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VPS hosting contains the best elements of shared hosting and dedicated hosting services. Like shared hosting, VPS hosting puts your website on a server that also has other sites running on it, except that there are fewer sites per server. The sites share the cost of running on the server, which results in a monthly or yearly charge that's less than the relatively high price tag of dedicated hosting.
VPS hosting is an affordable option when your business requirements outgrow the capacity of shared hosting. It's the next logical step: substantially more capacity and control for just a bit more money. Users on a shared web hosting server share the computing resources for the websites, email, and databases. There is no guarantee of CPU, RAM on shared web hosting. VPS hosting provides dedicated compute resources for your websites, files, email, databases, etc. keeping you in complete control. In addition, you get more control with root level access to perform configurations not possible in shared hosting.
VPS hosting sits in the intersection of shared hosting and dedicated servers. Unlike shared hosting, VPS is an isolated environment with dedicated resources, which means you have more processing power and your site loads faster. VPS is best suited for applications that have seasonal traffic spikes or RAM-heavy/CPU-intensive applications. If you are developing a new application that has a complex database and you're not sure how many visits you'll get, VPS hosting is the affordable option to start with before you upgrade to a dedicated server.
In particular, Web Hosting Hub uses BoldGrid as a site builder. BoldGrid is actually an add-on to WordPress, so there's no lock-in. This overcomes the major problem of most site builders: you're locked into that host and that tool, often requiring you to completely rebuild your site if you want to expand. By using a WordPress-based solution, all of the rather considerable power of WordPress is available for future expansion.
You get other customer-centric bonuses for 24/7 customer support, including 24/7 toll-free callback phone support, free domain name and SSL certificates for as long as you remain a MochaHost customer, a website builder with 500 free templates (and a service that will custom-design your site if you need) and a site migration service. In addition, all plans are e-commerce ready and come with free shopping cart software.
Rather than storing data on physical servers, cloud customers are hosted on a connected network of virtual machines. The service is software-independent and perfect for folks who benefit from high availability and on-demand, pay-as-you-go resources. Ranked #1 for cloud-based VPS hosting, InMotion offers performance-enhancing SSDs, free data backups, and managed server options.
This is unfortunate because, these days, owning a website is becoming a crucial part of running a successful business, and more and more folks are establishing a web presence for their personal brand as well. You can use hosting to sell online, store and share your portfolio, or even publish your freelance writing samples and resumé. Yet, even the basics — What is web hosting? — can be lost on the average web user.
A Content Distribution Network (CDN) is a network of servers distributed geographically. The purpose of a CDN is to deliver content on the internet much faster. If a user in Toronto is trying to access your website, the closest server to them will take care of this. But someone coming to your website from Sydney, Australia, will be catered to by a server closest to them. The main host server holds your website content, and all the other servers have a cached version each.
Running a website doesn't have to be hard work! Our customer service experts & our Papa Squad are here for you at every stage of your website development. Need help registering a domain and configuring your email? We have everything covered! We'll make sure that your website runs smoothly, loads quickly and is ready to handle the constantly evolving industry of internet marketing. Websites contain more data than ever before, and you need to know your web hosting company can provide you with the power and speed you need to keep your website running smoothly.
Unlike shared or VPS hosting, dedicated hosting makes your website the lone tenant on a server. To extend the housing metaphor, having a dedicated server is like owning your own home. The means that your website taps the server's full power, and pays for the privilege. If you're looking for a high-powered site—an online mansion for your business—dedicated hosting is the way to go. That said, many dedicated web hosting services task you with handling backend, technical issues, much as homeowners have manage maintenance that renters generally leave to their landlords.
Why? Because word of mouth only gets you so far in the internet era. People discover new businesses—even local business—via Bing, Google, and Yahoo. The days when they'd just look you up in the yellow pages are long gone. If you don't have a sharable website address, your chances of building online word of mouth via social networking plummet, too. In other words, no website, no discoverability, no money. Of course, web hosting isn't just for businesses. You may want to host a personal website or blog, too. Either way, the services here have you covered.
Many web hosting services offer so-called unlimited or unmetered service for whatever amount of bandwidth, disk storage and sites you use. It's important to understand that most terms of service actually do limit the definition of "unlimited" to what's considered reasonable use. The bottom line is simple: if you're building a pretty basic website, unlimited means you don't need to worry. But if you're trying to do something excessive (or illegal, immoral or fattening), the fine print in the terms of service will trigger, and you'll either be asked to spend more or go elsewhere.
That said, not all web hosts offer email. WP Engine, for example, does not. In such instances, you must email accounts from a company other than your web host. GoDaddy, for instance, sells email packages starting at $3.49 per user, per month. That might sound like a hassle, and just one more thing to keep track of, but there are actually some webmasters who feel that separating your email hosting and web hosting services is smart. That way, one provider going offline won't completely bork your business.
When you sign up for SiteGround, you get a proactive chat from a live agent offering to call you, inquiring if you need any help setting everything up. Customer support includes tutorials, live chat, tickets, and phone calls. All are available 24/7 - and when you call on a Saturday night and get connected to an agent in 10 seconds, you know you're getting your money's worth.
We're often asked whether a virtual private server is faster than shared hosting options. Unfortunately, it's impossible to provide a general answer. There are powerful hosting plans with faster computing resources than that of a VPS. The difference, however, lies in the fact that our VPS services offer guaranteed resources (computing power), whereas shared hosting products are influenced by the number of resources available to other customers and ultimately only represent theoretical values.
Disclaimer: Great efforts are made to maintain reliable data on all offers presented. However, this data is provided without warranty. Users should always check the offer provider's official website for current terms and details. Our site receives compensation from many of the offers listed on the site. Along with key review factors, this compensation may impact how and where products appear across the site (including, for example, the order in which they appear). Our site does not include the entire universe of available offers. Editorial opinions expressed on the site are strictly our own and are not provided, endorsed, or approved by advertisers.
HostGator gained points for uptime monitoring and regular backups, along with free cPanel or Plesk. We liked that SSH is available for the more technically inclined site operators and that dedicated IP addresses could be purchased. We did take points off because it's sometimes difficult to tell what the price is once promotional plans run their course. That said, with a generous 45-day money back guarantee, there's a hosting solution for almost everyone at HostGator.
Domain name registrars function as marketplaces to buy and sometimes host your website name. Just like with any online purchase, you'll need to go through the registration and check out process. Once you have a domain name, you'll need to point your domain information to your hosting provider. If that sounds too baffling - contact the support. They'll surely know how to do it.
If you aim to have a web presence, you've got to have email. It's a convenient way for potential customers and clients to send you a message, Word document, or other files. Thankfully, most web hosts include email in the price of their hosting plans. Some web hosts offer unlimited email account creation (which is great for future growth), while others offer a finite amount. You, naturally, should want unlimited email.
Web hosting is a service that allows organizations and individuals to post a website or web page onto the Internet. A web host, or web hosting service provider, is a business that provides the technologies and services needed for the website or webpage to be viewed in the Internet. Websites are hosted, or stored, on special computers called servers. When Internet users want to view your website, all they need to do is type your website address or domain into their browser. Their computer will then connect to your server and your webpages will be delivered to them through the browser.
The PRO web hosting will be the right fit for most small business websites. It includes automatic SSL protection, a free domain name registration, and delivers better performance (double the RAM, CPU, Disk throughput), enabling faster page loads and more simultaneous traffic to your website. It's a great choice for WordPress, Joomla or Drupal sites expecting to receive several hundreds of visitors per day. It also includes Premium features such as Anycast DNS.
WordPress Support WordPress is an online website creation tool. It is a powerful blogging and website content management system, which is a convenient way to create and manage website. WordPress powers over 25% of websites on the internet. Most hosting providers will tell you right away if their plans are WordPress-compatible or not. The simple requirements for hosting your WordPress websites include: PHP version 7 or greater; MySQL version 5.6 or greater.
You could think of the sites that share your server as your roommates; there's really not that much separating you from them. Sure, you can close the bedroom door, but they can still cause nightmares for you in the kitchen and the bathroom. In web hosting terms, all the sites share a single server's resources, so huge traffic spike on Site A may impact the neighboring sites' performances. It's even possible that another site could take down the shared server altogether, if it crashed hard enough.
Please be objective. Please remove A2 from your blog. They encountered serious issue with their Windows server. Site down again, same thing happened on past April, have a look at https://windowswebhostingreview.com/a2hosting-infected-malware-looking-for-a2-asp-net-hosting-alternative/. I think they will recover so I give chance for them. But not now, I will take legal action to them. I subscribed their Windows hosting plan for 12 months * Have had major outage in past April and then it happened again. Right now, my site still down. * The web server process is being killed after five to ten minutes idle time, which is resulting in a minimum 40 seconds extra wait time for most users entering the website. I RECOMMEND to avoid A2.
VPS hosting sits in the intersection of shared hosting and dedicated servers. Unlike shared hosting, VPS is an isolated environment with dedicated resources, which means you have more processing power and your site loads faster. VPS is best suited for applications that have seasonal traffic spikes or RAM-heavy/CPU-intensive applications. If you are developing a new application that has a complex database and you're not sure how many visits you'll get, VPS hosting is the affordable option to start with before you upgrade to a dedicated server.
Excellent performance, great features, and helpful tech support propel InMotion Hosting to second place in our roundup of the best web hosting services 2020. InMotion has been around since 2001, and is one of the world's largest independent web hosting companies. It offers several shared hosting plans, with prices starting at a very attractive monthly cost.
For over a decade we've helped business owners, entrepreneurs, bloggers and digital enthusiasts make their online vision a reality with award winning web hosting services. For both personal and professional websites, we are the Canadian web hosting company you can rely on. We have the technology to ensure your site runs smoothly and we're here for you every hour of everyday. Want to succeed online? Use HostPapa web hosting services to power your website.
While you will share some hardware resources with other VPS customers on your server, nothing your neighbours do will affect your container and vice versa. You get many of the benefits of your own dedicated server at a fraction of the cost. On a shared server, you rely on the performance of the platform provided. By choosing VPS, you get to select a configuration best suited for your applications, requirements, and growth.
Whether I'm geeking out with Ryan, Laura, and the team, or sketching out server diagrams to explain the basics to my grandmother, talking about a career in web hosting is always a creative endeavor, and I love it. Hopefully, this guide has cleared up some of the common confusions of hosting services, and you're ready to sign up with the web host that best fits your upcoming site needs.
Do you plan to sell products or services? If so, you'll want to invest in a web hosting service that offers Secure Sockets Layer (SSL). An SSL connection encrypts the financial information that shuttles between users' web browsers and your website, thus safeguarding the transmission of the purchasing information. Some hosting companies include a free SSL certificate when you sign up for a web hosting plan, while others charge close to $100. You can save some money by shopping around for services that offer the cheaper SSL plans.
Our web hosting plans are designed for Canadian small businesses and entrepreneurs wishing to build, grow, and publish their website quickly and efficiently without breaking the bank, and include great tools such as Site Builders, WordPress, email, visitor statistics, and more. They are easy enough to use even if you have no technical experience, while being powerful and versatile enough to meet the needs of advanced software developers and power users.
Web hosting is a service that allows organizations and individuals to post a website or web page onto the Internet. A web host, or web hosting service provider, is a business that provides the technologies and services needed for the website or webpage to be viewed in the Internet. Websites are hosted, or stored, on special computers called servers. When Internet users want to view your website, all they need to do is type your website address or domain into their browser. Their computer will then connect to your server and your webpages will be delivered to them through the browser.
All the features I've detailed to this point are valuable to the web hosting experience, but none matches the critical importance of site uptime. If your site is down, clients or customers will be unable to find you or access your products or services. It doesn't matter how great the features are, or how good it looks; if your site is down, it might as well not exist.
Finally, we were quite happy with their money-back guarantee. They offer a full 90-day guarantee (except for domain names you buy) and a pro-rated refund for the rest of your time. So if you want to cancel after 18 months and have six months left in your term, you'll get back the cost of the six remaining months. It's clear, it's understandable, and it's the straightforward way we'd like to see most providers use as a model.
CloudLinux helps protect websites in a shared website hosting environment. If one of the websites on the servers gets attacked or gets a sudden growth in popularity and a lot of unexpected traffic, it will start draining all of the server resources, affecting other websites. CloudLinux makes sure this doesn't happen. It will either slow down the website to stop it from hogging all resources or it will temporarily shut it down.
For more than a decade, Jeffrey L. Wilson has penned gadget- and video game-related nerd-copy for a variety of publications, including 1UP, 2D-X, The Cask, Laptop, LifeStyler, Parenting, Sync, Wise Bread, and WWE. He now brings his knowledge and skillset to PCMag as Senior Analyst. When he isn't staring at a monitor (or two) and churning out web hosting, music, utilities, and video game copy, Jeffrey makes comic books, mentors, practices bass and Jeet Kune Do, and appears on the odd podcasts or convention panel. He also collects vinyl and greatly enjoys a craft brew. You can a find Jeffrey online at jeffreylwilson.net, or send him a tweet at @jeffreylwilson
The web hosting provider got bonus points for its policy of performing regular daily backups, even on the lowest-priced shared hosting accounts. Be aware, though, that the promotional price on the low-cost shared hosting does go up after the promotional period. That said, Bluehost offers 24/7 phone support, a 30-day money-back guarantee and SSH access for certain plan options.
On the plus side, SiteGround offers free automatic daily backups, access to the Cloudflare CDN, high-performance SSDs for all plans, unlimited email accounts and integration of the free LetsEncrypt SSL certificate into sites. The company does limit bandwidth and storage, but even those who claim to offer so-called unlimited bandwidth and storage really have some limits in its terms of service.
A Content Distribution Network (CDN) is a network of servers distributed geographically. The purpose of a CDN is to deliver content on the internet much faster. If a user in Toronto is trying to access your website, the closest server to them will take care of this. But someone coming to your website from Sydney, Australia, will be catered to by a server closest to them. The main host server holds your website content, and all the other servers have a cached version each.
We use this analogy on our web hosting page. Shared hosting is like an apartment building – lots of residents in the same building, sharing the same resources, but also very affordable. Dedicated servers are like having your state-of-the-art house, where you'll pay a lot more, but you also don't have to share the space or utilities with anyone. VPS is in the middle, like a townhouse, where you have a few residents in the same building, but you each have dedicated resources and are in full control of your home. It costs less than a dedicated server, but still gives you much of the same benefits.
Partitioning a single server to appear as multiple servers has been increasingly common on microcomputers since the launch of VMware ESX Server in 2001. The physical server typically runs a hypervisor which is tasked with creating, releasing, and managing the resources of "guest" operating systems, or virtual machines. These guest operating systems are allocated a share of resources of the physical server, typically in a manner in which the guest is not aware of any other physical resources save for those allocated to it by the hypervisor. As a VPS runs its own copy of its operating system, customers have superuser-level access to that operating system instance, and can install almost any software that runs on the OS; however, due to the number of virtualization clients typically running on a single machine, a VPS generally has limited processor time, RAM, and disk space.[2]
Excellent performance, great features, and helpful tech support propel InMotion Hosting to second place in our roundup of the best web hosting services 2020. InMotion has been around since 2001, and is one of the world's largest independent web hosting companies. It offers several shared hosting plans, with prices starting at a very attractive monthly cost.
HostingAdvice.com is a free online resource that offers valuable content and comparison services to users. To keep this resource 100% free for users, we receive advertising compensation from the hosts listed on this page. Along with key review factors, this compensation may impact how and where hosts appear on the page (including, for example, the order in which they appear). HostingAdvice.com does not include listings for all web hosts.
A Virtual Private Server (VPS) is an independent, virtual partition on a physical server. Think of a server with different "containers" within it. When you sign up for HostPapa VPS hosting, you get one of these containers to manage as you wish—install applications or scripts, and arrange your web files the way you want. It's private, secure, and flexible.


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Card Type Edit
Limit Per Deck Edit
Original Text Edit
(You may banish this card from your hand to stop a physical or energy attack.) Physical attack. DAMAGE: AT +4 stages. Attach to your MP. While attached, once per combat as an action you may place into play a Setup or Drill from your hand. Banish this card when you change levels.
Erratum Text Edit
Official Clarifications Edit
Community Clarifications Edit
If you use the parenthetical text of this card it does not attach to your MP.
Game Rules Links Edit
To be added
Community content is available under CC-BY-SA unless otherwise noted.

subset: commoncrawl


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45 Minutes - An experience with Sleep Apnea
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What happens if you stop breathing in your sleep? Why does it occur?
Understanding, Preventing and Treating Sleep Apnea | Clinilabs Drug Development Corporation
Learn about some of the potential causes, including sleep apnea , and what short- and long-term consequences may occur. Breathing disturbances can commonly be observed during sleep. The most frequently noted is snoring. When the tissues of the upper airway vibrate during breathing, this causes the sound of snoring.
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It is also possible for breathing to completely stop, but what causes this? These events by definition last at least 10 seconds but they can last up to several minutes. Most often, this is due to obstructive sleep apnea OSA. OSA occurs when the tissues of the upper airway — the tongue, soft palate, and uvula — collapse and disturb normal airflow. There may still be an effort to breathe, with the chest and abdomen moving, but the air is not normally moving past the obstruction within the throat.
As a result, airflow is reduced or is simply not observed to be moving through the nose or mouth during these periods. There are other potential causes of disturbed breathing in sleep to consider. Less commonly, the breathing pattern may be abnormal due to a failure of the brainstem to stimulate breathing. This occurs in central sleep apnea , Cheyne-Stokes respiration, and congenital central hypoventilation syndrome.
The first two conditions may be present in heart failure, with narcotic use, or near death. The latter disorder is rarely present in some children at birth.
- US Army, Technical Manual, TM 5-3805-281-24-1, HYDRAULIC EXCAVATOR JOHN DEERE MODEL 330LCR.
- A Comprehensive Guide to Sleep Apnea | Updated for | studcupobipa.cf?
- The Importance of Sleep!
- Severe sleep apnea: Understanding severe apnea risks?
This occurs with a burst of cortisol hormone. This stress response spikes the heart rate and blood pressure and can lead to other problems over the long term. The person experiencing it may wake up gasping or choking and an observer may witness a loud snort and movement of the body. The severity of sleep apnea varies. It is thought to be normal for pauses in breathing to occur up to 5 times per hour in adults and once per hour in children. These events may even occur as part of normal sleep stage transitions. If breathing disruptions occur more frequently, sleep apnea may be diagnosed with a sleep study.
Severe sleep apnea: Understanding severe apnea risks
The following categories are used to classify sleep apnea based on the apnea-hypopnea index AHI :. It is also important to recognize the degree of oxygen deprivation that occurs with these events. In the setting of heart or lung disease, the oxygen levels may drop drastically with each apnea event. As a result, there can be greater stress placed on the body overnight. Chronic oxygen deprivation may lead to both short-term and long-term effects.
Snoring and Sleep Apnea
It is extremely unlikely that a single witnessed pause in breathing would be the last breath the person takes. Instead, the event is likely to end as described above.
- Search Harvard Health Publishing.
- How Does NightLase® Work?!
- Stories and Social Media: Identities and Interaction (Routledge Studies in Sociolinguistics).
- Managing Projects (Institute of Learning & Management Super Series).
- Oral Appliances.
- Sleep Apnea - studcupobipa.cf.
When apnea occurs chronically, it can lead to other health problems , but can it lead to sudden death? Sleep apnea increases the risk of sudden death in sleep. It can also lead to atrial fibrillation, heart attack myocardial infarction , and even stroke. Although usually short, sleep terrors can last up to 45 minutes. Nightmares and sleep terrors are more common in children than adults.
In children, they are rarely caused by a physical or mental illness. Both nightmares and sleep terrors are more common in people with other sleep problems, such as obstructive sleep apnea.
How Does NightLase® Work?
Sleepwalking, also known as somnambulism, is a disorder in which a person partially wakes up during the night and walks around without realizing it. The sleepwalker might make repetitive movements, such as fumbling with clothing, get out of bed and stroll around, or even talk to you. Sleepwalking is usually not a cause for concern.
Most children will outgrow sleepwalking by their teenage years. Lakeland Locations. Health Library. Rights and Privacy. Lakeland News. Southwest Michigan. About Lakeland. Get Fit in the Mitt. Community Resources. Menu About Us. Our Services.
- Sleep Apnea | Associates in Periodontics | South Burlington VT.
- Computer Awareness Reference Manual!
- Wetwares: Experiments In Postvital Living (Theory Out Of Bounds)!
- The Other Life of Billy the Kid.
- Love in Luwangwa!
- Brass Quartet Recital Album Trumpet 1.
- When Your Breathing Stops in Your Sleep?
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Eli Smith, banjo player and co-founder of the Brooklyn Folk Festival, kicked off St. Ann and the Holy Trinity Church's new weekly Summer Music Concert Series on Thursday, June 18, at 5:30 pm. The 30-minute concerts are held in the newly landscaped Peace Garden on Clinton Street and feature musicians performing every Thursday in different genres. Passersby took up the invitation to listen from the sidewalk while keeping safe social distance. The concerts will be recorded and will be posted on the parish website for all who cannot attend to enjoy.
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Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF-α signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF-α signaling pathways converge at a point upstream of NF-κB activation and involve phosphorylation and degradation of the NF-κB inhibitory molecule IκBα. The HCMV inhibition of IL-1 and TNF-α pathways corresponded to a suppression of NF-κB activation. Analysis of IκBα phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-κB activation, which occurred upstream from the point of convergence of the IL-1 and TNF-α pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.
Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α) signaling pathways of the innate immune response are critical antimicrobial responses that have been highly conserved (37). In higher vertebrates, these pathways have evolved to serve as a critical interface between the innate and adaptive immune system (25). IL-1 and TNF-α signaling is an area of extensive research that has resulted in a considerable understanding of the signaling events involved in these pathways (1, 9). Activation of IL-1 and TNF-α pathways is initiated by proinflammatory cytokines IL-1α/β and TNF-α binding their cognate receptors following release from activated macrophages and T lymphocytes at sites of infection. Signal transduction via IL-1 and TNF-α pathways involves activation of pathway-specific receptor proximal kinases and ubiquitin E3 ligases. IL-1 and TNF-α signaling pathways converge more distally at the level of mitogen-activated protein kinase kinase kinases (MAP3Ks), leading ultimately to the activation of MAPKs (p38, c-Jun NH2-terminal kinase [JNK], and extracellular signal-regulated kinase) and the transcription factor nuclear factor-κB (NF-κB) (47, 55). Together, MAPKs and NF-κB induce expression of inflammatory chemokines (e.g., IL-8, IL-6, growth-regulated oncogene alpha [GRO-α], and monocyte chemoattractant protein 1 [MCP-1]), as well as adhesion and costimulatory molecules that recruit and activate various aspects of the innate and adaptive immune response.
MAPKs are activated by phosphorylation on specific threonine and tyrosine residues in a conserved "activation loop" of the MAPK kinase domain (34). Following activation, MAPKs induce the expression of IL-1- and TNF-α-responsive genes by phosphorylating a number of transcription factors, including Jun, Fos, and ATF family members. NF-κB is a critical transcription factor that serves as a major modulator of inflammation, immune regulation, differentiation, and apoptosis (19, 24). NF-κB induces target gene expression by binding to NF-κB binding sites in the promoter region of NF-κB-responsive genes. The NF-κB molecule is a homo- or heterodimer of Rel family members, with the most common form being comprised of a heterodimer between RelA (p65) and p50. In nonactivated cells, NF-κB resides in an inactive cytoplasmic complex with an inhibitory molecule, IκBα, which masks the NF-κB nuclear localization sequence. NF-κB activation results from the phosphorylation, ubiquitination, and subsequent proteosomal degradation of IκBα. IκBα degradation appears to represent the rate-limiting step in activation of NF-κB, and following release from IκBα, NF-κB translocates to the nucleus to directly induce target gene expression (24).
The importance of the IL-1 and TNF-α pathways for infectious organisms can be seen by the large number of bacteria (13) and viruses (23) that have evolved mechanisms to modulate (both activate and suppress) these signaling pathways. Cytomegaloviruses (CMVs), a family of ubiquitous DNA viruses, have coevolved with their host (36), which has resulted in a considerable level of coadaptation between host and virus. A critical aspect of this coevolution appears to be the ability of CMV to modulate components of the IL-1 and TNF-α signaling pathways. To date, most studies have focused on the activation of these pathways by human cytomegalovirus (HCMV) infection. At early times after infection, numerous studies have shown that HCMV activates signaling components of both IL-1 and TNF-α, including NF-κB (32, 45, 56, 57, 59) and various MAPKs (29, 30, 43, 56). This activation occurs in two distinct phases, with initial activation being mediated by virus attachment and entry (6, 12, 57). A second phase of activation occurs following virus entry and is mediated, at least in part, by major transactivators of the virus, IE1 and IE2 (10, 45, 59). These activation events have been proposed as necessary for the production of a cellular environment conducive to initial viral replication. IL-1 and TNF-α pathway activation may also facilitate virus dissemination through recruitment of HCMV-susceptible cells by increasing the secretion of chemokines (IL-8, IL-6, RANTES, and MCP-1) (2, 4, 12, 18, 20, 22, 40, 41).
More recent studies are beginning to reveal an ability of HCMV to suppress IL-1 and TNF-α signaling pathways at later times of infection (3, 22). Specifically, HCMV has been shown to decrease TNF-α-mediated signaling, which corresponded to a down-modulation of TNFR1 surface expression and decreased activation of the MAPK JNK (3). Similarly, HCMV has been shown to block both IL-1β- and TNF-α-induced expression of the chemokine MCP-1 at the level of transcription (22). The precise mechanisms involved in these inhibitory effects of HCMV on the IL-1 and TNF-α pathways are not completely resolved, and the breadth of the suppression on the proinflammatory response has not been investigated. However, these observations suggest a requirement for suppression of proinflammatory signaling pathways at later stages of the HCMV replication cycle.
In the current study, we have further examined the ability of HCMV to modulate the IL-1 and TNF-α proinflammatory signaling pathways. Our results show that HCMV suppresses both IL-1β and TNF-α signaling pathways at late times of infection. This inhibitory effect corresponded to impaired IL-1β- and TNF-α-induced IκBα phosphorylation and NF-κB activation and decreased expression of multiple inflammatory chemokines. Surprisingly, HCMV infection resulted in a differential effect on the IL-1 pathway compared to the TNF-α pathway, with a greater effect on IκBα phosphorylation and NF-κB activation observed for the IL-1β signaling pathway. This differential inhibition of IL-1β and TNF-α signal transduction suggests that HCMV modulates these pathways at distinct positions upstream from the point of pathway convergence. A number of studies indicate that endothelial cells represent an important site of HCMV persistent replication in vivo (16, 21, 42, 49, 50). The observation that HCMV suppression of IL-1 and TNF-α signaling pathways occurs at late times of infection within this cell type suggests that the ability of HCMV to regulate these pathways may represent a critical adaptation to enable virus persistence within the host.
Cells and virus.HCMV laboratory strains Towne and AD169 were obtained from the American Type Culture Collection (Manassas, VA). HCMV strain TR is a low-passage clinical isolate obtained from an AIDS patient that we have cloned as a bacterial artificial chromosome. TR retains all characteristics of a parental virus, including tropism for endothelial cells and macrophages. HCMV strains were propagated and titrated in normal human foreskin fibroblast (HFF) cells by standard methods (27). HFF cells were cultured at 37°C in an atmosphere of 5% CO2 in complete medium (Dulbecco modified Eagle medium containing 10% fetal bovine serum and supplemented with 4 mM l-glutamine, 200 μg/ml penicillin G, and 200 μg/ml streptomycin sulfate). Primary human umbilical vein endothelial cells (HUVECs) (Cascade Biologics, Portland, OR), retinal (HREC-181) (Cell Systems Corporation, Kirkland, WA) endothelial cells, and telomerase life-extended tAEC-5A endothelial cells were cultured in microvascular endothelial growth medium (EGM-2 MV; Cambrex Bioscience Rockland, Inc., Rockland, ME).
Analysis of IL-1β- and TNF-α-induced chemokine mRNA levels by quantitative real-time reverse transcription-PCR (qRT-PCR).Subconfluent HFF cells cultured in six-well plates were either infected with HCMV at a multiplicity of infection (MOI) of 3 or mock infected. At day 3 postinfection (p.i.), cells were treated with IL-1β (1 ng/ml) (R&D Systems, Inc., Minneapolis, MN) or TNF-α (10 ng/ml) (R&D Systems, Inc.). Cytokines were added to the cells directly into the media overlying the cells either with or without changing the media, depending on the experiment. However, in any particular experiment, all wells were treated in an identical fashion other than for the addition of the proinflammatory cytokine. At 1 and 3 h posttreatment, cells were harvested using TRIzol (Invitrogen, Carlsbad, CA), duplicate wells were pooled, and RNA was extracted using the manufacturer's protocol. RNA was quantified by absorbance at an optical density at 260 nm, and 2 μg of DNase-treated RNA was reverse transcribed using OmniScript (Sigma-Aldrich, St. Louis, MO) and oligo(dT) primers (Invitrogen, Carlsbad, CA). The resulting cDNA product was amplified using the ABI Prism 7700 sequence detector in the presence of 2× SYBR green master mix (Applied Biosystems, Foster City, CA) and 0.25 μM of forward and reverse primers, resulting in 120-bp amplicons. For each primer set, gene amplification in 40 cycles of PCR was measured by absolute quantification and then normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels to determine induction (n-fold). The denaturing and annealing/extension conditions for each cycle of PCR were 95°C for 15 s and 60°C for 1 min, respectively. The primer sets used for each gene of interest were as follows: GAPDH, 5′-TGACCTCAACTACATGGTTTACATGT-3′ and 5′-AGGGATCTCGCTCCTGGAA-3′; IL-8, 5′-CAACACAGAAATTATTGTAAAGCTTTCT-3′ and 5′-GAATTCTCAGCCCTCTTCAAAAA-3′; MCP-1, 5′-TCGCTCAGCCAGATGCAAT-3′ and 5′-ATGGTCTTGAAGATCACAGCTTCTT-3′; IL-6, 5′-GGATTCAATGAGGAGACTTGCC-3′ and 5′-ACAGCTCTGGCTTGTTCCTCAC-3′; and GRO-α, 5′-CCGAAGTCATAGCCACACTCA-3′ and 5′-TGGATTTGTCACTGTTCAGCATC-3′.
ELISA analysis of IL-8 levels.HFF cells and HUVECs were infected with HCMV at an MOI of 3 or mock infected, followed by treatment with IL-1β or TNF-α at day 3 p.i. After 6 h, supernatants were harvested, clarified by centrifugation, and stored at −80°C prior to analysis. The concentration of IL-8 released from cells was determined by enzyme-linked immunosorbent assay (ELISA) using the human IL-8 screening set (Endogen Inc., Rockford, IL) following the manufacturer's protocol. Plates were analyzed at 492 nm using a SpectraMax 190 plate reader (Molecular Devices Corp., Sunnyvale, CA), and levels of IL-8 released were determined by comparison to a standard curve.
Western analysis of viral and cellular proteins.Following IL-1β and TNF-α treatment, cell monolayers were washed twice with Dulbecco's phosphate-buffered saline and harvested at the times indicated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing a protease inhibitor cocktail (0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 2 μg/ml aprotinin, 10 mM phenylmethylsulfonyl fluoride) with 10 mM Na orthovanadate and 50 mM NaF. In designated experiments, the proteosome inhibitor epoxomicin (1 μM) (Peptide Institute, Osaka, Japan) was added to cells 1 h prior to IL-1β or TNF-α treatment. Proteins were separated by SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), and blocked. Viral and cellular proteins were detected using the following primary antibodies (Abs): rabbit anti-immediate-early HCMV IE2 (R638; 1:1,000) (15), rabbit anti-IκBα (C-15; 1:400) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-phospho-IκBα (B-9; 1:150) (Santa Cruz Biotechnology, Inc.), rabbit anti-p65 NF-κB (C-20; 1:2,000) (Santa Cruz Biotechnology, Inc.), and rabbit anti-p38 (C-20; 1:2,000) (Santa Cruz Biotechnology, Inc.). Primary Abs were detected with species-specific horseradish peroxidase-conjugated secondary Abs (1:1,500 to 1:2,500) (Amersham, Piscataway, NJ) and visualized using West Pico chemiluminescence substrate (Pierce, Rockford, IL).
IF microscopy.After fixation in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline, localization of viral and cellular proteins in cells treated with IL-1β and TNF-α was determined by indirect immunofluorescence (IF) microscopy, as previously described (26). Cells were harvested at 6 h posttreatment for analysis of IL-8 and 30 min posttreatment for analysis of NF-κB translocation. Primary Abs used were as follows: rabbit anti-IE2 (R638; 1:300), rabbit anti-p65 NF-κB (C-20; 1:300), mouse anti-gB (27-156) (1:150) (27), and mouse anti-IL-8 (clone 6217; 1:100) (R&D Systems, Inc.). After incubation with the appropriate fluorescently labeled species-specific secondary Ab, fluorescence was visualized using an Olympus confocal microscope.
HCMV inhibits IL-1β- and TNF-α-mediated induction of multiple inflammatory genes.A unique characteristic of HCMV is the ability of the virus to persist within the host, even in the presence of vigorous inflammatory responses, such as those observed in the tissues of transplanted organs during chronic rejection and transplant vascular sclerosis (28). We hypothesized that HCMV possesses an ability to modulate the innate immune response, which may be critical for persistence of the virus within such harsh proinflammatory environments. To examine whether HCMV was able to modulate the inflammatory environment by interfering with signaling pathways that contribute to cellular responses to soluble mediators of inflammation, we determined the level of inflammatory chemokine induction following treatment of HCMV-infected cells with the two major proinflammatory mediators IL-1β and TNF-α. HFF cells were infected with HCMV strain TR at an MOI of 3 or mock infected. At 3 days p.i., cells were treated with either IL-1β (1 ng/ml) or TNF-α (10 ng/ml), harvested at 1 and 3 h postinduction, and analyzed for the level of chemokine mRNA induction by quantitative real-time RT-PCR. For these studies, we focused on the induction of four inflammatory chemokines (IL-8, IL-6, MCP-1, and GRO-α), which are major mediators of the inflammatory response, being involved in both the recruitment and activation of multiple components of the immune system (5). The level of chemokine induction was normalized to GAPDH, and results are presented as induction (n-fold) compared to basal levels prior to IL-1β or TNF-α addition. The GAPDH levels were comparable in HCMV- and mock-infected cells and were not affected by treatment with either IL-1β or TNF-α (data not shown). Treatment of mock-infected cells with either IL-1β or TNF-α induced mRNA expression of all inflammatory chemokines studied (IL-8, IL-6, MCP-1, and GRO-α), consistent with the known positive regulation of the expression of these chemokines by IL-1β and TNF-α (Fig. 1). In contrast, cells infected with HCMV showed a decrease in response to both proinflammatory cytokines (IL-1β and TNF-α), as observed by the minimal induction of IL-8, IL-6, MCP-1, or GRO-α. At the time of harvest, HCMV-infected cells were >90% viable by trypan blue exclusion, and absolute basal levels of IL-8, IL-6, MCP-1, and GRO-α prior to IL-1β and TNF-α treatment were comparable to those of mock-infected cells (data not shown). These results indicate that the effect of HCMV on chemokine induction in infected cells did not result from either increased cell death or a maximal chemokine expression making cells refractory to IL-1β or TNF-α chemokine induction. Together, these results reveal an ability of HCMV to suppress the induction of multiple inflammatory chemokines by the two major proinflammatory cytokines IL-1β and TNF-α.
HCMV inhibition of IL-1 and TNF-α proinflammatory pathways corresponds to a decreased release of IL-8 from infected cells.To further investigate the characteristics of the HCMV inhibition of proinflammatory pathways, we focused on the effect of HCMV on IL-8 expression. IL-8 was selected as an IL-1β- and TNF-α-inducible gene marker, since this chemokine is an extensively studied prototypical inflammatory chemokine that is highly induced by both proinflammatory cytokines (24) and whose expression following induction with IL-1β and TNF-α was observed to be decreased in HCMV-infected cells (Fig. 1). To determine whether the effect of HCMV on IL-8 expression observed by qRT-PCR corresponded to a decrease in IL-8 release, we used an ELISA to measure the levels of IL-8 released into the supernatant of HCMV-infected cells following IL-1β and TNF-α induction. Since endothelial cells are a biologically relevant cell type for HCMV infection that represent a site of persistent HCMV infection within the host, the effect of HCMV infection on IL-8 release was analyzed in HUVECs as well as HFF cells. Cells were infected with HCMV TR at an MOI of 3 or mock infected. At day 3 p.i., cells were treated with either IL-1β or TNF-α, and the concentration of IL-8 released into the supernatant was quantified at 6 h postinduction. As shown in Fig. 2, treatment of mock-infected cells with either IL-1β or TNF-α resulted in a significant release of IL-8 from both HUVECs and HFF cells. However, consistent with the results from the qRT-PCR, both HCMV-infected HUVECs and HCMV-infected HFF cells released reduced levels of IL-8. Together, these results show that the effect of HCMV on proinflammatory cytokine induction of chemokine expression corresponds to a decrease in the release of chemokines from infected cells and that this effect appears to be independent of cell type.
HCMV inhibition of IL-1 and TNF-α proinflammatory pathways occurs at later times after infection.Typical of all herpesviruses, HCMV genes can be characterized into three kinetic classes (immediate early [IE], early [E], and late [L]) based on time of viral gene expression. To determine the stage of the viral life cycle that corresponded to the HCMV-mediated inhibition of the proinflammatory pathways, we analyzed the induction of IL-8 in infected cells by IF microscopy. Cells were infected with HCMV TR at an MOI of 0.5 and induced at day 3 p.i. for 6 h with either IL-1β or TNF-α. Cells were then fixed and stained for IL-8 as well as HCMV IE (IE2) and L (gB) antigens. The lower MOI utilized in these studies ensured the presence of HCMV-infected cells at different stages of the viral life cycle secondary to the spread of HCMV from infected to surrounding noninfected cells. Figure 3A shows a typical IF micrograph displaying the characteristic IL-8 expression in HCMV-infected cells (HUVECs) following proinflammatory cytokine induction (in this case, IL-1β). A similar effect of HCMV on IL-8 expression was observed following TNF-α treatment and in a number of different cell types (HFF and primary retinal microvascular [HREC-181] and telomerase life-extended aortic macrovascular endothelial cells [tAEC-5A]) following IL-1β or TNF-α treatment (data not shown). In all cases, IL-8 expression was evident in both noninfected and HCMV-infected cells expressing only the IE antigen IE2. In contrast, HCMV-infected cells that had progressed to the stage of E/L gene (gB) expression were negative for IL-8 following induction with either IL-1β or TNF-α. These results show that the inhibitory effects of HCMV on the proinflammatory cytokine pathways occurred at later times of HCMV infection, corresponding to the time of gB expression. Since the inhibitory effect was restricted to only a defined population (i.e., gB-expressing cells) within the culture, these results also show that the suppressive effect of HCMV required direct infection of the host cell. This latter observation lessens the likelihood of a paracrine mechanism mediated by a soluble factor released from HCMV-infected cells.
HCMV inhibition of IL-1 and TNF-α proinflammatory pathways is HCMV strain independent.To determine whether the HCMV-mediated inhibition of proinflammatory pathways was unique to the TR strain or was a general characteristic of HCMV, the ability of additional HCMV strains to prevent IL-8 induction was assessed by IF microscopy as described above. HFF cells were infected at an MOI of 0.5 with two laboratory strains (AD169 and Towne). Cells were then fixed and stained for IL-8, IE2, and gB after induction with IL-1β and TNF-α at day 3 p.i. All viruses exhibited an inhibitory effect on IL-1β (Fig. 3B) and TNF-α (data not shown) induction of IL-8. Similar to the TR strain, the effect on IL-8 induction by these viruses corresponded to gB expression at later times of infection. These results indicate that HCMV inhibition of proinflammatory pathways is a general characteristic of HCMV that is conserved between multiple strains.
HCMV inhibition of IL-1 and TNF-α pathways corresponds to an inhibition of NF-κB activation.The IL-1β and TNF-α signaling pathways converge at the level of the MAP3Ks to activate the downstream transcription factor NF-κB. In noninduced cells, NF-κB is retained in the cytoplasm by an association with the inhibitory molecule IκBα. Following proinflammatory cytokine induction, IκBα is phosphorylated, ubiquitinated, and degraded. This targeted degradation of IκBα releases NF-κB to translocate to the nucleus and activate gene expression (for a review, see reference 19). To determine whether the HCMV-mediated inhibition of proinflammatory pathways corresponded to impaired activation of NF-κB, NF-κB activation was measured in HCMV-infected cells by visualization of NF-κB translocation to the nucleus. Figure 4 shows the effect of HCMV infection on the nuclear translocation of NF-κB following 30 min of IL-1β induction. In the absence of induction, NF-κB was localized to the cytoplasm regardless of HCMV infection status. However, HCMV infection had a significant effect on the subsequent activation of NF-κB by IL-1β. In both noninfected cells and HCMV-infected cells expressing only the IE marker IE2, treatment with IL-1β caused rapid relocalization of NF-κB to the nucleus. In contrast, in HCMV-infected cells expressing the E/L marker gB, NF-κB activation was impaired as shown by the continued localization of NF-κB to the cytoplasm. These results show that HCMV suppresses the ability of proinflammatory cytokines to activate the transcription factor NF-κB. The effects of HCMV on chemokine induction and NF-κB activation closely paralleled one another (compare Fig. 3 and 4), with both events requiring direct infection and occurring in infected cells only at later times of infection. This observation suggests that the suppressive effect of HCMV on IL-1 and TNF-α pathways is mediated, at least in part, through an inhibitory effect on the nuclear translocation of NF-κB.
Interestingly, although HCMV inhibited the nuclear translocation of NF-κB following induction with either IL-1β or TNF-α, the effect on TNF-α activation of NF-κB appeared to be less than complete, with an intermediate phenotype being observed (Fig. 5). This differential effect of HCMV on NF-κB activation mediated by IL-1β compared to TNF-α suggested that HCMV may be modulating these two proinflammatory pathways independently at distinct points upstream (receptor proximal) of the convergence of these two pathways. The IF-based assay used to analyze NF-κB nuclear translocation is inherently qualitative. To determine more definitively whether HCMV was modulating these two pathways independently, we analyzed the degradation of IκBα following induction with IL-1β or TNF-α by Western blotting. The rationale behind this experiment was that independent regulation of these two pathways, occurring upstream of the convergence point, would be suggested by distinct characteristics of IκBα degradation following IL-1β induction compared to TNF-α induction. HCMV- or mock-infected cells were induced at day 3 p.i. with either IL-1β or TNF-α. Cells were harvested at increasing times postinduction, and the degradation of IκBα was measured in cell lysates by Western analysis (Fig. 6A). In mock-infected cells, IL-1β and TNF-α induction resulted in comparable and rapid degradation of the IκBα protein. The small shift in size of IκBα occurring at 5 min postinduction corresponds to phosphorylation of IκBα, prior to ubiquitination and degradation. Importantly, the comparable rates in loss of IκBα observed in mock-infected cells (see Fig. 6A) following treatment with either IL-1β or TNF-α indicate that the rates in signal transduction resulting in IκBα degradation are similar for these two pathways in this cell type. Dose-response analysis between cytokine level and IκBα degradation also indicated that the concentrations of IL-1β and TNF-α used were subsaturating (data not shown). HCMV infection had a significant effect on the ability of IL-1β or TNF-α to induce IκBα degradation. However, although HCMV-infected cells displayed a decreased rate of IκBα degradation following induction with either IL-1β or TNF-α, the block in IκBα degradation was complete following IL-1β but not TNF-α treatment. This observation suggests that the effect of HCMV on these signaling pathways is distinct. In the absence of IL-1β or TNF-α treatment, basal levels of total IκBα were comparable in HCMV-infected and mock-infected cells (Fig. 6A) and were independent of virus strain (Fig. 7). These results revealing distinct characteristics of IκBα degradation suggest that HCMV affects the IL-1β or TNF-α pathway independently at a position upstream from the point of signaling pathway convergence.
To further examine the differential effect of HCMV on the IL-1β or TNF-α signaling pathway, the experiment whose results are shown in Fig. 6A was repeated in the presence of the highly specific proteosome inhibitor epoxomicin (38), which serves to stabilize the phosphorylated form of IκBα. In mock-infected cells, IL-1β and TNF-α induction resulted in comparable and rapid phosphorylation of the IκBα protein, as shown by Western analysis using a phospho-IκBα-specific Ab (Fig. 6B). Stabilization of the phosphorylated form was also observed by the small shift in IκBα size occurring at 5 min postinduction. Consistent with the inhibitory effect of HCMV on the degradation of IκBα (Fig. 6A), phosphorylation of IκBα was also significantly decreased in HCMV-infected cells. However, the characteristics of the effect on IκBα phosphorylation following IL-1β induction compared to TNF-α induction were distinct, with an absence of IκBα phosphorylation being observed only following IL-1β induction. This absence of IκBα phosphorylation and degradation following IL-1β induction (compared to a partial block following induction with TNF-α) closely paralleled the effect on NF-κB nuclear translocation following induction with IL-1β and TNF-α, respectively (Fig. 5). Together, these results demonstrate that HCMV suppresses both the IL-1β and TNF-α pathways, resulting in impaired inflammatory chemokine induction, which corresponds to inhibition of NF-κB activation. HCMV appears to modulate IL-1β or TNF-α signaling independently at a level upstream from the point of convergence of these two pathways.
The viral gene(s) responsible for inhibition of IL-1 and TNF-α proinflammatory pathways is expressed with L gene kinetics.The observation by IF microscopy that the inhibitory effect of HCMV on proinflammatory pathways and NF-κB activation corresponded to times of gB expression suggested that the viral gene(s) mediating these effects was expressed with either E or L kinetics. To precisely define the kinetic class of the viral gene(s) mediating the inhibitory effect, we assessed the effect of foscarnet, an inhibitor of viral L gene expression, on IκBα degradation. HFF cells were infected at an MOI of 3 with HCMV and maintained in the presence or absence of foscarnet. At 3 days p.i., cells were treated with either IL-1β or TNF-α and harvested at increasing times postinduction for Western analysis of IκBα degradation, as described above. Foscarnet treatment overcame the inhibitory effect of HCMV on both the IL-1β- and TNF-α induced degradation of IκBα (Fig. 8). This finding indicates that the viral gene(s) responsible for inhibition of NFκB activation, and presumably chemokine induction, is expressed with L gene kinetics.
In the current study, we have observed an ability of HCMV to inhibit the IL-1 and TNF-α proinflammatory pathways at late times after infection. This inhibitory effect of HCMV resulted in reduced induction of multiple IL-1- and TNF-α-responsive target genes, including the major inflammatory chemokines IL-6, IL-8, GRO-α, and MCP-1. Inhibition of these signaling pathways corresponded to impaired activation of NF-κB, a transcription factor essential for expression of target genes through these pathways. Analysis of phosphorylation and degradation of IκBα, a negative regulator of NF-κB, showed that HCMV prevented the normal destruction of IκBα in response to IL-1β and TNF-α, consistent with the observed block in NF-κB activation. However, characteristics of the HCMV modulation of IκBα destruction following treatment with IL-1β, compared to treatment with TNF-α, suggested that inhibition of these two closely related proinflammatory pathways was mediated by HCMV at distinct positions upstream from the point of pathway convergence.
Although most previous studies have focused on the ability of HCMV to activate components of these pathways at earlier times of infection (29, 30, 32, 43, 45, 56, 57, 59), more recent studies have indicated that HCMV down-modulates aspects of the proinflammatory response, primarily at later times (3, 22). Specifically, HCMV has been shown to inhibit IL-1β and TNF-α induction of MCP-1 expression (22) and TNF-α activation of JNK (3). The viral genes involved in the inhibition have not been identified; yet, the effect on MCP-1 was mediated at the level of MCP-1 gene transcription. The effect on TNF-α activation of JNK corresponded to a down-modulation of TNFR1 surface expression, resulting from an internalization of the receptor from the cell surface. However, since the ability of TNFR1 expression to "rescue" the effect of HCMV on JNK activation was not determined, the precise role of receptor down-modulation in mediating inhibition of the TNF-α signaling pathway is unclear. Our finding that HCMV inhibits proinflammatory pathways further suggests an importance for suppression of signaling components, as well as inflammatory mediators, involved in the IL-1 and TNF-α pathways at later stages of the HCMV replication cycle.
IL-1 and TNF-α signaling involves closely related pathways that are composed of receptor proximal pathway-specific signaling events that converge at the level of MAP3Ks. Following MAP3K activation, a common signaling pathway is utilized for IL-1 and TNF-α activation of NF-κB (9). In the current study, the differential characteristics of the inhibition of the IL-1 pathway compared to that of the TNF-α pathway observed for IκBα phosphorylation and degradation, as well as for nuclear translocation of NF-κB, suggests that the HCMV-mediated inhibition of these pathways occurs receptor proximal from the point of pathway convergence at the MAP3Ks (Fig. 9). A number of possible mechanisms could be responsible for the HCMV-mediated inhibition. One possibility is that inhibition is due to independent down-modulation of IL-1R1 and TNFR1 surface expression. By fluorescence-activated cell sorter analysis, we observed only background levels of IL-1R1 (and TNFR1) surface expression regardless of infection (data not shown), which prevented the assessment of the effect of HCMV infection on IL-1R1 expression. However, since near-undetectable levels of IL-1R1 expression have been shown to result in normal levels of signaling via IL-1R1 (48), a complete block in signal transduction (as we observed for the IL-1 pathway) via receptor down-modulation would be unexpected. It is possible that the more intermediate inhibitory level of HCMV on TNF-α signaling is mediated by the previously identified effect of HCMV infection on TNFR1 surface expression (3). In this earlier study, the effect of HCMV on signaling events following TNF treatment was only partial (assayed in an in vitro JNK assay using glutathione S-transferase-Jun as a substrate). Hence, TNFR1 down-modulation as a mechanism for the inhibition of TNF signaling is consistent with our observation of a partial effect on NF-κB signaling after TNF treatment, but other explanations could also account for these data.
The comparable kinetics of inhibition, occurring at late times of infection for both IL-1 and TNF-α signaling pathways, suggest that inhibition of both pathways may be mediated by a single viral factor. Although the IL-1 and TNF-α pathways utilize distinct signaling molecules upstream from the convergence of these pathways, a number of these components are closely related. For example, both utilize members of a family of closely related TRAF (TNFR-associated factor) proteins (9), with the IL-1 and TNF-α pathways utilizing TRAF 6 (35) and TRAF 2/5 (52), respectively. TRAFs are a family of ubiquitin E3 ligases that catalyze a number of essential ubiquitination events required for signal transduction (11, 31). TRAFs contain conserved sequence and structural motifs, including an N-terminal RING domain, several zinc finger motifs, and a C-terminal TRAF domain. Hence, the differential effect on IL-1 and TNF-α signaling may be the consequence of a single viral factor exhibiting a difference in affinity for individual TRAF family members. A previous study indicated that a viral tegument protein, pp65, was able to suppress the induction of inflammatory genes that occurs in the absence of cytokine addition, which is observed very early (6 h) following infection (7). This inhibition was suggested to be due to an inhibitory effect of pp65 on NF-κB (7). However, the UL83 gene that encodes pp65 is expressed with E kinetics (8). In our study, the ability of foscarnet, which prevents L viral gene expression, to overcome the inhibitory effect of HCMV on IκBα degradation (Fig. 8) suggests that the inhibitory phenotype at late times of infection is independent of pp65. A more recent study has shown that the immediate-early viral transactivator IE2 can dampen the induction of inflammatory genes that accompanies HCMV infection (53). However, in our studies, the normal response of cells expressing IE2 alone in terms of IL-8 induction and activation of NF-κB following cytokine treatment, combined with the ability of foscarnet to reverse the inhibitory effect of HCMV on these signaling pathways, indicates that cells expressing IE2 in the absence of E/L genes have intact responses to IL-1β and TNF-α.
The similarity between the inhibitory effects of HCMV on IL-1β- and TNF-α-induced inflammatory chemokine expression and NF-κB nuclear translocation suggests that the inhibition of chemokine induction results, at least in part, from inhibition of NF-κB activation. However, the comparable suppression levels of IL-1β- and TNF-α-induced inflammatory chemokine expression by HCMV infection (Fig. 1) were surprising given the difference in the level of inhibition of HCMV on IL-1β induction compared to that of TNF-α induction of NF-κB nuclear translocation (Fig. 4 and 5). In addition to NF-κB activation, MAPKs have been shown to be critical for maximal induction of chemokine expression via IL-1 and TNF-α pathways (24, 33, 51). Consequently, the comparable inhibitory effects of HCMV on chemokine induction via the IL-1 and TNF-α pathways may result from additional viral effects on MAPK activity. The effects of HCMV on MAPK activation via IL-1 and TNF-α signaling pathways are unclear. In studies investigating the effect of HCMV infection on basal activity of MAPKs (in the absence of proinflammatory cytokine stimulation), infection has been shown to induce activation of both extracellular signal-regulated kinase (43) and p38 (29). In contrast, a recent study identified a biphasic effect of HCMV on expression of the inflammatory chemokine IL-6, with induction at early times of infection being followed by suppression at later times. This suppression corresponded to a decrease in the stability of IL-6 chemokine mRNA transcript (18), which is regulated by p38 MAPK (17). The inhibitory effect of HCMV on TNF-α signal transduction, which was correlated to TNFR1 down-regulation, corresponded to a block in JNK activation (3).
The difference between the level of chemokine suppression and inhibition of NF-κB nuclear translocation following TNF-α treatment may, alternatively, indicate additional levels of HCMV-mediated modulation of NF-κB activity. Recent studies have shown that NF-κB activity is regulated at multiple levels, with IκBα-mediated cytoplasmic localization of NF-κB representing only one tier of regulation (14, 44, 46, 60). Direct phosphorylation of the NF-κB molecule at multiple sites has been shown to both positively and negatively regulate NF-κB activity. Phosphorylation of NF-κB is mediated by a variety of kinases (54), which could serve as potential targets for additional HCMV-mediated modulation of the pathway.
The apparent differential effect on the IL-1 signaling pathway compared to that on the TNF-α signaling pathway is intriguing. This differential effect may reflect the proapoptotic signaling associated with TNF-α, but not IL-1, signal transduction pathways in the absence of NF-κB activation. Recently, the requirement for NF-κB activity was shown to result from NF-κB-mediated induction of antiapoptotic molecules that regulate cell survival following activation of the TNF-α signaling pathway (39). TNFR1 engagement was shown to result in the formation of two distinct complexes: a prosurvival complex (complex I) and a proapoptotic complex (complex II or death-inducing signaling complex). Complex I formed rapidly following receptor engagement and resulted in NF-κB activation. In contrast, complex II assembled more slowly and resulted in apoptosis. Importantly, NF-κB activity was shown to be required for the induction of antiapoptotic molecules, primarily FLIPL, which modified the function of complex II and prevented cell death. Hence, in our study, the intermediate effect of HCMV on NF-κB activity following TNF-α may reflect the requirement for a necessary level of NF-κB induction of antiapoptotic molecules to prevent cell death. In contrast, since apoptotic signals are not associated with IL-1 signaling pathways, a complete blockade of NF-κB activation can be induced without any negative apoptotic ramifications. In both cases, the virus has evolved to maximize the level of NF-κB suppression within the constraints of a requirement to avoid induction of apoptosis.
In the current study, the inhibitory effect of HCMV on IL-1 and TNF-α signaling pathways corresponded to an inhibition of NF-κB activation. This finding was surprising given the numerous studies showing NF-κB activation following HCMV infection. These previous studies have focused primarily on early times of infection and led to the development of a model wherein HCMV mediates a biphasic activation of the NF-κB pathway (58). The first phase occurs during virus entry (12, 56, 57) and is mediated by the major viral envelope glycoproteins gB and gH (56, 57). The second phase requires protein synthesis and is mediated by induction of de novo NF-κB synthesis (32, 58). Our current study indicates a further complexity in HCMV-mediated modulation of components of the IL-1 and TNF-α signaling pathways, with an importance for HCMV down-modulation of these pathways at later times of infection. The requirement for NF-κB inhibition at later stages of the HCMV life cycle is probably multifactorial but may be important for functions such as immune evasion and viral persistence. However, given the central role of NF-κB in many cellular processes, such as inflammation, immune regulation, differentiation, and apoptosis (19, 24), this HCMV-mediated inhibition of NF-κB may also be responsible for various aspects of viral pathology, including developmental and immune dysregulation and tumorigenesis.
We are grateful to Sherry Farley and Veselina Korcheva from the B.E.M. laboratory for technical support and Andrew Townsend at Extreme Images for assistance with graphics illustrations.
This work was supported through funding from the NIH to J.W. and B.E.M. (A1059335), M.A.J. and J.A.N. (AI21640), and W.J.B. (AI35602).
- Received 9 January 2006.
- Accepted 15 March 2006.
- Copyright © 2006 American Society for Microbiology


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If you have any problems, feel free to reply that you think didn't work. I'd love an open ended 3 to 5 mission additional story. But we prefer the stealthy nature of this game, that is why we will be playing as stealthily as possible and beat the story. Please note this, our Download is secured by a gateway to prevent bots and spammers from downloading our tool and sharing it everywhere. Unleash the power of the most lethal agent to ever exist. But the stealth is well balanced in this new game. Some of the typical scenarios that we see in Blacklist were completely absent in the previous version which left every fan of the series disappointed.
Ubisoft today released the Homeland downloadable content for , bringing new co-op missions, weapons, and gadgets to the action game. This was such a disappointing outcome for the stealth fans who loved the series previously. This has to be one of the best stealth games. This combines both stealth and action elements to perfection. I'd also love added missions to Grim, Charlie, and Kobin. With the introduction of plenty of ducts to crawl through and plenty of pipes to hand on to you will be moving unnoticed all the time if you prefer. Note : This download protected by small survey to prevent from Spams and Bots.Next
So at this time we are offering 1650 downloads only. Splinter Cell: Blacklist launched in August. If you fail, millions will die. There are two sections of Paladin, med bay and cargo bay, that have nothing in them but you can still go in them. Unleash the power or the most lethal agent to ever exist. When you have your code, redeem it in the Steam Game Stores.Next
Your Splinter Cell Blacklist Game download will start immediately. If you prefer the all-guns-blazing kind of action then you can play that way. After you generate the Redeem code you can visit Playstation store and Redeem it. Painkillers makes my non-lethal Ghost Style approach perfect. There are new aesthetic options too, like new Mercs and Spy skins, and gold, amber and white goggles for players who just want to mix things up a bit.Next
When you have your code, redeem it in the PlayStation Store. It doesn't force you into playing a specific way, and it provides all the tools necessary to accommodate your playstyle, retaining the agility of its predecessor without its restrictions. The Dead Coast and Billionaire's Yacht cooperative maps -- which came in various pre-order and special edition forms at launch -- give you new spaces to use a sleeping gas-only crossbow. New products, exclusive collectors editions and amazing promotions all year round: only the best from Ubisoft! Open your Xbox 360 and enter the code in the Xbox Live marketplace. Also, the way the campaign ends, and the team conversations after, seem to indicate there could easily be more. About This Content As leader of the newly formed 4th Echelon, Sam Fisher must hunt down merciless terrorists before The Blacklist countdown reaches zero. I know there were some bonus 4E missions if you preordered, sure they'll be made available for purchase eventually.Next
New goggle colors -- gold, white, and amber -- also come alongside a new multiplayer skin for your spy and merc. Ghost Recon got one with Raven Strike. You've been given the ultimate freedom to protect innocents against the Blacklist terror attacks known as -- the freedom to use limitless force, to break every law, and to become the globe's deadliest operative. I think the new 4E missions should be like double agent. When you are revived your code, write it down on a piece of paper or some place. Sam Fisher is the leader of the newly formed 4th Echelon unit: a clandestine unit that answers solely to the President of the United States. With season passes, merchandise and apparel, enjoy the complete gaming experience.Next
When you click download now button just complete simple quick survey to unlock your download. Sam and his team must hunt down these terrorists by any means necessary, and stop the Blacklist countdown before it reaches zero. Tom Clancy's, Splinter Cell, Splinter Cell Blacklist, Sam Fisher, the Soldier Icon, Ubisoft and the Ubisoft logo are trademarks of Ubisoft Entertainment in the U. After doing the correct step, you will have completed the process. I really have a good feeling that there will be more co op missions including Victor character. I don't think you can wait around too long. You have many different ways in which you can complete any given mission.Next
But what I really wants is more. The second co-op mission is called Dead Coast and has players taking out a terrorist cell in a remote part of the Middle East. Gold, amber, and white goggles and new skins for the game's Spies vs. Com The United States has a military presence in two thirds of countries around the world. A terror group known as the Blacklist is threatening the United States.Next


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Since the election of the Conservative-Liberal Democrat coalition in May the political debate has centred around their determination to reduce the deficit – and to do so in the main by cutting what George Osbourne describes as "wasteful spending". Of course, the collective left are having a field day, with multiple campaigns against the cuts though Labour (if you still recognise them as a party of the left) recognise the need for some cuts. I guess I'm somewhere in the middle (which means in this post I'm going to piss off both left and right and lose any friends I've made in the last few weeks).
The reason I'm writing this post is my reaction to this piece by Caron. Basically, she has reacted rather angrily to the way the Daily Mail has covered this story. Granted, it doesn't really take much to get angry at a Daily Mail article, but I'm not as annoyed by it as Caron is. Indeed, I'm more annoyed at the subject of the article.
As background (and there may well be more to this story than has been made public – especially since it is a Daily Mail story!) the couple in question have recently had twins (born early September) which brings their family to six children. Neither parent works and they live in a small council house – and have "demanded" to use the Daily Mail's terminology a larger (council) home. Caron argues – not entirely unjustifiably – that the Mail should be emphasising the need for better and more affordable housing rather than criticising them for not working and naming their children after celebrities. I'm not immune to this at all.
But I do think we need a little perspective as well – and this is where the "cuts" debate above comes into play. Having debated with Caron on the issue, its clear that she thinks if we don't help the children in this situation we are actively punishing them for their parents' choices. My argument was and is that there is a distinction to be made between actively providing support (and thereby helping the children) and withholding support (which would not help them but equally we wouldn't actively be hurting them). Absolutely, it isn't the kids fault their parents are irresponsible. But it also isn't the taxpayers' responsibility to play parent in their place. Obviously, its a tough situation, and I have sympathy with them. But they made choices to have 6 children knowing well they could not afford them.
As a young, recently-wed, (unfunded!) postgrad I well understand the pressure we're under to try to buy a property. But there's no way I could afford it at the moment. So I rent. And I have done for three years. And the flat is one bed and a boxroom – its not like I'm a rich brat living in a four-bed mansion here. And because I'm still a student, we cut back – we don't do all the things we'd like to do, we buy only the shopping we need and cut out the luxuries. We did, however, get a cat. After a lot of considering whether we could afford it, eventually we decided we could. Granted, a cat is not as costly as a baby (or 6) but that's not really the point. We realise our limits and have made decisions based on them. In short, I like to think we've been responsible.
Now, obviously my situation is different – and perhaps I've been given better opportunities than the parents (and definitely the children) in the case highlighted above. Â I'm not immune to that argument. Â All I'm saying is that 6 kids when you don't have a job just isn't responsible. And I'm just not convinced that the state should pick up the slack – I wouldn't expect them to if it was me.
Maybe it is unfair to pick on this family (especially the way the Daily Mail has). But maybe its time for some tough love. Let's support these people, but do it in a way that isn't simply a hand-out for people who don't have a job but still think they can support a family of six because the government will pay for them. Let's help them make the right choices, be more responsible with their decision-making (and family planning!) and let's not just give them state handouts when they want a bigger house.
For me, when you hear people moaning about folk scrounging off benefits when "I'm out working my fingers to the bone" this is why. It's why people are apathetic about politics and disengaged with politicians. Parties continually go into elections with pledges to "get Britain working", to get people off benefits and into jobs, and yet when it comes to it, the jobs just aren't there.
I couldn't agree more with what Simon Heffer says here: "welfare should be a safety net, and not a way of life." In the meantime, let's not vilify as "right-wing" those who have recognised this and are trying to change it.  Otherwise we'll be forever stuck with a system which seems to suggest that if you want a bigger house just have six kids and the government will pay for it.


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Explore 15+ million radio shows, DJ mixes and podcasts made by passionate creators.
30 Apr 2015
This upload features tracks from Nick Jagger, Funky Truckerz and more.
This week, PaKo played AN ALL HOUSE CLASSIC SET! 45 Minutes of well known huge House tunes! And beside that, of course a lot of new stuff in the Featu...
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Matthew Currie Holmes (1974 - )
Film DeathsEdit
- The Fog (2005) [Sean Castle]: He is killed when a ghost throws a knife at him which enters his brain through the eye. We see him later in a berth looking all grey and with eyes disfigured when Tom Welling discovers him.
- Wrong Turn 2: Dead End (2007) [Michael, referred to as M] Tethered to a chair and decapitated with a large meat cleaver by Ashlea Earl. (Thanks to Brian).
TV DeathsEdit
- Dead Like Me: Pilot (2003) [Brandon Marshall Moore]: Neck snapped or skull fractured when he slips and falls into a revolving door just as a rescue worker runs into the bank, causing the door to close on his head.
Community content is available under CC-BY-SA unless otherwise noted.


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It's summer here. It's hot. It's time for cold brew.
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We've got a super simple cold brew coffee recipe that you can make. Successfully. In the comfort of your own home.
Here's what you need:
The amounts of water and coffee suggested here makes for a ratio of 1:4 (1 part coffee to 4 parts water), which means you're making a concentrate. For reference, an espresso is usually 1:2 and a filter coffee is 1:16.
Here's what you do:
Here's some drinks inspo:
So you gonna give this a try?
Or do you already have a cold brew lab in your kitchen?
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✌❤☕ - Holly
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text: KeelansGlove 0 Posted August 24, 2010 I really don''t think some of you deserve a club to support.What would you have said if we sold Holt and Hoolahan and didnt replace them ? The money has been invested in the extra seats because casual fans were moaning they couldn''t get in, they need to be paid for.If we do not sell out every game so what , we are in the company of most other clubs in the country. reduce the prices and that is a slap in the faces of those that made a commitment for the season making season tickets poorer value for money.As I have explained elsewhere I bought my season tickets knowing I couldn''t get to every game but not wanting to be in the situation I was last year (first year for 20 years without a season ticket and struggling to get a ticket for some matches)You have three choices, buy a season ticket , pay the going price for a ticket , go and do something else instead.If we were charging QPR prices I could understand. Get over it Share this post Link to post Share on other sites


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masanobu ando wife
About Us
Her name is Misako Honda and she used to work for preschool as a teach after graduating from high school until she got married to Keisuke. They trace the assassination order back to Yamaoka (Yasuko Matsuyuki), who is willing to hand them over the killers. He is an actor, known for Battle Royale (2000), Kizzu ritân (1996) and Sukiyaki Western Django (2007). His career took off in Takeshi Kitano's Kid's Return and he became infamous even more in Kinji Fukasaku's Battle Royale. Help us build our profile of Masanobu Andô! This November, Tortoise is thrilled to welcome back the celebrated Japanese ceramist, Masanobu Ando. Soccer Player Keisuke Honda's Beautiful Wife|Photos and 4 Facts About Her . Tweet. Masanobu Andô, Actor: Batoru rowaiaru. Masanobu Ando travels a lot, so it is particularly difficult to sustain stable relationships. He has been married to unknown since 2014. Contribute. The place I'm writing about is Gallerie Momogusa – the gallery and café of Masanobu Ando and Akiko Ando, his wife, propped in the middle of lush green woods in Tajimi, Japan. For his second film, in 1996, he won the Film Academy of Japan's Best New Actor Award, starring in Takeshi Kitano's Kids Return.
Our last exhibition with Ando was in September 2014.
chinese tea ceremony with ceramicist and japanese tea master, masanobu ando.
Emma Tsuchida; October 19, 2018; Saturday November 24th and Sunday November 25th 11:00am-12:30pm and 2:00pm-3:30pm. Fact 2) Keisuke and Misako were Highschool Sweethearts .
Masanobu Ando (安藤 政信, Andō Masanobu, born May 19, 1975) is a Japanese actor.. Ando has appeared in films such as Takeshi Kitano's Kids Return (1996), Kinji Fukasaku's Battle Royale (2000), Takashi Miike's Sukiyaki Western Django (2007), and Chen Kaige's Forever Enthralled (2008). Login to add information, pictures and relationships, join in discussions and get credit for your contributions.
Masanobu Ando, was once a popular young actor in the late 90's, especially after Kids Return (1996) and Battle Royale (2000). And? One of the dead guys is a certain yakuza boss who has been killed by the infamous assassins Vertebrae (Masanobu Ando) and Viscera (Ryushin Tei). Has Masanobu Ando got married? Masanobu Ando Japanese actor, born in May 19 1975. In 1988, he remodeled an original Japanese ceremonial teahouse into a gallery called Gallerie Momogusa. Following this period, he channeled his influence derived from Zen culture into his works and began creating functional objects for daily life. Kirin ga Kuru (麒麟がくる, Awaiting Kirin) is a 2020 Japanese historical drama television series starring Hiroki Hasegawa as Akechi Mitsuhide, a samurai and general during the Sengoku period.The series is the 59th NHK taiga drama, premiering in January 19, 2020.
He continues to be married to unidentified since 2014. Weddings are normally certainly one of the main causes for the conflicts that arise in a couple. Masanobu And? It is the first regular taiga drama to be fully shot in 4K resolution. They will have one child.
This can be complicated to know given the secrecy of his private life.
They have one child. Masanobu Ando; Chiaki Kuriyama; Riki Takeuchi; Novel Manga Manga Sequel ... (it's implied the government chased her father thus he divorced his wife to protect Mitsuko). Pocket.
We are the first tumblr dedicated to making gifs of Masanobu Ando! This was the setting that welcomed us as we entered a very special day in the life of the lauded ceramic artisan.
Masanobu Ando, born 1957, started his career creating contemporary art and studying Buddhism. Masanobu Andô is a member of the following lists: 1975 births, Japanese film actors and Japanese television actors.. But slowly after that, Masanobu seemed to "disappear".


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USFDA issues 2 observations after inspection of Lupin's Pithampur unit
The inspection of the company's Pithampur Unit-I facility by the USFDA has been completed.
The inspection of the company's Pithampur Unit-I facility by the USFDA has been completed.
In a regulatory filing to BSE, Ipca Laboratories said the USFDA had conducted the inspection of the its formulations manufacturing unit situated at Piparia (Silvassa) from August 19 - 23.
Zydus Cadila has received the final approval from the United States Food and Drug Administration to market Chlorthalidone tablets.
Paleja has informed the company's board of his decision to resign as the vice chairman and managing director effective May 31, 2019 for embracing challenges outside of Novartis, the company said in a regulatory filing.
The company's wholly-owned subsidiary has entered into a stock purchase agreement to acquire 534,194 shares of common stock of biotechnology firm Inipharm Inc USA, for a cash consideration of USD 534.20 per share, Jubilant Life Sciences said in a filing to BSE.
The company had posted a net profit of Rs 204 crore for the corresponding period of the previous fiscal, Torrent Pharmaceuticals said in a statement.
The company said it had submitted a detailed compliance report to the US Food and Drug Administration (USFDA) within the stipulated period in response to the Form 483 containing eight observations issued to it by the regulator.
The inspection at Pithampur was conducted by the FDA from July 23, to July 27, 2018 and the inspection at Roha was conducted by the FDA from July 30, to August 3, 2018, it added.
The tablets are generic versions of Bristol-Myers Squibb Company's Glucophage tablets in the same strengths, it added.
Suven Life said it has been granted "one product patent from the US corresponding to the new chemical entities (NCEs) for the treatment of disorders associated with neuro-degenerative diseases".
The company said its board has appointed Gopal Daptari as additional independent director with immediate effect up to the conclusion of the ensuing annual general meeting.
The Bengaluru-based firm had inked a pact to acquire 51 per cent stake in Australia's Generic Partners Holdings Co for AUD 15 million and a commitment of AUD 10 million to fund future R&D.
The company had posted a net profit of Rs 14.34 crore during the same quarter of the previous fiscal as it was hit by a one-off item of Rs 1,513 crore paid for settlement of a legal dispute.
Natco Pharma today said its active pharmaceuticals ingredient plant at Chennai has suspended operations due to flooding caused by rains.
"We plan to invest Rs 200 crore, excluding the land cost, to open two plants by middle of 2016. One of them will be in Sikkim and the other one at Behror in Rajasthan," Mankind Pharma Managing Director Sheetal Arora told .
Drug firm Sanofi India has sold its commercial property, land along with a building known as 'Aventis House' in Mumbai, for Rs 111 crore.
Drug firm Vivimed Labs has signed a pact for sale of some of products in its speciality chemicals division to Clariant Chemicals India.
It added: "The fund raising is subject to shareholders' approval and such other statutory approvals as may be required including that of Foreign Investment Promotion Board."