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PrimeHealth MD Atlanta has truly changed my life. For years, I attempted to get healthy and lose weight. I would think and talk about it constantly, lose most of the weight then slowly gain it back, slipping back into my old habits. This summer, I was at my heaviest weight ever and decided I had to do something ASAP. I finally ran out of excuses, my knees and back hurt and I was extremely tired most of the day. I started researching online, found their website, and nervously called to setup my initial appointment not knowing exactly what to expect but I was desperate and ready for a permanent lifestyle change. The last 13 weeks have been far from easy but so doable and necessary. I have lost over 40lbs and gained so much knowledge on the science of weight loss. What makes this time different? I am committed, and I have PrimeHealth. The staff, especially Sara and Hillary are my biggest supporters and really make this challenging experience exciting! You can tell it is not just a job for them, they make you feel extremely comfortable, provide you with the knowledge to be successful, and hold you accountable. I referred my co-worker and she is also having great success. I have 40 more lbs to reach my goal weight but I am confident with their coaching, we will be celebrating my amazing transformation in no time!
I have had the best experience at Prime Health. I have been trying my whole life to lose weight. I have tried every diet imaginable. This summer I not only reached my goal weight but I am below it!!! It was all in thanks to hard work and AMAZING support from Prime Health. I have been able to maintain the weight and feel so much better. I have even referred 3 people there who are also having good experiences. Sara the Nurse Practitioner is awesome. She really knows her stuff and is there to support you and help you reach your goals. I am not going to say the program is easy. This is not a diet program it is a NEW and IMPROVED way of life. That is never easy but once you make the commitment you will not regret it and with the support of Prime Health MD you will be successful!!!
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be spread by different routes to humans. And if by chance the person is not healthy, this may be a problem."
The analysis was conducted in Portugal between 2019 and 2020. While the study focused on dog food brands currently available in Europe, the researchers noted that some are also marketed internationally.
Freitas said she cannot speak to the popularity or availability of raw dog food in the United States. However she noted that the U.S. Centers for Disease Control and Prevention plainly states on its website that it generally "does not recommend feeding raw diets to pet[s]," as the bacteria found in such raw food has the potential to make not only pets sick but also their owners.
"Your family also can get sick by handling the raw food or by taking care of your pet," the CDC stresses. People who still choose to bring raw pet food into the home are advised to wash their hands with soap and water when handling such food, and to "clean and disinfect all surfaces that the raw food touched, like countertops, microwaves, refrigerators and objects like knives, forks, and bowls."
Freitas and her colleagues will report their findings this week at a virtual meeting of the European Congress of Clinical Microbiology and Infectious Diseases (ECCMID). Such research is considered preliminary until published in a peer-reviewed journal.
Gene link between dogs, owners
Freitas' study was not the only research presented at the meeting on the relationship between dogs, their owners and antibiotic-resistant bacteria.
A gene that confers resistance to a last-resort antibiotic can be passed from dogs to their owners, another group of Portuguese researchers reported.
The mcr-1 gene -- first reported in China in 2015 -- provides resistance to colistin, an antibiotic of last resort. There are fears the gene could combine with drug-resistant bacteria to spawn an untreatable type of infection.
To find out if household pets may be acting as a reservoir of the gene and helping it spread, the researchers analyzed fecal samples from 126 healthy people living with 102 cats and dogs in 80 households in Lisbon. They found the gene in four people and two pet dogs. In two cases, both the dog and its owner had the gene. No cats were carrying the gene.
While transmission in both directions is possible, it is thought that in at least one case, the gene passed from the dog to its owner.
The findings raise concerns that pets can act as reservoirs for the gene and help spread resistance to last-line antibiotics, the researchers noted.
"Colistin is used when all other antibiotics have failed -- it is a crucial treatment of last resort. If bacteria resistant to all drugs acquire this resistance gene, they would become untreatable, and that's a scenario we must avoid at all costs," study author Juliana Menezes, from the University of Lisbon's Centre of Interdisciplinary Research in Animal Health, said in an ECCMID news release.
It's an issue that is definitely worth being concerned about, said Dr. Amesh Adalja, a senior scholar with the Johns Hopkins Center for Health Security in Baltimore.
"Antibiotic-resistant bacteria are one of the most pressing public health challenges that exists," said Adalja. "It threatens to take us to a pre-penicillin world.
"This finding is not surprising," he said. "We can find similar resistant bacteria in raw human food as well, and [it] reflects the use of antibiotics in agriculture."
On that latter point, Freitas concurred, noting that "genetically identical [antibiotic-resistant] bacteria have also been found in farm animals and wastewater across the UK."
That does not mean that raw pet food must be banned altogether, she said, "but it should be better controlled" via a periodic inspection regimen that screens for antibiotic resistance. Meanwhile, Freitas echoed the CDC's advice, adding that "dog owners can always cook this food to guarantee its quality."
There's more on risks of raw dog food at the U.S. Centers for Disease Control and Prevention.
SOURCES: Ana Freitas, PharmD, PhD, investigator, UCIBIO@REQUIMTE, and secretary, EFWISG-ESCMID, lab of microbiology, faculty of pharmacy, University of Porto, Portugal; Amesh Adalja, MD, senior scholar, Johns Hopkins Center for Health Security, Baltimore; European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), July 9-12, 2021, virtual meeting and news release
Bacterial Skin Infections
Common Bacterial Skin Infections
Transverse Myelitis
Bacterial Skin Infections in Children
A Faster, Cheaper Test to Gauge the Risk of Premature Delivery?
Antibiotics Quiz

redpajama_set_name: RedPajamaCommonCrawl


Added on February 12, 2015 by sage hammond.
Congratulations to Teri and Cayne!!! The exchanged their wedding vows at Dreams Sugar Bay Resort. The first full moon of the year made the perfect backdrop for their wedding photos!


Start by drying your chicken with a paper towel, this will make the spices stick better, and in the ned, give your chicken a better flavor.
Spread the olive oil and lemon juice over the chicken.
Rub the spices over the chicken.
Set the chicken in the air fryer basket, at 360 degrees F, for 10 minutes, after 10 minutes, flip them, and cook for another 10 minutes.
Remove from the air fryer, I cut up some lemons and herbs and placed them on a bed, ready for serving.


Dona Onete, Stella Chiweshe, Noisettes, Petite Noir and Shingai.
Debbie Golt takes African music and wider arts and culture as her starting point. This week, ahead of International Women's Day, Debbie welcomes the tour de force that is Shingai (https://www.instagram.com/shingai/), as she embarks on a solo career. Well known as the frontwoman of The Noisettes, Shingai now brings her Zimbabwean roots forward. Visit outerglobe.co.uk for more information. Tweet to @outerglobe.


We don't only develop solutions for our customers, but we are continually evolving as a company.
Time does not stand still, and we face new manufacturing, material, and product challenges almost daily. These are defined and recorded in our quality management processes. Through regular monitoring (certifications) and a continuous improvement process, we ensure consistent quality standards.
Our traditional business has been awarded numerous certifications which attest to our high quality standards. In addition to the appropriate TÜV certificates in the field of packaging plastics, natureOffice has also awarded us a certificate on the topic of "sustainable environmental protection". This is due to the fact that, as their partners, we have been offering carbon-neutral printing for our transparent boxes since 2012. A clear signal for environmental protection!


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Q: ASP.NET issue with System.Web.Security.ActiveDirectoryMembershipProvider: The network address is invalid I have an Azure VM running Windows Server 2008 with Active directory instance listing test users. I do also have Azure SQL test database. I would like to access this DB and the AD from a ASP.NET MVC app running in IIS in my local PC.
I am able to log in as a user that is listed in the Azure VM AD (ADRoleConnectionString is working) but then System.Web.Security.ActiveDirectoryMembershipProvider Initialize throws an error when I try to get some additional information about the users for this app referenced in the DB.
Stack trace:

---> System.Runtime.InteropServices.COMException: The network address is invalid.
at System.DirectoryServices.DirectoryEntry.Bind(Boolean throwIfFail)
     at System.DirectoryServices.ActiveDirectory.DirectoryContext.IsContextValid(DirectoryContext context, DirectoryContextType contextType)
     --- End of inner exception stack trace ---
     at System.DirectoryServices.ActiveDirectory.DirectoryContext.IsContextValid(DirectoryContext context, DirectoryContextType contextType)
     at System.DirectoryServices.ActiveDirectory.DirectoryContext.isServer()
     at System.DirectoryServices.ActiveDirectory.Domain.GetDomain(DirectoryContext context)
     at System.Web.Security.DirectoryInformation.InitializeDomainAndForestName()
     at System.Web.Security.ActiveDirectoryMembershipProvider.Initialize(String name, NameValueCollection config)
     at System.Web.Configuration.ProvidersHelper.InstantiateProvider(ProviderSettings providerSettings, Type providerType)
     --- End of inner exception stack trace ---
     at System.Web.Configuration.ProvidersHelper.InstantiateProvider(ProviderSettings providerSettings, Type providerType)
     at System.Web.Configuration.ProvidersHelper.InstantiateProviders(ProviderSettingsCollection configProviders, ProviderCollection providers, Type providerType)
     at System.Web.Security.Membership.InitializeSettings(Boolean initializeGeneralSettings, RuntimeConfig appConfig, MembershipSection settings)
     at System.Web.Security.Membership.Initialize()
     at System.Web.Security.Membership.get_Provider()

The connection strings are:
<connectionStrings>
<add name="ADConnectionString" connectionString="LDAP://address-to-azure:389/OU=users,OU=myservice,DC=servicedomain,DC=com" />
<add name="ADRoleConnectionString" connectionString="LDAP://address-to-azure:389/OU=users,OU=myservice,DC=servicedomain,DC=com" />

...

<add name="MY_ADMembershipProvider" type="System.Web.Security.ActiveDirectoryMembershipProvider, System.Web, Version=4.0.0.0, Culture=neutral, PublicKeyToken=b03f5f7f11d50a3a" enableSearchMethods="true" connectionStringName="ADConnectionString" attributeMapUsername="sAMAccountName" connectionProtection="None" connectionUsername="testUser@servicedomain.com" connectionPassword="password" applicationName="MyTestApp" />

This works when I deploy the app to the Azure VM IIS but not from my local PC IIS. I have port 389 open both TCP and UDP and I am able to list view users by using Active directory explorer tool without problems. My local active directory instance (localhost:389) works also without problems.

*

*I have tried to change the IIS application pool identity but it did not help.

*If I change ADConnectionString path OU=users,OU=myservice,DC=servicedomain,DC=com to something that does not exist in the AD I get an error: The container specified in the connection string does not exist. This proofs that the network address is valid as such because it is aware of the Azure AD path existence.
I am totally out of ideas what might be the issue. Does anyone have idea what might be the problem?

redpajama_set_name: RedPajamaStackExchange


Rays of sun beamed on tomato plants all aligned in rows at Lewis Educational Agriculture Farm (L.E.A.F.) as workers prepped for the new growing season.
This year marks the second year in which L.E.A.F. has provided the C.S.A (Community Supported Agriculture) program to local consumers, allowing them to buy a "share" of fresh produce each week. Last year, the goal was to reach a C.S.A. membership of 25 people, but the farm exceeded that goal with 50 members. This year, the goal is to reach 200 members.
From 24 types of tomatoes to 36 types of peppers to broccoli, eggplant and kale, L.E.A.F. grows 200 varieties of the fresh vegetables for nine months of the year on the farm's fertile fields and greenhouses. Ramsay and his team started seeding in March, and so far have not only planted tomatoes, but also onions, herbs, lettuce, kale, collards, radishes and carrots, among other vegetables.
Through its C.S.A. program, Ramsay said consumers develop a direct relationship with the farmer while keeping their money in the community and getting to know where the food was produced.
Under the C.S.A. program, members have access to the freshest, high-quality flavored seasonal products, said Ramsay. Before the season begins, C.S.A. members buy a "share," which is a box of freshly picked sustainable grown produce. The size of each box depends on the size of the share a member has bought, such as a full share, half share, or one-third share. Every full share box has six to ten different kinds of produce based on the season and the weather, and each full share will have a value of at least $37.50 per week, according to L.E.A.F.'s website.
Members of C.S.A. have the option to sign up for several different shares: a 16-week share, a 21-week share, and a four-season share. The 16-week share begins July 11 and ends October 25, and the 21-week share begins June 14 and ends November 1, according to L.E.A.F.'s website. After July 11, members can pick up their share box at the L.E.A.F. site itself or at the Southington Farmers Market. For most members, the 21-week share is preferred, said Ramsay.
Located at 65 Blueberry Lane, L.E.A.F. "promotes education through agriculture on the historic Lewis Family Farm by using practices that are socially and ecologically sustainable," as the first part of its mission statement goes. Whether Ramsay is growing onions, carrots and cucumbers in the greenhouse or planting cold crops like cabbage, kale and radishes, he has adopted sustainable farming practices throughout the years. For the past 25 years the Lewis family has added and tilled in decomposing leaf mulch (through the town's leaf collection program). This has built up the organic matter in the soil, which has helped the produce to grow faster and easier.
Ramsay said another key component of L.E.A.F.'s sustainable practices includes Integrated Pest Management (I.P.M.) Through I.P.M, farmers find potential harmful pests in the vegetable fields. Ramsay said the best course of action is determined when a pest reaches a certain threshold.
Chris Daley, a college student at the University of Hartford, has helped out in the fields for two consecutive summers. A vegetarian, Daley said he has learned a lot working with Ramsay, and feels proud to know where his food comes from.
Looking at his rows of tomato plants, Ramsay said he hopes to start a winter C.S.A. this year, and is also currently working with the Southington Community YMCA on implementing a winter farmers market. If all goes according to plan, Ramsay said L.E.A.F. will grow produce in the greenhouses from November to January.
"We're going to grow things that can take 50-degree temperatures," said Ramsay, adding that he also plans to start adding a link to different recipes on the website and hopes to conduct cooking demonstrations with C.S.A. members.
For more information, visit http://www.leafct.com/.
Lewis Farm supports Community Supported Agriculture (C.A.S.).


This week, the Trump administration announced tariffs on Chinese imports. Now, increasingly, the president's trade policies are putting many Republican politicians and business leaders in a bit of an awkward spot. Many see merit in the president's arguments and want to give his tough policies a chance to work. But they're also seeing some negative impact on businesses in their communities. And one person in the crosshairs of all of this is Ted Pitts. He's president and CEO of the South Carolina Chamber of Commerce and joins us this morning. Mr. Pitts, welcome.
TED PITTS: Thank you, David.
GREENE: I just want to get to a case study in your state. Element Electronics - they make TVs. They announced that they're going to have to lay off all 126 of their workers. They say they need parts from China. They can't afford the tariffs on these parts. What is your message to this company right now?
PITTS: You know, basically, if you look at South Carolina as a whole, we've been successful in diversified manufacturing. And Element Electronics is one of those examples. When Governor Haley was in charge, she and Secretary Hitt helped recruit Element Electronics to South Carolina under a Walmart program that was made in America. They were moving manufacturing to the U.S. to sell American-made goods in their stores. You know, obviously, with the tariff issue going on, some components that go into the television that Element made, you know, had a tariff put on them. So therefore, the cost went up. And you know, they can't deliver the product to Walmart at the negotiated price. So you know, they're a bystander of the trade conflict we find ourselves in.
GREENE: So what are you going to do for them? Or what can be done for them?
PITTS: You know, when you talk about what can be done for them, I mean, the business model has to work. And that's really what comes down to this trade issue and the conflict we see with multiple countries around the world. You know, South Carolina businesses have been treated unfairly. So there has been unfair trade practices for a long time. And China cheats, and people know that. It's well-documented.
GREENE: So you're saying the president provoking these trade wars has not been an approach that you feel is working.
PITTS: You know, what I would say is the president's approach some have praised and said that's only the way you're going to get people to the table. You know, I think companies that are affected by this approach would tell you they would much rather have a direct negotiated approach where the U.S. and China sit down and negotiate fair trade agreements and we hold China accountable. But in the big picture, I would tell you that the state is uniformly aware that there are unfair trade practices need to be dealt with.
Where there is some difference of opinions is how you deal with those unfair trade practices. Some have said this is the only approach - the president's approach is the only approach that's going to get a response. And others have said, you know, there's going to be too much pain through this process and that we need to have a country leadership - the administration - that will sit down and deal with the bad actors one on one and make sure that we have a more fair process as goods go back and forth between China, for example.
GREENE: Can I just ask you about the political dynamic here? I mean, Congressman Mark Sanford recently lost his primary. Many believe it was because of his criticism of President Trump. I mean, are there political pressures here? Is it tough for a congressional delegation - and even business leaders like yourself - to outspokenly criticize the president because you don't want to get on the wrong side of this White House?
PITTS: I don't believe that Congressman Sanford lost directly because of this. I mean, there's a lot of things at play that go. Katie Arrington's a good candidate. What I would tell you is politically it is very difficult for politicians. You do not want to - even companies - you do not want to criticize this president because he attacks you back. So companies and politicians have chosen to use a probably more productive approach, which is work behind the scenes with the administration to make their case. And that's where we think you get the best results. And South Carolina's congressional delegation as well as South Carolina companies are doing that.
GREENE: Ted Pitts is the president and CEO of the South Carolina Chamber of Commerce. Thanks a lot. We appreciate it.
PITTS: Thanks for having me. Transcript provided by NPR, Copyright NPR.


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Downbeach Events
Local artists invited to participate in plein air event at county parks
Published by Nanette LoBiondo Galloway on August 23, 2018
Last year's first place plein air winner, Walking Path by Doreen Serago Khebzou of Galloway Township.
The Atlantic County Parks System has put out a call for artists of any skill level or experience to participate in the second annual Plein Air event hosted at all Atlantic County parks Saturday, Sept. 15 (raindate Sept.16).
Participants will be able to sketch and paint outdoors at any one of the county parks including Gaskill and Lake Lenape Parks in Mays Landing, the park at Estell Manor, Weymouth Furnace, Oscar E. McClinton Waterfront Park in Atlantic City, Leeds Point Nature Area in Galloway, the 7.5-mile county bikeway and more.
"Last year's inaugural event was very well received so we are pleased to host it again. Our parks are a tremendous resource that provide enjoyment to so many," Atlantic County Executive Dennis Levinson said. "We are grateful for the support of Friends of the Parks and Atlantic Cape in showcasing these amenities as well as the talents of our citizens."
All participants must register the day of the event beginning at 8 a.m. at the Atlantic County Park Nature Center, 109 Route 50, Estell Manor and submit completed artwork by 6:30 p.m. that evening. There is a $20 fee to participate for up to two pieces.
Honorable mention, Weymouth Furnance by Linda Reardon Gazsi of Somers Point.
The event is co-sponsored by the Friends of the Parks, Inc., a non-profit citizens group established in 1985 to help promote Atlantic County parks, and Atlantic Cape Community College which will hold an exhibit of submitted works Sept. 27 through Oct. 27 at the college's art gallery on the Mays Landing campus.
Monetary prizes will be awarded to the top three selections at the plein air exhibit opening reception 6-8 p.m. Thursday, Sept. 27 at the Atlantic Cape Art Gallery.
For more information, see www.aclink.org/parks or Atlantic County Friends of the Parks on Facebook.
Categories:	Atlantic County
Nanette LoBiondo Galloway
Award winning journalist covering news, events and people of Atlantic County for more than 20 years.
Atlantic Electric can help with your energy bills
MAYS LANDING – Behind on utility bills? Atlantic City Electric is reminding customers who may be financially impacted by the COVID-19 pandemic, to contact the company now to establish payment arrangements and get connected to Read more…
Grants awarded to Atlantic County arts and history organizations
The Atlantic County Office of Cultural and Heritage Affairs has awarded a total of $70,000 to 14 local non-profit arts organizations and $4,208 to two local non-profit history organizations through a competitive grant process. These Read more…
Ventnor City loses another resident to COVID-19
Atlantic County Health Department Saturday, Jan. 16 reported six new deaths, including a Ventnor City man, and 287 new COVID-19 positives. The six deaths include two Absecon men, a 79-year-old from a long-term care facility Read more…
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Home > News > Andy Hageman named director of the Center for Ethics and Public Engagement at Luther College
Andy Hageman named director of the Center for Ethics and Public Engagement at Luther College
Andy Hageman, associate professor of English, has been named the new director of the Center for Ethics and Public Engagement (CEPE) at Luther College.
"I am thrilled and honored to be taking the role of director of the CEPE," said Hageman. "My training, research, and teaching focus on the power of narrative, the ethical opportunities and challenges as new technologies emerge and shape our cultures, and the role science fiction can play in creatively addressing climate change and racism. I look forward to leading the center with attention to ethics in the stories we create, circulate, and consume about justice, from algorithms and athletics to ancient aquatic arthropods."
Founded in 2006, the CEPE's mission is to extend conversation beyond the classroom by examining assumptions, exchanging ideas, and encouraging responsible action in our world. By encouraging and supporting deep reflection about ethical matters and responsible citizenship, the center helps students connect the pursuit of learning with what it means to live a meaningful life.
This mission is supported through programming such as guest speakers and residencies, discussions, reading groups, news hour gatherings, and panel discussions. A theme guides each year's focus. This year the theme is The Ethics of Leadership.
Hageman teaches courses in the English department, environmental studies program, Paideia first-year experience, and capstones on ethics. He promotes the rigorous imagining of how and why an interdisciplinary liberal arts education prepares students for a future of life, work, and civic engagement. He collaborates with students to shape their core competencies in creative analysis and design, ethical judgment, and the power of narrative.
"The CEPE is a portal through which Luther students, faculty, and staff can share their innovative and interdisciplinary work with broader communities. At the heart of my own work is embracing the entanglement of different disciplines. I see the center as a way to connect projects and pursuits–a space where scholars in English, environmental studies, and data science, for example, design projects together. Likewise, the center will provide students opportunities to connect their academic work to the paths they will move along after graduation," said Hageman.
Hageman will begin his five-year term as director of the CEPE in February 2022. He succeeds Victoria Christman who was recently appointed director of the Center for Global Learning at Luther College.


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Respiratory Research
Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells
Sixin Jiang1 &
Dale D. Tang1
Respiratory Research volume 16, Article number: 93 (2015) Cite this article
Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that has been implicated in the regulation of mitosis. In addition, the activation of mitogen-activated protein kinase (MAPK) is a key event in the early stage of the growth factor response. The role of Plk1 in MAPK phosphorylation in cells has not been investigated.
Immunoblot analysis was used to evaluate Plk1 and MAPK phosphorylation in cells upon stimulation with platelet-derived growth factor (PDGF). We also generated stable Plk1 knockdown (KD) cells to assess the role of Plk1 in MAPK activation and cell proliferation. Furthermore, we used a non-phosphorylatable Plk1 mutant to determine the function of Plk1 phosphorylation in these processes.
Treatment with PDGF increased Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in human airway smooth muscle cells. Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 as well as cell proliferation. However, phosphorylation of Raf-1 and AKT upon stimulation with PDGF was not reduced in Plk1 KD cells. Furthermore, the expression of T210A Plk1 (alanine substitution at Thr-210) inhibited the PDGF-stimulated MEK1/2 phosphorylation, ERK1/2 phosphorylation and cell proliferation.
Together, these findings suggest that Plk1 is activated upon growth factor stimulation, which may control the activation of MEK1/2 and ERK1/2, and smooth muscle cell proliferation.
Airway smooth muscle cell proliferation contributes to the pathogenesis of airway remodeling, a key characteristic of chronic asthma [1]. However, the mechanisms that regulate smooth muscle cell proliferation are not fully understood.
Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that has been implicated in cell-cycle-associated processes such as centrosome maturation, mitotic spindle assembly, sister chromatid cohesion and cytokinesis [2, 3]. Knockdown (KD) or depletion of Plk1 induces mitotic arrest in various nonmuscle cell types [2, 4, 5]. During mitosis and cytokinesis, the functions of Plk1 are regulated by its expression, spatial localization, and activation [2, 5].
The activation of Plk1 is largely regulated by phosphorylation at Thr-210 located in the catalytic domain of the kinase [2, 6]. In vitro biochemical studies suggest that phosphorylation at Thr-210 increases Plk1 activity [6, 7]. In addition, Plk1 is first activated in G2 and reaches maximal activity in mitosis, coincident with the kinetics of Thr-210 phosphorylation [4]. By phosphorylating distinctive substrates, activated Plk1 regulates centrosomal maturation, spindle organization and cytokinesis [2, 3].
The mitogen-activated protein kinase (MAPK) pathway plays an essential role in regulating various cellular functions including cell proliferation [8–10]. In response to stimulation with growth factors within minutes, MEK1/2 (MAPK kinase) gets phosphorylated by Raf-1 [10, 11], which in turn phosphorylates and activates ERK1/2. Activated ERK1/2 phosphorylates several protein kinases, transcription factors, and other proteins to promote cell proliferation eventually [8–11].
In addition to mitosis and cytokinesis, Plk1 has been implicated in the DNA damage response, development [3], cancer cell invasion [12], apoptosis and autophagy [13]. However, the role of Plk1 in the early stage of the growth factor response has not been investigated.
Here, we find that stimulation with platelet-derived growth factor (PDGF) induces Plk1 phosphorylation at Thr-210. KD of Plk1 inhibits the PDGF-induced phosphorylation of MEK1/2, ERK1/2 and smooth muscle cell proliferation. Furthermore, phosphorylation at Thr-210 is required for the Plk1-mediated activation of MEK1/2 and ERK1/2. Thus, we propose that Plk1 is a critical molecule that regulates the activation of MEK1/2 in smooth muscle cells during the cellular responses to growth factor stimulation.
Human airway smooth muscle (HASM) cells were prepared from human bronchi and adjacent tracheas obtained from the International Institute for Advanced Medicine [8, 14–16]. Human tissues were non-transplantable and consented for research. This study was approved by the Albany Medical College Committee on Research Involving Human Subjects. Briefly, muscle tissues were incubated for 20 min with dissociation solution [130 mM NaCl, 5 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 10 mM Hepes, 0.25 mM EDTA, 10 mM D-glucose, 10 mM taurine, pH 7, 4.5 mg collagenase (type I), 10 mg papain (type IV), 1 mg/ml BSA and 1 mM dithiothreitol]. All enzymes were purchased from Sigma-Aldrich. The tissues were then washed with Hepes-buffered saline solution (composition in mM: 10 Hepes, 130 NaCl, 5 KCl, 10 glucose, 1 CaCl2, 1 MgCl2, 0.25 EDTA, 10 taurine, pH 7). The cell suspension was mixed with Ham's F12 medium supplemented with 10 % (v/v) fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin). Cells were cultured at 37 °C in the presence of 5 % CO2 in the same medium. The medium was changed every 3–4 days until cells reached confluence, and confluent cells were passaged with trypsin/EDTA solution [8, 17, 18]. HASM cells (passage 3–10) from four non-asthmatic donors were used for experiments. They were serum starved for 24 h before treatment with PDGF.
Immunoblot analysis
Cells were lysed with RIPA 150 (50 mM Tris–HCl, pH7.6, 150 mM NaCl, 2 mM sodium pyrophosphoate, 0.5 % sodium dexoycholate,1 % NP-40, 0.1 % SDS, 5 mM EDTA) with protease and phosphatase inhibitors (1 mM benzamidine, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 2 mM sodium orthovanadate, 5 mM sodium fluoride). Cell lysates were mixed with SDS sample buffer composed of 1.5 % dithiothreitol, 2 % SDS, 80 mM Tris–HCl (pH 6.8), 10 % glycerol and 0.01 % bromophenol blue, and were boiled for 5 min and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane. The membrane was blocked with bovine serum albumin or milk for 1 h and probed with use of primary antibody followed by horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific). Proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) using the LAS-4000 Fuji Image System. Antibodies used were anti-phospho-Plk1 (Abcam), anti-Plk1 (EMD Millipore), anti-phospho-MEK1/2 (Santa Cruz Biotech.), anti-MEK1/2 (Santa Cruz Biotech.), anti-phospho-ERK1/2 (Cell signaling), anti-ERK1/2 (Cell signaling), anti-phospho-Raf-1 (Santa Cruz Biotech.), anti-Raf-1 (Santa Cruz Biotech.) and anti-GAPDH (Ambion). The levels of total protein or phosphoprotein were quantified by scanning densitometry of immunoblots (Fuji Multigauge Software). The luminescent signals from all immunoblots were within the linear range [14, 19–21].
Assessment of cell proliferation
Cells were seeded in 24-well plates with the F12 medium with 10 % FBS for at least 18 h. Cells were then serum starved for 24 h. They were subsequently treated with PDGF-BB (20 ng/ml) in the medium containing 0.25 % FBS. Numbers of viable cells were counted using the trypan blue exclusion test [9]. Briefly, cell suspension was incubated with 0.4 % trypan blue for 5 min at room temperature. Unstained cells (viable cells) were then counted.
The BrdU (5′-bromo-2′-deoxyuridine) cell proliferation assay kit (Millipore) was also used to evaluate DNA synthesis. BrdU is an analog of thymidine, which is able to incorporate into newly-synthesized DNA. Cells in 96-well (1.2x 104 cells/well) were treated with BrdU for 24 hrs. They were then fixed and reacted with BrdU antibody for 1 h, followed by incubation with secondary antibody conjugated with peroxidase. They were reacted with peroxidase substrates, and the reaction was detected using a Promega GloMax-Multi Microplate reader.
Generation of stable Plk1 KD cells
We used lentivirus-mediated shRNA [14–16, 20] to generate stable Plk1 knockdown (KD) cells. Briefly, lentiviral particles encoding Plk1 shRNA (sc-36277-V) or control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology. HASM cells were infected with control shRNA lentiviruses or Plk1 shRNA lentiviruses for 12 h. They were then cultured for 3–4 days. Positive clones expressing shRNAs were selected by puromycin. Immunoblot analysis was used to determine the protein levels of Plk1 in these cells. Plk1 KD cells and cells expressing control shRNA were stable at least five passages after initial infection.
In vitro kinase assay
Active Plk1 (40 ng, Millipore) and 2 μg MEK1 (unactive, Millipore) were placed in 20 μl kinase buffer containing 20 mM HEPES (pH7.5), 60 mM NaCl, 2 mM MgCl2, 5 mM EGTA and 100 μM ATP. Kinase reaction mix was incubated at 30 °C for 30 min, and stopped by the addition of the SDS sample buffer [18, 22]. The samples were boiled for 5 min and separated by SDS-PAGE followed by membrane transfer. The membrane was probed with phospho-MEK1/2 antibody, stripped, and reprobed with MEK1/2 antibody.
Mutagenesis, plasmid purification and cell transfection
T210A Plk1 (alanine substitution at Thr-210) was generated by using the Quick change II XL site-directed mutagenesis kit (Agilent Technologies). The template plasmid pcDNA3 3xFlag-Plk1 was kindly provided by Dr. David Stern (Yale University). The primers were designed using the online service PrimerX (http://www.bioinformatics.org/primerx/index.htm) and were synthesized by Invitrogen. The 5′ primer was 5′ –GGGGAGAGGAAGAAGGCCCTGTGTGGGACTC- 3′. The 3′ primer was 5′- GAGTCCCACACAGGGCCTTCTTCCTCTCCCC- 3′. The PCR product was digested with Dpn I at 37 °C for 2-3 h to remove the parental DNA, and was transformed into XL10- Gold Ultracompetenet cells (Agilent Technologies). Plasmids were purified by using the Pureklink Quick Plasmid Miniprep kit (Invitrogen). DNA sequencing was performed by Genewiz Life Sciences.
All statistical analysis was performed using Prism 6 software (GraphPad Software, San Diego, CA). Comparison among multiple groups was performed by one-way analysis of variance followed by Tukey's multiple comparison test. Differences between pairs of groups were analyzed by Student-Newman-Keuls test or Dunn's method. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.
PDGF stimulation induces phosphorylation of Plk1, MEK1/2 and ERK1/2 in smooth muscle cells
As described earlier, Plk1 has been implicated in mitosis and cytokinesis in nonmuscle cells [2, 4, 5]. The role of Plk1 in the early phase of the cellular responses to growth factor stimulation is largely unknown. We evaluated the effects of PDGF stimulation on Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) [6]. Human airway smooth muscle cells were serum starved for 24 h, which synchronizes the cells in G0-G1 [9, 20]. Cells were then stimulated with 20 ng/ml for different time periods, or left unstimulated. Plk1 phosphorylation at Thr-210 in the cells was assessed by immunoblot analysis. In unstimulated cells, the level of Plk1 phosphorylation at Thr-210 was relatively low. Stimulation with PDGF increased Plk1 phosphorylation, which was evident as early as 2 min after PDGF stimulation, and persisted for the 40-min duration of the stimulation (Fig. 1a).
Stimulation with PDGF induces the phosphorylation of Plk1, MEK1/2 and ERK1/2 in smooth muscle cells. Human airway smooth muscle (HASM) cells were stimulated with 20 ng/ml PDGF for different durations or left unstimulated. The phosphorylation of Plk1 (a), MEK1/2 (b) and ERK1/2 (c) in these cells was evaluated by immunoblot analysis. The phosphorylation levels of the proteins in stimulated cells were normalized to corresponding unstimulated cells. * Significantly higher phosphorylation levels in stimulated cells compared with unstimulated cells (P < 0.05). Values represent mean ± SE (n = 4)
Because the MAPK pathway has been implicated in growth factor-mediated signaling [8–10], we also determined the phosphorylation of MEK1/2 and ERK1/2 in smooth muscle cells. The phosphorylation of MEK1/2 and ERK1/2 was significantly increased 5 min after PDGF stimulation and maintained 40 min after the stimulation with PDGF (Fig. 1b and c).
KD of Plk1 attenuates the phosphorylation of MEK1/2 and ERK1/2 upon PDGF stimulation
To reveal the functional role of Plk1 in the growth factor-associated process, we evaluated the effects of Plk1 KD on MEK/1/2 and ERK1/2 phosphorylation in smooth muscle cells. Stable Plk1 KD cells were generated by using lentivirus-mediated shRNA. Immunoblot analysis showed that the protein level of Plk1 in cells infected with viruses for Plk1 shRNA was lower compared to control cells. However, the protein level of GAPDH was similar in these cells. Ratios of Plk1/GAPDH were lower in Plk1 KD cells than in control cells (Fig. 2a).
Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not Raf-1 phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE (n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA (P < 0.05). b Cells expressing control shRNA or Plk1 shRNA were treated with 20 ng/ml PDGF for 10 min, or they were not stimulated. Protein phosphorylation was then evaluated by immunoblotting. MEK1/2 phosphorylation induced by PDGF was significantly reduced in Plk1KD cells compared to cells treated with control shRNA (*P < 0.05). NS, basal MEK1/2 phosphorylation in Plk1 KD was not significantly different from cells expressing control shRNA (P > 0.05). Values are mean ± SE (n = 8). c PDGF-induced ERK1/2 phosphorylation was significantly reduced in Plk1 KD cells (*P < 0.05, n = 7). NS, basal ERK1/2 phosphorylation in Plk1 KD was not significantly different from cells expressing control shRNA (P > 0.05). d PDGF-stimulated Raf-1 phosphorylation at Ser-338 was comparable in Plk1 KD cells and cells expressing control shRNA. NS, Raf-1 phosphorylation in Plk1 KD was not significantly different from cells expressing control shRNA (P > 0.05, n = 8). Protein phosphorylation under various treatments was normalized to the levels in unstimulated cells expressing control shRNA
Plk1 KD cells and cells expressing control shRNA were stimulated with 20 ng/ml PDGF for 10 min or left unstimulated. The phosphorylation of MEK1/2 and ERK1/2 in the cells was determined by immunoblot analysis. In smooth muscle cells expressing control shRNA, the phosphorylation of MEK1/2 and ERK1/2 was increased in response to stimulation with PDGF. However, the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 was reduced in Plk1 KD smooth muscle cells (Fig. 2b and c). Basal phosphorylation of MEK1/2 and ERK1/2 was not affected by Plk1 knockdown.
Raf-1 phosphorylation is not reduced in Plk1 KD cells
Because Raf-1 is known to regulate MEK1/2 phosphorylation, we also evaluated the effects of Plk1 KD on Raf-1 phosphorylation in smooth muscle cells. Blots of cells treated with 20 ng/ml PDGF for 10 min were probed with antibodies against phospho-Raf-1 and total Raf-1. Raf-1 phosphorylation upon PDGF stimulation was similar in cells expressing control shRNA and Plk1 KD cells (Fig. 2d). Basal Raf-1 phosphorylation was also comparable in cells expressing control shRNA and Plk1 KD cells.
Plk1 catalyzes MEK phosphorylation in Vitro
As described above, KD of Plk1 mediates the phosphorylation of MEK1/2 and ERK1/2 without affecting Raf-1 phosphorylation. Because ERK1/2 is phosphorylated by the dual-specificity protein kinase MEK1/2, and Plk1 is not a dual-specificity protein kinase [8, 10], it is unlikely that Plk1 catalyzes ERK1/2 directly. To verify whether Plk1 directly mediates MEK phosphorylation, purified active Plk1 was added to purified unactive MEK1 in kinase buffer. MEK phosphorylation was determined by immunoblot analysis. The addition of Plk1 to the kinase buffer containing MEK1 increased the phosphorylation of MEK (Fig. 3). The results suggest that Plk1 directly catalyzes MEK phosphorylation.
Plk1 catalyzes MEK phosphorylation in vitro. Purified active Plk1 (40 ng) and 2 μg purified unactive MEK1 were placed in kinase buffer. MEK phosphorylation was determined by immunoblot analysis 30 min after the initiation of the reaction. a Representative immunoblots illustrates Plk1-mediated MEK phosphorylation. b MEK phosphorylation catalyzed by Plk1 was normalized to the level of MEK phosphorylation in the absence of Plk1 (n = 4, *P < 0.05)
KD of Plk1 inhibits smooth muscle cell proliferation
We assessed the effects of Plk1 KD on cell proliferation. Smooth muscle cells were treated with PDGF for 24–72 h. The numbers of smooth muscle cells were then evaluated. Compared to cells expressing control shRNA, the numbers of Plk1 KD cells were reduced (Fig. 4a).
Effects of Plk1 KD on smooth muscle cell proliferation. a Smooth muscle cells expressing control shRNA or Plk1 shRNA were treated with 20 ng/ml PDGF for 24–72 h. The numbers of viable cells were then determined. Plk1 KD inhibits smooth muscle cell proliferation. Data are mean ± SE (n = 4, * P < 0.05, **P < 0.01). b Smooth muscle cells expressing control shRNA or Plk1 shRNA were treated with or without 20 ng/ml PDGF for 24 h. DNA synthesis in these cells was evaluated using the BrdU incorporation assay. Newly-synthesized DNA was reduced in Plk1 KD cells. Data are mean ± SE (n = 6, ** P < 0.01)
We also used the BrdU incorporation assay to assess the effects of Plk1 on DNA synthesis. Basal BrdU incorporation was not different between cells expressing control shRNA and Plk1 KD cells. However, BrdU incorporated into newly synthesized DNA was lower in Plk1 KD cells than in cells expressing control shRNA (Fig. 4b).
Plk1 silencing does not affect AKT phosphorylation in smooth muscle cells
In addition to MEK1/2 and ERK1/2 activation, exposure to growth factors also activates AKT/PKB in smooth muscle cells [9, 23]. To determine whether AKT activation is regulated by Plk1, we assessed the effects of Plk1 KD on AKT phosphorylation in cells. Stimulation with PDGF induced a significant increase in AKT phosphorylation in cells expressing control shRNA and Plk1-deficient cells (Fig. 5). The average increase in AKT phosphorylation in these cells was not significantly different 10 min after PDGF stimulation.
AKT phosphorylation stimulated by PDGF is not reduced in Plk1 KD smooth muscle cells. Stable Plk1 KD cells and cells expressing control shRNA were treated with 20 ng/ml PDGF for 10 min, or left untreated. a Representative immunoblots illustrating the effects of Plk1 KD on AKT phosphorylation. b Basal and the PDGF-induced AKT phosphorylation (Ser-473) is similar in Plk1 KD cells and cells producing control shRNA (P > 0.05). Stimulated AKT phosphorylation was normalized to the basal level of cells expressing control shRNA. Values are mean ± SE (n = 4)
The expression of mutant T210A Plk1 inhibits the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation
As described earlier, PDGF stimulation induces Plk1 phosphorylation at Thr-210 (Fig. 1a). To determine the role of Thr-210 phosphorylation, we assessed the effects of mutant T210A Plk1 (alanine substitution at Thr-210) on MEK1/2 and ERK1/2 in smooth muscle cells. Immunoblot analysis verified the expression of wild type (WT) or T210A Plk1 in smooth muscle cells (Fig. 6a). In cells expressing WT Plk1, PDGF stimulation induced the phosphorylation of MEK1/2 and ERK1/2. In contrast, the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 was reduced in cells expressing T210A Plk1 (Fig. 6b and c).
Expression of mutant T210A Plk1 inhibits the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation. a Smooth muscle cells were transfected with plasmids encoding Flag-wild type (WT) or T210A Plk1. Blots of cell extracts were detected with antibodies against Flag and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells transfected with plasmids were normalized to untransfected cells. Values are mean ± SE (n = 4, *P < 0.01 vs. untransfected cells). b and c Cells expressing WT or T210A Plk1 were treated with 20 ng/ml PDGF for 10 min, or they were not treated. Protein phosphorylation was then evaluated by immunoblotting. MEK1/2 phosphorylation (n = 5) and ERK1/2 phosphorylation (n = 4) induced by PDGF was significantly reduced in cells expressing T210A Plk1 compared to cells expressing WT Plk1 (*P < 0.05). Protein phosphorylation under various treatments was normalized to the levels in unstimulated cells expressing WT Plk1. Values are mean ± SE. NS, basal MEK1/2 or ERK1/2 phosphorylation in Plk1 KD was not significantly different from cells expressing control shRNA (P > 0.05). d Smooth muscle cells expressing WT or T210A Plk1 were treated with 20 ng/ml PDGF for 24–72 h. The numbers of viable cells were then determined. Data are mean ± SE (n = 4, *P < 0.05, **P < 0.01)
Furthermore, we also evaluated the effects of T210A Plk1 on cell proliferation. In cells expressing WT Plk1, PDGF treatment induced cell proliferation. However, the expression of T210A Plk1 attenuated the PDGF-induced cell proliferation (Fig. 6d).
The protein kinase Plk1 has been implicated in various cellular functions including mitosis and cytokinesis [2, 5]. The physiological properties of Plk1 in the early stage of the growth factor response have not been investigated. In the present study, Plk1 underwent phosphorylation at Thr-210 in quiescent smooth muscle cells within minutes of PDGF stimulation. Structural analysis reveals that Plk1 is composed of a common N-terminal catalytic domain and a C-terminal regulatory domain with highly-conserved sequences named polo-box domain (PBD), and an interdomain linker [7]. PBD is involved in an autoregulatory mechanism and spatial localization [2, 7]. When inactive, PBD binds to the catalytic domain, which inhibits the access of substrates to the catalytic domain. Plk1 phosphorylation at Thr-210 may induce the dissociation of PBD from the catalytic domain, increasing kinase activity [2, 6, 7, 24]. Thus, the stimulation with the growth factor is able to activate Plk1 in smooth muscle cells.
The activation of the MAPK pathway is a key event in the early phase of cellular responses to growth factor stimulation. Our recent studies showed that the MAPK pathway in airway smooth muscle is regulated by Raf-1 [8], which is a known target of Ras [10, 11]. Thus, it is likely that the MAPK cascade is mediated by the Ras-Raf pathway in smooth muscle. Although the role of Plk1 in cell division/proliferation is well recognized, the studies on Plk1 are largely focused on the stages of mitosis and cytokinesis [2, 4, 5]. In this report, stimulation with PDGF also induced the phosphorylation of MEK1/2 and ERK1/2, which is consistent with previous results [8, 9, 25]. Furthermore, Plk1 activation preceded MEK/ERK activation in smooth muscle cells upon PDGF stimulation. More importantly, Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2. To the best of our knowledge, this is the first evidence to suggest that Plk1 is necessary for the activation of the MAPK pathway in smooth muscle cells upon growth factor stimulation.
In response to activation with growth factors, ERK1/2 gets phosphorylated at Thr-202/Tyr-204, which is catalyzed by the dual-specificity protein kinase MEK1/2 [8, 10]. Since Plk1 is a serine/threonine protein kinase [2, 5], it is unlikely that Plk1 catalyzes ERK1/2 directly. Previous studies by others suggest that MEK1/2 is phosphorylated at Ser-218/Ser-222 and activated by Raf kinase during activation with growth factors [10, 11]. Moreover, in response to cell adhesion to fibronectin, p21-activated kinase mediates MEK1/2 phosphorylation at Ser-298, which subsequently promotes phosphorylation of Ser-218/Ser-222 and activates MEK1/2 [26]. In the present study, Plk1 KD did not affect Raf-1 phosphorylation in smooth muscle cells. Plk1 directly catalyzed MEK phosphorylation at Ser-218/Ser-222 as evidenced by the in vitro kinase assay. Thus, Plk1 may regulate the ERK1/2 activation by controlling MEK1/2 phosphorylation in smooth muscle cells during stimulation with growth factors.
As described earlier, the MAPK pathway is activated within minutes in response to growth factor stimulation, which phosphorylates and activates downstream targets and promotes cell proliferation eventually (takes days) [8–11]. In this report, Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, DNA synthesis and cell proliferation. The results support the concept that Plk1-mediated MAPK activation (within minutes) ultimately affects smooth muscle cell proliferation.
AKT phosphorylation and activation may facilitate cell survival in response to growth factor stimulation [9, 23]. Although Plk1 regulates the MAPK pathway, KD of Plk1 did not affect the PDGF-induced phosphorylation of AKT. The results suggest that Plk1 differentially regulates the activation of MEK1/2 and AKT in smooth muscle cells. AKT phosphorylation may be regulated by PI3 kinase, PTEN, the PIKK (PI3 kinase related kinase) superfamily, mTORC2 (mammalian target of rapamycin complex 2) and DNA-PK [23].
In this report, the expression of T210A Plk1 diminished the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 as well as smooth muscle cell proliferation. The results suggest that phosphorylation at Thr-210 is required for the activation of MEK1/2 and ERK1/2, and cell proliferation. Phosphorylation at this residue may increase Plk1 activity, which in turn phosphorylates MEK1/2 and activates the downstream events.
ERK1/2 has been shown to control transcription factors (Elk-1, Ets-2), ribosomal S6 kinase, MNK kinase, and cPLA2, which regulate gene transcription, protein translation and other processes that facilitate cell proliferation [27, 28]. Future investigations are required to assess whether these molecules are involved in the Plk1/MAPK-regulated proliferation in smooth muscle cells. Moreover, asthmatic airway smooth muscle cells display different proliferative properties compared to non-asthmatic smooth muscle cells [1]. Since Plk1 regulates non-asthmatic airway smooth muscle cell proliferation, it would be interesting to understand whether the Plk1/MAPK pathway is altered in asthmatic airway smooth muscle cells in future studies.
How PDGF stimulation induces Plk1 phosphorylation at Thr-210 upon growth factor stimulation is currently unknown. Ste20-like kinase (SLK) is a serine/threonine protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis [29–31]. In vitro studies suggest that SLK may catalyze Plk1 phosphorylation at Thr-210, which may facilitate G2/M transition [29, 32]. In addition, the kinase Aurora-A and its cofactor Bora are pivotal for Plk1 phosphorylation at Thr-210 during mitosis of human osteosarcoma U2OS cells [4]. Furthermore, phosphatidylinositol 3-kinase (PI3K) has been implicated in Plk1 activation during mitotic phase of Hela cells [33]. Future studies are needed to evaluate whether SLK, Aurora-A/Bora and PI3K are upstream regulator of Plk1 in smooth muscle cells in response to growth factor stimulation.
Plk1 is a serine/threonine protein kinase that has been implicated in mitosis and cytokinesis. Our present study unveils a new and novel role that Plk1 plays in the regulation of smooth muscle cell proliferation. In response to activation of growth factors, Plk1 gets phosphorylated at Thr-210 and activated. Activated Plk1 may promote the phosphorylation and activation of MEK1/2, which subsequently activates ERK1/2 and smooth muscle cell proliferation. Thus, we propose that Plk1 controls cell proliferation at multiple cellular stages including the activation of MEK1/2, mitosis and cytokinesis.
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We thank Ruping Wang and Brennan D. Gerlach for technical assistance. This work was supported by NHLBI Grants HL-110951 (to Dale D. Tang) and HL-113208 (to Dale D. Tang) from the National Institutes of Health.
The Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, MC-8, Albany, NY, 12208, USA
Sixin Jiang
& Dale D. Tang
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Correspondence to Dale D. Tang.
SJ performed cellular, biochemical and molecular studies, and drafted the manuscript. DT designed the experiments, analyzed data and revised the manuscript. All authors read and approved the final manuscript.
Jiang, S., Tang, D.D. Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells. Respir Res 16, 93 (2015) doi:10.1186/s12931-015-0257-8
Smooth Muscle Cell
Smooth Muscle Cell Proliferation
Airway Smooth Muscle Cell
Growth Factor Stimulation
Human Airway Smooth Muscle


Up until a little over a year ago, I considered all computers to be the ultimate expression of Evil with a capital "E" - one of the main causes of everything that is wrong with this world. In this age when children are born wired to the Internet, I suppose I must explain my blatant "Ludditism."
Be it indeed a situation of temporal circumstance or just the fact that I've always felt I was born ten years too late, I got just enough of the Sixties in my bloodstream to make my existence in the Eighties and Nineties pretty much an unbearable case of genuine 1984-esque culture shock.
I found comfort in that which came slightly before me. I understood that tubes sounded more "real" to my ears than transistors. Similarly, an early model vintage VW had an "air" about it that later (post-'67) Bugs didn't. Everything on a vintage VW was finely crafted and had a purpose, and it fulfilled that purpose with a sense of simple elegance that defied and mocked that which came after it. When things were later changed from sturdy metal to cheap plastic to appease the demands of the masses, the VW (for me) lost its integrity somewhere in the process.
My "Dynaco Stereo 70" tube amp, built from a kit around the time I was born, had a warm sound with a presence that said "High Fidelity" to my ears. All my friends' new solid-state Panasonic amps sounded pale and flat in comparison, even with their many-slidered graphic equalizers as opposed to my simple bass and treble pots. I have always appreciated that sense of "simple elegance" I discovered in the vintage VW, where functionality blended with inspired design yielded something truly unique and wonderful: a machine that one could actually bond with. In short, a truly "Fahrvernügen" experience turns my proverbial crank.
Around the beginning of 1999 I was asked to do a bit of anarchic writing for a friend's website. I prided myself as I noisily clacked out page after page on my old Brother word processor. How clever I was to avoid the very technology my words would end up being displayed by! Ah, the self-righteous and smug expressions that I'm certain crossed my face as that little glorified typewriter did it's thing.
I eventually hung out with my friend enough to see him reboot his Power Mac 7100/80AV countless times (not the most astute troubleshooter, this guy, but that's another story!). After one of his "Three-Finger-Salutes," I took notice of the cute little "Happy Mac" that showed up on the screen at reboot. Something in me started to soften. My thick Luddite skin started to crack and peel just a bit. If ever there was a way for me, a confirmed hater of "high technology," to finally enter the computer age, then this had to be it. Anything that smiled at you as if to say "Hi! I'm happy! Let's have fun!" when it turned itself on had to be worth checking out.
In February of last year, around my birthday, a friend of mine who had just gotten a WallStreet PowerBook asked if I wanted to buy her Power Mac 7200/120. She would even take payments.
I couldn't refuse, and on February 7, 1999, I brought home "Rocco," my first Mac. Decked out with 128 MB of memory, a 4.2 GB Seagate Medalist hard drive, and OS 8.5.1, Rocco certainly served as a great intro to the Mac experience. Being a "fixit-guy" at heart, I immediately started digging, rooting about on every Mac-related website I could find. I dug into my system folder. I dug into the Apple TIL. I dug into archived news messages on university BBS's from the "old days." I found that the Mac thought very much the same way I did. I "grokked" it in a way that I didn't have to think about.
I achieved the same sort of "Fahrvernügen" with my 7200 that I had with my Bug almost immediately. Within a couple of months I was doing things with Photoshop that veteran MacHeads were agog at. I built websites using only my homemade Photoshop graphics and Netscape Communicator's "composer" feature. I found an old Apple Service Source CD and read all the Service Manuals I could find. I popped open my 7200 and figured out where everything was. I diagnosed a defective 16 MB DIMM within the first few months of having the machine. I learned to troubleshoot most common problems with the help of a plethora of Mac books, especially past editions of the wonderful old Mac Bible series, so plentiful in used book stores in Austin. I found myself eagerly absorbing as much as I could about something that I had abhorred (and wanted nothing to do with) only a short time before.
Put plainly, I absorbed as much of the Macintosh Mythos as I could in as short a time as I could. I found the "underdog/underground" aspect of the Mac irresistible: If ever there was a computer with an actual soul, one even a grumpy old Hippie like me could actually love, this was it. "Macintosh Venceramos!" I bought the whole thing, as they say, hook, line, and sinker. I considered myself a "MacHead;" armed with my 7200, I could now enter the fray of the Modern World. I settled into a smug and comfy state of MacUserhood, secure in my new knowledge. Little did I know this was actually just the first stage of a progressive (and seemingly incurable) new affliction.
My true descent into incurable Macoholism occurred a few months later when I was thrift-shopping with the girlfriend one Saturday afternoon. We found a couple of little compact Macs in great shape for next to nothing. I'd stumbled upon the Low End Mac and Jags House websites (those bastions of Old Mac Love-n-Lore) and had been itching to find an old compact to play with. Sitting for five minutes with a Mac Plus running System 6.0.8, booted from a single 800k floppy disk, and I felt I'd finally found the real Macintosh. The Power Mac paled in my sight. Franki Valli and the Four Seasons sang in my head: "Workin' my way back to yooooou, babe!" That "simple elegance" thing was blasting at me full volume with every mouse-click - utter MacFahrvernügen.
Here was the "vintage VW" of the Macintosh world! I found an upgraded 512k for six bucks a few weeks later and fell deeper in love with a machine than I ever had before. Of course, having found solace in Tolkien's Middle Earth as a disaffected youth, I had to name the Macs after Hobbits. It seemed the only correct thing to do.
I began to collect Macs of all flavors. They came to me. People gave them to me. I dug into every "new" model with a vengeance: Where were the RAM slots? Can I slap another hard drive in there?
It occurred to me that I really was unearthing history as I went. I took to using the signature "MacArchaeologist" on all my list postings; it was the best way I could describe myself to this newfound community of really cool people I'd discovered on the Low End Mac lists. The Compact Macs list continues to be my main group of "homies," people who understand the effect these little machines can have on you, people who encourage and support you in your addiction! (Hi, guys! ;-) The parallel between vintage VW's and vintage Macs rings even truer when you consider "Mac Folks" are the same kinda crowd as "VW Folks," always ready to help solve problems, give you goodies you've been searching for, and generally aid and abet your disease.
During the holidays this past year I collected, tweaked, and gave away six Li'l Macs to folks who (like myself) had never had any use for (or had been scared by) computers their whole lives, tossing some weekly "MacLessons" into the deal, consisting of the tips/tricks I'd found out on my own so far, and through the excellent resource that the Low End Mac email lists are. Seeing the looks on their faces when their Li'l Macs talked to them for the first time, or when they actually wrote their first letter and printed it out on their very own printer . . . well, it gave me warm fuzzies. Those folks are still happily playing with their Pluses and SEs, and are about to get their first color Macs, thanks to a friend who's office caught "upgrade-itis".
The thought that I can set up a compact Mac and a printer for someone for basic pocket change, sign them up for a free email account, and help them get empowered in a way they never would have taken the steps to be themselves, well, it gives me a charge. It makes me feel like some sort of crusader.
Cut to scene of a windswept hilltop. b.b. stands at the summit, "Indiana Macintosh"-style-hat cocked at a jaunty angle, a chiselled Art-Deco-30's-Worker-Poster look about him. Copland's "Fanfare for the Common Man" plays in the background as he tosses L'il Macs into the outstretched arms of the waiting Proletariat. The title of our little film rolls across the screen (in 3D Photoshop text, of course): "The Adventures of Johnny MacintoshSeed."
I can hear all the grizzled old MacHeads out there chuckling. They know I've fallen victim; they know I cannot refuse the call!
I began to see how each model of Mac has its own personality, its own characteristics. It just rocks my socks to know that a fifteen year old machine still does exactly what it was intended to do.
That's another thing about the whole Mac Experience that I can appreciate: If you understand the limitations and abilities of the machine, there is no reason to be disillusioned with its performance. I don't expect to go 85 m.p.h. in a 40-horsepower Bug, and I likewise don't sit down at my SE or 512k and expect to do 3D graphics or play the latest game. Instead, I appreciate the little bugger for what it can do, what it still does so efficiently and so elegantly. I love reading email on the SE. It gives me a sense of "having been there in the Old Days." Yes, that "Mac Mythos" has me by the short and curlies, and I am really not complaining.
Faramir, dual-floppy SE with "engineered" 80 MB internal HD!
Master Samwise, the 512k, is currently under the weather. A couple of evenings with Larry Pina's legendary book and a soldering iron, and he'll be feeling sprightly again soon!
Given away during the holidays: Frodo, Merry, Pippin, Pearl, Priscilla, and The Gaffer.
Most all are LocalTalked together with simple phone wire (need more phone wires!), able to print to a pristine LaserWriter Plus given to me by the same friend whose office caught a bad case of "upgrade-itis." Their loss, my rockin' crispy prints! I am getting people I don't even know calling me to "fix" their Macs. People who have been Mac users for years are asking me what to do when their Mac crashes. I've downloaded everything I could click on (and learned some hard but valuable lessons about Virus Protection Software!). I have sent Macs and MacStuff all over the globe, and have also received Mac Goodies from around the world, sent to me by others afflicted with Macoholism, just for love of the machine. I correspond regularly with fellow MacHeads in England and further.
I have been "assimilated" - and I love it.
To say that getting my first Macintosh changed my life might sound overly dramatic, but it's true. Macs came along at a time in my life when I was totally disillusioned about "career choices" and feeling like there was nothing I could do for a living that wouldn't (in the final analysis) cause some sort of harm to myself, someone else, or the planet in general. Yesterday, a lady smiled and handed me $80 for spending two hours helping her get to know her new iMac better. She was happy that I could tell her things she didn't know. She was happy I knew what I knew, and I was happy knowing that she appreciated that fact. A very "clean" exchange, and one that carried no "bad money vibes," and that totally rocks in this age of "dirty dollars!"
And guess what: after all is said and done, I still get to be a "Luddite" of sorts!
"G4s? We don' need no stinkin' G4s!"


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Home » Sport » Vitória de Guimarães official transfer of Pedro Henrique
Vitória de Guimarães official transfer of Pedro Henrique
The 29-year-old centre-back played more than 150 games for Vitória, between 2015 and 2020, and will now continue his career in Singapore.
Photography: Octavio Passos/Global Images
pink melody
Vitória de Guimarães made official this Thursday the transfer of Pedro Henrique to a club in Singapore, confirming the news in a timely manner advanced by O JOGO.
"Vitoria Sport Clube – Futebol, SAD informs that it has agreed to terminate the relationship with the player Pedro Henrique after an agreement with Lion City Sailors, which pays 1.3 million euros for the hiring of the Brazilian defender", reads the note published on the website. official of the club from Guimarães.
The 29-year-old centre-back played more than 150 games for Vitória, between 2015 and 2020, and will now continue his career in Singapore, after loans to Al-Wehda, from Saudi Arabia, and Atlético Goianense, from Brazil. Last week, Adson Batista, president of Atlético Goianiense, had already announced the decision of Vitória de Guimarães. "His club sent a document asking for the return. The player informed me that he was sold to a club in Singapore", said, at the time, the president of the Brazilian emblem.
Topics: I League, National Football, OJ, the game, V. Guimarães
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← Call Center Blues: Does Credibility Matter?
Do you feel you are somehow getting better service when someone at a call center in India speaks with an English Accent? Is it better to speak with someone who speaks perfect clear English but who is not energetic or helpful? Which person gets your respect and trust? These questions actually go right to the heart of our attitudes and beliefs about customer service.
Americans are notorious for complaining about companies who use callers from call centers in India because they are hard to understand, do not understand the other person's accent, and do not inspire confidence. The conventional wisdom is that if you are calling native English speakers, your call center will do better if you have callers who speak with an English or American accent. Call centers in India that call people in the U.S. generally do better when they hire native English speakers. This is what the mainstream media generally reports.
It is possible, though, that Americans have become suspicious of foreigners and just aren't willing to compromise on what they want. They feel uncomfortable with accents and immediately remember their jobs are being taken away be "these people." According to a 2011 Q&A session with professor Steven Neuberg in Scientific American, "People who are foreign to us are more likely to pose certain kinds of threats" and are not seen as having our interests at heart. They are more likely "to take more than their fair share" and to cheat us. Currently, the Better Business Bureau and many other organizations that are reputable and influential have blogs that include consumer comments on "foreign" accents. The FTC itself has a website blog "Fraud Affects 25 million people" that includes many consumer comments demonstrating suspicion about foreigners. If foreigners are cool– should we condone such comments? If outsourcing is going to work–shouldn't we be extra careful about what we say and publish? The spin on foreign accents always seems negative, never positive.
18 ways to boost your social media marketing in 10 minutes per day!
That expression, by the way, is an English slang expression meaning "the top" or "the best." Just how important is your accent?
According to most surveys, neutral English accents are preferred; people do not mind talking with someone from a call center in the Philippines, for example, because that person can speak clear English. That is a major reason why call centers in the Philippines are getting so much work lately: native English speakers want to interact with those who speak English.
However– what if you are a highly motivated and reliable worker at a call center in India and you do not speak perfect English? A call center manager who employs callers in various parts of the globe told us that if your Indian accent is not particularly heavy–but you understand the person's questions, can explain everything clearly, and make a good impression–you can do just as well as native English speakers if you just do your job.
Doing your job at a call center in India means conveying the message, getting a positive response from the person at the other end of the line, maybe making a sale or giving good advice about a product, and not having that person hang up on you or complain. If the person has gotten good information, he or she will not feel the caller with a foreign accent has been dishonest or unreliable in any way.
If you are able to convince the person you are calling that you understand what is being said, and if you can respond in an upbeat manner with good suggestions or comments, you may do just fine.
I would rather hear from someone at a call center in India who is positive and smart than someone who sounds lazy and stupid but speaks perfect English. I don't care what the person's name is if I can understand what is being said and I get some good information. I am not worried that this person will take jobs away from the U.S, because that person is a human being who is trying his or her best…and where the jobs go is beyond my control. The success of the conversation depends only on whether the person is doing the job.
If you work at a call center in India, you owe it to yourself to try to speak clearly and give the very best information you can. Be natural and helpful, and don't worry about the person on the other end of the phone. Just do your job!
(1) Is it more important to have an English accent or to just speak clearly and do your job?
(2) Many Indians think a UK accent is the way to get ahead. Being helpful counts more!
This entry was posted in Call Center, Popular Posts and tagged call center, Call Centers, Call centers in India. Bookmark the permalink.


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Queens County Federal Savings & Loan Assn.
Address: 89-61 162nd Street
Neighborhood: Jamaica
Architect: Heller, Simeon
Current Status: Significantly Altered
Award: Bronze Plaque for Rehabilitations and Alterations
Typology: Bank
Not the only Queens Modern winner to be converted into a discount store, this former bank branch is now largely obscured by awnings and unfortunate painting over of much of the Regal Blue and Alabama White marble on the front facade. Passerby can mostly identify the building by the still prominent flagpoles at the roofline and the metal grill above the door. The interior had teak paneling (now gone) and an acoustic tile ceiling with recessed lighting, which is still visible in the commercial space. This project won an award as a combining and rehabilitation of two existing buildings and not as a new structure.
Learn About the Architect


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Home > Comment > People are sharing too much online
People are sharing too much online
13th May 2014 Comment
Instagram is the perfect place to share the finer details of your life, but some things were always considered too personal to share with the world – until now. A recent viral trend on Instagram has people take a selfie immediately after having sex, in a departure from the perfect models we usually see sexualised in media. Around the same time, a Facebook group called "Women Who Eat on Tubes" gained popularity, in which members are invited to send in any photos they've snapped of women grabbing a snack on the move. Both of these trends are perfect examples of society going too far in documenting their daily lives.
Excessive sharing has caused a number of problems. A particularly harmful effect is that it makes people feel ashamed of things that aren't the least bit shameful. Take, for example, the after-sex selfie phenomenon, which is making people who don't have sex, or don't want to share their bedroom experience with the world, feel uncomfortable or ashamed. On the other hand, we're constantly told that being open about sex and sexuality is a positive thing because it liberates us to talk about an aspect of human life that has a huge bearing on our health and social welfare. However, taking a picture – one of the more graphic ways of opening up – can come across as bragging and leaves some people feeling as though they're going against the norm, particularly when a trend such as this goes viral.
"Women Who Eat on Tubes", though, is shameful as well as shaming. Whereas the after-sex selfies don't set out to make anyone feel bad about themselves, that seems to be the very point of the Facebook page. By taking pictures of women eating with the intent to share it with a community based on bullying, people are making others feel ashamed of a common biological function. I'm eating as I write this because it's lunchtime and that's exactly what lunchtime is for. In fact, I eat at least three times a day, as do most people. Who cares if I eat in the comfort of my own home or while I'm moving from one place to the next? If we're looking for things like this to share, then we're clearly sharing too much.
Whatever an article argues, nobody really has the right to tell you how much you can post. But if you have to invade someone else's privacy to get material, you're clearly scraping at the bottom of the barrel and violating social boundaries as you do. One woman who found herself featured on the "Women Who Eat on Tubes" page said that she felt "hurt and humiliated", but we often don't think about the subject of our images as a real person when sharing them with the world – we're all guilty of it, whether it's a photo you've taken or a comment you've made on someone else's. Although we may regret doing so later, and I hope the people shaming others online feel the regret soon. When you put something on the internet, there's no taking it back.
It's not just other people's privacy we're ignoring either – it's our own. The after-sex selfies and many selfies in general are clear examples of people wilfully giving up their privacy in exchange for a few likes, or just the knowledge that somebody wants to see. Overall, this trend is less damaging than the images of women on the tube. Though, as the selfie-takers are their own subjects, they can control how they're represented and make sure they're happy with the result before posting it to the world, whereas the women being publicly shamed don't have a say. However, it is depersonalising one of the most intimate acts that we do; you probably wouldn't ask your Instagram followers to come, sit down, and physically watch, so why are we happy to share such personal details with them online? We are in some cases leaving nothing to the imagination.
Remember back to when you were young: you had to ask your parents or grandparents to get the photo albums down to look back through important events like birthdays and holidays. There were perhaps a handful of images for each, yet from those you could visualise the whole occasion. You didn't need more. So why do we now feel compelled to post 20 pictures when we buy a new top? Hitting the shutter button is now a reflex that has left nothing private or special anymore.
Internet, photography, posts, privacy, selfie, sharing, social life, social network
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MSU researchers ask what steak eaters crave
The information presented on this page was originally released on October 28, 2010. It may not be outdated, but please search our site for more current information. If you plan to quote or reference this information in a publication, please check with the Extension specialist or author before proceeding.
Beef, Family, Food
By Justin Ammon
Mississippi Agricultural and Forestry Experiment Station
MISSISSIPPI STATE – W. Harris of Starkville takes his time when searching for steaks to feed his family. He carefully inspects the meat at the grocery store and often ends up with a pack of thin-cut New York strips.
"The strips make my family happy," Harris said. "You can see the juice in that cut before it's even cooked."
Mississippi State University researchers from the Department of Food Science, Nutrition and Health Promotion, found that Harris is not the only American who considers a variety of factors when choosing a steak.
A research team, led by Mississippi Agricultural and Forestry Experiment Station assistant research professor Jason Behrends, put up a tent in MSU's Junction while fans tailgated before a home football game. Their intention was to gauge the public's willingness to pay extra for a thicker, heartier steak.
"It was a perfect day," Behrends said. "It was 40 degrees: refrigeration-like temperatures. We roped an area off, started recruiting people and attracted more than 300 participants."
Behrends, along with associate professor Mark Schilling and doctoral student Christine Leick, both in MSU's food science, nutrition and health promotion department, and Ty Schmidt, assistant professor in the Department of Animal and Dairy Sciences, set up three tables. Each table displayed a different type of steak: ribeye, New York strip and top sirloin. The steaks were available in three categories of thickness, and each category had a different price.
The thinnest steaks were the cheapest, costing $8.99 per pound for ribeye and strip steaks and $4.99 per pound for sirloin steaks. The mid-thickness steaks were tagged at $9.99 per pound for ribeye and strip steaks and $5.99 per pound for sirloin. The thickest steaks cost $10.99 per pound for ribeye and strip steaks and $6.99 per pound for sirloin.
Participants were asked to choose three steak packages from each table. They were told that every steak, regardless of thickness, all weighed exactly the same—ribeyes and strips weighed 12 ounces, and sirloins weighed 10 ounces—and their choices should be based on price, color, marble, texture and thickness.
What most participants did not realize, however, was that every steak was cut from the same quality of carcass, which was low choice, the most common grade of grocery store steak. The only noticeable difference between the meats was in thickness.
"We wanted to look at thickness as the primary goal. We focused on that and kept everything else constant," Behrends said. "We wanted to see if consumers would pay more for it."
Behrends and his team found that most participants did not highly value thickness. Only 23 percent of consumers listed thickness as the most important factor of a ribeye, making it only the third most important attribute. Meanwhile, 30 percent thought that marbling was the most important attribute of ribeye, making it the most important factor of those tested.
Thickness defeated only marbling for first place with the sirloin steaks.
Less than 17 percent of participants listed price as the overriding selection factor for any steak type, though participants' annual income ranged from $20,000 to $60,000.
When price was completely removed from consideration, consumers still did not choose the thicker steaks, which were originally marked as the most expensive in the experiment.
"We thought all consumers would pick the thick steaks. That's what people want, right?" Behrends asked. "That was the biggest surprise to me. People still didn't pick thick steaks even when we took price out of it."
Some people chose thinner steaks because the larger surface area made them appear bigger, despite the fact that they were told they all weighed the same, Behrends said.
Behrends said that even if consumers are not willing to pay more for thickness, most of them are willing to pay more for characteristics such as color and marbling.
"No matter what you do, you eat with your eyes first," he said. "When consumers select something, they are predetermining what it's going to taste like."
Harris, who previously worked in kitchens from New Orleans to Atlanta, said he knows how to find a steak his entire family can enjoy.
"I can't tell you what a perfect steak is. I don't know who can," he said. "But I know what everyone wants: a reasonable steak with lots of flavor at a reasonable price."
Contact: Dr. Jason Behrends, (662) 325-3200
Released: October 28, 2010
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http://www.ext.msstate.edu/news/feature-story/2010/msu-researchers-ask-what-steak-eaters-crave


"Mr. Spritz Goes to Washington"
Horace Wilcox was the former congressman of Springfield and member of the Springfield Republican Party.
The Simpson family went to see him about the planes going over their house, as the noise made it impossible to sleep, or do other things. He had agreed to help, but died of a heart attack before he could do anything. With no one to turn to, the Simpsons decided to get Krusty to replace Wilcox as congressman.
According to Kent Brockman, Wilcox was a Titanic survivor.
Episode – "Mr. Spritz Goes to Washington"
Modified on January 7, 2016, at 19:23.


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