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Q:
How is this question on topic?
Where in the solar system is the most viable place to put my colonists, after Mars and Luna?
I get that this is a fun question that allows people to contribute and that is why it gets so many votes. But that question feels like it is totally against the whole spirit of this site. It is not about world building but rather just a fun question for people to speculate on. But it really does not belong here. And if we continue to allow these types of questions this site will become cluttered with the type of nonsense that most internet forums are. I would hope that is a fate that we want to avoid.
A:
The text of the question (at present) is:
Mars and Earth's moon already feature in a lot of fiction about space colonization. After these two planets, and assuming that other solar systems are too far away, what is the best place within this solar system to colonize?
The first sentence establishes the context of fiction about space colonization, so I think it's pretty clear that this question is asking about building out a fictional scenario. That's on-topic. But it could be stronger; it would be better to cast the question not as "which is the most viable place..." but, rather, "where in the solar system can I put my colonists to minimize risks to them?". That places the emphasis more clearly on the problem to be solved -- I have colonists I want to put somewhere in this solar system.
I don't think the current question is off-topic. I'm going to propose the edit (of the title) that I've just described here. (Update: edit accepted.)
A:
It's on topic - for the simple reason that it's about creation of a setting. If someone is creating a setting involving colonizing the solar system then part of building that world is deciding where would next be colonized...
| 2024-01-05T01:27:17.760571 | https://example.com/article/6204 |
Keep Unwanted Guests Out of Your Home This Fall with the Best Pest Control in Orillia
There is a lot you can do to keep away pests from your home. But, if push comes to shove, you can just give the best pest control in Orillia to do it for you.
As summer fades into fall, everyone begins to prepare for the season to stay warm and cozy. Our furry counterparts are doing the same. Rodents begin searching for a nice place to call home to weather the storms and brisk temperatures that are approaching us. The best way to solve a pest problem is by proactively preventing one. If you have the best in pest control in Orillia, you can have a few ways to prepare your home for pest invasions before they get the chance to sneak in.
Remove Ideal Habitats Close to Your Home.
The further you can keep them away the better. Rodents are sure to migrate towards an area that will provide them with shelter to hide from predators, give them access to food and areas that provide warmth and moisture for reproduction. | 2024-05-28T01:27:17.760571 | https://example.com/article/9831 |
Q:
Unable to start container using kubectl
I am learning kubernetes and using minikube to create single node cluster in my ubuntu machine. In my ubuntu machine Oracle Virtualbox is also installed. As I run
$ minikube start
Starting local Kubernetes v1.6.4 cluster...
...
$ cat ~/.kube/config
apiVersion: v1
clusters:
- cluster:
certificate-authority: /root/.minikube/ca.crt
server: https://192.168.99.100:8443
name: minikube
...
$ kubectl run hello-minikube --image=gcr.io/google_containers/echoserver:1.4 --port=8000
error: failed to discover supported resources: Get https://192.168.99.100:8443/api: Service Unavailable
I am not getting that what is causing this error. Is there some place we can check for logs. I cannot use kubectl logs as it requires container to mention which is not creating at all. Please provide any possible solution to problem.
A:
You can debug using these steps:
kubectl talks to kube-apiserver at port 8443 to do its thing. Try curl -k https://192.168.99.100:8443 and see if there's a positive response. If this fails, it means kube-apiserver isn't running at all. You can try restarting the VM or rebuilding minikube to see if it comes up properly the 2nd time round.
You can also debug the VM directly if you feel brave. In this case, get a shell on the VM spun up by minikube. Run docker ps | grep apiserver to check if the kube-apiserver pod is running. Also try ps aux | grep apiserver to check if it's run natively. If both don't turn up results, check the logs using journalctl -xef.
| 2023-10-08T01:27:17.760571 | https://example.com/article/4069 |
Find a Business Near: Monona, WI
Banks - All In Monona
Banks - All
Below is a list of businesses which provide Banks - All services. If you do not see your business in the list, you can submit it for addition to this list. Adding your business will feature your listing above Standard listings.
Helpful Definition for: Banks - All
Banks act as payment agents by conducting checking or current accounts for customers, paying cheques drawn by customers on the bank, and collecting cheques deposited to customers' current accounts. A bank is an institution that deals in money and its substitutes and provides other financial services.
In Monona, a bank can generate revenue in a variety of different ways including interest, transaction fees and financial advice. A bank is a financial organization where people deposit their money to keep it safe. The whole banking system around the world is connected. Banks in Monona, is committed to the highest standards of business ethics and corporate governance, and has been recognized for years in these areas.
Recent News:
Firm Settles Violations with EPA; Provides Equipment to Maricopa County Clini...
Lee Ann Rush
SAN FRANCISCO – Today, the U.S. Environmental Protection Agency (EPA) announced a settlement with True View Windows & Glass Block, Inc. for violations of the federal Lead Renovation, Repair and Painting Rule. The agreement requires True View, which operates in Arizona and Colorado, to pay a $15,060 penalty and spend $14,940 on blood lead analyzers and test kits for six Maricopa County, Arizona. health clinics. “Exposure to lead-based paint is one of the most common ways children develop lead poisoning,” said EPA Pacific Southwest Regiona...
EPA Marks Cleanup Milestone at Former Synergy Site in Claremont, N.H.
Lee Ann Rush
BOSTON – The U.S. Environmental Protection Agency announced that the former Synergy manufactured gas facility in Claremont, N.H., is now suitable for reuse and redevelopment after a successful hazardous waste cleanup at the site. EPA and the New Hampshire Department of Environmental Services oversaw the cleanup, which began in 2015 and concluded in July 2018. On October 11, AmeriGas will transfer ownership of the property to the City of Claremont. “Today’s milestone is a testament to how strategic partnerships can clean up pollution whil...
U.S. EPA, California Settle with UC Regents Over Davis Superfund Site Cleanup
Lee Ann Rush
SAN FRANCISCO –The U.S. Environmental Protection Agency (EPA) and the California Department of Toxic Substances Control (DTSC) have reached a settlement with the Regents of the University of California (University) to begin an estimated $14 million cleanup of contaminated soil, solid waste, and soil gas at the Laboratory for Energy-related Health Research/Old Campus Landfill Superfund site in Davis, Calif. Contaminants found at the site include carbon-14, polychlorinated biphenyls, pesticides, solvents, such as chloroform, and metals, such...
EPA and Camden, New Jersey Tackle Illegal Dumping
Lee Ann Rush
NEW YORK, NY – On 10/04/2018, the U.S. Environmental Protection Agency (EPA) announced that Center for Family Services, Inc. in Camden, New Jersey was selected to receive $120,000 in funding through the 2018 Environmental Justice Collaborative Problem-Solving (EJCPS) Cooperative Agreement Program. Ten organizations nationwide were selected to receive a total of $1.2 million in funding. Center for Family Services, Inc. is a non-profit organization working to address public health threats and environmental problems caused by illegal dumping ...
EPA Finalizes Nearly $7 Million Plan to Clean Up Lead-Contaminated Soil at Re...
Lee Ann Rush
NEW YORK, NY – The U.S. Environmental Protection Agency (EPA) has finalized its plan to clean up lead-contaminated soil at approximately 28 residences that are impacted by the former Flintkote Plant property at the Eighteen Mile Creek Superfund Site, in Lockport, N.Y. As part of a multi-phased, comprehensive cleanup of the Eighteen Mile Creek Site, EPA will remove and transport approximately 14,000 cubic yards of contaminated soil for off-site disposal at facilities licensed to handle the waste. The excavated areas will be restored with cl... | 2024-07-05T01:27:17.760571 | https://example.com/article/9815 |
{% set version = "1.12.0" %}
{% set name = "BioCor" %}
{% set bioc = "3.11" %}
package:
name: 'bioconductor-{{ name|lower }}'
version: '{{ version }}'
source:
url:
- 'https://bioconductor.org/packages/{{ bioc }}/bioc/src/contrib/{{ name }}_{{ version }}.tar.gz'
- 'https://bioarchive.galaxyproject.org/{{ name }}_{{ version }}.tar.gz'
- 'https://depot.galaxyproject.org/software/bioconductor-{{ name|lower }}/bioconductor-{{ name|lower }}_{{ version }}_src_all.tar.gz'
md5: 4a0e124d712e680f626d4d6579980d25
build:
number: 0
rpaths:
- lib/R/lib/
- lib/
noarch: generic
# Suggests: reactome.db, org.Hs.eg.db, WGCNA, methods, GOSemSim, testthat, knitr, rmarkdown, BiocStyle, airway, DESeq2, boot, targetscan.Hs.eg.db, Hmisc, spelling
requirements:
host:
- 'bioconductor-biocparallel >=1.22.0,<1.23.0'
- 'bioconductor-gseabase >=1.50.0,<1.51.0'
- r-base
- r-matrix
run:
- 'bioconductor-biocparallel >=1.22.0,<1.23.0'
- 'bioconductor-gseabase >=1.50.0,<1.51.0'
- r-base
- r-matrix
test:
commands:
- '$R -e "library(''{{ name }}'')"'
about:
home: 'https://bioconductor.org/packages/{{ bioc }}/bioc/html/{{ name }}.html'
license: 'MIT + file LICENSE'
summary: 'Functional similarities'
description: 'Calculates functional similarities based on the pathways described on KEGG and REACTOME or in gene sets. These similarities can be calculated for pathways or gene sets, genes, or clusters and combined with other similarities. They can be used to improve networks, gene selection, testing relationships...'
license_file: LICENSE
| 2024-03-06T01:27:17.760571 | https://example.com/article/4740 |
name: Post-update master jobs
on:
push:
branches:
- master
jobs:
release_image:
name: Build and publish to Docker Hub
runs-on: ubuntu-latest
steps:
- uses: actions/checkout@v2
with:
fetch-depth: 1
- name: Build the image
run: docker build -f "Dockerfile" -t "kiwicom/schemathesis:latest" .
- name: Login to registry
run: docker login -u $DOCKER_USERNAME -p $DOCKER_PASSWORD
env:
DOCKER_USERNAME: ${{ secrets.DOCKERHUB_USERNAME }}
DOCKER_PASSWORD: ${{ secrets.DOCKERHUB_PASSWORD }}
- name: Publish latest image
run: docker push kiwicom/schemathesis:latest
| 2023-09-29T01:27:17.760571 | https://example.com/article/4489 |
Discuss the latest comic book news and front page articles, read or post your own reviews of comics, and talk about anything comic book related. Threads from the two subforums below will also show up here. News Stand topics can also be read and posted in from The Asylum.
"Why are you pointing your screwdrivers like that? They're scientific instruments, not water pistols.""Oh, the pointing again! They're screwdrivers! What are you going to do? Assemble a cabinet at them?""Are you capable of speaking without flapping your hands about?"""Timey" what? "Timey wimey"?"
IvCNuB4 wrote:The Old Doctor is Cat-Scratch ?Well that explains a lot :lol:
A friend just sent to me along with and image I have to figure out how to send it to you.
Your type of thing.
He's a big manga fan and comes across this sort of stuff and sends me a copy since he knows I'm a fan.
"Why are you pointing your screwdrivers like that? They're scientific instruments, not water pistols.""Oh, the pointing again! They're screwdrivers! What are you going to do? Assemble a cabinet at them?""Are you capable of speaking without flapping your hands about?"""Timey" what? "Timey wimey"?"
IvCNuB4 wrote:The Old Doctor is Cat-Scratch ?Well that explains a lot :lol:
"Why are you pointing your screwdrivers like that? They're scientific instruments, not water pistols.""Oh, the pointing again! They're screwdrivers! What are you going to do? Assemble a cabinet at them?""Are you capable of speaking without flapping your hands about?"""Timey" what? "Timey wimey"?"
IvCNuB4 wrote:The Old Doctor is Cat-Scratch ?Well that explains a lot :lol:
"Why are you pointing your screwdrivers like that? They're scientific instruments, not water pistols.""Oh, the pointing again! They're screwdrivers! What are you going to do? Assemble a cabinet at them?""Are you capable of speaking without flapping your hands about?"""Timey" what? "Timey wimey"?"
IvCNuB4 wrote:The Old Doctor is Cat-Scratch ?Well that explains a lot :lol:
I'm starting to think that would make a good anime/manga style statue of PG.
"Why are you pointing your screwdrivers like that? They're scientific instruments, not water pistols.""Oh, the pointing again! They're screwdrivers! What are you going to do? Assemble a cabinet at them?""Are you capable of speaking without flapping your hands about?"""Timey" what? "Timey wimey"?"
IvCNuB4 wrote:The Old Doctor is Cat-Scratch ?Well that explains a lot :lol:
"Why are you pointing your screwdrivers like that? They're scientific instruments, not water pistols.""Oh, the pointing again! They're screwdrivers! What are you going to do? Assemble a cabinet at them?""Are you capable of speaking without flapping your hands about?"""Timey" what? "Timey wimey"?"
IvCNuB4 wrote:The Old Doctor is Cat-Scratch ?Well that explains a lot :lol: | 2024-03-18T01:27:17.760571 | https://example.com/article/7029 |
Display panels are normally applied to display output information of electronic products. The display panel generally includes an array substrate which includes a plurality of display pixels. A self-luminous display pixel is advantageous among kinds of display pixels due to no need of backlight source. A display panel with the self-luminous display pixel also becomes an important development trend of existing display panels due to its advantages like light in weight and power-saving and so on.
However, structures and functions of the existing self-luminous display pixels still remain to be improved.
In order to improve the structures and functions of the existing self-luminous display pixels, new designs and optimization of the self-luminous display pixels are required. | 2023-11-10T01:27:17.760571 | https://example.com/article/5879 |
Alexandra Thinks About Things
Add it on!
I spent a rather large part of my working hours on the internet: reading feeds, monitoring microblogging sites or sharing blogs and articles via bookmarking services. To keep it short: I live in my web browser.
Extensions to my rescue
The smartest thing about Firefox is its extendability. There is a ton of more or less useful add-ons available on Mozilla’s Add-on site that solve even those issues you have hardly thought about before finding the extension.
I collected a list of extensions I use more or less every day for my colleagues who will jump to my side on the internet and I thought it might be of interest to more people.
It’s a huge list that covers a lot areas from the usual Google enhancements to more juicy things like Geasemonkey scripts and I will split it up into several blog postings over the next weeks.
About me
A Girl Gone Geek.
Discovered the Usenet in 94 and has been lost ever since. Started toying with web sites when browsers were called Lynx. Contributed to KDE. Spent 10 years living opera somewhere in the middle of it all. Learnt a lot. | 2024-03-12T01:27:17.760571 | https://example.com/article/1951 |
1. Field
This disclosure relates generally to a touchscreen and, more specifically to techniques for controlling operation of a device with a virtual touchscreen.
2. Related Art
Traditionally, a touchscreen has been a display that is configured to detect a presence and location of a touch (or near touch) input to an area of the touchscreen. Many touchscreens are configured to sense contact by an instrument, such as a stylus or a finger. Other touchscreens are configured to sense both passive objects (e.g., a finger, a pencil eraser, or a passive stylus) and active objects (e.g., an active stylus such as a digitizer pen) that may not physically contact the touchscreen. Touchscreens may be configured to concurrently sense a single point of contact or multiple points of contact. In general, touchscreens facilitate user interaction with what is displayed directly on the touchscreen, as contrasted with indirect interaction through, for example, a mouse or a touchpad.
Touchscreens are frequently incorporated within devices such as personal digital assistants (PDAs), satellite navigation equipment, point-of-sale systems, kiosk systems, automatic teller machines (ATMs), portable gaming consoles, mobile phones, smart phones, etc. A wide variety of different technologies may be employed in touchscreens. For example, traditional touchscreens may implement resistive, surface acoustic wave (SAW), capacitive, infrared, strain gauge, optical imaging, or dispersive signal technologies, among other technologies, depending on an application. A tablet personal computer (PC) is an example of a mobile computer system that may employ a touchscreen to facilitate user input (via a stylus, digital pen, fingertip, or other instrument) to operate the tablet PC.
A wide variety of systems (e.g., notebook computer systems (notebooks), personal digital assistants (PDAs), laptop computer systems (laptops), and portable media players) have implemented touchpads. A touchpad is a pointing device that can translate motion and position of a finger of a user to a relative position on a display screen. When implemented in a notebook, touchpads have usually been incorporated on a surface of the notebook, adjacent a keypad. Touchpads (and associated buttons) are frequently provided as a substitute for a mouse. Touchpads vary in size but are normally smaller than three square inches. Touchpads are relative motion devices, similar to a mouse, with a cursor on a display screen moving in a same direction as a finger moving across a surface of the touchpad. In a typical touchpad implementation, buttons adjacent (e.g., below or above) the touchpad serve as standard mouse buttons.
Depending on the touchpad and associated driver, a tap of a finger on the touchpad may correspond to a mouse click. Touchpad drivers may enable the use of multiple fingers to facilitate other mouse buttons (e.g., two-finger tapping for a center mouse button). Some touchpads also implement hotspots (i.e., locations on the touchpad that indicate user intentions other than pointing). For example, on certain touchpads, moving a finger along an edge of the touchpad initiates a scroll wheel for scrolling an in-focus window vertically or horizontally, depending on which edge of the touchpad is stroked. Certain touchpad drivers also allow for tap zones (i.e., regions in which a tap executes a function). In this case, tapping in a tap zone may, for example, pause a media player or launch an application. | 2023-12-10T01:27:17.760571 | https://example.com/article/6681 |
♥ SS501 * FOREVER LOVE ♥
Monday, August 9, 2010
“My character is an easily annoyed person. In one word, he’s a hopeless fella. September will soon become a hopeless month”.
Singer and actor Kim Hyun Joong created buzz with his explosive remarks made without hesitation. He was on MBC ‘Section TV Entertainment Station’ recently that airs on August 6 and shooting for his new drama ‘Playful Kiss’ was revealed too, when he was asked to introduce a little, ”In one word, I’m hopeless. (I will turn) September will soon turn into a hopeless month”, which aroused much laughter from the surrounding people (Jung Somin, Lee Taesung).
In the upcoming MBC drama ‘Playful Kiss’, Kim Hyun Joong plays the male protagonist who has an easily annoyed personality Baek Seung-jo. This character can learn up to 100 times faster about anything at all than any other person; he is a genius flower boy. And in the drama, HyunJoong will be romancing bitter-sweet love story alongside rookie actress Jung Somin.
fr: http://consumertimes.net/news/article.html?no=30460&
~~~~~~~~~~~
Lee Taesung who appears in the drama as one who likes Jung Somin single-heartedly, ”(Somin’s eyes) always look like it’s puffy and swollen, but it looks pretty/nice”, showing praise for her.
Upcoming MBC new drama that is to be aired on September 1, “Playful Kiss” male lead Kim Hyun Joong revealed his ‘secret trick’ to digesting his high-school student role.
On MBC entertainment programme ‘Section TV, Entertainment Station’ broadcasted on August 6, Kim HyunJoong stated, ”For the sake of this high-school student role, I had been taking care of my moustache diligently (shaving)”, which attracted attention. He adds on, “In a day, I do that (shaving of moustache) almost 3 times and more” so as to give the role more solidness, as he laughingly put it across. On the other hand, Kim HyunJoong talks about his role this time, ”Always lucky, yet charismatic hopeless brat. September will become a hopeless month (I will let it)”. He replenishes, ”In my three works — “Can Love be refilled”, “Boys over Flowers” and “Playful Kiss”, all my first scenes are kissing ones. Now, I’m just taking it naturally in my stride”. | 2023-08-13T01:27:17.760571 | https://example.com/article/6784 |
John J. Moran Medium Security Facility
The John J. Moran Medium Security Facility is a medium-security state men's prison in Cranston, Rhode Island, owned and operated by the Rhode Island Department of Corrections. The facility opened in 1992, and has an operational capacity of 1006 prisoners.
References
External links
John J. Moran Medium Security Facility
Category:Prisons in Rhode Island
Category:Buildings and structures in Cranston, Rhode Island
Category:1992 establishments in Rhode Island | 2024-03-28T01:27:17.760571 | https://example.com/article/7480 |
1. Introduction {#sec1-ijms-20-05008}
===============
Nemaline myopathy (NM) causes weakness and poor tone (hypotonia) in the muscles of the face, neck, and upper limbs, and often greatly affects the respiratory muscles \[[@B1-ijms-20-05008],[@B2-ijms-20-05008]\]. NM is a congenital neuromuscular disorder that is caused by mutations in genes that play critical roles in muscle contraction, in particular genes related to the structure and function of the thin filament \[[@B3-ijms-20-05008],[@B4-ijms-20-05008]\]. NM mutations in the nebulin gene (*NEB*) make up a large fraction of cases \[[@B5-ijms-20-05008],[@B6-ijms-20-05008]\], but to date many other genes have also been implicated, in particular actin alpha 1 (*ACTA1*) and in more rare cases tropomyosin 3 (*TPM3)*, tropomyosin 2 (*TPM2)*, troponin T1 (*TNNT1)*, cofilin 2 (*CFL2)*, kelch repeat and BTB domain containing 13 (*KBTBD13)*, kelch like family member 40 (*KLHL40)*, kelch like family member 41 (*KLHL41)*, leiomodin 3 (*LMOD3)*, myopalladin (*MYPN),* ryanodine receptor 3 *(RYR3),* or Myosin 18B (*MYO18B)* \[[@B7-ijms-20-05008],[@B8-ijms-20-05008]\]. Muscle weakness in NM involves force-deficits at the sarcomere level \[[@B9-ijms-20-05008]\], while other NM characteristics include the presence of nemaline rods (dense accumulations of proteins inside the fibers), hypotrophy, and fiber type switching \[[@B10-ijms-20-05008]\]. Currently, no cures exist for NM. Respiratory failure is thought to be an important cause of death in NM \[[@B11-ijms-20-05008],[@B12-ijms-20-05008]\] and, thus, insights in diaphragm contractility in NM are needed for fully elucidating the pathology of NM and for developing treatment strategies. Yet, diaphragm muscle has been less studied than peripheral muscles, due to the limited availability of diaphragm biopsies. Here we studied how the function of the diaphragm is altered in a mouse model of NM, the recently developed tissue-specific and conditional nebulin KO model, the of nebulin-based NM (Neb cKO) mouse, that phenocopies a severe form of NM \[[@B13-ijms-20-05008]\].
Nebulin is a giant filamentous protein (\~800 kDa) that is an integral component of the skeletal muscle sarcomere \[[@B14-ijms-20-05008],[@B15-ijms-20-05008]\]. It winds around the actin filament and spans from the Z-disk to near the pointed end of the thin filament \[[@B14-ijms-20-05008],[@B16-ijms-20-05008]\]. Nebulin is known as a thin filament stabilizer that regulates the length of the thin filament \[[@B16-ijms-20-05008],[@B17-ijms-20-05008]\]. It plays important roles in skeletal muscle where it is involved in laterally aligning myofibrils \[[@B18-ijms-20-05008]\] and in regulating contraction \[[@B19-ijms-20-05008],[@B20-ijms-20-05008],[@B21-ijms-20-05008],[@B22-ijms-20-05008],[@B23-ijms-20-05008]\]. Mutations in nebulin cause alterations in thin filament structure and function that contribute to the force deficit, via reduced thin filament length \[[@B24-ijms-20-05008]\], reduced thin filament stiffness, and altered tropomyosin and troponin behavior \[[@B25-ijms-20-05008]\]. These changes depress force generation due to reduced thin filament activation and altered cross-bridge cycling kinetics \[[@B24-ijms-20-05008],[@B26-ijms-20-05008],[@B27-ijms-20-05008],[@B28-ijms-20-05008]\]. Contractile weakness in respiratory muscle, relative to peripheral skeletal muscle, is considered to lead to respiratory failure, which is the main cause of death in NM patients \[[@B11-ijms-20-05008]\]. Joureau et al. \[[@B29-ijms-20-05008]\] have shown that contractile weakness of permeabilized diaphragm muscle fibers of a NM mouse model was more pronounced than that in soleus muscle fibers. To date, no intact muscle experiments have been performed on the NM diaphragm and, additionally, there are no effective treatment options to improve contractile weakness in NM.
As a possible therapeutic option in this work we focused on *tirasemtiv*, a small molecule that selectively activates the fast skeletal muscle troponin complex by increasing its sensitivity to calcium \[[@B30-ijms-20-05008]\]. *Tirasemtiv* has been investigated as a potential therapy for neuromuscular disorders, such as Amyotrophic Lateral Sclerosis (ALS) and, myasthenia gravis \[[@B31-ijms-20-05008],[@B32-ijms-20-05008],[@B33-ijms-20-05008],[@B34-ijms-20-05008],[@B35-ijms-20-05008]\]. Considering that FSTAs increase the calcium sensitivity of permeabilized muscle fibers of nebulin-deficient skeletal muscle \[[@B36-ijms-20-05008]\], we hypothesized that *tirasemtiv* has a beneficial effect on contractile performance of electrically stimulated diaphragm muscle from the Neb cKO NM mouse model. Additionally, we investigated the effect of *tirasemtiv* on in vivo peripheral muscle strength in the NM mouse model and focused this part of our study on the gastrocnemius complex.
We found a large specific-force deficit in the electrically stimulated diaphragm of Neb cKO muscle, relative to control muscle, that was largest at low to intermediate stimulation frequencies (\~70% at 1--60 Hz). *Tirasemtiv* significantly reduced the force deficit of Neb cKO diaphragm (from \~70% to \~40%). Similar findings were made in the in vivo gastrocnemius study. Load-clamp experiments on diaphragm muscle showed that *tirasemtiv* increased the shortening velocity at all loads, which resulted in significant reduction in the power deficit of cKO. Thus, *tirasemtiv* significantly improves muscle function in a mouse model of nebulin-based NM.
2. Results {#sec2-ijms-20-05008}
==========
2.1. Isometric Force Production by Diaphragm Muscle {#sec2dot1-ijms-20-05008}
---------------------------------------------------
At all stimulation frequencies, the specific force of diaphragm muscle strips was lower in Neb cKO mice than in ctrl mice ([Figure 1](#ijms-20-05008-f001){ref-type="fig"}A, [Supplementary Table S1](#app1-ijms-20-05008){ref-type="app"}). The maximal tetanic force (stimulation frequency 200 Hz) of the cKO diaphragm was reduced by 57%, from 200 mN/mm^2^ to 86 mN/mm^2^ (*p* \< 0.001) ([Table 1](#ijms-20-05008-t001){ref-type="table"}). Note that in both genotypes there were only small increases in tetanic force at stimulation frequencies beyond 100 Hz. Dividing force at each stimulation frequency by the maximal tetanic force revealed relative force--frequency curves that were distinct in the two genotypes ([Figure 1](#ijms-20-05008-f001){ref-type="fig"}B), with the most severe force reduction in cKO diaphragm at stimulation frequencies \<\~30 Hz. The Hill-fits to the relative force--frequency relation showed in cKO diaphragm a significantly higher frequency needed to elicit half-maximal force (F~50~ frequency) 33 vs 38 Hz and a Hill coefficient that was significantly higher than in ctrl, 1.5 vs 2.5 ([Table 1](#ijms-20-05008-t001){ref-type="table"}) for ctrl and Neb cKO, respectively.
The force of the cKO relative to ctrl is shown as a function of frequency in [Figure 1](#ijms-20-05008-f001){ref-type="fig"}C. The force, relative to ctrl, is depressed most at frequencies ≲20 Hz and the difference becomes smaller at higher frequencies. This unique frequency-dependent force deficit behavior is also apparent in earlier results \[[@B13-ijms-20-05008]\] obtained on intact extensor digitorum longus (EDL) and soleus muscle (results reproduced in [Figure 1](#ijms-20-05008-f001){ref-type="fig"}C). Thus in both diaphragm and peripheral muscles, nebulin-deficiency more severely affects force at low--intermediate stimulation frequencies. It is also noteworthy that comparison between diaphragm and peripheral muscle types (all intact muscles, stimulated electrically) show that the force deficit in cKO (relative to ctrl) was less severe in diaphragm than the peripheral muscles ([Figure 1](#ijms-20-05008-f001){ref-type="fig"}C).
2.2. Effect of Tirasemtiv on Diaphragm Muscle Strip {#sec2dot2-ijms-20-05008}
---------------------------------------------------
The effect of the FSTA *tirasemtiv* on specific force was evaluated in ctrl and NM diaphragm muscle. *Tirasemtiv* increased specific force, in both genotypes and at all stimulation frequencies ([Figure 2](#ijms-20-05008-f002){ref-type="fig"}A, [Table 1](#ijms-20-05008-t001){ref-type="table"}, [Suppl Table S1](#app1-ijms-20-05008){ref-type="app"}). Similarly, the maximal speed of specific force rise (dF/dt max) increased in both genotypes ([Figure 2](#ijms-20-05008-f002){ref-type="fig"}B). The increase in both specific force and speed of force rise was low at the lowest tested frequencies, increased sharply with frequency to reach a peak at \~20 Hz, and then fell again ([Figure 2](#ijms-20-05008-f002){ref-type="fig"}C,D). Although this general pattern was similar in the two genotypes, the average increase in specific force and speed of force rise were much higher in the cKO. In particular, the average specific force increase due to *tirasemtiv* across all frequencies was 72 ± 21% in cKO and 27 ± 7% in ctrl. The maximal difference between the genotypes peaked at \~20 Hz where *tirasemtiv* increased specific force by 219 ± 24% in cKO and 67 ± 6% in ctrl.
The force deficit of the cKO diaphragm relative to ctrl (no *tiramsemtiv*) is shown in [Figure 3](#ijms-20-05008-f003){ref-type="fig"}. *Tirasemtiv* reduced the force deficit of cKO, averaged across all frequencies, from 62.5 ± 2.4% in absence of *tirasemtiv* to 45.0 ± 2.7% with *tirasemtiv*. The largest effect was found at 20 Hz where the deficit fell from 76% to 29%. Thus, *tirasemtiv* greatly improves isometric force production, especially at intermediate stimulation frequencies.
Load-clamp experiments were performed in which the diaphragm was tetanized at a 50 Hz stimulation frequency (this frequency was selected because it is the lowest stimulation frequency that results in a fused tetanus, avoiding velocity oscillations during the load-clamp period) and when force was maximal, the force was abruptly decreased and held constant at a preset value, allowing isotonic shortening speed to be measured and mechanical power to be calculated (as the product of force and velocity of shortening, see Methods for details). The shortening velocity of cKO diaphragm was much lower than that of ctrl diaphragm, both with force expressed on an absolute scale ([Figure 4](#ijms-20-05008-f004){ref-type="fig"}A) and with force normalized to the isometric force ([Figure 4](#ijms-20-05008-f004){ref-type="fig"}B). Power curves ([Figure 4](#ijms-20-05008-f004){ref-type="fig"}C) were much lower in cKO, as expected, with a reduction in maximal power of 62% in cKO relative to ctrl ([Table 1](#ijms-20-05008-t001){ref-type="table"}). *Tirasemtiv* resulted in an increase of shortening velocity in both ctrl and cKO ([Figure 4](#ijms-20-05008-f004){ref-type="fig"}A,B). The *tirasemtiv*-based shortening velocity increase was larger in the ctrl (average at all load clamp levels: 48.9 ± 3.5%) than in the cKO diaphragm (average at all load clamp levels: 26.2 ± 2.8%). Importantly, the maximal power deficit (relative to ctrl in the presence of vehicle) was reduced from 62% without *tirasemtiv* to 42% with *tirasemtiv* ([Table 1](#ijms-20-05008-t001){ref-type="table"}). Thus, *tirasemtiv* increases the shortening velocity in both genotype and reduces the power deficit of cKO diaphragm.
2.3. Effect of Tirasemtiv on in Vivo Muscle Strength of Gastrocnemius Complex {#sec2dot3-ijms-20-05008}
-----------------------------------------------------------------------------
A separate group of mice was used to test the effect of *tirasemtiv* on in vivo gastrocnemius muscle performance (see Methods). Specific force was lower in cKO muscle at all stimulation frequencies. The maximal tetanic force was 0.81 ± 0.04 mN/mg in Neb cKO vs. 2.50 ± 0.07 mN/mg in ctrl, i.e., a reduction of 68% in the cKO. *Tirasemtiv* left-shifted the force--frequency curves in both ctrl and Neb cKO mice, due to the large force increase at low--intermediate stimulation frequencies and the minimal effect at frequencies that give rise to (near) maximal tetanic force levels ([Figure 5](#ijms-20-05008-f005){ref-type="fig"}A,B). The frequency dependence of the force increase is shown in [Figure 5](#ijms-20-05008-f005){ref-type="fig"}C. The average force increase at stimulation frequencies of 10--60 Hz was 95 ± 19% in ctrl and 65 ± 21% in cKO mice. Because of the frequency-dependent *tirasemtiv* effect, the frequency for half-maximal tension (F~50~ frequency) was significantly reduced by *tirasemtiv*, in both genotypes from 58 to 39 Hz in ctrl and 37 to 27 Hz in Neb cKO ([Table 1](#ijms-20-05008-t001){ref-type="table"} and [Figure 5](#ijms-20-05008-f005){ref-type="fig"}A,B insets).
The effect of *tirasemtiv* on the force deficit of the cKO gastrocnemius complex relative to ctrl (no *tirasemtiv*) is shown in [Figure 5](#ijms-20-05008-f005){ref-type="fig"}D. *Tirasemtiv* reduced the force deficit from 55 ± 6% in absence of *tirasemtiv* to 30 ± 11% with tirasemtiv, averaged across 10--60 Hz. The largest *tirasemtiv* effect was found at 30 Hz where the deficit fell from 42% to 4%. Thus, at low--intermediate frequencies, *tirasemtiv* greatly improves the in vivo strength of the gastrocnemius complex.
3. Discussion {#sec3-ijms-20-05008}
=============
Respiratory failure due to diaphragm dysfunction is considered a main cause of death in NM \[[@B2-ijms-20-05008]\], necessitating a need to understand how NM affects diaphragm function. Here we studied both isometric force and isotonic shortening velocity of diaphragm muscle strips in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was found in nebulin-deficient intact muscle that is frequency dependent, with the largest deficits at low--intermediate stimulation frequencies. The effect of the troponin activator *tirasemtiv* as a possible therapeutic was examined. *Tirasemtiv* had no effect at maximal stimulation frequencies, but greatly reduced the force deficit of cKO diaphragm at sub-maximal activation. Similar effects were found in the gastrocnemius muscle complex. Below we discuss these findings in detail.
3.1. Contractile Performance in Neb cKO Diaphragm {#sec3dot1-ijms-20-05008}
-------------------------------------------------
Nebulin deficiency results in a large force deficit in the diaphragm strips, between \~70% (low stimulation frequencies) and \~55% (high stimulation frequency). This frequency dependence can be explained by the reduced calcium sensitivity of force generation (increased calcium level required to reach half-maximal force) that has been found in nebulin-deficient permeabilized muscle fibers \[[@B21-ijms-20-05008],[@B29-ijms-20-05008]\]. This is expected to reduce force in intact cKO muscle at submaximal stimulation frequencies. Because the reduction in force of permeabilized fibers in cKO only occurs at submaximal activation levels, altered calcium sensitivity is unlikely to play a role in intact muscle that is maximally stimulated (200 Hz). The large reduction in maximal tetanic force (\~55%) is likely due to other factors including a reduced thin filament length \[[@B13-ijms-20-05008],[@B20-ijms-20-05008],[@B37-ijms-20-05008]\], a reduced number of force generating cross-bridges \[[@B13-ijms-20-05008],[@B24-ijms-20-05008],[@B26-ijms-20-05008],[@B27-ijms-20-05008],[@B38-ijms-20-05008],[@B39-ijms-20-05008]\], and less force per attached cross-bridge \[[@B40-ijms-20-05008]\]. Changes in cross-bridge cycling are also evident from the lower shortening velocity that was found in this study ([Figure 4](#ijms-20-05008-f004){ref-type="fig"}). Some of these changes could in principle be due to fiber type switching but unlike in other studied muscle types of Neb cKO mice, fiber type switching in diaphragm muscle is minimal \[[@B13-ijms-20-05008]\] and is unlikely to play a major role in our diaphragm study. In summary, a large force deficit occurs in electrically stimulated diaphragm strips of Neb cKO mice that can be explained by structural and functional changes in the myofilaments with a frequency dependence that is likely due to reduced calcium sensitivity at submaximal activation levels.
Previous studies on biopsies of NM patients indicated that the diaphragm is more affected than peripheral skeletal muscles \[[@B41-ijms-20-05008]\], a conclusion based on the presence of more nemaline rods in the diaphragm than in non-respiratory muscles. Earlier functional studies on permeabilized fibers from a nebulin-deficient mouse model (Neb^ΔExon55^ model) tested this notion \[[@B29-ijms-20-05008]\]. Maximally activated fibers from the diaphragm had a higher force deficit than soleus muscle fibers \[[@B29-ijms-20-05008]\], lending support to the view that the diaphragm is unusually sensitive to NM-causing mutations. However, our present study on intact muscle reveals that the force deficit of the diaphragm is less than that of the EDL and soleus muscle ([Figure 1](#ijms-20-05008-f001){ref-type="fig"}C), particularly at submaximal stimulation frequencies, and less than in the gastrocnemius, as measured in vivo ([Figure 5](#ijms-20-05008-f005){ref-type="fig"}). The explanation for the differences in results between the present and earlier study \[[@B29-ijms-20-05008]\] is unclear but might involve the more severe pathology in Neb^ΔExon55^ mice (mice die within the first week of life) and the milder pathology in the Neb cKO model, with survival into adulthood. Although the diaphragm might not be more sensitive to dysfunction than peripheral muscles, the severe contractility deficit of the diaphragm is a likely major cause of weakness in NM patients. The diaphragm is the main muscle of inspiration, taking up some seventy percent of the work of breathing in humans \[[@B29-ijms-20-05008]\] and, furthermore, the diaphragm is highly active with a duty cycle (ratio of active to inactive time) of \~35% \[[@B42-ijms-20-05008]\], several-fold higher than for peripheral muscles \[[@B43-ijms-20-05008]\]. Thus, obtaining therapeutic approaches that increase diaphragm strength is critically important.
3.2. Tirasemtiv Effect on Diaphragm {#sec3dot2-ijms-20-05008}
-----------------------------------
*Tirasemtiv* is a small molecule that selectively activates fast skeletal muscle by slowing the rate of calcium release from the troponin complex \[[@B30-ijms-20-05008],[@B44-ijms-20-05008]\]. It increases the stability of the open conformation of the fast skeletal muscle troponin/tropomyosin complex and enhances cross-bridge formation at a given submaximal calcium concentration \[[@B44-ijms-20-05008]\]. *Tirasemtiv* has been investigated as a potential therapy for neuromuscular disorders, such as Amyotrophic Lateral Sclerosis (ALS) and, myasthenia gravis \[[@B31-ijms-20-05008],[@B32-ijms-20-05008],[@B33-ijms-20-05008],[@B34-ijms-20-05008],[@B44-ijms-20-05008]\]. CK-2066260, another FSTA \[[@B22-ijms-20-05008],[@B35-ijms-20-05008]\] from a similar chemical class as *tirasemtiv*, has also been studied in permeabilized fibers activated with exogenous calcium, using fibers from the Neb^ΔExon55^ mouse model \[[@B36-ijms-20-05008]\] and from NM patients \[[@B36-ijms-20-05008]\]. In those studies, calcium sensitivity was significantly increased by *tirasemtiv* as reflected by the increased submaximal force levels. Here we studied the effect of *tirasemtiv* on electrically stimulated ctrl and Neb cKO diaphragm muscle. *Tirasemtiv* enhanced the specific force of diaphragm muscle strips in both genotypes. This effect reached a maximum at \~20 Hz (219% ± 24%) and then gradually decreased as stimulation frequencies were increased ([Figure 2](#ijms-20-05008-f002){ref-type="fig"}C). *Tirasemtiv* also increased the maximal speed of force rise (dF/dt max) in both genotypes with a similar frequency dependence to that of specific force ([Figure 2](#ijms-20-05008-f002){ref-type="fig"}B,D); increasing both the speed of force rise and the final force level are likely to positively impact breathing function \[[@B45-ijms-20-05008]\]. Although *tirasemtiv* affects both the ctrl and Neb cKO diaphragm, the magnitudes of the effects are much greater in nebulin-deficient muscle than in ctrl muscle ([Figure 2](#ijms-20-05008-f002){ref-type="fig"}C,D). This might be explained by the reduced calcium sensitivity of Neb cKO muscle, relative to ctrl, which results in a lower fraction of attached cross-bridges during submaximal activation \[[@B21-ijms-20-05008],[@B24-ijms-20-05008],[@B26-ijms-20-05008],[@B27-ijms-20-05008]\], providing more 'range' for additional activation by *tirasemtiv*. In summary, *tirasemtiv* greatly improves the contractility of the diaphragm and the larger effect in Neb cKO diaphragm is fortuitous.
3.3. Contractility of Gastrocnemius Complex and Effects of Tirasemtiv {#sec3dot3-ijms-20-05008}
---------------------------------------------------------------------
The in vivo contractility of the gastrocnemius complex was severely depressed in the Neb cKO mouse with a maximal specific force that was reduced by 68% ([Table 1](#ijms-20-05008-t001){ref-type="table"}). The frequency for half-maximal force (F~50~ frequency) was reduced in the gastrocnemius (from 58 Hz in ctrl to 37 Hz in Neb cKO), in contrast to the diaphragm where an increase was found (from 33 Hz to 38 Hz). This reduction in F~50~ frequency of the gastrocnemius might reflect the fiber-type switch that occurs in this muscle from \<5% type-I in ctrl mice to \~40% type-I in Neb cKO and the lower F~50~ frequency of type I fibers, compared to type II fibers \[[@B13-ijms-20-05008]\]. This lower F~50~ frequency of type I fibers is likely due to their higher calcium sensitivity \[[@B46-ijms-20-05008],[@B47-ijms-20-05008]\]. *Tirasemtiv* significantly increased the contractile force of the gastrocnemius complex in both genotypes at stimulation frequencies less than 60 Hz ([Figure 5](#ijms-20-05008-f005){ref-type="fig"}C). However, unlike in the diaphragm, the effect was not larger in the Neb cKO muscle relative to ctrl. As discussed above, the increased sensitivity of the Neb cKO diaphragm to *tirasemtiv* is likely due to the reduced myofilament calcium sensitivity of this muscle type, but this effect might be negated in the gastrocnemius muscle complex due to fiber type switching away from type II to type I fibers (where *tirasemtiv* does not have an effect). Regardless, there is a robust effect of *tirasemtiv* on muscular strength of the gastrocnemius complex resulting in a much reduced force deficit in Neb cKO mice ([Figure 5](#ijms-20-05008-f005){ref-type="fig"}D). Considering that skeletal muscles, including diaphragm muscle, operate at submaximal activation levels during normal activity (between 10% and 65% of its maximal capacity) \[[@B47-ijms-20-05008],[@B48-ijms-20-05008],[@B49-ijms-20-05008]\], the beneficial effect of *tirasemtiv* at submaximal activation levels suggest that *tirasemtiv* might be useful for treatment of nebulin-based NM patients.
3.4. Conclusion {#sec3dot4-ijms-20-05008}
---------------
Intact muscles from Neb cKO mice stimulated either electrically (diaphragm) or via its innervating nerve (gastrocnemius complex) produce, relative to ctrl muscles, much reduced levels of force. FSTA *tirasemtiv* robustly enhances contractility in sub-maximally stimulated intact muscles and has therefore therapeutic potential in diaphragm as well as in peripheral muscle. Whether these findings in the mouse can be extrapolated to other species, including human, will depend on whether fast fibers (type II fibers) are expressed at relatively high levels. Slow fibers (type I fibers) and fast fibers are present in approximately equal proportion in the adult human diaphragm while intercostal muscles contain a higher proportion of fast fibers \[[@B50-ijms-20-05008]\]. Peripheral muscle in humans contain between 25% and 50% type II fibers \[[@B51-ijms-20-05008]\]. Thus, the fiber type composition of human muscles is favorable for a significant *tirasemtiv* effect. An additional requirement for *tirasemtiv* to work is a physiological activation level that is submaximal. It has been previously shown that during normal breathing, the diaphragm muscle is activated far below its maximal activation level and generates \~25% of its maximal force \[[@B45-ijms-20-05008],[@B52-ijms-20-05008]\] and evidence exists that peripheral muscle are also submaximally activated during normal daily activities \[[@B49-ijms-20-05008]\]. Thus, *tirasemtiv* and other FSTAs are likely to augment breathing as well as peripheral muscle function in mouse and human. *Reldesemtiv,* a next generation FSTA, is currently under clinical investigation in ALS and spinal muscular atrophy, studies that include measures of respiratory and peripheral muscle function. Overall, fast skeletal muscle troponin activators might also be suitable for treating diaphragm dysfunction in patients with nebulin-based NM, where reduced myofilament calcium sensitivity is a hallmark characteristic of the underlying muscle dysfunction.
4. Material and Methods {#sec4-ijms-20-05008}
=======================
4.1. Animal Model {#sec4dot1-ijms-20-05008}
-----------------
The conditional nebulin knockout (Neb cKO) mouse that was used has previously been described \[[@B13-ijms-20-05008]\]. All mice are on a C57BL/6J background. Floxed mice were bred to a MCK-Cre strain that expresses Cre recombinase under control of the Muscle Creatine Kinase (MCK) promoter. Mice that were positive for MCK-Cre and homozygous for the floxed nebulin allele were nebulin deficient and are referred to as Neb cKO. Mice with at least one nebulin WT allele and being either MCK-Cre positive or negative were grouped as control (ctrl) mice. The experiments were approved by the University of Arizona Institutional Animal Care and Use Committee (Project number 09-057) and were in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals.
4.2. Tirasemtiv {#sec4dot2-ijms-20-05008}
---------------
*Tirasemtiv* was provided by Cytokinetics (South San Francisco, CA, USA). For the ex-vivo diaphragm study, *tirasemtiv* was dissolved in dimethylsulfoxide (DMSO) to make a stock solution, and then it was added directly to the experimental solution to prepare the final desired concentration of *tirasemtiv* (3 μM) in 1% DMSO. The concentration of *tirasemtiv* (3 μM) was chosen based on previous studies that showed that this concentration effectively increases sub-maximal force in skeletal muscle \[[@B31-ijms-20-05008],[@B32-ijms-20-05008]\]. In the in vivo studies to measure in vivo function of the gastrocnemius complex, a single dose (10 mg/kg) of *tirasemtiv* dissolved in 10% DMA, 50% PEG400 and a 40% Cavitron cyclodextrin formulation at a concentration of 3 mg/mL was delivered via intraperitoneal (IP) injection. A separate control group received vehicle injections for comparison.
4.3. Ex-Vivo Diaphragm Muscle Mechanics {#sec4dot3-ijms-20-05008}
---------------------------------------
### 4.3.1. Muscle Experiments {#sec4dot3dot1-ijms-20-05008}
Four month old Neb cKO (136 ± 2 days old) and control (ctrl) (137 ± 1 days old) mice were used for this study. Mice were anesthetized with isoflurane and sacrificed by cervical dislocation. Immediately, the whole diaphragm muscle was excised and a small muscle strip from central tendon to ribcage on the left side of diaphragm was isolated with parallel fiber alignment. Muscle strips were mounted to the isolated muscle test system (1200A, Aurora Scientific Inc., Aurora, ON, Canada) that has been described previously \[[@B53-ijms-20-05008]\]. In brief, tendon and floating rib were tied with 7-0 silk sutures, and the muscle was attached between the lever arm of dual servomotor-force transducer (300C, Aurora scientific, Aurora, ON, Canada) and a fixed hook. The experimental bath was filled with Krebs-Ringer solution containing (in mM) 137 NaCl, 5 KCl, 1 MgSO~4~, 2 CaCl~2~, 1 NaH~2~PO~4~, 24 NaHCO~3~, 11 Glucose with 95%/5% O~2/~CO~2~ supply (pH = 7.4). The temperature during the experiments was controlled at 30 °C. The muscle was placed between platinum electrodes connected to an electrical stimulator (701C, Aurora scientific, Aurora, ON, Canada) for muscle activation. The optimal muscle length (L~0~) was determined by adjusting overall muscle length until maximal force was generated with a 400 ms 20 Hz stimulation (pulse duration of 200 μs with biphasic polarity). The muscle length at L~0~ was measured using a digital caliper. At L~0~, an initial tetanic contraction at 150 Hz stimulation frequency was imposed to evaluate the quality of the muscle preparation. Measured forces were normalized to the cross-sectional area (CSA) to obtain specific force (mN/mm^2^). CSA (in mm^2^) was calculated using muscle mass (in g), fiber length (in mm), and muscle density (1.056 g/mm^3^); CSA = muscle mass / (muscle length × muscle density).
### 4.3.2. Protocols {#sec4dot3dot2-ijms-20-05008}
#### Force--Frequency Protocol
To establish the force--frequency relation, active forces at various stimulation frequencies were measured. 15 ctrl and 18 Neb cKO mice were used for the force--frequency protocol. The muscle was stimulated at 1, 5, 10, 20, 30, 40, 60, 100, 150, and 200Hz and waiting for 30, 30, 60, 90, 90, 120, 120, 180, 180, and 180 s, respectively, in between each stimulation. The durations of stimulation were 0.4 s for 1--40 Hz and 0.8 s for 60--200 Hz of stimulation. The first force--frequency protocol was performed in the vehicle solution, and the experimental solution was then switched to *tirasemtiv* solution. *Tirasemtiv* was dissolved in dimethylsulfoxide (DMSO) and directed added to the experimental solution to prepare desired final concentration (3 μM). The muscle was incubated in *tirasemtiv* solution for 10 min with 30 Hz stimulation frequency every minute to monitor changes in force. When the increase in force was stable, the force--frequency protocol in *tirasemtiv* solution was measured. In order to limit the chance on a false negative result due to the incomplete removal of *tirasemtiv*, we consistently performed the vehicle condition first and then the *tirasemtiv* condition.
#### Force--Velocity Protocol
To determine the force-velocity relation, load-clamp experiments were performed. Shortening velocities were measured during isotonic contraction against loads 85%, 70%, 55%, 40%, 25%, and 10% of maximal force at 50 Hz of stimulation frequency. Each contraction started out with a 200 ms isometric phase (to reach the plateau force), and then a 150 ms load-clamp was applied. A series of load-clamps was performed in solution with vehicle and the same protocol was then repeated in *tirasemtiv* solution. Before the protocol was repeated with *tirasemtiv*, the muscle was contracted with a 30 Hz stimulation frequency (0.4 s in duration) every minute until tension stopped to increase. After completion of each experiment, the sutures and tendons were carefully removed, and the muscle mass was then measured.
#### Analysis
The force--frequency curve was fit with a Hill equation to calculate the frequency that results in 50% of maximal force (F~50~ frequency) and the Hill coefficient (n~H~), an index of the steepness of the curve. The maximal speed of specific force rise was analyzed as the maximum of the derivative of force with respect to time (dF/dt) during the force rise phase. Shortening velocity (in mm/sec) was measured as a slope of linear portion of the shortening period (\~40 ms duration) after force was stable. For each preparation, shortening velocity was normalized by muscle fiber length. Muscle power production was calculated by multiplying the shortening velocity and the applied load. Force--power curves were fitted by 2nd order polynomial non-linear regression, and the maximal power production was determined from the apex of the force--power curve.
4.4. In Vivo Strength Measurement of Gastrocnemius Muscle Complex {#sec4dot4-ijms-20-05008}
-----------------------------------------------------------------
Two month old Neb cKO (54 ± 2 days old) and ctrl (53 ± 2 days old) mice were tested using an in vivo muscle analysis apparatus (model 1300A; Aurora scientific, Aurora, ON, Canada). In order to test the effect of *tirasemtiv* on the in vivo muscle strength, we selected the gastrocnemius muscle complex due to the large population of fast skeletal muscle fibers and the convenience of in situ force measurement. Subcutaneous needle electrodes were positioned along the sciatic nerve, distal to the peroneal nerve to induce plantar flexion. The minimum current required to induce maximal force was used to avoid stimulating antagonist muscle groups. Contractile force of the gastrocnemius muscle complex was measured through the movement of the foot that was attached to the footplate force measurement system. Mice were first sedated under anesthesia using 1--2% of isoflurane with O~2~ supply and placed on the experiment platform. Body temperature was maintained with a circulated water bath system. Hair around the experimental area of the right leg was removed with an electric razor. The leg was immobilized using a blunt knee clamp and the ankle joint positioned at a 90° angle with the foot secured to the force transducer footplate using adhesive tape. Two needle electrodes were placed distal to the peroneal nerve to stimulate the gastrocnemius muscle complex. The optimum pulse phase polarity was determined using a 10 Hz stimulation frequency at 3mA and the current set using a series of twitches repeated every 10 s until maximum forces were obtained. Separate groups of mice were used to test the effect of *tirasemtiv* on in vivo strength of the gastrocnemius complex. The treatment group received a single dose of *tirasemtiv* (10 mg/kg dissolved in 50% PEG400: 40% (40% g/mL Cavitron in water): 10% DMA) acutely via intraperitoneal (IP) injection, while a vehicle solution (same carrier as used for *tirasemtiv*) was administered to the control group for comparison. Pre- and post- treatment tests were also conducted to serve as internal controls. Following the procedure, animals were euthanized and the gastrocnemius complex (soleus, plantaris and gastrocnemius) were dissected and weighed for the normalization of measured force (mN/mg).
### Protocols
To establish the force--frequency relation, gastrocnemius complex muscle forces at various stimulation frequencies were measured. 9 ctrl and 11 Neb cKO mice were used for the force--frequency protocol in both the treatment and vehicle groups. The gastrocnemius complex was stimulated at 10, 20, 30, 40, 60, 100, 125, 150, and 200 Hz, waiting for 30, 30, 30, 60, 60, 120, 120, and 120 s, respectively, in between each stimulation. A pulse width of 200 µs and 350 ms duration was used for all tetanic stimulations. The first force--frequency protocol was performed under baseline conditions. Following a brief rest period, each mouse received an IP injection containing either a 10 mg/kg dose of *tirasemtiv* or vehicle only. A 30 Hz stimulation frequency was repeated every minute for a minimum of 10 min to monitor changes in force. When the increase in force was stable, the force--frequency protocol was repeated. The force--frequency curve was fit with a Hill equation to calculate the F~50~ frequency and the Hill coefficient (nH), an index of the steepness of the curve.
4.5. Statistical Analysis {#sec4dot5-ijms-20-05008}
-------------------------
Data are presented as mean ± SD. We used a repeated measure two-way ANOVA with post-hoc tests using the Sidak method with multiple comparisons correction, unless indicated otherwise. In order to identify the effect of *tirasemtiv* at each genotype, the two factors for analysis were frequency and treatment (vehicle or *tirasemtiv*; [Figure 2](#ijms-20-05008-f002){ref-type="fig"}A,B, [Figure 3](#ijms-20-05008-f003){ref-type="fig"}, [Figure 4](#ijms-20-05008-f004){ref-type="fig"}B, [Figure 5](#ijms-20-05008-f005){ref-type="fig"}A,B,D). For comparison of genotype, the two factors for analysis were frequency and genotype (ctrl vs. cKO; [Figure 1](#ijms-20-05008-f001){ref-type="fig"}A,B, [Figure 2](#ijms-20-05008-f002){ref-type="fig"}C,D, [Figure 5](#ijms-20-05008-f005){ref-type="fig"}C). *p* \< 0.05 was considered to be statistically significant. GraphPad Prism 6 was used for statistical analysis.
We are grateful to Luan Wyly and Xiangdang Liu for important support of this work.
######
Click here for additional data file.
Supplementary materials can be found at <https://www.mdpi.com/1422-0067/20/20/5008/s1>.
Conceptualization, E.-J.L., H.L.G., and D.T.H.; Methodology, E.-J.L.; Data Curation, E.-J.L. and J.K.; Formal Analysis, E.-J.L. and J.K.; Project Administration, H.L.G.; Funding Acquisition, H.L.G.; Supervision, H.L.G. and F.I.M.; Resources, F.I.M.; Writing---Review and Editing, E.-J.L., J.K., D.T.H., F.I.M., and H.L.G.
Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR053897. This study was also supported by a grant from A Foundation for Building Strength.
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
![Force--frequency relations in control (ctrl) and of nebulin-based NM (Neb cKO) diaphragm muscle strips. (**A**) Force--frequency curves. Forces are shown as specific force (force normalized to the muscle's cross-sectional area). Standard two-way ANOVA reveals that genotype significantly (*p* \< 0.0001) affects specific force. A post-hoc test (Sidak) corrected for multiple comparisons reveals that at all stimulation frequencies, Neb cKO produces significantly less force than ctrl (two lowest frequencies *p* \< 0.01, all other frequencies *p* \< 0.0001). (**B**) Relative force--frequency curves. For each genotype, force is normalized to the maximal force at 200 Hz. The Neb cKO curve has a different shape than the ctrl curve. Standard two-way ANOVA reveals that genotype significantly (*p* \< 0.0001) affects relative force. A post-hoc test (Sidak) corrected for multiple comparisons reveals that Neb cKO produces significantly less relative force than ctrl at 1--40 Hz (\*\* *p* \<0.01, \*\*\*\* *p* \< 0.0001). (**C**) Force of Neb cKO muscles relative to that of ctrl diaphragm (this study) and EDL and soleus (data from Li et al. \[[@B13-ijms-20-05008]\]), which uses the same mouse model (at 6 months old) and experimental protocols as the present study). Force normalized to the force of ctrl muscle at each frequency. Results from 15 ctrl and 18 Neb cKO mice. Mean ± SD are shown in A and B and only mean in C. Note that the x-axis uses a log-scale and that at many frequencies SD lines fall within the symbols.](ijms-20-05008-g001){#ijms-20-05008-f001}
{#ijms-20-05008-f002}
{#ijms-20-05008-f003}
{#ijms-20-05008-f004}
{#ijms-20-05008-f005}
ijms-20-05008-t001_Table 1
######
Effect of *tirasemtiv* on muscle performance of ctrl and Neb cKO mice.
Ctrl cKO
------------------------------------- -------------- --------------------- --------------------- ---------------------------
Diaphragm *n* = 15 *n* = 15 *n* = 18 *n* = 18
Specific force (mN/mm^2^) at 200 Hz 200.0 ± 12.0 207.6 ± 11.7\*\*\* 86.8 ± 7.0\#\#\#\# 94.4 ± 6.9\*\*\*,\#\#\#\#
F~50~ frequency (Hz) 33.4 ± 2.7 20.3 ± 2.3\*\*\*\* 38.3 ± 2.4\#\#\#\# 21.1 ± 2.1\*\*\*\*
nH 1.47 ± 0.1 1.05 ± 0.05 2.49 ± 0.13\#\#\#\# 1.38 ± 0.09\*\*\*\*
Velocity @ 0.1Fmax(Lo/sec) 2.39 ± 0.1 4.05 ± 0.2\*\*\*\* 1.75 ± 0.13\#\#\#\# 2.47 ± 0.18\*\*\*\*
Max Power((mN/mm^2^)x(Lo/s)) 65.0 ± 5.4 115.7 ± 7.9\*\*\*\* 24.6 ± 3.7\#\#\#\# 37.5 ± 5.3\*\*\*\*
Gastrocnemius complex *n* = 9 *n* = 9 *n* = 11 *n* = 11
Specific force (mN/mg) at 200 Hz 2.50 ± 0.07 2.39 ± 0.08 0.81 ± 0.04\#\#\#\# 0.85 ± 0.03\#\#\#\#
F~50~ frequency (Hz) 57.9 ± 1.3 39.1 ± 2.7\*\*\*\* 37.2 ± 1.4\#\#\#\# 27.3 ± 1.5\*\*\#
nH 4.9 ± 0.5 2.7 ± 0.5\*\* 3.6 ± 0.5\# 2.5 ± 0.5\*
Asterisk: significant difference between *tirasemtiv* and vehicle; hashtag: significant difference between genotypes.
| 2024-04-09T01:27:17.760571 | https://example.com/article/3561 |
using System;
using System.Collections.Generic;
using System.Windows.Forms;
namespace WpfUserControlHost
{
static class Program
{
/// <summary>
/// The main entry point for the application.
/// </summary>
[STAThread]
static void Main()
{
Application.EnableVisualStyles();
Application.Run(new Form1());
}
}
} | 2024-01-15T01:27:17.760571 | https://example.com/article/2023 |
Close Margins Less Than 2 mm Are Not Associated With Higher Risks of 10-Year Local Recurrence and Breast Cancer Mortality Compared With Negative Margins in Women Treated With Breast-Conserving Therapy.
The 2014 Society of Surgical Oncology-American Society for Radiation Oncology consensus suggested "no ink on tumor" is a sufficient surgical margin for invasive breast cancer treated with breast-conserving surgery (BCS). Whether close margins <2 mm are associated with inferior outcomes remains controversial. This study evaluated 10-year outcomes by margin status in a population-based cohort treated with BCS and adjuvant radiation therapy (RT). The subjects were 10,863 women with invasive cancer categorized as pT1 to T3, any N, and M0 referred from 2001 to 2011, an era in which the institutional policy was to re-excise close or positive margins, except in select cases. All women underwent BCS and whole-breast RT with or without boost RT. Local recurrence (LR) and breast cancer-specific survival (BCSS) were examined using competing-risk analysis in cohorts with negative (≥2 mm; n = 9241, 85%), close (<2 mm; n = 1310, 12%), or positive (tumor touching ink; n = 312, 3%) margins. Multivariable analysis and matched-pair analysis were performed. The median follow-up period was 8 years. Systemic therapy was used in 87% of patients. Boost RT was used in 34.1%, 76.9%, and 79.5% of patients with negative, close, and positive margins, respectively. In the negative, close, and positive margin cohorts, the 10-year cumulative incidence of LR was 1.8%, 2.0%, and 1.1%, respectively (P = .759). Corresponding BCSS estimates were 93.9%, 91.8%, and 87.9%, respectively (P < .001). On multivariable analysis, close margins were not associated with increased LR (hazard ratio, 1.25; 95% confidence interval 0.79-1.97; P = .350) or reduced BCSS (hazard ratio, 1.25; 95% confidence interval 0.98-1.58, P = .071) relative to negative margins. On matched-pair analysis, close margin cases had similar LR (P = .114) and BCSS (P = .100) to negative margin controls. Select cases with close or positive margins in this population-based analysis had similar LR and BCSS to cases with negative margins. While these findings do not endorse omitting re-excision for all cases, the data support a policy of accepting carefully selected cases with close margins for adjuvant RT without re-excision. | 2024-07-11T01:27:17.760571 | https://example.com/article/1660 |
Q:
Confusion about function composition in Haskell
Consider following function definition in ghci.
let myF = sin . cos . sum
where, . stands for composition of two function (right associative). This I can call
myF [3.14, 3.14]
and it gives me desired result. Apparently, it passes list [3.14, 3.14] to function 'sum' and its 'result' is passed to cos and so on and on. However, if I do this in interpreter
let myF y = sin . cos . sum y
or
let myF y = sin . cos (sum y)
then I run into troubles. Modifying this into following gives me desired result.
let myF y = sin . cos $ sum y
or
let myF y = sin . cos . sum $ y
The type of (.) suggests that there should not be a problem with following form since 'sum y' is also a function (isn't it? After-all everything is a function in Haskell?)
let myF y = sin . cos . sum y -- this should work?
What is more interesting that I can make it work with two (or many) arguments (think of passing list [3.14, 3.14] as two arguments x and y), I have to write the following
let (myF x) y = (sin . cos . (+ x)) y
myF 3.14 3.14 -- it works!
let myF = sin . cos . (+)
myF 3.14 3.14 -- -- Doesn't work!
There is some discussion on HaskellWiki regarding this form which they call 'PointFree' form http://www.haskell.org/haskellwiki/Pointfree . By reading this article, I am suspecting that this form is different from composition of two lambda expressions. I am getting confused when I try to draw a line separating both of these styles.
A:
Let's look at the types. For sin and cos we have:
cos, sin :: Floating a => a -> a
For sum:
sum :: Num a => [a] -> a
Now, sum y turns that into a
sum y :: Num a => a
which is a value, not a function (you could name it a function with no arguments but this is very tricky and you also need to name () -> a functions - there was a discussion somewhere about this but I cannot find the link now - Conal spoke about it).
Anyway, trying cos . sum y won't work because . expects both sides to have types a -> b and b -> c (signature is (b -> c) -> (a -> b) -> (a -> c)) and sum y cannot be written in this style. That's why you need to include parentheses or $.
As for point-free style, the simples translation recipe is this:
take you function and move the last argument of function to the end of the expression separated by a function application. For example, in case of mysum x y = x + y we have y at the end but we cannot remove it right now. Instead, rewriting as mysum x y = (x +) y it works.
remove said argument. In our case mysum x = (x +)
repeat until you have no more arguments. Here mysum = (+)
(I chose a simple example, for more convoluted cases you'll have to use flip and others)
A:
No, sum y is not a function. It's a number, just like sum [1, 2, 3] is. It therefore makes complete sense that you cannot use the function composition operator (.) with it.
Not everything in Haskell are functions.
A:
The obligatory cryptic answer is this: (space) binds more tightly than .
Most whitespace in Haskell can be thought of as a very high-fixity $ (the "apply" function). w x . y z is basically the same as (w $ x) . (y $ z)
When you are first learning about $ and . you should also make sure you learn about (space) as well, and make sure you understand how the language semantics implicitly parenthesize things in ways that may not (at first blush) appear intuitive.
| 2024-04-19T01:27:17.760571 | https://example.com/article/5572 |
[About the encapsulation of maupasina weissi (Nematoda, Subuluroidea) in the experimental intermediate host Locusta migratoria (Insecta, Orthoptera) (author's transl)].
Infective larvae of Maupasine weissi (Nematoda, Subuluroidea) are encapsulated in the adipose tissue of the Insect intermediate host where they elicit a hemocytic reaction. This encapsulation is ambiguous when compared with those of Subulurid and Spirurid Nematodes. It shows a defective adaptation of the parasite to the intermediate host. | 2024-05-04T01:27:17.760571 | https://example.com/article/4025 |
More Lanes Added for L.A. Cycling Enthusiasts December 26th, 2014 at 8:30 am Tweet
In 2010, Los Angeles passed a plan to add 1,684 miles of bikeways throughout the city. Over the years, the city has added hundreds of miles and continues to add more each year. Initially, the plan was to add 200 miles a year. The main goals of this project were to encourage more people to use a bicycle as their preferred form of transportation, decrease traffic and make bicycle commuting safer. Connecting a vast network of bike lanes allows people to get all over the city conveniently by bike.
With improvements to the bike lane infrastructure, more people will be motivated to get outside and bike. Bike paths make biking safer and help to reduce the rate of cyclist injuries. In addition, they also increase safety for motorists. Biking instead of driving is great for both your own personal health as well as the environment.
However, this project is not without its controversies. Some in Los Angeles argue that increasing bike lanes could actually make traffic worse, not better. It’s thought that some of the planned lanes will reduce flow on already tightly packed streets. In some areas, traffic is already reduced to one lane due to parking lanes and it’s unclear exactly how the bike lanes will fit into the equation.
Currently, no measurement is being completed to determine who is right in this debate. City planners are working on how to best measure, track and analyze how transportation is affected where the new bikeways have been added. The Transportation Department is making continued improvements to their analysis process and looking for additional budget-friendly tracking options.
To see a diagram of how the city’s expanded bikeways year by year, check out this graphic from the L.A. Times. | 2024-05-03T01:27:17.760571 | https://example.com/article/8144 |
Serum protein binding of tolbutamide in patients treated with antiepileptic drugs.
The possible development of a displacement interaction involving tolbutamide, in epileptic patients, has been explored by studying the serum protein binding of this drug in vitro in 199 samples of sera from patients treated with antiepileptic agents included in a programme of therapeutic drug monitoring. 82 of the samples were from patients receiving a single drug, 86 from patients treated with 2 drugs, and 31 from patients treated with 3 drugs. The free fraction of tolbutamide was higher in serum from patients treated with antiepileptic drugs than in serum from untreated 'normal' volunteers. The increase was more marked the greater the number of antiepileptic drugs administered. Valproate appeared to be the most powerful displacing agent. | 2024-03-27T01:27:17.760571 | https://example.com/article/9833 |
James Franco (Picture: AP)
Marina Abramovic, the Grand Dame of Performance Art, is making a new film about the life of 34-year-old Oz The Great and Powerful star James Franco.
The Serbian artist is a personal friend of Franco, a modern day ‘renaissance man’ who juggles life as a poet, painter, photographer and PhD student alongside a successful Hollywood career, and has dubbed him ‘the most interesting actor of the moment.’
Last year Abramovich was herself the subject of the film documentary Marina Abramovic: The Artist Is Present, which focused on her life as she prepared for a retrospective at the Museum of Modern Art in New York. The exhibition included a 736.5 hour performance in which she sat totally still and silently at a table, while visitors including Lady Gaga lined up to perch opposite her. Some callers, including Girls star Jemima Kirke, were moved to tears by the experience.
Abramovic has previously joined forces with Antony Hegarty, Willem Dafoe and legendary avant-garde director Robert Wilson to create an autobiographical piece of musical theatre, The Life and Death of Marina Abramovich. It remains to be seen if she will collaborate with any big creatives names on this new project, which will be her first film.
Advertisement | 2023-08-27T01:27:17.760571 | https://example.com/article/1471 |
Continuing in an improbable but pleasantly surprising new role, I’ll be serving as an assistant track coach this spring, specializing in sprinting and long jump.
Varsity Girls Basketball made BFA history last weekend by coming in second, in a nail-biting overtime, in DoDDS European Championships!
Curriculum for March: English: The Great Gatsby, modern poetry; Canadian History: World War II, the Cold War.
I’m thankful for:
The study of literature, which so often facilitates conversations about wisdom, art, love and truth, as we ponder together what it means to look for Christ in a world that often tries to hide Him.
Great conversations in the last weeks with friends, roommates, students, colleagues and parents. I’ve been so blessed by the perspectives and stories of others lately, and continue to be thankful for the diversity of this place.
Warm days in winter, allowing me to spend afternoons in the sun, running or grading or basking, loving life.
The Munich US Consulate who processed my passport in just under two weeks. Brilliant! Truly an answer to prayer.
Please Be in Prayer For:
High School Winter Retreat: Pray for students and staff to have a restful time of community, fun and spiritual renewal.
Health: Students and staff have been hard-hit by various illnesses lately. While I’ve been spared so far, pray for healing and rest for all.
Small Group: Emily and I will be spending a great deal of intentional time with our ninth grade girls’ small group this weekend. Pray that this is a time of relationship-building, both among the girls and with us.
If you have questions, want information about supporting financially, or want to share news or prayer requests of your own, please email me at kristi.dahlstrom@gmail.com. I’d love to hear from you! | 2023-11-26T01:27:17.760571 | https://example.com/article/7552 |
The sassy look of these dark green boots by Bettie Page isn't meant to be kept all to yourself! From their floral cutouts and bow-tied shafts, to their tapered heels and leather linings, these personality- packed kicks beg to be debuted here, there, and ev
These L'Artiste by Spring Step Fab Pumps are French designed and nature inspired with a unique and fun style for everyday wear. They showcase high quality full grain leather uppers, a beautiful back strap with laser cutouts, etching leatherwork, and open toe and heel, and a hook and loop closure. They feature a soft memory foam foot bed, durable rubber outsoles, and a 3-inch stacked wooden heel, with a 1/2-inch platform. Spot clean only.
These L'Artiste by Spring Step Fab Pumps are French designed and nature inspired with a unique and fun style for everyday wear. They showcase high quality full grain leather uppers, a beautiful back strap with laser cutouts, etching leatherwork, and open toe and heel, and a hook and loop closure. They feature a soft memory foam foot bed, durable rubber outsoles, and a 3-inch stacked wooden heel, with a 1/2-inch platform. Spot clean only.
Up for sale is a pair of BRAND NEW, NEVER WORN J SHOES SARDINIA FAB COLOR: SOFT GREY / BONE MEN'S SIZE 10.5 FREE DOMESTIC SHIPPING MONEY BACK GUARANTEE. If for any reason you are not completely satisfied with the item, please return it for a full refund.
These L'Artiste by Spring Step Fab Pumps are French designed and nature inspired with a unique and fun style for everyday wear. They showcase high quality full grain leather uppers, a beautiful back strap with laser cutouts, etching leatherwork, and open toe and heel, and a hook and loop closure. They feature a soft memory foam foot bed, durable rubber outsoles, and a 3-inch stacked wooden heel, with a 1/2-inch platform. Spot clean only.
These L'Artiste by Spring Step Fab Pumps are French designed and nature inspired with a unique and fun style for everyday wear. They showcase high quality full grain leather uppers, a beautiful back strap with laser cutouts, etching leatherwork, and open toe and heel, and a hook and loop closure. They feature a soft memory foam foot bed, durable rubber outsoles, and a 3-inch stacked wooden heel, with a 1/2-inch platform. Spot clean only.
These pull on boots with a soft sole and cozy and adorable. Like all Robeez Soft Sole shoes, this footwear features a non-slip suede outsole, which protects your child from slipping, and an elasticized ankle band which will keep this shoe securely on your baby's foot. If your child can't go barefoot, choose Robeez. Size Chart:.
These L'Artiste by Spring Step Fab Pumps are French designed and nature inspired with a unique and fun style for everyday wear. They showcase high quality full grain leather uppers, a beautiful back strap with laser cutouts, etching leatherwork, and open toe and heel, and a hook and loop closure. They feature a soft memory foam foot bed, durable rubber outsoles, and a 3-inch stacked wooden heel, with a 1/2-inch platform. Spot clean only.
Prices are provided by the merchants. We assume no responsibility for accuracy of price information provided by merchants. Please alert us to any pricing discrepancies and we will alert the merchant. Sales taxes are estimated at the zip code level. Shipping costs are estimates. Please check store for exact shipping costs. To learn more about why certain stores are listed on the site, click here
Product specifications are obtained from third parties, and while we make every effort to assure the accuracy of product information, we do not assume any liability for inaccuracies. Store ratings and product reviews are written and submitted by online shoppers to assist you as you shop. They do not reflect our opinions. We take no responsibility for the content of ratings and reviews submitted by users. | 2024-03-27T01:27:17.760571 | https://example.com/article/3022 |
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EngineeringInsurance
Engineering Insurance from Coversure :Let Us Engineer The Ideal Protection For Your Business
Running an engineering firm means facing a wide range of complex insurance risks. Whether your business is focused on creating design specifications or offers an end-to-end design and build service, is light or heavy, hi-tech or traditional, there are many areas of your business that need effective protection. This is where engineering insurance from Coversure comes in. Our comprehensive engineering insurance cover can be tailored to help protect all aspects of your company on a single, affordable policy.
Engineering Insurance From Coversure
Coversure's engineering cover is inclusive, highly-competitive and can be designed around the specific risks of firms both large and small. A typical policy includes the following essential protections:
Machinery and sudden unforeseen damage - including loss of profits
Machinery accidental damage cover - damage caused by breakdown and accidental damage and gives you the flexibility to insure all of the machinery on the premises
Deterioration of stock and of stored materials - covering loss of or damage by deterioration or putrefaction to goods stored in any cold storage spaces
Computers and I.T. cover - including the cost of recovering lost design or client data as well as providing replacement hardware
Coversure's Flexible Payment Options
To make getting the best engineering cover even easier, Coversure can offer you a range of flexible payment methods including payment by instalment plans.
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If you'd like to know more about our flexible payment options for engineering cover or would like to receive a highly-competitive engineering insurance quote that's tailored to your business's needs, then please contact your local Coversure office by clicking here.
Find your local Office
If you want our best price for your insurance or just want to make sure you have exactly the cover you need, it’s best to talk to your local Coversure office who will have access to our latest schemes and discounts.
Simply enter your postcode and find the contact details for your local office.
PLEASE NOTE: Coversure will save details on this computer regarding your postcode and the location of your nearest Coversure office. This will help us display relevant information to you on future visits. For more information please review our cookie and privacy policies. | 2024-05-17T01:27:17.760571 | https://example.com/article/9309 |
Maendeleo Chap Chap’s Titus Lolmogut Leokoe was third with 663 votes, followed by Tobias Chodow Lenguro of ODM (309) and Tom Ebukut Ekale of Frontier Alliance Party (FAP) with 247 votes.
Alex Ltamuni Louwa of Ford-Kenya trailed from behind with 147 votes.
The Thursday mini-election registered a low turnout in what many have termed as a lack of interest.
CHALLENGES
Laisamis Sub-County electoral commission IEBC Returning Officer Amina Jarso also decried logistical challenges including the vastness of the ward in arid northern Kenya, which she said ought to be a constituency on its own.
The Loiyangalani MCA seat fell vacant following the sudden death of FAP's ward representative Marko Ekale on April 29, 2019 in Mombasa.
Mr Nakeno termed the election process as fair and well conducted in all the polling stations, which he said he had visited.
“I am humbled to be considered worthy of this win by the Loiyangalani Ward electorates. I promise to deliver to my very best as soon as I get to office,” said the MCA-elect.
All the other candidates conceded defeat and congratulated Mr Nakeno for his victory. | 2024-04-07T01:27:17.760571 | https://example.com/article/2752 |
My Vodou Apprenticeship
I was not in Haiti, nor were the drums pounding. No ceremony had been performed. A friend, who said she was a medium, told me about the lwa: archetypal ancestors who speak through the living.
Bored, I told her I wasn’t interested; that didn’t sound likely to me. She said she could prove it.
Incense was lit, the lights were darkened and she began her spasms as the “spirit” took hold. I sat across the room shaking my head. Was this the best performance she could muster?
Then everything changed.
The Baron came through, one of the death spirits of Vodou. The shift in the room itself was palpable. I was jolted by a presence that even I could feel, and sat as straight as a roof-pillar for the next eighty seconds.
This began a lifelong interest in the beautiful faith of the diaspora, Vodou.
Vodouns after Fête Gede 2012. Picture by Saumya Haas.
La Source Ancienne
In the past I’ve found closed doors around Vodou. While my scholarly knowledge grew, my practical experience remained at a standstill, blocked by my lack of Haitian or even African ancestry.
But that changed with the friendship of Urban (Conversion or Initiation?) and Saumya (What is Voodoo?) and with La Source Ancienne, the community of Vodouns who initiated them in New Orleans. Open to people of all races, their house (temple) practices a very public, very community-oriented flavor of Vodou.
Vodou ceremony is a swirl of music, dance, laughter and visionary trance. It is experiential, and nothing you have read here or elsewhere gives you any sense of what that means.
I would enjoy simply being lost in it every week.
But the community can use more than that. During my six months here, my mission is to learn as many songs as I can—so that I can contribute another voice and, in turn, carry the ancient words to others.
And I’m chasing down the drummers to learn the beats, too.
I’m not formally apprenticed. There is no formal apprenticeship here. You just learn by doing and if you stay around long enough, you know something. Very different from the European way of my other religion.
This is New World, and that’s where we live. The Caribbean lies ahead of me, many more gods along the way. It’s time to see the rest of my heritage.
I really look forward to reading more on your experiences in Voodoo. Thank you for sharing. Especially since there is so much misunderstanding of Voodoo. Hopefully with more people talking about it on blogs like yours and elsewhere that the stereotypes and stigma around Voodoo will diminish. | 2023-08-03T01:27:17.760571 | https://example.com/article/3122 |
Cablemás
Cablemás was a Mexican regional telecommunications company, owned by Grupo Televisa. The company had a triple play-based service, consisting on subscription television, phone line and broadband internet offered in a single package deal. The company had its headquarters in Colonia Juárez, Cuauhtémoc, Mexico City.
In 2015, Cablemás's operations were rebranded as izzi Telecom following a corporate decision by Televisa to unify its regional-based TV providers, such as Cablecom and Cablevisión, under one sole brand.
History
It is the second-largest cable operator in Mexico after Megacable. It has offered cable since the late 1980s, and broadband Internet since the late 1990s with approximately 953,000 cable TV customers, 325,000 high speed internet customers, and 180,000 telephony lines in 49 cities in Mexico (July 2010). Cablemas is also the first company to distribute VoIP in Tijuana, Baja California. VoIP is the spearhead of Cablemas's new promotionals.
Due to the previous laws in Mexico, in the past, telecommunication companies cannot provide more than two services. An example of this is the company Telnor, which provides telephones and Internet. Another example is Megacable, a company that provides cable and broadband Internet. To provide more services, Cablemas made an alliance with Axtel, a new telephone company operating from Monterrey, and the new Cablemas-Axtel Alliance was created with initially nearly 1,000 customers. Nowadays, Cablemás offers also fixed telephony at its own.
Cablemas bases its primary operation in the city of Tijuana, which is also the city that produces the highest influx of money into the company.
From 2015, Cablemás disappears as a trademark to make way for Izzi Telecom, following Televisa's strategy to unify its cable TV operations throughout the country under a single brand (just as Cablevisión).
Controversy
Ongoing issues with service on Isla Mujeres and in parts of Cancún and Mérida have led to allegations of fraud against Cablemás. Tests performed by local municipalities show that advertised rates of 10Mbit/s are actually capped as low as .5 or even .3 Mbit/s, or less than 5% of advertised speeds. The lack of a stable Internet connection, coupled with the company's virtual monopoly in isolated regions like Isla Mujeres, has resulted in unrest among businesses and residents. In December 2014, the municipality of Isla Mujeres vowed to investigate Cablemás and recommend sanctions to the governor of Quintana Roo.
References
External links
Official Webpage
Press Releases
Annual Financial Statements
Issues with Cablemas
Category:Telecommunications companies of Mexico
Category:Telecommunications companies established in 1968
Category:Televisa subsidiaries
Category:1968 establishments in Mexico
Category:Technology companies disestablished in 2015
Category:2015 disestablishments in Mexico | 2024-05-11T01:27:17.760571 | https://example.com/article/1204 |
# Get Smart About Nicotine
A Hazelden Quick Guide
Hazelden Publishing
Center City, Minnesota 55012
800-328-9000
hazelden.org/bookstore
© 2013 by Hazelden Foundation
All rights reserved. Published 2013.
No part of this publication may be reproduced in any form or by any means without the express written permission of the publisher. Failure to comply with these terms may expose you to legal action and damages for copyright infringement.
Library of Congress Cataloging-in-Publication Data [to come]
eISBN 978-1-61649-518-3
Editor's notes:
The names, details, and circumstances may have been changed to protect the privacy of those mentioned in this publication.
This publication is not intended as a substitute for the advice of health care professionals.
Alcoholics Anonymous is a registered trademark of Alcoholics Anonymous World Services, Inc.
17 16 15 14 13 1 2 3 4 5 6
Cover design by Theresa Jaeger Gedig
Interior design and typesetting by BookMobile Design & Digital Publisher Services
Developmental editor: Peter Schletty
# About Hazelden Quick Guides
Hazelden Quick Guides are short, accessible e-books that draw on the original work and best practices of leading experts to help readers address common addiction recovery and emotional health issues. The Get Smart collection describes each of the major drugs, its historical, cultural, and legal context, its addictive qualities, and information on prevention and treatment.
# Contents
Introduction
History
How It Works
The Tobacco Plant
Combustible Tobacco Products
Smokeless Tobacco Products
Addictive Properties
Biological Factors
Genetic Factors
Environmental Factors
Use, Abuse, and Addiction
Christine's Story
Tobacco Use Disorder
Effects of Nicotine and Tobacco Use
Health Consequences and Risks for the User
Health Consequences and Risks for Secondhand Smoke
Economic Effects
Turning It Around—The Benefits of Quitting (Reversing Harms)
Treatment for Nicotine Addiction
Methods of Quitting
Programs for Youth
Quitting Tobacco—What to Expect
Relapse
Prevention Practices
Youth Tobacco Prevention
Notes
# Introduction
People have been using tobacco products in some manner or another since the early 1500s, though the history goes well beyond that. At one point, smoking was the norm in our culture, even a coming-of-age transition. A culture shift that began fifty or more years ago, backed by science and research, has dramatically reduced the number of people who use tobacco in the United States. Yet the problem persists, striking costly blows to our health and economy, and every new user is likely to become hooked on nicotine. The recent introduction of new smokeless forms of tobacco presents new challenges to the antitobacco movement. This e-booklet will explore the long history of tobacco, its cultural influences, and the nature of nicotine addiction. We'll also discuss different routes to recovery and why prevention is so critical with this drug.
## History
### Tobacco: America's Gift to the World
History tells us that Columbus and other early explorers were searching for a new route to Asia, as well as riches such as gold and spices, when they stumbled upon the New World. Who knew that one discovery of this era, the tobacco plant, would have such a profound effect on the world—and such devastating health consequences. Columbus is said to have had his first glimpse of tobacco when he stepped onto a small island in the Bahamas and the locals offered him dried tobacco leaves. Again and again, he and his crew witnessed natives using this plant and decided to bring it back with them to Spain.
A plant native to the Americas, tobacco was in use long before the European explorers caught sight of it. Archaeologists trace tobacco to the Andes area of South America, where it was first cultivated between 5000 and 3000 BCE. From there, the plant spread northward. Drawings carved in stone reveal that the Mayans used tobacco somewhere between 600 and 900 AD. By the time Columbus arrived in the region in 1492, tobacco use had spread throughout the Americas and to the Caribbean Islands.
When tobacco was introduced to Europeans, the medicinal value of the plant was of keen interest. This was an era where doctors would typically treat patients with various oils or herbs. And, unlike most plants that were thought to heal a specific ailment, tobacco was tested as a remedy for maladies and injuries of virtually all forms. Among other applications, it was ground up and swallowed or placed upon wounds. Before long, Europeans considered it a panacea and used it to treat everything from tumors to ringworm to the plague. In 1560, a French ambassador was introduced to tobacco while in Portugal; he brought the plant back with him and gave it to the queen, who suffered from migraines. The ambassador's name was Jean Nicot, and cultivated tobacco eventually took on the scientific name Nicotiana tabacum.
Tobacco was not limited to medicinal use. Explorers to the New World had seen the natives smoking tobacco in pipes of various shapes and sizes. With this in mind, they began smoking the dried leaves as well; eventually, they also chewed it and later rolled and smoked it. By the mid-1500s tobacco was well known for its pleasurable effects.
Yet, even from the beginning, tobacco had its critics. King James I of England was one. In 1604, he raised the import tax on tobacco forty-fold and wrote a book lambasting its effects, A Counterblaste to Tobacco. In 1610, Sir Francis Bacon wrote about its habit-forming characteristics that were difficult to break. In time, doctors began questioning how effective tobacco was as a therapy. By the end of the seventeenth century, its perceived value as an effective pharmaceutical was in decline, though the pharmacies of the day still made it available. Still, cure-all or not, tobacco was to retain a prominent role on the continent and beyond.
Native Americans and Tobacco
Throughout history Native American peoples have used tobacco in their spiritual ceremonies and rituals. They have also used it for medicinal purposes. And though Columbus and his companions witnessed Native American peoples enjoying tobacco as a part of their rituals, they did not smoke it every day.
Most Native Americans smoked tobacco in a pipe, which calls to mind the peace pipe that is used during ceremonies and symbolizes respect for one another. Smoking tobacco in a pipe still remains an important part of many Native American ceremonies, but many Native Americans now smoke cigarettes and use other recreational forms of tobacco as well.
When King James granted the Virginia Company of London permission to become the first English colony in the New World, profit was one of the things on his mind. The colonists arrived in 1607 but were unable to make use of the riches of this new land. It wasn't until 1610 with the arrival of John Rolfe, who would eventually marry Pocahontas, that the colonists turned their efforts toward growing and exporting tobacco. As a lucrative, in-demand crop, tobacco was also one of the keys to the colony's survival.
Tobacco continued to grow in importance in the Virginia colony. The rise of tobacco plantations, and the necessary labor for growing and harvesting the crop, would even be a factor in the development of slavery.
While the Virginia Company may have been the first to profit from tobacco in the New World, others were soon to follow. Today, the United States' largest producers of tobacco are North Carolina, Kentucky, Virginia, and Tennessee. Although small family tobacco farms have become rare and tobacco production in the United States has declined since the 1950s, it has grown throughout the world. The production of cigarettes increased with advances in technology and agriculture. Today, tobacco is produced in almost every nation, with an annual output of approximately six trillion cigarettes.
As smoking gained popularity, tobacco companies grew and became more powerful. Beginning in the 1920s, advertisers began targeting women, who took up the habit in droves. Cigarette companies such as Lucky Strike reached women through ad campaigns that emphasized the connection between smoking and weight control. Women were happy to oblige. The nineteenth amendment had just been passed, granting women the right to vote, so women were eager to exercise their newfound freedom. Of course, men still continued to smoke, and in increasing numbers. In World Wars I and II, soldiers were issued tobacco and rolling papers—or rolled cigarettes—as part of their rations, a move that was intended to offer stress relief but undoubtedly encouraged use that led to addiction.
By the midseventies, tobacco and its products—especially cigarettes—seemed to be everywhere. Kids made ashtrays for their parents in art class; the Marlboro Man greeted us on roadside billboards as we drove to and from the office; and whether you were at a baseball game, a medical clinic, or on an airplane, you or the person next to you may have been lighting up and taking deep drags. Cigarette use had become a ubiquitous and established part of our culture. While some restaurants featured "no-smoking sections," you certainly couldn't step inside a bar and expect to breathe clean air. But science was catching up with the behavior of the masses, and it wouldn't be long before the smoking culture and the tobacco industry were in for a shock.
### Hear, Hear—News from the Surgeon General
The U.S. Surgeon General is charged with providing leadership in medicine, and protecting and advancing the health of the nation. So when he or she releases a report, even on something as mundane as tobacco use, people stop and listen. The first Surgeon General's report on smoking was released in 1964—on a Saturday, so as not to send the stock market into a tailspin—and it confirmed that tobacco use was hazardous to our health and even led to cancer. All of a sudden, the country was shaken out of its hazy stupor.
That first Surgeon General's report on smoking was created by an advisory committee on smoking and health that had been brought together to investigate the health ramifications of smoking. Although the report stopped short of calling nicotine an addictive drug, it pronounced a definite connection between smoking and lung cancer and also asserted that the risk increased with the amount of cigarettes smoked.
The report marked the beginning of a shift in consumers' beliefs about tobacco as well as in their behavior, if only in small ways at first. Science had spoken, and now the government stepped in to take action. As a first move, Congress ordered that cigarette packaging contain a health warning. Next, it banned tobacco advertising on television and radio.
For their part, the tobacco companies responded to the public's newfound fear of their products by creating new, "safer" cigarettes. Now, and for many years to come, consumers could walk into a gas station and find "low," "light," and "mild" versions of their favorite cigarettes. Advertisements claimed, among other things, that their products were low-tar, ultra low-tar, or the lowest tar available, all while being "kindest to your taste." One advertisement stated, "You don't cut down on your smoking...The big duke filter does it for you." These product claims implied that smoking these cigarettes meant less risk. Yet research did not bear this out—reducing the level of tar and nicotine was not proven to safeguard users from the hazards of smoking.
### Lawsuits
The evidence that smoking endangered our health and that the tobacco industry was well aware of this continued to mount. Starting in 1994 with Mississippi, states began filing lawsuits against the tobacco industry for the public health costs associated with tobacco use. The lawsuits contended that the tobacco industry had engaged in consumer fraud, conspired to manipulate nicotine levels in cigarettes, and marketed their products to minors.
Prior to these state lawsuits, individuals with lung cancer and other diseases had sued cigarette manufacturers, making claims of fraud and that the companies knew their products were hazardous but had not warned the users. In these cases, the tobacco companies denied such claims, arguing that tobacco was neither harmful nor addictive and that smokers knew the risks they were taking. Whether in the initial rulings or in appeals court decisions, tobacco always came out the winner in these lawsuits.
But lawsuits with states were a different matter, and the tobacco industry would not prevail this time; instead, they would settle. The first states to reach agreements with the industry, in early 1998, were Minnesota, Mississippi, Texas, and Florida. The terms of each settlement differed by state, but each included awards in the billions. During the Minnesota lawsuit, in particular, the industry was forced to turn over internal documents it had previously withheld—35 million pages worth—and the content of those reports and memos incriminated the industry as well as gravely damaged its credibility.
An article published in the Journal of the American Medical Association summarized the significance of what was unearthed: "These documents reveal that for decades, the industry knew and internally acknowledged that nicotine is an addictive drug and cigarettes are the ultimate nicotine delivery device; that nicotine addiction can be perpetuated and even enhanced through cigarette design alterations and manipulations; and that 'health-conscious' smokers could be captured by low-tar, low-nicotine products, all the while ensuring the marketplace viability of their products." These documents would become a key part of the settlement that was reached six months later with the remaining states and territories that had sued the industry.
The clandestine reports and memos included the following statements:
* "Very few consumers are aware of the effects of nicotine, i.e., its addictive nature and that nicotine is a poison." —1978 Brown and Williamson memo
* "Goal—Determine the minimum level of nicotine that will allow continued smoking. We hypothesize satisfaction cannot be compensated for by psychological satisfaction. At this point smokers will quit, or return to higher T&N [tar and nicotine] brands." —February 13, 1980, Lorillard memo on the RT Information Task Force from Richard E. Smith to Alexander Spears, Minnesota Trial Exhibit #10170
* "Moreover, nicotine is addictive. We are, then, in the business of selling nicotine, an addictive drug effective in the release of stress mechanisms." —Addison Yeaman, B&W general counsel, 1963 {1802.05, p.4} (included in book The Cigarette Papers, by Stanton A. Glantz, John Slade, Lisa A. Bero, Peter Henauer, and Deborah E. Barnes)
* "We don't smoke that s**t, we just sell it. We reserve that right for the young, the poor, the black and the stupid." —R.J. Reynolds, as quoted in The Times of London, August 2, 1992
### The Master Settlement Agreement
The settlement between the five largest tobacco companies and the remaining forty-six states plus five territories and the District of Columbia was the largest recovery in the history of the United States. The historic $206 billion settlement would be paid out in yearly installments for twenty-five years, followed by annual payments for an indefinite period. The monies would help pay states for treating tobacco-related illnesses and would fund prevention programs.
In addition to the financial award to states and territories, the agreement included a host of terms limiting the sales and marketing of tobacco, especially to youth. Through numerous regulations, the settlement essentially aimed to prevent young people from taking up the habit and to protect those who were already users. The settlement agreement brought permanent relief to consumers by
* prohibiting youth targeting
* banning the use of cartoons in marketing
* eliminating outdoor and transit advertising
* limiting sponsorship (including concerts, sporting events, and other events attended by youth)
* banning the practice of making payments to the media
* banning tobacco-related merchandise
* prohibiting the practice of offering youth free samples and banning proof-of-purchase gifts to youth
* limiting tobacco lobbying
* establishing a minimum cigarette pack size of twenty
Fifteen years later, have these restrictions and regulations been effective? American Legacy Foundation, a national foundation whose creation was mandated in the terms of the Master Settlement Agreement, studies and educates the public about the impact of tobacco. The foundation reported that in the first ten years since the settlement, consumption of cigarettes in the United States decreased by approximately 28 percent, or 135 billion cigarettes. Yet in the past few years, the rate of tobacco use has stalled. Even so, government efforts to reduce smoking in the United States are continuing.
### The Tobacco Buyout
One such effort is to reduce the amount of tobacco grown. Toward that end, President George W. Bush signed the Fair and Equitable Tobacco Reform Act into law in October 2004, thereby ending the tobacco quota program for farmers that had been in place since the Depression. The quota program, which had dictated the amount of tobacco farmers could grow and sell and guaranteed prices, was replaced by the Tobacco Transition Payment Program—otherwise known as the tobacco buyout. This new program lifted restrictions on how much tobacco farmers could grow while eliminating price support. It was designed to both reduce the amount of tobacco grown in the United States and to aid growers whose profits had declined due to less demand for tobacco and competition from growers outside the country. The buyout program agreed to pay farmers, based on previous tobacco yields, annual payments for ten years as they transitioned to other crops or other activities.
The program has been an undeniable success. In Maryland alone, 86 percent of eligible farmers took the deal, with 75 percent of those growers signed up in the first year it was offered. In the eyes of some, this program has cost more than the settlements paid to farmers; another price was the end of a three-hundred-year tradition of tobacco farming. According to the 2007 Census of Agriculture, the number of tobacco farms in the United States dropped from more than 93,000 in 1997 to just more than 16,000 in 2007.
### FDA Control
Legislation has continued to place greater controls on "big tobacco" in an effort to protect the nation's health. The Family Smoking Prevention and Public Tobacco Control Act, passed in 2009, was viewed as a major breakthrough in the regulation of tobacco product. With it, the FDA finally gained the authority to regulate tobacco products, including distribution and marketing. What's more, the legislation banned most flavored cigarettes (except menthol and natural tobacco) and raised federal excise taxes. It also banned new vending machines with tobacco products and restricted those already in use to places where only people over age twenty-one can frequent. The act prohibited tobacco companies from using so-called reduced-harm claims on their product labels—no more low-tar, low-nicotine, and so on—or advertising as of June 2010. It also called for new, larger warning labels on tobacco products—the first update in twenty-five years. However, in 2011, the big tobacco companies filed a lawsuit against the FDA over the proposed new warning labels, arguing that the larger size of the warnings and the accompanying graphics would cover too much of their product labels. In 2012, an appeals court ruled in favor of the tobacco companies, agreeing that the proposed labels violated free speech protection.
### Cultural Shift
With the staggering amount of scientific evidence that seems to link smoking and other tobacco use with a variety of diseases, and with the unprecedented controls and restriction on smoking, the culture has shifted away from smoking and largely embraced tobacco-free living. Indeed, today it is not uncommon to see smokers banished to the back door or beyond, as more and more workplaces and public locations ban smoking on their premises. In a culture that by and large prides itself on tolerance, smoking seems to be the exception. Children who wrinkle up faces in disgust or complain loudly when they happen to smell cigarette smoke are rarely scolded by parents (who perhaps want to promote that antismoking attitude). The days of smoking at one's office Mad Men style are long gone.
Who Is Still Smoking?
The number of smokers has dropped sharply in the past forty years, falling more than 50 percent from 1965 to 2009. Still, cigarettes and other tobacco products have not gone away, with an estimated 46.6 million adults—about one in five people—still smoking as of 2009. Here's a look at who is still shelling out five to twelve dollars for a pack of cigarettes:
Men are smoking more than women: About 23.5 percent of men compared to 17.9 percent of women smoke.
White people are smoking more than any other group: More than 22 percent of non-Hispanic whites smoke followed by 21 percent of non-Hispanic blacks, 14.5 percent of Hispanics, and 12 percent of Asians in the United States.
Adolescents: The 2012 Monitoring the Future study found that 10.6 percent of students in grades eight, ten, and twelve had smoked within the past thirty days.
Pregnant women: As of 2005, 10.7 percent of all women smoked during pregnancy, down almost 45 percent from 1990.
Worldwide, even as tobacco use is going down among countries with higher standards of living, it is increasing as a whole. According to the World Health Organization, nearly 80 percent of the world's one billion smokers now live in low- and middle-income countries.
Over the past one hundred years, tobacco use in the United States has seemed to ride a roller coaster of highs and lows. While use skyrocketed after World War I and World War II, it has declined rapidly in the forty years since the Surgeon General's first report on the deadly effects of tobacco. The lawsuits by individual states and their historic settlements have changed the industry forevermore, as have the resulting restrictions and move to government oversight. Yet, the story of tobacco is far from over. The health consequences of new forms of smokeless tobacco are still being examined, and the youth culture's gravitation toward what's new and novel is still being played out.
# How It Works
We know that all tobacco products contain dried leaves from the tobacco plant in various states, but now let's take a closer look at the plant itself and how it is transformed into the products that warrant so much attention. This section will also explore the various tobacco products and discuss what makes them unsafe.
## The Tobacco Plant
Although the tobacco plant is native to the Americas (and parts of Australia), it grows well in many climates and regions of the world. Today, tobacco is grown in almost every nation, especially China, Australia, Cuba, the Dominican Republic, Jamaica, and Puerto Rico. As production has scaled back in the United States, Mexico, and Canada, it has increased elsewhere, particularly in Asia and Africa. The World Health Organization reports that Brazil, China, the United States, India, and Turkey together produce two-thirds of the world's tobacco.
Tobacco's genus, Nicotiana, includes more than twenty-one species. Today, the most frequently grown and used species are Nicotiana tabacum and Nicotiana rustica. Burley, flue-cured, dark air, and dark fire are the most common types of tobacco grown in the United States.
Even with modern technologies, tobacco farming is labor intensive. Ten to eleven weeks after they are transplanted, the plants are mature and harvesting begins with the leaves that are ripened; the plants ripen from the bottom up.
After harvesting, the tobacco leaves are dried and cured. This drying process takes anywhere from one to eight weeks, depending on the method used: flue-cured, air-cured, or fire-cured. The curing process not only removes moisture from the tobacco leaves, but also enhances its color and aroma. The tobacco used in cigarettes is then ready to be shredded, rolled into paper, and packaged. Tobacco used for cigars is also aged and fermented after it is cured.
Now let's take a closer look at each of the main types of tobacco products.
## Combustible Tobacco Products
### Cigarettes
Traditional cigarettes are formed by rolling shredded or ground tobacco in paper or another material that is not made out of tobacco. Most manufactured cigarettes on the market today include filters. Cigarettes are sometimes called smokes, fags, heaters, cigs, butts—even cancer sticks.
### Herbal Cigarettes
Herbal cigarettes—thin hand-rolled cigarettes wrapped in plant leaves—come in multiple flavors and do not include filters. Bidis are imported from India and other Southeast Asian countries. They may be flavored to taste like chocolate, licorice, or fruit. Kreteks are another type of herbal cigarette; these come from Indonesia and are flavored with cloves. As of 2009 with the passage of the Family Smoking Prevention and Tobacco Control Act, flavored cigarettes are not legal for sale in the United States, though it is not against the law to use such cigarettes. Users now typically purchase them from other countries through websites.
Bidis and kreteks contain high levels of nicotine and tar. Herbal cigarettes are becoming popular with young people. Though they may be portrayed as a safer alternative to traditional cigarettes, their use poses the same if not an increased level of risk.
### Cigars
A third type of smoked tobacco is the cigar. To meet the legal definition of a cigar, these products must be wrapped in tobacco leaves or another product containing tobacco. While the word cigar still evokes images of a proud new father handing out a box cigars to celebrate, today they come in a variety of forms and are used on more than special occasions.
There are small cigars that closely resemble, and are typically used as, cigarettes; moderate-size cigars that match the image from days of yore; and large cigars that contain as much nicotine as an entire pack of cigarettes and take an hour or more to smoke. Cigars seem quite trendy at the moment, but they are also increasing in use because they are taxed at a lower rate than cigarettes and are not subject to the same FDA regulations. This means that cigars can be flavored (they are available in a wide range of flavors, including grape, strawberry, and apple) and claims of being "low-tar" or "lite" can still be made on their packaging. Even so, cigars have not been found to be safer than cigarettes; they contain the same basic ingredients, though most lack filters. Cigar smokers may be at an even higher risk of harming their health, as the lengthy fermentation process involved in making cigars means they develop higher levels of nitrosamines, the chemical compounds that are known as carcinogens. When the larger cigars are smoked, the user is also exposed to these dangerous chemicals for a greater length of time.
### Pipe and Loose Tobacco
Smokers who prefer to roll their own cigarettes using special cigarette papers use loose tobacco. This is often done because of the preferred taste, more control over the size and strength of the cigarette, or because it's cheaper than prepackaged cigarettes. Pipe tobacco is similar, but is often flavored and moist, needing to be kept in an airtight container. While pipe and loose tobacco are slightly different from one another, users have lately been substituting pipe tobacco to roll their own cigarettes, since the excise taxes for pipe tobacco is substantially less than for either loose tobacco or cigarettes. As a result, sales of pipe tobacco have increased dramatically in the past few years.
## Smokeless Tobacco Products
### Pipe and Loose Tobacco—Hookah
Loose tobacco that is not smoked can be used in a water pipe, often called a hookah. (The most famous depiction is that of the Caterpillar character in Alice in Wonderland.) With this pipe, charcoal-heated air passes through flavored tobacco and into a water-filled chamber. From there, the user inhales the "smoke" or vapor through a tube and mouthpiece. Water pipes, which originated in Persia and India, are becoming more popular with young people in the United States, especially college students and young urban professionals. Hookah "smoking" is considered a social activity, and restaurants and special hookah bars across the country offer groups a place to use together. (As with other forms of smokeless tobacco, the regulations on water pipe usage are currently less stringent than for tobacco that is smoked.) Even though the smoke inhaled while using a hookah passes through water, it poses the same risks as other forms of tobacco use. According to the American Cancer Society, social users may be at higher risk for additional health problems, as they can contract infectious diseases by sharing hookahs.
### Chewing Tobacco
Chewing tobacco is also called spit, dip, chew, pinch, or snuff. It is sold, dry or moist, in small packets/pouches or tins, and comes in wads, leaves, or plugs. Users place the sweetened tobacco between their cheek and gum or between their lower lip and gum. It may also be chewed. Nicotine is absorbed through the skin when the person's saliva mixes with the tobacco. The tobacco juice is then often spit out. Dry snuff may also be sniffed up one's nose. Users may keep chewing tobacco in place for up to an hour.
### Snus
Snus is sold in teabaglike pouches that users place between their cheek and gum or under their upper lip. This tobacco is sold in many flavors, including licorice, peppermint, vanilla, mint, and natural. Snus is considered a spitless form of smokeless tobacco and is advertised as tobacco that can be used "in any social situation." Snus bags are generally held in place for about thirty minutes and then discarded.
### Dissolvable Tobacco
Dissolvable tobacco products are finely milled tobacco that is pressed into various shapes, including tablets or sticks. The shapes and the packaging that contain this tobacco bear a strong resemblance to mint and candy; they have even been referred to as "nicotine candy." As their name implies, they dissolve completely in the mouth, usually in five to fifteen minutes, depending on their shape. They are often flavored—mint, java, and natural, for example—and also deliver a dose of nicotine, ranging from 1.5 to 4 milligrams. (By comparison, smoking a cigarette typically delivers between 1 and 2 milligrams of nicotine.) And because dissolvable tobacco can be used so discreetly, the website for Camel dissolvables is correct when it proclaims, "Dissolvables are not subject to smoking restrictions."
Are Smokeless Tobacco Products Safe Alternatives to Smoking?
If most of the toxins found in tobacco are released in its smoke, then smokeless tobacco should solve all our problems...right? After all, this would allow people to receive all the pleasures of nicotine without any of the health risks. Unfortunately, while the smokeless tobacco companies might want to spread such a message, researchers don't agree. The National Cancer Institute, for example, reports that chewing tobacco and snuff contains twenty-eight cancer-causing compounds and increases your risk of developing oral cancers, such as cancer of the mouth, throat, and tongue. Smokeless tobacco also increases the risk of other cancers, heart disease, and stroke—just like combustible tobacco does. In addition, the Surgeon General points out that the overall health of the nation could be compromised if these new smokeless tobacco products attract people to tobacco who would not otherwise use, or if it stops some from quitting use completely.
Some experts contend that these smokeless products work better than the smoking cessation medications, such as the nicotine patch, for people who are trying to quit smoking. A cancer center in Kentucky has even started a campaign urging smokers in one city to "switch and quit." The University of Louisville–based campaign includes the use of billboards that pronounce: "Cancel your date with lung cancer. Switch to smoke-free." The FDA has not officially weighed in on the safety of smokeless tobacco products, but the Kentucky campaign nonetheless proposes that switching to smokeless tobacco or e-cigarettes moves nicotine addicts one step closer to quitting and is especially helpful for heavy smokers who haven't been able to quit other ways. The campaign compares the benefits to a drinker who shifts from consuming a six-pack daily to cutting back to just two drinks. Smokeless tobacco products contain about the same amount of nicotine as smokers receive in cigarettes; smoking cessation medications, on the other hand, contain a much lower level of the drug.
While electronic cigarettes do not contain tobacco, most provide a dose of nicotine that is mixed with other chemicals. The number of toxins released in the vapor of e-cigarettes is much lower than that of traditional cigarettes, but researchers warn that the long-term effect of such products is yet unknown. Because they don't produce smoke, e-cigarettes are permitted in bars, restaurants, and other places where smoking is prohibited. But this convenience factor may actually work against efforts to become nicotine free and instead promote use. One person interviewed for a New York Daily News article on e-cigarettes reported that since he was able to "vape" anywhere, he ended up using e-cigarettes more than he had ever smoked.
### E-cigarettes
Electronic cigarettes, commonly called e-cigarettes, are battery-powered and produce a vapor "smoke" that contains nicotine but not tar or carbon monoxide. E-cigarettes typically resemble cigarettes, cigars, or pipes, though some are made to look like common items such as pens or USB memory sticks. They include three elements: a cartridge filled with liquid nicotine and other chemicals, a battery that is used to heat the liquid, and an atomizer that turns the mixture into a vapor that the user inhales. E-cigarettes are available in either disposable or rechargeable form.
The FDA does not regulate or currently oversee the manufacturing of e-cigarettes. Thus the safety, contents, and potential benefits of these nicotine products are yet unknown. When the FDA did analyze a sample of e-cigarettes, it found inaccurate labeling—some products labeled as nicotine-free actually contained nicotine—and inconsistency among the amount of nicotine released in the vapor of various brands that, according to the label, contained the same amount of nicotine.
Critics of e-cigarettes argue that the industry is trying to skirt regulations, particularly against marketing their products to young people and claiming that e-cigarettes can help people stop smoking. In September 2010, the FDA sent a letter to e-cigarette manufacturers in the United States, warning them to stop making tobacco cessation claims and to clean up their manufacturing practices. Drugs that are lawfully marketed to help people quit tobacco need to be able to substantiate such claims—prove the drugs are safe and effective—before the FDA approves them. They also need to be manufactured so the products are consistent in strength, quality, and purity.
# Addictive Properties
Nicotine, an alkaloid found in the nightshade family of plants, has been called one of the most addictive drugs and harder to kick than heroin. We've all heard stories about the person using an oxygen tank for respiratory failure who still lights up, or the lung cancer patient whose first request upon waking up from surgery is for a cigarette. These stories tell us much about the vicelike grip that nicotine has on its faithful users—the addicts. To understand why nicotine hooks its users, we need to explore biological, genetic, and environmental factors.
## Biological Factors
Nicotine addiction has been called a "brain disease expressed as a compulsive behavior." Let's begin with a basic discussion of how the brain works. Here's how the authors of Drugs and the Developing Brain explain how the brain sends messages and controls our body:
The brain is made of billions of nerve cells, called neurons. Most neurons contain three parts:
1. a central cell body that directs all activities of the neuron;
2. dendrites, or short fibers, that receive messages from other neurons and relay them to the cell body; and
3. an axon, a long single fiber that sends messages from the cell body to other neurons or to other body structures, such as muscles.
Nerve cells communicate by transferring messages from the axon of one nerve cell to the dendrites of another. This transfer is called is neurotransmission. Neurotransmission is an essential part of how the brain performs its many important functions.
When nerve cells communicate by neurotransmission, axons and dendrites do not come into direct contact. Rather, communication happens mainly when a chemical substance is released into the space between the axon and dendrites of other neurons. This space is called the synapse.
When neurons send a message—in the form of an electrical impulse—it travels down an axon and toward a synapse. There it triggers the release of chemicals called neurotransmitters from the axon into the synapse. The neurotransmitters then move across the synapse and bind to special molecules called receptors. The receptors are located within the cell membrane that encloses the dendrites of the neighboring nerve cell. All of this then either stimulates or inhibits a new electrical response in the receiving neuron.
When we're thinking about the brain activity that causes addiction, the neurotransmitters and receptors are key players. Here's how it works: When nicotine enters the body with a puff of a cigarette, it enters the lungs, moves to the bloodstream (as would nicotine absorbed in the lining of the cheek or saliva), and then quickly travels to the brain—within eight seconds of taking that puff our brains feel it. Once in the brain, nicotine stimulates cell receptors—particularly alpha(4)beta(2) nicotinic acetylcholine receptors—and triggers the release of neurotransmitters. The neurotransmitters that are activated include dopamine as well as endogenous opioids, norepinephrine, 5-hydroxytryptamine, acetylcholine, gamma-aminobutyric acid (GABA), glutamate, and endocannabinoids. Taken as a whole, these neurotransmitter systems work to produce feelings of pleasure (a rewarding property), help develop tolerance and dependence, and lead users to seek out the drug again and again, even when they want to quit.
In essence, bringing nicotine into the body is like introducing a nonnative invasive plant species into a new region—the new plant affects the integrity and functioning of the existing ecosystem. With nicotine, and any other type of drug use, the natural order of brain activity is disrupted because of its presence, and the effects extend throughout the body's central nervous system.
The feel-good neurotransmitter dopamine has long been known as a key factor in the development of addiction. When it's around, our mood improves and we experience feelings of pleasure. Dopamine is also released through natural, everyday events in our lives, like having sex, exercising (think of the so-called "runner's high"), and even having a hearty belly laugh. But opening a pack of cigarettes and smoking them throughout the day means twenty dopamine hits a day—and let's be honest, how many of us have sex or even laugh that often? This reinforcement is part of what makes smoking addictive. Other mood-altering drugs, such as alcohol and heroin, act the same way on the brain.
Going back to our analogy of nonnative plants in a new environment, when those plants are invasive, the results can include disruptions to the nutrients and water available to native plants, as well as increase the risk for wildfires. The other plants must adapt or die. Likewise, when an invasive, continual drug such as nicotine invades the brain environment, the existing system of the brain may change. The brain can adapt to the flood of dopamine that comes with nicotine use and develop more nicotine receptors. This is believed to account for the strong cravings that come when nicotine is stopped. What's more, the brain can be "trained" to receive its hit of nicotine, and thus dopamine, at regular times, such as upon waking in the morning or after a meal, which is also a factor in cravings. The good news is that science is showing that the number of nicotine receptors begins to decline within six weeks of cessation.
How Nicotine Addiction Differs from Other Drug Addictions
Although they are classified the same way in the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5), and nicotine affects the brain in the same way as other mood-altering drugs to create addiction, nicotine does not produce a "high," only a feel-good state that either causes the user to feel more alert or, conversely, more relaxed and less anxious. It does not substantially affect a person's judgment or impair their motor control. Also, unlike many other drugs whose effects last anywhere from two hours and up to an entire day, the effect ends shortly after the drug supply stops, usually within ten to thirty minutes.
## Genetic Factors
What about genes? Can we place the blame fair and square on our parents and other blood relations for our addiction to nicotine? Science shows that genes do play a role in the development and maintenance of nicotine addiction, just as they make certain individuals more susceptible to other addictions.
With nicotine, twins and adoption studies have found that genes matter about half the time. This means that if two closely related people smoke a cigarette, one may experience little pleasure and have a take-it-or-leave-it attitude toward smoking, while the second person may enjoy the experience immensely and have the desire to smoke again as soon as possible.
Knowing you have a genetic history of nicotine addiction is often the first step towards prevention or quitting. Seeing a parent, grandparent, or other loved one suffer health problems or die as a result of smoking can be a powerful motivator to quit or never pick up the first cigarette. On the other hand, simply being exposed to smoking at a young age can have a powerful effect on a child's attitude towards smoking, making it seem normal and cool. According to the American Lung Association, parents who smoke are more likely to have children who smoke.
## Environmental Factors
But our brains and genes do not account for all tobacco use or nicotine addiction. The environment we grew up in, having friends or family members who currently smoke or not, and the cultural attitudes toward tobacco influence whether or not we pick up the habit. If you are the only one at a party not blowing out smoke rings, for example, you might feel a little left out. Likewise, abstaining by your lonesome when the rest of your family lights up regularly might lead you to try "just one" or go back to smoking if you have quit. Fortunately, the cultural and environmental influence to use tobacco has decreased dramatically as more and more people turn away from smoking.
# Use, Abuse, and Addiction
Smoking has long been called a habit, and that's certainly part of what smokers live out. Some people are able to limit their tobacco use to occasional social settings, such as being at a party or out at a bar. Other people do get in the habit of smoking or chewing at particular times but don't mind skipping it either. But for the majority of users, the real reason that they continue to smoke, especially in this day and age with antismoking messages everywhere, is singular: addiction.
Are You Addicted to Nicotine?
If you use tobacco or smoke, do you think you could be addicted? Answer the following twelve questions as you consider this possibility.
YES NO
1. _ _ Have you ever tried to quit smoking but couldn't?
2. _ _ Do you smoke now because it is too difficult to quit?
3. _ _ Have you ever felt like you were addicted to tobacco?
4._ _ Do you have strong cravings to smoke?
5._ _ Have you ever felt like you really needed a cigarette?
6._ _ Is it difficult to keep from smoking in places where you are not supposed to smoke?
7. _ _ Has it been difficult to keep from smoking when you have tried to stop smoking?
8. _ _ Has it been difficult to keep from smoking when you haven't used tobacco in a while?
9._ _ Have you found it difficult to concentrate because you couldn't smoke?
10. _ _ Have you ever felt irritable because you couldn't smoke?
11. _ _ Do you feel a strong need or urge to smoke?
12._ _ Have you ever felt nervous, restless, or anxious because you couldn't smoke?
If you answered yes to one or more of these questions, you may be experiencing nicotine addiction.
## Christine's Story
Let's talk about what draws people into smoking in the first place. Take the story of Christine, a young woman who tried smoking a cigarette just once in her life. It was shortly before she graduated from college and Christine panicked at the thought of all the "wild" things she hadn't done over her college career. Smoking a cigarette was on her list. So she found a friend who smoked (that was easy) and asked her to teach her how to properly light up and inhale. Soon after, a small group of friends met one sunny day outside, and then a lot of coughing ensued as Christine and her friends strutted along waving their cigarettes around. Christine reports having had a fun time that day, but that, other than having the opportunity to act out her vision of a cool smoker with the real thing—compared with the candy cigarettes she had done this with as a kid—the nicotine hit did absolutely nothing for her.
What would have happened if Christine kept at it, say bought her own pack—or even a carton—of cigarettes rather than bumming one off a friend? Undoubtedly, with continued use, her smoking life would have taken off. That's because smoking produces a number of effects. And, strange as it sounds, those effects can be polar opposites, both soothing and stimulating.
New smokers also discover that dependence on nicotine develops rather quickly. First, the user experiences tolerance to nicotine, meaning the effect is less pronounced and more is needed to achieve the same effect. Eventually, dependence and addiction take hold.
## Tobacco Use Disorder
What does it mean to be diagnosed as a nicotine addict? Officially, there is no such diagnosis, but the current Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5) includes "tobacco use disorder" under the umbrella of "substance use disorder"; in previous editions of the diagnostic manual, these were separated into diagnoses of "substance abuse" and "dependence." To be officially diagnosed with this disorder, users must meet at least two or three criteria for "mild" disorder, four to five criteria for "moderate" disorder, and six or more criteria to have a "severe" disorder.
Here are the criteria for tobacco substance use disorder outlined in the DSM-5:
* using more tobacco or for longer periods of time than intended
* persistent desire or unsuccessful efforts to stop or cut down on use
* significant time spent in activities to obtain or use tobacco
* craving or urge to use tobacco
* recurrent use that results in failure to fulfill obligations at home, work, or school
* continued use despite negative interpersonal consequences (such as arguments about use)
* giving up or reducing important social, work-related, or recreational activities in favor of tobacco use
* repeated use in situations where it is dangerous (such as smoking in bed)
* continued use despite physical or psychological problems likely to have been caused or exacerbated by tobacco
* tolerance
* withdrawal
Severity of the DSM-5 substance use disorders is based on the number of criteria endorsed: two to three criteria indicate a mild disorder; four to five criteria, a moderate disorder; and six or more, a severe disorder.
Nicotine as a Gateway Drug
Once a teen or young person crosses the threshold of smoking or using other forms of tobacco, the gate seems to slide open to other drug experimentation and use. The Johns Hopkins Bloomberg School of Public Health, for example, studied results from a survey on different types of drug use and found a link between those who began smoking early in life and later drug use. Smokers were seven times more likely to use marijuana than nonsmokers. When compared with nonsmokers, smokers were also more likely to use cocaine (again seven times more likely), crack (fourteen times more likely), and heroin (sixteen times more likely). The younger the smoker is upon first smoking, the more likely he is to move on to these other drugs.
While the reasons for this correlation have not been firmly established, one study on adolescent tobacco users found that their brains metabolize nicotine at a slower rate than the brains of adults do. This longer exposure may make them more susceptible to addiction. Researchers now believe that 25 percent of adolescents who smoke become hooked in just one month. In addition, animal studies have found that when mice are exposed to tobacco, their brains are more prone to cocaine addiction.
## Effects of Nicotine and Tobacco Use
When tobacco is burned, the resulting smoke contains more than 7,000 chemicals and chemical compounds; at least 250 of these are toxic. When we smoke, chew, or vaporize nicotine, the effects extend through our central nervous system and include feelings of pleasure, reduced stress, and mental alertness. In this way, nicotine acts as both a stimulant and a sedative on the body. This explains why people smoke both to kickstart their day and to wind down as the day draws to a close. The stimulant effect occurs when the adrenal glands are activated and trigger a rush of epinephrine, or adrenaline. This rush of adrenaline leads to the release of glucose and raises our blood pressure, heart rate, and breathing. Let's take a closer look at how tobacco use can affect our bodies over the long term.
## Health Consequences and Risks for the User
Tobacco use and smoking-related diseases lead to more than 393,000 deaths in the United States each year; another 50,000 each year are attributed to disease from secondhand smoke. But tobacco use doesn't always kill users quickly, and many develop chronic and debilitating diseases that affect them for years before their untimely deaths finally occur.
The harm caused by smoking begins immediately and progressively gets worse with continued use. However, upon quitting, the risk of developing many of the diseases associated with smoking goes down over time. Let's take a closer look at the most common diseases caused by tobacco use.
### Cancer
Lung cancer, once considered a rare disease before smoking rates exploded beginning in the late 1800s, is now viewed as an epidemic. In 2009, more than 200,000 people were diagnosed with lung cancer in the United States. This widespread disease is also a deadly one, killing more people than any other type of cancer. The connection between smoking and lung cancer has been firmly established, and smoking causes more than 85 percent of lung cancers in the United States.
Smoking has also been found to cause many other cancers—about one-third of all cancers—including cancer of the throat, larynx, tongue, esophagus, mouth, bladder, cervix, kidney, pancreas, stomach, and leukemia, a cancer of the blood and bone marrow. Because smoking also weakens the body's immune system, smokers have a more difficult time fighting cancer.
### Respiratory Conditions
In addition to causing lung cancer, smoking leads to other problems and diseases in the respiratory system. It irritates and damages the airways. This can cause complications with asthma and lead to the development of chronic obstructive pulmonary disease (COPD), which includes bronchitis (inflammation of the lungs) and emphysema. The problems may start with wheezing and coughing that produces phlegm and progress to shortness of breath and difficulty exhaling as the lungs become more and more incapacitated.
### Heart Disease, Heart Attack, and Stroke
Research shows a causal relationship between smoking and heart disease. That's because smoking damages your blood cells and blood vessels in a way that increases the risk of developing atherosclerosis, a disease that causes plaque buildup in your arteries. When arteries are clogged with plaque, blood is more likely to clot—which can lead to heart attacks, stroke, or sudden death.
### Diabetes
Although tobacco use has not been shown to cause diabetes, if you smoke and already have diabetes, your risk for dangerous complications from the disease increase. For example, smoking makes you two to three times more likely to develop kidney disease than diabetics who don't smoke. Diabetics who smoke also increase their risk for heart disease, eye disease and blindness, and nerve damage.
### Tooth Decay and Gum Disease
Smokeless tobacco increases your risk for developing oral health problems. Chewing tobacco contains sugar, which contributes to cavities and tooth decay. The coarse particles in chewing tobacco can also damage your gums and tooth enamel. Sugar and irritants can also make you more likely to develop gum disease, as gums may pull away from affected teeth and areas where you chew. If untreated, this may eventually lead to bone and tooth loss.
### Sleep Apnea
People who have sleep apnea repeatedly stop and start breathing in their sleep. Smoking poses a significant risk for the most common type of sleep apnea, called obstructive sleep apnea. This means that the person's throat muscles relax too much during sleep, creating an obstruction for airflow and wakening the person to breathe. People with sleep apnea may get a full night's sleep but not feel rested due to this constant wake-sleep cycle. Researchers estimate that smokers are three times more likely to struggle with sleep apnea than people who do not smoke. Higher levels of inflammation and fluid retention in the upper airway are thought to be the cause.
### Pneumonia
Smoking is the largest single risk factor in developing pneumonia in otherwise healthy, nonelderly individuals. Pneumonia is a lung infection caused by either bacteria or a virus that may be mild or severe, but it has the potential to lead to complications, especially in smokers. Those who have been smoking the longest and who smoke more are at greater risk. What's more, the risk for pneumonia is increased for nonsmokers who breathe secondhand smoke as well as for the smokers themselves.
### Buerger's Disease
One little-known side effect of smoking is thromboangiitis obliterans, otherwise known as Buerger's Disease. In some smokers and users of tobacco, the effect on their arteries and veins causes poor circulation in the hands and feet that can become gangrenous if left untreated. This is often treated with medications, costly and painful bypass surgeries, or even amputation in extreme cases.
Overdosing on Nicotine?
Did you know that nicotine can be lethal in high doses? That's right, it's possible to die from too much nicotine. This issue has been raised quite frequently with the introduction of new smokeless tobacco products, especially those that resemble candy and mints. The lethal dose of nicotine for children is much lower than for adults, and some worry that kids may inadvertently get into an older sibling's or parent's stash of these products, as well as nicotine patches and gum.
Symptoms of an overdose of nicotine include abdominal cramping, rapid or difficulty breathing, confusion, coma, fainting, or vomiting. If you suspect nicotine poisoning, do not make the person throw up. Instead, seek immediate medical attention, call 911, or call the National Poison Control Center at 800-222-1222.
### Pregnant Mothers and Reproductive Effects
According to a report by the Surgeon General on tobacco and women, women who smoke are at risk for a number of consequences related to their own health, their ability to have children, and the health of their unborn children. Smoking compromises fertility, and smoking during pregnancy increases the risk for complications including premature birth, low-birth-weight infants, stillbirth, and infant mortality. Research also shows a link between smoking by mothers and sudden infant death syndrome (SIDS).
What's more, smoking may also lead to more painful or irregular menstrual periods or the absence of menstruation.
Smoking can have a negative effect on male fertility as well.
Could Tobacco Really Produce Some Health Benefits?
Let's recall that Native Americans originally used tobacco for ceremonial and medicinal purposes. Then, when it was brought to Europe, it continued to be used in the treatment of various ailments, with some supposed success. Yet in today's world, only the hazards of tobacco use are spoken of, but research is showing some benefits as well. Here are some examples:
Pregnant women who smoke in the latter half of their pregnancy reduce their risk for developing preeclampsia and gestational hypertension. This is most likely a byproduct of the carbon monoxide in cigarette smoke; smokeless tobacco has not been shown to produce the same benefits.
In 2011, the journal Arthritis & Rheumatism reported on a study that found male smokers were less likely to need joint replacements than men who didn't smoke—50 percent less likely to need new knees or hips. The decreased risk seems to be related to a number of factors: the lower weight of most smokers; smokers' reduced activity level, which reduces the wear and tear on their joints; and nicotine's effect in activating cells that increase cartilage in our joints.
Researchers are looking for ways to use tobacco in the developing of health care products. A California-based company, for example, is using an antibody from a tobacco leaf extract in a product that would help prevent cavities. The product is currently undergoing clinical trial.
## Health Consequences and Risks for Secondhand Smoke
Also called environmental tobacco smoke or involuntary smoke, secondhand smoke has consequences greater than leaving an odor on your clothes or causing you to cough. We now know that the consequences can be just as deadly for the person who breathes secondhand smoke as it is for the smoker. According to the National Cancer Institute, people who are breathing secondhand smoke are ingesting at least sixty-nine known carcinogens. While restrictions on smoking tobacco in workplaces and public places have certainly lowered the exposure most people receive, the problem has not been eliminated since no level of exposure has been deemed safe.
Secondhand smoke can be caused by burning tobacco products or by the smoke that a user exhales. The following paragraphs show how secondhand smoke causes harm.
### Effects on Children
Because children's bodies are generally smaller than adults' and are still developing, they are especially susceptible to the negative effects of breathing tobacco smoke. According to the U.S. Surgeon General, secondhand smoke has been linked to SIDS in infants, bronchitis and pneumonia in young children, and more frequent and intense asthma attacks in children who have asthma; plus, it may cause respiratory symptoms such as coughing, wheezing, and breathlessness in school-age children and may increase their risk of ear infections. Some evidence suggests it may even cause cancer, leukemia, or brain tumors, though this is not yet conclusive. In the United States, 34 million children and youth are regularly exposed to secondhand smoke.
### Cancer
Breathing secondhand smoke has been linked to lung cancer, increasing your risk by 20 to 30 percent. Some evidence suggests that secondhand smoke may also cause breast cancer and increase the risk for developing nasal sinus cancer.
### Cardiovascular System
When adults are exposed to secondhand smoke, the effects can include harm to the heart, blood, and vascular systems and thereby increase your risk of having a heart attack. Breathing secondhand smoke increases your risk of developing heart disease by 25 to 30 percent.
### Respiratory Effects
Evidence suggests that breathing secondhand smoke may damage your airways and exacerbate asthma symptoms.
### Reproductive Effects
Evidence shows that when mothers are exposed to secondhand smoke, their babies may weigh slightly less at birth and may be born earlier than they otherwise would.
### Other Effects
In addition to the health effects described above, adults exposed to secondhand smoke may deal with nasal irritation as well as simply be bothered by the odor.
Sights and Smells—How Smoking Affects Our Appearance
It seems ironic that many people begin smoking because they think it makes them look cool, that they may continue smoking because it helps to suppress their appetite and control their weight, and thus make them look good, and yet the conclusion of continued use over the long term is anything but attractive. With time, smoking is said to speed up the aging process. The skin becomes dry and dull looking; premature wrinkles develop around the lips and eyes; teeth become discolored; and fingers and fingernails become stained. And if smoking-related disease sets in, a person's appearance will undoubtedly decline even further.
The good news is that quitting cigarettes can improve your appearance—and do so rather quickly. In as little as five days, your skin may start to look better as your circulation improves. Plus, the odor of smoke will fade from your hair, clothes, vehicle, and home. These are smells that you may not have even been aware of, even though those around you certainly smelled it.
### Fire Risk
According to the U.S. Fire Administration, almost one thousand people are killed each year in fires caused by cigarettes and other smoking materials. This is the number one cause of preventable fire deaths in the United States. If you smoke in your home, be sure to do the following:
* Put out the cigarette before walking away.
* Be alert.
* Never smoke in bed.
* Use sturdy ashtrays.
* Smoke outside whenever possible and always be sure to put out the cigarette completely.
## Economic Effects
### Personal Financial Costs
While the cost of a pack of cigarettes varies widely across the United States—federal excise taxes are the same everywhere, but states and localities set their own tax rates—the national average for a pack of cigarettes is about six dollars. Using this figure, let's do the math for what a pack-a-day smoker would pay for cigarettes.
Over a month, that's a tank of gas each week. Over a year, that's enough to take an extra vacation each summer. And over twenty years, that's enough to buy your dream sports car for retirement. It's easy to see why the tobacco industry's goal is reaching new smokers and keeping them for life.
### Societal Financial Costs
The significant financial cost to governments and society are what led to the state lawsuits against the tobacco industry in the first place. Cigarette smoking costs the nation $96 billion in direct medical costs and $97 billion in lost productivity annually. Secondhand smoke is estimated to cost roughly $5 billion in direct medical costs and another $5 billion in indirect costs.
## Turning It Around—The Benefits of Quitting (Reversing Harms)
If you've smoked for thirty years, are you a lost cause? Everyone around you may now be touting the benefits of a smoke-free lifestyle, and even though most of the places where you formerly smoked with abandon now prohibit smoking, you keep at it—going outside even in the bitter cold to light up. Perhaps you doubt it would do any good to quit now. You would be wrong in this assessment. Let's look at what research is showing us about the reversal of damage and how the body regains health.
In addition, the American Cancer Society reports that the improvements to your blood vessels that come with quitting reduce the risks for diabetics. Whether you quit after a few months or years of use, or after decades of use, your body will benefit and you may live longer.
# Treatment for Nicotine Addiction
With the current knowledge on the health and financial cost of tobacco use, prevalence of antismoking messages, and the general cultural stigma that is now placed on smokers, it's no wonder that more people are trying to quit—or are being pressured to do so. The stakes for successfully quitting are high for not only the user and her family, but also for the user's employer, health insurer, and government. The Centers for Disease Control (CDC) reports that more than 68 percent of current smokers would like to quit and more than half of all smokers have tried to do so within the past year. How many have been successful? Slightly more than 6 percent of smokers have succeeded in quitting within the past year.
## Methods of Quitting
There is no one-size-fits-all approach when it comes to quitting tobacco and nicotine. And there's no wrong way to quit either. Here are the most common approaches:
* Quitting on your own by stopping all at once—that is, going "cold turkey."
* Counseling. This includes a wide range of possibilities, from your doctor suggesting that you quit to entering an inpatient smoking cessation treatment program.
* Smoking cessation medications. This includes nicotine replacement therapies and prescription cessation medications without nicotine.
* Using a combination of treatments. This means using a medication for smoking cessation combined with therapy; this has been shown to increase your chance of success in quitting.
Whatever method you choose to try, we know that people succeed at quitting most often when they learn how to tolerate symptoms of withdrawal from nicotine, develop strategies to use when dealing with triggers, and receive support and encouragement. Motivation is another key factor in whether or not smokers succeed in their efforts to quit.
Paying for Smoking Cessation
Research shows that when people have insurance coverage for smoking cessation treatment, they are more likely to get treatment and thus more likely to be successful in quitting.
The Affordable Care Act passed in 2010 requires insurance companies and employers to cover smoking cessation treatment. Every state Medicare program is now required to cover smoking cessation treatment for pregnant women, and in 2014 will no longer be able to exclude smoking cessation medications, whether prescription or over the counter, from their drug coverage; this applies to all beneficiaries. Currently, six states cover all of the smoking cessation treatments that are recommended by the Public Health Service.
### Quitting on Your Own
Some people are able to wake up one day and simply decide to stop smoking, and so they do, quitting cold turkey. Those who use this method of quitting succeed 5 percent of the time. People who quit cold turkey will likely face an array of physical and psychological consequences. On the physical side, they should be prepared for symptoms of withdrawal and cravings to use. These will pass in time, but can be uncomfortable and include anxiety, nausea, irritability, and insomnia. People who quit cold turkey will also have to deal with the psychological implications of quitting tobacco, which may include encountering various triggers to use throughout their day and feelings of loss. Willpower alone is often not sufficient to keep a smoker from going back to tobacco. Having a solid support system may help those who choose to quit without medication or counseling.
Why Quitting Is So Hard
Imagine that you have spouse, partner, or best friend in life who is available to you 24/7. This is someone you can depend on in times of celebration and times of sorrow, times of boredom and times of stress and anxiety. Being with this person always leaves you feeling a bit better—whether you needed a pick-me-up or a nod to relax. What's more, you meet with this person at the same time each and every day, usually several times a day. The connection forged between you and this person is ultra-deep and you never fear that he or she will leave you, no matter what. Oh, by the way, this "person" is actually a cigarette—your most reliable and trusted "friend" in life. And now you want to walk away from it all?
With such a devoted friend as this cigarette (or cigar, pipe, or form of smokeless tobacco), is it any wonder why quitting smoking is so difficult? And the more a person smokes per day—the more times he stops to light up, or step outside, or meet a coworker for a cigarette break—the harder it is to quit. Smoking has been called a habit for good reason. Most other drug addicts do not turn to their drug of choice as many times a day as a smoker does.
What does it mean to "break the habit" then? Well, for starters most smokers will have to begin and end their day a different way, as well as what they do on their breaks at work, what they hold in their hand with a cup of coffee or a drink, and what they do when they're stuck in traffic. This might also entail spending less time with one's work buddies, since joining them over their cigarette breaks will be all but impossible. When you stop to think about how much this "habit" has been incorporated into the fabric of a smoker's life, you realize the extent of what it means to quit. All of these social aspects—and then there is the physical addiction and withdrawal process! It can be a lot for one person to handle. That's why having a support system can make all the difference.
### Counseling, Groups, and Other Support
For those who want or need help quitting, the number of counseling options has never been more plentiful, from one-on-one sessions to group classes. Though the types of available counseling vary widely, effective counseling usually includes skills and problem-solving training and social support.
### Brief Interventions
Today, we recognize that even brief interventions may help some people quit smoking. This may mean that your doctor asks about your tobacco use during a routine physical exam and gives you a pamphlet on the benefits of quitting. For this reason, clinicians are now advised to counsel all patients who use tobacco to quit, whether or not they have the desire to quit. This and education about the dangers of smoking may help motivate some people to quit.
Telephone counseling research has shown that telephone counseling can be very helpful to people who are trying to quit and to those who have already quit. In some cases, it is even as effective as face-to-face counseling and support groups. Telephone counseling sessions are often more convenient to people than traditional counseling programs. They can call from their own homes when it works best for their schedule, including nights and weekends. Trained counselors can offer advice on medications, nicotine replacement therapy, and support programs or classes. Twelve Step support groups such as Nicotine Anonymous offer people an opportunity to attend meetings and to listen to others who are going through the same process of learning to live without tobacco and nicotine. Anyone who wants to gain freedom from nicotine, including people using smoking cessation programs or medications, is welcome to attend the free meetings. Meetings are run by the participants, who offer one another experience, strength, and hope. To find a local meeting, go to www.nicotine-anonymous.org or call 877-879-6422.
Here are some resources and hotlines for support when quitting tobacco:
National Network of Tobacco Cessation Quitlines
Toll-free hotline: 1-800-QUITNOW (1-800-784-8669)
www.naquitline.org
American Cancer Society
Toll-free hotline: 1-800-ACS-2345 (1-800-227-2345)
www.cancer.org
American Lung Association
Toll-free hotline: 1-800-LUNGUSA (1-800-586-4872)
www.lungusa.org
National Cancer Institute
Toll-free hotline: 1-877-44U-QUIT (1-877-448-7848)
www.smokefree.gov
Centers for Disease Control and Prevention
How to Quit web pages provide a variety of cessation tips, tools, and resources.
www.cdc.gov/tobacco/quit_smoking/how_to_quit/quit_tips/
### Online Support Groups
Especially helpful for people living in areas without traditional support groups or for those who cannot find the time to attend such meetings, online groups and support networks provide invaluable guidance and advice—and they offer it 24/7.
What's more, today you can use apps to help you in your journey to nicotine-free living. Some apps help users create a personalized plan for quitting and offer motivational tips, while others tally how much quitting is increasing your lifespan and saving you money. You can even receive support from others through chat rooms connected to the apps. Facebook also features many pages related to smoking cessation efforts.
### Smoking Cessation Programs
Smoking cessation programs are available through many groups and locations, including hospitals and clinics, insurance providers and workplaces, and national organizations. Programs motivate users to quit and help them develop strategies to use for dealing with triggers and withdrawal symptoms.
While a handful of inpatient smoking cessation programs are now available, most programs designed to help you quit and stay quit are conducted on an outpatient basis. Programs may be held at a local clinic or community center, or they may be available exclusively online.
Research shows that the intensity of a program—how often it meets and how long the sessions are—corresponds with its rate of success. The American Cancer Society recommends looking for more intense programs, where
* each session lasts at least 15–30 minutes
* there are at least four sessions
* the program lasts at least two weeks—longer is usually better
* the leader is trained in smoking cessation
### Holistic Therapies
Some people report that alternative therapies such as meditation, acupuncture, or hypnosis are helpful in dealing with withdrawal symptoms and cravings for tobacco. Although the science backing up the effectiveness of these therapies in smoking cessation is limited, they seem to be most effective when combined with other methods of tobacco cessation.
### Medications
Let's look at the medications that are currently available to help people as they quit nicotine. These include five nicotine-based medications and two that do not include nicotine; all help with cravings and withdrawal symptoms.
### Nicotine Replacement Medications
Nicotine replacement medications contain low doses of nicotine, usually one-third to one-half the dose of traditional cigarettes. They are also smokeless and so do not expose users to chemicals that come with smoking. Nicotine replacement medications take longer to reach the brain than other tobacco products and are thus not viewed as posing a risk of dependence. The dose of nicotine during the cessation process starts off higher and is decreased as time goes on, and the person's withdrawal symptoms diminish and she prepares to be abstinent from all nicotine products. The packaging on most nicotine replacement medications suggests that they be used for six to sixteen weeks. Nicotine replacement medications have been approved by the FDA for use by tobacco users age eighteen and over.
Nicotine replacement has been found most effective for people who smoke more, which is defined as fifteen or more cigarettes per day. Tobacco users who want to quit are urged to consult with their health care provider about which, if any, nicotine replacement medication may be best for them.
Note: In April 2013, the FDA announced that the packaging of nicotine replacement therapies (NRT) will change in the near future, removing warnings about using NRTs with other forms of nicotine. This change is intended to reassure users that even if they slip and smoke a cigarette, they can still safety return to using this therapy in their next attempt to quit.
Below is a summary of the various types of nicotine replacement therapies, their brand names, and whether or not they require a prescription.
### Medications That Do Not Contain Nicotine
The FDA has approved two medications that do not contain nicotine for smoking cessation:
* bupropion SR (Zyban): A drug originally developed for treating depression, bupropion has been shown to reduce cravings for nicotine and ease symptoms of withdrawal.
* varenicline (Chantix): This drug helps ease withdrawal symptoms and, if the person relapses, reduces pleasurable feelings from smoking.
Research shows that medications work best when they are part of a more comprehensive smoking cessation plan rather than used on their own. Of note, a recent study by Harvard School of Public Health and University of Massachusetts–Boston found that people who used nicotine replacement therapies relapsed just as often as people who quit cold turkey.
### Combo Treatments
Research shows that using evidence-based smoking cessation tools—including counseling, support groups, and nicotine replacement products—doubles or even triples the odds of success. When counseling and nicotine replacement therapies are used together, success rates are between 20 and 40 percent.
## Programs for Youth
Gaining freedom from nicotine and learning how to live a tobacco-free lifestyle is not so different for adolescents than it is for adults. However, counseling may take on a more important role for youth, as those who are under eighteen cannot legally purchase nicotine replacement medications. (However, doctors can prescribe these medications for minors who are under their care if they are dependent on nicotine.)
Programs that are found to be most successful for youth are fun and engaging. Most include an educational component, focusing on the dangers of continued tobacco use. They also teach young people practical skills for staying off nicotine, including refusal skills.
One smoking cessation program for youth, the ASCENT program, uses some of the principles from the Stages of Change Model that was developed by James Prochaska and Carlo DiClemente in the 1980s. This model recognizes that people move through different stages as they prepare for and make behavior changes towards quitting smoking, drinking, or using other drugs. The idea behind this way of looking at behavior change is that people may be at different places in their readiness to change and may have more or less motivation to change. The ASCENT program, then, is designed to help adolescents wherever they are in the process of change. Here are the five stages:
1. Precontemplation: A stage of exploration, looking at pros and cons of quitting
2. Contemplation: Considering making change, a stage of motivation and encouragement
3. Decision and determination: Making the decision to change, developing strategies for quitting
4. Action: Utilizing plan and strategies to quit; providing support
5. Maintenance: Moving forward with a plan, using coping strategies and support as needed; if relapse occurs, getting back on course
## Quitting Tobacco—What to Expect
What should you expect when you quit? It's normal to experience symptoms of withdrawal and triggers to use. Tobacco use is an oral habit, so it's not uncommon for people to use food as a substitute for smoking during the early stages of quitting and to experience some minor weight gain.
### Withdrawal
Uncomfortable withdrawal symptoms may appear within a few hours of your last cigarette. Remember, when you stop feeding your brain the drug it has gotten used to, the corresponding feel-good results screech to an abrupt halt and your body cries out for more. Here's what that entails:
### Short-term Symptoms
* first few hours: fidgety, mild craving for a cigarette, thinking about a smoke
* four to eight hours: restless, can't concentrate, stronger urges
* eight to twelve hours: tired, nervous, craving a cigarette
* twelve to twenty-four hours: headache, fatigue, irritability, really want to smoke
### Long-term Symptoms
* one to two days: dizziness, tingling in hands and feet, anger
* three to five days: tightness in the chest, coughing, dry throat, sore throat, desire to smoke
* six to ten days: constipation, gas, depression, dreams about smoking, feeling a little better
* ten days to two weeks: most physical withdrawal symptoms are gone, but a person may still feel irritable, have difficulty sleeping and concentrating, be hungry, and crave a cigarette when around other smokers
Withdrawal symptoms are generally more intense the longer you smoked and the more you smoked. In time, your body and brain will learn to go on without nicotine and will begin to heal itself. Most people report that the worst withdrawal is over after the first week and the symptoms continue to decline over the first month. Smoking cessation medications may help lessen the intensity of withdrawal symptoms.
### Triggers
Beyond physical withdrawal symptoms, breaking the habit of smoking or using another form of tobacco includes dealing with the triggers to use in daily life. Whether someone has been a regular smoker for one, ten, or thirty years, habits form around that use. For example, a person may light up every time he:
* wakes up
* has coffee
* drives to work
* has a break
* eats lunch
* celebrates
* has a drink
* goes fishing
* goes hunting
* plays cards
* rides bike
* finishes exercising
* goes out in a boat
* gets in a car
* returns home
* mows the yard
* watches TV
* prepares for bed
These are, of course, just examples. Individual smokers may experience different triggers depending on when they usually smoked. Most experts recommend trying to identify your triggers and then make plans to avoid them, perhaps by changing your routine or by finding ways to lessen their effect. Preparation and awareness seem to be the key here.
Tips on Quitting
Drink lots of water!
Distract yourself from smoking by chewing a piece of gum, brushing your teeth, or calling a friend.
The next time you have a craving to smoke, try to delay for five minutes.
Remember, the craving goes away whether you smoke the cigarette or not!
Be prepared for the "after a meal" cigarette craving. Instead of smoking, brush your teeth, gargle with mouthwash, and find something else to do.
Change your routine and break habits that usually go with smoking cigarettes. If you like to drink beer, soda, or coffee with a cigarette, try a different drink, such as fruit juice or water.
Practice taking a few deep breaths when feeling the urge to smoke.
Write down a list of your most common triggers with coping strategies for each and carry it with you to reference when you encounter a tough situation.
Try to exercise for at least fifteen minutes every day.
Practice stress management exercises such as deep breathing, muscular relaxation, delay plus behavioral alternatives, imagery, thinking straight, and self-talk to help with urges to smoke.
If you have a slip and smoke a cigarette, do not give up. Relapse is common, and you just have to renew your commitment and get back on track.
Reward yourself for staying smoke free for one week, one month, three months, and so on.
Be patient! Frequently remind yourself why you quit smoking and that quitting smoking gets easier every day.
See yourself as a person who chooses not to smoke anymore.
Calculate how much money you are saving each week that you remain smoke free.
## Relapse
Perhaps more than with any other drug, quitting nicotine is hard to get right the first time. Relapse is common and it often takes several attempts before a person succeeds at quitting. For that reason, it's important to view quitting as a process—do not feel defeated if you slip up and smoke or relapse into your old using behavior. Instead, it's best to learn from your mistakes and be prepared to start again.
Many experts agree that success occurs when people take these four steps:
1. Make the decision to quit.
2. Pick a day to quit and make a plan for quitting.
3. Stick to your plan and work through withdrawal.
4. Make a plan to stay tobacco-free.
If you don't succeed with one quit plan, try another. Also, develop a relapse prevention plan that includes strategies to manage stress and get support. This plan might include a list of your common triggers, a list of alternative behaviors to deal with cravings, a list of people you can call for support, and your motivation for quitting (your health, children, etc.). Keep this plan handy and reference it throughout your first weeks of quitting.
# Prevention Practices
The vast majority of tobacco users began using during adolescence or young adulthood. Indeed, a 2012 U.S. Surgeon General report indicated that 99 percent of daily smokers began smoking by age twenty-six; 88 percent of these smokers started by the time they were just eighteen. These statistics lead to the obvious conclusion that reducing the deadly effects of tobacco means preventing young people from using in the first place. And it's true. If we can prevent teens and young adults from starting to smoke—and each day nearly 3,800 young people under age eighteen have their first cigarette—we can reduce the number of tobacco users, health problems, and financial costs in the future.
Yet, prevention encompasses much more when it comes to nicotine addiction. The Centers for Disease Control and Prevention recommends a comprehensive, statewide program for tobacco control to prevent tobacco's "staggering toll" on society. More specifically, such programs would have the following goals:
* preventing initiation among youth and young adults
* promoting quitting among adults and youth
* eliminating exposure to secondhand smoke
* identifying and eliminating tobacco-related disparities among population groups
The structure proposed by the CDC for this program includes numerous elements that work in concert. They include state and community interventions, health communication interventions, cessation interventions, program surveillance and evaluation, and effective and fiscally sound administration and management. This section will focus on preventing tobacco use in youth.
## Youth Tobacco Prevention
Preventing tobacco use among youth would fall in the "state and community interventions" category. The recommendations put forth in the independent Task Force on Community Preventive Services' Guide to Community Preventive Services include the following measures for youth tobacco prevention:
* increasing the unit price of tobacco products
* conducting mass media education campaigns combined with other community interventions
* mobilizing the community to restrict minors' access to tobacco products when combined with additional interventions (stronger local laws directed at retailers, active enforcement of retailer sales laws, retailer education with reinforcement)
* implementing school-based interventions in combination with mass media campaigns and additional community efforts
It's clear that delaying the onset of tobacco use, or preventing it completely, has enormous benefits to people's health as well as the economy: consider the costs of the associated health care. In recent years, youth prevention efforts have become more and more comprehensive, involving schools, parents, and communities. If it really takes a village to raise a child, it may also take one to keep that child free from the hazards of tobacco.
Although prevention in these early years is critical and plays an important role in overall tobacco control efforts, this is no easy task. As we've discussed, young people are uniquely susceptible to problematic tobacco use, due to their brain development (the impulse control and judgment part of the brain is not fully developed until around age twenty-two), the way their brains respond to nicotine, and to the effects of peer pressure.
Is Eighteen Too Young to Smoke?
While some contend that the number of options to further reduce use among minors is limited, others are now advocating that the legal age to buy tobacco products be raised to twenty-one. According to the American Lung Association, 85 percent of smokers begin smoking by age twenty-one. Several towns already have established twenty-one as the minimum age to buy tobacco products, and in a handful of states, nineteen is the minimum age. This effort is intended to reduce smoking by youth and to prevent them from picking up the habit later. Though eighteen has long been viewed as one benchmark of adulthood, the age when people acquire the right to vote, science is now indicating that true adulthood occurs later. Brain research confirms that the brain is still developing at eighteen—and continues until a person reaches her midtwenties. In particular, the last area of the brain to develop is the prefrontal cortex, the area where impulse control is housed. The legal age to drink was raised in the 1980s with the National Minimum Drinking Age Act of 1984 and now some efforts are under way to do the same with tobacco. In other countries around the world, the minimum age to use and buy tobacco ranges from fifteen to twenty-one.
### Protective Factors and Risk Factors
At its core, youth tobacco prevention is about decreasing risk factors and increasing protective factors in youth.
### Risk Factors
The more risk factors an adolescent has, the more likely he is to fall into the grips of nicotine use and addiction, and from there, possibly other drug use. Risk factors can include personal details, such as not doing well at school, or factors related to one's family, such as having parents who smoke. An adolescent's friends and environment can also set her up to begin using tobacco.
Here are some risk factors that the U.S. Office of Adolescent Health identified as increasing the likelihood that an adolescent will smoke:
* Older age. Smoking and tobacco use is more common among older adolescents.
* Being male. Most girls smoke fewer cigarettes a day than boys do.
* Being white. White teens are more likely to use tobacco than their black or Hispanic counterparts.
* Lacking college plans. Youth who plan to attend college smoke less than teens without such ambitions.
* Having parents who are not college educated. Adolescents whose parents had little or no college education are more likely to smoke than those whose parents graduated from college.
* Experiencing highly stressful events. The more stressful events that teens experience, the greater their risk of smoking. Highly stressful events include abuse, whether as a witness or victim, parental separation, or living with a family member who is mentally ill or incarcerated.
### Protective Factors
Having more protective factors makes it less likely that an adolescent will begin smoking. These include
* high parental expectations
* participation in religious activity
* involvement with sports programs
* volunteering
* parental involvement
Factors that protect all youth against the perils of tobacco include the restrictions that have been placed on advertising in the past fifteen years. Increased taxes, and thus higher prices, have also made tobacco products unaffordable for many young people. These are all part of tobacco prevention.
### Refusal Skills: How Do You "Just Say No"?
As we discussed in the treatment section, having refusal skills or knowing how to say no to offers of tobacco is key for adolescents who want to avoid tobacco use. Educators and parents offer youth these examples of positive and effective ways to say "no."
### Youth Tobacco Prevention Programs
A recent review of forty-nine studies on tobacco prevention programs used in schools found that they are highly effective, especially with youth who have never used up to that point. Comprehensive programs include classroom-based education, parental involvement, and community-wide efforts.
In these programs, students learn problem-solving and decision-making skills, learn about the short-term and long-term effects of tobacco use, and receive skills training. Individual programs are targeted at different age ranges, beginning in elementary school and continuing through college.
Some prevention programs include representatives from many sectors of the community, including police, school personnel, business owners, and religious leaders.
### Parental Influence
Studies show that what parents say matters. When they express their thoughts about tobacco and other drug use, their kids do hear them and are affected. In fact the National Crime Prevention Council has found that parental influence is the main reason that kids don't use tobacco or other drugs—they don't want to disappoint their parents. Even parents who smoke can let their children know that they want and expect something different for them. They can explain that they are addicted to nicotine and are trying to quit, if that is indeed the case.
Here are some tips for parents on ways to prevent their teens from using tobacco:
* Talk to your child about tobacco.
* Discuss information about the dangers of tobacco. Share stories about people you know who have lost loved ones due to tobacco or who are struggling to quit. Emphasize that most young people who smoke wish they hadn't started, and most didn't realize tobacco was so addictive.
* Invite your child to talk about his views on tobacco.
* It's important to have a two-way conversation with your child about tobacco. Allow your child to express her opinions about tobacco and to talk about the pressure she feels to use it. Avoid criticizing or arguing with your child during this conversation.
* Develop clear, consistent rules with appropriate consequences.
* Regardless of your child's age, he still needs boundaries. Set no-smoking rules for your family and enforce those rules with reasonable consequences. Clearly state those rules up front and be prepared to follow through on the consequences.
* Encourage your child to use you as an excuse if she is ever invited to use tobacco. "My mom or dad will ground me" is a great excuse.
* Monitor your child's activities and know his friends.
* Most adolescents are pressured to use tobacco by their peers. Make it a point to meet your child's friends, know where your child is, and know what she is doing with these friends.
* Know how your child is using his money.
* If you give an allowance or your child has a job, make sure that your child is not using that money to buy tobacco. If you find out that a local store is selling tobacco to minors, report it to the store manager or the police.
* If you use tobacco, make sure it isn't easily available to your child. Don't ask your child to get tobacco products for you.
* Recognize that, as a parent, you are a role model.
* Two of the greatest tools you can use to help your child avoid tobacco use are love and support. If an adolescent feels good about herself, she is less likely to give in to the pressure to use tobacco.
* Seek outside help if your child is already using tobacco.
Also, it's important to be aware that children are exposed to tobacco marketing and other societal influences. Talk to your kids about advertising and media regarding tobacco use. Get involved in community programs to reduce youth tobacco use and support efforts to place further restrictions on tobacco.
## Notes
### Introduction
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### How It Works
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### Addictive Properties
T. P. Condon, "The Role of Nicotine Addiction in Tobacco Use," Neurobiology of Nicotine Addiction: An Overview, December 8, 2008, CDC website, www.cdc.gov/tobacco/icsh/meetings/summary120808/neurobiology/.
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### Use, Abuse, and Addiction
From J. A. Hoffman and S. Nemes, ASCENT: A Program for Adolescent Smoking Cessation (Center City, MN: Hazelden, 2002) and adapted from "Hooked on Nicotine Checklist," from J. R. DiFranza, N. A. Rigotti, A. D. McNeill, J. K. Ockene, J. A. Savageau, D. St. Cyr, and M. Coleman, "Initial Symptoms of Nicotine Dependence in Adolescents," Tobacco Control 9 (2000): 313–19.
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### Treatment for Nicotine Addiction
"Quitting Smoking among Adults—United States, 2001–2010," Morbidity and Mortality Weekly Report 60, no. 44 (2011): 1513–19.
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Hoffman and Nemes, ASCENT.
Ibid.
### Prevention Practices
U.S. Surgeon General, Preventing Tobacco Use Among Youth and Young Adults: A Report of the Surgeon General, Executive Summary (Rockville, MD: U.S. Department of Health and Human Services, 2012), 3.
Centers for Disease Control and Prevention, Best Practices for Comprehensive Tobacco Control Programs—2007 (Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, October 2007).
Ibid.
See, for example, L. Steinberg, "Raise Age to Buy Cigarettes to 21," CNN, April 18, 2011, www.cnn.com/2011/OPINION/04/18/steinberg.smoking.teens/index.html.
U.S. Department of Health and Human Services, Office of Adolescent Health, Risk, and Protective Factors, accessed May 13, 2013, www.hhs.gov/ash/oah/adolescent-health-topics/substance-abuse/tobacco/risk-and-protective-factors.html.
T. S. Schepis and U. Rao, "Epidemiology and Etiology of Adolescent Smoking," Current Opinions in Pediatrics 17, no. 5 (October 2005): 607–12.
Adapted from C. L. Perry, R. V. Luepker, D. M. Murray, M. H. Smyth, Minnesota Smoking Prevention Program (Center City, MN: Hazelden, 2004, 2013).
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| 2023-12-24T01:27:17.760571 | https://example.com/article/1331 |
---
abstract: 'Using genetically engineered light-powered [*E. coli*]{} whose locomotion can be reversibly controlled by light, we study the emergence of collective bacterial swimming, a phenomenon often refereed to as bacterial turbulence. Bacterial turbulence is triggered in our experiments by adjusting light intensity, allowing for real-time imaging of the onset of collective motions in concentrated bacterial suspensions. We systematically map the phase diagram of the 3D flow of bacterial suspensions over a large control parameter space spanned by bacterial concentration, the swimming speed of bacteria and the number fraction of active swimmers and delineate the phase boundary between the disordered phase and the turbulent phase. The diagram quantitatively agrees with a simple model balancing hydrodynamic interactions with rotational diffusion of bacteria. We further identify two different kinetic pathways towards bacterial turbulence. Near the phase boundary, one-step transitions with long incubation periods are observed, whereas two-step transitions driven by a long-wavelength instability are found deep inside the turbulent phase. The microscopic origin of these unusual kinetics is finally discussed. Our experiments verify the key predictions of existing theories on transition points and the mode of instability and reveal unexpected kinetic features of nonequilibrium phase transitions in active matter.'
author:
- Yi Peng
- Zhengyang Liu
- Xiang Cheng
title: 'Imaging the emergence of bacterial turbulence using light-powered [*Escherichia coli*]{}'
---
I. INTRODUCTION {#i.-introduction .unnumbered}
===============
Collective motions of biological systems such as bird flocks, fish schools and bacterial swarms are the most vivid examples of the emergent behaviors of active matter [@Vicsek12; @Toner98; @Marchetti13]. While moving independently at low density, self-propelled units in active matter can move collectively at high density, giving rise to coherent flows at length scales much larger than the size of individual units. In bacterial suspensions, these coherent flows manifest a chaotic pattern of intermittent vortices and jets, reminiscent of turbulent flows at high Reynolds numbers. Hence, the flows induced by bacterial collective swimming are often called as active or bacterial turbulence [@Wolgemuth08; @Ishikawa11; @Wensink12; @Dunkel13; @Kaiser14; @Linkmann19].
Extensive theoretical and numerical studies have been conducted in understanding the physical principles underlying the nonequilibrium transition between the disordered and the turbulent states in bacterial suspensions [@Toner98; @Marchetti13; @Linkmann19; @Baskaran09; @Ramaswamy10; @Koch11; @Saintillan15; @Takatori15; @Hernandez05; @Aranson07]. Particularly, mean-field kinetic theories have been developed by extending the classic models of suspensions of passive rod-shaped particles [@Koch11; @Saintillan15; @Stenhammar17]. Subramanian and Koch showed that the isotropic disordered state of suspensions of pusher swimmers like [*E. coli*]{} becomes unstable above a threshold concentration $n_c$ that is inversely proportional to the activity of swimmers as quantified by their swimming speed $v$ [@Koch11; @Subramanian09]. Independently, Saintillan, Hohenegger and Shelley showed that a long wavelength hydrodynamic instability drives the transition to active turbulence in pusher suspensions [@Saintillan15; @Saintillan08; @Saintillan08_2; @Hohenegger10]. The transition kinetics was further studied beyond the linear regime by numerically solving kinetic equations [@Saintillan08_2] and simulations [@Saintillan12].
{width="1\linewidth"}
In comparison, definitive experiments on bacterial suspensions that can quantitatively verify theoretical predictions are still few and far between [@Koch11; @Saintillan15; @Schwarz-Linek16]. Pioneering experiments on suspensions of [*Bacillus subtilis*]{} first showed the emergence of bacterial turbulence at high concentrations in absence of bioconvection and chemotaxis [@Dombrowski04; @Cisneros07; @Sokolov07; @Sokolov09; @Cisneros11]. Various physical properties of bacterial turbulence such as density fluctuations [@Zhang10], coherent and defect structures [@Sokolov12; @Ryan13; @Guo18; @Li19_1] and mass and momentum transports [@Wu00; @Peng16; @Yang16; @Lopez15; @Gachelin13; @Liu19; @Saintillan18] have been subsequently studied. While most of these studies focused on the rise of bacterial turbulence with increasing bacterial concentrations, few experiments have considered the effect of other important factors such as the swimming speed of bacteria [@Sokolov12; @Ryan13] and the presence of defective immobile bacteria on the bacterial turbulent transition. A detailed mapping of the phase diagram of 3D bacterial flows over a large control parameter space is still lacking. Such a phase diagram is crucial to quantitatively verify the theoretical predictions on the transition point including $n_c \sim 1/v$ from the kinetic theories [@Koch11; @Stenhammar17]. Particularly, the study of the effect of doping immobile cells on the phase dynamics will provide important evidence to resolve the long-standing controversy over whether hydrodynamic or steric interactions play the dominant role in inducing the collective motion of swimming microorganisms [@Koch11; @Saintillan15; @Aranson07; @Ryan13]. While the presence of immobile bacteria would reduce the mean active hydrodynamic stress in bacterial suspensions and thus suppress the hydrodynamics-induced collective motion, the steric interaction arising from the rigid rod-shaped body of immobile cells would promote the collective motion at a given concentration of active particles as demonstrated by experiments on dry active granular matter [@Kumar14].
In addition to the phase dynamics, the kinetic pathway towards the collective motion of swimming microorganisms also remains largely unexplored. Kinetics of a phase transition affect both the transition rate and the structure of intermediate states and is of great importance in understanding the phase dynamics of equilibrium systems [@Wang12; @Peng15]. It plays an equally important role in a nonequilibrium phase transition, which can provide the crucial information on the rise of the nonequilibrium order from interparticle interactions and reveal the nature of the transition. Hence, resolving the route to bacterial turbulence—a premier example of collective motions—addresses the central question in active matter: how do random self-propelled units self-organize into large-scale dynamic structures?
{width="0.85\linewidth"}
II. RESULTS AND DISCUSSION {#ii.-results-and-discussion .unnumbered}
==========================
In this study, we fill the important knowledge gap and experimentally investigate both the phase dynamics and kinetic route of bacterial turbulence. Our study provides not only the most comprehensive phase diagram of the flow of 3D bulk bacterial suspensions hitherto, but also the detailed quantitative characterization of the kinetics of the bacterial turbulent transition. In order to explore a large control parameter space, we use three different strains of [*E. coli*]{} in our experiments, which all share similar body plans (Materials and methods). In addition to a wild-type strain (BW25113), a strain of tumblers with [*cheZ*]{} knocked out (RP1616) is used as immobile passive particles. Moreover, a strain of light-powered [*E. coli*]{} (BW25113 with proteorhodopsin, a light-driven transmembrane proton pump [@Walter07; @Arlt18]) is also used, whose mobility varies with green light of different intensities (Fig. S1 in [@Supplementary]). By mixing different strains of bacteria and controlling light intensity, we explore the phase diagram of 3D bacterial flows spanned by the total bacterial concentration, the average swimming speed of bacteria and the number fraction of active swimmers (Materials and methods).
A. Transition to bacterial turbulence {#a.-transition-to-bacterial-turbulence .unnumbered}
-------------------------------------
The collective motion of bacteria leads to bacterial turbulence with characteristic intermittent jets and whirls, whose lengths and speeds are much larger than those of individual bacteria (Figs. \[Figure1\]a, b and SI Video 1). To quantify bacterial turbulence, we calculate the orientational correlation of local velocities, $c_i=\min_{j=1..4}(\hat{v}_i\cdot\hat{v}_j)$, where $\hat{v}_i$ is the unit vector along the direction of the local velocity at position $i$ and $\hat{v}_j$ is the direction of the velocity in one of its four neighboring boxes adopted in Particle Imaging Velocimetry (PIV) (Materials and methods). We identify a local region with high velocity orientational correlation when $c\geq0.9$. The area fraction of these highly correlated regions, $\phi$, is used in our study as an order parameter quantifying the rise of bacterial turbulence (Fig. \[Figure1\]c). This choice is similar to that used in previous studies [@Cisneros11]. Quantitatively similar results can also be obtained if the average orientational correlation $\langle c \rangle$ or the velocity correlation length is used as the order parameter (Fig. S2 in [@Supplementary]). In addition to the velocity orientational order, we also measure the strength of bacterial flows via energy density, $E=\langle v_x^2+v_y^2 \rangle/2$, where $\vec{v}=(v_x,v_y)$ is the in-plane velocity of 3D bacterial flows.
A transition to bacterial turbulence is observed as we increase bacterial concentration, $n$ (Fig. \[Figure2\]a). A sharp increase of $\phi$ occurs around $n = 10n_0$, where $n_0 = 8 \times 10^8$ ml$^{-1}$ is the [*E. coli*]{} concentration at OD$_{600}$ of 1.0 close to the natural bacterial concentration in the intestinal tract [@Berg04]. The transition point, $n_c$, is then obtained from the inflection point of the error function fitting of $\phi(n)$. Coincidentally, the flow energy $E$ also increases sharply around $n_c$. We find similar sharp transitions to bacterial turbulence when increasing $v$ and $f$ at fixed $n$ (Figs. \[Figure2\]b, c). Particularly, bacterial turbulence is observed at $f = 20\%$ for the mixture of wild-type [*E. coli*]{} ($v = 28.7$ $\mu$m/s) and immobile bacteria at $n = 100n_0$. Such a low $f$ demonstrates the robustness of the collective flow of bacterial suspensions, greatly surpassing the state-of-the-art design of active robotic systems [@Li19]. The high tolerance to malfunctioning units likely arises from long-range hydrodynamic interactions, lacking in dry active matter [@Marchetti13].
B. 3D phase diagram {#b.-3d-phase-diagram .unnumbered}
-------------------
Systematic measurements over a thousand bacterial samples under different combinations of control parameters yield a full 3D phase diagram of bacterial flows, where bacterial turbulence emerges at large $n$, $v$ and $f$ (Fig. \[Figure2\]d). It should be emphasized that, although the transition to bacterial turbulence has been investigated with increasing $n$ and $v$ in different experiments [@Sokolov07; @Sokolov09; @Cisneros11; @Sokolov12; @Ryan13], to the best of our knowledge, systematic measurements over such a large parameter space in the same experimental system have not been achieved previously. This comprehensive 3D phase diagram not only allows us to quantitatively verify theoretical predictions that has not been possible before, but also sets up an indispensable framework for exploring the kinetics of the transition in the next section.
We estimate the phase boundary between disordered and turbulent phases based on a simple hydrodynamic model, where two-body hydrodynamic interactions that promote local bacterial alignment compete with random rotations that disorientate bacteria [@Drescher11]. To consider the presence of immobile bacteria, we have also included the higher-order hydrodynamic interactions from reflection flows in the model [@Supplementary]. Transition concentration $n_c$ is then predicted as $$\frac{n_c}{n_0} - n_0 r_0^3 \left(\frac{n_c}{n_0}\right)^2 = \frac{1}{f} \frac{v_0}{v}, \label{tp}$$ where $r_0 = 1.6$ $\mu$m and $v_0 = 296$ $\mu$m/s are geometric and dynamic factors related to the shape and activity of bacteria [@Supplementary]. Eq. \[tp\] allows for the collapse of the transition boundaries under different control parameters and shows a quantitative agreement with experiments (Fig. \[Figure2\]e).
We compare our experiments with kinetic theories. First, since $n_0 r_0^3 \sim 0.003$, the square term on the left of Eq. \[tp\] is negligible when $n_c/n_0$ is not too large, which leads to $n_c \sim 1/v$, directly confirming the prediction of kinetic theories [@Koch11; @Stenhammar17]. Second, at $f = 1$, we find $n_c$ ranges between $12n_0$ and $40n_0$ for different bacterial velocities. The average distance between bacteria at $n_c$ is about 4 to 6 $\mu$m, comparable to the size of a single bacterium with its extended flagellar bundle [@Berg04]. Short-range hydrodynamic and steric interactions are supposed to be important at such small scales [@Aranson07]. Hence, it is rather surprising that the far-field dipolar interaction adapted in our simple calculation is sufficient to describe the experimental phase boundary quantitatively, which indeed supports the basic assumption of kinetic theories [@Koch11; @Saintillan15; @Stenhammar17]. Consistent with this finding, increasing the fraction of immobile bacteria at either a fixed total concentration $n$ or a fixed concentration of active swimmers $nf$ suppresses bacterial turbulence in our experiments, rather than promoting the collective motion as observed in dry active matter with purely short-range steric interactions [@Kumar14]. Lastly, it is worth noting that the size of samples in our experiments is fixed with the minimal dimension between 150 $\mu$m and 170 $\mu$m, much larger than the size of single bacteria. The volume of the samples is on the order of several microliters, which contains more than $10^7$ bacteria at $n=10n_0$. Simulations and kinetic theories have shown that $n_c$ is independent of the system size at such large scales [@Stenhammar17]. Our previous experiments have shown that the correlation length of collective flows saturates for large systems above 140 $\mu$m [@Guo18]. More recent experiments have also shown that the onset of collective motions and bacterial superfluids is insensitive to the system size above 170 $\mu$m [@Martinez20]. Thus, the phase diagram shown in Fig. \[Figure2\]d should approximate the phase behavior of bacterial suspensions in the bulk limit.
![Transition kinetics. (a) Velocity orientational order $\phi$ and flow energy $E$ as a function of time through the turbulent transition. $t = 0$ corresponds to the time when light intensity is ramped up. Bacterial concentration $n = 40n_0$, speed $v = 10.5$ $\mu$m/s and swimmer fraction $f = 1$, which is near the phase boundary shown in Fig. \[Figure2\]d. (b) Spatiotemporal evolution of the local velocity orientational correlation $c$ (indicated by color) and the local flow energy $E$ (indicated by height) over the transition shown in (a). (c) $\phi$ and $E$ as a function of $t$ for $n = 100n_0$, $v = 10.5$ $\mu$m/s and $f = 1$ deep inside the turbulent phase. (d) Spatiotemporal evolution of $c$ and $E$ over the transition shown in (c). \[Figure3\]](Fig3.eps){width="1\linewidth"}
C. Transition kinetics {#c.-transition-kinetics .unnumbered}
----------------------
Next, using the phase diagram as a roadmap, we explore the kinetics of bacterial turbulent transition, which reveals the mode of instability and the transient structure of bacterial suspensions through the transition. To study the kinetics, we trigger the onset of bacterial turbulence by suddenly ramping up light intensity at $t = 0$, which increases bacterial swimming speed from that below the phase boundary to high $v$ above the boundary at given $n$ and $f$ (SI Video 2). Although individual bacteria in dilute suspensions recover their swimming speeds within a couple of seconds (Fig. S1 in [@Supplementary]), the emergence of collective flows can take much longer times depending on the specific control parameters. In the region above but close to the phase boundary, the transition exhibits a surprisingly long incubation period with low velocity orientational order $\phi$ and flow strength $E$ ($\sim 100$ s in Fig. \[Figure3\]a). During incubation, localized regions with relatively large velocity orientational correlation (high $c$) nucleate within the disordered phase (Fig. \[Figure3\]b). The incubation period witnesses the slow growth and the fluctuation of these high-$c$ regions. After incubation, the high-$c$ regions grow quickly and eventually percolate the entire system.
The incubation period becomes increasingly short as we move away from the phase boundary. Far above the boundary, the increase of the velocity orientational correlation is almost instantaneous across the entire system after the light ramp (Figs. \[Figure3\]c, d), whereas the energy of the flow remains low at beginning and grows to a steady-state plateau at a much later time. Thus, the system exhibits a transient state with high velocity orientational order but low flow energy (the middle two pictures at $t=10$ and 30 s in Fig. \[Figure3\]d).
A smooth crossover from the single-step transition to the two-step transition with the transient intermediate state can be seen from the $E-\phi$ plot (Fig. \[Figure4\]a): $E$ increases linearly with $\phi$ near the phase boundary at low $n$ indicating the concurrent increase of the two quantities, whereas L-shaped $E-\phi$ relations are observed for the two-step transition deep inside the phase boundary at high $n$. The fast increase of $\phi$ at early times results in the flat region of the curves at low $E$.
We further quantify the time and length scales associated with transition kinetics. The transition rate is measured by the incubation time, $\tau_{in}$, defined in analogy of nucleation and growth processes in equilibrium phase transitions (Fig. S3 in [@Supplementary]). $\tau_{in}$ decreases with $n$ and reaches a low plateau of $\sim 10$ s above $60n_0$ (Fig. \[Figure4\]b), comparable to the time it takes for a single bacterium recovering its swimming speed in the dilute limit. The presence of the transient state with high velocity orientational correlation and low flow energy is characterized by the time difference, $\Delta \tau = \tau_E-\tau_\phi$, where $\tau_E$ and $\tau_\phi$ are the times when $E$ and $\phi$ reach their steady states, respectively. $\Delta \tau$ increases with $n$ and can be as large as 65 s above $90n_0$ deep inside the phase boundary. Note that although we discuss transition kinetics with increasing $n$, qualitatively similar trends are also observed when $v$ and $f$ are varied (Figs. \[Figure4\]c-f).
![From one-step to two-step transitions. (a) Flow energy $E$ versus velocity orientational order $\phi$ at different bacterial concentrations $n$ and a fixed bacterial speed $v = 10.5$ $\mu$m/s and swimmer fraction $f=1$. $E$ is normalized by the maximal energy density at the steady state, $E_{max}$. (b) Incubation time, $\tau_{in}$, and the time difference, $\Delta \tau$, as a function of $n$ for the same $v$ and $f$. The error bars reflect the standard error of three to twelve experimental runs for each data points. (c) $E/E_{max}$ versus $\phi$ at different $f$ and a fixed $n = 60n_0$ and $v = 10.5$ $\mu$m/s. (d) $\tau_{in}$ and $\Delta \tau$ at different $f$ for the same $n$ and $v$. (e) $E/E_{max}$ versus $\phi$ at different $v$ and a fixed $n = 60n_0$ and $f = 1$. (f) $\tau_{in}$ and $\Delta \tau$ at different $v$ for the same $n$ and $f$. \[Figure4\]](Fig4.eps){width="1\linewidth"}
The length scales associated with the transition kinetics are revealed by the energy spectrum of bacterial flows, $E(k)$, where $k$ is the wavenumber [@Ishikawa11; @Wensink12] (Materials and methods). $E(k)$ is related to the energy density $E$ through $E = \int_0^\infty E(k)dk$. Near the phase boundary, the increase of $E(k)$ initiates at large $k$ (or short wavelengths) and then propagates to small $k$ over time (Fig. \[Figure5\]a), consistent with the scenario of nucleation and growth. In contrast, the spectrum increases significantly faster at small $k$ deep inside the phase boundary at early times (Fig. \[Figure5\]b), indicating a long-wavelength instability. Such a long-wavelength instability qualitatively explains the two-step transition. First, the instability at small $k$ naturally leads to a [*long-range*]{} velocity orientational correlation and, therefore, the fast increase of $\phi$ at early times. Second, since $E$ is determined by the integral of $E(k)$ over all $k$, the early-time increase of $E(k)$ at small $k$ does not substantially affect the flow energy, which shows strong increase only when $E(k)$ at high $k$ starts to grow at later times. As such, our kinetic measurements directly confirm the key prediction of kinetic theories on the existence of a long wavelength instability in suspension of pusher swimmers [@Saintillan08; @Saintillan08_2; @Hohenegger10; @Saintillan12]. The existence of the long incubation period near the phase boundary is, however, not captured by the kinetic theories. The presence of two different transition kinetics is consistent with the prediction of the phase diagram with binodal and spinodal regions based on the concept of swim pressures [@Takatori15].
![Energy spectrum through bacterial turbulent transition. (a) Energy spectrum $E(k)$ near the phase boundary with bacterial concentration $n = 40n_0$, speed $v = 10.5$ $\mu$m/s and swimmer fraction $f = 1$ at different times. (b) $E(k)$ deep inside the turbulent phase with $n = 100n_0$, $v = 10.5$ $\mu$m/s and $f = 1$ at different times. Since $E(k)$ shows a $k^{-4/3}$ scaling at high $k$, we plot $E(k)(k/k_l)^{4/3}$ to make the curves flat at high $k$, where $k_l = 2\pi/3$ $\mu$m$^{-1}$ is the wavenumber for the size of single bacteria. The dashed arrows indicate different increasing trends of $E(k)$. \[Figure5\]](Fig5.eps){width="1\linewidth"}
{width="0.75\linewidth"}
D. Microscopic view {#d.-microscopic-view .unnumbered}
-------------------
Lastly, we examine the evolution of the long-wavelength instability from the perspective of the dynamics of single bacteria using high-resolution fast confocal microscopy (Materials and methods). We find that the two-step transition deep inside the phase boundary can be understood microscopically from the development of the orientational order of bacteria in emerging turbulent flows. Directly tracking the orientation of individual bacteria in dense 3D suspensions is subject to large experimental errors. In order to quantify the local nematic order of bacteria without tracking individual bacteria, we perform Fast Fourier Transform (FFT) on local regions ($20 \times 20$ $\mu$m$^2$) of confocal images. The degree of the local nematic order can be characterized by the anisotropy of the resulting FFT patterns, $F$ (Fig. \[Figure6\]b inset) (Materials and methods). Our measurements show that the local bacterial nematic order is linearly proportional to the flow energy $E$, but decouples from the velocity orientational order $\phi$ in the two-step transition (Figs. \[Figure6\]c, d).
The finding suggests the following physical picture. At early times, a slight deviation from the isotropic distribution of bacterial orientations induced by the long wavelength instability is sufficient to establish a strong velocity orientational correlation, where bacteria appear to orientate randomly in the emergent turbulent flow as shown in Fig. \[Figure6\]a. This picture further corroborates the exceptional robustness of bacterial turbulence identified from the phase diagram. Over time, the flow energy gradually increases as bacteria align more and more with the local flows and their neighbors (Fig. \[Figure6\]c). This observation directly verifies the numerical solution of the kinetic equations in the nonlinear regime [@Saintillan08_2], where the alignment of pusher swimmers with the direction of local flows develops progressively through the transition and the increasing alignment then further enhances the velocity of local flows. A strong steady-state turbulence is finally established when bacteria well align with their neighbors as shown in Fig. \[Figure6\]b, which gives rise to a large flow energy and a high local bacterial nematic order. Thus, our confocal measurements provide a microscopic view of the two-step kinetics induced by a long wavelength instability: the disordered phase (low $\phi$, low $E$, low $F$) $\to$ the disordered turbulent phase (high $\phi$, low $E$, low $F$) $\to$ the ordered turbulent phase (high $\phi$, high $E$, high $F$).
III. CONCLUSIONS {#iii.-conclusions .unnumbered}
================
We experimentally studied the emergence of the collective motion of bacterial suspensions, the so-called bacterial turbulence. By using three different [*E. coli*]{} strains and examining over a thousand of bacterial suspensions at different concentrations, swimming speeds and fractions of active swimmers, we systematically mapped the phase diagram of 3D bulk bacterial flows over a large parameter space in one single experimental system. Based on a simple model balancing the hydrodynamic interactions and the random diffusion of bacteria, we presented a quantitative description of the phase boundary between the disordered and the turbulent phases and drew a detailed comparison with theoretical predictions that has not been possible before. Our experiments supported the basic assumption of the kinetic theories that the formation of the collection motion of swimming microorganisms is dominantly driven by the hydrodynamic interactions between bacteria, where the presence of immobile cells suppress, instead of promoting, the collective swimming in bulk bacterial suspensions.
Using the light-powered [*E. coli*]{}, we triggered the onset of bacterial turbulence and imaged the kinetics of bacterial turbulent transition in real time. Two distinct kinetic pathways were discovered, which exhibit different transition rates and structures of the transient state. Near the phase boundary, the transition shows a feature of nucleation and growth with a long incubation period. Deep inside the phase boundary, the transition takes two steps with a transient state of high velocity orientational order but low flow energy. Moreover, we illustrated the existence of a long wavelength instability in pusher suspensions deep inside the phase boundary and thus confirmed the key prediction of the kinetic theories. Our microscopic measurements further revealed the development of the instability in terms of the dynamics of single bacteria and showed that the transient state in the two-step transition is characterized by a low bacterial nematic order. These results demonstrated the remarkable robustness of bacterial turbulent flows and verified the kinetic theories from both macroscopic and microscopic perspectives. Taken together, our study provided a solid experimental benchmark on the collective motion of swimming microorganisms and addressed fundamental issues on the kinetics of nonequilibrium phase transitions in active matter.
Finally, our study also opens important questions for future experimental and theoretical development. First, it is still unclear on the microscopic origin of the long incubation period in the one-step transition near the phase boundary. Our preliminary study indicates that the incubation process may be influenced by the formation of bacterial clusters in suspensions. Nevertheless, the incubation time can be significantly longer than the time scale associated with the dissolving of bacterial clusters close to the phase boundary, suggesting the generic nature of the phenomenon. To understand the nucleation and growth process with long incubation requires prolonged experiments beyond the typical time of our experiments due to the problem of photobleaching and poses an experimental challenge for future studies. On the other hand, how to incorporate such an incubation process in kinetic theories presents perhaps an even more challenging theoretical question. Second, the long wavelength instability observed in our experiments indicates a system-size dependence of phase dynamics in confined systems. The change of the phase diagram and the kinetic pathway with the system size needs to be further confirmed and quantitatively measured in future experiments. Lastly, it is certainly worth of investigating the kinetic route to collective motions in other active fluids such as active cytoskeletons and suspensions of colloidal swimmers and examining whether the kinetic features observed in our experiments are generic for active matter or unique for microbiological systems.
ACKNOWLEDGMENTS
===============
We thanks Yi-Shu Tai, Kechun Zhang, Seunghwan Shin, Xinliang Xu, Kevin Dorfman and Stefano Martiniani for fruitful discussions and help with experiments and data analysis. The research is supported by NSF CBET-1702352, Packard Foundation and DARPA YFA-D16AP00120.
APPENDIX A: MATERIALS AND METHODS
=================================
1. Bacteria {#bacteria .unnumbered}
-----------
Three strains of [*E. coli*]{} are used. ([*i*]{}) A wild-type [*E. coli*]{} K-12 strain (BW25113), which shows normal run-and-tumble motions with an average swimming speed $v = 28.7$ $\mu$m/s [@Berg04]. ([*ii*]{}) A “tumbler” strain, which is made by knocking out [*cheZ*]{} of a wild-type [*E. coli*]{} strain (RP1616). The $\Delta$[*cheZ*]{} mutant shows constant tumbling motions. We use tumblers as passive particles. To deactivate the bacteria, we remove the flagella of tumblers by pipetting tumbler suspensions through a narrow pipette tip a few tens of times. ([*iii*]{}) A light-powered [*E. coli*]{}, whose swimming speed can be reversibly controlled by light intensity. We introduce a light-driven transmembrane proton pump, proteorhodopsin (PR), to wild-type [*E. coli*]{} (BW25113) by transforming the bacteria with plasmid pZE-PR encoding the SAR86 $\gamma$-proteobacterial PR-variant [@Walter07]. The activity of PR is directly correlated with the light intensity. Thus, we can control the swimming speed of bacteria using light of different intensities. The quantitative relation between the light intensity (in terms of the power of the light source) and the average swimming speed of bacteria is measured by tracking the motion of bacteria in dilute suspensions (Fig. S1a in [@Supplementary]). The response time of the bacteria is fast within a couple of seconds after the change of the light (Fig. S1b in [@Supplementary]). Note that the highest speed of the light-powered bacteria at high light intensity is about 10 to 11 $\mu$m/s (Fig. \[Figure2\]c), which is smaller than that of the wild-type strain. Three strains share a similar body plan with the semi-major axis of the bacterial body $a = 1.4 \pm 0.2$ $\mu$m and the semi-minor axis $b = 0.5 \pm 0.2$ $\mu$m based on direct imaging of bacteria. The errors reported here are the standard errors, instead of the standard deviations, of the measurements.
All the three strains are cultured using a similar procedure. They are first cultured at 37.0 $^\circ$C with a shaking speed at 250 rpm for 14-16 hours in terrific broth (TB) culture medium \[trypotone 1.2$\%$ (w/v), yeast extract 2.4$\%$ (w/v), and glycerol 0.4$\%$ (v/v)\]. The saturated culture is then diluted 1:100 in TB culture medium and grown at 30 $^\circ$C for 6.5 hours. PR expression is triggered by 1 mM isopropyl $\beta$-D-thiogalactoside and 10 $\mu$M methanolic all-trans-retinal, which are supplemented in the mid-log phase for the synthesis of proteorhodopsin. The bacteria are harvested by gentle centrifugation (800$g$ for 5 min) in the late log phase. After discarding the upper medium, we resuspend bacteria with motility buffer MB (0.01 M potassium phosphate, 0.067 M NaCl, 10 M EDTA, PH 7.0). The suspension is finally washed twice and adjusted to the target concentration. To create the mixture of tumblers and swimmers, we separately prepare suspensions of tumblers and swimmers both at the targeted concentration. We then mix the two suspensions at a predetermined ratio to control the fraction of active swimmers $f$.
2. Sample preparation and video microscopy {#sample-preparation-and-video-microscopy .unnumbered}
------------------------------------------
For the wild-type bacteria, oxygen is necessary to maintain the swimming of bacteria. We deposit a 2 $\mu$l wild-type bacterial suspension on a microscope coverslip, which forms a free suspension-air interface on the top. The droplet is millimetric in the lateral directions ($x$-$y$) normal to the imaging plane and about 150 $\mu$m in height ($z$). The coverslip and the suspension are further enclosed in a humid chamber of $ \sim 1000$ mm$^3$ to reduce evaporation as well as perturbation due to ambient air flows. An inverted microscope is used to image bacterial motions 70 $\mu$m above the coverslip, which shows bacterial flows in the 2D $x-y$ plane inside the 3D suspension (SI Video 1).
For the light-powered bacteria, it is important to shut down the metabolic pathway of aerobic respiration, so that the locomotion of bacteria is solely controlled by the PR pump. We inject a suspension of light-powered bacteria into a sealed cell of $18 \times 3 \times 0.17$ mm$^3$. For concentrated bacterial suspensions above $35n_0$, bacteria stop swimming after a few minutes in the cell due to the depletion of oxygen. Microscope illumination is then used to switch on/off and control the activity of the PR pump for bacterial swimming. 2D bacterial flows are imaged 80 $\mu$m above the bottom wall.
For most of our experiments, 2D flow fields are imaged through an inverted bright-field microscope using a $10\times$ (NA 0.3), $20\times$ (NA 0.5) or $40\times$ (NA 0.6) objectives. The field view of images ranges from $640 \times 640$ $\mu$m$^2$ to $160 \times 160$ $\mu$m$^2$. To measure the velocity orientational order and the flow energy in steady states, one-minute videos are taken 15 minutes after wild-type [*E. coli*]{} samples are loaded or 2 minutes after a new light condition is applied to light-powered [*E. coli*]{} samples. These times are sufficient for the sample to reach the steady state. Five-minute videos are taken for transition kinetics after light ramping. All the videos are recorded at 30 frames per second by a sCMOS camera. To control bacterial velocity, light intensity is tuned by the voltage of the light source. Three to twelve independent measurements are taken for each set of control parameters of our experiments.
To measure the dynamics of individual bacteria during the turbulent transition, we image fluorescent-labeled [*E. coli*]{} with an inverted fast confocal microscope using a $60\times$ objective (NA 1.4). The green fluorescent protein expressed in bacteria is excited by a 488 nm laser. The field view of the images is $180 \times 120$ $\mu$m$^2$. Bacterial flows are imaged 10 $\mu$m above the coverslip. Five-minute videos are taken at 10 frames per second.
3. Image processing and data analysis {#image-processing-and-data-analysis .unnumbered}
-------------------------------------
The velocity field of bacterial suspensions is obtained from raw videos using standard Particle Imaging Velocimetry (PIV). For each pair of neighboring frames, the interrogation window size shrinks from $19.6 \times 19.6$ $\mu$m$^2$ to $4.8 \times 4.8$ $\mu$m$^2$ in three iterations. The final lattice spacing of the velocity field is 2.4 $\mu$m. The velocity field in Fig. \[Figure1\]a shows the velocity vectors of every other lattices.
Energy spectrum quantifies the energy distribution over different length scales, $\lambda = 2\pi/k$, where $k$ is the wavenumber. To obtain the energy spectrum, we first calculate the Fourier transform of the 2D velocity field $v_x(x,y)$ and $v_y(x,y)$ to obtain $u_k(k_x,k_y)$ and $v_k(k_x,k_y)$. The point-wise kinetic energy density in $k$-space is then computed $E(k_x,k_y)= \langle u_k(k_x,k_y) u_k^*(k_x,k_y)+v_k(k_x,k_y) v_k^*(k_x,k_y)\rangle/2$, where \* represents the complex conjugate. Finally, the energy spectrum $E(k)$ is obtained by summing up $E(k_x,k_y)$ at a constant $k=(k_x^2+k_y^2)^{1/2}$. An alternative way to calculate $E(k)$ is through the Fourier transform of the two-point velocity correlation function $\langle \vec{v}(\vec{r}_0) \cdot \vec{v}(\vec{r}_0+\vec{r})\rangle_{\vec{r}_0}$, which yields quantitatively similar results.
To extract bacterial nematic order, we perform Fast Fourier transformation (FFT) on the local regions ($20 \times 20$ $\mu$m$^2$) of confocal images. The FFT patterns are first smoothed with 3 neighboring pixels (0.15 $\mu$m$^{-1}$) and then fitted with ellipses. The aspect ratio of the ellipses, $a/b$, quantifies the local nematic order of bacteria (Fig. \[Figure6\]b inset). The average of the aspect ratio is finally taken over the whole field of view, $F=\langle a/b \rangle$. To calibrate our method, we show numerically that the anisotropy of the FFT pattern, $a/b$, is approximately linearly proportional to the local nematic order parameter of bacteria $S=\frac{1}{N} \sum_{j=1}^{N} (3\cos^2\alpha_j-1)/2$ within the range of our experiments (Fig. S4 in [@Supplementary]), where $\alpha_j$ is the angle between the orientation of bacterium $j$ with respect to the mean orientation of all the $N$ bacteria in the local region in 3D.
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See Supplemental Materials for our experimental procedures, theory and figures.
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| 2024-05-15T01:27:17.760571 | https://example.com/article/1548 |
import React, { FunctionComponent } from "react";
import ReactSelect from "react-select/async";
import { ValueType } from "react-select/src";
import fetch from "isomorphic-fetch";
import { config } from "config";
import RegionSelectStyles from "./RegionSelectStyles";
import { Region } from "helpers/datasetSearch";
const loadOptions = (props: PropsType) => async inputValue => {
if (!props.steRegion) return [];
const queryStr = inputValue.trim();
const res = await fetch(
`${config.searchApiUrl}regions?type=SA4${
props.steRegion && props.steRegion.regionId
? `&lv2Id=${encodeURIComponent(props.steRegion.regionId)}`
: ""
}${queryStr ? `&query=${encodeURIComponent(queryStr)}` : ""}`
);
if (res.status !== 200) {
throw new Error("response.statusText");
}
const data = await res.json();
if (!data || !Array.isArray(data.regions)) {
throw new Error("Invalid server response");
}
if (props.regionId && !props.value) {
// --- set initial prepopulated value
data.regions.forEach(region => {
if (region.regionId === props.regionId) {
typeof props.onChange === "function" &&
props.onChange(region, true);
}
});
}
return data.regions;
};
interface PropsType {
countryRegion?: ValueType<Region>;
steRegion?: ValueType<Region>;
value?: ValueType<Region>;
regionId?: string;
onChange?: (
option: ValueType<Region>,
notResetOtherRegions?: Boolean
) => void;
}
const RegionSelect: FunctionComponent<PropsType> = props => {
const { countryRegion, steRegion } = props;
let placeHolderText = "Please select region...";
if (!countryRegion && !steRegion) {
placeHolderText = "Please select country and state first.";
} else if (!steRegion) {
placeHolderText = "Please select state first.";
}
const isDisabled = countryRegion && steRegion ? false : true;
return (
<div className="region-select">
<ReactSelect<Region>
key={(() => {
const keyParts = [
props.regionId ? props.regionId : "",
countryRegion ? countryRegion.regionId : "",
steRegion ? steRegion.regionId : ""
];
return keyParts.join("|");
})()}
isClearable
cacheOptions
defaultOptions
value={props.value}
loadOptions={loadOptions(props)}
getOptionLabel={option => option.regionName as string}
getOptionValue={option => option.regionId as string}
styles={RegionSelectStyles}
placeholder={placeHolderText}
isDisabled={isDisabled}
onChange={option =>
typeof props.onChange === "function" &&
props.onChange(option)
}
/>
</div>
);
};
export default RegionSelect;
| 2023-12-18T01:27:17.760571 | https://example.com/article/9504 |
Bitumart
The Petrosil Bitumen Report is published in Bitumart (www.bitumart.com). The Bitumart Report is published on a fortnightly basis. Subscribers receive reports by e-mail and a password to the site to access prices, price trends, archived reports, demand and supply issues, directory databases of buyers and sellers and other bitumen statistics and information. The Bitumart Report currently enjoys a subscriber base of over 300 global subscribers who cover the entire value chain from refiners, traders, research organizations, logistics companies and other industry players. | 2024-03-27T01:27:17.760571 | https://example.com/article/6498 |
Alcaligenes infection in cystic fibrosis.
The aim of this study was to investigate the effect of chronic Alcaligenes species infection of the respiratory tract on the clinical status of patients with cystic fibrosis. We conducted a retrospective case-controlled study. The microbiological records of all patients attending the Leeds Regional Pediatric and Adult Cystic Fibrosis Units from 1992-1999 were examined. Chronic Alcaligenes infection was defined as a positive sputum culture on at least three occasions over a 6-month period. These patients were compared with controls matched for age, gender, respiratory function, and Pseudomonas aeruginosa infection status. Respiratory function tests, anthropometric data, Shwachman-Kulczycki score, Northern chest x-ray score, intravenous and nebulized antibiotic treatment, and corticosteroid treatment were compared from 2 years before to 2 years after Alcaligenes infection. From a clinic population of 557, 13 (2.3%) fulfilled the criteria for chronic infection. The median age at acquisition of infection was 17.2 years (range, 6.5-33.6). There was no significant difference in the changes of percentage predicted values for FEV(1), FVC, FEF(25-75), or Shwachman-Kulczycki and Northern chest x-ray scores, or in weight, height, and body mass index z-scores between Alcaligenes-infected cases and controls. There was also no significant difference in the use of antibiotics (intravenous and nebulized) or corticosteroids (inhaled and oral). We conclude that in our clinic, chronic infection with Alcaligenes species was uncommon. Chronically infected patients showed no excess deterioration in clinical or pulmonary function status from 2 years before to 2 years after primary acquisition. | 2024-03-02T01:27:17.760571 | https://example.com/article/1420 |
Monophysite Churches, 1 Definition(s)
Monophysite Churches means something in Christianity Check out some of the following descriptions and leave a comment if you want to add your own contribution to this article:
1 Definition(s) from various sources:
These churches broke away from the rest of Christianity after the Council of Chalcedon (451) declared their distinctive belief about Jesus' nature incorrect. Whereas the Council decided that Jesus was a combination of two natures (human and divine), the monophysites believe that he had only one nature, namely, a divine one. The Coptic, Ethiopic and Armenian Church broke away and still hold monophysite beliefs. | 2023-11-19T01:27:17.760571 | https://example.com/article/7290 |
import { Alert, List, ListItem } from '@hospitalrun/components'
import { mount, ReactWrapper } from 'enzyme'
import { createMemoryHistory } from 'history'
import React from 'react'
import { act } from 'react-dom/test-utils'
import { Router } from 'react-router-dom'
import NotesList from '../../../patients/notes/NotesList'
import PatientRepository from '../../../shared/db/PatientRepository'
import Note from '../../../shared/model/Note'
import Patient from '../../../shared/model/Patient'
describe('Notes list', () => {
const setup = async (notes: Note[]) => {
const mockPatient = { id: '123', notes } as Patient
jest.spyOn(PatientRepository, 'find').mockResolvedValueOnce(mockPatient)
const history = createMemoryHistory()
history.push(`/patients/${mockPatient.id}/notes`)
let wrapper: any
await act(async () => {
wrapper = await mount(
<Router history={history}>
<NotesList patientId={mockPatient.id} />
</Router>,
)
})
wrapper.update()
return { wrapper: wrapper as ReactWrapper, history }
}
it('should render a list of notes', async () => {
const expectedNotes = [
{
id: '456',
text: 'some name',
date: '1947-09-09T14:48:00.000Z',
},
]
const { wrapper } = await setup(expectedNotes)
const listItems = wrapper.find(ListItem)
expect(wrapper.exists(List)).toBeTruthy()
expect(listItems).toHaveLength(expectedNotes.length)
expect(listItems.at(0).find('.ref__note-item-date').text().length)
expect(listItems.at(0).find('.ref__note-item-text').text()).toEqual(expectedNotes[0].text)
})
it('should display a warning when no notes are present', async () => {
const expectedNotes: Note[] = []
const { wrapper } = await setup(expectedNotes)
const alert = wrapper.find(Alert)
expect(wrapper.exists(Alert)).toBeTruthy()
expect(wrapper.exists(List)).toBeFalsy()
expect(alert.prop('color')).toEqual('warning')
expect(alert.prop('title')).toEqual('patient.notes.warning.noNotes')
expect(alert.prop('message')).toEqual('patient.notes.addNoteAbove')
})
it('should navigate to the note view when the note is clicked', async () => {
const expectedNotes = [{ id: '456', text: 'some name', date: '1947-09-09T14:48:00.000Z' }]
const { wrapper, history } = await setup(expectedNotes)
const item = wrapper.find(ListItem)
act(() => {
const onClick = item.prop('onClick') as any
onClick({ stopPropagation: jest.fn() })
})
expect(history.location.pathname).toEqual(`/patients/123/notes/${expectedNotes[0].id}`)
})
})
| 2024-02-08T01:27:17.760571 | https://example.com/article/5142 |
Functional and structural characterization of 2B viroporin membranolytic domains.
Nonstructural 2B viroporin is an intracellularly produced pore-forming protein required for effective enteroviral and rhinoviral replication. The sequence of 2B displays two putative interconnected transmembrane domains, which are predicted to insert into the negatively charged membranes of target organelles forming an integral hairpin. The use of an overlapping peptide library that spanned the complete 2B sequence has recently allowed the mapping of the cell plasma membrane porating activity to the partially amphipathic, amino-terminal transmembrane domain (TM1, residues 35-55). We describe here that although the TM1 peptide was effective in permeabilizing uncharged membranes, it induced marginal lysis of anionic bilayers. In fact, only the peptide representing the highly conserved carboxy-terminal transmembrane domain (TM2, residues 59-82) reproduced the capacity of the full 2B protein to efficiently permeabilize bilayers made of anionic phospholipids. Insertion into lipid monolayers and circular dichroism determinations were, however, consistent with penetration of the TM1 helix into both anionic and zwitterionic membranes, while TM2 interacting with membranes assumed a mixture of conformations. Moreover, addition of TM1 strongly stimulated TM2-induced permeabilization of the anionic membranes. In combination, TM1 and TM2 formed a complex that had structural properties, including a high proportion of extended nonhelical secondary structure, that were distinct from those of the individual peptides. Finally, a comparison of antimicrobial and hemolytic activities further underscored the TM1 domain's cytolytic character. Overall, our data support the idea that the cytolytic activity of TM1 in the negatively charged cell endomembranes targeted by 2B viroporin requires the cooperation of both transmembrane domains. | 2024-07-05T01:27:17.760571 | https://example.com/article/9077 |
# Copyright 2015 The Chromium Authors. All rights reserved.
# Use of this source code is governed by a BSD-style license that can be
# found in the LICENSE file.
import parser
import symbol
import sys
import token
import tokenize
from py_utils.refactor import offset_token
class Snippet(object):
"""A node in the Python parse tree.
The Python grammar is defined at:
https://docs.python.org/2/reference/grammar.html
There are two types of Snippets:
TokenSnippets are leaf nodes containing actual text.
Symbols are internal nodes representing higher-level groupings, and are
defined by the left-hand sides of the BNFs in the above link.
"""
@property
def type(self):
raise NotImplementedError()
@property
def type_name(self):
raise NotImplementedError()
@property
def children(self):
"""Return a list of this node's children."""
raise NotImplementedError()
@property
def tokens(self):
"""Return a tuple of the tokens this Snippet contains."""
raise NotImplementedError()
def PrintTree(self, indent=0, stream=sys.stdout):
"""Spew a pretty-printed parse tree. Mostly useful for debugging."""
raise NotImplementedError()
def __str__(self):
return offset_token.Untokenize(self.tokens)
def FindAll(self, snippet_type):
if isinstance(snippet_type, int):
if self.type == snippet_type:
yield self
else:
if isinstance(self, snippet_type):
yield self
for child in self.children:
for snippet in child.FindAll(snippet_type):
yield snippet
def FindChild(self, snippet_type, **kwargs):
for child in self.children:
if isinstance(snippet_type, int):
if child.type != snippet_type:
continue
else:
if not isinstance(child, snippet_type):
continue
for attribute, value in kwargs:
if getattr(child, attribute) != value:
break
else:
return child
raise ValueError('%s is not in %s. Children are: %s' %
(snippet_type, self, self.children))
def FindChildren(self, snippet_type):
if isinstance(snippet_type, int):
for child in self.children:
if child.type == snippet_type:
yield child
else:
for child in self.children:
if isinstance(child, snippet_type):
yield child
class TokenSnippet(Snippet):
"""A Snippet containing a list of tokens.
A list of tokens may start with any number of comments and non-terminating
newlines, but must end with a syntactically meaningful token.
"""
def __init__(self, token_type, tokens):
# For operators and delimiters, the TokenSnippet's type may be more specific
# than the type of the constituent token. E.g. the TokenSnippet type is
# token.DOT, but the token type is token.OP. This is because the parser
# has more context than the tokenizer.
self._type = token_type
self._tokens = tokens
self._modified = False
@classmethod
def Create(cls, token_type, string, offset=(0, 0)):
return cls(token_type,
[offset_token.OffsetToken(token_type, string, offset)])
@property
def type(self):
return self._type
@property
def type_name(self):
return token.tok_name[self.type]
@property
def value(self):
return self._tokens[-1].string
@value.setter
def value(self, value):
self._tokens[-1].string = value
self._modified = True
@property
def children(self):
return []
@property
def tokens(self):
return tuple(self._tokens)
@property
def modified(self):
return self._modified
def PrintTree(self, indent=0, stream=sys.stdout):
stream.write(' ' * indent)
if not self.tokens:
print >> stream, self.type_name
return
print >> stream, '%-4s' % self.type_name, repr(self.tokens[0].string)
for tok in self.tokens[1:]:
stream.write(' ' * indent)
print >> stream, ' ' * max(len(self.type_name), 4), repr(tok.string)
class Symbol(Snippet):
"""A Snippet containing sub-Snippets.
The possible types and type_names are defined in Python's symbol module."""
def __init__(self, symbol_type, children):
self._type = symbol_type
self._children = children
@property
def type(self):
return self._type
@property
def type_name(self):
return symbol.sym_name[self.type]
@property
def children(self):
return self._children
@children.setter
def children(self, value): # pylint: disable=arguments-differ
self._children = value
@property
def tokens(self):
tokens = []
for child in self.children:
tokens += child.tokens
return tuple(tokens)
@property
def modified(self):
return any(child.modified for child in self.children)
def PrintTree(self, indent=0, stream=sys.stdout):
stream.write(' ' * indent)
# If there's only one child, collapse it onto the same line.
node = self
while len(node.children) == 1 and len(node.children[0].children) == 1:
print >> stream, node.type_name,
node = node.children[0]
print >> stream, node.type_name
for child in node.children:
child.PrintTree(indent + 2, stream)
def Snippetize(f):
"""Return the syntax tree of the given file."""
f.seek(0)
syntax_tree = parser.st2list(parser.suite(f.read()))
tokens = offset_token.Tokenize(f)
snippet = _SnippetizeNode(syntax_tree, tokens)
assert not tokens
return snippet
def _SnippetizeNode(node, tokens):
# The parser module gives a syntax tree that discards comments,
# non-terminating newlines, and whitespace information. Use the tokens given
# by the tokenize module to annotate the syntax tree with the information
# needed to exactly reproduce the original source code.
node_type = node[0]
if node_type >= token.NT_OFFSET:
# Symbol.
children = tuple(_SnippetizeNode(child, tokens) for child in node[1:])
return Symbol(node_type, children)
else:
# Token.
grabbed_tokens = []
while tokens and (
tokens[0].type == tokenize.COMMENT or tokens[0].type == tokenize.NL):
grabbed_tokens.append(tokens.popleft())
# parser has 2 NEWLINEs right before the end.
# tokenize has 0 or 1 depending on if the file has one.
# Create extra nodes without consuming tokens to account for this.
if node_type == token.NEWLINE:
for tok in tokens:
if tok.type == token.ENDMARKER:
return TokenSnippet(node_type, grabbed_tokens)
if tok.type != token.DEDENT:
break
assert tokens[0].type == token.OP or node_type == tokens[0].type
grabbed_tokens.append(tokens.popleft())
return TokenSnippet(node_type, grabbed_tokens)
| 2024-05-16T01:27:17.760571 | https://example.com/article/2819 |
This invention relates to telephones and, more particularly, to telephone handset holders.
Telephone construction, operation, and design have changed dramatically since the invention of the telephone by Alexander Graham Bell in U.S. Pat. No. 174,465. Early telephones were cumbersome, crank operated and utilized a separate earpiece connected by a cord to stationary telephone box. The telephone box was mounted on a wall and had a speaker cone providing a mouthpiece. In the 1950s, black bulky telephones were in use with a stationary base on which was mounted a rotatable dial. A movable handset with an earpiece and mouthpiece was connected by a cord to the stationary base. In the 1980s, lighter weight white and colored handsets were developed with pushbuttons between the earpiece and mouthpiece or with pushbuttons on the base. Pushbutton telephones have generally replaced rotary telephones.
Pay telephones or telephone pay station included several slots for the deposit of coins of varying denominations. A typical arrangement allowed the deposit of nickels, dimes and quarters so that payment of various amounts could be utilized in connection with the pay station. This arrangement was first used with manual telephones in which the initial deposit of a single coin would signal the operator that a call was to be placed. Additional coins were added for payment of calls to long distance locations. Internal arrangements of gongs transmitted various sounds back to the operator and gave the operator an indication of proper coin deposit. Eventually, telephone pay stations became dial operated, and an operator would normally not be required in order to place local calls. Various arrangements for actuating the equipment and for accepting and returning coins were developed, including electronic totalizers that were able to count and accurately register the number and denomination of coins deposited. Such totalizers also facilitated the development and utilization of single slot pay stations in which a single slot accepted coins of various denominations.
Some coin operated telephones and public telephones were developed with a slot or card swipe to receive a telephone identification credit card, or other card with a magnetic strip to enable the user to make calls without the need for cash/coins, by charging the telephone call to the user's account. Telephones that accept credit cards represent a much more recent development that has found widespread use. Pay telephones that accept credit cards are sometimes found in airports, railroad stations, etc., and have been well received by travelers.
Recent development and adaptation of the pay station have permitted use in commercial aircraft. Credit card-accepting pay stations have been incorporated into commercial aircraft where, by means of radio-telephone links, telephone service can be made by the user from the commercial aircraft to ground-based telephone equipment. Aircraft telephones themselves have typically been installed on bulkheads located adjacent to flight-attendant stations. This arrangement requires the user to leave the passenger seat to go to the location of the on board pay station to initiate the call. If the pay station is equipped with a wireless telephone unit, the flight attendant often becomes involved in providing the user with the hand-held telephone.
Wireless aircraft telephones have been provided with a credit card and reader. In this type of system, the user has to walk to the phone station, insert and leave the credit card in the handset mount to release the handset, then carry the handset back to the passengers seat, and after use, replace the handset and retrieve the credit card. However, with this type of system the passenger cannot obtain the handset during turbulence or at other times when passengers are required to remain in their seats. Furthermore, it is inconvenient to obtain a handset during the time meals are being served and in particular when ones seat is not located on the aisle. Such arrangements fail to afford the convenience and safety, an aircraft passenger might desire in connection with the placement of a call.
In the 1980s, personal computers (PCs) were mass produced for businessmen, word processing personnel, financial analysts, bankers, and other consumers. Such personal computers were generally more compact then their predecessors, had greater memory capabilities, and faster processing times. Lap top computers and other portable computers were also produced. Computers can be hooked up with data bases and other computers at distant or remote locations via a modem.
Over the years a variety of telephone handsets, holders, and other telecommunication equipment have been suggested. Typifying these prior art telephone handsets and telecommunication equipment are those shown in U.S. Pat. Nos. 2,557,393; 4,252,994; 4,488,009; 4,587,379; 4,661,659; 4,672,661; 4,727,569; 4,788,420; 4,811,387; Des. 276,723; Des. 280,986; Des. 297,839; Des. 299,136; Des. 299,137; and Des. 301,034. These prior art telephone handsets, holders, and telecommunication equipment have met with varying degrees of success.
It is, therefore, desirable to provide a telephone handset and holder assembly with improved features. | 2023-09-06T01:27:17.760571 | https://example.com/article/9698 |
// @flow
import ArrowMenu from './ArrowMenu';
import Backdrop from './Backdrop';
import Breadcrumb from './Breadcrumb';
import Button from './Button';
import ButtonGroup from './ButtonGroup';
import Checkbox, {CheckboxGroup} from './Checkbox';
import CircularProgressbar from './CircularProgressbar';
import CroppedText from './CroppedText';
import DatePicker from './DatePicker';
import Dialog from './Dialog';
import Divider from './Divider';
import DropdownButton from './DropdownButton';
import Email from './Email';
import FileUploadButton from './FileUploadButton';
import Form from './Form';
import GhostIndicator from './GhostIndicator';
import Grid from './Grid';
import Heading from './Heading';
import Icon from './Icon';
import ImageRectangleSelection from './ImageRectangleSelection';
import InfiniteScroller from './InfiniteScroller';
import Input from './Input';
import Loader from './Loader';
import Masonry from './Masonry';
import Matrix from './Matrix';
import Menu from './Menu';
import MultiItemSelection from './MultiItemSelection';
import MultiSelect from './MultiSelect';
import Navigation from './Navigation';
import Number from './Number';
import Overlay from './Overlay';
import Phone from './Phone';
import Popover from './Popover';
import PublishIndicator from './PublishIndicator';
import ResourceLocator from './ResourceLocator';
import SingleSelect from './SingleSelect';
import Table from './Table';
import Toggler from './Toggler';
import Toolbar from './Toolbar';
import Url from './Url';
import withContainerSize from './withContainerSize';
export {
ArrowMenu,
Backdrop,
Breadcrumb,
Button,
ButtonGroup,
Checkbox,
CheckboxGroup,
CircularProgressbar,
CroppedText,
DatePicker,
Dialog,
Divider,
DropdownButton,
Email,
FileUploadButton,
Form,
GhostIndicator,
Grid,
Heading,
Icon,
ImageRectangleSelection,
InfiniteScroller,
Input,
Loader,
Matrix,
Menu,
Masonry,
MultiItemSelection,
MultiSelect,
Navigation,
Number,
Overlay,
Phone,
Popover,
PublishIndicator,
ResourceLocator,
SingleSelect,
Table,
Toggler,
Toolbar,
Url,
withContainerSize,
};
| 2023-10-21T01:27:17.760571 | https://example.com/article/5105 |
Map & Location
Street View
Property Description
INVESTORS.. PRICE TO SELL ASAP! Great rental opportunity near the old historic district of Seminole Heights. This charming bungalow offers 3 units for lease.. one in the main house 3/1, 2/1 in the back and an efficiency as well. Close to the river, I275, I4, shopping, dining and so much more! Bring all offers!
In the Community
Nearby Schools
106 W Curtis St, Tampa, FL 33603 (MLS# T2852803) is a
Single Family property with 5 bedrooms and 4 full bathrooms.
106 W Curtis St is currently listed for $168,900 and was received on December 01, 2016.
Want to learn more about 106 W Curtis St?
Do you have questions about finding other
Single Family
real estate for sale
in Tampa?
You can browse all Tampa real estate or
contact a Coldwell Banker agent to request more information.
Quick Links
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Stay Updated
Information deemed reliable but not guaranteed. Information is provided, in part, by MFRMLS (My Florida MLS). This information being provided is for consumer's personal, non-commercial use and may not be used for any other purpose other than to identify prospective properties consumers may be interested in purchasing.
Boundaries provided by Maponics LLC
Home Partners of America and A New Path to Homeownership are registered trademarks of Home Partners of America LLC.
Real estate agents affiliated with Coldwell Banker are independent contractor sales associates and are not employees of Coldwell Banker. | 2023-09-23T01:27:17.760571 | https://example.com/article/2511 |
Three-dimensional corrective osteotomy using a patient-specific osteotomy guide and bone plate based on a computer simulation system: accuracy analysis in a cadaver study.
The accuracy of three-dimensional (3-D) corrective osteotomy using a patient-specific osteotomy guide and bone plate based on computer simulation was investigated. Six fresh-frozen cadaver upper limbs were used. A patient-specific osteotomy guide designed to realize a preplanned osteotomy was set on the distal humerus and distal radius, and the error in the setting location was evaluated. After the osteotomy, the surgical site was fixed using a patient-specific bone plate designed to exactly fit the anatomical shape of the postoperative bone model. The postoperative results were compared with the preoperative simulation. The errors in the guide location on the humerus and radius were <1.5° and 1.0 mm and <1.0° and 1.0 mm, respectively. The plate fixation errors of the humerus and radius were <2.0° and 1.5 mm and <1.0° and 1.0 mm, respectively. The system is sufficiently feasible to realize precise 3-D deformity correction of a limb. | 2024-05-04T01:27:17.760571 | https://example.com/article/4247 |
Great Day to finish my NH 48 4K on Adams. Hike with my friend Dave who nabbed his 1st NH 4K. After hitting Adams we leisurely traveled over to Quincy Adams as the day was to nice to head straight back down. We came up Airline and headed back down Valley Way. Enjoyed the falls along Fallsway on our way out.
see Madison post. After a rough night left my tent around Noon and quickly climbed Adams which proved fairly calm and very sunny. Got views from the Presidential Range for the first time in 3 trips. Well worth it. Descended through Thunderstorm Junction and the Edmond's Col to Jefferson.
all excited to do this trip. It started raining when we got to craig camp. As it cleared, we decided to summit...clearish up until about Thunderstorm Junction. By the time we reached the summit...50 mph winds, "sleet" (honest...), and so foggy we hit to wrong trail down TWICE. I think I have PTMD...post traumatic mountain disorder! But...won't be my last climb...of that I am sure.
Climbed with Madison and Jefferson via the Gulfside Trail from Lakes of the Clouds. A really tough trail that almost entirely runs across boulder fields and talus slopes. The fog eventually burnt off as we continued our traverse of the Northern Presidentials. Great views from the summit. | 2024-01-09T01:27:17.760571 | https://example.com/article/8981 |
// @flow
/* eslint-env mocha */
import assert from 'power-assert'
import requiredArgs from '.'
describe('requiredArgs', function() {
function wrapperFn(required) {
// $ExpectedMistake
return function() {
requiredArgs(required, arguments)
}
}
const twoArgsRequired = wrapperFn(2)
context('with correct number of passed arguments', function() {
it('does not throw an error', function() {
assert.doesNotThrow(() => twoArgsRequired(1, 2))
})
})
context('with wrong number of arguments', function() {
it('throws correct error message', function() {
assert.throws(
function() {
twoArgsRequired(1)
},
function(err) {
return (
err instanceof TypeError &&
err.message === '2 arguments required, but only 1 present'
)
}
)
})
})
})
| 2023-08-30T01:27:17.760571 | https://example.com/article/3189 |
*n - n + 3*n)*(2*n**3 - 2*n + 2*n).
24*n**4 + 13*n**3
Expand (-69*c + 8 + 34*c + 30*c)*(-11 - 8 - 15).
170*c - 272
Expand (-131 - 24 + 65)*(-58*z - 20 + 107*z - 56*z).
630*z + 1800
Expand (-5*a**2 + 3*a**2 + 3*a**2)*(-3*a**3 + 4*a**3 - 4*a**3) - 9698*a**3 + 9698*a**3 + 1013*a**5.
1010*a**5
Expand (259 + 5*k - 259)*(-204*k**2 - 274*k**2 + 417*k**2).
-305*k**3
Expand 2*g - 4*g + 3*g + (-4 - 5 + 0)*(-7432 - 128*g + 7432).
1153*g
Expand (4*b - 4*b + 2*b)*(-2*b + 2*b + 3*b**2) + (-4*b**2 + 4*b**2 - 2*b**2)*(14*b - 12*b + 4 + 5*b) + 15*b**3 + 18*b**3 - 10*b**3 + b**3 - 3*b**3 + 0*b**3.
13*b**3 - 8*b**2
Expand (13*v + 21*v - 25*v)*((0 - 2 + 0)*(4*v - 3*v - 2*v) + 0*v - v - 3*v)*(2222 - 2222 + 14*v).
-252*v**3
Expand (z - 4*z + 0*z)*((z - 2*z - z)*(-4 + 5 - 187) + 0*z - 3*z + z).
-1110*z**2
Expand (-61*c + 61*c - 108*c**2)*(2*c - 3*c + 8*c)*(0*c - 2*c - 2*c).
3024*c**4
Expand (-2*j + j - 8*j)*((-j - 2*j + 2*j)*(-2*j - 3*j + 4*j) + 5 + j**2 - 5 - 6*j - 17*j**2 + 0*j**2 - 9*j**2).
216*j**3 + 54*j**2
Expand (2 - 2 + 2)*((-a + 2*a - 3*a)*(0 - 2 + 3) - 7944 - 4985*a + 7944).
-9974*a
Expand (l + 0*l + l)*(1 - 2 + 3)*(-38*l + 18*l + 76*l).
224*l**2
Expand -b**4 + 5*b**4 - 5*b**4 + (93 - 50 - 20)*(-2*b + 2*b - 2*b**4) + (9*b**2 - 9*b**2 + 3*b**4)*(-1 + 5 - 3).
-44*b**4
Expand (4*d**2 + d**2 - 2*d**2)*(-3*d + 0*d + d)*(d + 7 - 28 - 2*d).
6*d**4 + 126*d**3
Expand -2*d**3 - d**3 + d**4 - 3*d**4 + (d - d - d)*(263*d**3 - 588*d**3 + 94*d**3).
229*d**4 - 3*d**3
Expand l + 2*l**3 - l + (0 + 0 - 2)*(-l**3 + 4 - 4) + 4*l - 443*l**3 - 2*l + 416*l**3.
-23*l**3 + 2*l
Expand (-2*h**2 + 72*h - 72*h)*(-73 + 73 - 22*h)*(0 + h + 0)*(2*h - 2 + 0 - 2).
88*h**5 - 176*h**4
Expand (o**2 - o**2 - 6*o**3)*(54*o - 123*o - 134*o) + o - o - 3*o**4 + 2*o**4 - o**4 + 0*o**4 + (o**2 - 6*o**2 + 3*o**2)*(-3*o**2 + 0*o**2 + o**2).
1220*o**4
Expand (-15 - 2 - 1)*(1816*x**2 - 1816*x**2 + 8*x**3).
-144*x**3
Expand (3 - q - 3)*(0*q**2 + 0*q**2 - 2*q**2) + 96*q**3 - 15*q**3 + 46*q**3 + (-q + 4*q - 2*q)*(-q**2 + 2*q**2 + q**2).
131*q**3
Expand (t + 2 - 3*t + t)*(-2*t + 2*t + t + (0 + 4 - 2)*(-3*t + 3*t + 2*t))*(252 - 194*t + 388*t - 195*t).
5*t**3 - 1270*t**2 + 2520*t
Expand 1241*q + 0*q**5 - q**5 - 1389*q - 3*q**5 + q**5 + 0*q**5 + (-2*q - q + q)*(2*q**4 - q**3 + q**3) + 0*q**5 - q**5 + 18*q**5.
10*q**5 - 148*q
Expand (2393*o - 1021*o + 16*o + 423*o + 2702*o + 2256*o)*(0*o**3 - 4*o**3 + 3*o**3).
-6769*o**4
Expand ((2 + 1 - 5)*(31*f**2 + 44*f**2 + f**2) - f**2 + 2*f**2 + 0*f**2 + 49*f + 14*f**2 - 49*f + 3 - 3 + f**2)*(f + 1 + f**3 - 1).
-136*f**5 - 136*f**3
Expand a**5 - a**5 - a**5 + (2*a**4 + 3*a**4 - 4*a**4)*(1 - 1 - a) - 1 - 1 - 1103*a**5 + 11 - 1688*a**5.
-2793*a**5 + 9
Expand (4 - 4 + 3*w)*(0*w + w + w)*(-894 - 2572 + 483).
-17898*w**2
Expand 1937*k**3 - 330*k**3 + 638*k**3 + (2*k**2 - 3*k**2 + 3*k**2)*(k - 2*k + 3*k) + k**3 + 1 - 1.
2250*k**3
Expand (0*h**2 + 0*h**2 + h**2)*(1 - 2 + 2) + 27 + 25*h**2 - 28 - 3*h - 114*h**2.
-88*h**2 - 3*h - 1
Expand (0 + 5 + 4)*(-4*z + 4*z + 3*z)*((39*z**2 + 37*z**2 - 28*z**2)*(-2*z + 3*z + z) + 3 - 3 + 2*z**3).
2646*z**4
Expand (-h**3 + 6*h**3 - 2*h**3)*(-10788 - 804*h**2 - 62*h**2 + 5396 + 5392).
-2598*h**5
Expand (-3 + 1 + 7)*(-4*z + 4*z - 2*z + (1 + 1 + 0)*(3531 + 710*z - 3531)).
7090*z
Expand 3*f**4 - 2*f**2 + 2*f**2 + (-1 + 2 + 1)*(2*f**4 - 2 + 2) + (-54*f**2 + 15*f**2 - 37*f**2)*(2*f**2 + f**2 - 5*f**2).
159*f**4
Expand (-1 - 2 + 4)*(-54*w + 125*w - 73*w - 4*w**3 - 1).
-4*w**3 - 2*w - 1
Expand (-6*r + 2*r - r)*(2*r**2 - r + r + (0*r + 11*r + 4*r)*(-r + 0 + 0 + 2 - r - 2 + (-5 + 1 + 3)*(-1 + 1 + r))).
215*r**3
Expand (-15 - 2*p + 8 - 9)*(3 - 3 - 3*p)*(19*p - 6*p + 11*p)*(p - p + 2*p).
288*p**4 + 2304*p**3
Expand (1 + 13*c - 10*c - 7*c)*(248 - 189 - c - 153).
4*c**2 + 375*c - 94
Expand (-v**2 - 5*v**2 + 2*v**2)*(4*v - 3*v - 2*v) - 1235*v**2 + 1235*v**2 + 65*v**3.
69*v**3
Expand (-1 - 3*f + 1)*(-1 + 0 - 4) + (-306 + 405 + 227)*(-2*f + 2*f - 2*f).
-637*f
Expand (-d**2 + 8*d**2 + 4*d**2)*(364*d**2 + 1091*d - 1091*d) + 3 - 3 - 2*d**4.
4002*d**4
Expand (21*k**2 - 184*k**2 + 850*k**2)*(-2 - 4 - 5 + 2).
-6183*k**2
Expand (4 - 3 - b + 0*b)*(126*b + 47*b + 33*b).
-206*b**2 + 206*b
Expand (-2*y**4 + 5*y**4 - 4*y**4)*(3 - 3 - 1) - 525 + 22*y**4 + 525 - 5*y**2.
23*y**4 - 5*y**2
Expand -2*n**2 + n**2 + 2*n**2 + (-n + 3 - 3)*(4*n + 2*n - 3*n) + (-2*n**2 + 6*n**2 + 3*n**2)*(3 + 3 - 1) + (-1 + 1 - 3)*(-2*n**2 - 4*n**2 + 5*n**2).
36*n**2
Expand ((-5 + 2 + 1)*(-2 - 2 + 3) - 3 + 5 + 2)*(-12*b + 12*b - 4*b**3 + (b**2 + b**3 - b**2)*(1 + 1 + 0))*(-2 + 2 + 1)*(-9 + 4 - 7).
144*b**3
Expand -2*n**5 - 4*n**5 - n**5 + (-2*n**3 + 4 - 4 + (5 - 5 - 7*n)*(n + 2*n**2 - n))*(2*n**2 - 5 + 5).
-39*n**5
Expand (-2*a - 2*a + 2*a + (0 + 0 + 2*a)*(0 + 0 + 2))*(854*a - 310 - 1716*a + 860*a).
-4*a**2 - 620*a
Expand -2*y**5 + 0*y**5 + y**5 + (-16468 + 16468 + 205*y**5)*(-1 - 3 + 1).
-616*y**5
Expand (-3 + 2 + 3)*(-4*b**2 + 3*b**2 - 2*b**2) + (198*b + 199*b - 489*b)*(-4*b + 3*b + 2*b).
-98*b**2
Expand (-555*h**2 - 420*h**2 + 112*h**2)*(2*h + h + 2 - 4*h).
863*h**3 - 1726*h**2
Expand (-15*o + 33*o + 7 - 16*o)*(5*o - 2 + 2) + 0*o + 0*o - 2*o**2 + (-3*o + 2*o + 2*o)*(-12 - 3*o + 12) + 9 + o**2 - 9.
6*o**2 + 35*o
Expand y**2 + 5*y**2 + 21*y**2 + (2*y**2 + 4*y**2 - 5*y**2)*(3 - 6 + 1) + 0*y**2 - 3*y**2 + 4*y**2 - 8*y**2 + 11*y**2 - 19*y**2 + 2*y - 2*y - 2*y**2.
8*y**2
Expand (1 - a**2 - 1)*(-13 + 39 - 13 - 13 - 6515*a + 104334*a).
-97819*a**3
Expand (6 - 6 + 11*k + (0 + 2 - 4)*(k - 13*k + 10*k) + (-k + 0*k + 3*k)*(6 - 1 - 3))*(-2 - 2 + 5).
19*k
Expand (1 - 2 + 0)*(-328 - 576 + 173)*(0*a - 3*a + a)*(-1 - 3*a**2 + 1).
4386*a**3
Expand (-3 + 3 - 1 + (-3 + 6 + 2)*(-1 + 0 + 0))*(-2 + 2*r + 2).
-12*r
Expand 631*f**2 - 644 + 644 + (-5 + 1 + 3)*(-5*f**2 + 4*f**2 + 3*f**2).
629*f**2
Expand (213343 + s**2 - 106833 - 5*s - 106696)*(0*s + s + s).
2*s**3 - 10*s**2 - 372*s
Expand (974 - 974 + 31*k**4)*((-1 - 1 + 0)*(-3 + k + 3) + 3*k + 4 - 4) + 8*k**5 - 2*k**5 - 5*k**5.
32*k**5
Expand (-3 + 3 - 5*m)*(23 + 25 - 97)*(515 - 515 + 26*m).
6370*m**2
Expand (-19 - 31 - 8)*(-2 + 4 - 4)*(-3 + i + 3).
116*i
Expand (1 + 0 + 2 + 2 - 4 + 1 + (-4 + 3 - 4)*(0 - 2 + 0))*(173*i - 449*i - 829*i).
-13260*i
Expand -2*s + 5*s + 3*s + 2*s + 0*s - 3*s + (-2*s - s + 2*s)*(0 + 3 - 5) + 0 + 0 + s + 5*s - 69 + 69 + 70*s - 2403 + 2403.
83*s
Expand -1 + 1 + 227*p**2 + 1709*p**2 + (-3 + 2 + 2)*(p**2 + 8*p**2 - 3*p**2).
1942*p**2
Expand (28*w + 26*w + 2*w)*(-2*w + 5 - w + w + w - 5*w + 2*w + (-2*w - w + 2*w)*(-1 + 2 + 0)).
-280*w**2 + 280*w
Expand -6*x**4 + x**4 + 2*x**4 + (-439*x**3 + 13*x**3 + 71*x**3)*(0 - x + 0) - 2*x + 0*x - x**4 + 3*x.
351*x**4 + x
Expand (1 - 1 - 5*o)*(3 - 4 + 0) + 13242*o - 6666*o - 6991*o + 1.
-410*o + 1
Expand (3*b - 2*b - 2*b)*(2*b - b - 3*b) + (-4*b - b + 2*b)*(110*b - 12 - 53*b - 60*b).
11*b**2 + 36*b
Expand (0 - 2 + 4 + 3 - 3 - 1 + (0 + 0 - 2)*(0 - 2 + 0) + 0 - 1 + 3)*(0*n - n + 3*n)*(0*n - 2*n + 4*n) + (2*n - 3*n + 5*n)*(7282 - 7282 - 165*n).
-632*n**2
Expand l**4 + 0*l**4 - 4*l**4 + (-4 - 2*l**2 + 4)*(0*l + l**2 + 0*l) - l**4 + 3*l**4 - 4*l**4 - 3279*l**3 + 3279*l**3 + 759*l**4.
752*l**4
Expand 2 - 1 + 1 + 2*w**3 + (3*w**2 + 2*w**2 - 2*w**2)*(10*w + 23*w - 9*w).
74*w**3 + 2
Expand -19*m**3 - 27*m**3 + 15*m**3 + 2*m**3 - 4*m**3 + m**3 + 4*m**3 + m**3 - 4*m**3 + (-2*m**2 + m**2 - m**2)*(3*m - 6*m + 2*m) + 4*m**3 + 5*m**3 + 17*m**3.
-3*m**3
Expand (2233 - 62*m**4 - 2233)*(10 - 5 + 7)*(0 + 1 + 0) + (m - m + 2*m)*(0*m**3 + m**3 - 2*m**3).
-746*m**4
Expand (58*y**2 + 129*y**2 + 77*y**2)*(4 - 4 - 1)*(-28*y + y - 23*y).
13200*y**3
Expand (6*u + 2*u + 12*u + (2*u - 2*u + u)*(0 - 1 + 0) - 4*u + 3*u + 0*u)*(7*u + 19*u - 5*u)*(u - 2*u - u).
-756*u**3
Expand -x**4 + 0 - 2*x**2 - 2 + 0*x**2 + (16*x**2 + 27*x**2 + 8*x**2)*(30 - 12*x**2 - 30).
-613*x**4 - 2*x**2 - 2
Expand (o + o - 3*o)*(320 + 116 - 119 + 2*o).
-2*o**2 - 317*o
Expand (-2*z + 12 - 12)*(-3 - 20 - 2)*(0*z - 3*z + z) - z**2 + 1 - 1.
-101*z**2
Expand -31*x**3 + 17*x**3 + 16*x**3 + (x + 1 - 1)*(0*x + 0*x + 3*x**2) - x**3 - 9*x**3 + 3*x**3 + 3*x**2.
-2*x**3 + 3*x**2
Expand (-85 + 53 + 46)*(-2*h**3 - 5*h**3 + 3*h**3) + 2*h**3 + h**2 - h**2 + (0 - h + 0)*(-2*h**2 + 4*h - 4*h).
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Expand -4 - 5*u + 0 + 4*u + (-210*u + 61*u + 98*u)*(0 - 3 + 27) + 3 - u - 3.
-1 | 2023-12-16T01:27:17.760571 | https://example.com/article/3656 |
Russian billionaire Mikhail Prokhorov has now decided he wants to own ALL of the Brooklyn Nets
Lucinda Shen
Jul. 16, 2015, 4:42 PM
Presidential candidate Mikhail Prokhorov (right) talks with Russian Prime Minister Vladimir Putin during their meeting in Moscow on March 5, 2012. Alexsey Druginyn/RIA Novosti/Pool/Reuters Earlier this year, Brooklyn Nets majority shareholder Mikhail Prokhorov was looking to sell his share of the team and their stadium, but now it looks like the Russian oligarch has changed his mind.
The word is that the 10th wealthiest man in Russia might want to settle a debt of an undisclosed amount that Bruce Ratner, the team's minority shareholder, owes him — though no one can be for sure.
The team, which is currently juggling debt due to a bad television deal and high payroll for players, is worth about $1.5 billion, reported Forbes.
The sense within the industry is that Prokhorov wants to buy the team to sell it at a higher profit later, the Daily News reported.
"We can confirm that Onexim Sports and Entertainment is looking at various ways to restructure debt owed to the company in connection with its US assets," said a spokesperson for Prokhorov and his holding company, ONEXIM, over email to the Daily News. "One possibility is the conversion of such debt into equity and consolidation of shares, but there are other scenarios also under consideration by Onexim and its partners, and no decision has yet been made."
In other words, it's a proposed debt-to-equity swap.
Prokhorov owns 80 percent of the team, and 45 percent of the stadium.
At the time of purchase, Prokhorov agreed to pay 80 percent of the team's $220 million debt, ESPN reported. Russia's own tenuous economic situation may also be a factor in Prokhorov's decision. | 2023-11-27T01:27:17.760571 | https://example.com/article/8522 |
799 P.2d 364 (1990)
BENNETT COLLEGE, Bennett College Foundation, Inc., and Pace University, Petitioners,
v.
UNITED BANK OF DENVER, NATIONAL ASSOCIATION, Charles W. Ennis, St. John's Episcopal Cathedral, Dumb Friends League Animal Shelter, Metropolitan Opera Guild, Inc., and Marist College, Respondents.
No. 89SC297.
Supreme Court of Colorado, En Banc.
September 10, 1990.
Rehearing Denied November 13, 1990.
Ireland, Stapleton, Pryor & Pascoe, P.C., Monte Pascoe, Margaret L. Toal-Rossi, Jennifer A. Ostom, Denver, for petitioners.
Davis, Graham & Stubbs, Richard W. Daily, Susan K. Morath, Denver, for respondents St. John's Episcopal Cathedral, Dumb Friends League Animal Shelter, and Metropolitan Opera Guild, Inc.
No Appearance, on behalf of respondents United Bank of Denver, Nat. Ass'n., Charles W. Ennis, and Marist College.
Justice VOLLACK delivered the Opinion of the Court.
Bennett College, Bennett College Foundation, Inc., and Pace University (the Colleges) *365 petitioned for certiorari review of the court of appeals decision in United Bank of Denver v. St. John's Episcopal Cathedral, Nos. 87CA1050 & 87CA1051 (Colo.App. March 23, 1989) (unpublished opinion). In United Bank of Denver, the court of appeals affirmed the Denver probate court's summary judgment entered against the Colleges. We granted certiorari to consider whether the court of appeals properly applied the doctrine of collateral estoppel.[1] We affirm.
I.
A.
In 1970, Margaret Collbran, an alumna of Bennett College, established a trust, with the United Bank of Denver as the trustee (the Collbran Trustee), providing that upon her death the remainder of the trust would be "divided into equal parts and distributed outright to each of the following organizations" in existence at the time of her death: St. John's Episcopal Cathedral, Dumb Friends League Animal Shelter, Metropolitan Opera Guild, Inc., and Bennett College.
In May 1977, Bennett College, an all-women's college, filed for bankruptcy in federal district court, which adjudicated Bennett College bankrupt in October 1977. By March 1978, the bulk of Bennett College's assets had been liquidated and disbursed to creditors. The bankruptcy estate was closed in 1984.
Following Collbran's death on February 22, 1986, the Collbran Trustee petitioned the Denver Probate Court for instructions on whether or how it should distribute Bennett College's interest in the trust corpus. In the probate proceedings, St. John's Episcopal Cathedral, Dumb Friends League Animal Shelter, and Metropolitan Opera Guild (the Charities) argued that Bennett College's interest in the trust corpus should be distributed equally among them because Bennett College is no longer in existence.[2] Bennett College Foundation, Inc., (Bennett Foundation), and Bennett College, which has not received students since 1977 although the state of New York has not divested the college of its school charter, and Pace University[3] argued that Bennett College was still in existence at the time of Collbran's death.
The Charities subsequently filed two motions for summary judgment against the Colleges, arguing that they were collaterally estopped and judicially estopped from asserting that Bennett College was still in existence at Collbran's death. The motions for summary judgment were based on previous New York court cases following the adjudication of Bennett College's bankruptcy in which the college's entitlement to funds unrelated to the Collbran trust was questioned. The probate court granted the Charities' motions for summary judgment, and the court of appeals affirmed.
B.
Because of the limited nature of our review in this case, see note 1, supra at 365, we consider only the New York case on which the probate court and court of appeals relied in holding that the Colleges were collaterally estopped from asserting that Bennett College was still in existence at the time of Collbran's death.
In 1937, Margaret Gage, a New York resident and alumna of Bennett Junior College, *366 established a trust fund and named Bennett College as its beneficiary.[4] The trust provided that another charitable beneficiary could be named "[i]n the event that [Bennett College] cease[s] to exist, whether by reason of dissolution, merger, consolidation or otherwise." Bankers Trust Company (the Gage Trustee) eventually was named the trustee. The Gage Trustee stopped disbursing trust moneys to Bennett College in September 1977, after the college had filed for bankruptcy in May 1977.
In May 1979, the Gage Trustee initiated a proceeding in the Supreme Court of New York for, inter alia, a ruling that Bennett College had ceased to exist by virtue of the college's bankruptcy and the subsequent sale of its assets, and that the Gage Trustee was no longer obligated to disburse trust moneys to Bennett College. Notice of the proceeding was served on Bennett College through the college's last president, J.W. Nystrom, the bankruptcy trustee for Bennett College (the Bankruptcy Trustee), and the New York Attorney General.[5]
In July 1979, the Gage Trustee, the Bankruptcy Trustee, and apparently the New York Attorney General entered into a stipulation by which the Gage Trustee agreed to pay $3,668.22 to the Bankruptcy Trustee.[6] The amount represented the net moneys that were payable to Bennett College but had not been paid because of the Gage Trustee's concern over whether the college continued to exist as required by the Gage trust. Also in July 1979, the Supreme Court of New York issued a memorandum decision finding that Bennett College had "ceased to exist" and permitting the Gage Trustee to substitute other beneficiaries to the Gage trust in accordance with the trust's terms.
In September 1979, the Supreme Court of New York approved the July 1979 stipulation, and, after referring to its July 1979 memorandum decision, "ORDERED, ADJUDGED AND DECREED that Bennett College ... has for the purposes of [the Gage] Trust ceased to exist."
II.
The Colleges argue that the court of appeals and probate court erroneously applied the doctrine of collateral estoppel in this case. We disagree.
Collateral estoppel, or issue preclusion, bars relitigation of an issue that was determined at a prior proceeding if: (1) the issue precluded is identical to an issue actually determined in the prior proceeding; (2) the party against whom estoppel is sought was a party to or was in privity with a party to a prior proceeding; (3) there was a final judgment on the merits in the prior proceeding; and (4) the party against whom the doctrine is asserted had a full and fair opportunity to litigate the issue in the prior proceeding. E.g., City of Colorado Springs v. Industrial Comm'n, 749 P.2d 412, 414 (Colo.1988); Industrial Comm'n v. Moffat County School Dist. RE No. 1, 732 P.2d 616, 619-20 (Colo.1987); United States v. Jesse, 744 P.2d 491, 503-04 (Colo.1987). The issue on which preclusion is asserted must have been actually litigated and necessarily adjudicated in the prior proceeding. Jesse, 744 P.2d at 503.
A.
We first consider whether the issue of Bennett College's existence that was decided in the New York proceeding is identical to the issue before the Denver Probate Court.
*367 The Gage trust provided that another charitable beneficiary could be named "[i]n the event that [Bennett College] cease[s] to exist, whether by reason of dissolution, merger, consolidation or otherwise." In its "Petition for Voluntary Accounting and Construction of Deed of Trust," the Gage Trustee specifically requested that the New York Supreme Court determine "for purposes of the administration of said trust [that] Bennett College ... ceased to exist on or about August 8, 1977."
The Collbran trust provided that upon Collbran's death the trust would be "divided into equal parts and distributed outright to the [named] organizations that are in existence" at the time of her death. In its "Petition for Order Ascertaining Beneficiaries of Trust," the Collbran Trustee stated that the Charities had requested distribution of the trust corpus to the Charities only, because Bennett College was not in existence at the time of Collbran's death. The Collbran Trustee also stated in the petition that it was "uncertain" whether it was required to distribute a one-fourth share to Bennett College because, inter alia, the Gage Trustee had learned that Bennett College had been adjudicated a bankrupt, that the college had not conducted classes or functioned as an academic institution since fall 1977, and that in the Gage trust court proceedings the New York Supreme Court had found that, for purposes of the Gage Trust, the college had ceased to exist. The Collbran Trustee requested through the petition that the Denver Probate Court determine the beneficiaries of the trust and instruct the Collbran Trustee on how to distribute the trust assets to the beneficiaries.
The Colleges argue that the issues in the two court proceedings are different because "[e]ach trust is unique, which requires that each be examined independently." Specifically, the Colleges argue that the issue in the Gage trust court proceeding must have involved a different issue because the document creating the Gage trust defined cessation of existence as "dissolution, merger, consolidation or otherwise," and the document governing the Collbran trust does not define "existence." We disagree.
A comparison of the facts in the Gage trust court proceedings and the court proceedings in this case demonstrates that the issue before the New York Supreme Court was identical to the issue before the Denver Probate Court: whether or not Bennett College was in existence. The Gage trust deed's definition of "exist" did not cause the issue in the New York court proceedings to be different from the issue before the Denver Probate Court. The deed's definition of "exist""dissolution, merger, consolidation or otherwise"is too broad to differentiate the Gage trust deed's use of "exist" from the Collbran trust instrument's use of "existence." Cf. Webster's Third International Dictionary 796 (1986) ("exist" means "to have actual or real being" or to "have being in space and time").
B.
The Charities argue, and the probate court and court of appeals held, that the Colleges were in privity with Bennett College's bankruptcy trustee during the 1977-1978 bankruptcy proceedings of the college. We agree.
For purposes of collateral estoppel, a privy is one who, after rendition of judgment, has acquired an interest in the subject matter affected by the judgment through or under one of the parties, as by successor, inheritance, or purchase. See Murphy v. Northern Colorado Grain Co., 30 Colo.App. 21, 24, 488 P.2d 103, 104 (1971) (citing Bernhard v. Bank of America Nat'l Trust & Sav. Ass'n, 19 Cal.2d 807, 810, 122 P.2d 892, 894 (1942)), cited with approval in Pomeroy v. Waitkus, 183 Colo. 344, 350, 517 P.2d 396, 399 (1973).
The bankruptcy trustee is considered to be the successor in interest to the debtor and the debtor's property during the bankruptcy proceeding, 1B J. Moore, J. Lucas & T. Currier, Moore's Federal Practice ¶ 419[3.-1], at 634 (2d ed.1988) [hereinafter 1B Moore's Federal Practice], to collect, liquidate, and ultimately distribute the trustee's estate to the creditors. Bankruptcy *368 Act of 1898, ch. 541, 30 Stat. 544, 557 [hereinafter Bankruptcy Act] (1978 repeal effective 1979), 1979 version reprinted in 11 U.S.C.A.App. at 141, 180-81 (West 1979) (current version at 11 U.S.C. §§ 101 to 1330 (1979 and Supp.1990)).[7] Although under the Bankruptcy Act a trustee is considered a representative of the creditors, the trustee for many purposes is deemed to be in privity with the debtor and stands in the debtor's shoes. 2 D. Cowans, Bankruptcy Law and Practice § 604 (2d ed. 1978); accord In re Ahead By A Length, Inc., 100 B.R. 157, 173 (Bankr.S.D.N.Y. 1989); see also Heiser v. Woodruff, 327 U.S. 726, 734, 66 S.Ct. 853, 857, 90 L.Ed. 970 (1946) (holding that issue of fraud litigated between creditor and bankrupt, and litigated between creditor and bankruptcy trustee, "who represents the bankrupt and [the bankrupt's] creditors," is res judicata and cannot be further litigated in the bankruptcy proceeding); 4 Collier on Bankruptcy ¶ 704.02 n. 2 (L. King 15th ed. 1979) ("Rights of action arising upon the contracts or property of the debtor, not yet resolved into suit, pass to the trustee, and [the trustee] should assert them in the proper tribunal whenever necessary for the collection or preservation of the estate.") (citing In re Bowling Green Milling Co., 36 F.Supp. 785, 786 (W.D.Ky.1941)).[8]
For purposes of the application of collateral estoppel on the issue of whether Bennett College was in existence at the time of Collbran's death, we conclude that the Bankruptcy Trustee was a privy to Bennett College.
C.
The Colleges argue that the third requirement for the application of collateral estoppelfinal judgment on the merits in the prior proceedingcannot be satisfied in this case, because the issue of Bennett College's existence was not actually and necessarily decided by the New York Supreme Court in the Gage trust court proceedings because the parties reached a settlement.
Under certain circumstances a stipulation between parties to central questions of fact and law or a stipulation to questions of fact may render a judgment not "final" as to those questions, and in a subsequent proceeding collateral estoppel should not prevent the litigation of those questions unless the parties in the original lawsuit intended the questions to be settled as to future actions. See Restatement (Second) Judgments § 27 comment e (1980); 1B Moore's Federal Practice ¶ 0.444[4].
In this case, the "stipulated fact" doctrine is inapplicable. Because the trustee for the Gage trust placed in issuethrough its petition for accounting and construction of deed of trustBennett College's existence, and because the issue of the college's existence was crucial to the trustee's decision on how it would disburse the trust corpus, the New York Supreme Court could not avoid entering an order deciding the issue.
The question, then, is whether the parties to the Gage trust proceedings reached a stipulation of fact on the question of Bennett College's existence. We conclude that the parties did not reach such a stipulation. The New York Supreme Court order indicates that the parties to the Gage trust proceedings stipulated only to the amount owed and payable to Bennett College during the period from September 6, 1977, to October 14, 1977.[9] Additional evidence *369 indicating that the parties had not reached a stipulation on the question of Bennett College's existence is that the parties' July 13, 1979, stipulation was preceded by the New York Supreme Court's July 2, 1979, "Memorandum Decision," which provided in relevant part:
Upon the foregoing papers there being no objection, this application to settle trustee's account and for a construction granting petitioner permission to substitute as beneficiary in place and stead of Bennett College, which has ceased to exist, ... one or more ... organizations... is granted.
(Emphasis supplied). The New York court's "Final Judgment on Intermediate Accounting" incorporated the Memorandum Decision, and "ORDERED, ADJUDGED AND DECREED that Bennett College ... has for the purposes of said Trust ceased to exist."
We hold that the 1979 stipulation does not prevent the application of collateral estoppel to the issue of Bennett College's existence.
D.
The Colleges' final argument is that they were deprived of a full and fair opportunity to litigate Bennett College's existence in the Gage trust proceeding.[10] We disagree.
As we have stated, the Bankruptcy Trustee was in privity with Bennett College, and the Bankruptcy Trustee is under a fiduciary duty to preserve the debtor's estate. See 2 D. Cowans, supra p. 11, § 604; see also 4 Collier on Bankruptcy, supra p. 11, ¶ 704.02 n. 2. Thus the issue we must resolve is whether the Bankruptcy Trustee was given a full and fair opportunity to litigate the issue of Bennett College's existence in the Gage trust proceeding.
Factors determinative of whether a party has been given full and fair opportunity to litigate include whether the remedies and procedures in the first proceeding, or the proceeding itself, is substantially different from the proceeding in which collateral estoppel is asserted, see Salida School Dist. R-32-J v. Morrison, 732 P.2d 1160, 1164 (Colo.1987); whether the party in privity in the first proceeding has sufficient incentive to vigorously assert or defend the position of the party against which collateral estoppel is asserted, see id.; and the extent to which the issues are identical, 18 C. Wright, A. Miller & E. Cooper, Federal Practice and Procedure § 4423, at 221 (1981). See generally Wright, Miller & Cooper, supra, § 4423.
Applying these factors, we can find nothing in the record suggesting that the Colleges were denied a full and fair opportunity to litigate through the Bankruptcy Trustee. The New York Supreme Court proceedings were substantially similar, and the Colleges have not argued that the New York trial procedures are substantially different from Colorado's trial procedures. With more than $465,000 at stake in the trust corpus, we conclude that the Bankruptcy Trustee, who was obligated to preserve Bennett's College's assets, had adequate incentives to assert in the New York court the existence of Bennett College. Finally, the issue in the Gage trust proceedings and the issue in the Collbran trust proceedings were identical.
III.
We hold that the probate court and court of appeals properly applied the doctrine of collateral estoppel on the issue of Bennett College's existence.
Judgment affirmed.
Justice LOHR dissents, and Justice KIRSHBAUM joins in the dissent.
Justice LOHR dissenting:
This case involves the distribution of funds pursuant to a trust established by *370 Margaret Collbran in 1970. Under the terms of the trust, upon Collbran's death the remainder was to be divided equally and distributed to "the following organizations that are in existence at the time of [Collbran's] death": St. John's Episcopal Cathedral, Dumb Friends League Animal Shelter, Metropolitan Opera Guild, Inc., and Bennett College, Collbran's alma mater.
On October 14, 1977, Bennett College was adjudicated bankrupt. During the several months that followed, most of Bennett College's assets were liquidated and the proceeds distributed to its creditors. Pace University subsequently assumed many of Bennett College's administrative obligations, including administering student loans and scholarship funds, maintaining Bennett College records and raising funds for the Bennett College Foundation. Pace accepted all of the Bennett College students and hired many of the faculty and staff formerly employed by Bennett. Pace also allowed Bennett's alumnae office to operate on its grounds. The Bennett College charter was never relinquished or revoked, and in 1986 Bennett College and Pace University were formally consolidated by the New York State Board of Regents.
Collbran died on February 22, 1986. The trustee petitioned the Denver Probate Court for instructions regarding Bennett College's interest in the trust. The share at issue is valued at more than $7,000,000. The other three trust beneficiaries contended that Bennett College was no longer in existence. The three beneficiaries filed two motions for summary judgment seeking dismissal of the claim of Bennett College, Bennett College Foundation, Inc. and Pace University (referred to collectively as Bennett) to be beneficiaries of the Collbran trust. The moving parties contended that Bennett was both collaterally and judicially estopped from asserting that Bennett College was in existence at the time of Collbran's death. The Denver Probate Court granted the motions for summary judgment and the Colorado Court of Appeals affirmed. The majority affirms the judgment of the court of appeals on the ground of collateral estoppel. I do not agree that collateral estoppel is applicable in this case.
The court of appeals held that Bennett was collaterally estopped from litigating the issue of Bennett College's existence by the Gage Trust litigation, which occurred before a New York Supreme Court, a trial court. Margaret Gage created a trust with Bennett College named as the beneficiary. The Gage Trust provided in pertinent part:
In the event that the beneficiary hereinabove named shall at any time cease to exist, whether by reason of dissolution, merger, consolidation or otherwise, I reserve to myself during my lifetime, and to my trustees after my decease, the right and privilege of substituting as beneficiary in the place and stead of the beneficiary hereinabove named, one or more religious, charitable, eleemosynary, educational or benevolent organizations or institutions wholly charitable in nature and purpose and not operated for profit.
In May 1979, the trustee of the Gage Trust filed an action in a Supreme Court of New York, seeking a ruling that Bennett College had ceased to exist, that the trustee need not continue disbursing trust funds to Bennett College and that the trustee could substitute a new beneficiary for Bennett College. The trial court issued an order to show cause why Bennett College had not ceased to exist. On July 2, 1979, the trial court issued a memorandum opinion granting the Gage trustee's application to construe the trust to permit the trustee to substitute a new beneficiary for Bennett College, "which has ceased to exist." On July 13, 1979, the trustee and the Bennett College bankruptcy trustee entered into a stipulation in which they agreed that Bennett College's claim against the Gage Trust would be fully satisfied by a payment of $3,668.22, the amount of income accrued up to the date on which Bennett College was adjudicated bankrupt. On September 7, 1979, the court entered its final judgment, which repeated the rulings found in the memorandum decision and referred to the parties' stipulation.
In order for the litigation of any issue to be barred by collateral estoppel, four conditions must be met: (1) the issue for which litigation is precluded must be identical to an issue actually determined in a prior proceeding; *371 (2) the party against whom preclusion is sought must have been a party to, or in privity with a party to, the prior proceeding; (3) there must have been a final judgment on the merits in the prior proceeding; and (4) the party against whom preclusion is sought must have had a full and fair opportunity to litigate the issue in the prior proceeding. City of Colorado Springs v. Industrial Commission, 749 P.2d 412, 414 (Colo.1988).
The first requirement, identity of issue, is not present in this case. In order for collateral estoppel to apply, "the same identical matter must have been in issue in the former suit, and the precise fact determined by the former judgment." Hickey v. Anheuser-Busch Brewing Assoc., 36 Colo. 386, 389, 85 P. 838, 838 (1906).
The Gage Trust specified that the beneficiary, Bennett College, could "cease to exist" by such means as "dissolution, merger, consolidation or otherwise." Under this expansive definition, the New York court found that after the sale of Bennett College's assets and its subsequent consolidation with Pace University, Bennett College had ceased to exist.
The Collbran Trust, however, contains no definition of the term "existence." It merely states that in order to benefit from the trust, Bennett College must have been "in existence" at the time of Collbran's death. The meaning of the term "existence" in this context is not self-evident but is ambiguous. Cf. Pepcol Mfg. Co. v. Denver Union Corp., 687 P.2d 1310, 1314 (Colo.1984) (whether an ambiguity exists is a question of law). Extrinsic evidence should be admissible, therefore, to determine its meaning. See, e.g., id. After a trial, a court might find that Collbran intended to leave money to Bennett only if it continued to operate as it had when she was a student. On the other hand, a court might determine that Collbran merely wanted to leave a portion of her estate to an educational institution that was closely tied with her alma mater by corporate consolidation. Whatever a trial court might find, it is evident that the resolution of whether Bennett College is in existence for purposes of the Collbran trust is not controlled by the New York court's determination that it was not in existence under the definition controlling in the Gage Trust litigation. See Blue Cross of Western New York v. Bukulmez, 736 P.2d 834, 839 (Colo.1987) ("[s]ummary judgment should not be granted where there appears to be any controversy concerning material facts").
The majority reasons that the Gage Trust's "definition of `exist''dissolution, merger, consolidation or otherwise'is too broad to differentiate the Gage trust deed's use of `exist' from the Collbran trust instrument's use of `existence.'" Maj. op. at 367. This reasoning misconstrues the terms of the Gage Trust. The Gage Trust does not define "exist" broadly. It defines the phrase "cease to exist" broadly and, therefore, by necessary implication defines "exist" very narrowly. The Collbran Trust does not define existence and it remains a disputed issue of material fact whether Collbran intended to employ the narrow concept of existence used in the Gage Trust.
Accordingly, I would reverse the court of appeals' judgment that litigation regarding the issue of Bennett College's existence was barred by collateral estoppel.
Justice KIRSHBAUM joins in this dissent.
NOTES
[1] We also granted certiorari on the question of whether the court of appeals erred in applying the doctrine of judicial estoppel. Because we conclude that the court of appeals did not err in applying the doctrine of collateral estoppel, we need not, and do not, consider whether the court of appeals erred in its application of judicial estoppel. See, e.g., Industrial Comm'n v. Moffat County School Dist. RE No. 1, 732 P.2d 616, 620 (Colo.1987).
[2] Bennett College's one-quarter interest in the trust corpus is worth more than $7 million.
[3] After Bennett College filed for bankruptcy, Pace University, located in New York City, agreed inter alia to assume Bennett College's student loan administration obligations, maintain the college's records, raise funds for the Bennett Foundation, administer a Bennett College scholarship fund, and permit the college's alumnae office to operate on the university's property.
[4] Although Gage named Bennett School and Bennett Junior College, Bennett College's predecessors, as the trust beneficiaries, we refer to the school and junior college as "Bennett College." The parties do not dispute that Bennett College is not in privity with Bennett School and Bennett Junior College.
[5] Under New York law, in certain charitable trust proceedings the attorney general is deemed to be the representative of the state of New York and charitable trust beneficiaries, and notice of such trust proceedings must be served on the attorney general. N.Y.Est. Powers & Trusts Law § 8-1.1(g)-(h) (McKinney 1967); 1971 N.Y. Laws 2641-42.
[6] The trust corpus in 1979 was worth $465,983.37.
[7] The Bennett College bankruptcy proceedings, initiated in May 1977, were governed by the Bankruptcy Act. See Bankruptcy Reform Act of 1978, Pub.L. 95-598, § 403(a), 92 Stat. 2549, 2683 (1978).
[8] To the extent that they are relevant to this case, the cases cited by the Colleges are consistent with our recognition that the Bankruptcy Trustee was in privity with Bennett College, the debtor in bankruptcy, in the Gage trust court proceedings. For example, DePinto v. United States, 407 F.Supp. 5, 7 (D.Ariz.1976), aff'd, 585 F.2d 405 (9th Cir.1978), states that a bankruptcy trustee has a "duty to represent the creditors and to realize the maximum profit on the bankruptcy estate."
[9] The trustee suspended trust payments to Bennett College on September 6, 1977, because of concerns about whether the college continued to exist, as the Gage trust deed required. Bennett College was adjudicated bankrupt on October 14, 1977.
[10] We note that, as the Colleges acknowledge, Bennett College was served with notice that the New York Supreme Court had issued an order to show cause directing Bennett College, and the other parties interested in the Gage trust, to appear before the court to show cause why the court should not make a determination of whether Bennett College had ceased to exist. However, no personal representative from the college appeared at the proceeding to show cause.
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The gist of the NYT story was that Haspel persuaded Trump to take strong action by showing him pictures of poisoned children and dead ducks, tagged as collateral damage in the attack on the Skripals. The gist of my post was that there were no dead ducks or poisoned children, the Brits never claimed that there were, and the real question was who was lying to whom.
" Russiagate and the missing ducks " addressed a New York Times story (April 16) about Trump's reaction to a briefing by CIA director Haspel (then the deputy) about the (alleged) Skripal poisoning in Britain.
The CIA has never responded to charges that its head lied to the president on a matter of great national importance, nor did the NYT, until last Wednesday (June 5). Then, the reporter used a nine-part tweetfest to explain that no one had deceived anyone. Early reports mentioned poisoned ducks and children, and these were shared with the U.S. in the interests of full disclosure, but there were no pictures. The pictures shown the president were from Syria.
Whether the president was shown the early, erroneous reports about ducks and children was left unclear by the carefully drafted tweets, as was any information as to when, if at all, these were corrected.
The correction illustrates the old aphorism that the cover-up can be worse than the crime.
There were in fact no early reports of poisoned ducks and children, at least not public ones. How did such information get into the system, and when was it corrected? Children had been feeding ducks near the Skripals, but none were affected, and the authorities did not even check up on them for two weeks.
The original NYT story was changed to say, "Ms Haspel displayed pictures illustrating the consequence of nerve agent attacks, not images specific to the chemical attacks in Britain." Note that this leaves open the possibility that Haspel said that such collateral damage occurred in Britain and then illustrated it with Syrian pictures. Indeed, given the artfulness of the denials, a cynic might say such a conclusion is compelled.
A Russophile (but always interesting) blog called The Duran takes the cynical view: "NYT covers up CIA Skripal lie with even worse CIA Skripal lie":
So in other words, Ms Haspel couldn't show any pictures from Salisbury to persuade the sceptical Mr Trump, because there weren't any to show. So she showed him pictures from other nerve agent attacks, which were presumably sufficiently bad to turn him from his scepticism, to expelling 60 diplomats. Even though nothing like that happened in Salisbury.
Moon of Alabama also has a serious, and skeptical, discussion of the matter, noting the squishiness of the sourcing:
The original report was sourced on 'people briefed on the conversation'. The corrected version is also based on 'people briefed on the conversation' but adds 'a person familiar with the intelligence'. Do the originally cited 'people' now tell a different story? Are we to trust a single 'person familiar with the intelligence' more than those multiple 'people'? What kind of 'research' did the reporter do to correct what he then and now claims was told to him by 'people'? Why did this 'research' take eight weeks?
The whole Skripal Affair remains a riddle covered in a mystery wrapped in an enigma. And it remains possible that it is connected to the Steele Dossier and the rest of Russiagate.
Once again, unleash the attorney general.
James V DeLong lives in the Shenandoah Valley. | 2024-07-08T01:27:17.760571 | https://example.com/article/3900 |
Q:
Wave behavior of particles
When people say that every moving particle has an associated wave, do they mean that the particles will move up and down physically, for example when we say that a moving electron has a wave associated with it, does the electron physically oscillate? Or is it some other wave, like a probability wave? I really don't understand the latter.
A:
I agree with Pieter, especially when he refers to QED, writing
Feynman explains this (in his little book QED, I recommend to read
that) with electrons having dials that turn around. Or one could
represent phase as color.
It must be remarked however that Feynman was a strong opponent of the
so-called "wave particle duality. Let me quote from p. 15:
I want to emphasize that light comes in this form - particles. It is
very important to know that light behaves as particles, especially for
those of you wo have gone to school, where you were probably told
something about light behaving as waves. I'm telling you the way it
does behave - like particles.
And in note 3 at p. 23:
No reasonable model could explain this fact, so there was a period
for a while in which you had to be clever: You had to know which
experiment you were analyzing in order to tell it light was waves or
particles. This state of confusion was called the "wave-particle
duality" of light, and it was jokingly said that light was waves on
Mondays, Wednesdays, ans Fridays, it was particles on Tuesdays,
Thursday, and Saturdays, and on Sundays, we think about it! It is the
purpose of these lectures to tell you how this puzzle was finally
"resolved".
I entirely agree with Feynman. His purpose was to show how the
wave-like behaviour of particles could be modeled by assigning to each
particle an "amplitude" - a complex number, which Feynman depicts as
an arrow.
In some notes of mine I further elaborated this point. Here is a
translation (original is in italian):
Conviction about wave character of light was based on a series of
experimental facts [...] we only recall by their names:
interference and diffraction. It was known, and it is easy still
today to verify in the laboratory, that all known waves give rise to
interference and diffraction: this holds true for surface waves on a
liquid as well as for sound waves. From here it was concluded: since
waves exhibit interference and diffraction and light too exhibits
interference and diffraction, it follows that light is made of waves.
It is easy to see the on logical grounds such conclusion is
unwarranted: all fishes live in water, dolphins live in water, then
dolphins are fishes? We don't mean that for a century physicists had
fallen into a so trivial logical error, but simply that it was an
induction, not a deduction. It was very plausible, on the ground
of the analogy with wavelike phenomena, to induce that light too were
made of waves. Experimental sciences proceed this way very often, and
are often successful. But it may also happen that ensuing research
shows the induction was unjustified. This is what happened with light.
Our reasoning cannot stop at this point however, because if we
assert light is made of particles (photons) we still have to explain
how it can behave in ways we are used to attribute to waves. At least
we must be prepared to accept that if we are dealing with particles,
they are particles sui generis, very different from the idea of
particle our everyday experience could suggest.
| 2024-07-07T01:27:17.760571 | https://example.com/article/3724 |
Fergie can still buy
SIR Alex Ferguson will have a substantial transfer kitty to dip into this summer. That's the view of a financial expert in the wake of United's decision to issue bonds to ease the club's crippling debt. It will come as comfort to increasingly worried Reds fans. Despite Ferguson's insistence he would have funds to spend big if he wanted, the United support has largely been sceptical.
SIR Alex Ferguson will have a substantial transfer kitty to dip into this summer. That's the view of a financial expert in the wake of United's decision to issue bonds to ease the club's crippling debt.
It will come as comfort to increasingly worried Reds fans.
Despite Ferguson's insistence he would have funds to spend big if he wanted, the United support has largely been sceptical.
Fans were dubious that the champions could still compete in the transfer market with the astonishing £700m debt weighing the club down.
Ferguson has barely touched the £80m world record fee banked from the sale of Cristiano Ronaldo to Real Madrid and insists he wouldn't be doing business this January.
It fuelled fears that the Old Trafford boss could only strengthen a faltering United on the cheap.
And Reds followers have been alarmed and angry since the Glazers saddled the club with the £700m debt when they took over in 2005.
Interest on a colossal £509m loan the American owners took out to buy the club cost the Reds a mammoth £42m in interest in the last financial year.
Cash
Though the club announced profits of £48.2m, had it not been for the cash raked in for Ronaldo, United would have been announcing a loss of £31.8m.
For a club in profit up to the takeover, it is a reality that is hard for many to swallow.
Many have bitten the bullet while United have remained a trophy-winning super power able to pay record transfer fees, as in the past, for the likes of Rio Ferdinand, Wayne Rooney and Dimitar Berbatov.
With the squad looking increasingly like it needs costly rebuilding work, supporters have become scared that United are teetering on the brink.
However, the issuing of the £500m worth of bonds to restructure the debt is predicted to be a lifeline that should ensure the Reds boss won't have his hands tied in the summer.
"I would be very surprised if there wasn't a reasonable pot of money available for Sir Alex Ferguson in the summer, if not a substantial one," Vinay Bedi of financial experts Brewin- Dolphin told M.E.N. Sport. "It might not be the full £80m that is supposed to be available from the Ronaldo sale but it could be a considerable sum.
Spending
"If the manager was thinking of spending around £50m then I believe he could do that and there might be even more available."
The success of the £500m bond issue will determine the size of the pot.
But experts believe the Glazers are likely to be on a winner with the decision to restructure the debt in this way.
"The bond issue is going to be attractive to investors," Bedi added.
"It is a very sensible strategy. Basically, what an investor might do is buy £10m worth of the bonds at, for example, an interest rate of seven per cent.
"Every year for the length of the loan agreement, which maybe seven years, they will get seven per cent interest on their investment. At the end of the period of the loan they will also get the £10m back.
"It is a better investment than the savings rate at the moment so it is attractive to investors and it is a more attractive proposition for United than the situation they are currently in.
"The target is £500m but I think it will go down well and they could reach £600m or £700m. If that happens the club will be in an even better financial position and there will be more money available for transfers.
"This will all happen pretty quickly so fans will know how successful it has been soon. We are not talking six months I think we will have a good idea in three or four weeks how good it has been." | 2024-06-29T01:27:17.760571 | https://example.com/article/9032 |
Nonprofit Eatbch Shows How Every Little Microtransaction Helps
Last April news.Bitcoin.com reported on the Bitcoin Cash-powered nonprofit organization ‘Eatbch’ and since then the group has not only been feeding people in Venezuela, but has also expanded its outreach to help citizens in South Sudan. Eatbch continues its progress by helping those in need, and this past week during the BCH Stress Test Day they received over 3 BCH ($1,600+USD) in microtransactions.
Also Read: Bitcoin ATM Maker Develops Payment Solution for Cannabis Dispensaries
Eatbch Receives Over 2,000 Microtransactions Worth 3 BCH on Stress Test Day
The nonprofit Eatbch is definitely one of the Bitcoin Cash (BCH) community’s favorite charities. The reason for this is because the peer-to-peer electronic cash system is doing what many believe, it was intended to do — Spread economic freedom, and help others in an un-censor-able fashion. When fees become a burden for a cryptocurrency payment network, they essentially neuter certain applications like microtransactions, and this type of ‘fee market’ censors people in countries suffering from economic hardships. In contrast to a network with unreliable dynamic fees, the BCH chain has shown people that the chain can utilize microtransactions in a variety of ways. It happened this week during the BCH Stress Test Day when the nonprofit Eatbch received thousands of small transactions adding up to over 3 BCH worth of funds.
Feeding People in Venezuela
Eatbch has operations in Venezuela, and South Sudan by utilizing bitcoin cash to feed people in need. Both countries and the citizens are suffering from serious economic hardships and food, medicine, and basic supplies are hard to come by in the region for most people. Eatbch has three Twitter handles so people can follow them and see how they are helping people in need multiple times a week.
Eatbch Venezuela’s page shows the group continues to feed families, mothers, elderly, children, and anyone in need of nourishment by using BCH donations sent in by the community. Back on August 26 when the region suffered from some earthquakes Eatbch Venezuela had some issues, but still managed to feed people.
“Some of our locations couldn’t do the meals this weekend due to the recent earthquakes, power outages, and closed stores,” the group details.
But here are some pics of those that did could give away meals.
Feeding People in South Sudan
The Eatbch South Sudan is also helping a lot of families and people who are in need of basic nourishment. For instance, on August 28 the Eatbch South Sudan group published pictures of residents in Mahad and displaced people in a camp in Juba. That day the nonprofit said people received “staples (maize flour, oil, beans) brought by youth in their community, purchased using bitcoin cash.”
The day before on August 27 the group states:
Distribution of food bought with BCH donations in Mahad, an informal displaced persons camp in Juba housing over 3000 people displaced by conflict in (former) Jonglei. We distributed it to elderly women, who will share it in the community.
Both South Sudan and Venezuela have severe economic problems, underdeveloped infrastructure, and governments with horrible central planning. Bitcoin cash is showing that this technology can help people in a permissionless fashion and Eatbch is just one example of how cryptocurrencies and microtransactions can bolster economic freedom.
What do you think about the Eatbch project? What do you think about cryptocurrencies helping people in need? Let us know your thoughts on this subject in the comment section below.
Images via the various Eatbch Twitter, and Shutterstock.
Now live, Satoshi Pulse. A comprehensive, real-time listing of the cryptocurrency market. View prices, charts, transaction volumes, and more for the top 500 cryptocurrencies trading today. | 2024-03-29T01:27:17.760571 | https://example.com/article/9583 |
Uber CEO Travis Kalanick and Pepsi CEO Indra Nooyi will also join the President’s Strategic and Policy Forum.
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The transition team's announcement comes in advance of Trump’s Wednesday meeting with top tech leaders, including Musk, at Trump Tower.
Other key tech players expected to be at the meeting include Facebook’s Sheryl Sandberg; Eric Schmidt and Larry Page of Alphabet, Google's parent company; and Jeff Bezos, the founder of Amazon who also owns The Washington Post. A representative from Uber will not be in attendance.
“I look forward to engaging with our incoming President and this group on issues that affect our riders, drivers and the 450+ cities where we operate,” Kalanick said in a statement.
Musk, Kalanick and Nooyi will join 13 other members of the President’s Strategic and Policy Forum who are expected to “meet with the President frequently to share their specific experience and knowledge as the President implements his economic agenda.”
The forum will be chaired by Stephen A. Schwarzman, the CEO and co-founder of Blackstone, one of the world’s largest private equity firms.
“America has the most innovative and vibrant companies in the world, and the pioneering CEOs joining this Forum today are at the top of their fields,” Trump said in a statement.
“My Administration is going to work together with the private sector to improve the business climate and make it attractive for firms to create new jobs across the United States from Silicon Valley to the heartland.”
This story was updated on Dec. 14 at 9:59 a.m. | 2024-05-08T01:27:17.760571 | https://example.com/article/1533 |
books.google.com - This accessible overview of classical and modern moral theory with short readings provides comprehensive coverage of ethics and unique coverage of rights, justice, liberty and law. Real-life cases introduce each chapter. While the book's content is theoretical rather than applied ethics, Beauchamp consistently...https://books.google.com/books/about/Philosophical_Ethics.html?id=mEzXAAAAMAAJ&utm_source=gb-gplus-sharePhilosophical Ethics
Philosophical Ethics: An Introduction to Moral Philosophy
This accessible overview of classical and modern moral theory with short readings provides comprehensive coverage of ethics and unique coverage of rights, justice, liberty and law. Real-life cases introduce each chapter. While the book's content is theoretical rather than applied ethics, Beauchamp consistently applies the theories to practical moral problems. Aristotle, Hume, Kant, and Mill are at the book's core and they are placed in the context of moral philosophical controversies of the last 30 years. In this edition one-third of the reading selections are new and all the selections in chapter 8 on rights are new. Chapter 7 on Hume has been heavily reshaped. Chapter 1 has been reduced to get students past introductory material and into the philosophers.
About the author (2001)
A philosopher and writer in the field of biomedical ethics, Thomas Beauchamp was born in Austin, Texas. He was educated at Southern Methodist University, where he received his B.A. and M.A. degrees; at Yale University; and at Johns Hopkins, where he earned a Ph.D. in 1970. That same year Beauchamp joined the philosophy department at Georgetown University, becoming professor of philosophy in 1979. He also served as senior research scholar with the Center for Bioethics at Georgetown's Kennedy Institute. Beauchamp is an influential leader and teacher in the emerging field of biomedical ethics, which deals with the interrelationship of ethics and the complex technology of modern medicine. His concerns have embraced the philosophical and ethical dimensions of health care, therapeutic practice, and medical and biological research. His best known work, Principles of Biomedical Ethics (1979), was the first of an impressive series of studies he produced in the field of bioethics. | 2023-11-08T01:27:17.760571 | https://example.com/article/8097 |
Bitcoin Slides Beneath $7K Dragging Altcoins With it. Jonathan Ganor 2019-12-17 04:23:54 591 views
Will Bitcoin Rebound Shortly or Are We in For A Dark December?
Bitcoin has enjoyed a period of untypical stability for the fast several weeks. Its price has been fluctuating between upper resistance levels above $7,500 and bottom support levels of $7,000. This lack of volatility is a bit odd for Bitcoin which is known for wild fluctuations.
It seems like this period of stability might be over as Bitcoin has dipped below the $7000 support levels. The original cryptocurrency is now trading at $6,918 and could temporarily be heading even lower. This is the 1st time Bitcoin had dropped below $7000 in December of this year.
Nearly all altcoins are following Bitcoin's drop to an even greater extent. Many have reported losses typically ranging between 4%-7% in the last 24 hours as opposed to Bitcoin's -2.5% loss.
Decembers are known to be a wild time for Bitcoin. In 2017, Bitcoin reached its all-time high of just below $20,000. Just a year later and Bitcoin was trading at roughly $3,500 in 2018. Currently it seems that Bitcoin might be heading lower for December 2019.
One must keep in mind however that Bitcoin had a similar dip last month on November 25th. Bitcoin dropped from $7,100 to $6,600 only to recover back in less than 24 hours.
To check the possible depths of this drop, a look at the charts is necessary.
Bitcoin Weekly Review
BTC weekly review 17/12/2019 by Senin on TradingView.com
It seems that we have a historic moment for Bitcoin. It never went below EMA 200 on its weekly chart. We are not far from this point. This is going to create Second Leg pattern on 1W chart.
What it means for us? It means we need to be prepared for Bitcoin to take off soon.
Right now, Bitcoin is doing one of its best healthiest corrections which could include drops to $6,500. So, we expect 1D MACD will turn green and whole market will just explode. RSI is very carefully keeping its bullish position
All traders around the world are following Directional Trading index. And it shows us that bearish momentum is running out of its potential.
Please trade with care and follow our updates
 A special thanks to Senin for his Technical Analysis assistance
Back to Home | 2024-02-01T01:27:17.760571 | https://example.com/article/6747 |
“Making state in all three was kind of the goal,” Kamiak coach Chris Erickson said.
The Knights (3-0 in the league, 4-0 overall) easily defeated the Spartans 118-52 and then dispatched Lynnwood (117-53) and Meadowdale (119-51) in Wesco South meets Thursday, Dec. 13, at Lynnwood Pool.
Vlad Gilszmer made an impressive debut last season with a second place finish in the 500 freestyle at the 4A state championships. He also placed seventh in the 200 freestyle.
“I think I could have improved my times, but I felt pretty good about what I did last year,” Gilszmer said.
The Kamiak sophomore’s times are ahead of where he was last year at this time.
“It’s started out pretty good so far,” Gilszmer said.
Gilszmer likely will focus on the same two races in the postseason – 200 and 500 freestyles.
“I’m just hoping to improve my times from last year,” he said.
Up until last year, Gilszmer was a multi-sport athlete, who played football and basketball his freshman year at Kamiak High School.
But he once he realized how good he was at swimming, he decided to devote all of his time to it.
“I started swimming about five years ago,” Gilszmer said. “At first it was just to stay in shape, but then as I started getting better at it, it became my main sport that I focus on.”
Gilszmer doesn’t have to look far for a training partner. Slava, who placed eighth in the 500 freestyle and 13th in the 100 backstroke, provides good competition for Vlad.
“We push each other,” Vlad said. “We get to go faster every day competing against each other in practice.”
The Gilszmer brothers weren’t the only Knights to come home with individual medals from the state meet.
Fang finished third in the 200 freestyle and seventh in the 100 butterfly. Two other Knights who are back and should be state contenders are Park and Alex Kirby. Park finished 14th in the 50 freestyle and Kirby was 10th in the 100 freestyle.
Kamiak’s 200 medley relay team finished second and the 400 freestyle team was sixth. The 200 freestyle relay squad took seventh. The Knights came home with a second-place trophy, the best finish for the school since 2013.
“We have a bunch of kids that need to drop a couple of seconds if they’re going to make state,” Erickson added. “They’re in the ballpark.”
Erickson said Kamiak should be able to make another strong run at the state meet with a top 5 finish a realistic goal.
“When it gets down to the nitty gritty, we should be in the running,” Erickson said. | 2023-11-29T01:27:17.760571 | https://example.com/article/7878 |
Last year, in March 2018, then Attorney General Jeff Sessions enlisted his subordinates to lie on his behalf that he did not know he was under federal investigation when he fired then Deputy FBI Director Andrew McCabe—an investigation initiated by McCabe and overseen by him until it was taken over by Special Counsel Robert Mueller.
By doing so, Sessions and his allies succeeded in shutting down a major controversy. If it had been made public that the attorney general had knowingly fired the deputy FBI director (and later, acting director) who’d opened and supervised that criminal investigation, the resulting scandal would have engulfed Sessions and would likely have even more seriously threatened his already tumultuous tenure as attorney general. Besides the scrutiny of the media and Congress, Sessions might have faced an inquiry by one of the Justice Department’s internal watchdog agencies, the Department’s Inspector General or Office of Professional Responsibility, as well as incurring the wrath of an already embittered president.
A government official with first-hand knowledge of the matter told me that the attorney general therefore instructed aides to make false statements in briefings to the press. The official’s account of what was at stake for Sessions is corroborated in part by a mass of evidence in the Mueller Report detailing the special counsel’s investigation of the former attorney general.
In addition to firing McCabe, Sessions, while under investigation, played a leading role in more broadly carrying out President Trump’s relentless campaign to undermine and discredit the FBI. Several leading experts on legal ethics have told me that Sessions’s simultaneous and dual roles almost certainly violated the American Bar Association’s Model Rules of Professional Conduct and federal government conflict of interest regulations. That demonstrates both the utility of Sessions’s misstatements, and the gravity of the situation he faced.
In early 2017, Sessions had been investigated for perjury and lying to the Senate Judiciary Committee during his confirmation hearing to be attorney general about his contacts with a Russian diplomat during the 2016 presidential campaign. Sessions’s testimony was demonstrably misleading, but as the Mueller Report, released April 18, noted of its investigation of the matter: “evidence was insufficient to… obtain or sustain a conviction for perjury or false statements.”
Sessions had claimed that he had not known he’d been investigated for perjury until sometime during March 2018, the month he fired McCabe, when prosecutors had informed Sessions’s personal attorney that there was “insufficient” evidence to charge him. In fact, Sessions had been interviewed by prosecutors and FBI agents working for the special counsel some two months earlier, on January 17, 2018. Although Sessions was asked about a variety of subjects during that interview, there was a special emphasis on Sessions’s contacts with the former Russian ambassador, Sergey Kislyak, and Sessions’s subsequent Senate testimony.
During my reporting for this story, I spoke to more than half a dozen current and former federal law enforcement officials and criminal defense attorneys, with no direct involvement in the Mueller investigation. They told me that any skilled and experienced attorney would, in the case of an interview of such consequence, have asked the special counsel’s office beforehand whether their client was a “witness,” “subject,” or “target” of the investigation. Sessions’s attorney, Chuck Cooper, had been an assistant attorney general for the Office of Legal Counsel during the Reagan administration, and has over thirty-five years of legal experience. I could not learn if he asked the special counsel about Sessions’s status before he was interviewed, and if he did, what he was told. The special counsel declined to comment, and Cooper also refused to disclose this information or to comment on any other matter reported here.
A witness to a federal criminal investigation is a person not under investigation themselves, but who may have valuable information for the authorities. A “subject” of an investigation is someone under suspicion, but who the government wants to investigate further to see if there is evidence of a provable crime. The Justice Department defines a “target” as “a person as to whom the prosecutor or the grand jury has substantial evidence linking him or her to the commission of a crime and who, in the judgment of the prosecutor, is a putative defendant.”
According to the government official familiar with the matter, whatever Cooper asked or might have been told, the interview itself left little doubt that the special counsel was scrutinizing this particular issue. “Even if they did not tell Sessions he was under investigation,” this person told me, “both [Sessions and Cooper] would have understood that to be the case based on the nature and sheer number of questions.” A former federal law enforcement official not involved in the case further explained to me: “A witness’s status in a case like this is a very fluid thing, anyway. They can be a witness when they come in, and a subject when they leave, or vice versa.”
Another former federal prosecutor explained to me why they considered it to be “implausible” that Sessions and Cooper did not understand that Sessions was under investigation when questioned: the special counsel’s investigation’s mission, they pointed out, was to determine the relationship between the president’s campaign advisers and the Russian government; Sessions was one such campaign adviser Russia targeted to influence. Two US Senators had asked the FBI to investigate Sessions for perjury. And in an order outlining the jurisdiction of the special counsel’s investigation, Rosenstein referenced “allegations that [Sessions] made false statements to the United States Senate.”
At issue were statements Sessions had made at his confirmation hearing, claiming that he’d had no contacts with Russian diplomats during the 2016 presidential election when he was advising Trump. Sessions, in fact, had twice met with Kislyak. It was after The Washington Post disclosed details of these meetings that Sessions recused himself from overseeing the DOJ and FBI investigations into Russian interference in the 2016 election. Sessions’s recusal led to Deputy Attorney General Rod Rosenstein’s taking over supervision of those investigations and appointing Robert Mueller as special counsel.
Sessions was questioned at length by the special counsel about his contacts with Kislyak, according to portions of the Mueller Report in both the body of the document and in its footnotes. The same government official familiar with the matter confirmed to me the focus and substance of the Mueller investigators’ interview with Sessions. In particular, Sessions was asked about a September 8, 2016, meeting that Sessions had had with Kislyak in Sessions’s Senate office. Two senior aides to Sessions were also questioned by the special counsel about this meeting, something Sessions almost certainly learned either directly from them or because their attorneys informed his attorney, Cooper, about it.
The Mueller Report further relates: “During the meeting, Kislyak invited Sessions to further discuss US–Russia relations with him over a meal at the ambassador’s residence.” Sessions’s legislative director told the special counsel that she’d advised Sessions against accepting the one-on-one meeting with Kislyak, whom she assessed to be an “old school KGB guy.” Additionally, Sessions was questioned at length to account for the portions of his Senate testimony that appeared to be demonstrably false.
There is little doubt that Sessions purposely misled the senators considering his confirmation. But the threshold to prosecute anyone for perjury is extraordinarily high—as it should be. To bring and sustain a case, a prosecutor must not only prove that the witness lied, but also that what they said was literally not true.
Sessions was asked, for example, during his confirmation hearing, if he knew whether “anyone affiliated with the Trump campaign communicated with the Russian government.” He replied that he was “not aware of any of those activities,” further noting, that he had “been called a surrogate at a time or two in that campaign.” Sessions had, in fact, met with Kislyak in his Senate office during the campaign. Sessions argued to the special counsel that his testimony was literally true, even if misleading, because he had met Kislyak in his capacity as a US senator, not as a representative of the Trump campaign. Mueller accepted this rationale.
Further evidence comes via the order authorizing the investigation of Sessions on October 20, 2017, from Deputy Attorney General Rod Rosenstein. Having first appointed Mueller as special counsel on May 17, 2017 “to investigate Russian interference with the 2016 presidential election and related matters,” the October order clarified that the special counsel’s jurisdiction included several other matters—one of which was to investigate “allegations that [Attorney General Jeff Sessions] made false statements to the United States Senate.”
*
Sessions fired McCabe early on the morning of March 16, 2018. The firing was controversial for a number of reasons: McCabe, as the FBI’s second-highest-ranking official under former FBI Director James Comey, was a witness in the special counsel’s investigation of whether the president had obstructed justice in firing Comey. Trump and his legal team—as well as Sessions—would have known that if Trump was charged with obstruction upon leaving office, McCabe would almost certainly be a crucial prosecution witness against the president, as well as a potential witness against the president in any impeachment proceedings.
In early 2018, at the president’s direction, according to a report by Axios, Sessions pressured FBI Director Christopher Wray to fire McCabe, a request that Wray considered so improper that he threatened to resign if Sessions’s demands continued. Some law enforcement officials believed that Trump’s attempts to have McCabe fired, using his own attorney general, might constitute a possible obstruction of justice by one or both men. McCabe’s termination appeared particularly vindictive—a little over twenty-four hours before he was due to retire from the FBI and become eligible for a full pension earned during a twenty-one-year career at the agency. President Trump had earlier taken a personal interest in the matter, browbeating McCabe on Twitter two days before Christmas: “FBI Deputy Director Andrew McCabe is racing the clock to retire with full benefits. 90 days to go?!!!”
On March 14, 2018, however, the FBI’s Office of Professional Responsibility concluded that the deputy director had allegedly misled the agency’s investigators about having an “unauthorized” contact with reporters for the Wall Street Journal. Sessions now unexpectedly had the perfect pretext—indeed a legitimate reason—that he did not have before, to fire McCabe.
The government official with first-hand knowledge of this episode told me that among Sessions’s motives was his concern that he would appear to have a grave conflict of interest in firing an FBI official who had been leading an investigation of Sessions himself. Especially given his earlier recusal from overseeing the Russian investigation, Sessions would face a storm of questions about why he had not also recused himself in this matter. Sessions’s conduct in firing McCabe could also be construed as retaliating against the FBI official. Above all, though, Sessions’s action in firing McCabe might constitute an obstruction of justice.
Why, in this case, did Sessions do what Trump sought of him—and mislead Congress—risking his reputation and career, and further investigation? The answer is easy enough. The president was so enraged at Sessions’s initial recusal that shortly after Mueller’s appointment as the special counsel on May 17, 2017, Trump demanded Sessions’s resignation. Sessions accordingly submitted his letter of resignation. Only the intercession of the president’s top advisers, fearing a crisis that could threaten Trump’s presidency, prevented the president from following through.
But after agreeing not to fire Sessions, the president did not return the letter to him. According to the Mueller Report, then White House Chief of Staff Reince Priebus warned Sessions that the president was holding back on returning the letter as a sort of “shock collar” to have the “DOJ by the throat.” During a trip to the Middle East, aboard Air Force One, that same week, Trump pulled Sessions’s resignation letter from his pocket to show it to others, according to the Mueller Report. Only on May 30, almost two weeks later, did Trump finally return the letter to Sessions.
Two months later, on July 21, 2017, President Trump once again demanded that Sessions resign. In contemporaneous notes made by Priebus, the president told him he “need[ed] a letter of resignation on [his] desk immediately.” Priebus told the special counsel that he too feared that he would be fired if he failed to “carry out the President’s directive.” Although Mueller concluded that the president wanted to fire Sessions because he had recused himself from the Russia probe, Trump attempted to use the pretext of a newly discovered allegation of impropriety by Sessions as his public rationale for doing so.
Earlier that same day, The Washington Post had revealed that intercepted conversations between Kislyak and his superiors in Moscow indicated that he and Sessions had “discussed campaign-related matters, including policy issues important to Moscow,” contrary to Sessions’s public assertions otherwise. After he was told that firing Sessions would likely lead to mass resignations at the Justice Department, Trump backtracked. But the episode demonstrates that Trump was looking for any pretext to fire Sessions, particularly one regarding wrongdoing. It is reasonable to assume that if Trump had learned that Sessions was under criminal investigation for perjury, the president may have fired him. Sessions would not have wanted anyone—least of all the president of the United States—to know that he was under investigation for perjury, and that he was firing McCabe when he knew this to be the case.
Sessions, then, had to navigate the president’s constant pressure to impede the FBI’s and special counsel’s investigations, as well as attempts made by the president to have the Justice Department investigate his political adversaries. Sessions pushed back or quietly frustrated many of the president’s most outlandish and seemingly legally dubious demands—tempering some before implementing them, while at other times simply accommodating the president’s worst impulses.
The president’s efforts to impede the special counsel’s investigation, and even to fire Mueller, were inextricably linked with his campaign to have a new special counsel appointed to investigate the FBI and others Trump deemed his political adversaries. But it would have been a severe abuse of power, not seen since Watergate, for the Justice Department to begin a criminal investigation, let alone a special counsel’s inquiry, without any evidence or the necessary predicate required to do so.
In May 2018, four months after Sessions learned he was under investigation by the FBI, while berated by Trump over his refusal to appoint a special counsel to probe alleged FBI malfeasance, he and Rosenstein agreed to ask the Justice Department’s Inspector General to investigate instead. Sessions also agreed to have the US Attorney from Utah, John Huber, review whether the FBI had done anything wrong. If either the IG or Huber uncovered any wrongdoing, a special counsel might then be named, Sessions and Rosenstein assured the president, while acknowledging privately, according to a senior law enforcement official I spoke to, that this was unlikely. This week, Attorney General William Barr assigned a third prosecutor, John Durham, the US Attorney for Connecticut, to examine the origins of the FBI’s Russia investigation.
Around this time, the Justice Department initially refused to turn over sensitive DOJ and FBI files to congressional Republicans about the Russia investigation, saying that their disclosure could “compromise ongoing investigations,” hamper ongoing intelligence operations, and lead to the “potential loss of human lives.” But after Trump pressured them to do so, Sessions and Rosenstein reversed the decision and turned over the records.
During the entire time that Sessions engaged in these various activities to undermine the FBI, most notably his firing of McCabe, Rosenstein knew that Sessions was under investigation for lying to Congress. Rosenstein appointed Mueller as special counsel in May 2017, who then took over the investigation of Sessions from the FBI. That October, Rosenstein wrote in an order that Mueller was to investigate Sessions for making false statements to Congress. It is entirely possible that Rosenstein did not then tell Sessions that he was under investigation: prosecutors often do not want those under investigation to know that they face legal jeopardy.
In January 2018, however, the special counsel interviewed Sessions. Rosenstein was briefed about the interview by the special counsel, and likely was told about it by Sessions himself. According to a person close to Sessions, Rosenstein never once advised Sessions not to involve himself in the firing of McCabe, or in other matters related to the FBI and its Russia investigation.
I asked a senior federal law enforcement official who worked closely with Sessions at the time why Sessions did not recuse himself from oversight regarding investigations into the FBI. This person provided me with a brief explanation: “He wanted to keep his job.” The official added: “Sessions’s most valuable use to the president was controlling the Russia investigation. This is all that he had left.”
*
It was on March 21, 2018, five days after Sessions fired McCabe, that ABC News first disclosed that a year earlier McCabe “oversaw a federal criminal investigation into whether Sessions lacked candor when testifying before Congress.” In response to this news report, Sessions ordered an aide to deny that Sessions ever knew he was under investigation. This denial led ABC then to report: “One source told ABC News that Sessions was not aware of the investigation when he decided to fire McCabe.” (Notably, though, ABC pointed out, “an attorney representing Sessions declined to confirm that.”)
Representing Sessions, Chuck Cooper provided ABC and other news organizations with this statement:
The Special Counsel’s office has informed me that after interviewing the attorney general and conducting additional investigation, the attorney general is not under investigation for false statements or perjury in his confirmation hearing testimony and related written submissions to Congress.
That day or the next, many of the nation’s most influential news organizations quoted not only Cooper’s statement in full, but also Sessions’s own assertions that he did not know that he was under investigation for perjury or making false statements when he fired McCabe. Fox News, CNN, The New York Times, The Washington Post, USA Today, the New York Daily News, and others all repeated Sessions’s denial in virtually identical words, attributing them to either “a source close to” or “a person close to” Sessions himself.
Two of the nation’s most highly respected legal ethics experts say that the former attorney general’s actions likely violate federal conflict of interest regulations, as well as the legal profession’s formal ethical rules. Deborah Rhode, a law professor at Stanford University, told me that she believes that Sessions’s firing of McCabe, and more broadly his undermining of the FBI, while Sessions himself was under FBI investigation, may have violated the American Bar Association’s Model Rules of Professional Conduct in regards to conflict of interest. The American public, she says, deserves Attorneys General whose decisions are “competent” and “disinterested” and who do not “undermine the legitimate and independent” actions of federal law enforcement for reasons of self-interest. Kathleen Clark, a law professor at Washington University in St. Louis, Missouri, told me that Sessions’s actions might be contrary to federal government ethics rules that a government employee should not involve themselves in any matter if the circumstances would cause a reasonable person “to question his impartiality in the matter.”
Explaining his firing of Andrew McCabe, Sessions directed his aides to issue a statement in his name, saying: “The FBI expects every employee to adhere to the highest standards of honesty, integrity, and accountability.” | 2024-03-26T01:27:17.760571 | https://example.com/article/3749 |
Donald Trump, Jr., and Kimberly Guilfoyle hit the campaign trail in Ohio together on Thursday, conducting a series of events for Republicans in the Buckeye State designed to hold the House majority, add to the Senate majority, and keep the governor’s mansion in GOP hands in the critical 2018 midterm election.
A source familiar with their campaigning in Ohio told Breitbart News that Trump, Jr. and Guilfoyle held a lunchtime fundraiser in Powell, Ohio, for the GOP’s U.S. Senate nominee in the state, Rep. Jim Renacci (R-OH), then traveled to Delaware, Ohio, for a meet-and-greet with Rep. Troy Balderson (R-OH).
The Renacci fundraiser pulled in more than $50,000 for the GOP candidate, and the Balderson event drew more than 100 people. Balderson won the special election in Ohio’s 12th district earlier this year, and faces a rematch against his Democrat opponent in November in a critical must-hold House district for Republicans to retain the majority in the U.S. House of Representatives.
From the Balderson event, Trump, Jr., and Guilfoyle traveled to Salem, Ohio, to headline a fundraiser for GOP gubernatorial nominee former U.S. Sen. Mike DeWine (R-OH) where they raised more than $250,000 for the ticket.
“With the exception of of President Trump, I’m not sure there’s anyone in Republican politics today that can light a crowd up like Don and Kim can. They’re an amazing one-two punch,” a Republican strategist working on the midterms this year told Breitbart News.
This was the first time on the campaign trail together for Trump, Jr., and Guilfoyle. The pair, who are dating, comprise the president’s eldest son and the former Fox News host who now works for the pro-Trump super PAC America First Policies.
“We are on path to making America great again,” Trump, Jr., told Breitbart News. “From the Supreme Court to tax cuts and record breaking job creation. We can’t let radical liberals stop our momentum or reverse the current course. Keeping Ohio in GOP hands is vital in making America great again!”
“I was thrilled to return to Ohio to support conservative candidates who will ensure President Trump’s agenda is not reversed,” Guilfoyle added. “Over the next 50 days I look forward to doing my part to keep Congress in conservative hands.”
Ohio is one of the key battlegrounds in 2018, in that DeWine faces former Barack Obama administration Consumer Financial Protection Bureau (CFPB) chief Richard Cordray at the top of the ticket in the governor’s race. Recent polling has put DeWine well ahead of the hard leftist Cordray.
In the Senate race, Renacci faces the hard leftist Sen. Sherrod Brown (D-OH) in a closer and tougher race. Polling has shown Brown with a slight lead all season, but Renacci has been running a fierce campaign aggressively challenging Brown in a manner many political pundits and prognosticators did not expect.
“I was thrilled to welcome Donald Trump Jr. back to Ohio today,” Renacci said in a statement on Thursday. “Don, President Trump, and Vice President Pence have been staunch supporters of my campaign, and I appreciate all their efforts to help me defeat liberal career politician Sherrod Brown in November.”
Trump, Jr., and Guilfoyle were joined on the trail in Ohio by Republican National Committee (RNC) co-chair Bob Paduchik, who told Breitbart News that the Balderson event was very important as well.
“Don is such an important leader and spokesman on Second Amendment rights,” Paduchik told Breitbart News. “It was great having him campaign for Troy Balderson’s re-elect where his opponent Danny O’Connor wants to not only ban guns but have the government confiscate them.”
If Republicans do well at the top of the ticket in the banner Senate and Governor races in Ohio–and for that matter other states too–they could have a shot at holding the House majority in November by helping candidates like Balderson hold their seats. The Democrats are challenging dozens of races, but only need to take 24 net seats in November to give House Minority Leader Nancy Pelosi back the Speaker’s gavel. | 2023-09-25T01:27:17.760571 | https://example.com/article/6604 |
Q:
In android ListView last element is repeatedly showing
I have created a custom listview with two text fields. showing name and age.
I have given a sample array of three objects. but every time it is only showing the last element in the array for all positions of the ListView.
Here is my main activity:
package com.sridatta.listview;
import android.support.v7.app.AppCompatActivity;
import android.os.Bundle;
import android.widget.ArrayAdapter;
import android.widget.ListView;
public class MainActivity extends AppCompatActivity {
@Override
protected void onCreate(Bundle savedInstanceState) {
super.onCreate(savedInstanceState);
setContentView(R.layout.activity_main);
details[] objects={new details("datta",19),new details("siva",20),new details("kd",18)};
CustomArrayAdapter adapter=new CustomArrayAdapter(this,R.id.listtype,objects);
ListView lv=(ListView)findViewById(R.id.list_view);
lv.setAdapter(adapter);
}
}
here is my Custom ArrrayAdapter class:
public class CustomArrayAdapter extends ArrayAdapter<details> {
public CustomArrayAdapter(@NonNull Context context, int resource, @NonNull details[] objects) {
super(context, resource, objects);
}
@NonNull
@Override
public View getView(int position, @Nullable View convertView, @NonNull ViewGroup parent) {
if(convertView==null){Context context=getContext();
LayoutInflater inflater=(LayoutInflater) context.getSystemService(Context.LAYOUT_INFLATER_SERVICE);
convertView=inflater.inflate(R.layout.list_type,null);
}
TextView tv1=(TextView)convertView.findViewById(R.id.name);
TextView tv2=(TextView)convertView.findViewById(R.id.age);
tv1.setText(getItem(position).name);
tv2.setText(Integer.toString(getItem(position).age));
return convertView;
}
}
here is details class
public class details {
static String name;
static int age;
public details(String name,int age){
this.name=name;
this.age=age;
}
}
A:
You are using recycling view, which will generate when android think you may see it on screen. For example, when scroll up, the last item may be hidden from screen, it will reuse the last item to show. And you only setText when initial the layout. You should change the code like this:
@NonNull
@Override
public View getView(int position, @Nullable View convertView, @NonNull ViewGroup parent) {
if(convertView==null){Context context=getContext();
LayoutInflater inflater=(LayoutInflater)context.getSystemService(Context.LAYOUT_INFLATER_SERVICE);
convertView=inflater.inflate(R.layout.list_type,null);
}
//update the text when view show up
TextView tv1=(TextView)convertView.findViewById(R.id.name);
TextView tv2=(TextView)convertView.findViewById(R.id.age);
tv1.setText(getItem(position).name);
tv2.setText(Integer.toString(getItem(position).age));
return convertView;
}
just notice your object class, you want them as different objects, but the static will meet their value to same memory position. you should remove the static in order to store them as unique object.
public class details {
private String name;
private int age;
public details(String name,int age){
this.name=name;
this.age=age;
}
}
| 2023-09-09T01:27:17.760571 | https://example.com/article/2665 |
Harvest Moon: Magical Melody Release Dates - Wii
Harvest Moon: Magical Melody on Wii was released on Tuesday 25 August 2009. You can now play Harvest Moon: Magical Melody on your Wii in the USA. View below for more Harvest Moon: Magical Melody release date details. | 2024-02-09T01:27:17.760571 | https://example.com/article/6970 |
Desert Sun staff
Editor's note: Bryan Reyes is the police chief of the Palm Springs Police Department, which recently lost officers Jose Gilbert Vega and and Lesley Zerebny. At Tuesday's memorial, Reyes urged the community to watch for "red flags" that could help prevent "senseless acts of violence." These are his full statements.
Good morning.
I say ‘good morning’ because, although we are all gathered here today because of the tragic loss of peace officers Gil Vega and Lesley Zerebny, today people throughout our country will learn how wonderful these two Palm Springs guardians were, the way they lived and what they left behind.
My heartfelt condolences go out to Lesley’s husband, Zach, their 4-month-old daughter Cora and the entire Zerebny family, Lesley’s father David, mother Luanne and her extended family.
My heartfelt condolences go out to Gil Vega’s wife, Susana, your 9-year-old daughter Vanessa, Gil’s seven adult children, and the entire Vega family.
SOLEMN MEMORIAL: Thousands say goodbye to fallen officers
The city of Palm Springs and the Palm Springs Police Department appreciate the time that all of you have taken out of your lives to come to our wonderful city to support the families of our fallen heroes and our police department.
It is my understanding that we have agencies present from throughout the United States, so I will start off with providing you with an understanding of our police department and our community.
We are allocated 96 sworn officers, 41 non-sworn support staff. We have a permanent population just shy of 50,000 that can double on any given day given that our city is a very popular tourist destination.
VISITING COPS: Mourners came from as far as New York and D.C. for Tuesday's memorial
The city subscribes to the belief that the people are the city. “Like no place else” is our brand. We have an office of neighborhood involvement that coordinates the effort of 42 organized neighborhoods within the city limits of Palm Springs. Two officers, a sergeant and a lieutenant are assigned to every single neighborhood. We attend quarterly, bi-annual and annual meetings.
We have a tremendous relationship with our community and our local tribe, the Agua Caliente Band of Cahuilla Indians. Our police department has a Citizens Police Academy, LGBT Outreach Committee, Police Advisory Board, and we collaborate with the Human Rights Commission to create equality for all who live, work and visit this great city.
We do not experience much gang-related crime, and we did not experience any homicides this entire year – up until this tragedy. So, with all this in place, how does such a tragedy occur in such an environment with so many people working together every day to make this city a better place for all to enjoy.
All I can say is this: The threat to law enforcement throughout our country is very real. No community is exempt to such a tragedy.
The subject – and I say subject because I refuse to identify him by name. He doesn’t meet the definition of a man in my book, so therefore I will proceed with subject. The subject responsible for this tragedy grew up within Palm Springs, attended our schools and lived a life of criminal behavior.
On Oct. 8, he murdered two of our officers and injured a third. What a very troubling time this is in that the moral compass has shifted to the degree in that a subject such as this one chose to arm himself with a rifle and murder officers simply because they responded to a call for service regarding a family disturbance
Enough is enough.
Our law enforcement brothers and sisters throughout the country are growing tired of this unacceptable behavior. People need to start taking responsibility for their own households and stop ignoring the signs of escalating violent behavior.
Interrupted by applause.
Start paying attention to the red flags that are evident in all tragic events. The Pulse Nightclub Tragedy in Florida. The terrorist attack in San Bernardino, California. The murder of our Palm Springs police officers and countless other tragedies. They all had red flags that could have been brought to the attention of local law enforcement before it escalated to these levels.
For example, reading some of the Facebook posts of this subject responsible for the assassination of our officers, clearly depict – in my mind – a ticking time bomb. His friend was contacted by a local news outlet and quoted as saying "He was known to do drugs and carry weapons." When asked about the killing of our officers, we was also quoted as saying "Believe me, it don’t surprise no one that knew him."
Quit ignoring the signs. Give a call to local law enforcement and give them the opportunity to address these subjects before it turns tragic. Keeping the community safe is a shared responsibility.
Now more than ever, our officers are trying to maintain peace and order during some of the most difficult times in policing history. We need to work harder with our communities to identify potential threats, because if we do not, we will continue to see grieving families. Children left behind without a mother or a father. And forever-changed police officers who are growing tired of these senseless acts of violence.
I want to take the time to address the Palm Springs Police Department retirees seated to the front right hand side of our room. Retired chiefs, captains, lieutenants, sergeants, detectives, officers and non-sworn staff – as you might imagine, I have a good understanding of the circumstances our officers found themselves in on Oct. 8. I understand their approach, their reaction when they are fired upon, their tactical movements in an effort to re-position, cover each other, assess the threat and engage in a fierce firefight, develop a plan to extract downed officers and subsequently arrange for the transport of the three injured officers.
The majority of this occurred with line-level officers that ranged from Officer Vega’s 35 years of experience to an officer that just completed the field training program and, in fact, he was on his fourth day on his own. No one of the officers had a SWAT background, yet every single officer responded in the same manner – as a team. Tactics were on point. Decisions were quick. And they were all in, until the end. That says an awful lot about our field training officers and our line-level supervision and preparing our field personnel to address to most dangerous situations they can possibly face.
Sit there proudly knowing that we all walk in the footsteps of those who came before us. And know we all now walk in the footsteps of two heroes of our chosen profession.
Shortly, you are all going to hear from family and friends of Officer Vega and Officer Zerebny, You are going to learn that they are far more than police officers. They had a family just like you. Take the time to absorb what you see, what you hear and what you feel. Make a pledge to yourself that you will do a better job in your effects to strengthen the relationships you have with those around you all in an effort to never see, hear or feel the pain that we were all experiencing today.
To all my brothers and sisters, in our chosen profession, you are all supported, you are all loved and you are all appreciated. May you be blessed, for you are trying to keep the peace in the most difficult time in the history of policing.
Thank you. | 2024-06-05T01:27:17.760571 | https://example.com/article/8245 |
CALL NOW TO HAVE YOUR CASE EVALUATED
AVAILABLE 24 HOURS, 7 DAYS A WEEK203-982-8396
LAW OFFICE OF BRYAN
MCENTEE
19 Mitchell Ave, 2nd Floor
Waterbury, CT 06710
Phone: (203)
982-8396
Fax: (203) 413-2925
FAMILY LAW
Divorce can be an extremely complex matter.
There are issues involving alimony, the division
of property, child custody, child support, tax
issues, retirement income, and numerous others.
Attorney McEntee understands that these issues
can weigh on your
mind and create an enormous amount of stress.
Attorney McEntee is sensitive to the stress and
anxiety you are
experiencing. It is important that you have a
capable, competent, experienced, and zealous
attorney acting on your behalf during the
divorce process in order to obtain the most favorable
out come possible given all of the facts and
evidence. Attorney
McEntee is available for help 24
hours a day, 7 days a week. Attorney
McEntee can be reached for a free consultation
at(203) 982-8396.
DIVORCE
In the State of Connecticut you begin a divorce
by filing a complaint and summons. If you are
thinking of filing a divorce these forms may
seem complex and confusing to you. If you are
the receiving end of these forms, you may be
extremely angry. Attorney McEntee may be able to help you
regardless of what category you fall into.
Attorney McEntee has represented both Husbands and Wives; both Plaintiffs
and Defendants.
Once the divorce process has been initiated,
Connecticut law requires a 90 day waiting period
before divorce can become final. If no agreement
can be reached during this 90 day waiting period
then the parties begin discovery and exchange
important financial documents. It is also
possible during this time to go to family
relations counseling, mediation, arbitration, special
masters conference, or judicial pre-trial
conference. If these judicial processes do not
result in a settlement, the case is put down for
trial. Attorney McEntee can help you reach
a fair and just settlement with your spouse
during negotiations, or represent you zealously
at trial.
While your case is awaiting final disposition
the time period is referred to as Pendente
Lite. During the Pendente Lite
period it is possible to have your spouse start
paying you alimony, child support, and household
expenses almost immediately in certain
situations. Contact Attorney McEntee to learn whether this
would apply in your case.
If your spouse is
asking you to pay alimony and child custody
Attorney McEntee
may also be able to offer you assistance. There
are several defenses that can be raised to
motions for alimony, child support, or the
payment of household expenses. If you are on the
receiving end of these documents, contact
Attorney McEntee to learn how to mount a
defense..
CHILD SUPPORTIn Connecticut
the income and expenses of both parents are
analyzed to determine the amount of child
support that must be paid. This formula for
making a determination can be difficult and
confusing for some clients. Clients may not
understand how to calculate child support
guidelines, determine the amount of child
support due when there is a split custody
situation, the amount owed for day care expenses
and health care expenses of the child, or how to
calculate the amount of past due child support. Additionally, clients may not
understand how to obtain the other parent's
financial and work information if they suspect
that the other parent is hiding income. In some
situations, the Court will even require a parent
who is unemployed to pay child support.
It may also be possible to get the Court to
issue an order relating to the payment of
college expenses of the child at the time a motion for child
support is argued.
Child support can be modified if there is a 15%
change in income since the last court order
relating to child support was entered. If your
child's parent has recently gotten a raise or a
bonus at work, won money at the casino, or
received an inheritance then you may be entitled
to a higher amount of child support than you are
currently receiving. If this applies to your
situation, Attorney McEntee can evaluate whether or not
you are owed additional money.
CHILD CUSTODY
There are
numerous factors that the Court considers when
making a child custody decision. Some of the factors
that the Court will consider when making a child
custody decision are listed under Connecticut
General Statutes, Section 46b-56(a). No one
factor is more important than any other factor.
The underlying consideration is what is in the
best interests of the child. If you contact
Attorney McEntee he will begin analyzing your case and gathering
evidence to make the strongest argument possible
for why you should have custody of your
children.
ATTORNEY'S FEES
Don't let Money be an issue. It you are unable
to pay attorney's fees it may be possible in
Connecticut to get your spouse to pay your
attorney's fees. It may also possible to have
your spouse start paying you alimony, child
support, and household expenses almost
immediately in certain situations. Contact me to
learn whether this would apply in your case.
Please be aware that the Client is always
responsible for paying all costs and expenses
regardless of what side pays Attorney fees.
The information on this
Law Office of Bryan McEntee Website is for general information purposes only.
Nothing on this or associated pages, documents, comments, answers,
e-mails, or other communications should be taken as legal advice for any
individual case or situation. Information on this website is not intended to
create, and receipt or viewing of this information does not constitute or
create, an attorney-client relationship. There is no Attorney-Client
relationship created without a signed retainer agreement. Attorney cannot
guarantee any particular outcome. Client is responsible for paying all costs and
expenses.
Waterbury
photographs of the
Clock Tower at Union Station and Carrie Welton
Fountain copyright Daniel M. Lynch. Used with
Permission. | 2024-04-22T01:27:17.760571 | https://example.com/article/8762 |
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Doru Frantescu, director VoteWatch Europe | 2024-01-13T01:27:17.760571 | https://example.com/article/8164 |
Q:
Windows Forms, Set method throws string.isnullorempty exception? C#
This a class which holds the value of first and last name.
Another class called attendee inherates from person.
class Person
{
static private string FirstName;
static private string LastName;
static public string firstname
{
get
{
return FirstName;
}
set
{
if(string.IsNullOrEmpty(FirstName))
{
throw new ArgumentException("First name must not be blank!");
}
FirstName = value;
}
}
This is in my GUI class and the method retrieves the value of the first name and updates it within the Person class.
private void BtnSet_Click(object sender, EventArgs e)
{
Attendee.firstname = FirstName.Text;
... //more code (not relevant)
}
For some reason this throws the exception from the Person class but I do not understand why?
A:
You check the FirstName field which is initially null in the setter of firstName property, use instead
set
{
if(string.IsNullOrEmpty(value))
{
throw new ArgumentException("First name must not be blank!");
}
FirstName = value;
}
| 2024-02-22T01:27:17.760571 | https://example.com/article/3585 |
http://1conn.com/?post_type=team It’ll assist in the sending of information securely – and be considered a large gain in the combat against hackers – however purpose extensive situation that the secretive communist state is pioneering its use. | 2024-07-26T01:27:17.760571 | https://example.com/article/7180 |
You are here
2015 Governors-elect Candidates' Bios and Statements
Colorado Chapter
The ballots for the current Governors-elect election have been
mailed. The voting membership (Masters, Fellows, Members, and
Resident/Fellow Members with an electdate of 9/1/2013 or earlier)
can vote online at(www.directvote.net/ACP/) or
mail-in ballot. You will need your mailed ballot with your
ACP Member Number and Election Passcode in order to make a
vote. Below are the bios and statements of the candidates
that are running in this election. Online voting began on September
21, 2015 and ends on November 9, 2015 at 11:59 PM Eastern. Please
be sure to vote!
PRESENT POSITION: full time Clinical Primary Care Internist,
independent practice, Littleton, CO; Clinical Associate Professor
of Medicine, University of Colorado School of Medicine, includes
clinical, classroom, workshop, and small group teaching
CANDIDATE STATEMENT: I hope to help further ACP's primary
missions: educating physicians, promoting the High Value Care
initiative, and educating state and national government officials
on public policy issues effecting patients and internists. We have
a unique, once in a generation, opportunity to help shape
implementation of the Affordable Care Act. ACP's input on
regulatory and bureaucratic policy, as well as state pilot programs
for health care delivery reform, payment reform, and mental health
care integration, is essential. Also, I'd like to bring my
experience and interest in organizational leadership and non-profit
board development to the ACP Board of Governors, after serving on
over a dozen health and non-health related Boards. Lastly, I will
bring perspective and experience from the daily immersion in
clinical practice.
Alwin F. Steinmann, MD, FACP
EDUCATION: Manhattan College (BS - Chemistry): 1976-80 New York
University School of Medicine (MD): 1980-84
CANDIDATE STATEMENT: I am truly honored to run for Governor of
the Colorado Chapter of the ACP, and am particularly humbled that
as a relative newcomer, I have been given this wonderful
opportunity. In this time of disruptive change and rapid evolution
of health care, it is critical that the ACP serves as a voice for
Internists and our patients. I believe my experience in health
policy would help me serve you in the coming years as we navigate,
and hopefully help shape, the changing landscape of our health care
system. We should also continue to listen to our members regarding
what educational and advocacy services they need to help them
practice medicine. The Colorado ACP is a vibrant, engaged and
committed chapter with a long tradition of excellence. It is the
sacred trust of anyone who serves as Governor to ensure the Chapter
continues this tradition. | 2023-08-21T01:27:17.760571 | https://example.com/article/5119 |
Q:
ASP.NET core persisting values between Get and Post error validation
I'm new to web development so I don't know a good way on how to persist data between requests.
This is my site so far:
The elephant title is being fetched from an API on the GET request, according to the titleId query parameter. When I press login, model validations are being run, for example that email and password must have been entered. However, when error page is returned, elephant text is empty since that value was not persisted. What are the best approaches to persist that value so that is still visible when POST error is returned? Does it has to be included in the POST data? I don't want to request the API again.
Code behind:
public class IndexModel : PageModel
{
private string apiTitle;
public string ApiTitle { get { return apiTitle; } set { apiTitle = value; } }
// Bind form values
[BindProperty]
public User user { get; set; }
public Task<IActionResult> OnGetAsync(string titleId)
{
if (!string.IsNullOrEmpty(titleId))
{
ApiTitle = await GetTitleFromApiAsync(titleId);
}
return Page();
}
public async Task<IActionResult> OnPostLoginAsync()
{
if (!IsLoginFormValid())
{
// When this is returned, for example if no password was entered,
// elephant title goes missing since apiTitle is null?
return Page();
}
var user = await LoginEmailAsync();
return RedirectToPage("/Profile");
}
}
Html:
@page
@model IndexModel
@{
ViewData["Title"] = "Home page";
}
<link rel="stylesheet" href="css/index.css">
<script src='http://cdnjs.cloudflare.com/ajax/libs/jquery/2.1.3/jquery.min.js'></script>
<div class="login-page">
<span id="label_api_title">@Model.ApiTitle</span>
<div class="form">
<form class="login-form" method="post" asp-page-handler="Login">
<input type="text" placeholder="Email" asp-for="User.Email"/>
<span asp-validation-for="User.Email" class="text-danger"></span>
<input type="password" placeholder="Password" asp-for="User.Password1" />
<span asp-validation-for="User.Password1" class="text-danger"></span>
<button>login</button>
<p class="message">Not registered? <a href="#">Create an account</a></p>
</form>
</div>
</div>
<script src="js/index.js"></script>
A:
Yes. What you see is the expected behavior. Remember, Http is stateless. You are making 2 separate http calls, one for the GET and one for POST. The second call has no idea what the first call did ( or even there was first call at all!)
If you want to have a way to read the ApiTitle property value in the Post call and return that to the view, you need to persist it somewhere so that it is available between http calls. But in your case, all you need is to include that in the form post and have the framework bind it for you.
In your case, you can simply use a public property (Which is settable and gettable) for this. No need to keep a private variable. Decorate your property with BindProperty attribute so the model binder will bind the data on this property.
public class CreateModel : PageModel
{
[BindProperty]
public string ApiTitle { get; set; }
//Your existing code goes here
}
Now inside your form tag, have an input hidden element for the ApiTitle. This way, when the form is submitted, the value of ApiTitle property will be send in the request data.
<form class="login-form" method="post" asp-page-handler="Login">
<input type="hidden" asp-for="ApiTitle"/>
<input type="text" placeholder="Email" asp-for="User.Username"/>
<!--Your other existing form elements -->
<button>login</button>
</form>
Now in your OnPostLoginAsync method, you can read the ApiTitle value if needed. When you return the Page (when validation fails), the UI will display the ApiTitle property value in your span element.
public async Task<IActionResult> OnPostLoginAsync()
{
var title = this.ApiTitle; // If you want to read this value
if (!ModelState.IsValid)
{
return Page();
}
return RedirectToPage("/Profile");
}
| 2023-12-29T01:27:17.760571 | https://example.com/article/6334 |
Foam Polishing & Finishing Pads
World-class car polishers aren’t the only thing RUPES specialise in! Your polisher, compounds and polishes are only as good as the pads you’re using them with. This is especially true with the RUPES Big Foot Polishers because of the enormous stroke each tool makes. RUPES has spent a considerable amount of time developing foam pads that would not only work with the massive stroke of the Big Foot Polishers, but actually take advantage of it to deliver best results.
RUPES Big Foot Polishing and Finishing foam pads are available in 40mm, 70mm, 100mm, 150mm and 180mm variations. | 2024-01-17T01:27:17.760571 | https://example.com/article/5105 |
Kono Pizza plans multi-unit deal in NY, CA
EDISON, N.J. (January 13, 2013) – It looks like a newly introduced delicacy of pizza served in cones is here to stay. Just six months after making its American debut with a corporate-owned location in New Jersey, Kono Pizza, a quick-service franchise that sells pizza in a unique cone-shaped crust filled with fresh, Italian ingredients, has announced the signing of multiple franchise deals from California to New York. In all, Kono Pizza has 15-plus additional units in the pipeline as part of its newly launched U.S. expansion.
Kicking off the expansion is a multi-unit franchise agreement that will bring three new Kono Pizza restaurants to San Diego. The California expansion will be led by franchisees Jonathan Scoggins and Craig Dewitt, who plan to open their first Kono Pizza location in the Gaslamp Quarter by the end of 2014, followed by additional future locations along the West Coast. Scoggins, co-founder and vice president of Synthonix, and Dewitt, ex-special operations forces for the U.S. Marines Corps, chose Kono Pizza to further diversify their business development and management skills.
“San Diego is the perfect location for us to expose a new business –– with the sunny weather year-round there’s always people walking around,” stated Scoggins. “The foot traffic in the downtown area coupled with the proximity to a large convention center will set us up to gain a huge following.”
In addition to the California growth, Kono Pizza’s co-founders Carlo Ruggiero, David Ragosa and Greg Kinlaw will open a corporate-owned location in Raleigh, North California, by the end of the First Quarter of 2014. The trio opened their first corporate Kono Pizza in Edison, New Jersey, in August 2013. They plan to expand the concept through franchising at least 10 franchise locations in Long Island and at least one location in downtown Philadelphia.
Kono Pizza embraces original Italian recipes and ingredients inspired by the overseas locations. Served in a cone and filled with fresh, quality ingredients, some imported straight from Italy, the brand offers consumers a healthier, on-the-go alternative to one of their favorite foods. Featuring a thinner crust and low-moisture cheese, Kono Pizza cones are only 250-280 calories each, compared to 450 calories in the average traditional pizza slice.
“In a flooded market with more than 70,000 pizzerias and fast-casual pizza concepts, we stand alone with a unique pizza product like no other franchise in the U.S.,” stated David Ragosa, co-founder of Kono USA. “We expect our success in California and Long Island to act as a catalyst for further expansion across the country, offering entrepreneurs the opportunity to get in on the ground floor with an innovative franchise in the quick-service pizza segment.”
Kono USA has its sights set on opening locations across the country both stand-alone and kiosk formats. Their mission is to keep everything simple and affordable for franchisees by offering a streamlined, manager-driven business model with a low start-up cost of $250,000-$400,000.
About Kono Pizza
Founded in Edison, New Jersey., Kono USA was established in the United States in 2013 by David Ragosa, Carlo Ruggiero and Greg Kinlaw, who took the already popular European concept and brought it to America. Originated by Italian culinary genius, Rossano Boscolo, Kono Pizza cones are filled with fresh, quality ingredients inspired by original Italian recipes, giving consumers an on-the-go, healthier way to enjoy their favorite food. There are currently 130 Kono Pizza locations operating in 20 countries. | 2024-03-23T01:27:17.760571 | https://example.com/article/5064 |
Gov. Hogan: Cummings 'Could Do A Lot More,' But Other Federal Help Needed
Gov. Larry Hogan said Thursday that Rep. Elijah Cummings could be doing more for Baltimore, but that so could everybody else in Washington.
Hogan spoke to Fox News several days after telling WBAL's C4 that President Donald Trump's tweets criticizing Cummings and Baltimore City were "outrageous and inappropriate."
"I don't want to characterize Elijah Cummings. I don't think you can put all the blame of Baltimore City on Elijah Cummings, but I also think he could do a lot more to help us," Hogan said. "I'd love to have more help from the White House and from the Congress, frankly, to help the state go in there and help the city. It's really a city problem, but they can't do it by themselves."
Hogan credited federal officials with helping tackle the state's opioid crisis, including a slew of federal indictments announced Thursday by U.S. Attorney Robert Hur. Hogan said that if there's a "silver lining" to Trump's comments, it could be in drawing attention to the city's continuing needs.
He highlighted state investment in Baltimore schools and the demolition of vacant homes. He reiterated criticism of state lawmakers for their failure to pass legislation mandating stricter sentences for repeat violent offenders.
"We've failed in our urban areas across the country and Baltimore certainly is a perfect example of that and the leadership has failed for decades and decades," Hogan said. "But instead of just focusing on the bad stuff... let's figure out how do we figure out how to fix it."
Hogan took office last week as chair of the National Governors Association. A moderate who neither endorsed Trump nor voted for him in 2016, Hogan earlier flirted with the idea of challenging Trump in the Republican primary before publicly ruling it out. He told Fox News he "never really gave it a lot of consideration," but said he plans to use his new post as a platform for national issues.
Hogan is set to appear early Friday on "Late Night with Seth Meyers," which will air at 12:35 a.m. on WBAL-TV 11. | 2023-12-24T01:27:17.760571 | https://example.com/article/9058 |
Washington: George E Curry, a journalist, civil rights activist and publisher whose syndicated column ran in hundreds of black-owned newspapers around the United States, has died. He was 69.
Curry, of Laurel, Maryland, died suddenly on Saturday at Washington Adventist Hospital in Takoma Park, Maryland, after he was taken to the emergency room there, his sister, Charlotte Purvis, said on Sunday.
Curry's syndicated column was carried in more than 200 African American-owned newspapers, and he served two stints as editor-in-chief of the National Newspaper Publishers
Association, a news service for black papers.
Goerge Curry. Twitter. /@currygeorge
He also served as editor-in-chief of Emerge magazine in the 1990s, and within the past year, he had been raising money to relaunch Emerge as a digital magazine covering racial injustice and other issues important to the black community.
He became the first African-American to be elected president of the American Society of Magazine Editors.
He was a frequent commentator on Black Entertainment Television and on the Rev Al Sharpton's radio show. "I am saddened beyond words upon hearing of the death of George Curry," Sharpton tweeted yesterday. "He was a giant and trailblazer. RIP."
I am saddened beyond words upon hearing of the death of George Curry, Publisher of Emerge Magazine. He was a giant and trailblazer. RIP.
A native of Tuscaloosa, Alabama, Curry graduated from Knoxville College in Tennessee, where he edited the school paper and played football. He worked as a reporter for the St Louis Post-Dispatch, Sports Illustrated and the Chicago Tribune.
"George E Curry was a giant in journalism," Rep G K Butterfield, D-N C, the chairman of the Congressional Black Caucus, said in a statement. "He stood on the front lines of the civil rights era and used his voice to tell our stories when others would not."
While at Emerge, he led a four-year campaign to win the release of Kemba Smith, a woman sentenced to 24 years in prison for her minor role in a drug ring. Emerge ran a cover story on Smith in 1996 called "Kemba's Nightmare." President Bill Clinton pardoned Smith in 2000.
In addition to his sister, Curry is survived by his son, Edward Curry, and granddaughter, Neyah Curry. | 2024-05-31T01:27:17.760571 | https://example.com/article/7350 |
Former Senate Democratic Leader Harry Reid is expected to be called to testify in the corruption trial of Sen. Robert Menendez, a source with knowledge of the situation told Fox News on Friday.
Menendez is accused of accepting campaign donations, gifts and vacations from Florida ophthalmologist Dr. Salomon Melgen. In return, Menendez, D-N.J., allegedly used his Senate powers to lobby on behalf of Melgen’s business interests.
Reid's name re-emerged weeks ago in the case, when prosecutors said the former congressional leader was “first enlisted” by Menendez in November 2011 to advocate for Melgen in the ongoing dispute the doctor had with the Centers for Medicare and Medicaid Services (CMS).
Speculation has focused on a possible trial appearance by Reid following testimony this week from another former senator, Democrat Tom Harkin. The former Iowa senator said he attended a meeting in 2011 facilitated by Menendez, in which he and Melgen discussed an $8.9 million Medicare dispute.
Reid, D-Nev., already has met with the Justice Department and the FBI in connection with the investigation, back in 2015.
MENENDEZ CORRUPTION AND BRIBERY TRIAL: WHAT TO KNOW
Should Reid be called, prosecutors have already hinted at what they might want to ask about.
Court documents claim that Reid reached out to the White House deputy chief of staff in 2011 about Menendez being “upset about how a Florida ophthalmologist was being treated” by CMS. Prosecutors said the White House deputy chief of staff “demurred” as it involved a “dispute between a single doctor and an administrative agency, not a policy matter.”
In June 2012, Melgen also flew Reid on his company’s private plane from Washington to Boston and back, but Menendez was not present, according to a source with knowledge of the situation.
MENENDEZ SET UP MEETING ABOUT PAL'S MEDICARE DISPUTE, EX-SENATOR HARKIN TESTIFIES
According to a trial brief filed by federal prosecutors last month, on Aug. 2, 2012, Reid and Menendez met with then-Health and Human Services Secretary Kathleen Sebelius in Reid’s office on Capitol Hill, where, according to witnesses in the meeting, Menendez allegedly “argued vehemently” for Melgen’s position on the billing dispute between Melgen’s company, Vitreo-Retinal Consultants, and the federal government.
The documents said Menendez “personally pressured” Sebelius to “intervene in the billing dispute.”
JUDGE DENIES 'SPECIAL TREATMENT' FOR SEN. MENENDEZ AS CORRUPTION TRIAL SET TO BEGIN
“Harry Reid’s been implicated, as has Kathleen Sebelius, so it’s clear the corruption extended beyond Menendez’s office,” Republican National Committee spokesman Michael Ahrens told Fox News. “Who knows exactly who else will testify, but it’s just as stunning that no Senate Democrat has said Menendez should resign immediately if convicted.”
Menendez has proclaimed his innocence in the case. When the trial opened earlier this month, his lawyers said the case is not about corruption or bribery but about a long-term friendship.
The Justice Department declined to comment on whether Reid will testify.
A message left on Reid's voicemail was not immediately returned. Fox News has also reached out to a representative for Reid for comment.
The source also suggested to Fox News that prosecutors could seek Sebelius' testimony as well. According to a Politico report in 2015, Sebelius met with federal investigators to discuss the meeting in 2014. | 2024-03-20T01:27:17.760571 | https://example.com/article/6647 |
Barclay Goodrow was traded from the San Jose Sharks to the Tampa Bay Lightning on Feb. 24. Seventeen days into his tenure with his new team, the NHL shut down because of the coronavirus outbreak.
Goodrow had just settled into his condo with his girlfriend, Madison. They could have gone back home to Toronto, but Goodrow was wary of traveling during a pandemic.
"Going through an airport when we didn't need to isn't something we wanted to do," the 27-year-old forward said. So they stayed in Florida.
"We were watching the news one night and they said shelters were pretty much at capacity with animals," Goodrow said. "And a lot of shelters were being shut down."
The couple had both grown up with dogs and always wanted one of their own; being bound to their apartment in a new city for an indefinite period felt like as good an opportunity as any to fulfill that desire.
So they applied to foster two puppies from the Humane Society of Tampa Bay.
"We showed up to pick up our two dogs, and as we went into the room, [Victor] Hedman and his wife were sitting there to pick up their two dogs, as well," Goodrow said. "It was a total coincidence, but we were getting four dogs from the same litter."
Once the Humane Society of Tampa began posting photos of Goodrow and Hedman and their 6-week-old foster puppies -- believed to be collie-retriever mixes -- teammate Braydon Coburn became interested. Coburn's family came in the next day, and they're fostering the puppies' three other siblings because they didn't want to separate them.
Lightning defenseman Victor Hedman is fostering two dogs from the Humane Society of Tampa Bay. Courtesy of the Humane Society of Tampa Bay
"They're calling it the Lightning litter," Hedman said. "At this point, we want to come closer to a solution for the virus, obviously follow the rules as much as we can and be positive as much as we can. But having these puppies at home with us really helps time move faster."
Said Goodrow: "When you're stuck inside a pretty small apartment, the days can go by pretty slowly. These two guys have provided a lot of excitement. And something to do. They definitely help lighten the mood. It's a joy. They're probably sick of walking three times a day, but there's nothing else to do."
NHL players, like the rest of us, are homebound for the foreseeable future. Without morning skates, team meetings, travel and games to fill their days, many have adopted dogs to give them companionship during this uncertain time.
"Everyone is getting a dog right now," Vancouver Canucks forward Brock Boeser said. "It's the perfect time to get a dog."
Boeser already had Coolie, an Australian Cattle mix he -- coincidentally -- adopted in Tampa Bay during the NHL All-Star Weekend in 2018. Boeser wanted to adopt another dog last summer, "but I had too much going on," he said. Right before Boeser was set to make the 27-hour drive from Vancouver to Minnesota after the NHL's coronavirus pause, he spotted a dog available for adoption online. Boeser applied, got accepted and upon returning to Minnesota, Coolie had a little brother, Milo, who Boeser believes is either a lab-shepherd mix or a husky-shepherd mix.
"I feel like it's easier right now to train him because I'm at home the whole day, I can't leave," Boeser said. "I let him out every two hours, so he's only had a few accidents in the house. I have him sleep in the kennel at night from 12 to 7, then I let him out and let him sleep with me the rest of the morning. He's been a beauty so far."
Blue Jackets defenseman Scott Harrington is quarantined in his Columbus apartment with his girlfriend, Maddie.
"About a week in, once we realized we were going to be here for a while, we decided to get a dog," Harrington said. "It wasn't a situation where it was like, 'Oh my god, we're so bored we need something to entertain ourselves.' We were probably going to get a dog this summer, but we saw this pause as a good opportunity to do it earlier. It's been a bonus that she keeps us busy."
Harrington now has an 11-week-old French bulldog named Josie.
"I think we spend 99 percent of the day just watching her, waiting for her to go to the bathroom in the house and try to catch her before it happens," Harrington said. "I have to say, she's been a great addition to our house, especially since we don't have much to do. She always wants to be around us, she sleeps on top of my girlfriend's head, it's cute."
Raising a dog with social-distancing guidelines isn't easy. Harrington ordered dog food online that was supposed to arrive within 48 to 72 hours, but instead took a week and a half. Luckily, Harrington's teammate, Pierre-Luc Dubois, also has a French bulldog, and Maddie happened to dogsit for Dubois when the Blue Jackets were on a road trip earlier this season.
"So we had a bunch of leftover food at our apartment for the same breed, which held us over until our shipment came," Harrington said.
"I have to say, she's been a great addition to our house," Blue Jackets defenseman Scott Harrington says of Josie, his 11-week old French bulldog. Courtesy Scott Harrington
Even though Victor Hedman, Goodrow and Coburn live in the same city and are fostering the same family of dogs, they haven't seen each other since the adoption.
"We're pretty strict about our stay-at-home business," Hedman said. "We haven't ventured out too much. I've seen some of my neighbors, talking from a safe distance, but that's it. We have a fenced-in backyard, so [the dogs] can run around, so there's no need for us to go on unnecessary hikes."
Hedman and his wife, Sanna, already had a bulldog named Harry. They always wanted to foster or adopt another dog, but it's already difficult arranging for one dog to travel internationally to their native Sweden every offseason. Plus, if they got a new dog in season, Hedman knows his wife would take on a brunt of the work, thanks to his travel schedule.
"We saw fostering these puppies for three weeks as a great opportunity for us to help out how we could," Hedman said. "You form a relationship with them, and hopefully then can go to a good home after."
It appears Harry, right, is all good with sharing some real estate with his new foster siblings. Courtesy of Victor Hedman
And then there's Capitals forward Tom Wilson who, fortuitously, adopted a black lab mix named Halle two weeks before the NHL's coronavirus shutdown. Wilson's girlfriend, Taylor, is a professional beach volleyball player and also pursuing her MBA at George Washington University.
"We've wanted a dog, but obviously, our busy schedules didn't permit," Wilson said. "And then we saw Halle on a shelter's Instagram one day, and we figured this might be the best time, heading into summer."
Wilson said raising a puppy while still playing was "hard" and "a lot of work." When he was on a road trip, and Taylor was away for a day, teammate Michal Kempny's girlfriend, Nicola, watched Halle for a day. "It helps having teammates, and when you have a cute puppy, people are usually happy to help." (Halle did pee on Kempny's couch, which Wilson said was "the only casualty.")
The transition to stay-at-home parent was also a struggle.
"We went from wanting to spend as much time with her as possible to having to make sure we put her in her pen or crate so she doesn't get too attached, or develop doggie anxiety when she's away from us," Wilson said.
The toughest part for Wilson now is that Halle has all of her shots and is at the age when she should be socializing.
"It's time for her to be at the park and meeting other dogs, so that sucks for her a little bit," Wilson said. "We have the Dowds [teammate Nic Dowd and his wife, Paige] come over with their golden in the backyard and we let them play. So it's good she realizes there's other furry animals out there, it's not just me and Taylor."
Halle is already so popular on the internet, the Capitals have arranged to sell bobbleheads featuring her and Wilson (with a portion of the proceeds benefiting Wolf Trap Animal Rescue, from which Halle was adopted).
A TOM AND HALLE WILSON BOBBLEHEAD!? Yes, please.
Support a great cause with this @tom_wilso and Halle Wilson Washington Capitals Rescue Dog Bobblehead.
A portion of proceeds will benefit the Wolf Trap Animal Rescue!
Pre-Order Now: https://t.co/7bUEX3w36G#ALLCAPS // @FOCOusa pic.twitter.com/oCH27TNphz — Washington Capitals (@Capitals) April 3, 2020
Meanwhile, in Tampa Bay, Goodrow is getting used to life as a temporary puppy dad.
"They're high-energy, they eat well and they're constantly growing," Goodrow says. "Then as soon as 10 [p.m.] hits, they don't want to go to bed. They're up every hour wanting to pee. When you're a 4-pound dog, you don't have the biggest bladder. There's been a lot of sleepless nights for sure."
The Humane Society named Goodrow's puppies Eddie and Tundra, but he renamed them Norman and Winston. Since they are hockey dogs, Goodrow of course assigned them nicknames: Normie and Winnie. | 2023-10-06T01:27:17.760571 | https://example.com/article/6721 |
Man loses benefits because he had heart attack during work capability assessment
A man with heart problems who couldn’t complete a work capability assessment because he had a heart attack during the interview had his benefits stopped by the job centre for ‘withdrawing’ from the interview.
Reblogged this on Political Pip Spit or Swallow its up to You and commented:
Excellent item from Pride’s Purge. I think when I get around to categorizing the different subjects on this blog this one will have to go in the ‘You Couldn’t Make it Up’ category’.
At the risk of repeating myself …. this absolutely beggars belief! I’ve reposted the link on my facebook page with the following comment “Are you sheeple or people? Vote this f**ckers out at the next election. Why? because this could happen to you!”
I think that Poe’s Law these days has come more generally to mean anything that can’t been identified as satire because it could be real, extremism or not! Just like the UK Government. Another reason I ran away to New Zealand!
I to am not shocked I should be but lets just say de sensatisation properganda in full force . But is it wrong 1001% what society will our children friends ect grow up in if this dose become the norm . ??? Not one i wish to be apart of . gross
I got my fit for work letter today ive claimed ESA for less than 20wks my white cells are abnormal they rushed me through atos quicker than NHS test what employer will take me on six months to live ????? more blood tezts ect while as a single mum applying for jobs ect Im sure the pure stress will do me wonders x
your not thick hun its me being sarcastic ..lol how long or what ive got still on going ..like my friend i survived treatment in 2007.reason for ESA claim was ongoing tests but yes today im fit for work same day white blood cells abnormal .
debs
oh,i get what you mean….atos miracles…a surreal sideshow of benefit games…now you see them now you don’t, eventually one ceases to be shocked and a numb desensitisation takes over…our vitals of life in their hands…
Mt wife has blood cancer I cannot believe what this government is doing and most people are unaware of this they want to make out all people on sick benefit are scroungers I do hope your condition goes into remission
thankyou Anthony.
If I’d of known back in the day I would of take out a life cover insurence although it would of been a struggle but to pull the rug from under people last minute when there isnt a thing you can do about it is almost criminal .
Having to sign on look for work go to interwiews while attending blood test doctor appointments hospital appointments as well as care for my young child and being tierd all the time is an extreme situation that Im praying I can find the strength for.
It’s all very well complaining about how unfair and immoral this Work Capability Assessment system is, and all caring people will agree, but does anyone have any idea about how to get rid it? Voting Labour isn’t going to help, because they introduced it. Obviously voting Tory or Lib Dem isn’t. So what’s left?
This is getting worse! IDS has been recalled for more questions on Monday 3rd February but he’ll get away with it again. The man should be hung, drawn and quartered – but if not, he should be sacked!
I don’t know how the Atos demonstrations will go later this month but the one thing that we can do is keep on writing to him and to MP’s and Lords.
One thing I do know is that none of my facebook friends are interested any more – that disgusts me and I’ll be closing the stupid account soon. I cannot understand how so called friends can turn away from the messages and the petitions when they know just how I am with my disabilities – they have seen the daily struggles myself and my husband go through each and every day yet all they care about is themselves. They know damn well that it could be them today or tomorrow in the same situation, but they close their eyes to it! Sickening!
OBD – I think you’re right that if the whole medical profession was united about this then the government might listen (though I wouldn’t stake my life on it!). But I’m afraid they are not united, because if they were then I think they would have done something already.
I have no answers to this… other than move to Scotland. By the way, did you know that the devolved Scottish Parliament is going to ask Westminster for the powers to repeal the bedroom tax in Scotland? Westminster will say f*ck off, but at least they’re trying. In the long term my faint hope is that an independent Scotland can show the rest of the UK the way to go.
Reblogged this on Beastrabban’s Weblog and commented:
More evidence that this government is a sick travesty, just as IDS and the DWP are shabby caricatures of human beings. It’s no point asking how low they can go, as just when you think they really, really can’t go any further, they go just that little bit deeper beyond any semblance of humanity or rationality or even basic decency …
I used to live in England, and I’m convinced that on average the people up here just have their heads screwed on better! It’s clearly reflected in how they vote. Also, if there’s something which has to be done, they just roll up their sleeves and get on with it. I don’t know why there’s such a difference between England and Scotland, but there is.
labour also opened 90 odd remploy factories which enabled disabled people to live independently, look at who shut them all down, thus causing a lot of hardship!! I’ll give you a clue: It wasn’t Labour!
I’ll quote a friend who I think knows what he’s talking about – “There are just more w@nkers down south”. Not as poetic as Burns, but still true!
Something significant changes over the border, and like you I really don’t know what ‘it’ is. The political, moral and practical differences are plain to see for someone like me who moved during adulthood. Unless you have seen the differences for yourself then maybe you can’t understand it. Of course not everything is good, far from it, but I have a fundamental confidence in the people of this country which I NEVER had in England.
Joanna – Are you kidding??? The Remploy closures began in 2007! 30 were closed in 2008 under a LABOUR government, throwing 2,500 disabled people out of work! This current government has only carried what the Blair government started.
I grow my own tatties and much more besides. I go fishing (including on Sundays) and I eat what I catch. You should try my smoked trout! I burn wood grown on my own land to heat my house. I support my local economy and Scotland as a whole. I’m sure most Scots would call me English, but I feel something about this country and this people which I never felt in England.
@ FinkFurst I’ve not forgotten who introduced it; but introducing something is not the same as implementing it to such a degree that people who go through the process and who are already poorly suffer in this way and in some cases (according to DWP own findings) die as a result. | 2024-02-14T01:27:17.760571 | https://example.com/article/5885 |
In a pervasive computing environment, the availability of accurate and up-to-date maps of buildings and their internal spaces is essential for enabling location based commercial and emergency services. However, these maps are expensive to make and maintain. Traditional methods of mapping buildings rely on blue prints or drawings. Some problems with prior art methods are the difficulty of obtaining an accurate map to begin with, and the high cost of maintaining an accurate map over time. For example, employees in an office building frequently change their office or cubicle locations, certain offices may be physically eliminated, corridors may be added or removed, and new offices are continually added. The net result is that using physical maps to route users through a building will nearly always be inaccurate and out of date. The problem is especially acute in large, multi-story buildings such as government offices, corporate headquarters, etc. New employees or visitors who are not familiar with the layout of a facility have a particularly difficult time navigating these buildings. Although navigational systems for use in outdoor environments, such as hiking, driving, etc. have been commercially available, they all rely on manually digitized maps that are input to the system. While the input of such a large amount of data is a daunting task, it is a task that only needs to be performed once, as buildings, roads, cities, contains and other large physical structures in the outdoor environment rarely, if ever, move or disappear. However, this is not the case for the indoor environment, where scale is smaller and geography is dynamic. It would be a desired addition to the art if an easy, inexpensive method of dynamically creating map data could be used to guide individuals through buildings.
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity showing only those specific details that are pertinent to understanding the embodiments of the present invention. | 2024-07-23T01:27:17.760571 | https://example.com/article/2500 |
[Articular and muscular manifestations of bacterial endocarditis. 11 cases (author's transl)].
We report 11 cases of bacterial endocarditis with muscular and articular manifestations seen over the past ten years. There was arthralgia in 7 cases, vertebral pain in 7 cases and myalgia in 3 cases. Arthritis consisted of a monoarthritis of the ankle in 2 cases and oligoarthritis in 2 cases. There were also 2 cases of lumbar spondylodiscitis and 1 of finger clubbing in the series. The underlying heart disease was a valvular lesion of the left side of the heart in 10 cases out of 11 and the organism isolated by blood culture was a streptococcus in 9 cases and a staphylococcus in 11. We emphasis the need for early diagnosis and appropriate antibiotic therapy, in the absence of which the course may be fatal in the short term, as it was the case in one of our own patients. | 2023-08-19T01:27:17.760571 | https://example.com/article/4040 |
All relevant data and instructions to repeat the simulations are within the paper. The weather data can be obtained from [www.sws.uiuc.edu/warm](http://www.sws.uiuc.edu/warm). A synthetic data set is also included as supplemental material.
Introduction {#sec001}
============
Genomic selection \[[@pone.0130855.ref001]\], enabled by whole genome prediction (WGP) methods, is revolutionizing plant breeding \[[@pone.0130855.ref002]\]. Since its inception, attempts to improve prediction accuracy have focused on: developing improved and specialized statistical models \[[@pone.0130855.ref003]--[@pone.0130855.ref006]\], increasing the marker density used \[[@pone.0130855.ref007]--[@pone.0130855.ref009]\], increasing the size and defining optimal designs of estimation sets \[[@pone.0130855.ref010]--[@pone.0130855.ref013]\] and better understanding the genetic determinants driving prediction accuracy \[[@pone.0130855.ref014], [@pone.0130855.ref015]\].
In-silico phenotypic prediction, enabled by dynamic crop growth models (CGMs), dates back to the late 1960's \[[@pone.0130855.ref016]\] and it has constantly evolved through inclusion of scientific advances made in plant physiology, soil science and micrometeorology \[[@pone.0130855.ref016], [@pone.0130855.ref017]\]. CGMs used in plant breeding are structured around concepts of resource capture, utilization efficiency and allocation among plant organs \[[@pone.0130855.ref018]--[@pone.0130855.ref021]\] and are used to: characterize environments \[[@pone.0130855.ref022], [@pone.0130855.ref023]\], predict consequences of trait variation on yield within a genotype × environment × management context \[[@pone.0130855.ref024]\], evaluate breeding strategies \[[@pone.0130855.ref025]--[@pone.0130855.ref027]\], and assess hybrid performance \[[@pone.0130855.ref002]\].
Early attempts to extend the use of CGMs to enable genetic prediction have focused on developing genetic models for parameters of main process equations within the CGM \[[@pone.0130855.ref021], [@pone.0130855.ref028], [@pone.0130855.ref029]\]. Linking quantitative trait locus (QTL) models and CGMs for complex traits motivated adapting CGMs to improve the connectivity between physiology and genetics of the adaptive traits \[[@pone.0130855.ref021], [@pone.0130855.ref027], [@pone.0130855.ref030]\]. However, despite a tremendous body of knowledge and experience, CGMs were largely ignored for the purpose of WGP.
There is ample evidence for the importance of epistasis in crops, including for economically important traits such as grain yield in maize \[[@pone.0130855.ref031]--[@pone.0130855.ref033]\]. Yield and other complex traits are the product of intricate interactions between component traits on lower hierarchical levels \[[@pone.0130855.ref019], [@pone.0130855.ref034]--[@pone.0130855.ref037]\]. If the relationship among the underlying component traits is nonlinear, epistatic effects can occur on the phenotypic level of complex traits even if the gene action is purely additive when characterized at the level of the component traits \[[@pone.0130855.ref033]\]. This phenomenon was first described for multiplicative relationships among traits by Richey \[[@pone.0130855.ref038]\] and later quantified by Melchinger et al. \[[@pone.0130855.ref039]\]. CGMs, which explicitly model these nonlinear relationships among traits, have therefore the potential to open up novel avenues towards accounting for epistatic effects in WGP models by explicit incorporation of biological knowledge.
The target population of environments for plant breeding programs is subject to continuous re-evaluation \[[@pone.0130855.ref002]\]. To select for performance in specific environments, genotype by environment (G×E) interactions have to be predicted. Genomic prediction of G×E interactions is therefore of great interest for practical applications of breeding theory. Previous attempts incorporated G×E interactions in WGP models through environment specific marker effects \[[@pone.0130855.ref040]\] or genetic and environmental covariances \[[@pone.0130855.ref041]\]. Later Jarquín et al. \[[@pone.0130855.ref042]\] and Heslot et al. \[[@pone.0130855.ref043]\] developed WGP models that accounted for G×E interactions by means of environmental covariates.
While these previous attempts are promising, they are purely statistical in nature and do not leverage the substantial biological insights into the mechanisms determining performance in specific environments. CGMs are an embodiment of this biological knowledge and might serve as a key component in novel WGP models for predicting G×E interactions. In fact, Heslot et al. \[[@pone.0130855.ref043]\] recognized this potential for CGMs. However, they employed them only for computing stress covariates from environmental data, which were subsequently used as covariates in purely statistical WGP models.
Given the potential merits of integrating CGMs in WGP, the question arises of how to combine the two in a unified predictive system. The ever increasing computational power of modern computing environments allows for efficient simulation from the most complex of models, such as CGMs \[[@pone.0130855.ref027]\]. This computational power is leveraged by *approximate Bayesian computation* (ABC) methods, which replace the calculation of a likelihood function with a simulation step, and thereby facilitate analysis when calculation of a likelihood function is impossible or computationally prohibitive. ABC methods were developed in population genetics, where they helped solve otherwise intractable problems \[[@pone.0130855.ref044]--[@pone.0130855.ref047]\]. However, ABC methods were rapidly adopted in other scientific fields, such as ecology \[[@pone.0130855.ref048]\], systems biology \[[@pone.0130855.ref049]\] and hydrology \[[@pone.0130855.ref050]\]. Recently, Marjoram et al. \[[@pone.0130855.ref051]\] proposed using ABC methods for incorporating the biological knowledge represented in gene regulatory networks into genome-wide association studies, arguing that this might present a solution to the 'missing heritability' problem.
Here we make the case that ABC may hold great promise for enabling novel approaches to WGP as well. Thus, the objective of this study is to provide a proof of concept, based on synthetic data sets, for using ABC as a mechanism for incorporating the substantial biological knowledge embodied in CGMs into a novel WGP approach.
Materials and Methods {#sec002}
=====================
CGM and environmental data {#sec003}
--------------------------
We used the maize CGM developed by Muchow et al. \[[@pone.0130855.ref052]\], which models maize grain yield development as a function of plant population (PPOP, plants *m* ^−2^), daily temperature (°*C*) and solar radiation (*MJ* *m* ^−2^) as well as several genotype dependent physiological traits. These traits were total leaf number (TLN), area of largest leaf (AM), solar radiation use efficiency (SRE) and thermal units to physiological maturity (MTU). Details on the calculation of trait values for the genotypes in the synthetic data set are provided later. However, the values used were within typical ranges reported in the literature. The simulated intervals for TLN, AM, SRE and MTU were \[6, 23\] \[52, 53\], \[700, 800\] \[52, 54\], \[1.5,1.7\] \[55\] and \[1050, 1250\] \[56--58\], respectively, with average values at the midpoints of the intervals.
We chose Champaign/Illinois (40.08° N, 88.24° W) as a representative US Corn Belt location. Temperature and solar radiation data were obtained for the years 2012 and 2013 (Data provided by the Water and Atmospheric Resources Monitoring Program, a part of the Illinois State Water Survey (ISWS) located in Champaign and Peoria, Illinois, and on the web at [www.sws.uiuc.edu/warm](http://www.sws.uiuc.edu/warm)). The sowing date in 2012 was April 15th and in 2013 it was May 15th. We modified the original CGM of Muchow et al. \[[@pone.0130855.ref052]\] by enforcing a maximum length of the growing season, after which crop growth simulation was terminated, regardless of whether the genotype reached full physiological maturity or not. The length of the growing season in 2012 was 120 days from sowing and in 2013 it was 130 days from sowing. Both durations are within the range typically observed in the US Corn Belt \[[@pone.0130855.ref059]\]. In 2012 PPOP was 8 plants *m* ^−2^ and in 2013 PPOP was 10 plants *m* ^−2^. The 2012 and 2013 environments therefore differed not only in temperature and solar radiation but also in management practices. The temperature and solar radiation from date of sowing is shown in [Fig 1](#pone.0130855.g001){ref-type="fig"}. Typical total biomass and grain yield development curves for early, intermediate and late maturing genotypes in the 2012 and 2013 environments are shown in [Fig 2](#pone.0130855.g002){ref-type="fig"} and corresponding curves for development of total and senescent leaf area in [S1 Fig](#pone.0130855.s003){ref-type="supplementary-material"}.
{#pone.0130855.g001}
{#pone.0130855.g002}
The CGM can be viewed as a function *F* of the genotype specific inputs (the physiological traits) and the environment data $$F\left( y_{TLN_{i}},y_{SRE_{i}},y_{AM_{i}},y_{MTU_{i}},\Omega_{k} \right)$$ where *y* ~*TLN*~*i*~~ etc. are the values of the physiological traits observed for the *i* ^*th*^ genotype and the weather and management data of environment *k* are represented as Ω~*k*~. To simplify notation, we will henceforth use *F*(⋅)~*ik*~ to represent the CGM and its inputs for genotype *i* in environment *k*.
Approximate Bayesian Computation (ABC) {#sec004}
--------------------------------------
ABC replaces likelihood computation with a simulation step \[[@pone.0130855.ref044]\]. An integral component of any ABC algorithm is therefore the simulation model operator $\text{Model}(y_{ik}^{*} \mid \theta)$ which generates simulated data $y_{ik}^{*}$ given parameters *θ*. In our proof of concept study, the crop growth model *F*(⋅)~*ik*~ represents the deterministic component of $\text{Model}(y_{ik}^{*} \mid \theta)$, to which a Gaussian noise variable distributed as $\mathcal{N}(0,\sigma_{e}^{2})$ is added as a stochastic component. If $\text{Model}(y_{ik}^{*} \mid \theta)$ is fully deterministic, the distribution sampled with the ABC algorithm will not converge to the true posterior distribution when the tolerance for the distance between the simulated and observed data goes to zero \[[@pone.0130855.ref050]\].
The weather and management data Ω~*k*~ was assumed to be known, the physiological traits, however, were unknown and treated as latent or hidden variables, that were modeled as linear functions of the trait specific marker effects $$\begin{aligned}
y_{TLN_{i}} & {= \mu_{TLN} + \mathbf{z}_{i}\mathbf{u}_{TLN}} \\
y_{AM_{i}} & {= \mu_{AM} + \mathbf{z}_{i}\mathbf{u}_{AM}} \\
y_{SRE_{i}} & {= \mu_{SRE} + \mathbf{z}_{i}\mathbf{u}_{SRE}} \\
y_{MTU_{i}} & {= \mu_{MTU} + \mathbf{z}_{i}\mathbf{u}_{MTU},} \\
\end{aligned}$$ where **z** ~*i*~ is the genotype vector of the observed biallelic single nucleotide polymorphism (SNP) markers of genotype *i*, *μ* ~*TLN*~ etc. denote the intercepts and **u** ~*TLN*~ etc. the marker effects. For brevity, we will use *θ* to denote the joint parameter vector \[*μ* ~*TLN*~,..., *μ* ~*MTU*~,**u** ~*TLN*~,...,**u** ~*MTU*~\].
We used independent Normal distribution priors for all components of *θ*. The prior for *μ* ~*TLN*~ was $\mathcal{N}(m_{TLN},\sigma_{\mu_{TLN}}^{2})$. To simulate imperfect prior information, we drew the prior mean *m* ~*TLN*~ from a Uniform distribution over the interval $\lbrack 0.8 \cdot \overline{TLN},1.2 \cdot \overline{TLN}\rbrack$, where $\overline{TLN}$ is the observed population mean of TLN. The average difference between *m* ~*TLN*~ and $\overline{TLN}$ then is 10% of the latter value. The prior variance $\sigma_{\mu_{TLN}}^{2}$, which represents the prior uncertainty, was equal to 2.25^2^. The prior means of AM, SRE and MTU were obtained accordingly and the prior variances $\sigma_{\mu_{AM}}^{2}$, $\sigma_{\mu_{SRE}}^{2}$ and $\sigma_{\mu_{MTU}}^{2}$ were 150^2^, 0.3^2^ and 225^2^, respectively.
The prior for the marker effects **u** ~*TLN*~ was $\mathcal{N}(0,\sigma_{\mu_{TLN}}^{2})$, which corresponds to the *BayesC* prior \[[@pone.0130855.ref060]\]. In BayesC, the prior variance of marker effects $\sigma_{\mu_{TLN}}^{2}$, which introduces shrinkage, is the same across markers. For simplicity, we set this variance to a constant value and did not attempt to estimate it. Also in this case we simulated imperfect information by drawing the value of $\sigma_{\mu_{TLN}}^{2}$ from a Uniform distribution over the interval \[0.8 ⋅ *var*(*TLN*)/*M*,1.2 ⋅ *var*(*TLN*)/*M*\], where *M* is the number of markers and *var*(*TLN*) the observed population variance of TLN. The prior variances of marker effects of the other traits were obtained accordingly.
The value of $\sigma_{e}^{2}$, the variance of the Gaussian noise variable that is part of the model operator $\text{Model}(y_{ik}^{*} \mid \theta)$, was drawn from a Uniform distribution over the interval \[0.8 ⋅ *v* ~*e*~,1.2 ⋅ *v* ~*e*~\], where *v* ~*e*~ is the residual variance component of the phenotypic grain yield values used to fit the model.
Algorithm 1 in [Table 1](#pone.0130855.t001){ref-type="table"} shows pseudocode for the ABC rejection sampling algorithm we used. As distance measure between the simulated and observed data we used the Euclidean distance. The tolerance level *ϵ* for the distance between the simulated and observed data was tuned in a preliminary run of the algorithm to result in an acceptance rate of approximately 1 ⋅ 10^−6^. The number of posterior samples drawn was 100. We will refer to this ABC based WGP method that incorporates the CGM as *CGM-WGP*. The CGM-WGP algorithm was implemented as a C routine integrated with the R software environment \[[@pone.0130855.ref061]\].
10.1371/journal.pone.0130855.t001
###### Pseudocode of ABC rejection sampling algorithm.
{#pone.0130855.t001g}
-----------------------------------------------------------------------------------------
**while** *x* \<= no. posterior samples **do**
**while** *d* \> *ϵ* **do**
draw candidate *θ*\* from prior(*θ*)
**for All** *i* = 1, 2, ..., *N* **do**
generate simulated data $y_{ik}^{*}$ from $\text{Model}(y_{ik}^{*} \mid \theta^{*})$
**end for**
compute $d = \sqrt{\sum_{i = 1}^{N}{(y_{ik} - y_{ik}^{*})}^{2}}$
**end while**
accept and store *θ*\*
increment *x*
**end while**
-----------------------------------------------------------------------------------------
Basic ABC rejection sampling algorithm to sample from the approximate posterior distribution of *θ*.
Synthetic data set {#sec005}
------------------
To test the performance of CGM-WGP, we created a biparental population of 1,550 doubled haploid (DH) inbred lines in silico. The genome consisted of a single chromosome of 1.5 Morgan length. The genotypes of the DH lines were generated by simulating meiosis events with the software package hypred \[[@pone.0130855.ref062]\] according to the Haldane mapping function. On the chromosome, we equidistantly placed 140 informative SNP markers. A random subset of 40 of these markers were assigned to be QTL with additive effects on either TLN, AM, SRE or MTU. Each physiological trait was controlled by 10 of the 40 QTL, which were later removed from the set of observed markers available for analysis.
The additive substitution effects of the QTL were drawn from a Standard Normal distribution. Raw genetic scores for each physiological trait were computed by summing the QTL effects according to the QTL genotypes of each DH line. These raw scores were subsequently re-scaled linearly to the aforementioned value ranges. Finally, phenotypic grain yield values were created as $$y_{ik} = F\left( \cdot \right)_{ik} + e_{ik},$$ where *e* ~*ik*~ is a Gaussian noise variable with mean zero and variance *v* ~*e*~. The value of *v* ~*e*~ was chosen such that the within-environment heritability of *y* ~*ik*~ was equal to 0.85. We generated 50 synthetic data sets by repeating the whole process. An example synthetic data set is available as supplemental material ([S1 Dataset](#pone.0130855.s002){ref-type="supplementary-material"}).
Estimation, prediction and testing procedure {#sec006}
--------------------------------------------
The models were fitted using *N* = 50 randomly chosen DH lines as an estimation set. The remaining 1500 DH lines were used for testing model performance. Separate models were fitted using the 2012 and the 2013 grain yield data of the estimation set lines. The environment from which data for fitting the model was used will be referred to as *estimation environment*. Parameter estimates from each estimation environment were subsequently used to predict performance of the lines in the test set in both environments. Predictions for the same environment as the estimation environment will be referred to as *observed environment predictions* (e.g., predictions for 2012 with models fitted with 2012 data). Predictions for an environment from which no data were used in fitting the model will be referred to as *new environment predictions* (e.g., predictions for 2013 with models fitted with 2012 data).
As a point estimate for predicted grain yield performance in a specific environment, we used the mean of the posterior predictive distribution for the DH line in question. The posterior predictive distribution was obtained by evaluating *F*(⋅)~*ik*~ over the accepted *θ* samples, using the weather and management data Ω~*k*~ pertaining to that environment.
Prediction accuracy was computed as the Pearson correlation between predicted and true performance in the environment for which the prediction was made. The true grain yield performance was obtained by computing *F*(⋅)~*ik*~ with the true values of the physiological traits.
As a performance benchmark we used genomic best linear unbiased prediction (GBLUP \[[@pone.0130855.ref001]\]). The model is $$y_{ik} = \beta_{0} + \mathbf{z}_{i}\mathbf{u} + e_{i}$$ where *β* ~0~ is the intercept, **u** the vector of marker effects and *e* ~*i*~ a residual. As before, **z** ~*i*~ denotes the marker genotype vector. The GBLUP model was fitted with the R package rrBLUP \[[@pone.0130855.ref063]\]. GBLUP and BayesC are comparable in their shrinkage behavior because both use a constant variance across markers. For GBLUP, predicted values were computed according to [Eq (4)](#pone.0130855.e025){ref-type="disp-formula"} as *β* ~0~+**z** ~*i*~ **u**. Note that because the conventional GBLUP model does not utilize information about the environment for which predictions are made, observed and new environment predictions are identical.
Results and Discussion {#sec007}
======================
Predicting performance in observed environments {#sec008}
-----------------------------------------------
The accuracy of observed environment predictions achieved by CGM-WGP was considerably larger than that of the benchmark method GBLUP in both environments ([Table 2](#pone.0130855.t002){ref-type="table"}, [Fig 3](#pone.0130855.g003){ref-type="fig"}, [S2 Fig](#pone.0130855.s004){ref-type="supplementary-material"}). This superiority of CGM-WGP over GBLUP can be explained by the presence of non-additive gene effects which cannot be captured fully by the latter. In the example scenario we studied, the non-additive gene effects on grain yield are a result of nonlinear functional relationships between the physiological traits and grain yield, which was particularly pronounced for TLN ([Fig 4](#pone.0130855.g004){ref-type="fig"}).
10.1371/journal.pone.0130855.t002
###### Accuracy of grain yield predictions of DH lines in the test set.
{#pone.0130855.t002g}
Estimation Env. Prediction Env. CGM-WGP GBLUP
----------------- ----------------- --------- -------
2012 2012 0.77 0.54
2013 0.48 0.10
2013 2012 0.42 0.08
2013 0.75 0.62
Prediction accuracy for grain yield of DH lines in the test set, averaged over 50 replications.
{#pone.0130855.g003}
{#pone.0130855.g004}
For any point in time (*t*) during the maize growth cycle, dry matter growth (DM~*g*~) results from the interception of solar radiation (SR) and its conversion into mass with efficiency SRE. Light interception in turn depends on the size of the canopy, which is determined by the leaf area per plant (LAPP) and the plant population (PPOP), and the distribution of light within the canopy, which is modeled using a coefficient of light extinction (*k*). The relationship between DM~*g*~, SR, SRE, LAPP and PPOP is non-linear \[[@pone.0130855.ref052]\] $$\text{DM}_{g_{t}} = \text{SR}_{t} \times \text{SRE} \times \left( 1 - e^{- k \times \text{LAPP}_{t} \times \text{PPOP}} \right).$$ Because LAPP is determined by TLN and AM \[[@pone.0130855.ref052]\], DM~*g*~ increases with increasing AM but only up to a point when canopy size maximizes light interception. Because grain yield is a fraction of the integral of DM~*g*~ over the growing season, there is a non-linear relationship between AM and grain yield, which can often be detected as a weak correlation ([S3 Fig](#pone.0130855.s005){ref-type="supplementary-material"}). From [Eq (5)](#pone.0130855.e026){ref-type="disp-formula"} one can see that an increase in SRE is always beneficial for DM~*g*~, which was reflected by the more or less linear relationship between SRE and grain yield ([S3 Fig](#pone.0130855.s005){ref-type="supplementary-material"}). The longer the length of the period between silking and physiological maturity, the more time the genotype has for grain filling. However, the end of the growing season can forestall exploitation of the longer grain filling periods of high MTU genotypes. Thus, increasing MTU beyond a point determined by the growing season length will have no further effect on grain yield. Such a saturation curve was indeed observed in 2013 ([S3 Fig](#pone.0130855.s005){ref-type="supplementary-material"}). The relationship between MTU and grain yield tended to be less clear in 2012, where many genotypes did not reach physiological maturity because of the shorter growing season. The relationships between grain yield and the physiological traits AM, SRE and MTU, were generally weak, however, and not obvious in all data sets. We will therefore focus on discussing the relationship between grain yield and TLN, which was very distinct and consistent.
TLN is closely related with the maturity rating of genotypes \[[@pone.0130855.ref052]\]. The higher it is, the later the onset of the reproductive phase and the later the maturity. Late genotypes have a higher yield potential than earlier genotypes because of a greater leaf area ([S1 Fig](#pone.0130855.s003){ref-type="supplementary-material"}). However, if the growing season is too short, they cannot realize this yield potential because of their slower development and later onset of the generative phase ([Fig 2](#pone.0130855.g002){ref-type="fig"}). Very early genotypes on the other hand, have a low leaf area and do not make use of the full growing season. As a consequence, their realized yield is low, too. The relationship between TLN and grain yield therefore follows an optimum curve ([Fig 4](#pone.0130855.g004){ref-type="fig"}). This was particularly pronounced in 2012, which had the shorter growing season and therefore penalized the late maturing genotypes more. The more decidedly nonlinear relationship between grain yield and TLN in 2012 also explains why the difference in prediction accuracy between CGM-WGP and GBLUP was greater in this season than in 2013 (0.23 points in 2012 compared to 0.13 points in 2013, on average).
The scenario we studied is an example of a particular case of epistasis, which might be called *biological epistasis*, that can arise even if the gene effects on the physiological component traits underlying the final trait of interest (grain yield in our case) are purely additive \[[@pone.0130855.ref033]\]. We accounted for nonlinear functional relationships among traits with the CGM. This enabled us to capture biological epistasis through simple linear models relating marker genotypes to the unobserved underlying physiological traits. Previously developed WGP models attempted to capture epistasis by directly fitting nonlinear marker effects to the final trait of interest \[[@pone.0130855.ref064]--[@pone.0130855.ref066]\]. While these models showed some promise, they have not been adopted by practitioners on a larger scale. By combining statistics with biological insights captured by CGMs, CGM-WGP takes a fundamentally different approach and presents a potentially powerful alternative to purely statistical WGP models.
Predicting performance in new environments {#sec009}
------------------------------------------
New environment prediction accuracy was considerably lower than observed environment prediction accuracy, for both prediction methods ([Table 2](#pone.0130855.t002){ref-type="table"}, [Fig 3](#pone.0130855.g003){ref-type="fig"}, [S2 Fig](#pone.0130855.s004){ref-type="supplementary-material"}). The average prediction accuracy for performance in 2012 when using the 2013 estimation environment was 54% (CGM-WGP) and 15% (GBLUP) of the respective prediction accuracy achieved when using the 2012 estimation environment. The corresponding values for the accuracy of predicting performance in 2013 were 64% (CGM-WGP) and 16% (GBLUP). Thus, CGM-WGP still delivered a decent accuracy for predicting performance in new environments, while GBLUP largely failed in this task. The prediction accuracy of GBLUP was in fact negative, and sometimes strongly so, for close to 50% of the synthetic data sets ([S2 Fig](#pone.0130855.s004){ref-type="supplementary-material"}). For CGM-WGP negative accuracies were observed in only 14% (2012) and 4% (2013) of the cases.
The genetic rank correlation between true performance in 2012 and 2013 was only 0.54 (averaged over 50 synthetic data sets), which indicated the presence of considerable G×E interactions, including changes in rank ([S4 Fig](#pone.0130855.s006){ref-type="supplementary-material"}). A genetic correlation of 0.54 between environments is within the range typically observed in plant breeding data sets \[[@pone.0130855.ref067]\] and crossover interaction between environments is a common phenomenon in plant breeding \[[@pone.0130855.ref034], [@pone.0130855.ref068], [@pone.0130855.ref069]\].
The interaction between the environment and TLN again explains the occurrence of G×E to a large degree. In the shorter 2012 season, the late maturing genotypes cannot realize their growth and yield potential and are outperformed by the genotypes with early and intermediate maturity (Figs [2](#pone.0130855.g002){ref-type="fig"} and [4](#pone.0130855.g004){ref-type="fig"}). In the 10 day longer growing season of 2013, however, the late maturing genotypes can realize their greater yield potential better and outperform the early maturing genotypes and have a similar performance as genotypes with intermediate maturity. This dynamic leads to crossover G×E interactions between the 2012 and 2013 environments.
That new environment prediction under the presence of G×E interaction is considerably less accurate than observed environment prediction was expected and already observed in other studies \[[@pone.0130855.ref011], [@pone.0130855.ref070]\]. It is encouraging that the reduction in accuracy for CGM-WGP was considerably less severe than for the conventional benchmark method GBLUP because this indicates that the former method did succeed in predicting G×E interactions to some degree.
Predicting G×E interactions in new environments for which no yield data are available, requires WGP models that link genetic effects (e.g., marker effects) with information that characterizes the environments. Jarquín et al. \[[@pone.0130855.ref042]\] accomplished this by fitting statistical interactions between markers and environmental covariates. A similar approach was taken by Heslot et al. \[[@pone.0130855.ref043]\], who in addition used a CGM to extract stress covariates from a large set of environmental variables. CGM-WGP takes this approach a step further by making the CGM and the environmental data that inform it, an integral part of the estimation procedure.
Nonetheless, while novel prediction methods might succeed in narrowing the gap between new and observed environment prediction, the former should always be expected to be less accurate than the latter. Field testing should therefore be performed in environments of particular importance for a breeding program to achieve the maximum attainable prediction accuracy for these. The same applies for target environments in which G×E interaction effects are expected to be particularly strong. CGMs can help to identify such environments and to inform experimental design and utilization of managed environments \[[@pone.0130855.ref027], [@pone.0130855.ref029]\]. However, the range of the target population of environments of modern plant breeding programs is much too large for yield testing across the whole breadth \[[@pone.0130855.ref002]\]. Predicting performance in new environments will therefore always be required and novel methods like CGM-WGP are anticipated to be instrumental for enabling and enhancing success in this particularly daunting task.
Areas of further research and development {#sec010}
-----------------------------------------
### Alternatives to CGM-WGP {#sec011}
With continual technology improvements for phenotyping traits it is becoming increasingly feasible to assay phenotypic variation for many of the physiological traits underlying the CGM \[[@pone.0130855.ref019]\]. There would then be no need to treat them as latent, hidden variables as in CGM-WGP. Such improved precision phenotyping capabilities thus open up possible alternatives and extensions to the CGM-WGP methodology introduced here.
One alternative that can be considered is a two-step procedure, in which (1) physiological traits are predicted based on QTL identified in dedicated mapping experiments and (2) the so obtained physiological trait values are used to parametrize CGMs and predict the expected yield performance of novel genotypes in the same or different environments \[[@pone.0130855.ref071]--[@pone.0130855.ref073]\]. Using WGP instead of QTL mapping in step 1, could further enhance that procedure.
One shortcoming of this approach is that all relevant physiological traits have to be measured for all genotypes in the estimation set. This may prove to be unfeasible in practice, particularly when done on an industrial scale (i.e., for many populations and repeated year after year). The situation is exacerbated when more sophisticated models like APSIM \[[@pone.0130855.ref074]\], which can model plant-soil interactions related to water and nutrient uptake, are used. The set of relevant physiological traits for these CGMs includes root traits for example \[[@pone.0130855.ref075]\], which are particularly difficult to measure in a routine, high-throughput fashion \[[@pone.0130855.ref076]\].
A key novelty of CGM-WGP is that it can accommodate partially or fully unobserved physiological traits by treating them as hidden variables. It could thus facilitate incorporating a CGM in WGP even when phenotyping all relevant physiological traits is not feasible.
However, CGM-WGP and the described two-step approach have a common objective, which is to apply a suitable CGM to capture non-linear relationships among traits and the environment to succeed in the crucial but challenging task of predicting yield in future environments. At this stage it is premature to suggest one approach ahead of these or other possibilities. However, the results of the present study indicate that there are opportunities to improve predictions for quantitative traits influenced by epistasis and G × E interaction, through effective integration of appropriate CGMs into the genetic prediction methodology.
### More sophisticated CGMs {#sec012}
For this first proof of concept study, we assumed that the CGM used in the estimation process fully represented the systematic component of the data generating process, besides the random noise. This was clearly a 'best case scenario'. However, decades of crop growth modeling research have provided the know-how necessary to approximate real crop development to a high degree of accuracy \[[@pone.0130855.ref017], [@pone.0130855.ref030], [@pone.0130855.ref077]\]. Advanced CGMs such as *APSIM*\[[@pone.0130855.ref074]\], for example, model functional relationships between various crop parameters and external factors such as water and nutrient availability, soil properties as well as weed, insect and pathogen pressure. Thus, tools are principally available for applying CGM-WGP in more complex scenarios than the one addressed in this study.
With multiple possible CGMs to choose from, model selection becomes an issue. The ABC algorithm underlying CGM-WGP could in principle be used to perform model selection simultaneously with parameter estimation \[[@pone.0130855.ref049], [@pone.0130855.ref051], [@pone.0130855.ref078]\]. It could thus provide a statistically formal way of comparing the fit of several CGMs.
### Stochastic CGMs {#sec013}
There are examples of the use of fully deterministic model operators in ABC \[[@pone.0130855.ref078], [@pone.0130855.ref079]\]. However, with fully deterministic model operators the sampled distribution would not converge to the true posterior when the tolerance level *ϵ* goes to zero \[[@pone.0130855.ref050]\] and instead reduce to a point mass over those parameter values that can reproduce the data. The CGM we used was fully deterministic. We therefore followed the example of Sadegh and Vrugt \[[@pone.0130855.ref050]\], who constructed a stochastic model operator by adding a random noise variable, with the same probabilistic properties as assumed for the residual component of the phenotype, to the deterministic functional model. A more elegant and possibly superior solution, however, would be to integrate stochastic processes directly into the CGM. While the vast majority of CGMs are deterministic \[[@pone.0130855.ref016], [@pone.0130855.ref017]\], there are examples of stochastic CGMs \[[@pone.0130855.ref080]\]. In addition to incorporating inherently stochastic processes of development \[[@pone.0130855.ref081]\], stochastic CGMs could also serve to account for uncertainty in the parameters of the functional equations comprising the model \[[@pone.0130855.ref082]\].
### Advanced ABC algorithms {#sec014}
For this proof of concept study we used the basic ABC rejection sampling algorithm \[[@pone.0130855.ref044], [@pone.0130855.ref045]\]. Considerable methodology related advances have been made, however, over the last decade that have led to algorithms with improved computational efficiency. Of particular interest here are population or sequential Monte Carlo algorithms, which are based on importance sampling \[[@pone.0130855.ref078], [@pone.0130855.ref083], [@pone.0130855.ref084]\]. These algorithms can dramatically increase acceptance rates without compromising on the tolerance levels. They achieve this by sampling from a sequence of intermediate proposal distributions of increasing similarity to the target distribution. Unfortunately, importance sampling fails when the number of parameters gets large, because then the importance weights tend to concentrate on very few samples, which leads to an extremely low effective sample size \[[@pone.0130855.ref085]\]. In the context of sequential Monte Carlo, this is known as particle depletion and was addressed by Peters et al. \[[@pone.0130855.ref084]\]. We implemented their approach, but were not able to overcome the problem of particle depletion. The number of parameters we estimated was 404 (100 marker effects per physiological trait plus an intercept), which seems well beyond the dimensionality range for importance sampling \[[@pone.0130855.ref085]\].
Another interesting development is *MCMC-ABC*, which incorporates ABC with the Metropolis-Hastings algorithm \[[@pone.0130855.ref086]\]. MCMC-ABC should result in high acceptance rates if the sampler moves into parameter regions of high posterior probability. However Metropolis-Hastings sampling too can be inefficient when the parameter space is of high dimension.
The greatest computational advantage of the original ABC rejection algorithm over Monte Carlo based ABC methods is that it generates independent samples and therefore readily lends itself to 'embarrassingly' parallel computation \[[@pone.0130855.ref086]\]. The computation time thus scales linearly to the number of processors available. Using the ABC rejection algorithm therefore allowed us to fully leverage the high performance computing cluster of DuPont Pioneer. In the era of cloud computing \[[@pone.0130855.ref087]\], high performance computing environments are readily available to practitioners and scientists in both public and private sectors. Generality, scalability to parallel computations, and ease of implementation make the basic rejection sampler a viable alternative to more sophisticated approaches.
### Using prior information {#sec015}
We used mildly informative prior distributions, the parameters of which were derived from the population means and variances of the physiological traits. In practice, the required prior information must be obtained from extraneous sources, such as past experiments or from the literature \[[@pone.0130855.ref080]\]. Such information is imperfect and only partially matches the true population parameters of the population in question. We determined the prior parameters from the population itself, but perturbed them considerably to simulate erroneous prior information. Specifically, the average relative discrepancy (bias) between the prior parameter used and the true population parameter was 10%. When we increased the relative discrepancy to 25% (i.e., a maximum discrepancy of 50%), prediction accuracy dropped somewhat ([S1 Table](#pone.0130855.s001){ref-type="supplementary-material"}). The reduction was only slight for observed environment prediction but more pronounced for new environment prediction. However, CGM-WGP was still considerably more accurate than the benchmark GBLUP. Thus, CGM-WGP seems to be relatively robust to moderate prior miss specification, as long as the value range supported by the prior distribution is not out of scope. In the ideal case of no prior bias, on the other hand, new and observed environment prediction accuracy increased slightly as compared to a bias of 10%.
In the synthetic data sets we generated, the component traits were controlled by QTL with independent effects and thus uncorrelated. In practice, however, the traits might be correlated, because of pleiotropic QTL, for example. In this situation, marker effects are correlated too. We modeled the marker effects as *a priori* independent and note that this does not preclude posterior correlation if CGM-WGP in its current implementation is applied to a scenario with correlated component traits. However, it is possible to model whole genome marker effects as *a priori* correlated. This was explored for the purpose of fitting WGP models with breed specific but correlated marker effects in animal breeding \[[@pone.0130855.ref088]\]. Modeling a correlation structure could allow information sharing across traits. It could also improve computational efficiency of CGM-WGP, because the prior distribution would be closer to the posterior and thereby result in fewer rejected samples.
Lastly, CGM-WGP can be modified to use 'BayesB' \[[@pone.0130855.ref001]\] or 'BayesC*π*' \[[@pone.0130855.ref060]\] as priors of marker effects, which would allow marker effects to be exactly zero. By allowing the effects of markers to be zero, marker effect estimation and implicit SNP model selection are done simultaneously. This could present an interesting compromise between a continuous WGP approach (BayesC) and a discrete, QTL based approach to prediction.
### Number of markers {#sec016}
We applied CGM-WGP to a biparental population, which is by far the most common population type in commercial plant breeding programs \[[@pone.0130855.ref089]\]. Previous WGP studies found that marker density is typically not the most limiting factor in these type of populations and that densities achievable with around 200 genomwide markers suffice for accurate predictions \[[@pone.0130855.ref013], [@pone.0130855.ref090]--[@pone.0130855.ref092]\]. This proof of concept showed that computations for CGM-WGP are feasible for 100 markers. Thus, while more challenging, we expect that computations can be facilitated for the numbers of markers required for biparental populations. However, applying CGM-WGP to data sets with tens of thousands of markers is likely not possible with current ABC algorithms, in particular if more sophisticated CGMs are used that require specification of more physiological traits. Technow and Melchinger \[[@pone.0130855.ref012]\] showed that using a more realistic but complex WGP model with a lower marker density can result in a higher prediction accuracy than using a less realistic WGP model with a higher marker density. Thus, the greater realism of CGM-WGP might compensate for the fact that it can currently be applied only at low to intermediate marker densities.
In contrast to the complex trait of interest, component physiological traits may be realistically modeled based on a relatively simple genetic architecture, and for such traits, QTL explaining a sizable proportion of genetic variance can be mapped and characterized \[[@pone.0130855.ref071], [@pone.0130855.ref093]--[@pone.0130855.ref096]\]. In fact, such component trait QTL have been successfully used to parametrize CGMs for studying genotype dependent response to environmental conditions \[[@pone.0130855.ref028], [@pone.0130855.ref029], [@pone.0130855.ref073], [@pone.0130855.ref094], [@pone.0130855.ref095]\]. Knowledge about the location and effect of such QTL, or of transgenes \[[@pone.0130855.ref097]--[@pone.0130855.ref099]\], could be incorporated as an additional source of prior information. Then, instead of estimating marker effects for the whole genome, CGM-WGP could focus on genomic regions of particular importance. This reduces the dimensionality of the parameter space dramatically and enables CGM-WGP to be used in settings that traditionally required high marker densities, such as WGP in diverse germplasm \[[@pone.0130855.ref100]\].
### Identifiability {#sec017}
It is possible that the CGM generates the same yield for two or more sets of component trait values. There are often several possible biological strategies with equivalent outcome, so this does not necessarily indicate model missspecification. In this situation, however, it is not possible to identify from the observed yield data alone which set of trait values is more appropriate. By extension, the same applies to the sets of marker effects of which the component trait values are linear functions. This is referred to as likelihood nonidentifiability (short 'nonidentifiability') and is a known problem in biological modeling with hidden variables \[[@pone.0130855.ref101]\]. When analyses are conducted under the Bayesian statistical paradigm, nonidentifiability does not necessarily preclude inference and estimation, because informative prior distributions can identify the parameters nonetheless \[[@pone.0130855.ref102], [@pone.0130855.ref103]\]. This is another argument in favor of using informative prior distributions. Nonidentifiability also does not preclude prediction, because the posterior predictive distribution is a function that is identified even if the parameters are not \[[@pone.0130855.ref104]\]. If prediction is the sole purpose, the nonidentified parameters can be viewed as nuisance variables \[[@pone.0130855.ref101]\], that are averaged over in the posterior predictive distribution. In fact, Gianola \[[@pone.0130855.ref104]\] argues that in a predictive setting, parameters are merely 'tools enabling one to go from past to future observations'. However, nonidentifiability can be associated with computational problems \[[@pone.0130855.ref102]\] and is of course an issue if the latent variables are of interest themselves. A special case of nonidentifiability occurs when the parameters are not identifiable for the estimation data set at hand, out of sheer coincidence \[[@pone.0130855.ref101]\]. However, when applied to new observations, i.e., for prediction, the parameters might be identifiable, with one set of parameters being more appropriate than the others. If this is the case, nonidentifiability might lead to a reduction in prediction accuracy.
In addition to using informative prior distributions, increasing the informativeness of the data with respect to the parameters is a direct way to improve their identifiability. In our case, this can be achieved by using additional response variables next to final grain yield. One possible choice is to use grain yield measurements from multiple environments, because several sets of component trait values might generate the same grain yield in one environment, but not in the others. Other possible choices are intermediate traits generated by the CGM, such as early biomass development or leaf area index, which can be measured non-destructively and with high-throughput \[[@pone.0130855.ref105]--[@pone.0130855.ref107]\].
Actually measuring the underlying physiological traits obviates the need to treat them as latent, hidden variables. This would obviously guarantee identifiability. As mentioned before, it might be possible to measure at least some of the traits. If these are key traits in the development of grain yield, observing them would identify the unobserved traits, too. One way of exploiting the information from observed physiological traits in CGM-WGP is to treat them as constants in the estimation procedure. In this framework, physiological trait values of new genotypes have to be predicted from conventional QTL or WGP models, as described above. However, CGM-WGP could also be extended to estimate marker effects for observed and hidden physiological traits simultaneously.
### Other applications {#sec018}
The idea of incorporating biological insights into WGP models is not limited to CGMs. Plant metabolites are chemical compounds produced as intermediate or end products of biochemical pathways. They are seen as potential bridges between genotypes and phenotypes of plants \[[@pone.0130855.ref108]\] and are therefore of particular interest in plant breeding \[[@pone.0130855.ref109]\]. Metabolic networks model the interrelationships between genes, intermediate metabolites and end products through biochemistry pathways \[[@pone.0130855.ref110]\]. Elaborate metabolic network models are available today that allow studying and simulating complex biochemical processes related to crop properties, such as flowering time, seed growth, nitrogen use efficiency and biomass composition \[[@pone.0130855.ref097], [@pone.0130855.ref111]--[@pone.0130855.ref113]\]. Liepe et al. \[[@pone.0130855.ref049]\] demonstrated how ABC can be used for parameter estimation with metabolic and other biochemical networks. Using the principles outlined here for CGM-WGP, metabolic networks might add valuable biological information for the purpose of WGP, too.
Despite ever increasing sample sizes and marker densities, most of the genetic variance of complex traits remains unaccounted for in genome-wide association studies \[[@pone.0130855.ref114]\]. Marjoram et al. \[[@pone.0130855.ref051]\] argued that signal detection power could be increased by augmenting the purely statistical association models used thus far with biological knowledge. They demonstrated their approach by using ABC for incorporating gene regulatory networks into their analysis. Here we showed that the same principle can be applied to WGP by using ABC for integrating a CGM in the estimation of whole genome marker effects. Yield is a product of plant genetics and physiology, the environment and crop management and integrating information pertaining to these components will ultimately enable us to better predict it \[[@pone.0130855.ref115]\]. While this study is only a first step and many questions remain, we conclude that CGM-WGP presents a promising novel path forward towards a new class of WGP models that integrate genomics, quantitative genetics, and systems biology and thereby increase prediction accuracy in settings that have proved challenging for plant breeding and applied genetics.
Supporting Information {#sec019}
======================
###### Accuracy of grain yield predictions of test DH lines with increased error in prior parameters.
(PDF)
######
Click here for additional data file.
###### SNP genotypes and trait phenotypes of synthetic maize doubled haploid lines.
The data set is a representative example of the 50 synthetic data sets used in the study.
(CSV)
######
Click here for additional data file.
###### Simulated development of total and senescent leaf area.
The early, intermediate and late maturing genotypes had a total leaf number (TLN) of 6, 14.5 and 23, respectively. The values for the other three traits were 750 for AM, 1.6 for SRE and 1150 for MTU and in common for all genotypes. The full and dotted vertical lines indicate the end of the 2012 and 2013 growing season, respectively.
(PDF)
######
Click here for additional data file.
###### CGM-WGP vs. GBLUP prediction accuracy in 50 synthetic data sets.
(PDF)
######
Click here for additional data file.
###### Relationship between physiological traits and total grain yield.
Data shown are a random sample of 1000 genotypes from a representative example replication.
(PDF)
######
Click here for additional data file.
###### Distribution of simulated grain yield in 2012 and 2013 environments.
The grey lines indicate the performance of specific genotypes in both environments. Data shown is from a representative example replication.
(PDF)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors are employed by DuPont Pioneer, which owns a pending unpublished patent application covering concepts disclosed in the manuscript. The authors have no financial interest in the patent application. Part of the authors' employment responsibilities at DuPont Pioneer include developing systems and methods disclosed in the manuscript. The authors declare they are unaware of any competing interests. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
[^2]: Conceived and designed the experiments: FT CDM LRT MC. Performed the experiments: FT. Analyzed the data: FT. Contributed reagents/materials/analysis tools: FT CDM LRT MC. Wrote the paper: FT CDM LRT MC.
| 2023-10-06T01:27:17.760571 | https://example.com/article/5278 |
Q:
Incluir link em um container
Tenho um container no meu código e preciso que ele seja clicável, com href, vamos supor que ao clicar ele direcione para "Google.com". Como posso fazer isso?
<div class="project-2 v-center">
<div><!--v-center-->
<div class="container">
<h1>COMPADRE IPSUM</h1>
<p>Eiiitaaa Mainhaaa!! Esse Lorem ipsum é só na brincadeira!!.</p>
</div>
<!--/.container-->
</div>
<!--/v-center-->
A classe:
.container {
position: relative;
z-index: 2;
}
A:
A troca do DIV por um A permite o uso do href tradicional, e funciona mesmo sem JS:
CSS:
.container {
display:block;
position: relative;
z-index: 2;
text-decoration:none;
... outras estilizações necessárias ...
}
HTML:
<div class="project-2 v-center">
<div><!--v-center-->
<a class="container" href="http://google.com">
<h1>COMPADRE IPSUM</h1>
<p>Eiiitaaa Mainhaaa!! Esse Lorem ipsum é só na brincadeira!!.</p>
</a>
<!--/.container-->
</div>
<!--/v-center-->
Esta solução só passa na validação em HTML5.
Em HTML4, não eram válidos elementos de bloco dentro de <A>. Na prática funcionavam, mas não passavam na validação, portanto, um risco de efeitos colaterais a ser evitado.
Leitura complementar: Block level links and accessibility(en)
Esta resposta é equivalente à segunda sugestão da resposta do @Guilherme Bernal, que considero melhor que a primeira, e por isso elaborei com os detalhes relevantes.
| 2023-10-11T01:27:17.760571 | https://example.com/article/4200 |
Long Beach, Calif.
— Three to four times a week, Jayme Bassett rides her 20-year-old Huffy bike five miles from home to the local metro-train stop. She uses a magnetized key to open the back door of a free-standing, concrete-and-glass building with the word "Bikestation" painted on the front window. The modern structure is ensconced in palm trees and raised above a cement pond, all just yards from platforms to buses and trains.
"Biking is the life," says Ms. Bassett, a teachers' credit union employee who takes a bus or train to complete her trip. "With the price of gas shooting up, smog, congestion, and all the rest, why do we need more cars, SUVs, and Hummers on the road?"
It's a refrain being heard across the United States as advocates push for more bike paths – and cities begin to build them.
There's just one catch. For all the growth in two-wheel travel that some cities are experiencing, a nationwide surge in nonrecreational cycling has yet to materialize. US sales of bicycles peaked in 2005 then fell last year to their lowest level since 2001, according to projections by the National Bicycle Dealers Association. The share of adults bicycling to work was only 0.4 percent in 2005, according to census figures released last week, exactly the same percentage as in 1990 and 2000.
That has not stopped many cities from beefing up their biking infrastructure. For example:
•In Austin, Texas, $250 million worth of trails are planned for the metro area, including 32 miles directly through the town center.
•Dallas/Fort Worth has $900 million in bike trails planned. "Texas is reaching out to embrace biking like never before," says Robin Stallins of the Texas Bicycle Coalition. "That says a lot about a shift of cultural values."
•Chicago has unveiled a plan for 500 miles of designated bike routes.
•New York Mayor Michael Bloomberg has called for 200 miles of bike paths throughout the city by 2009.
•Seattle Mayor Greg Nickels released a 10-year master plan in April to "make the city a world-class city for bicycling." It commits $240 million over the next decade for 452 miles of marked routes, bike facilities, roadway crossings and bridges, parking, and maps.
•San Francisco Mayor Gavin Newsom last month set a goal for residents to use bikes for at least 10 percent of their trips by 2010. In 2005, 1.8 percent of workers age 16 or over commuted to work by bike, census data show.
Of course, such sweeping plans often run into fierce opposition when plans affect specific neighborhoods, bike advocates note. Others see bikers as special interest groups whose limited numbers don't deserve the new attention.
"Before they completely redesign the streets of San Francisco for the 1.8 percent of commuters who use bikes, they should at least consider what the wider impacts of their plan will be," says Rob Anderson, an activist who sued the city for failing to submit a required environmental-impact statement with its recent five-year bike plan. A court agreed, forcing a two-year study. "This is Progressive Land, where they do things for political reasons and not necessarily rational ones," adds Mr. Anderson.
Still, some cities report a rise in cycling. Seattle saw downtown bike traffic increase 57 percent during the morning rush hour between 1992 and 2000. San Francisco saw a 27 percent rise in Bike to Work Day participants this year compared with 2006.
If cities make biking easier and safer, proponents say, more residents will do it.
"One of the big deterrents to bicycle use as transportation is finding convenient, theft-proof parking places," says Andréa White, who heads Bikestation. "This trend has been building for years and is now poised to explode."
The Santa Barbara Bikestation – an $80,000 self-parking garage with showers, a changing area, and a bathroom – is an attempt to meet that need. A decade after the first US facility, Bikestations have sprung up in several California cities (San Francisco, Palo Alto, Santa Barbara, Berkeley, and Fruitvale) in addition to Seattle and Chicago. Many others are on the way.
Another measure of the move to biking is the growth of Thunderhead Alliance – a coalition of state and local bike and pedestrian organizations that help strengthen local advocacy groups. They have grown from 12 member organizations in 1996 to 128 coalitions in 49 states. The alliance's Complete the Streets Campaign has helped win legislation in 23 cities and nine states, which requires that streets be designed to be safe and accessible for all users.
"Roads have been viewed as places for cars only, but complete-streets policies say roads are for all people including bicyclists and pedestrians," says Kristen Steele of Thunderhead Alliance.
The group is working with organizations in 15 more states this year that will lobby Congress for a federal complete-streets policy. | 2024-05-09T01:27:17.760571 | https://example.com/article/5399 |
Activity of the botanical aphicides 1,5-diphenyl-1-pentanone and 1,5-diphenyl-2-penten-1-one on two species of Aphididnae.
1,5-Diphenyl-1-pentanone (A) and 1,5-diphenyl-2-penten-1-one (B) are natural products extracted for the first time from Stellera chamaejasme. Laboratory bioassay showed that the two products have strong contact activity and very good anti-feedant activity against Aphis gossypii and Schizaphis graminum. Both products showed dose-dependent relationships for both forms of activity against the two aphids, the contact activity of B being about twice that of A. Both products were inferior to methomyl in contact activity but superior in anti-feedant activity against the two aphids. This is the first report of aphicidal activity in these two compounds, which may represent a new class of aphicide. | 2023-09-11T01:27:17.760571 | https://example.com/article/3020 |
An Indiana nail salon customer unhappy with her manicure allegedly dragged an employee across the parking lot with her car, according to officials.
Charley Fowler, 28, is accused of walking out without paying Saturday in a bizarre meltdown at Diamond & Nails Spa in Valparaiso, the Times of Northwest Indiana reported.
Nail salon employees said the woman complained about her manicure then refused an offer to have it redone.
When she reportedly refused to cough up the $30 she owed, the salon contacted police. During the call to authorities, Fowler allegedly fled the spa and got into her white BMW, according to the workers.
Surveillance video reportedly showed a male employee chasing after Fowler before her vehicle dragged him across the parking lot.
She told police that she planned to wait for officers to arrive but employees began banging on her windows.
Fowler, who works as a mental health therapist, was arrested on suspicion of theft and criminal recklessness, according to the newspaper. | 2024-03-12T01:27:17.760571 | https://example.com/article/6552 |
WINSTON-SALEM, N.C. (AP) - Chris Paul wanted to make sure he made an immediate impact the first time he met Skip Prosser.The star guard was a high school player at an AAU tournament several years ago in Florida when, in a desperate attempt to catch the Wake Forest coach's eye, he walked directly into Prosser's path.``I bumped into him purposefully so Coach would get to know who I am,'' Paul said through tears Tuesday night during Prosser's funeral. ``At the time, I never knew this man would change my life forever. He gave us a chance. He cared about us, and he helped us not only grow as basketball players but also as men.''Prosser, who died suddenly last Thursday of an apparent heart attack, was remembered five days later as an engaging, congenial coach who brought integrity to the sport and reignited the students' passion for Wake Forest basketball by leading Paul's final Demon Deacons team to the first No. 1 ranking in school history.``His integrity was unwavering,'' Wake Forest associate head coach Dino Gaudio said. ``Those of us that were blessed to be under his charge will carry that with us for the rest of our lives.''Former Pittsburgh Steelers coach Bill Cowher, Michigan State's Tom Izzo, former Virginia coach Pete Gillen and ex-Wake Forest guard Muggsy Bogues joined the 11 other Atlantic Coast Conference coaches - including Tobacco Road rivals Mike Krzyzewski of Duke and Roy Williams of North Carolina - in packing the Catholic church where Prosser's funeral was held.``This is the very last day of the recruiting period, and I see so many coaches,'' Gaudio said with a smile. ``If he couldn't be on the road recruiting, you guys couldn't be on the road recruiting either.''Penn State coach Ed DeChellis, who shared ties to western Pennsylvania with the Pittsburgh-born Prosser, called him ``my dearest, closest and most trusted friend.'' He got to know him through the network of assistant coaches and saying they called each other several times each week.``The man I count on for direction is gone,'' DeChellis said. ``My compass in life has gone to another place.''Shortly after returning from his noon jog last Thursday, Prosser was found unresponsive and slumped on his office couch by an assistant coach. Medical personnel performed CPR and used a defibrillator, but Prosser was pronounced dead after being taken to Wake Forest University Baptist Medical Center.Prosser spent six seasons with the Demon Deacons, reaching the NCAA tournament in his first four seasons and winning an ACC regular-season title and the league's coach of the year award in 2003. The following year, Wake Forest went to No. 1 for the first time.``Wake Forest is profoundly grateful for the gifts this remarkable man spread in our midst,'' university president Nathan O. Hatch said.Before arriving in Winston-Salem, Prosser was head coach at Xavier for seven seasons and at Loyola of Maryland for one. He is the only coach to take three schools to the NCAA tournament in his first season at each.Prosser had a career record of 291-146, including 126-68 with Wake Forest, coaching future NBA stars Paul and Josh Howard.Along the way, he helped create Wake Forest's ``tie-dye nation,'' encouraging students to wear black-and-gold tie-dye T-shirts to games at Lawrence Joel Veterans Memorial Coliseum and dubbing them the ``Screaming Deacons.'' The team's glitzy lineup introduction - in which the lights go out, spotlights swirl and the school mascot revs a Harley-Davidson as he enters the court - was another of his creations.``But away from the games, he was the most understated man I know,'' athletics director Ron Wellman said.The funeral was simulcast at Wait Chapel on the Wake Forest campus, where several of the roughly 2,000 mourners wore tie-dye T-shirts in his memory.The makeshift shrine on the campus quad remained as a tribute to Prosser, with toilet paper still floating in the breeze from the ash trees after students ``rolled'' it in his honor soon after his death. Dozens of flowers were laid by signs near the entrance, and one bore one of Prosser's favorite mantras - ``If you can't be on time, be early. God called this play and Skip left early. Too early.''``If Coach were here, he'd be upset, because it was never about Skip,'' Gaudio said. ``It was about the guys.''---AP Sports Writer Aaron Beard in Winston-Salem, N.C., contributed to this report.
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About: TheSpread.com is the largest sports betting news site in the United States. We provide point spread news, odds, statistics and information to over 199 countries around the world each year. Our coverage includes all North American College and Professional Sports as well as entertainment, political and proposition wagering news. | 2023-08-30T01:27:17.760571 | https://example.com/article/5222 |
Receive the latest celebrity updates in your inbox
The husband of "Real Housewives of Beverly Hills" star Taylor Armstrong has committed suicide, according to authorities.
Russell Armstrong was found dead after L.A. firefighters were called to the estranged couple's home Monday night, NBCLA.com confirmed. No suicide note was found.
"I can confirm that Mr. Armstrong was found dead from an apparent hanging in a residence on Mulholland Drive," LAPD spokesman Richard French said.
The death of Armstrong, 47, left his celebrity wife despondent, according to TMZ. She "is hysterical, and can barely speak. She has no idea why Russell would do this. She doesn't know how she is going to be able to tell her daughter, Kennedy," a source told the site.
In Memoriam
A spokesperson for Bravo released the following statement: "All of us at Bravo are deeply saddened by this tragic news. Our sympathy and thoughts are with the Armstrong family at this difficult time."
Andy Cohen, Bravo executive vice-president, tweeted, "I am so very sad about today's news. My prayers and thoughts are with Taylor Armstrong and her family."
Taylor Armstrong, 40, had filed for divorce in July. The couple had been married for six years and had a 5-year-old daughter. Armstrong cited "irreconcilable differences" in the divorce petition, and the couple's marital struggles were seen on the reality show.
Taylor Armstrong had claimed that her husband had abused her by pulling her hair and shoving her, People magazine reported. He denied the hair-pulling but acknowledged he may have pushed her "in the heat of the moment," and said the TV show "has literally pushed us to the limit."
The couple was being sued for $1.5 million by MyMedicalRecords.com, according to People, and sources told Radaronline they were under extreme financial stress. | 2024-07-12T01:27:17.760571 | https://example.com/article/3214 |
Malignant transformation of human fibroblasts correlates with increased activity of receptor-bound plasminogen activator.
To determine whether a relationship exists among urokinase plasminogen activator (u-PA) activity, tissue plasminogen activator (t-PA) activity, and the malignant transformation of human fibroblasts, we measured receptor-bound and secreted u-PAs and t-PA activity in fibroblast cell strains of a unique cell lineage and compared the results with the values obtained in human fibrosarcoma-derived cell lines and control cell lines. The lineage consists of four nonmalignant, infinite life span cell strains, clonally derived from a finite life span, neonatal foreskin-derived cell line or one of its derivatives and 10 malignant cell strains clonally derived from that same derivative. Seven of the latter were malignantly transformed by K-, H-, or N-ras oncogene transfection, two were obtained following carcinogen treatment, and one arose spontaneously. All 10 malignant strains in this lineage exhibited significantly higher levels of activity of receptor-bound u-PA than was found in the cell strain from which they arose or the nonmalignant cell strains derived from it. The ras oncogene-transformed malignant strains also exhibited significantly higher levels of activity of receptor-bound t-PA than their cell strain of origin. The other three malignant strains showed undetectable levels, consistent with their attaining the malignant state by an alternate process. The five fully malignant fibrosarcoma-derived cell lines tested also showed high levels of receptor-bound u-PA and t-PA. The majority (greater than or equal to 80%) of the nonmalignant control cell lines did not do so. The 10 malignant cell strains in the lineage also exhibited higher levels of activity of secreted high molecular weight u-PA or t-PA than did their cell strain of origin and the nonmalignant cell strains derived from it, as did the malignant fibrosarcoma-derived cell lines. The data suggest that the malignant state of human fibroblasts is always associated with high levels of activity of receptor-bound u-PA, and in addition cells transformed to the malignant state are very likely to exhibit high levels of receptor-bound t-PA and secreted forms of plasminogen activators. | 2024-02-21T01:27:17.760571 | https://example.com/article/9832 |
Category: Rules Procedures
This publication addresses the classical yet still nonetheless arguable query of what the function is of judicial discretion in adjudication. Its major function is to debate the philosophical and epistemic foundations of 2 assorted criminal theories: Hartian positivism and Dworkin's interpretivism. making an allowance for the talk among realism and antirealism, the writer exhibits how Hartian positivism faces critical problems to prevent worldwide scepticism, and the matter of rule-following by way of easily connecting criminal determinacy with the lifestyles of settled conventions. by contrast, it truly is argued that Dworkin's interpretive concept can conquer scepticism through connecting criminal determinacy with the belief of the simplest interpretation, and via rejecting externalism. with a purpose to justifying this declare, the writer provides a reconstruction of Dworkin's philosophical place, which built alongside the traces of Putnam's inner realism and Rawls' reflective equilibrium. This e-book could be of curiosity to felony theorists, legal professionals, judges, and philosophers.
Scientific proof is normal in cutting-edge felony trials. From hair and handwriting research to ink and DNA fingerprints, scientists have introduced their international to endure at the justice system.
Combining political research, medical reasoning, and an in-depth examine of particular country perfect lawsuits, Black gowns, White Coats is an interdisciplinary exam of the culture of "gatekeeping," the perform of determining the admissibility of novel clinical facts. Rebecca Harris systematically examines judicial policymaking in 3 components forensic DNA, polygraphs, and mental syndrome proof to reply to the query: Why is clinical proof taken care of in a different way between a variety of jurisdictions? those judgements have very important implications for comparing our judicial process and its skill to appropriately advance clinical policy.
While the interplay of those professions happens as the white coats frequently enhance and be sure wisdom deemed very priceless to the black gowns, Harris concludes that the black gowns are good located to render applicable rulings and ensure the acceptability of harnessing a selected technology for felony purposes.
First booklet to systematically assemble and study judicial judgements on clinical admissibility
In contemporary years, tales of reckless attorneys and grasping electorate have given the felony process, and sufferers mostly, a foul identify. Many american citizens have come to think that we are living within the land of the litigious, the place frivolous complaints and absurdly excessive settlements reign. students have argued for years that this universal view of the wicked spoil of our civil felony approach is a delusion, yet their examine and data infrequently make the scoop. William Haltom and Michael McCann right here persuasively convey how popularized distorted understandings of tort litigation (or tort stories) were perpetuated via the mass media and reform proponents. Distorting the legislation lays naked how media assurance has sensationalized court cases and sympathetically portrayed company pursuits, helping giant enterprise and reinforcing damaging stereotypes of legislation practices. in response to vast interviews, approximately twenty years of newspaper assurance, and in-depth experiences of the McDonald's espresso case and tobacco litigation, Distorting the legislations deals a compelling research of the presumed litigation quandary, the crusade for tort legislation reform, and the an important position the media play during this strategy.
Many criminal theorists and judges agree on one significant premise within the box of legislation and faith: that faith clause jurisprudence is in a country of disarray and has been for a while. In Masters of phantasm, Frank S. Ravitch provocatively contends that either tough originalism (a strict specialize in the motive of the Framers) and neutrality are illusory in faith clause jurisprudence, the previous since it can't stay as much as its promise for each side within the debate and the latter since it is just most unlikely within the faith clause context. but those rules were utilized in virtually each very best courtroom determination addressing faith clause questions.Ravitch unpacks some of the rules of faith clause interpretation, drawing on modern debates corresponding to institution prayer and showing the 10 Commandments on courthouses, to illustrate that the neutrality precept doesn't paintings in a pluralistic society. whilst outlined through huge, overarching rules of equality and liberty, neutrality fails to account for adjustments among teams and contributors. If, although, the courtroom drew on a number of ideas rather than a unmarried idea of neutrality to make a decision even if legislation facilitated or discouraged non secular practices, the end result can be a extra equitable method of faith clause situations.
As gang violence keeps to upward push around the state and the realm, police departments, prosecutors, and group contributors are trying to find new the way to lessen the unfold of gang-related illegal activity. Civil gang injunctions became a starting to be characteristic of crime regulate courses in different states around the state. Gang Injunctions and Abatement: utilizing Civil treatments to minimize Gang-Related Crimes examines the effectiveness of this procedure and explores the accompanying constitutional controversies concerning freedom of speech, meeting, and different rights.
Questions raised through this thought-provoking quantity include:
What are the prices of gang violence to society?
Do civil treatments decrease violence within the groups the place they're implemented?
What components make a given injunction or abatement kind of effective?
What criminal and coverage concerns stand within the approach of gang injunctions and abatement?
Providing step by step directions on how you can determine a profitable injunction and abatement software, the e-book offers complete examine at the theoretical foundation for the tactic. It features a criminal and chronological development of exact situations and their results, describing weaknesses and successes in a number of courses. providing succinct instructions from classes realized, the ebook permits prosecutors, police corporations, and the general public to take steps towards removing gang actions of their communities.
The Psychiatrist as professional Witness, moment variation , by way of Thomas G. Gutheil, M.D., is a hugely readable and useful replace of his profitable first version. the point of interest of the recent quantity is to lead readers to turn into, increase, and effectively marketplace a occupation as a psychiatric specialist witness. A professor of psychiatry on the Beth Israel-Deaconess scientific heart, Harvard clinical tuition, Gutheil attracts on his a long time of expertise within the court and numerous newbie s errors to aid readers steer clear of pitfalls. whereas of significant worth to novices to the sector, the booklet bargains insights and counsel to early-career and professional professional witnesses alike. This quantity has been generally revised and up-to-date from the 1st version, which has been utilized in approximately all forensic psychiatric education courses within the usa and Canada. As within the first variation, this quantity explores the position of the specialist witness, ethical concerns, uncomplicated ideas, depositions and trials, writing for the courtroom, and moral advertising. moreover, it features a thesaurus, improved definitions of key innovations, a lengthened dialogue of bias in testimony, new illustrative examples, a version consent shape for forensic exam, and situations that experience arisen because the first version.
For the 1st time in our background, U.S. prisons condominium over one million inmates, adequate to populate a urban higher than San Francisco. development prisons is the hot progress undefined, because the American public reacts to a perceived raise in violence and politicians take a difficult line towards crime. yet this eagerness to build extra prisons increases simple questions about what the group desires and should tolerate and what the best court docket will sanction.
In this well timed ebook, Norman Finkel appears to be like on the courting among the "law at the books," as set down within the structure and constructed in circumstances and judgements, and what he calls "commonsense justice," the standard citizen's notions of what's simply and reasonable. legislations is an basically human pastime, a set of mental theories approximately why humans imagine, believe, and behave as they do, and whilst and why we should always locate a few of them blameworthy and punishable. yet is it self sustaining of neighborhood sentiment, as a few may contend? Or, as Finkel indicates, do juries convey the community's judgment to undergo at the ethical blameworthiness of the defendant? while jurors make a decision that the legislation is bigoted, or the punishment irrelevant for a specific defendant, they've got occasionally nullified the law.
Nullification represents the jury's hope to not defeat yet "to excellent and whole" the legislations. it's the "no self assurance" vote of common-sense justice refusing to persist with the trail the legislations has marked out--and pointing to a brand new direction in line with what appear to be extra simply grounds. Finkel brings to lifestyles the tale at the back of the jury and judicial judgements, interweaving anecdotes, case legislations, and social technology study to offer a balanced and entire view of significant felony and social coverage issues.
The irreparable harm rule says that courts won't furnish an equitable therapy to avoid damage if it'd be sufficient to permit the damage take place and provide the felony treatment of cash damages. After surveying greater than 1400 situations, Laycock concludes that this historic rule is dead--that it virtually by no means impacts the result of situations. whilst a courtroom denies equitable aid, its actual purposes are derived from the pursuits of defendants or the felony method, and never from the adequacy of the plaintiff's criminal therapy. Laycock seeks to accomplish the assimilation of fairness, displaying that the law-equity contrast survives purely as a proxy for different, extra sensible differences. reading the true ideas for selecting treatments by way of those practical differences, he clarifies the complete legislation of treatments, from grand conception right down to the sensible info of particular instances. He indicates that there's no optimistic legislations help for an important functions of the legal-economic thought of effective breach of agreement. incorporated are wide notes and a close desk of instances prepared by way of jurisdiction.
In capital trials archival study files race-of-victim discrimination whereas the experimental learn helps a race-of-offender influence. utilizing interviews with jurors, Edelman sought to reconcile this clash and clarify how and while race results are inclined to ensue. White jurors have been prone to mitigating facts whilst the sufferer used to be white. White jurors empathized extra with a white than black sufferer. sufferer race additionally had an oblique influence at the review of the defendant. Killers of whites have been evaluated much less certainly than killers of blacks. those results recommend that white jurors have been much less more likely to carry a existence sentence place whilst a black defendant used to be convicted for murdering a white sufferer.
The worldwide unfold of transnational mining funding, which has been occurring because the Nineteen Nineties, has usually ended in unstable conflicts with neighborhood groups. This e-book examines the rules of those conflicts via nationwide, transnational, and native felony procedures. In doing so, it examines how felony authority is being redistributed between private and non-private actors, in addition to nationwide and transnational actors, due to globalizing forces. The ebook provides a case examine about the negotiation of land move and resettlement among a transnational mining company and indigenous peasants within the Andes of Peru. The case learn is used to discover the intensely neighborhood dynamics excited by negotiations among company and neighborhood representatives, and the function performed by means of felony ordering in those kin. particularly, the booklet examines the operation of a transnational felony regime controlled via the realm financial institution to treatment the social and environmental affects of initiatives which obtain international financial institution information. The publication explores the character and personality of the realm financial institution regime and the a number of outcomes of this projection of transnational legislations right into a neighborhood dispute. | 2023-11-29T01:27:17.760571 | https://example.com/article/6126 |
Q:
Test class make dynamically?
below is my test class.
@isTest
public class CaseUserHelperTest{
static testMethod void helperMethod(){
Datetime myDate = Datetime.newInstance(2017, 24, 3, 10, 50, 0);
Datetime myDate1 = Datetime.newInstance(2017, 24, 3, 12, 50, 0);
Test.startTest();
Case_User__c cuser1 = new Case_User__c(AvailableUsers__c='00546000000hZWK',Timings__c='2:00PM-11:00PM', SkillSet__c ='Product Support',Availability__c = TRUE,Last_Case_Allocated_Time__c = myDate );
Case_User__c cuser2 = new Case_User__c(AvailableUsers__c='00546000000hZUx',Timings__c='2:00PM-11:00PM', SkillSet__c ='Product Support',Availability__c = TRUE,Last_Case_Allocated_Time__c = myDate1 );
insert cuser1;
insert cuser2;
Case cas = new Case(Status ='New', Priority = 'Medium', Origin = 'Email',CaseRelatedTo__c='Product Support');
insert cas;
Test.stopTest();
cas = [Select OwnerId From Case where Id=:cas.id];
System.assertEquals(cas.OwnerId, 'u.id');
}
}
In the above test class Availableusers__c is a picklist when going to create a new Case_User__c object record.
for testing i'm passing the Availableusers__c is statically but help me to how a two new users creation and how i make that test class dynamically
Thanks
A:
create a method to add new user
public static user createTestUser(){
list<Profile> p = [SELECT Id FROM Profile WHERE Name='Standard User'];
if(p.size()>0){
User u = new User(Alias = 'testU', Email='stdusr@test.com',
EmailEncodingKey='UTF-8', LastName='Test', LanguageLocaleKey='en_US',
LocaleSidKey='en_US', ProfileId = p[0].Id,
TimeZoneSidKey='America/Los_Angeles', Username = 'testU@a.com' + System.currentTimeMillis());
insert u;
return u;
}
else return null;
}
so your above code can be changed to
@isTest
public class CaseUserHelperTest{
static testMethod void helperMethod(){
Datetime myDate = Datetime.newInstance(2017, 24, 3, 10, 50, 0);
Datetime myDate1 = Datetime.newInstance(2017, 24, 3, 12, 50, 0);
Test.startTest();
user usr1= createTestUser();
user usr2= createTestUser();
Case_User__c cuser1 = new Case_User__c(AvailableUsers__c=usr1.id,Timings__c='2:00PM-11:00PM', SkillSet__c ='Product Support',Availability__c = TRUE,Last_Case_Allocated_Time__c = myDate );
Case_User__c cuser2 = new Case_User__c(AvailableUsers__c=usr2.id,Timings__c='2:00PM-11:00PM', SkillSet__c ='Product Support',Availability__c = TRUE,Last_Case_Allocated_Time__c = myDate1 );
insert cuser1;
insert cuser2;
Case cas = new Case(Status ='New', Priority = 'Medium', Origin = 'Email',CaseRelatedTo__c='Product Support');
insert cas;
Test.stopTest();
cas = [Select OwnerId From Case where Id=:cas.id];
System.assertEquals(cas.OwnerId, 'u.id');
}
}
| 2024-01-03T01:27:17.760571 | https://example.com/article/8927 |
Opening Bell: Sensex down 100 points, Nifty tests 9,450
Benchmark indices on Friday kicked off with a negative start, as investors stay cautious ahead of the rollout of much-awaited Goods & Services Tax (GST). The BSE Sensex dropped over 100 points, while the Nifty50 was testing 9,450 level. | 2024-06-19T01:27:17.760571 | https://example.com/article/9862 |
extern "C" {
#include <libcgc.h>
#include <stdlib.h>
};
extern "C" {
extern void (*__init_array_start[])(void) __attribute__((__visibility__("hidden")));
extern void (*__init_array_end[])(void) __attribute__((__visibility__("hidden")));
};
void call_ctors(void) {
size_t len, i;
for (len = __init_array_end - __init_array_start, i = 0; i < len; i++) {
__init_array_start[i]();
}
}
| 2023-10-22T01:27:17.760571 | https://example.com/article/7553 |
1.. Introduction
================
The development of highly sensitive methods for detecting cancer biomarkers has great significance for predicting cancer in early stage in modern biochemical and biomedical research \[[@b1-sensors-12-17320]--[@b3-sensors-12-17320]\]. Generally, high sensitivity can often be obtained using a signal amplification procedure. Many signal amplification strategies have been reported for sensitive bioanalyte quantification, such as rolling circle amplification \[[@b4-sensors-12-17320]\], avidin-biotin amplification \[[@b5-sensors-12-17320]\], and exponential isothermal amplification \[[@b6-sensors-12-17320]\]. However, these approaches usually require extremely complex reagents and intensive protocols. Recently, amplification of immunoassay signals by exploiting the properties of gold nanoparticles (AuNPs) for catalytic growth has emerged as a field attracting significant attention. The commonly used example is silver enhancement, in which AuNPs promote the reduction of silver ions and their deposition on the surface of AuNPs, thereby resulting in further growth of the AuNPs \[[@b7-sensors-12-17320]\]. But it is sensitive to pH, natural light, and chloride ions \[[@b8-sensors-12-17320]\]. Instead of silver staining, using gold salts for catalytic enlargement of AuNPs has solved these issues \[[@b9-sensors-12-17320]\]. This alternative approach has been successfully exploited to develop the detection of IgG by the naked eye \[[@b10-sensors-12-17320]\], target DNA by electrochemical means \[[@b11-sensors-12-17320],[@b12-sensors-12-17320]\] and others \[[@b13-sensors-12-17320]--[@b15-sensors-12-17320]\].
Chemiluminscence immunoassay (CLIA), which was first reported in the late 1970s \[[@b16-sensors-12-17320]\], simultaneously combines the high sensitivity of chemiluminescence analysis and the good specificity of immunoassays. Since most biomolecules (such as tumor markers) have no native CL emission, CLIA quantitation is generally realized by indirectly measuring the intensity of chemiluminescence (CL) labels. Recently, methods using nanoparticles (NPs), in particular metal NPs, as CLIA labels have attracted considerable interest \[[@b17-sensors-12-17320]--[@b19-sensors-12-17320]\]. Among these, AuNPs have been the subject of wide research efforts directed at gene analysis \[[@b20-sensors-12-17320],[@b21-sensors-12-17320]\] and antibody or antigen detection \[[@b22-sensors-12-17320],[@b23-sensors-12-17320]\] because of their special properties, which include high density, large dielectric constant, and biocompatibility. It has been found that AuNPs can greatly enhance the CL intensity of the luminol-H~2~O~2~ system \[[@b24-sensors-12-17320]\]. Accordingly, a CLIA based on irregular AuNPs was first reported by Li and co-workers \[[@b25-sensors-12-17320]\]. However, the difficulty of controlling the synthesis of irregular nanoparticles may influence the repeatability among different batches and this limits the practical application of this method. Duan *et al*. \[[@b26-sensors-12-17320]\] established a microplate-compatible CLIA method for the determination of human IgG based on the luminol-AgNO~3~-gold nanoparticles CL system. The CL detection is readily influenced by the sediment from the mixture of basic luminol and AgNO~3~ and has poor sensitivity. In our work, a highly sensitive immunogold amplified FI-CL immunoassay for the detection of CEA is presented through the catalytic precipitation of gold on the immunogold nanoparticles, which combined the intrinsically high sensitivity and good repeatability of FI-CL with the signal enhancement of immunogold enlargement. The present strategy shows excellent promise for ultrasensitive detection of other cancer biomarkers and infectious agents in clinical analysis.
2.. Experimental Section
========================
2.1.. Reagents and Apparatus
----------------------------
Polystyrene 96-well microtiter plates (Costar, NY, USA) were used to perform the immunoreactions. CEA and anti-CEA antibody were purchased from R & D Systems (Minneapolis, MN, USA). Goat-anti-mouse IgG was purchased from Beijing Chengwen Company (Beijing, China). Bovine serum albumin (BSA), HAuCl~4~·3H~2~O (99.99%) was bought from Sigma-Aldrich (USA). Trisodium citrate, NaH~2~PO~4~, Na~2~HPO~4~, and NaCl are obtained from Beijing Chemical Reagents Company (Beijing, China). The luminol stock solution (2.5 × 10^−2^ M) was prepared by dissolving luminol (obtained from Sigma-Aldrich, USA) in 0.1 M NaOH solution and stored in a dark place. The buffers used were as follows: (A) coating buffer, 0.05 M carbonate/bicarbonate buffer solution, pH 9.6; (B) blocking buffer, 1% (w/v, g·mL^−1^) BSA in 0.01 M sodium phosphate buffered saline with 0.05% (v/v) Tween 20 (PBST-BSA), pH 7.4. The blocking buffer was stored at 4 °C and used within a week; (C) washing buffer, 0.01 M PBS with 0.05% (v/v) Tween 20, pH 7.4; In all the procedures, the water used was purified through an Olst ultrapure K8 apparatus (Olst, Ltd., China, resistivity \>18 MΩ). All other reagents were of analytical reagent grade and used as purchased without further purification. The CL intensity was measured and recorded with an Ultra-weak luminescence analyzer and software BPCL-T15 (Institute of Biophysics, Academic Sinica, Beijing, China). Two peristaltic pumps were used to deliver all the chemicals. A six way injection valve fitted with a 100 μL sample loop was used for the injection of the sample solution. PTFE tubing (1.0 mm i.d.) was used to connect all components in the flow system. Transmission electron microscopy (TEM) was performed with a JEOL-100CX electronmicroscope (Jeol, Japan) under 80 kV accelerating voltage.
2.2.. Preparation of Au Nanoparticles (NPs)
-------------------------------------------
A solution of 15 nm AuNPs was synthesized according to a literature procedure \[[@b27-sensors-12-17320]\] with slight modifications. Briefly, 5.0 mL of 1% trisodium citrate was quickly added to 100 mL of boiling 0.01% HAuCl~4~. Solution under reflux was stirred for 10 min, during which the color changed to red. After being slowly cooled down to room temperature, the solution was centrifuged to remove impurities and ions, and then diluted to 100 mL. The AuNPs have an average diameter of ca. 15 nm.
2.3.. Preparation of AuNPs-Labeled Goat-Anti-Mouse IgG
------------------------------------------------------
The preparation of AuNPs-labeled goat-anti-mouse IgG was performed according to the modification in literature \[[@b28-sensors-12-17320]\]. The pH of the AuNPs was adjusted to 8.0 with 0.1 M Na~2~CO~3~. Precisely, 1.0 mL of 550 μg·mL^−1^ goat-anti-mouse IgG (10% more than the minimum amount, which was determined using a flocculation test) was added to 5 mL of pH-adjusted AuNPs, followed by incubation at room temperature for 30 min. Afterward, 5% BSA was added to a final concentration of 1% with stirring about 5 min. To remove the excess of antibody, the conjugates were centrifuged at 10,000 rpm for 10 min, and the soft sediment was resuspended in 0.01 M PBS containing 1% BSA. It can be used directly or stored in 0.01 M PBS buffer with 50% glycerol for several months at −20 °C.
2.4.. Immunoassay Procedure
---------------------------
The assay was performed in a polystyrene 96-well microtiter plate. Initially, 200 μL of serially diluted human CEA dilutions in coating buffer were coated on wells of the plate overnight at 4 °C. The unbound antigen was washed off three times with 300 μL of washing buffer and the uncoated active sites of polystyrene substrate were saturated with 300 μL of blocking buffer for 1 h at 37 °C, in which BSA was used as a blocking agent to prevent nonspecific adsorption of the antibody in the next step. Afterward, 200 μL of 5 μg·mL^−1^ anti-CEA antibody was added into the wells and incubated for 2 h at 37 °C. The wells were washed three times with washing buffer followed by addition of 200 μL AuNPs-labeled goat-anti-mouse IgG. Finally the wells were washed thoroughly with washing buffer (three times) and pure water (three times). Then, the wells were immediately treated with 200 μL per well of a mixture of 0.01% HAuCl~4~ and 0.4 mM NH~2~OH·HCl in a dark for 2 min at 30 °C.
2.5.. Standard Procedures for the FI-CL Detection
-------------------------------------------------
Metallic gold on enhanced plates were washed three times with 300 μL of pure water, dried, and dissolved with 200 μL of 2.0% HNO~3~-3.4% HCl for three hours at room temperature to ensure that the Au nanoparticles were completely dissolved. Solutions were then transferred to 1.5 mL centrifuge tubes and 200 μL H~2~O and 75 μL 0.1 M sodium tartrate (C~4~H~4~O~6~Na~2~) were added to each sample in turn. Then 1.0 M NaOH was added to the AuCl~4~^−^ solutions to adjust pH. As shown in [Figure 1](#f1-sensors-12-17320){ref-type="fig"}, AuCl~4~^−^ was reacted with the mixture of luminol and H~2~O~2~ in the flow cell to produce the CL signal. The CL signals were monitored with a photomultiplier tube adjacent to the flow cell.
3.. Results and Discussion
==========================
3.1.. Principles of the Experiment
----------------------------------
A schematic representation of the detection principle of this noncompetitive CL immunoassay is shown in [Scheme 1](#f6-sensors-12-17320){ref-type="fig"}. The human CEA analyte is first immobilized on a 96-well polystyrene microtiter plate. The mouse-anti-CEA as primary antibody is then captured by the CEA and sandwiched by a goat-anti-mouse IgG secondary antibody labeled with colloidal gold. After that, we used a method for enlargement of colloidal Au nanoparticles called "seeding", based on the colloidal Au surface-catalyzed reduction of Au^3+^ by NH~2~·OH. While NH~2~·OH is thermodynamically capable of reducing Au^3+^ to bulk metal, the reaction is dramatically accelerated by Au surfaces. As a result, no new particle nucleation occurs in solution, and all added Au^3+^ goes into production of larger particles. In this case, Au NPs are specifically enlarged by HAuCl~4~ and NH~2~OH·HCl for 2 min \[[@b9-sensors-12-17320]\]. Next, the AuNPs are dissolved in a 2.0% HNO~3~-3.4% HCl solution and the gold ions (AuCl~4~^−^) were stripped out from the solid polystyrene substrate surface. The concentration of CEA was quantified based upon the concentration of dissolved AuCl~4~^−^, which was quantified by the CL intensity. A comparison of analytical performance before and after gold amplification was also investigated and the detailed optimization of the gold amplified FI-CL immunoassay is reported in the following sections.
3.2.. Dissolution of Gold Nanoparticles
---------------------------------------
The AuNPs used in this work before and after enlargement with HAuCl~4~ and NH~2~OH·HCl are shown in [Figure 2A,B](#f2-sensors-12-17320){ref-type="fig"}. To detect the enlarged colloidal gold label by CL, the gold was dissolved to form AuCl~4~^−^, which can catalyze the luminol-H~2~O~2~ CL reaction. As the *aqua regia* (16.0% HNO~3~-27% HCl) resulted in a high CL background, dilution of the *aqua regia* was used to dissolve the colloidal gold. Therefore, the effect of the dissolution reagent concentration on CL intensity was investigated. The signal/noise ratio increased with increasing dissolution reagent concentration and reached a maximum value at 1:7 dilution of *aqua regia* (2.0% HNO~3~-3.4% HCl), as shown in [Figure 3](#f3-sensors-12-17320){ref-type="fig"}. It is possible that AuNPs could not be completely dissolved when the concentration of the dissolution reagent was lower than the 1:7 dilution of *aqua regia*. The signal/noise ratio decreased gradually when it exceeded 1:7 and it is likely that the strong ionic strength in the solution induces an increase in the CL background intensity when a much higher concentration of dissolution reagent is used. Thus, the 1:7 dilution of *aqua regia* was used as the optimum dissolution reagent for the CL assay.
The catalytic activity of AuCl~4~^−^ on the luminol-H~2~O~2~ CL reaction is greatly influenced by the pH of the AuCl~4~^−^ solution. Therefore, the effect of AuCl~4~^−^ pH on CL intensity was investigated by using 1.0 M NaOH and 0.1 M C~4~H~4~O~6~Na~2~ to adjust the acidity of the solution after dissolving the AuNPs with 1:7 (v/v) *aqua regia*. As shown in [Figure 4](#f4-sensors-12-17320){ref-type="fig"}, the signal/noise ratio increased with increasing pH of sample solution because the blank CL intensity decreased with increasing sample pH below 4.0, reaching the highest value at pH 4.0. However, when the pH of the sample was higher than 4.0, the signal/noise ratio decreased. The cause is that the stability of AuCl~4~^−^ has been destroyed. Therefore, pH 4.0 was chosen as the optimum pH value for the CL measurement.
3.3.. Optimization of the CL Detection Conditions
-------------------------------------------------
To optimize the proposed CL assay, the effects of luminol and H~2~O~2~ pH on the CL intensities were studied. The optimization of the pH value of luminol was investigated over the pH range 10.0--13.0. The result indicates that the signal/noise ratio had a maximum at pH 12.5. The effects of luminol and H~2~O~2~ concentration on the CL intensity of the sample and blank were also examined. This indicated that the CL intensity increased as the luminol and H~2~O~2~ concentrations were increased but the CL intensity of the blank became very high when the concentration of luminol exceeded 1.0 × 10^−4^ M and led to poor reproducibility. Considering the CL intensity and the consumption of the reagents, 7.5 × 10^−5^ M and 0.01 M H~2~O~2~ were chosen for subsequent work (data not shown).
3.4.. Analytical Performance
----------------------------
Under the optimum conditions described above, the relationship between CEA concentration and CL intensity was investigated. The results showed that the CL intensities of luminol-H~2~O~2~-AuCl~4~^−^ increased with the increase of concentration of the CEA ranging from 10 pg·mL^−1^ to 1 μg·mL^−1^ ([Figure 5A](#f5-sensors-12-17320){ref-type="fig"}). The linear range for CEA was 100 pg·mL^−1^ to 1 μg·mL^−1^ with the equation of lg\[y\] = 3.8077 + 0.1215l g\[x\] (y is the CL intensity; x is the concentration of CEA, ng·mL^−1^; n = 3, R = 0.9892; [Figure 5B](#f5-sensors-12-17320){ref-type="fig"}). The relative standard deviations for 10 pg·mL^−1^ and 1 μg·mL^−1^ of CEA were 0.44% and 4.7%, respectively (n = 3). The detection limit was 20 pg·mL^−1^. [Table 1](#t1-sensors-12-17320){ref-type="table"} shows the comparison between the proposed CL method and general immunoassay formats for determination of CEA. It can be seen that the proposed CLIA is competitive with or better than other immunoassay formats and has the advantage of simple instrumentation.
4.. Conclusions
===============
The feasibility of a highly sensitive FI-CL immunoassay, based on the quantitative enlargement of immunogold tags, has been demonstrated. AuNPs are dissolved into Au^3+^, which catalyzes the luminol chemiluminescence (CL) reaction. The CL intensity, which is proportional to the amount of CEA, could be greatly enhanced. The response of this immunosensor was linear from 0.1--1,000 ng·mL^−1^ with a LOD of 20 pg·mL^−1^ (S/N = 3). The procedure involved in this work is simple, low cost and rapid. The proposed method can provide high sensitivity, a wide linear range and represents a new approach to the ultrasensitive determination of other bioactive molecules for early disease diagnosis.
This study was supported by Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality (PHR20100718).
{#f1-sensors-12-17320}
{#f2-sensors-12-17320}
{#f3-sensors-12-17320}
{#f4-sensors-12-17320}
{#f5-sensors-12-17320}
{#f6-sensors-12-17320}
######
Comparison of various immunoassay methods for CEA determination.
**Label** **Analytical method** **Detection limit (ng·mL^−1^)** **Reference**
----------- ------------------------------------------------------ --------------------------------- -----------------------------
HRP ELISA 0.048 \[[@b29-sensors-12-17320]\]
HRP Chemiluminescence immunoassay 0.5 \[[@b30-sensors-12-17320]\]
HRP Enzyme-linked immunoassay 3.0 \[[@b31-sensors-12-17320]\]
AuNPs ICPMS immunoassay after silver amplification 0.03 \[[@b32-sensors-12-17320]\]
AuNPs Electrochemical immunoassay 0.27 \[[@b33-sensors-12-17320]\]
AuNPs Electrochemical immunoassay 0.57 \[[@b34-sensors-12-17320]\]
AuNPs Chemiluminescence immunoassay after gold enlargement 0.02 this work
AuNPs Chemiluminescence immunoassay 1.0 this work
| 2024-03-08T01:27:17.760571 | https://example.com/article/1471 |
Q:
No output by insertion sort for a custom test case
I have this code for insertion sort which is failing to give an output for a test case..
import java.io.*;
import java.util.*;
public class Solution {
public static void insertionSortPart2(int[] ar)
{
// Fill up the code for the required logic here
// Manipulate the array as required
// The code for Input/Output is already provided
int n =ar.length;
for(int i=1;i<n;i++)
{int k= ar[i];
int j=i-1;
while(ar[j]>k && j>-1)
{
ar[j+1]=ar[j];
j--;
}
ar[j+1]=k;
printArray(ar);
}
}
public static void main(String[] args) {
Scanner in = new Scanner(System.in);
int s = in.nextInt();
int[] ar = new int[s];
for(int i=0;i<s;i++){
ar[i]=in.nextInt();
}
insertionSortPart2(ar);
}
private static void printArray(int[] ar) {
for(int n: ar){
System.out.print(n+" ");
}
System.out.println("");
}
}
The test case for which this does not return an output is
9 8 6 7 3 5 4 1 2
error: Exception in thread "main" java.lang.ArrayIndexOutOfBoundsException: -1
I cannot figure out why..
It works correctly for 1 4 3 5 6 2
A:
Issue is with your while loop and this is the line where you might be getting exception while(ar[j]>k && j>-1). You are trying to access array using negative index i.e. -1. You get this exception when you try to access elements outside of either lower or outer bound of an array.
When i is 1, j is 0 by j=i-1
You enter the while loop since a[1] > a[0] i.e. 9 > 8
You do j-- where value of j becomes -1
You come back to while (while(ar[j]>k && j>-1)) try to access a[j] ie.. a[-1] and hence you get ArrayOutOfBoudException.
You may need to check j > -1 before accessing array i.e. reverse your condition in while loop from
while(ar[j]>k && j>-1)
To
while(j>-1 && ar[j]>k)
| 2024-05-22T01:27:17.760571 | https://example.com/article/3189 |
Predictable progressive Doppler deterioration in IUGR: does it really exist?
An objective of the Prospective Observational Trial to Optimize Pediatric Health in IUGR (PORTO) study was to evaluate multivessel Doppler changes in a large cohort of intrauterine growth restriction (IUGR) fetuses to establish whether a predictable progressive sequence of Doppler deterioration exists and to correlate these Doppler findings with respective perinatal outcomes. More than 1100 unselected consecutive ultrasound-dated singleton pregnancies with estimated fetal weight (EFW) less than the 10th centile were recruited between January 2010 and June 2012. Eligible pregnancies were assessed by serial Doppler interrogation of umbilical (UA) and middle cerebral (MCA) arteries, ductus venosus (DV), aortic isthmus, and myocardial performance index (MPI). Intervals between Doppler changes and patterns of deterioration were recorded and correlated with respective perinatal outcomes. Our study of 1116 nonanomalous fetuses comprised 7769 individual Doppler data points. Five hundred eleven patients (46%) had an abnormal UA, 300 (27%) had an abnormal MCA, and 129 (11%) had an abnormal DV Doppler. The classic pattern from abnormal UA to MCA to DV existed but no more frequently than any of the other potential pattern. Doppler interrogation of the UA and MCA remains the most useful and practical tool in identifying fetuses at risk of adverse perinatal outcome, capturing 88% of all adverse outcomes. In contrast to previous reports, we have demonstrated multiple potential patterns of Doppler deterioration in this large prospective cohort of IUGR pregnancies, which calls into question the usefulness of multivessel Doppler assessment to inform frequency of surveillance and timing of delivery of IUGR fetuses. These data will be critically important for planning any future intervention trials. | 2023-10-24T01:27:17.760571 | https://example.com/article/3237 |
---
abstract: 'It is consistent that for every function $f:{\omega}\to{\omega}$ there is a graph with size and chromatic number $\aleph_1$ in which every $n$-chromatic subgraph contains at least $f(n)$ vertices ($n\geq 3$). This solves a \$ 250 problem of Erdős. It is consistent that there is a graph $X$ with ${{\rm Chr}}(X)=|X|=\aleph_1$ such that if $Y$ is a graph all whose finite subgraphs occur in $X$ then ${{\rm Chr}}(Y)\leq \aleph_2$ (so the Taylor conjecture may fail). It is also consistent that if $X$ is a graph with chromatic number at least $\aleph_2$ then for every cardinal ${\lambda}$ there exists a graph $Y$ with ${{\rm Chr}}(Y)\geq{\lambda}$ all whose finite subgraphs are induced subgraphs of $X$.'
author:
- |
Péter Komjáth[^1]\
Saharon Shelah[^2]
title: Finite subgraphs of uncountably chromatic graphs
---
Introduction
============
In [@erdhaj1] Erdős and Hajnal determined those finite graphs which appear as subgraphs in every uncountably chromatic graph: the bipartite graphs. In fact, not just that any odd circuit can be omitted, for every natural number $n\geq 1$ and infinite cardinal ${\kappa}$ there is a graph with cardinality and chromatic number ${\kappa}$ such that it omits all odd circuits up to length $2n+1$. They observed that the so-called $r$-shift graph construction has all but one of these properties; the vertex set of ${\rm Sh}_r(\kappa)$ is the set of all $r$-tuples from $\kappa$, with $\{x_0,x_1,\dots,x_{r-1}\}_<$ joined to $\{x_1,x_2,\dots,x_{r}\}_<$, then this graph omits odd circuits of length $3,5,\dots,2r+1$ and the Erdős-Rado theorem asserts that the chromatic number of ${\rm Sh}_r({\rm exp}_{r-1}(\kappa)^+)$ is at least $\kappa^+$.
The problem of determining the classes of finite graphs that occur in uncountably chromatic graphs seems to be much harder, and its investigation was strongly pushed by Erdős and Hajnal.
An early conjecture for example was the following. Every uncountably chromatic graph contains all odd circuits from some length onward. This was then proved by Erdős, Hajnal, and Shelah [@erdhajshe], and independently, by Thomassen [@thomassen].
In [@taylor2] and later in [@erdhajshe] the following problem was posed (the Taylor conjecture). If $\kappa$, $\lambda$ are uncountable cardinals and $X$ is a $\kappa$-chromatic graph, is there a $\lambda$-chromatic graph $Y$ such that every finite subgraph of $Y$ appears as a subgraph of $X$. Notice that the above shift graphs give some evidence for this conjecture—the finite subgraphs of ${\rm Sh}_r(\kappa)$ do not depend on the parameter ${\kappa}$. The authors of [@erdhajshe] remarked that even the following much stronger conjecture seemed possible. If $X$ is uncountably chromatic, then for some $r$ it contains all finite subgraphs of ${\rm Sh}_r(\omega)$. This conjecture was then disproved in [@hajkom].
One easy remark, the so called Hanf number argument gives that there is a cardinal ${\kappa}$ with the property that if the chromatic number of some graph $X$ is at least ${\kappa}$ then there are arbitrarily large chromatic graphs with all finite subgraphs appearing in $X$. This argument, however, does not give any reasonable bound on ${\kappa}$.
Another conjecture of Erdős and Hajnal if the maximal chromatic number of $n$-element subgraphs of an uncountably chromatic graph as a function of $n$ can converge to infinity arbitarily slowly as $n$ tends to infinity. It was mentioned in several problem papers, for example in [@erd1], [@erd4], [@erd5], [@erdhaj3], [@erdhajsze]. See also [@chunggraham], [@jensentoft]. The relevance of the above examples is that the chromatic number of the $n$-vertex subgraphs of (any) ${\rm Sh}_r(\kappa)$ grows roughly as the $r-1$ times iterated logarithm of $n$. Perhaps it was this fact that led Erdős and Hajnal to the above problem.
Erdős also tirelessly popularized the Taylor conjecture, he mentioned it e.g., in [@erd2], [@erd3], [@erd6], [@erdhaj2], [@erdhaj3], [@erdhajshe]. It is also mentioned in [@chunggraham], the book collecting Erdős’ conjectures on graphs. In [@komshe] we gave some results when the additional hypotheses $\vert X\vert =\kappa$, $\vert Y\vert
=\lambda$ was imposed. We described countably many different classes ${\cal K}_{n,e}$ of finite graphs and proved that if $\lambda^{\aleph_0}=\lambda$ then every $\lambda^+$-chromatic graph of cardinal $\lambda^+$ contains, for some $n$, $e$, all elements of ${\cal K}_{n,e}$ as subgraphs. On the other hand, it is consistent for every regular infinite cardinal $\kappa$ that there is a $\kappa^+$-chromatic graph on $\kappa^+$ that contains finite subgraphs only from ${\cal K}_{n,e}$. We got, therefore, some models of set theory, where the finite subraphs of graphs with $\vert X\vert={{\rm Chr}}(X) =\kappa^+$ for regular uncountable cardinals $\kappa$ were completely described.
Notice that the class of regular cardinals on which the above result operated excludes ${\omega}_1$, and in this paper we show the reason, by resolving the above Erdős-Hajnal conjecture: it is consistent that for every monotonically increasing function $f:{\omega}\to{\omega}$ there is a graph with size and chromatic number $\aleph_1$ in which every $n$-chromatic subgraph has at least $f(n)$ elements ($n\geq 3$). The possibility of transforming the proof into a ZFC argument will be checked in the forthcoming [@Sh:e], Chapter 9. An application of the method presented here gives the consistent existence of a graph $X$ with ${{\rm Chr}}(X)=|X|=\aleph_1$ such that if $Y$ is a graph (of any size) all whose subgraphs are subgraphs of $X$ then ${{\rm Chr}}(Y)\leq\aleph_2$. This gives a consistent negative answer to the Taylor conjecture. As for the positive direction we prove that it is consistent that if $X$ is a graph with chromatic number at least $\aleph_2$ then there are arbitrarily large chromatic graphs all whose finite subgraphs being induced subgraphs of $X$.
Theorems 1 and 2 were proved by S. Shelah and then P. Komjáth proved Theorems 3 and 4.
[**Notation.**]{} We use the standard axiomatic set theory notation. If $A$ is a set of ordinals, ${\beta}$ is an ordinal, then ${\beta}<A$, means that ${\beta}<{\alpha}$ holds for every ${\alpha}\in A$. Similarly for ${\beta}\leq A$, $A<{\beta}$, etc. If $f$ is a function, $A$ a set, then we let $f[A]=\{f(x):x\in A\}$. If $S$ is a set, $\kappa$ is a cardinal, $[S]^\kappa=\{X\subseteq S:|X|=\kappa\}$, $[S]^{<\kappa}=\{X\subseteq S:|X|<\kappa\}$. A [*graph*]{} is an ordered pair $(V,X)$ where $V$ is some set (the set of [*vertices*]{}) and $X\subseteq [V]^2$ (the set of [*edges*]{}). In some cases we identify the graph and $X$. The [*chromatic number*]{} of a graph $(V,X)$ is the least cardinal $\mu$ such that there exists a function $f:V\to\mu$ with $f(x)\neq f(y)$ for $\{x,y\}\in X$ (a good coloring). A [*path*]{} of length $n$ is a sequence $\{v_0,\dots,v_n\}$ of distinct vertices such that $\{v_i,v_{i+1}\}\in X$ holds for $i<n$. A [*cycle*]{} of length $n$ is a sequence $\{v_1,\dots,v_n\}$ of vertices such that for $1\leq i<n$ we have $\{v_i,v_{i+1}\}\in X$ and $\{v_n,v_{1}\}\in X$ also holds. If the vertices are distinct then we call it a [*circuit*]{}.
A large chromatic graph with small chromatic finite subgraphs
=============================================================
Let $f:{\omega}\to{\omega}$ be a strictly increasing function. Fix a sequence $\{C_{\alpha}:{\alpha}<{\omega}_1,\mbox{limit}\}$ such that $C_{\alpha}$ is an ${\omega}$-sequence converging to ${\alpha}$, and the whole sequence is a club guessing sequence, that is, if $C\subseteq{\omega}_1$ is a closed unbounded set, then $C_{\alpha}\subseteq C$ holds for some ${\alpha}$. Notice that the existence of a guessing sequence is an easy consequence of the diamond principle.
We are going to define the notion of forcing $(Q^f,\leq)$. Every condition $p\in Q^f$ will be of the form $p=(s,u,g,m,h_i,c)$ where $s\in [{\omega}_1]^{<{\omega}}$, $u\subseteq s$, consisting of limit ordinals, $g$ is a graph on $u$, for every ${\alpha}\in u$ we have an $m({\alpha})<{\omega}$, and then the ordinals $h_0({\alpha})<\cdots<h_{m({\alpha})-1}({\alpha})<{\alpha}$ which are $C_{\alpha}$-separated, that is, $\min(C_{\alpha})<h_0({\alpha})$ and between $h_{i}({\alpha})$ and $h_{i+1}({\alpha})$ there is an element of $C_{\alpha}$ ($h_i({\alpha})$ is undefined if $i\geq m({\alpha})$ or ${\alpha}\notin u$). $c:g\to{\omega}$ satisfies that if $\{x,{\alpha}\}\in g$, $\{y,{\alpha}\}\in g$ and $x,y<{\alpha}$ then $c(x,{\alpha})\neq c(y,{\alpha})$.
Given $p\in Q^f$ we define $$Y^p_r = \{e\in g:c(e)\geq r\}$$ for any natural number $r$.
We add the following stipulations.
1. If ${\alpha}\in u$ is incident to some $e\in g$ with $c(e)\geq r$ then $m({\alpha})\geq r$.
2. If $x$, $y$ are connected in $Y^p_r$ in $i$ steps then $h_{j-i}(x)<h_{j}(y)<h_{j+i}(x)$ holds for $i\leq j\leq f(r)-i$.
3. $Y^p_r$ does not contain odd circuits of length $\leq f(r)$.
We notice that although a condition $p=(s,u,g,m,h_i,c)$ is infinite (perhaps $p=(s,u,g,m,(h_i)_{i<{\omega}},c)$ or $p=(s,u,g,m,h_i,c)_{i<{\omega}}$ would be better notation) as all but finitely many of the partial functions $\{h_i:i<{\omega}\}$ are the empty function, every condition is really a finite object.
The partial order on $Q^f$ is defined the natural way. $p'=(s',u',g',m',h'_i,c')$ extends $p=(s,u,g,m,h_i,c)$ iff the following hold. $s'\supseteq s$, $u=u'\cap s$, $g=g'\cap [s]^2$. $m'({\alpha})\geq m({\alpha})$ holds for ${\alpha}\in u$, and $h'_i({\alpha})=h_i({\alpha})$ for $i<m({\alpha})$. Finally, $c'(x,{\alpha})=c(x,{\alpha})$ holds for $\{x,{\alpha}\}\in g$.
We call two conditions $p=(s,u,g,m,h_i,c)$ and $p'=(s',u',g',m',h'_i,c')$ [*isomorphic*]{} iff $|s|=|s'|$ and the unique order preserving mapping $\pi:s\to s'$ satisfies $u'=\pi[u]$, $g'=\pi[g]$, $m({\alpha})=m'(\pi({\alpha}))$ for ${\alpha}\in u$, $h'_i(\pi({\alpha}))=h_i({\alpha})$ and $c'(\pi(x),\pi({\alpha}))=c(x,{\alpha})$ hold whenever the right hand sides are defined. Notice that, as our conditions are finite structures, we have only countably many isomorphism types.
From a generic $G\subseteq Q^f$ we define the following graphs on ${\omega}_1$: $$X = \bigcup \left\{g:(s,u,g,m,h_i,c)\in G \right\},$$ $$X_n = \left\{ \{x,y\}: \{x,y\}\in g , c(x,y)=n,(s,u,g,m,h_i,c)\in G
\right\},$$
$$Y_r = X_r\cup X_{r+1}\cdots$$ and notice that $Y_r=\cup\{Y^p_r:p\in G\}$.
[**Lemma 1.**]{} [*For ${\alpha}<{\omega}_1$ the set $\{(s,u,g,m,h_i,c)\in Q^f:{\alpha}\in s\}$ is dense.*]{}
[**Proof.**]{} Straightforward.
[**Lemma 2.**]{} [*If ${\alpha}<{\omega}_1$ is limit, $(s,u,g,m,h_i,c)\in Q^f$, ${\alpha}\notin s$, then there is an extension $(s',u',g',m',h'_i,c')\leq (s,u,g,m,h_i,c)$ with ${\alpha}\in u'$.*]{}
[**Proof.**]{} Straightforward.
[**Lemma 3.**]{} [*If $(s,u,g,m,h_i,c)\in Q^f$, ${\alpha}\in u$, $n<{\omega}$ then there is an extension $(s',u',g',m',h'_i,c')\leq (s,u,g,m,h_i,c)$ with $m({\alpha})\geq n$.*]{}
[**Proof.**]{} It suffices to show that $m({\alpha})$ can be incremented by one. Given $h_{m({\alpha})-1}({\alpha})<{\alpha}$ if we choose $h'_{m({\alpha})}({\alpha})<{\alpha}$ large enough the condition on $C_{\alpha}$ will surely be satisfied.
[**Lemma 4.**]{} [*$(Q^f,\leq)$ is ccc.*]{}
[**Proof.**]{} Modulo standard arguments we have to show that $p=(s,u,g,m,h_i,c)$ and $p'=(s',u',g',m',h'_i,c')$ are compatible, assuming that they are isomorphic, and $s\cap s'$ is an initial segment of both $s$ and $s'$. Let $\pi:s\to s'$ be the order preserving structure isomorphism. We let $p''=(s'',u'',g'',m'',h''_i,c'')$ where we take unions in all coordinates.
In order to show that $p''$ is a condition we have to check properties (1.–3.).
(1.) is obvious.
For (3.) assume that $C$ is an odd circuit of length $\leq f(r)$ in $Y^{p''}_r$. If we replace every $e\in C$ that contains at least one vertex from $s-s'$ with $\pi(e)$ then we get an odd cycle $C'$ in $Y^{p'}_r$. $C'$ splits into circuits, at least one of them odd, so we get a contradiction.
For (2.) notice that it holds if $x$, $y$ are joined via a path going entirely in $Y^{p}_{r}$ or $Y^{p'}_{r}$. It suffices, therefore, to show that if some path $P$ between $x$ and $y$ of length $i$ is split by an inner point $z$ into the paths $P_0$ and $P_1$ between $x$ and $z$, and $z$ and $y$, respectively, and of the respective lengths $i_0$ and $i_1$ (so $i_0+i_1=i$) and the statement holds for $P_0$ and $P_1$ then it holds for $P$, as well. Indeed, for $i\leq j \leq f(r)-i$ we have $$h_{j-(i_0+i_1)}(x)<h_{j-i_1}(z)<h_{j}(y)<h_{j+i_1}(z)<h_{j+(i_0+i_1)}(x).$$
[**Lemma 5.**]{} [*${{\rm Chr}}(X)={\omega}_1$.*]{}
[**Proof.**]{} Assume that some $\overline{p}$ forces that $\underline{F}$ is a good coloring of $X$ with the elements of ${\omega}$. Select an increasing, continuous sequence of countable elementary submodels $ \overline{p}, \underline{F}, Q^f,
{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}\in N_0\prec N_1\prec\cdots N_{\alpha}\prec H(\lambda)$ with some large enough regular cardinal $\lambda$, for ${\alpha}<{\omega}_1$, such that ${\gamma}_{\alpha}=N_{\alpha}\cap{\omega}_1$ is an ordinal. The set $C=\{{\gamma}_{\alpha}:{\alpha}<{\omega}_1\}$ will be a closed, unbounded set, so by the guessing property there is some ${\delta}={\gamma}_{\alpha}$ such that $C_{\delta}\subseteq C$. Notice that all points of $\overline{p}$ are smaller than ${\delta}$.
Extend $\overline{p}$ to a $p'$ using Lemma 2., adding ${\delta}$ to the $u$-part, then let $p^*$ be a condition extending $p'$ such that $p^*{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}\underline{F}({\delta})=i$ holds for some $i<{\omega}$.
Let $n$ be some natural number that $n\notin \{c^*(x,{\delta}):\{x,{\delta}\}\in g^*, x<{\delta}\}$ where $p^*=(s^*,u^*,g^*,m^*,h^*_i,c^*)$. Extend $p^*$ using Lemma 3., to some condition $p$ with $p=(s,u,g,m,h_i,c)$ such that $m=m({\delta})\geq f(n)$ holds.
In $p$ we have the values $$h_{0}({\delta})<h_{1}({\delta})<\cdots<h_{m-1}({\delta})<{\delta}$$ and by our requirements on conditions there are elements ${\delta}_0,\dots,{\delta}_{m-1}$ of $C_{\delta}$ such that
$${\delta}_0<h_{0}({\delta})<{\delta}_1<h_{1}({\delta})<\cdots<{\delta}_{m-1}<h_{m-1}({\delta})<{\delta}$$ holds.
The values ${\delta}_0,\dots,{\delta}_{m-1},{\delta}$ break $s$ into disjoint parts: $s=s_0\cup\cdots\cup s_m\cup s_{m+1}$ with $$s_0<{\delta}_0\leq s_1<{\delta}_1\leq s_2<\cdots <{\delta}_{m-1}\leq s_m <{\delta}\leq s_{m+1}$$ (some of them may be empty).
[**Sublemma.**]{} [*There is a condition $p'$ on some $s'=s_0\cup s'_1\cup\cdots\cup s'_m\cup s'_{m+1}$ isomorphic to $p$ with $|s'_i|=|s_i|$, ${\delta}'=\min(s'_{m+1})$, $p'{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}\underline{F}({\delta}')=i$ and $$s_0<s'_1<{\delta}_0\leq s_1<s'_2<{\delta}_1\leq s_2<\cdots< s'_m
<{\delta}_{m-1}\leq s_m <s'_{m+1} <{\delta}\leq s_{m+1}$$ holds.*]{}
[**Proof.**]{} Let ${\theta}$ be the isomorphism type of $p$. For the ordered finite sets $\overline{x}_0,\overline{x}_1,\dots,\overline{x}_{m+1}$ let $\psi(\overline{x}_0,\overline{x}_1,\dots,\overline{x}_{m+1})$ denote the statement that $\overline{x}_0<\overline{x}_1<\cdots<\overline{x}_{m+1}$, $|\overline{x}_i|=|s_i|$ and for the (unique) condition $p$ on $\overline{x}_0\cup\overline{x}_1\cup\cdots\cup\overline{x}_{m+1}$ of type ${\theta}$ $p{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}\underline{F}({\delta})=i$ where ${\delta}=\min(\overline{x}_{m+1})$.
Set $$\varphi_{m+1}(\overline{x}_0,\overline{x}_1,\dots,\overline{x}_{m+1})=
\psi(\overline{x}_0,\overline{x}_1,\dots,\overline{x}_{m+1})$$ and for $0\leq i\leq m$ $$\varphi_i(\overline{x}_0,\overline{x}_1,\dots,\overline{x}_{i})=
\exists^*\overline{x}_{i+1}\varphi_{i+1}(\overline{x}_0,\overline{x}_1,
\dots,\overline{x}_{i+1})$$ where the quantifier $\exists^*$ denotes “there exist unboundedly many” which is expressible in the first order language of $({\omega}_1,<)$.
[**Claim.**]{} [*For $0\leq i \leq m+1$ the sentence $\varphi_i(s_0,s_1,\dots,s_{i})$ holds.*]{}
[**Proof.**]{} We prove the statement by reverse induction on $0\leq i \leq m+1$. We certainly have $\varphi_{m+1}(s_0,s_1,\dots,s_{m+1})$. If for some $0\leq i \leq m$ we had that $\varphi_{i+1}(s_0,s_1,\dots,s_{i+1})$ holds yet $$\varphi_i(s_0,s_1,\dots,s_{i})=
\exists^*\overline{x}_{i+1}\varphi_{i+1}(s_0,s_1,\dots,s_i,
\overline{x}_{i+1})$$ fails, then there was a bound, computable from $s_0,s_1,\dots,s_i$ for the minima of those sets $\overline{x}_{i+1}$ for which $\varphi_{i+1}(s_0,s_1,\dots,s_i,\overline{x}_{i+1})$ holds. Then this bound was smaller than ${\delta}_i$ (${\delta}$ for $i=m$) as there is an elementary submodel containing the ordinals $<{\delta}_i$ (or $<{\delta}$) but this contradicts the fact that $\varphi_{i+1}(s_0,s_1,\dots,s_{i+1})$ holds and ${\delta}_i\leq s_i$.
Using the Claim we can inductively select the sets $s'_1,\dots,s'_{m+1}$ such that for every $1\leq i \leq m+1$ we have $\varphi_i(s_0,s'_1,\dots,s'_{i})$ and $s_i<s'_{i+1}\leq {\delta}_i$, as required.
Using the Sublemma we create the following one-edge amalgamation $p''$ of $p$ and $p'$. $p''=(s'',u'',g'',m'',h''_i,c'')$ where $s''=s\cup s'$, $u''=u\cup u'$, $g''=g\cup g'\cup\left\{\{{\delta},{\delta}'\}\right\}$, $m''=m\cup m'$, $h''_i=h_i\cup h'_i$, $c''=c\cup c'$.
We have to show that $p''$ is indeed a condition, that is, we have to check if the properties (1.-3.) hold.
(1.) is obvious.
For (2.) we argue as in the proof of Lemma 4; every path in question is the union of paths for which this condition holds, plus possibly the path $\{{\delta},{\delta}'\}$ but (2.) also holds for this.
Assume finally, that $C$ is a circuit of length $2t+1\leq f(r)$ in $Y^{p''}_r$. Unless $C$ contains $\{{\delta},{\delta}'\}$, we can argue as in Lemma 4. So we are left with the case that $C$ contains $\{{\delta},{\delta}'\}$ and $r\leq n$. That is, ${\delta}\in s$ and ${\delta}' \in s'$ are joined in $Y^{p}_r\cup Y^{p'}_r$ in $2t$ steps, and this is only possible if the connecting path has vertices in $s\cap s'$. So we get that ${\delta}$ can be connected in $Y^{p}_r$ with some point in $s\cap s'$ in $\leq t$ steps. But this is impossible: if $x\in s\cap s'$ is such a point then $ h_0({\delta})<h_t(x)<h_{2t}({\delta})$ by condition (2.) and also $x<h_0({\delta})$, a contradiction.
As $p''$ forces that $F({\delta})=F({\delta}')=i$ yet they are joined in $X$, we are finished.
[**Theorem 1.**]{} [*The forcing $Q^f$ adds an uncountably chromatic graph $X$ on ${\omega}_1$ such that every subgraph on at most $f(r)$ vertices is at most $2^{r+1}$-chromatic.*]{}
[**Proof.**]{} As every $X_n$ is a circuitfree graph, it can be colored with two colors. Consider now a subgraph of $X$ induced by a set $S$ of at most $f(r)$ vertices. On $S$ all the graphs $X_0,\dots,X_{r-1}$ are bipartite, and so is $Y_r=X_r\cup\cdots$ (as it has no odd circuits of length $\leq f(r)$). So their union, $X$ restricted to $S$, can be colored by at most $2^{r+1}$ colors.
[**Theorem 2.**]{} [*It is consistent with CH that for every function $f:{\omega}\to{\omega}$ there is an uncountably chromatic graph $X$ on ${\omega}_1$ such that every sugraph of $X$ on $f(r)$ vertices is at most $r$-chromatic ($r\geq 2$).*]{}
[**Proof.**]{} Assume that $\diamondsuit$ holds in the ground model. Then we have CH and there is a club guessing sequence $\{C_{\alpha}:{\alpha}<{\omega}_1,\mbox{limit}\}$ as required for Theorem 1. We force with a finite support iteration $P=\{P_{\alpha},Q_{\alpha}:{\alpha}<{\omega}_1\}$. In step ${\alpha}<{\omega}_1$ we add $Q_{\alpha}=Q^{f_{\alpha}}$ for some increasing function $f_{\alpha}:{\omega}\to{\omega}$. As this will be a ccc forcing that preserves CH it is possible by bookkeeping to make sure that every suitable $f:{\omega}\to{\omega}$ occurs as some $f_{\alpha}$. Also, as $P_{\alpha}$, the iteration up to ${\alpha}$ is ccc, every closed, unbounded set $C$ in $V^{P_{\alpha}}$ contains a ground model closed, unbounded set $D$, and as there is some element $C_{\alpha}$ of the club guessing system that $C_{\alpha}\subseteq D$ we have $C_{\alpha}\subseteq C$, that is the club guessing system retains its property in $V^{P_{\alpha}}$.
Call a condition $p\in P$ [*determined*]{} if for every ${\alpha}<{\omega}_1$ the condition $p|{\alpha}$ completely determines $p({\alpha})$, that is, for every ${\alpha}$ coordinate $p({\alpha})$ is not just a name for a finite structure but it is actually a finite structure.
[**Lemma 6.**]{} [*The determined conditions form a dense set in $P$.*]{}
[**Proof.**]{} We prove by induction on ${\alpha}<{\omega}_1$ that the determined conditions form a dense subset of $P_{\alpha}$. This is obvious if ${\alpha}$ is limit, as we are considering finite supports. Assume that we have the statement for some ${\alpha}<{\omega}_1$ and we try to handle the case of ${\alpha}+1$. Let $(p,q)\in P_{{\alpha}+1}=P_{\alpha}\ast Q_{\alpha}$ be arbitrary. Extend $p$ to some $p'$ that completely determines $q$, that is, there is a finite structure $h$ that $p'{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}q=h$. Then extend $p'$ to a determined $p^*\in P_{\alpha}$. Now $(p^*,h)$ is a determined extension of $(p,q)$.
[**Lemma 7.**]{} [*For every ${\alpha}<{\omega}_1$, ${{\rm Chr}}(X_{\alpha})={\omega}_1$ holds in $V^P$.*]{}
[**Proof.**]{} By moving to $V^{P_{\alpha}}$ we can assume that ${\alpha}=0$. We imitate the proof of Lemma 5. By Lemma 6 we can work with determined conditions. We consider every such condition as a finite structure on some finite subset $s$ of $\omega_1$, here $s$ contains all points of all graphs $p({\beta})$ where ${\beta}$ is an arbitrary element of the support of $p$, and we also add the elements of the support to $s$. Assume that some $\overline{p}\in P$ forces that $\underline{F}$ is a good coloring of $X_0$ with the elements of ${\omega}$. With an argument like in Lemma 5 we get some natural number $n$, ordinals ${\delta}'<{\delta}<{\omega}_1$, and also ${\alpha}_1,\dots,{\alpha}_m,{\beta}_1,\dots,{\beta}_t, {\beta}'_1,\dots,{\beta}'_t$ and two isomorphic determined conditions $p$ and $p'$ with the respective supports $\{0,{\alpha}_1,\dots,{\alpha}_m,{\beta}_1,\dots,{\beta}_t\}$ and $\{0,{\alpha}_1,\dots,{\alpha}_m,{\beta}'_1,\dots,{\beta}'_t\}$ that $p{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}\underline{F}({\delta})=n$, $p'{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}\underline{F}({\delta}')=n$ hold, $p(0)$ and $p'(0)$ are isomorphic conditions that behave like $p$ and $p'$ in Lemma 5, for every ${\alpha}_i$ the structures $p({\alpha}_i)$ and $p'({\alpha}_i)$ are isomorphic with the common part preceding the tails, that is, it is possible to make a non-edge amalgamation. We can, therefore, take the union of $p$ and $p'$, and add the edge $\{{\delta},{\delta}'\}$ with color $n$ in coordinate 0.
The above Lemma concludes the proof of the Theorem.
The Taylor conjecture
=====================
[**Theorem 3.**]{} [*It is consistent that there is a graph $X$ with ${{\rm Chr}}(X)=|X|=\aleph_1$ such that if $Y$ is a graph with all finite subgraphs occurring in $X$ then ${{\rm Chr}}(Y)\leq \aleph_2$, that is, the Taylor conjecture fails.*]{}
[**Proof.**]{} Let $V$ be a model of GCH and $\diamondsuit$. Let $P$ be the notion of forcing that adds a Cohen real. It is well known that $P$ adds an undominated real, that is, a function $f:{\omega}\to{\omega}$ such that for no $g:{\omega}\to{\omega}$ in $V$ does $f(n)\leq g(n)$ hold for every $n<{\omega}$. Let $V'$ be the forced model. Notice that $V'$ still has GCH and club guessing (by the argument in the proof of Theorem 2.). Now force over $V'$ with the partial order $Q^f$, and get a graph $X$ with ${{\rm Chr}}(X)=|X|=\aleph_1$ such that every $n$-chromatic subgraph of $X$ has at least $f(n)$ elements ($n\geq 3$). This $X$ will be our graph. To show the property stated, assume that $Y$ is a graph in $V^{P,Q^f}$ whose every finite subgraph is a subgraph of $X$. We assume that the vertex set of $Y$ is some cardinal $\lambda$. We notice that every $n$-chromatic subgraph of $Y$ has at least $f(n)$ elements.
[**Lemma 8.**]{} [*If $Z\subseteq Y$ is a subgraph with $Z\in V$ then $Z$ is finitely chromatic.*]{}
[**Proof.**]{} Otherwise for every $n<{\omega}$ we can let $g(n)$ be the minimal size of an $n$-chromatic subgraph of $Z$. Now notice that $g\in V$ and also by the absoluteness of the set of finite subsets of $\lambda$ and the absoluteness of the cromatic number of a finite graph, $g$ denotes the same thing in $V$ and $V^{P\ast Q^f}$. This implies that $g(n)\geq f(n)$ holds for every $n$, but that obviously contradicts the fact that $f$ cannot be dominated by the the ground model ${\omega}\to{\omega}$ functions.
We finally need the following Lemma.
[**Lemma 9.**]{} [*If $R$ is a notion of forcing over some model $V$, $Y$ is a graph in the extended model on some ordinal ${\lambda}$ then $Y$ is the union of at most $|R|$ subgraphs which are elements of $V$.*]{}
[**Proof.**]{} Let $\tau$ be a name for (the edge set of) $Y$. Set $$Z_p=\{e\in [\lambda]^2:p{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}e\in\tau\}$$ for $p\in R$, then $Y=\bigcup\{Z_p:p\in G\}$ where $G\subseteq R$ is a generic set.
To finish the proof of the Theorem we remark that by Lemma 9 $Y$ decomposes into the union of $|P\ast Q^f|=\aleph_1$ graphs each being in $V$, therefore finitely chromatic, so we get ${{\rm Chr}}(Y)\leq 2^{\aleph_1}=\aleph_2$.
The followig argument gives that the Hanf number mentioned in the Introduction can be as small as $\aleph_2$.
[**Theorem 4.**]{} [*It is consistent that if $X$ is a graph with ${{\rm Chr}}(X)\geq\aleph_2$ then for every cardinal ${\lambda}$ there exists a graph $Y$ with ${{\rm Chr}}(Y)\geq{\lambda}$ all whose finite subgraphs are induced subgraphs of $X$.*]{}
[**Proof.**]{} Let $V$ be a model of GCH. Choose the regular cardinal ${\kappa}$ so large that the following holds. If $X$ is a graph with ${{\rm Chr}}(X)\geq{\kappa}$ and ${\lambda}$ is a cardinal then there is a graph with ${{\rm Chr}}(Y)\geq{\lambda}$ all whose finite subgraphs occur as subgraphs of $X$. Clearly, such a ${\kappa}$ exists.
Let $P={\rm Col}({\omega},{\kappa})$ be the collapse of ${\kappa}$ to $\aleph_0$, that is, the elements of $P$ are those functions of the form $p:n\to{\kappa}$ for some $n<{\omega}$ with $p\leq q$ iff $p$ extends $q$ as a function. Our claim is that if $G\subseteq P$ is generic then $V[G]$ models the statement of the Theorem. Notice that $|{\kappa}|=\aleph_0$ holds there and calculation shows that GCH still holds in $V[G]$.
Assume that $X$ is a graph in $V[G]$ with chromatic number at least $\aleph_2$ (that is, $\aleph_2^{V[G]}$). By Lemma 9., $X$ is the union of $|G|=\aleph_0$ ground model graphs. As $\aleph_1^{\aleph_0}=\aleph_1$, one of them, say $Y$ must have chromatic number at least $\aleph_2$. In $V$, $Y$ has chromatic number at least $\aleph_2^{V[G]}={\kappa}^{++}$. Assume that we are given some ${\lambda}>{\kappa}$. By the choice of ${\kappa}$, there is a graph $Z$ with ${{\rm Chr}}(Z)\geq{\lambda}$ such that every finite induced subgraph of $Z$ is an induced subgraph of $Y$.
[**Lemma 10.**]{} [*${{\rm Chr}}(Z)\geq{\lambda}$ holds in $V[G]$.*]{}
[**Proof.**]{} Otherwise let $\underline{F}$ be a name for a coloring with the ordinals less than $\tau<{\lambda}$. Then the coloring $x\mapsto (p,\xi)$ is a good coloring of the vertices of $Z$ with ${\kappa}+\tau<{\lambda}$ colors, where $p\in P$ is some element of $P$ with $p{{\,\parallel\joinrel\relbar\joinrel\relbar\,}}\underline{F}(x)=\xi$.
We are almost finished, the only problem is that the finite induced subgraphs of $Z$ are not induced subgraphs of $X$, they only are (edge-)subgraphs of induced subgraphs of $X$. The following Lemma is what we need.
[**Lemma 11.**]{} [*There is a graph $Z'$ on the vertex set of $Z$ with $Z'\supseteq Z$ and such that every induced subgraph of $Z'$ is an induced subgraph of $X$.*]{}
[**Proof.**]{} Let $S$ be the vertex set of $Z$. For every finite subset $s$ of $S$ there are some graphs on $s$, which on the one hand are isomorphic to induced subgraphs of $X$, on the other hand they are supergraphs of the graph $Z$ restricted to $s$. Call these graphs [*appropriate*]{} for $s$. Notice that there are finitely many appropriate graphs for every given $s$, and if $T$ is an appropriate graph for $s$ and $s'$ is a subset of $s$ then $T$ restricted to $s'$ is a graph appropriate for $s'$. We can therefore apply the Rado selection principle (or the compactness theorem of model theory) and get a graph $Z'$ on $S$ every induced subgraph of which is appropriate, so $Z'$ is a required.
As ${{\rm Chr}}(Z')\geq{{\rm Chr}}(Z)\geq{\lambda}$ holds we are done.
[99]{}
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[^1]: Research partially supported by Hungarian National Research Grant T 032455.
[^2]: This research was supported by the Israel Science Foundation. Publication 788.
| 2024-02-26T01:27:17.760571 | https://example.com/article/4783 |
What’s the Story Behind the ‘World’s Toughest Job?’
An interviewee reacts to American Greetings’ “job description.” The interviewer (inset) is an actor.
The advertising company that came up with the viral “World’s Toughest Job” video campaign for Greeting-card maker American Greetings, said the idea was based on a true story.
According to Mark Wenneker, chief creative officer for Mullen, the Boston ad firm, a member of his creative staff was at a family get-together a few months ago when she noticed her sister-in-law’s efforts at caring for her baby. It seemed like an extremely difficult job.
“So she stored that away like a squirrel” and later recalled the experience while working on the American Greetings project for promoting Mother’s Day, Wenneker said.
The resulting video appears to be a collection of job interviews conducted over Skype. The occupation, described as “Director of Operations” sounds promising at first but quickly heads downhill. As the interviewer gradually reveals increasingly unpleasant, outrageous details of the gig, the interviewees, who are not actors and were not aware of the prank, react with disbelief and disillusionment.
This is a 24-hour job that required heavy lifting, standing most of the time and bending over a lot. There are no breaks and worst of all, there is no pay. The audience soon finds this is the descriptions of a mother’s job. | 2023-08-10T01:27:17.760571 | https://example.com/article/9926 |
Apropos of Steve Sailer’s recounting of New York’s Hug Thug, a black man who offered "free hugs" to women in Times Square and sometimes punched them if they didn't tip him, I have a question: Why hasn’t the man or men in some woman’s family stopped this violent pervert?
The woman he assaulted with a can of water and iced coffee claimed she has a boyfriend. Even if that isn’t true, where were her father or brothers or male friends? Why hasn’t a sin gle man of this woman’s acquaintance put this dangerous weirdo in the hospital? Why hasn’t a man or men on the scene intervened? Jermaine Himmelstein has been arrested 12 times since 2012 for similar crimes. ['Free hug guy' accused in other attacks across New York City, abc7ny.com, May 13, 2016]
I’ve been asking the same question for years about O.J. Simpson. Why didn’t one of the men in Nicole Brown’s life stop him the very first time he beat her to a pulp? No one stopped him, and she wound up dead.
The answer to my question is this: Feminists and “anti-racists” have emasculated the American white man. He fears defending his women in general, and he fears defending them against black predators in particular — I suspect that most of the Hug Thug’s victims are white — partly because he fears being called a “racist.” That would be much worse than being called a coward.
This may have been what paralyzed the three white men who did not fight the black Carr brothers when they perpetrated the Wichita Massacre. [See The Real 'Wichita Horror': Media-Induced Passivity, by Sam Francis.] Maybe they were just scared, but one can’t help but think that those three Good Whites, and the two Good White women, believed that fighting the Carrs would have been racist. To prove one isn’t a racist, he must lay down his life.
The Hug Thug can be stopped. Question is, who’s going to do it? | 2024-05-01T01:27:17.760571 | https://example.com/article/7916 |
This invention relates to a method and apparatus for monitoring the movement of macro-organisms. More particularly the instant invention relates to a method and apparatus for tagging and retrieving fish.
For many years, biologists and fisheries managers have monitored the migratory habits of fish. In recent years, particular emphasis has been placed upon studying the migratory habits of anadromous and catadromous fish as these fish migrate past dams and hydroelectric facilities. This emphasis has focused, in part, on studying the effects which passage through a turbine or fishway may have on a fish population, as migratory and resident fish may suffer injury when passing through a hydroelectric facility. However, as discussed at length in Turbine Related Fish Mortality; Review and Evaluation of Studies AP-5480 Research Project 2694-4, Electric Power Research Institute 1987, no adequate or reliable methods for satisfactorily quantifying the effects of such facilities exists.
A hydroelectric generating area commonly consists of a dam and an associated reservior wherein a large quantity of river water is stored temporarily. The retained water passes through hydroelectric generating turbines and the like in a manner known to those skilled in the art. When a dam or other hydroelectric facility is constructed in a stream or river for such purposes as development of power or storage of water for industrial, municipal or irrigation purposes, certain types of fish may be attracted to or transported with the flow of water from the reservior through the turbines and other bypass routes. As these fish travel in and through the turbines, which rotate at high speeds, fish may suffer injury or be killed as they come into contact with rotating turbine blades or shear forces. Additionally, the fish may suffer injury or death as a result of the changes in water flow and pressure which occur during turbine passage.
Thus, although beneficial to man, some hydroelectric generation facilities have been implicated as being destructive to many types of fish. Overflow spillways may provide some method of escape from the foregoing, however, when there is a water shortage for hydroelectric or irrigation purposes, the spillways may only be opened infrequently, if at all. Moreover, some spillway configurations may be detrimental to a fish population where, for example, the fish are swept downwardly at a precipitous angle and dashed at high velocity against a concrete apron or into an extremely turbulent pool.
In the past there have been attempts to form fish passage structures for diverting fish both upstream and downstream, as well as to form conduits bypassing hydroelectric dams or the like for the intended purpose of attempting to protect fish from passing through hydroelectric turbines and the like. Attempts to save fish have also included the netting and catching of fish for transport downstream by way of tank trucks, barges or the like thereby averting passage through the dam. These and other methods for the diversion of downstream migrating fish are described in Koch, U.S. Pat. No. 4,437,431; Matthews, Park, Achord and Ruele, Static Seawater Challenge Test To Measure Relative Stress Levels in Spring Chinook Salmon Smelts, Trans. of Am. Fisheries Society, 115:236-244 (1986) and Matthews et al., Improved Bypass And Collection System For Protection Of Juvenile Salmon And Steelhead Trout At Lower Granite Dam, U.S. National Marine Fisheries Service, Marine Fisheries Review 39(2):10-14. The attempts made at saving fish described above have serious drawbacks, not the least of which is the significant cost of such measures. A fish way or other diversion device is costly to install. Additionally, where attempts have been made to divert fish via a downstream passage structure, large amounts of water, otherwise useful for hydroelectric generation are normally lost. Thus, the efficiency of the use of the water and of the facility is greatly reduced.
In light of the potential environmental impact which a hydroelectric site may have both upon migrating and resident fish populations and in light of the significant costs often associated with remedying such an impact, Federal and State regulatory and environmental authorities often require a developer or owner of a site to plan and conduct studies detailing the environmental effect which the hydroelectric site has on fish passing through the site. The primary object of these studies is often to measure the environmental impact of the facility by calculating the injury and mortality rate for resident and migrating fish passing through the facility. Federal and State environmental authorities often condition operating and renewal licenses for such facilities on the results of such studies and may deny an operating license to facilities which inflict an unacceptable level of harm on migrating and resident fish populations. Additionally, where the environmental impact has been determined to be high, a regulatory authority may require a developer or owner to implement one or more expensive remedial measures including the construction of fish ways or other diversions, as a precondition to the issuance of an operating permit or license. Such remedial construction measures can exceed tens of millions of dollars in cost.
Given the high cost of remedial measures and the fact that an operating license or permit may be denied if a particular facility has been determined to cause excessive harm to a certain fish population, the accuracy of the study evaluating the effect of the facility on populations which pass through it is of crucial importance. If, based upon such a study, a developer or owner can report that only a small percentage of fish are affected by passage through the facility, costly remedial measures may be avoided. Before such a report will be accepted by the appropriate governmental regulatory authority, however, the owner or developer may often need to explain the details of how the study was performed and to substantiate its accuracy.
Heretofore, in order to collect the mortality data needed for operating licenses or permits several techniques have been employed. These include tracking radio tagged fish past a hydroelectric facility as well as mass marking techniques. In mass marking (stains, fin clips, coded tags) large numbers of fish are introduced upstream of a facility and a portion of these are recaptured after their passage through the facility, usually in some type of nets or screening device. Methods for attaching identification or radio tags to fish are disclosed in U.S. Pat. No. 3,820,545 to Jefferts; U.S. Pat. No. 3,369,525 to Dbrotnic, et al.; U.S. Pat. No. 4,790,090 to Sharber; U.S. Pat. No. 4,646,455 to Gardner; and U.S. Pat. No. 3,313,301 to Jeffers et al.
Monitoring fish passage and associated mortality via radio telemetry normally requires that a miniature radio transmitter be fixed in a suitable waterproof enclosure. That transmitter is then either inserted into the fish's stomach, surgically implanted, or externally attached to the fish. The tagged fish are then introduced upstream prior to the hydroelectric facility. Radio receivers are then used to track the fish after passage through the turbines. The status of each fish is determined by monitoring its movements. A steady signal would indicate that the test specimen is stationary and is therefore presumed dead. This presumption introduces uncertainty into the study, as some stationary fish may be healthy, but simply inactive. In addition, in order to determine if there are any delayed effects due to turbine passage, tracking will have to transpire over a number of days, normally a 72-hour period. Additional uncertainty is also introduced, as there are instances when a test specimen is preyed upon during the tracking period.
One such method for telemetrically monitoring migration has been described in Knight, Marancik and Layzer, Monitoring Movements of Juvenile Anadromous Fish by Radio Telemetry, Prog. Fish Cult 39(3):148-150. The method disclosed by this reference suffers from a number of significant problems. First, the tagged fish are often difficult to locate quickly after passing through the turbines especially if they move into deep pools, as the strength of the signal will attenuate as the depth of the transmitter within the water increases. As a result, a third to a half of the tagged fish may go undetected. Of course, for each fish that goes undetected the uncertainty and margin for error introduced into the results of the study increases. In addition, it bears noting that the cost of each transmitter tag is substantial and typically few of these tags are ever recovered. Also, because of the inherent unreliability and unpredictability of this method, several seasons may be needed to complete a study. Finally, because of the man-power requirements of locating and tracking dispersed fish and because of the often protracted time period needed to determine delayed effects of turbine passage, the costs of conducting such studies are often substantial.
A number of modifications have been made to this known method, in an attempt to increase the percentage of fish recovered. While these modifications have to some extent increased the percentage of fish recovered, the modifications themselves have introduced additional uncertainty into the study with the result that the overall accuracy of the study has not been significantly enhanced. For example, in one such modification, nets are introduced in the tailwaters to collect the fish. Netting permits the recovery of a percentage of the test specimens but often the capture technique itself introduces additional uncertainty into the study. According to this method nets are deployed at the turbine port outlets or downstream a short distance so as to trap and recover the fish quickly after their turbine passage and before they may disperse. These nets themselves are expensive to construct and install and often require on-going maintenance and repair. More importantly, however, the fish are often injured as a result of the netting and when the fish are recovered it is often impossible to determine whether the injury resulted from the turbine passage or from the netting. As a result, those using this method typically assume that all fish recovered in a deceased state lacking visible damage were killed by the net and not by some other cause. This assumption ignores the possibility of suffocation, shock and other mortalities which do not manifest themselves visibly and externally, but which may occur as the fish passes through the turbine, thereby drawing into question the results of the study. Although water filled boxes with diminished water currents ("live cars") attached to the end of a collecting net have lessened the magnitude of injury associated with the capture net in some instances, these devices add to the cost in that they require considerable maintenance as well as a large sampling of fish. Additionally, high flow conditions and large debris loads in the water often make these live cars ineffective or inoperable.
Another modification which has been attempted is the attachment of buoyant floats, fishing floats, or the like to the fish prior to their introduction upstream. These floats tend to float and may bring the fish to the surface after their turbine passage thereby simplifying their recovery. However, the problem presented by this method is that there is no way to control the buoyancy of these floats. The float may inhibit or otherwise interfere with the natural movement of the fish as it approaches and passes through the turbine. Often, the fish surface before or during their turbine passage and may come into contact with stationary or moving parts of the turbine facility thereby causing severe, if not fatal injury to the fish. Since after recovery it is difficult to determine whether the injury resulted from factors associated with the float or from the turbine, the accuracy of this method is at best doubtful. Additionally, because of drag forces the float can be readily torn from the fish during passage.
Finally, it has been known to conduct fish migration studies wherein fish have been placed in sealed plastic bags containing water and some chemicals capable of producing a gas. The bagged fish are then introduced upstream of the dam as the chemicals in the bag react to form a gas. After passage through the turbines the formed gas forces the bags to the surface facilitating their recovery. This method has numerous drawbacks. By putting the fish in a sealed bag during the test fish may be deprived of oxygen thereby increasing the mortality rate. Furthermore, the movement of the fish within the bag may be difficult to control. Since this movement may increase the rate of any chemical/gas-producing reaction within the bag, it may cause inflation to incur prior to entry into the turbines. Finally, the bags themselves seriously impair the natural movement of the fish and its ability to propel itself within the water. To the extent this natural movement is altered by the surrounding bag, the accuracy and reliability of the study is drawn into question.
Of the known prior art methods discussed above, all have limitations which have been heretofore unsuccessfully addressed. Known methods have proved to be unreliable, inaccurate, expensive, and time-consuming. | 2023-08-20T01:27:17.760571 | https://example.com/article/7143 |
A federal judge has ruled that the Trump administration violated the law by failing to promptly resolve visa applications for thousands of Afghans and Iraqis who worked for American troops and diplomats, and ordered the government to fix the delays.
U.S. District Court Judge Tanya Chutkan of Washington, D.C., said the government offered no convincing explanation why it has failed to abide by 2013 legislation requiring authorities to deliver a decision on visa applications for Afghans and Iraqis within nine months. Instead, many applicants — who risked their lives working for U.S. troops or other government agencies — have had to wait for several years to get an answer on their visa requests, the court said in the ruling handed down on Friday.
"This ruling could finally bring relief to these men and women and their families who have been waiting in fear for far too long," said Deepa Alagesan of the International Refugee Assistance Project, one of the lawyers who argued the case.
"They served bravely in support of our missions abroad, and we promised them a pathway to safety in return. This ruling ensures that we keep our promise," Alagesan said.
Calling the government's arguments "untenable," the court ordered the Trump administration to produce a plan within 30 days on how it plans to remedy the problem with the visa applications.
A State Department official, who was not authorized to speak on the record, said, "We are aware of the court’s decision and are reviewing with our colleagues at the U.S. Department of Justice."
The ruling against the administration came as the White House weighs how many refugees — including former Iraqi interpreters — to admit to the U.S. in the next fiscal year starting in October. The Pentagon has argued against further reducing refugee admissions that are already at historic lows, and cited the plight of Iraqis who worked with U.S. soldiers and diplomats at great personal risk.
The Special Immigrant Visa (SIV) program was set up to help Iraqis and Afghans who worked for U.S. troops or diplomats to resettle in the U.S. But the program has been plagued by delays in both Republican and Democratic administrations. Frustrated lawmakers introduced a law in 2013 ordering the State Department and other agencies to process visa applications within nine months and to report back to Congress on any reason for delay.
"It is clear that Congress did not intend to give Defendants (U.S. government) an unbounded, open-ended timeframe in which to adjudicate SIV applications," the federal judge said in her ruling.
Administration lawyers had argued in court that evaluating visa applications was a complex task and officials had to weigh the need to safeguard national security and not only the deadline imposed by Congress.
But Judge Chutkan dismissed the administration's argument, saying the law adopted by Congress recognized the complexity of the process and that it allowed for exceptions in cases deemed to pose a "high risk."
The judge wrote that "the fact that the adjudication process is a complex one does not excuse delays of such magnitude, because Congress explicitly referenced that complexity in the 9-month provision."
Documents revealed during the course of the case showed virtually all SIV applicants have faced delays beyond the nine-month limit for an answer on their visas and that an applicant can expect about a four-year wait.
The class action suit was filed last year by a group of five unnamed Afghans and Iraqis against Secretary of State Michael Pompeo as well as officials leading the Department of Homeland Security and U.S. Citizenship and Immigration Services. The plaintiffs had said they were forced to flee their homes and faced death threats from militants due to their work with the U.S. government.
Ryan Crocker, a retired senior diplomat and former ambassador to both Afghanistan and Iraq, filed a brief in support of the Iraqi and Afghan plaintiffs, saying failure to resolve visa applications damaged the country's national security interests.
"The perception that our country cannot or will not honor its promises to those who have risked literally everything to help our soldiers and diplomats is by itself an irreparable harm to our operational capacities," Crocker wrote. "Moreover, those perceptions have already become a propaganda tool that will certainly risk diminishing our ability to recruit allies in future conflicts."
The number of SIV visas being issued has declined under the Trump administration, and refugee advocates believe it's a result of wider restrictions on immigration and refugee admissions.
After 2014, Iraqis could no longer qualify for the SIV program and now have to apply as refugees. Last year, the U.S. government only admitted two former Iraqi interpreters, despite a backlog of tens of thousands, NBC News previously reported.
Members of Congress have accused the administration of rebuffing requests for information on why applicants are in limbo, and several lawmakers have raised questions about the case of one Afghan interpreter, Muhammad Kamran, who has been denied a visa despite facing death threats. | 2024-07-04T01:27:17.760571 | https://example.com/article/3635 |
INTRODUCTION {#s1}
============
The t(10;11)(p13;q14) translocation leads to the fusion of the *CALM* (*PICALM*) and *AF10* genes \[[@R1]\]. *CALM/AF10* fusions are observed in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and in lymphoma \[[@R2]--[@R5]\], being very frequent in gamma/delta lineage T-acute lymphoblastic leukemias \[[@R6]--[@R8]\].
The expression of CALM/AF10 leads to the development of leukemia in murine bone marrow transplantation and transgenic models \[[@R9]--[@R12]\], and increasing evidence suggests that CALM/AF10 exerts its leukemogenic potential through transcriptional deregulation of target genes, including the HOXA gene cluster, therefore interfering with normal hematopoietic differentiation \[[@R9], [@R13]--[@R15]\], through increased genomic instability by reducing global histone H3K79 methylation \[[@R16], [@R17]\] and through a novel proposed mechanism mediated by the CRM1-dependent nuclear export pathway \[[@R18]\]. Identification of several CALM/AF10 interacting proteins (*e.g*. CATS (FAM64A), DOTL1, IKAROS, FHL2), has shed light on the molecular mechanism relevant for CALM/AF10-mediated leukemogenesis and the possible involvement of these proteins in malignant transformation \[[@R15], [@R19]--[@R22]\].
CATS (FAM64A; Gene ID: 54478) was initially identified as a CALM interacting partner \[[@R19]\]. The findings that expression of CATS (FAM64A) markedly increased the nuclear localization of CALM/AF10 \[[@R19]\] and that the murine *Cats (Fam64A)* transcripts were up-regulated in hematopoietic cells (B220^+^ lymphoid cells) transformed by CALM/AF10 in comparison to the same subpopulation from non-leukemic mice \[[@R10], [@R23]\], suggested that CATS (FAM64A) may play a role in CALM/AF10-mediated transformation. In agreement with that, CATS (FAM64A) functions as a transcriptional repressor \[[@R19]\] capable of antagonizing the transactivation activity of the leukemic fusion protein CALM/AF10 in a GAL4-based transactivation assay \[[@R24]\]. However, whether CATS (FAM64A) contributes to leukemogenesis remains to be determined.
In normal adult tissue, *CATS (FAM64A)* is predominantly expressed in the lymphoid compartment, whereas it is highly expressed in leukemia, lymphoma and tumor cell lines. The protein level of CATS (FAM64A) strongly correlates with cellular proliferation in both normal and malignant cells \[[@R23]\].
Zhao and co-workers reported that CATS (FAM64A) (referred to as RCS1 in their study), is a mitotic regulator that controls the metaphase-to-anaphase transition \[[@R25]\]. Additional roles for CATS as a neuronal protein that is co-expressed and interacts with the cellular prion protein (PrPC) have also been proposed \[[@R26], [@R27]\]. Most recently, CATS (FAM64A) was found among the three most upregulated genes, whose high expression is associated with poor prognosis of more aggressive triple-negative breast cancer (TNBC) \[[@R28]\].
In order to gain further insight into CATS function we performed an extensive analysis of CATS expression during differentiation of leukemia cell lines and investigated the effect of CATS silencing in the CALM/AF10-positive U937 leukemia cell line as well as the effect of Cats overexpression in murine primary bone marrow cells. Here we show that changes in CATS expression affect cell proliferation, cell cycle control and clonogenicity of hematopoietic cells.
RESULTS {#s2}
=======
CATS expression decreases during induced differentiation of leukemia cell lines {#s2_1}
-------------------------------------------------------------------------------
We first investigated CATS gene and protein expression during induced differentiation of leukemia cell lines into erythrocytes, megakaryocytes, monocytes and granulocytes (Figure [1](#F1){ref-type="fig"} and [Supplementary Figure S1](#SD1){ref-type="supplementary-material"}). *CATS* expression decreased during erythroid (by 60%), megakaryocytic (by 43%) and monocytic (by 65% at day 2, and by 96% at day 4) differentiation (Figure [1A-1C](#F1){ref-type="fig"}). However, *CATS* expression increased by 2 fold during granulocytic differentiation of both NB4 (at day 4) and U937 cells (at day 2) (Figure [1D](#F1){ref-type="fig"}). At day 4 of U937 granulocytic differentiation, CATS expression returned to its initial level. Expression of CATS protein followed the same pattern as its transcript levels (Figure [1D](#F1){ref-type="fig"}, lower panels).
{#F1}
CATS knockdown reduces cell proliferation of the U937 cell line *in vitro* {#s2_2}
--------------------------------------------------------------------------
U937 cells were transduced with lentiviral particles expressing a pool of multiple shRNAs for silencing CATS. This approach provides a selective reduction of off-target effects since the pool silence the intended target but have unique off-target signatures \[[@R29]\]. CATS silencing was efficiently achieved at both mRNA and protein levels in shCATS transduced cells when compared to shControl cells ([Supplementary Figure S2A](#SD1){ref-type="supplementary-material"}).
Cell number decreased by approximately 20% upon CATS depletion at two different time points: 24h (Figure [2A](#F2){ref-type="fig"}) and 48h ([Supplementary Figure S3A](#SD1){ref-type="supplementary-material"}), as assessed by MTT. No difference in apoptosis was encountered ([Supplementary Figure S3B](#SD1){ref-type="supplementary-material"}), leading us to believe the difference in viability was due to reduced cell proliferation. Accordingly, Ki-67 analysis revealed reduced proliferation of CATS silenced cells by 13% (Figure [2B](#F2){ref-type="fig"}). As shown by cell cycle analysis, a lower percentage (12% lower) of cells in the S phase of the cell cycle was observed in shCATS cells in comparison to shControl cells (Figure [2C](#F2){ref-type="fig"}). These results indicate a reduced proliferation of U937 cells upon CATS depletion. Alteration in the cell cycle control was further demonstrated by decreased expression of CYCLIN A, E and B1 in shCATS cell lysates, as demonstrated by Western blotting. Levels of CYCLIN D1 remained unaltered in CATS knockdown cells when compared to control cells (Figure [2D](#F2){ref-type="fig"}).
{#F2}
CATS knockdown does not interfere with growth of U937 cells in a xenotransplant model {#s2_3}
-------------------------------------------------------------------------------------
A xenotransplant model was used to assess whether CATS depletion would inhibit tumor growth *in vivo*. No difference was observed in volume or weight of shCATS and shControl tumors after 12 days of tumor growth (Figure [3](#F3){ref-type="fig"}). *CATS* knockdown was confirmed on excised tumors samples ([Supplementary Figure S4](#SD1){ref-type="supplementary-material"}).
{#F3}
CATS knockdown does not alter apoptosis of the U937 cells {#s2_4}
---------------------------------------------------------
Recently, we identified CATS interaction with proteins involved in apoptotic response (unplublished data), therefore we wanted to investigate whether CATS plays a role in apoptosis. shCATS and shControl cells were induced to cell death upon UV irradiation exposition and assessed for annexin-V/PI staining. No difference in the percentage of viable versus apoptotic cells was observed (Figure [4](#F4){ref-type="fig"}).
{#F4}
CATS depletion decreases migration of U937 cells {#s2_5}
------------------------------------------------
We performed a transwell chemotaxis assay in order to investigate whether CATS contributes to cell migration. For that purpose we analyzed shCATS and shControl cell migration towards three different chemotatic stimuli: FBS (Fetal Bovine Serum), CXCL12 and FBS with CXCL12. Approximately 20% fewer shCATS cells than shControl cells migrated towards 10% FBS and 10% FBS with CXCL12, whereas migration towards CXCL12 was unaltered (Figure [5A](#F5){ref-type="fig"}). Cell migration towards 0.5% BSA (used as a control for the experiment) was low and equal for both shControl and shCATS.
{#F5}
Next, we investigated expression of proteins involved in microtubule dynamics and known to control cell migration. Western blot analysis revealed decreased expression of α-TUBULIN, and STATHMIN1, as well as reduced overall α-TUBULIN acetylation and STATHMIN1 phosphorylation on residues Ser16 and Ser38 (Figure [5B](#F5){ref-type="fig"}).
CATS knockdown reduces clonogenicity of U937 cell line {#s2_6}
------------------------------------------------------
We employed a colony formation assay to assess the clonogenic potential of the CATS depleted U937 cells on semisolid medium in the absence of growth factors. A significant decrease of 66% in colony number was observed for shCATS transduced U937 cells compared to the shControl transduced cells (Figure [6A](#F6){ref-type="fig"}).
{#F6}
In order to investigate a possible mechanism involved in the reduced ability of CATS depleted cells to form colonies, we analyzed the expression of the self-renewal related genes *NANOG* \[[@R30], [@R31]\], *BMI-1* \[[@R32]\] and *GLI-1* \[[@R33]\] in the shCATS and shControl cells. Interestingly, while expression of *NANOG* and *BMI-1* were not altered, a reduction of *GLI-1* expression ranging between 18% and 75% was observed in CATS depleted U937 cells (Figure [6B](#F6){ref-type="fig"}).
Since CATS interacts with the CALM/AF10 fusion protein which is present in the U937 cells, we sought to investigated whether CATS depletion in U937 cells would affect the expression of the CALM/AF10-leukemia deregulated genes *HOXA5, HOXA7, HOXA9, HOXA10, HOXA11* and *MEIS1*. Expression of *HOXA7, HOXA9, HOXA10, HOXA11*, and *MEIS1* genes were unaffected in U937 upon CATS depletion. Although not statistically significant, a tendency towards increased *HOXA5* expression was consistently observed in different samples of CATS-depleted U937 cells (Figure [6B](#F6){ref-type="fig"}). In CALM/AF10 leukemia, *HOXA5* overexpression results from CALM/AF10 recruitment of hDOT1L and local H3K79 hypermethylation at the *HOXA5* locus \[[@R15]\], whereas a global reduction of this epigenetic marker also occurs \[[@R16]\]. We analyzed global H3K79 methylation in shCATS transduced cells and shControl cells by Western blotting. The global H3K79 methylation levels in U937 cells were not affected by CATS depletion ([Supplementary Figure S5](#SD1){ref-type="supplementary-material"}).
Cats overexpression reduces clonogenicity of mice primary bone marrow cells {#s2_7}
---------------------------------------------------------------------------
In order to investigate a possible role of Cats in the clonogenicity and/or differentiation potential of primary bone marrow cells, we retrovirally transduced murine progenitor cells to overexpress Cats ([Supplementary Figure S2B](#SD1){ref-type="supplementary-material"}) and performed colony forming cell assays. Hematopoietic progenitors overexpressing Cats (MIG-HA-Cats) formed considerably fewer GM (granulocytes and monocytes), BFU-E (erythrocytes) and GEMM (progenitors) colonies (60%, 76% and 67%, respectively) when compared to cells expressing MIG only (Figure [7A](#F7){ref-type="fig"}). Transduced cells expressing Cats showed a 45% decrease in the total number of colony forming units compared to control (MIG only) (Figure [7B](#F7){ref-type="fig"}) and formed 67% fewer secondary GM colonies than controls (Figure [7C](#F7){ref-type="fig"}).
{#F7}
DISCUSSION {#s3}
==========
To explore the possibility that CATS might play a role in leukemogenesis, we performed an extensive analysis of CATS expression during induced differentiation of leukemia cell lines and functional characterization of CATS in the CALM/AF10-positive U937 leukemia cells.
Expression analysis on leukemic cell lines showed that CATS expression drastically decreased when the cells ceased to proliferate upon exposure to a variety of differentiation agents. Since we have previously shown that CATS is a marker for proliferation, the reduction of its expression during the cellular differentiations was expected.
However, differently from what was observed for erythroid, megakaryocytic and monocytic differentiation, granulocytic differentiation resulted in a significant increase in CATS expression. CATS upregulation was not dependent on the cell line, as it was observed for both U937 and NB4 ATRA-treated cells, but not for U937 PMA-treated cells (monocytic maturation). These results suggest that CATS may perhaps play a role in granulocytic differentiation or could be an ATRA responsive gene. Further studies will be necessary to address a possible role of CATS in granulocytic differentiation.
Using a lentivirus-mediated shRNA knockdown, we demonstrated that CATS silencing reduced cell proliferation and lowered the percentage of cells in the S-phase of the cell cycle. The inhibitory effect of CATS silencing on proliferation was discrete; nevertheless the data clearly demonstrates an altered control of cell cycle progression, attested by diminished expression of the cell cycle regulators CYCLIN A, CYCLIN E and CYCLIN B1, responsible for cell entry and progression in the S-phase and mitosis, respectively \[[@R34]\]. These results are consistent with the described accumulation of CATS protein in S, G~2~ and G~2~/M phases of the cell cycle and its suggested function in these phases \[[@R23], [@R25]\].
The reduced proliferation *in vitro* could not be documented *in vivo*, since growth of xenografts from U937 cells did not show any significant difference in tumor growth depending on the presence or absence of CATS. Therefore, CATS silencing was not sufficient to hinder tumor growth in this model.
In a previous study we identified KIS and SIVA1 as CATS interacting proteins (\[[@R24]\] and data not published). These proteins were recently reported to negatively regulate STATHMIN and consequently impair cell migration through altered microtubule dynamics \[[@R35], [@R36]\]. The interaction between CATS and the two regulators of STATHMIN prompted us to investigate whether CATS contribute to cellular migration. CATS silencing indeed resulted in less migration. Notably, STATHMIN and α-TUBULIN expression were downregulated in CATS silenced cells. The reduced protein expression was accompanied by lower levels of acetylated α-TUBULIN, a posttranslational modification only present on microtubule polymers (for review \[[@R37]\]) and phosphorylated Ser16 and Ser38 of STATHMIN, target residues for the CATS interacting proteins SIVA1 and KIS, respectively \[[@R24], [@R35], [@R36]\]. These results suggest that the reduced migratory ability of CATS depleted cells are due to interference with microtubule dynamics in these cells.
The connection of CATS with microtubule dynamics is very intriguing. Of note is the report that CATS functions during mitosis as a regulator metaphase-anaphase transition \[[@R25]\], a time when mitotic spindles move chromosomes.
Our data demonstrated that CATS depleted U937 cells form fewer colonies than control cells, leading to the conclusion that CATS may play a role in the clonogenicity of the U937 cell line. In an attempt to identify possible mechanism through which CATS could exert this function, we analyzed expression levels of self-renewal genes. We showed that *GLI-1*, a positive effector of the Hedgehog (Hh) signaling pathway \[[@R38]\], was significantly reduced in CATS depleted cells. Recently, Wellbrock and colleagues demonstrated *GLI1* expression as negative prognostic factor in AML. In that report, the authors showed that shRNA-mediated *GLI1* targeted inhibition ranged from 45% and 69% caused antileukemic effects, resulted in a significantly elevated induction of apoptosis and significantly reduced proliferation and colony formation in all three AML cell lines analyzed \[[@R39]\]. Thus, our findings indicate that the *GLI1* downregulation observed in CATS depleted U937 cells might be functionally relevant, and the impairment of this self-renewal pathway could be involved in the reduced colony forming ability of the CATS depleted cells.
Interestingly, *Cats* overexpression in murine primary bone marrow cells also lead to a decreased ability to form different types of colonies and reduced total cell number in colony forming cell assays. Taken together, these results suggest that any disturbance in the optimal expression level of CATS protein might have an impact on cell proliferation and clonogenicity.
Interestingly, in a very recent work using single cell RNA-Seq analysis of individual hematopoietic stem cells (HSCs) *Cats* (*Fam64a*) was found to be highly expressed in the HSC population primed for proliferation. Single-cell colony forming assays confirmed the high proliferation and differentiation potential of the *Cats*-expressing HSC population \[[@R40]\].
We have previously hypothesized that through its interaction with CALM CATS could play a role in CALM/AF10-mediated leukemogenesis \[[@R19]\]. Therefore, we analyzed the expression of the *HOXA* gene cluster, its co-factor *MEIS1* and *BMI1*, all known to be deregulated in CALM/AF10-positive leukemias \[[@R9], [@R13]--[@R15]\]. We observed a tendency of *HOXA5* upregulation in CATS depleted U937 cells compared to control cells. In CALM/AF10 leukemia, *HOXA5* overexpression results from hDOTL1-dependent retention of CALM/AF10 in the nucleus and local H3K79 hypermethylation at the *HOXA5* locus \[[@R15]\]. Just like hDOTL1, CATS is able of markedly increase the nuclear localization of the CALM/AF10 fusion protein \[[@R19]\]. Thus, it is tempting to speculate that CATS and hDOT1L compete for CALM/AF10 interaction, and upon CATS depletion, more hDOT1L is recruited to methylate the *HOXA5* locus. In addition to local H3K79 hypermethylation at the *HOXA5* locus, CALM/AF10-positive leukemia is characterized by a global reduction of the H3K79 epigenetic mark \[[@R16]\]. However, global H3K79 methylation did not change upon CATS depletion in U937 cells. Additional, more locus specific analysis of H3K79 methylation status after CATS depletion are now required.
Here we targeted CATS in a CALM/AF10 positive leukemia cell line and revealed some features of its function yet we should bear in mind the limitations of using this only cell line in the study. Therefore we can neither assume that our findings are restricted to U937 nor dependent on CALM/AF10. Although we have some *in vitro* evidences supporting the possible role of CATS in CALM/AF10-mediated leukemogenesis (demonstrated here and in previous publications \[[@R19], [@R23], [@R24]\]), the present study does not provide evidence for a role of CATS in *in vivo* CALM-AF10 leukemia.
Nevertheless our functional analysis demonstrates that CATS plays a role in cellular processes involved in tumorigenesis, such as cell cycle control, migration, clonogenicity and possibly self-renewal. Hence, further support the notion that disruption of CATS function might contribute to tumorigenesis.
MATERIALS AND METHODS {#s4}
=====================
Leukemia cell lines {#s4_1}
-------------------
Leukemia cell lines U937, K562, NB4 and KU812, obtained from the American Type Culture Collection (ATCC, Manassas, USA) or the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) were grown according to the suppliers\' recommendations and used for cell differentiation, RNA and protein extraction, and virus transduction. All cell lines were tested and authenticated by STR matching analysis using the PowerPlex^®^ 16 HS system (Promega, Madison, WI, USA) and the ABI 3500 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). Additionally, in U937 CALM/AF10 expression was confirmed by RT-PCR (data not shown).
Differentiation of cell lines {#s4_2}
-----------------------------
RNA and protein samples of KU812, K562 and NB4 leukemia cells induced to differentiate were obtained from previous study \[[@R41]\] and *CATS* expression was assessed in the Hemin and Hydroxyurea (HE-HU)-induced erythroid differentiation of KU812, the phorbol-13 myristate-12 acetate (PMA)-induced megakaryocytic differentiation of K562 and the all-trans retinoic acid (ATRA)-induced granulocytic differentiation of NB4 cells.
U937 cells were induced to differentiate for 2 and 4 days into monocytes and granulocytes with PMA (20 nM) and ATRA (10^−6^ M) treatment, respectively. Differentiation was monitored by flow cytometry for positive staining of the cell surface markers CD11b, CD14 and CD15 and by qPCR for transcript levels of *CD15* and *GCSFR*. Cells were collected for morphology analysis and for RNA and protein extraction ([Supplementary Figure S1](#SD1){ref-type="supplementary-material"}).
Quantitative PCR {#s4_3}
----------------
Total RNA was isolated using the RNeasy^®^ Mini or Micro Kit (Qiagen, Hilden, Germany). DNAse I treated RNA was reverse transcribed with oligo dT primers and RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). Reactions were carried out with Maxima SYBR Green qPCR master mix, according to the manufacture\'s protocol (MBI Fermentas, St. Leon-Rot, Germany). The plates were run and analyzed by Mastercycler^®^ ep realplex 4 System (Eppendorf, Hamburg, Germany). Primer sequences are available upon request. *HPRT* and *GAPDH* were used as endogenous control and relative gene expression was calculated using the 2^−ΔΔC^ ~T~ equation \[[@R42]\].
Immunobloting {#s4_4}
-------------
Cellular lysates were electrophoresed on 10-12% SDS-PAGE and transferred to nitrocellulose membrane (Hybond™ ECL™, GE Healthcare, Buckinghamshire, UK). The membranes were blocked and probed with specific antibody, followed by detection with fluorescently labeled secondary antibodies. Membranes were visualized on Alliance 2.7 (UVItec, Cambridge, England). Primary antibodies were purchased from Santa Cruz Biotechnologies, unless otherwise specified: anti-ACTIN (I-19; sc-1616) (1:2000), anti-GAPDH (6C5; sc-32233) (1:4000), anti-CYCLIN E (E-4; sc-377100) (1:1000), anti-CYCLIN B1 (D-11; sc-7393) (1:1000), anti-CYCLIN D1 (A-12; sc-8396) (1:1000), anti-CYCLIN A (H-3; sc-271645) (1:2000), anti-α-TUBULIN (B-7; sc-5286) (1:2000), anti-SIVA (C-20; sc-7436) (1:1000), anti-pOP18 S16R (sc-12948) (1:2000), anti-pOP18 S38 (sc-101810) (1:1000), anti-OP18 (E-3; sc-55531) (1:2000), anti-H3 (C-16; sc-8654) (1:1000). Anti-H3K79 (ab3594) (1:1000) and anti-ACETYL-α-TUBULIN (6-11B-1; ab24610) (1:2000) were purchased from Abcam, Cambridge, England and anti-CATS 2C4 (1:250) was previously described \[[@R23]\]. Secondary antibodies were purchased from Life Technologies (Carlsbad, CA, USA): anti-mouse (1:5000), anti-rat (1:2000), anti-rabbit (1:10000) and anti-goat (1:4000). Quantitative analyses of the optical intensities protein bands were determined with UN-SCAN-IT gel 6.1 software (Silk Scientific, Inc., USA) and normalized by control protein.
Transduction of U937 cells {#s4_5}
--------------------------
U937 cells (American Type Culture Collection (ATCC, Manassas, U.S.A) were transduced with lentivirus particles expressing a pool of two short hairpin RNAs (shRNA) targeting the CATS sequence (FAM64A sc-93656-V; Santa Cruz Biotechnologies, CA, USA; siRNA target sequences GCUUCAUACUCAAGGAUGUtt and GAAGUGCUAGCAUCAGAUAtt) or nonspecific control target (sc-108080), named shCATS and shControl cells, respectively. Briefly, 2 × 10^5^ cells were transduced by spinoculation at multiplicity of infection (MOI) equal to 1 and stable polyclonal shCATS and shControl cell lines were generated after 15 days selection with puromycin (10 μg/ml).
Proliferation assays {#s4_6}
--------------------
For MTT assay cells were seeded in 96-well plates at density of 2.5 × 10^4^ cells/well. After 24 or 48 hours of incubation under normal culture condition, 10 μl of a 5 mg/ml solution of MTT was added per well and incubated for 4 hours. Reaction was stopped by adding 100 μl of 0.1N HCl/isopropanol and proliferation evaluated by measuring the absorbance at 570 nm with an automated plate reader.
Cell proliferation was further assessed by Ki-67 staining. ShCATS and control cells were stained with Ki-67 antibody according to manufacturer\'s instructions (Ki-67 PerCP-Cy5.5 clone B56; BD Bioscience, San Jose, CA, USA). The mean of fluorescence intensity (M.F.I) was obtained by FACS analysis using a FACSCalibur (Becton Dickinson, San Jose, CA, USA) and FlowJo software. IgG isotype was used as negative control for each condition and ten thousand events were acquired for each sample.
Cell cycle analysis {#s4_7}
-------------------
2.5 × 10^5^ cells were fixed in 70% ethanol for 30 min on ice, washed with PBS and stained with 20 μg/ml propidium iodide (PI) containing 10 μg/ml RNAse A for 30 min at room temperature (RT). Cell cycle analysis was performed using FACSCalibur (Becton-Dickinson, California, USA) and Modfit (Verify Software House Inc., USA).
Apoptosis assays {#s4_8}
----------------
For apoptosis evaluation, cells seeded in 12-well plates were treated with UV irradiation (40 J/m^2^) and collected at 0, 3, 6, 9 and 12 hours after UV exposure. At the indicated time points, cells were washed with ice-cold PBS and stained with annexin V and PI (BD Biosciences Pharmingen, California, USA) for 15 minutes at RT. Apoptosis analysis was performed using FACSCalibur (Becton-Dickinson) and FACSDiva software (BD Biosciences Pharmingen). Ten thousand events were acquired for each sample.
Xenograft model of tumor growth in NOD/SCID mice {#s4_9}
------------------------------------------------
For the xenograft tumor model, 1 × 10^7^ U937 shCATS or shControl transduced cells were implanted into the dorsal sub cutis of 8-12 week-old female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Tumors grew locally for 12 days, then they were excised, measured and weighted. Tumor measurements were converted to tumor volume (V) by the formula (V = W^2^ x L x 0.52), where W and L stands for smaller and larger diameters, respectively. RNA was extracted from tumors samples.
Migration assay {#s4_10}
---------------
Migration assays were performed in 12-well 8μm pore-sized transwell plates (Costar, Corning, NY, USA). Cells were seeded above the filters at a density of 1 × 10^5^ cells/well. The lower compartment was filled with the following media: 10% FBS/0.5% BSA; 200 ng/mL CXCL12/0.5% BSA (PeproTech, Rocky Hill, NJ, USA); 10% FBS/200 ng/mL CXCL12/0.5% BSA and 0.5% BSA was used as negative control. After 24 hours, the number of cells which migrated through the filter and reached the lower compartment was counted. Values were expressed as percentage of the input (cells applied directly to the lower compartment) set as 100%.
Colony forming assay {#s4_11}
--------------------
shControl and shCATS transduced U937 cells were plated in semisolid medium depleted of any growth factors (5 × 10^2^ cell/ml; MethoCult 4230; StemCell Technologies Inc., Vancouver, Canada). Colonies were detected after 8 days of culture by adding 200 μl of a 5 mg/ml MTT solution and scored by Image J quantification software (NIH, Bethesda, Maryland, USA).
Construction of retroviral vector and virus production {#s4_12}
------------------------------------------------------
Murine *Cats* hemagglutinin (HA)-epitope tagged cDNA was generated by PCR and subcloned into the multiple cloning site of the modified murine stem cell virus vector (MIG), upstream of the internal ribosomal entry site (IRES) and enhanced GFP (EGFP) gene (MIG-HA-Cats). The MIG vector carrying only the IRES-EGFP cassette was used as a control.
Production of high-titer helper-free recombinant retrovirus was carried out following standard procedures \[[@R43]\] by using the ecotropic 293-Phoenix cell line and the packing cell line GP^+^E86 \[[@R44]\]. Briefly, 293-Phoenix cells were first transfected with the MIG or MIG-HA-Cats plasmids. Supernatant was used to infect the GP^+^E86 cells, which were sorted based on GFP expression. Single cell culture was carried out in order to obtain monoclonal populations of GP^+^E86-MIG or MIG-HA-Cats viral producer cells. The viral titre was determined by transducing NIH3T3 cells.
Retroviral transduction of primary bone marrow cells {#s4_13}
----------------------------------------------------
Primary bone marrow cells were obtained from 8 to 12-week-old C57BL/6 mice pretreated with 5-fluorouracil for 5 days. Total bone marrow was harvested from femurs and cultured in DMEM, 20% FBS (Gibco by Life Technologies, Carlsbad, CA, USA) and cytokines (100 ng/mL stem cell factor (SCF), 10 ng/mL interleukin 6 (IL6) and 6 ng/ml interleukin 3 (IL3)) for 48 hours. For retrovirus transduction, 5 × 10^6^ cells were cocultured with 1.2 × 10^6^ virus-producing GP^+^E86 cells (GP^+^E86-MIG and -MIG-HA-Cats) in the presence of cytokines and protamine sulfate (5 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA). The transduction was stopped by removing the bone marrow cells from the GP^+^E86 cells and culturing them in afore mentioned media for another 48 hours to allow for GFP expression. Retrovirally transduced cells were sorted based on GFP expression on a FACSAria IIu (Becton-Dickinson). Sorted GFP-positive cells were further cultured for 48 hours in DMEM, 35% FBS plus cytokines and used for colony forming cell (CFC) assays and RNA extraction.
Colony forming cell assay {#s4_14}
-------------------------
2 × 10^4^ transduced cells were cultured in 1% myeloid-conditioned methylcellulose medium (MethoCult M3434; StemCell Technologies Inc., Vancouver, Canada) for 8 days, after which GM (granulocytes and monocytes), BFU-E (erythrocytes) and GEMM (progenitors) colonies were counted. The total number of cells was determined; cells were replated in the same concentration and cultured for an additional 10 days, after which secondary colonies were counted.
Animal care {#s4_15}
-----------
Animal care was conducted in accordance with the standard ethical guidelines. Protocols have been approved by the local ethical committee "Comissão de Ética no Uso de Animais (CEUA)/Unicamp" - Protocol n° 3165-1. Parental strain mice were bred and maintained at the University of Campinas Central Breeding Center (Campinas, SP, Brazil).
SUPPLEMENTARY MATERIALS FIGURES {#s5}
===============================
**CONFLICTS OF INTEREST**
The authors declare no conflicts of interest.
**GRANT SUPPORT**
This research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 101624/2011-5 to LFA), Coordenação de Aperfeiçoamento Pessoal de nível Superior (CAPES) and Fundação de Amparo à Pesquisa de São Paulo (FAPESP; 2011/18188-0 to IB and 2011/51959-0 to STOS). SKB is supported by Leukaemia and Blood Cancer New Zealand and the family of Marijanna Kumerich. The Hematology and Hemotherapy Center-UNICAMP is part of the National Blood Institute (INCT de Sangue CNPq/MCT).
[^1]: Present address: Department of Laboratory Medicine, Yale Stem Cell Center
[^2]: Present address: Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
| 2023-11-19T01:27:17.760571 | https://example.com/article/9091 |
Q:
Asymptotic convergence
I have a previous similar question. I'm working out that one with the answerer, but I'm trying to gain insight from a different angle, especially in approaching these problems.
I must establish that two vectors $A$ and $B$ are equal asymptotically (length of vectors, $n\to\infty$). I consider the error vector, $e=A-B$ and try to show that $e\to 0$ as $n\to\infty$.
I then show that for each element $e_i$ of the error vector, $\Vert e_i\Vert_2\to 0 $ as $n\to\infty$. How can I proceed from here? Is this sufficient to say that the two vectors are equal in some sense?
A:
Not sure I quite follow, but suppose $A$ is all zeros except for a one in its last component, and $B$ is all zeros except for a one in its next-to-last component. Then each component of the difference goes to zero but the difference doesn't go to zero as the number of components goes to infinity.
| 2023-09-04T01:27:17.760571 | https://example.com/article/2494 |
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of your laptops.
Category number 12 is
end-of-life treatment
of your sold products.
Sadly, most products that
we use end up as waste
and this emission source
is the carbon emissions
caused by, say for instance,
landfilling or incineration.
Category is 13 is
downstream leased assets.
This category includes emissions
from the operation of assets that you own,
but you lease that to other businesses
that are not already
included in your scope 1
or scope 2 inventory.
Category 14 is franchises.
This category includes emissions
from the operation of franchises
not included in your scope
1 or scope 2 inventory.
And finally category 15, investments.
This category includes
emissions associated
with your investments that
are not already included
in your scope 1 or scope 2 inventory.
Category 15 is primarily
designed for banks.
While this list can be a bit overwhelming,
not all emission sources will be relevant.
It's important to prioritize
your data collection efforts
and focus on your most significant
and relevant emission sources.
You can ask questions,
such as whether you
expect the emission source
to be large relative to your scope 1
and scope 2 sources,
or whether you have
influence over the activity
or whether your stakeholders deem
the emission source relevant.
In my next video I'll be
talking about the benefits
of calculating a scope 3 carbon inventory.
Thanks for watching!
| 2024-07-12T01:27:17.760571 | https://example.com/article/7274 |
st value in -4.82, -1/5, 5, 1, -1, -2, -18?
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-4
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-2.1
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What is th | 2024-04-25T01:27:17.760571 | https://example.com/article/2963 |
847 F.2d 368
William J. KLEIN, Plaintiff-Appellant,v.Lawrence RYAN and Frank Lombardo, Defendants-Appellees.
No. 87-2554.
United States Court of Appeals,Seventh Circuit.
Argued Feb. 9, 1988.Decided May 16, 1988.
Robert M. Hodge, Chicago, Ill., for plaintiff-appellant.
John B. Murphey, Rosenthal, Murphey, Coblentz & Janega, Chicago, Ill., for defendants-appellees.
Before BAUER, Chief Judge, WOOD, Jr., Circuit Judge, and ESCHBACH, Senior Circuit Judge.
HARLINGTON WOOD, Jr., Circuit Judge.
1
Plaintiff Klein brought this action against defendants Ryan and Lombardo under 42 U.S.C. Sec. 1983. Klein claims that the defendants violated his fourth and fourteenth amendment rights when they shot Klein as he was fleeing after committing a burglary. The district court granted the defendants' motion for summary judgment based upon their affirmative defense of qualified immunity. We affirm.
I. FACTUAL BACKGROUND
2
In April 1983, the owner of Family Pride Laundromat, located at 495 Roosevelt Road, Glen Ellyn, Illinois, complained to the Glen Ellyn Police Department that someone was taking money from the laundromat. Through the aid of photographic surveillance, the police determined that the man breaking into the laundromat was plaintiff William Klein. As part of their investigation, the Glen Ellyn police periodically placed the laundromat under surveillance.
3
Defendant Lawrence Ryan, a police officer with the Village of Glen Ellyn, participated in the laundromat investigation. During the course of his investigation, and prior to May 12, 1983, Ryan had been advised by a police officer in West Chicago that on an earlier occasion when Klein had been arrested in that city, he had been carrying a gun. Ryan had attempted to locate Klein from an address that Klein had given on previous arrests in the Village of Mount Prospect, but the address was fictitious.
4
Ryan and defendant Frank Lombardo, an auxiliary police officer with the Glen Ellyn Police Department, were assigned to the evening shift on May 12, 1983. At the beginning of that shift, the shift commander briefed the officers on the proposed stakeout of Family Pride Laundromat. In the course of the briefing, the shift commander told the officers of a prior incident where Klein had attempted to run someone over with an automobile.
5
Around 11:00 p.m. that evening, Ryan and his partner, Mark Tobias, checked on the laundromat and found the back door propped open with a rock. They closed the door and drove east on Roosevelt Road. As the policemen were driving away from the laundromat, they observed Klein a few feet away in another vehicle. Ryan recognized Klein from the surveillance photos taken at the laundromat in April.
6
Ryan and Tobias parked across the street south of the laundromat. Klein parked his car in the parking lot south of the laundromat, with his car facing west. Ryan observed Klein walk along the west side of the laundromat to the front (north) door, carrying a container. Klein opened the front door and entered the laundromat. After Ryan observed Klein remove coin boxes from the dryers, Ryan radioed for assistance.
7
While Klein was still inside, Ryan walked to the west wall of the laundromat and watched Klein through a window. Ryan did not bring his police radio with him.
8
In response to Ryan's request for assistance, Lombardo was assigned to go to the front entrance of the laundromat, in accordance with the original plan that the briefing officer had outlined. En route to the laundromat, Lombardo learned that Klein was parked in the rear of the building, necessitating a change in the plan. Upon arriving at the laundromat, Lombardo left his squad car and positioned himself behind a dumpster located in the rear parking lot of Arby's, a restaurant directly east of Family Pride Laundromat. Lombardo was armed with a handgun and a shotgun.
9
From his vantage point along the west side of the laundromat, Ryan observed Klein exit the building through the south door. When Ryan saw that there was no movement or action by other officers, Ryan claims that he shouted to Klein by name, identified himself as a police officer, and told Klein to halt. Lombardo also claims that he heard someone, whom he believed to be Ryan, identify himself as a police officer and order Klein to halt.
10
As he left the building, Klein was positioned between Ryan (to the west) and Lombardo (to the east). Klein claims that he had walked south about twenty feet toward his car when he heard someone shout, "What were you doing in the laundromat for a few minutes? We want to talk to you." Klein turned and saw a man in plain clothes pointing a gun at him.1 Klein became scared and ran toward his car, dropping a plastic bucket containing the stolen coins. While he was running, Klein claims that he heard two shots,2 but he was not hit. In his attempt to get away, Klein cut his wheels hard and backed up in a northerly direction, then made a hard left, heading his vehicle past Arby's, toward the east exit of the parking lot. As Klein was headed out of the parking lot, he heard some gunshots, mostly shotgun blasts, and felt some stings, but continued on to the street.
11
Lombardo asserts that after he heard someone shout for Klein to halt, he saw Klein running toward his car, which was approximately thirty to fifty feet from Lombardo's position. Lombardo moved out from behind the dumpster in the direction of Klein's car. As he approached Klein's car, he claims that it was "coming at me in reverse, attempting to run me down, and I was able to move out of the way to the south." Ryan also saw the car back up toward Lombardo, and Lombardo move out of the car's path.
12
After Lombardo evaded the car, Lombardo saw the car head east out of the parking lot. Lombardo then fired at least two rounds from his shotgun. Lombardo claims that he was aiming at Klein's rear right tire. Despite these shots, Klein continued out of the parking lot at a high rate of speed. Lombardo claims that he noticed that there were no police vehicles positioned to prevent Klein's escape from the east exit of the parking lot. Lombardo then fired another round at the rear of the vehicle.
13
As Klein was exiting the parking lot, Ryan also fired three shots with a handgun, allegedly aiming at Klein's right rear tire. None of Lombardo's or Ryan's shots had any effect on the tire, although the rear window was shot out. After Klein drove out of the parking lot, a number of police vehicles began to pursue him. After driving about five or six blocks, Klein abandoned his car and fled on foot, eluding capture.
14
Although the Glen Ellyn police were unsure whether Klein had been injured during his escape, Tobias alerted area hospitals to be on the lookout for a man fitting Klein's description with a gunshot wound. On May 15, 1983, Ryan received a call from St. Anne's Hospital in Northlake, Illinois, informing him that a patient fitting Klein's description with a gunshot wound was at the hospital. Klein had checked into the hospital under the alias of Michael Telleen, claiming that he had been injured during a fight in a bar.
15
Klein subsequently pleaded guilty to the charge of burglary and was sentenced to four years imprisonment. After being released from the penitentiary, Klein developed seizures as a result of his head wound. Klein then filed an action under 42 U.S.C. Sec. 1983, alleging that Ryan and Lombardo violated his fourth and fourteenth amendment rights when they shot him as he was fleeing from the scene of the burglary. The district court granted summary judgment for the defendants on the basis of qualified immunity. Klein appeals the district court's ruling. We have jurisdiction over this appeal under 28 U.S.C. Sec. 1291.
II. ANALYSIS
16
The doctrine of qualified immunity shields government officials performing discretionary functions from liability for civil damages. To overcome the defense of qualified immunity, the plaintiff must show that the officials violated "clearly established statutory or constitutional rights of which a reasonable person would have known." Harlow v. Fitzgerald, 457 U.S. 800, 818, 102 S.Ct. 2727, 2738, 73 L.Ed.2d 396 (1982).
17
A court should grant summary judgment when there is no genuine issue as to any material fact and the moving party is entitled to judgment as a matter of law. Fed.R.Civ.P. 56(c); Reardon v. Wroan, 811 F.2d 1025, 1027 (7th Cir.1987). As we have explained, "appellate review of a denial of summary judgment on the issue of qualified immunity is limited to the legal question of whether the law was well established at the time of the conduct." Wade v. Hegner, 804 F.2d 67, 70 (7th Cir.1986); see Mitchell v. Forsyth, 472 U.S. 511, 528, 105 S.Ct. 2806, 2816, 86 L.Ed.2d 411 (1985). "[I]f there are issues of disputed fact upon which the question of immunity turns, or if it is clear that the defendant's conduct did violate clearly established norms, the case must proceed to trial." Green v. Carlson, 826 F.2d 647, 652 (7th Cir.1987).
18
Klein has the burden of demonstrating that the defendants violated a constitutional right that was clearly established on May 12, 1983. Davis v. Scherer, 468 U.S. 183, 197, 104 S.Ct. 3012, 3020-21, 82 L.Ed.2d 139 (1984); Abel v. Miller, 824 F.2d 1522, 1534 (7th Cir.1987). For a right to be "clearly established,"
19
[t]he contours of the right must be sufficiently clear that a reasonable official would understand that what he is doing violates that right. This is not to say that an official action is protected by qualified immunity unless the very action in question has previously been held unlawful, but it is to say that in the light of preexisting law the unlawfulness must be apparent.
20
Anderson v. Creighton, --- U.S. ----, 107 S.Ct. 3034, 3039, 97 L.Ed.2d 523 (1987) (citation omitted). As this circuit has held, "[t]he right must be sufficiently particularized to put potential defendants on notice that their conduct probably is unlawful." Azeez v. Fairman, 795 F.2d 1296, 1301 (7th Cir.1986). In determining whether a right was clearly established, we often look to closely analogous case law. Lojuk v. Johnson, 770 F.2d 619, 628 (7th Cir.1985), cert. denied, 474 U.S. 1067, 106 S.Ct. 822, 88 L.Ed.2d 795 (1986).
21
The parties agree that the relevant law governing this case is the Illinois fleeing felon statute.3 In 1983, the statute stated:(a) A peace officer ... is justified in using force likely to cause death or great bodily harm only when he reasonably believes that such force is necessary to prevent death or great bodily harm to himself or such other person, or when he reasonably believes both that:
22
(1) Such force is necessary to prevent the arrest from being defeated by resistance or escape; and
23
(2) The person to be arrested has committed or attempted a forcible felony....
24
Ill.Rev.Stat. ch. 38, p 7-5 (1981).
25
The parties agree that Ryan and Lombardo reasonably believed that Klein had committed the forcible felony of burglary at the time of the shooting, thus satisfying the second prong of the statute. Our sole inquiry, then, is whether a reasonable officer in the circumstances confronting the defendants would have believed that the use of deadly force4 was necessary to prevent Klein from defeating the arrest by escape. In making this determination, we must consider all the undisputed facts in the record, viewing them in the light most favorable to Klein. Green v. Carlson, 826 F.2d at 650.
26
In Anderson v. Creighton, --- U.S. ----, 107 S.Ct. 3034, 97 L.Ed.2d 523 (1987), the Supreme Court authorized a fact-specific inquiry to determine whether an official's conduct violated clearly established law. In that case, several police officers conducted a warrantless search of the Creighton home. The Court recognized that law enforcement officers, on occasion, will reasonably but mistakenly conclude that probable cause is present. If an official's actions are objectively legally reasonable, the official should not be held personally liable. Id. 107 S.Ct. at 3039. In examining the issue of qualified immunity, the Court held that "[t]he relevant question ... is the objective (albeit fact-specific) question whether a reasonable officer could have believed Anderson's warrantless search to be lawful, in light of clearly established law and the information the searching officers possessed. Anderson's subjective beliefs about the search are irrelevant." Id. at 3040. This circuit has also recognized that "the test for immunity should be whether the law was clear in relation to the specific facts confronting the public official when he acted." Colaizzi v. Walker, 812 F.2d 304, 308 (7th Cir.1987).
27
Therefore, we must consider the specific facts confronting Ryan and Lombardo when they began shooting at Klein. Both Ryan and Lombardo knew that Klein had just committed a forcible felony. They also knew that Klein had carried a gun in the past.
28
In addition, the defendants reasonably believed that Klein heard an officer identify himself as a policeman and tell Klein to halt. Ryan asserts that he identified himself as a police officer and told Klein to halt. Lombardo also heard someone shout: "Klein, halt, police!" Klein denies having heard anyone identify themselves as police officers. Nevertheless, Klein does not present any evidence to dispute that Ryan did identify himself as a police officer;5 Klein merely says that he did not hear anyone do so. Thus, we find, as did the district court, that it is undisputed that Ryan identified himself as a police officer and ordered Klein to halt. The district court also found undisputed that it was late at night in a quiet suburban parking lot. Lombardo was thirty to fifty feet from Klein when he heard an officer order Klein to halt. Significantly, Klein was between Ryan and Lombardo when Lombardo heard the command to halt. Therefore, an officer in Lombardo's or Ryan's position reasonably could have assumed that Klein did hear the order to halt, but ignored it as he attempted to flee the scene.6
29
Viewing the undisputed evidence in the light most favorable to Klein, we will assume that Klein was unarmed and that Klein did not threaten the officers in any way. With these assumptions, we have reconstructed the following scenario. Police officers tell a person, who they reasonably suspect of having committed a forcible felony, to halt. They reasonably believe that the suspect heard them, but the suspect continues to flee. The suspect gets in his car and begins to drive away, with no resistance from any other officer. In this situation, a police officer could reasonably believe that deadly force was "necessary to prevent the arrest from being defeated by resistance or escape." Ill.Rev.Stat. ch. 38, p 7-5(a)(1) (1981). The decisions of Illinois courts in factually similar cases arising under this statute also support this conclusion. See Onesto v. Police Bd., 92 Ill.App.3d 183, 416 N.E.2d 13, 48 Ill. Dec. 118 (1st Dist.1980) (police officer reasonably used deadly force to prevent escape of fleeing felony suspect who failed to stop after warning was given); Moore v. Chicago Police Bd., 42 Ill.App.3d 343, 355 N.E.2d 745 (1st Dist.1976) (police officer acted reasonably in using force likely to cause death or great bodily harm against burglary suspect who resisted arrest); LaMonte v. City of Belleville, 41 Ill.App.3d 697, 355 N.E.2d 70 (5th Dist.1976) (police officer acted reasonably in shooting robbery suspect who, after being disarmed in struggle with police, tried to escape despite orders to halt).
30
Klein argues that the defendants improperly used deadly force in this instance because reasonable alternatives were available to them. See Fornuto v. Police Bd., 38 Ill.App.3d 950, 957, 349 N.E.2d 521, 527 (1st Dist.1976) (suggesting officer should first exhaust all reasonable alternatives to prevent escape by non-violent means); but cf. Moore, 42 Ill.App.3d at 348, 355 N.E.2d at 749 (because Illinois law authorizes the use of deadly force when a suspect is arrested for the forcible felony of burglary, the amount of force the police officer actually used could not be considered excessive). Klein contends that because the police knew the suspect, they simply could have allowed Klein to escape the scene of the burglary and arrested him at a later time. Klein recognizes that Ryan had checked out an address that was supposedly Klein's and found it to be fictitious. Nevertheless, Klein insists that, with "a bit more investigative work," the police could have located Klein. Klein provides no support for this surprising assumption; nor does Klein suggest what investigative techniques the police should have employed to find him. Contrary to Klein's assertions, however, the Glen Ellyn Police Department had no idea how to locate Klein. Ryan had earlier checked the only address the department had for Klein and found it to be a false lead. In fact, after Klein eluded the police on the night of the burglary, the police were only able to apprehend him because he appeared at an area hospital complaining of gunshot wounds. In addition, when he checked into the hospital, Klein was using an alias, compounding the problem of identification. Thus, Klein provides no support for his theory that the police could have easily apprehended him following the burglary. While we must review the undisputed evidence in the light most favorable to Klein, we need not accept conclusory allegations completely lacking evidentiary support. Anderson v. Liberty Lobby, Inc., 477 U.S. 242, 106 S.Ct. 2505, 2514, 91 L.Ed.2d 202 (1986) (party opposing summary judgment motion cannot rely on mere allegations, but must present affirmative evidence); Valentine v. Joliet Township High School Dist. No. 204, 802 F.2d 981, 986-87 (7th Cir.1986) (plaintiff's conclusory allegations, without supporting affidavits or other evidence, were not sufficient to establish a material issue of fact requiring a trial).7
31
Klein also relies on the fact that the chief of the Glen Ellyn Police Department believed that Ryan and Lombardo used excessive force and subsequently disciplined both of them. Lombardo and Ryan, however, were suspended for violating police department regulations. In this action, the plaintiff claims that the defendants violated state law; therefore, the local police department regulations are irrelevant in determining what was clearly established law. Davis v. Scherer, 468 U.S. 183, 194 & n. 12, 104 S.Ct. 3012, 3019 & n. 12, 82 L.Ed.2d 139 (1984) ("officials sued for violations of rights conferred by a statute or regulation ... do not forfeit their immunity by violating some other statute or regulation") (emphasis in original); see Brown v. City of Clewiston, 644 F.Supp. 1417, 1420-21 (S.D.Fla.1986).8
32
Finally, Klein insists that the defendants, particularly Lombardo, received inadequate training to allow them to assess when the use of deadly force was appropriate. Qualified immunity, however, involves an objective inquiry. The subjective beliefs of the defendants are not in issue. Anderson v. Creighton, 107 S.Ct. at 3040. We need only consider whether a reasonable officer in like circumstances would have acted as the defendants did. Klein's reliance on Malley v. Briggs, 475 U.S. 335, 106 S.Ct. 1092, 89 L.Ed.2d 271 (1986), is misplaced. In Malley, the Supreme Court stated that "the qualified immunity defense ... provides ample protection to all but the plainly incompetent or those who knowingly violate the law." Id. at 341, 106 S.Ct. at 1096. Klein insists that we must consider the defendants' training to determine whether they were "plainly incompetent." Klein is mistaken. The Malley Court explained: "Defendants will not be immune if, on an objective basis, it is obvious that no reasonably competent officer would have [acted as the defendants did]; but if officers of reasonable competence could disagree on this issue, immunity should be recognized." Id. If, as we have determined, a reasonably competent officer would have acted as Ryan and Lombardo did, they necessarily cannot be characterized as "plainly incompetent."
III. CONCLUSION
33
We certainly do not condone the unnecessary use of deadly force by police officers in any situation. The Supreme Court recently instructed courts that "[i]t is not better that all felony suspects die than that they escape. Where the suspect poses no immediate threat to the officer and no threat to others, the harm resulting from failing to apprehend him does not justify the use of deadly force to do so." Tennessee v. Garner, 471 U.S. 1, 11, 105 S.Ct. 1694, 1701, 85 L.Ed.2d 1 (1985). We recognize that the Glen Ellyn Police Department disproved of Lombardo's and Ryan's use of deadly force in trying to apprehend a burglar who, although fleeing the scene, did not have a weapon and did not physically threaten the officers.9 Nevertheless, we cannot rely on the 20/20 vision of hindsight; rather, we must consider the undisputed facts as they were known to the defendants at the time of the burglary and apply the state law as it existed at the time of the burglary. Under the clearly established law of Illinois on May 12, 1983, we find that reasonable police officers would have been justified in using deadly force to prevent the escape of a person they reasonably believed to have committed the forcible felony of burglary. Therefore, the defendants are qualifiedly immune from suit. The district court is
34
AFFIRMED.
1
On the night of the burglary, Ryan and his partner, Mark Tobias, were driving an unmarked car and were not wearing uniforms
2
Ryan denies having taken any shots before Klein was driving away in his automobile
3
Two years after the Family Pride Laundromat burglary, the Supreme Court held that police officers may not use deadly force to prevent the escape of an apparently unarmed felony suspect unless such force is necessary to prevent the escape and the officer has probable cause to believe that the suspect poses a threat of death or serious injury to the officer or others. Tennessee v. Garner, 471 U.S. 1, 11-12, 105 S.Ct. 1694, 1701, 85 L.Ed.2d 1 (1985). Following the decision in Garner, Illinois amended its fleeing felon statute to limit the use of deadly force to instances where the forcible felony "involves the infliction or threatened infliction of great bodily harm." Ill.Ann.Stat. ch. 38, p 7-5(a)(2) (Smith-Hurd Supp.1987)
On May 12, 1983, however, the existing Illinois fleeing felon statute was not considered unconstitutional. Although no case had specifically adjudicated the constitutionality of the Illinois statute, Illinois state courts and federal courts had applied the statute. See, e.g., Onesto v. Police Bd., 92 Ill.App.3d 183, 416 N.E.2d 13, 48 Ill. Dec. 118 (1st Dist.1980); LaMonte v. City of Belleville, 41 Ill.App.3d 697, 355 N.E.2d 70 (5th Dist.1976); Silverman v. Ballantine, 694 F.2d 1091 (7th Cir.1982); Rodriguez v. Schweiger, 534 F.Supp. 229 (N.D.Ill.1982). Because qualified immunity analysis requires us to apply the law that was clearly established on May 12, 1983, see Zook v. Brown, 748 F.2d 1161, 1164 (7th Cir.1984), Garner does not control this case. See Brown v. City of Clewiston, 644 F.Supp. 1417, 1420-22 (S.D.Fla.1986) (Florida statute in existence prior to Garner provided clearly established law in 1983 because no Florida court had ever held that statute unconstitutional); Lundgren v. McDaniel, 814 F.2d 600, 603 (11th Cir.1987) (citing Brown).
4
Section 7-8 defines deadly force:
(a) Force which is likely to cause death or great bodily harm, within the meaning of Sections 7-5 and 7-6 includes:
(1) The firing of a firearm in the direction of the person to be arrested, even though no intent exists to kill or inflict great bodily harm; and
(2) The firing of a firearm at a vehicle in which the person to be arrested is riding.
Ill.Rev.Stat. ch. 38, p 7-8 (1981).
5
Klein did not attempt to interview other officers at the scene to dispute Ryan's allegation that he shouted a warning to Klein to halt. See Ford v. Childers, 650 F.Supp. 110, 113 (C.D.Ill.1986) (plaintiff's self-serving statement that he did not hear the policeman's warning was insufficient to meet plaintiff's burden before a jury where plaintiff failed to interview the other officer present during the incident)
6
According to Klein, he had no idea that Ryan, who was wearing civilian clothing, was a policeman. Klein saw a man pointing a gun at him, and his only thought was to get away. Nevertheless, even if we accept Klein's version as true, we must consider the facts known to the defendants at the time of the shooting
7
Klein proposes other alternatives that would have avoided the need for deadly force. Klein suggests that deadly force would have been unnecessary if Ryan had arrested him inside the laundromat. This is mere speculation by the plaintiff. We will not second-guess an experienced police officer's decision as to the safest location in which to arrest a suspect. Klein also tries to persuade us that, as a reasonable alternative to shooting at him, the defendants could have chased him as he left the parking lot. Klein ignores the fact that neither Ryan nor Lombardo were in their cars as he was speeding away. Further, a high-speed chase through the streets of Glen Ellyn seems just as likely to endanger innocent bystanders as shooting at the suspect. Finally, we note that other officers did chase Klein and were unsuccessful in apprehending him. Thus, we are not persuaded that reasonable officers in the defendants' position would have utilized these alternatives. See Onesto, 92 Ill.App.3d at 186, 416 N.E.2d at 16, 48 Ill.Dec. at 121 (suggested alternatives to deadly force were "mere speculations with no reasonable basis in the evidence")
8
Klein also points to Ryan's deposition in which Ryan stated that, in retrospect, he also believed that both he and Lombardo used poor judgment in shooting at Klein. In determining whether a defendant violated clearly established law, however, the defendant's subjective beliefs are irrelevant. Anderson v. Creighton, 107 S.Ct. at 3040
9
At one point, Lombardo believed that Klein was trying to run him down with his car. When Klein first saw Ryan pointing a gun at him, Klein jumped in his car and hurriedly put the car in reverse, turning sharply. Unknown to Klein, he backed up his car in Lombardo's direction. (Klein maintains that he never saw Lombardo.) Lombardo had been told earlier that day that Klein had once tried to run over someone with his car. Therefore, an officer in Lombardo's position, with Lombardo's knowledge of Klein, might reasonably have believed that Klein was trying to hit him with the car. Nevertheless, Lombardo easily moved out of the path of the vehicle. Thus, this was not an incident in which deadly force was necessary to prevent death or great bodily harm to a peace officer or other person
| 2023-08-19T01:27:17.760571 | https://example.com/article/9750 |
Monday, October 30, 2006
Baba kekere has made himself a regular part of my life now. He comes to get me from work and has offered to take me to work on many occasions. I allow him to pick up from work but I refuse his offers to take me to work since I like to use that time to gather my thoughts and people watch.Well, after Baba Kekere asked me on many occasions if he could see my apartment I decided that I would let him to see it. Of course he wanted to stay the night after I gave him a small tour. When I told him that I would prefer if he went home he then proceeded to tell me how tired he was and how he did not think he would make it home in one piece. I relented and allowed to him stay the night.Wow, silly me, he proceeded to butter me up by telling me that he loves me a thousand times. I hope he does not think that I am stupid enough to fall for that shit. I think I have mentioned before how Naija men in Baltimore are famous for telling a woman soon after meeting, how much "they love them". African-American men DON'T do this( yes I know i am generalizing).This guy told me I love you so much that I wanted to tell him to shut his mouth before I stuff it with alligator pepper. Besides the I love you stuff, Small Daddy is hella hyper. I find it hard to believe that he is older than me. I know that he likes to take kola nut, but I would not be surprised if he has taken a hit off of a crack pipe. NO 30 YEAR OLD MAN SHOULD BE THAT DAM HYPER.Anyway, after I agreed to allow him to stay he preceded to take off all of his clothes but his boxers and his tank top. He started to complain about his back hurting and asked me to apply a heat wrap to his back. Maaan when he pulled that shirt up I was assaulted again by his hairy chest. I HAVE NEVER SEEN OR KNOWN A MAN TO HAVE DO MUCH HAIR ON HIS CHEST. No lie oooh, it look he had stapled a carpet down to his chest(or maybe he is related to bigfoot). That ish looks worse than anything I could imagine. I had to tell small daddy that he could not take his shirt off until he shaved his chest.So, after I applied the heat wrap to his back I turned on the TV so that I could watch a Nigerian movie. Within 20 minutes of starting the movie I feel something hard in the small of my back. I told Small Daddy that I refuse to play that game with him tonight. I told him that he would not be touching my oboo(pu#$y) before I allowed him to enter my apartment. He moved over for about 2 minutes and then he was on me once again. At this point I decided to remind him of his religion and how it is important that he adhere to its rule on sex before marriage. That worked for about a good 15 minutes, and once again I felt something in my back. At this point I guess Small Daddy was at his limit because he started to beg, YES BEG for a sample.Having sex with him was out of the question. I had no desire to sleep with him at all. Men are so weird/weak when it comes to sex. I refuse to beg any man for sex. Small Daddy had no problem though, he said " I beg you, I will do anything, please".His begging went on for about 15 minutes. He asked me to use my hand if I did not want to give him the real thing. I thought about it for a few minutes, and decided to use my hand.So Small daddy finally pulled off his shorts-ONLY. You know I was not playing about that undershirt. Oh my goodness, what did I see when he pulled of his shorts, a micro penis. I AM NOT MAKING THIS UP, his thing was soooooo small. It was so small that when I went to j@#k him off it keep slipping out of my hand. Now if you can remember I think I mentioned that there is a two handed move that I have when j#$king a man off than can make him scream like a baby. Well, Small daddy was to tiny for me to even do that. Ladies take your hand and make it into a fist with your thumb facing upwards, now you have the length of small daddy's manhood.I felt like someone had played a practical joke on me. How could a grown man have a penis sosmall. I mean his balls stuck out further than his di%k did(NO LIE). I guess he must have seen the disappointment of my face or maybe women in his past had insulted him because of his "micro peny", since he proceed to repeat many times that "it is small but I can work it". I got over my initial shock and proceeded to rock his world. All 31/2 inches of it. Small Daddy started to scream and twist and turn which did not help me at all because now I was losing my grip. Then it got so good for him that he wanted to sit up and watch me work my magic. When he sat up it was as if it was going to disappear because then I could only use two fingers to stimulate him. I had to push him back down flat in the bed so that I could finish. Finally, he reached his peak because he started to really twist and scream and he let out a hell of a load. He let off so much stuff that I'm sure he lost a pants size and ten pounds. It took him a few minutes to catch his breath because he was breathing so hard. I jumped up to wash my hands because I find that stuff nasty, which is why I don't think I can ever let a man sleep with me without a condom.So after finishing with his small parts, I tried to finish the movie and go to sleep. Throughout the night while I tried to sleep but Small Daddy woke me up with either his snoring or his shouting in Yourba on his cell phone (in a dialect that I could not understand, I think it was ekite). Since I got no sleep during the night, when he left early in the morning all I could do was take a short nap before I had to go to work.
Wednesday, October 25, 2006
Sorry, I've never gone one week without posting. I've just been too lazy to post.
Anyway, I know I like to write about ghetto girls and boys. Well, I figured that its time that I did a little something for all the trailer park trash. Please observe Beat them up Becky and her crew. They are sporting the finest in trailer park attire(mullets and stretch paints two sizes two small). Please turn up the volume so you can get the full effect.
Tuesday, October 17, 2006
I finally let Small Daddy talk me into visiting his house. He told me he owns his home(a plus). When I walked in I must say it is a clean and tidy place for a bachelor's home. There were no dirty clothes on the floor or dishes piled high in the sink. I sat in the living room and proceeded to watch a Nigerian movie(my fav) and ate a very late dinner( I was so wrong for eating that cheese steak). Small daddy was trying to rush me(eating) so that I could go to sleep in the bedroom with him. I told him that I have to stay up a while so that I could digest the food(garbage) ,otherwise my stomach would give me problems throughout out the night.Eventually Small Daddy grew tired of waiting for me because he went to sleep in his bedroom. I stayed up to watch the second part of the Nigerian movie. About midway through the movie I began to hear loud rumbling. I thought to myself"Oh God, hear we go again". You see, the Beast was a loud snorer too. The Beast snored so loud he could wake the dead. I never went into the bedroom. I decided to sleep on the couch because for one I was not giving up any couchie and two I did not want to have my ear drums to explode.Small daddy's snoring was so bad that it woke me up a few times during the night, preventing me from getting a good sleep. At about 7am I went to wake him so that he could take me home. I had a few things I needed to do that day, and I also needed to sleep(peacefully). Overall, I must say that so far Small Daddy has been a gentleman.
Saturday, October 14, 2006
You know you got some good pu#sy when you can make a man behave like this on his wedding day. May I one day learn the tricks and positions that this woman knows. I would not be surprised if she is Superhead's sister.
Wednesday, October 11, 2006
My new job has been taking its toll. I am so tired. I have little time to blog although I try to read as many of my favorite blogs as possible.
I met someone new. Another Nigerian(Yoruba) ,and he is a Baba kekere(small daddy). I don't know what is with me and short men lately. It seems as if that is all I am pulling to me. I think my little men have a serious case of Napoleon complex. This guy is another cab driver. That is another pattern for me, cab drivers. Maybe I should call a physic to figure out why I keep getting the interest of cab drivers. He is also in nursing school(nice money in the long run). He is not the best looking thing on the planet but he will do for now. Its not like I'm looking for a boyfriend. I am simply looking for someone to shoot the breeze with.Small daddy can be a bit annoying though. He calls me like know mans business. If I don't answer the phone when he calls he may call me two or three more times until I answer. Most of the time I looking at the phone as it is ringing but, decide not answer it simply because I don't feel like talking. Small daddy says he is 30 but he is very young acting at times. Sometimes he can get so hyper, and it doesn't help matters that has the size of a teenage boy(before puberty).He has been trying his hardest to get me to come over to his house to watch Nigerian movies with him. He came over last night and begged me to go over to his house or he said I should let him spend time with me in my apartment. He must think I am stupid as hell. I know what he was after, and the answer is no I did not let him come up to my apartment nor did I go to his house. He had the nerve to call me a little while later and start asking me about sex. He told me that he has never eaten pus^y. Now yall know if a man is not willing to lick me there is nothing that I can do anything with him. Once he said that I knew for sure small daddy won't get me anywhere alone. I refuse to be the first chic he goes downtown on. I heard nightmares from women who had men take the words "eating pu#%y" literally. I'm not trying to go to the emergency room with a chewed up coochie.
Tuesday, October 10, 2006
I I've never tried vanilla, but this guy right here will make me sample something new. If he was not a soccer player he could most def been a top male model. He is simply gorgeous. Posh Spice should thank God everyday for blessing her with such a good looking husband.
Thursday, October 05, 2006
10. Paris Hilton admits she is dumb. I think most of us figured that out already.9. Many women's clothing stores are now selling size 00. I thought Americans are getting bigger so why are they now cutting smaller sizes to sell.8. Why do some black men become angry when they see a black woman holding hands with a white man, yet have no problem dating said white man's sister Becky.7. Why do some women enjoy performing oral sex on men they have just met(one night stands).6. Forrest Whitaker as Idi Amin, I so can't wait for this movie to come out.5. I love the weather during this time of the year,not to warm and not to cold.4. I am seriously considering letting my hair grow back natural.3. Monique is the prettiest girl on this season's America's Next Topmodel, but girlfriend is seriously unhinged. There are a handful screws lose in that head of hers.2. Why are some men intimidated by women who are as sexually confident as Samantha Jones(from Sex and The City).1. I'm thinking of going to Amsterdam or London for Vacation. Which is better.
Monday, October 02, 2006
Lil man texted me on Friday night. The lil idiot wrote "Today can we have cool sex tonight." Of course I texted him immediately to tell the Little idiot that I don't do sex on the first date(not even with the Swinger). I then sent him a second text telling him not to send me anymore x-rated text messages. I have not had a another text or phone call from him since.It is very funny that he would want to have a one night stand with methis time around, when he claimed to be frightened when I asked him to come alone for our first date. Some men are crazy when it comes to pussy. They are willing to do anything for p@#$y, and take all types of risks to get it.If Lila man was so scared than why was he not scared this time. I could of took him somewhere and robbed his dumb ass or had a few of my "yo boy" associates beat him down and rob him. It is not like homeboy has any size to him. He is built more like a hobbit than a man. He could of been the lone black person in Lord Of The Rings.Please don't worry about me on the dating front because I'm not really looking for a serious boyfriend. I'm more in search of someone who can stimulate my mind and keep my company sometimes (and maybe give me my first vaginal orgasm).
So you want to know about Me
A very cute black chic who loves everything about fashion and beauty. I'm struggling through the ups and downs of being in my twenties. I am a recent graduate so I am doing everything on a really tight budget.That means no more Gucci or Louie Vuitton bags for now. I'll take a cat anyday over a dog.Travelling is my greatest love. I frequently discuss sex and how much I love a man with a average sized one. Mainly, this blog serves as a tool for me to get some things off my chest. | 2023-08-05T01:27:17.760571 | https://example.com/article/5053 |
Speaking on the occasion of NV Subhash, grandson of the former Prime Minister PV Narasimha Rao who joined the party on Friday, Kishan Reddy urged the Governor to take steps n a war-footing to ensure uninterrupted power supply to farmers. He said that people were joining the party in the interest of the country without aspiring any personal gains. | 2023-11-05T01:27:17.760571 | https://example.com/article/8053 |
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