the appropriately restricted PCR products were transferred into the Eco RV - Hind III sites of pCH-9/3091B, a previously constructed HBV vector carrying a deletion in the 5′-ε sequence [@pone.0072798-Feng1], thus restoring an analogous arrangement as in the wild-type HBV expressing vector pCH-9/3091 [@pone.0072798-Nassal4]. The pcDNA3.1--3FlagP expression vector for HBV P protein with an N-terminal 3×FLAG tag was constructed by insertion of the P protein coding sequence into the Hind III/Xho I sites of pcDNA3.1(+) (Invitrogen), followed by insertion of the 3xFLAG coding sequence between the Nhe I and Hind III restriction sites. pcDNA3.1-ε, expressing the HBV ε sequence [@pone.0072798-Jones1], was constructed by insertion of the wild-type ε sequence into the Kpn I/Apa I sites of pcDNA3.1(+). Vectors for mutant ε sequences were constructed by using pcDNA3.1-ε as PCR template for site-directed mutagenesis (QuikChange™ Site-Directed Mutagenesis Kit, Stratagene). All resulting constructs were confirmed by sequencing the relevant regions on the plasmids.
Transfection and isolation of core particles {#s5c}
--------------------------------------------
Transfections of HepG2 hepatoma cells with pCH-9/3091 and its derived constructs, and isolation of cytoplasmic core particles were performed as previously described [@pone.0072798-Feng1]. Transfections of HEK293T cells with pcDNA3.1-3FlagP alone, or together with pcDNA3.1--ε or individual pcDNA3.1-ε mutants were performed exactly as described in [@pone.0072798-Jones1].
Southern blotting {#s5d}
-----------------
To analyze HBV DNAs by Southern blotting, cytoplasmic core DNA was extracted, separated by agarose gel electrophoresis and, after blotting, hybridized to a ^32^P-labeled random-primed probe covering the whole HBV genome as previously described [@pone.0072798-Feng1].
To directly analyze viral DNA content of nucleocapsids, intact cytoplasmic core particles were separated by 1% native agarose gel electrophoresis (NAGE) before blotting on nylon membranes by capillary transfer. The membranes were first soaked with denaturing buffer (0.2 M NaOH, 1.5 M NaCl) for 15 sec to release DNA from capsids, and then were neutralized by exposure to 0.2 M Tris/HCl (pH 7.5) supplemented with 1.5 M NaCl for 15 sec. After washing with distilled water, the membranes were cross-linked by UV irradiation and hybridized to a (+)-polarity HBV probe generated by nonsymmetrical PCR amplification of nucleoties 837--1284 of the HBV DNA, using Eco RI linearized plasmid pCH-9/3091 as template, a (+)-primer (5′-CTAGACACTATTTACACACT-3′) and dNTP mix containing α-^32^P-dATP (3,000 Ci/mmol).
RNase protection assay (RPA) {#s5e}
----------------------------
To analyze encapsidated pgRNA, core pgRNA was extracted from an aliquot equivalent to four-fifth of the transfected cells from a 10 cm diameter plate as previously described [@pone.0072798-Kim1]. To analyze cytoplasmic pgRNA, total RNA from the remainder one-fifth transfected cells of a 10 cm diameter plate was extracted with Trizol (Invitrogen). Both RNA fractions were then subjected to RNase protection assays using the RPA II kit (Ambion). The probe consisted of a 314 nucleotide antisense transcript comprising about 270 nucleotides complementary to HBV positions 3271--3282/1--261, and 41 nucleotides of non-matching polylinker sequence. All steps were performed as recommended by the manufacturer.
Endogenous polymerase assay (EPA) {#s5f}
---------------------------------
EPA was performed as previously described [@pone.0072798-Feng1] by incubating cytoplasmic core particles at 37° C for 6h with α-^32^P-dCTP (3,000 Ci/mmol) or α-^32^P-dATP (3,000 Ci/mmol) supplemented with a mixture of the other three dNTPs. The reaction products were separated on a 1% agarose gel and then subjected to dry gel autoradiography.
In vitro priming assay {#s5g}
----------------------
Two days post-transfection, the FLAG-tagged P proteins, expressed alone or co-expressed with wild-type or mutant ε RNAs, were immobilized to anti-FLAG beads as described [@pone.0072798-Jones1]. An aliquot equivalent to one-third of the purified complex was used for in vitro priming as reported, with minor modifications. Briefly, 38 µL TMgNK buffer (20 mM Tris/HCl \[pH 7.0\], 15 mM NaCl, 10 mM KCl, 4 mM MgCl~2~) supplemented with protease inhibitors (1×EDTA-free protease inhibitor cocktail and 1 mM PMSF), DTT (4 mM) and RNasin Plus RNase inhibitor (1 U/ µL) was added after the FLAG lysis buffer was removed from the bead-bound P protein. 1 or 2 µL α-^32^P-dATP (3,000 Ci/mmol) was then added. After incubating at 25° C for 4 h with shaking, the beads were washed with TNK buffer (20 mM Tris/HCl \[pH 7.0\], 15 mM NaCl, 10 mM KCl) supplemented with protease inhibitors and β-ME. Next the beads were boiled in sample buffer and the released components were analyzed by SDS-PAGE in 10% polyacrylamide gels and subsequent dry gel autoradiography.
Western blotting {#s5h}
----------------
Western blotting for core protein and capsids was conducted as described in [@pone.0072798-Feng1].
Quantitative evaluations {#s5i}
------------------------
Signal intensities from Southern blots, endogenous polymerase assays and RNase protection assays were determined by phosphorimaging using OptiQuant 5.0 software (Perkin Elmer), and related to the respective signals from wild-type HBV transfected cells as detailed in the legends of the corresponding figures. Data were derived from at least two, and usually three independent transfections.
Supporting Information {#s6}
======================
######
**Limited impact of mutations at upper stem positions A1, A2, A9, A10, U13 and U15 on ε - φ base-pairing.**
(PDF)
######
Click here for additional data file.
######
**Oligonucleotides used in this study.**
(PDF)
######
Click here for additional data file.
We thank Ms. Weiwei Wang and Ms. Yun Gan for excellent technical assistance in cell culture and transfection, and Dr. Jianming Hu (Pennsylvania State University) for helpful experimental discussions regarding the *in vitro* priming assay.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: KH MN HF. Performed the experiments: HF PC FZ KH. Analyzed the data: KH MN HF. Contributed reagents/materials/analysis tools: KH MN PC FZ. Wrote the paper: MN KH HF.
[^3]: Current address: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
| 2024-01-17T01:26:19.665474 | https://example.com/article/7634 |
Q:
My code throws stackoverflowexception when i execute
I am working on a Windows Application Development in c# language.
I have a template form => Masterpage which is inherited by all my other forms.
It has menustrip to open all other forms as well:
my error reads:
System.StackOverflowException
HResult=0x800703E9
Source=<Cannot evaluate the exception source>
StackTrace:
<Cannot evaluate the exception stack trace>
my Masterpage code is:
namespace WindowsProject
{
public partial class Masterpage : Form
{
BookEditor bookE;
StudentEditor studentE;
ViewBorrowedBooks viewB;
IssueBook issueBook;
public Masterpage()
{
InitializeComponent();
bookE = new BookEditor();
studentE = new StudentEditor();
viewB = new ViewBorrowedBooks();
issueBook = new IssueBook();
}
private void exitToolStripMenuItem_Click(object sender, EventArgs e)
{
Application.Exit();
}
private void showForm(Form f)
{
bookE.Hide();
studentE.Hide();
viewB.Hide();
issueBook.Hide();
this.Hide();
f.Show();
}
private void booksToolStripMenuItem_Click(object sender, EventArgs e)
{
showForm(bookE);
}
private void studentsToolStripMenuItem_Click(object sender, EventArgs e)
{
showForm(studentE);
}
private void viewBorrowedBooksToolStripMenuItem_Click(object sender, EventArgs e)
{
showForm(viewB);
}
private void issueBookToolStripMenuItem_Click(object sender, EventArgs e)
{
showForm(issueBook);
}
}
}
all my other forms inherits from Masterpage
A:
Why does StackOverflowException occur?
StackOverflowException occurs because of the infinite recursion inside the constructor of the Masterpage.
Lets consider how this recursion occurs. BookEditor inherits Masterpage. (The following conclusions are also applicable to the other inheritors of the Masterpage). I suspect that BookEditor has a single constructor without parameters BookEditor(). In C# if you call constructor without parameters of the derived type it by default calls constructor without parameters of the base type (if you don't explicitly specify other constructor of the base type). Lets assume that we want to create an instance of the Masterpage. We call its constructor Masterpage(). Inside it we call constructor of the BookEditor. But BookEditor inherits Masterpage and therefore it calls constructor of the Masterpage. Constructor of the Masterpage again calls constructor of the BookEditor and etc. These sequence of the constructor calls leads to the infinite recursion and StackOverflowException as a result.
How should you fix this problem?
I think that more information is needed to thoroughly answer this question.
As a workaround you should create inheritors of the Masterpage outside of its constructor:
public partial class Masterpage : Form
{
private readonly IEnumerable<Form> _forms;
public Masterpage()
{
InitializeComponent();
}
// Now we set forms here, not in the constructor.
public void SetForms(IEnumerable<Form> forms)
{
_forms = forms;
}
private void showForm(Form f)
{
foreach (Form form in _forms)
form.Hide();
this.Hide();
f.Show();
}
// Other members of the Masterpage.
...
}
// Palce this code where you create and show Masterpage.
var bookE = new BookEditor();
var studentE = new StudentEditor();
var viewB = new ViewBorrowedBooks();
var issueBook = new IssueBook();
var forms = new List<Form> {bookE, studentE, viewB, issueBook};
bookE.SetForms(forms);
studentE.SetForms(forms);
viewB.SetForms(forms);
issueBook.SetForms(forms);
var masterPage = new Masterpage();
masterPage.SetForms(forms);
| 2023-11-11T01:26:19.665474 | https://example.com/article/4073 |
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