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The all-new BMW 8 Series appeared in its coupe avatar in the middle of 2018, and a few months down the line it is time for the family to grow. BMW has used the 8 Series name in the 1980s and 90s, but this cannot really be called a successor to that car. Still, if you want to, call it a second generation! The 8 Series convertible has also since arrived, and a 4-door Gran Coupe bodystyle will complete the family when it gets here by mid-2019.
WATCH: BMW 8 Series Convertible Review
The new 8 Series is nearly identical to the 6 Series in dimensions, but is tad wider and shorter in height & length
But let's stay focussed on the new 8 Series convertible shall we? The 8 Series effectively replaces the previous 6 Series family, but is being positioned as more premium and sporty. This is largely true, as you shall find out. The car is 4851 mm long, with a 2822 mm wheelbase, 1902 mm wide and 1345 mm high. That makes it near identical in dimensions to the previous 6 Series convertible. It's a tad wider, but is shorter in height and length, as well as wheelbase. But it is obviously more luxurious and better appointed. And looks sexier too.
Also Read: BMW Begins Production Of 8 Series Coupe
The grille is large and looks menacing while the headlamps are the slimmest in BMW's line-up
The car looks great to be honest. Low slung form, great proportions, and rather obvious width - especially in how the rear end has been styled. The hood and flanks are very muscular, and there are a couple of prominent character lines that emphasise its length, along the side. The grille is a bit large and wide, yet very well executed and kind of menacing. The headlights are the slimmest in BMW's line-up, and can be upgraded to Laserlight tech if you pay more.
The 8 Series Convertible misses out on gesture control but gains the BMW voice assistant instead
Material quality, equipment and yes even the gadgets are a step up for sure. Virtual cluster, head up display, bigger touchscreen with the latest iDrive 7.0 and the latest in connectivity and sound systems. Harmon Kardon is standard; Bowers & Wilkins diamond surround is an option. The convertible does not have gesture control (the hard tops have it), but does get the BMW personal assistant voice-controlled functionality. The start stop button, gear shifter and iDrive controller are finished in crystal glass like we saw on the X5 and X7. But this is the first non-X model to get what BMW calls 'crafted clarity'. The dash and door panels are all wrapped in leather. The car certainly exudes quality and class.
Also Read: BMW 8 Series Coupe: All You Need To Know
The wind deflector can be manually popped on or off over the rear seats, while roof folds up or down in just 15 seconds
There is a wind deflector that comes standard which can be manually popped on or off over the rear seats. The roof folds up or down in just 15 seconds, and can be operated at up to 50 kmph. The previous 6 convertible did it in 19 seconds and up to 40 kmph for what it's worth.
While the car is not then a 7 series based model, it does share its drivetrains. The M850i xDrive has the 520 bhp V8 petrol and the 3-litre, 314 bhp in-line six diesel that powers the 840d xDrive. Now BMW could bring us the convertible as a direct import in really small numbers. But you know what? It would also have to import in the wonderful clean air and the beautiful blue sky I've been lucky to have on my drive! That is why it is going to be the hard top that comes to us, and more likely not the coupe, but the Gran Coupe.
Yes it is likely we will only get the 8 Series Gran Coupe. But that car will only be revealed around June or July this year. So you might wonder why I am testing the convertible then? Well, three reasons why. First, because the engines we get will be shared across bodystyles, and so will be the same. Second. It is a chance to see where BMW has taken the 8 Series family, after all we never did drive the coupe as yet. And third - even though we may get a different bodystyle, a lot of the dynamics, performance and design elements will be similar, so it will still give us a good idea on what to expect. Satisfied? Then read on!
BMW is going through a major new vehicle offensive. New models, first time models and upgrades galore. While 2018 saw a lot of that action stay on the X or SUV side of things, 2019 seems to be about the sedans and sports cars. And the 8 Series, while not a sports car like the new Z4 is certainly very sporty and a lot of fun. Almost surprisingly so! And I have to say, having the powerful M850i with me today has been nothing short of fun! The car uses BMW's latest V8 petrol motor with 68 bhp more power than its predecessor. And peak torque is bumped up a massive 100 Nm to 750 Newton Metres. Two twin-scroll turbos on this V8 engine by the way. The first thing that strikes you when you start the engine and begin your drive is how quickly the power reaches the wheels. So despite its size this thing can move!
BMW has certainly made a huge effort to really come back to the centre of what the brand was always all about. Yes it seemed to have lost its way a few years ago, but like I have said, its come roaring back, and is once again making cars that are great to drive. The good part though is that they don't knock you around, they are not that stiff anymore, so while you get all the dynamics that you want, you still get a fair amount of comfort, and an obvious sense of luxury. And especially for a car like this, that is important.
So yes the 8 series betters the performance seen on the previous 6 Series by miles. The car belies its size and is quick, adept and very happy to be pushed through sharp corners and yet maintains superb control. The handling on this car is easily right up there with the best, and the steering is on point. The 8 Series gets active steering and all-wheel drive or xDrive as standard. The car's athleticism comes from there too. Active steering means the rear wheels also turn (either with or against the front wheels depending on the situation or speed) and give you the sense of driving a more compact car. The all-wheel drive system works well, as does the 8-Speed Steptronic gearbox - that is also common to both engine types.
The acoustic levels are amazing despite being a top down. You can hear the music very well and talk to the person next to you
Now it's an amazing kind of dynamics that the engineers these days have been able to achieve, the car's acoustic levels inside, even with the top down are amazing, you can hear the music really well, you can talk to the person next to you, take a phone call on the system as well, and in my case - does not mess up your hair way too much. That's important!
The car is fun to drive for sure, and can take a fair amount of punishment. And that leads me to wonder, no salivate, at the prospect of the M8 that is also expected later this year. Yes, more so than the Gran Coupe even though from a market point of view I should care about that more! On the downside the cabin does feel a touch cramped, especially at the back. And given it's a new model that is not the next generation of a 6 Series model line - and is also meant to rival the likes of the Mercedes-Benz S-Cabriolet, it could have been a bit larger on the inside. But in every other aspect the car certainly impresses. And that is why the prospect of India getting the longer wheelbase Gran Coupe then, works. Expect that to only happen around the end of this year, or perhaps February 2020.
For the latest auto news and reviews, follow carandbike on Twitter, Facebook, and subscribe to our YouTube channel.


text:
Story From The Field
Teacher Space: A Forum for Informal Feedback
Build an online community for giving and receiving video-based feedback.
How To Guide
Teacher Space: A Forum for Informal Feedback
Based on Ideas by
Missy C, Samantha S, Robert W
Build an online community for giving and receiving video-based feedback.
What You'll Need
A video recording device, like a Smartphone. A computer with Internet connection. Also, a YouTube account (free); Access to Google Hangouts, or other online meeting platform
How To
Form a Teacher Space group.Recruit participants from within your school or professional networks.Keeping a group to three to five teachers will ensure that everyone gets feedback every few weeks.If there's enough interest to form multiple groups consider organizing them around different interests(e.g.by subject matter,grade level,or instructional focus,such as how to check for student understanding.)When recruiting,explain how each group will work by summarizing the steps that follow.After testing the Teacher Space feedback process with individual teachers,Swanson recruited five participants from the 30 elementary math teachers she coaches in her role at the Academy for Urban School Leadership(AUSL).She says,"As a coach,I will go from one school to another in the same day,and actually be helping different teachers on the same thing.This is a way to connect teachers who might not otherwise be connected,but who might really be working on similar things."
Norm the group's expectations for feedback. Based on her review of best practices literature, Swanson drafted this short set of guidelines for giving feedback: Focus on behaviors, not on people. Be specific. Use "I" statements, not "you" statements. State observations, not interpretations. Remember the 3 Cs: Caring: Be kind in your comments; Content: Base comments on teacher's specific questions and objectives; and Conventions: Try to make your language as clear and easy to read as possible. Make sure feedback is actionable. With just these guidelines, however, Swanson found that participants varied widely in the tone and style of their feedback. She recommends each group kick off with a discussion—in person, or via Google Hangout or similar—to reach consensus about the type of feedback they want (e.g. do they want questions that prompt them to further reflection, or just specific suggestions.) She also says participants should use this discussion to share any context that may be important for others to know when providing them feedback. This may include the type of community the school serves, if the teacher is using a particular curriculum, and the teachers' past experience. Explains Swanson: "Once you make that connection with others in your group you want to come back and continue the conversation."
Record, review, and upload a video of teaching. A member of the group captures part of a lesson that's relevant to the area of practice he or she wants to improve. Keep it to no more than 10 minutes. Before uploading the video to a YouTube channel shared by the group, the teacher reviews the video and prepares an open-ended question (not yes/no) for the viewers to guide their feedback. Along with a question, the teacher includes any brief context essential to understand what's shown. Also helpful are timestamps, notes about the exact time in in video when something relevant occurs (Timestamps added at the front of comments in YouTube let a viewer skip right to that moment). Says Swanson: "The teacher's going through the first round of learning by observing themselves." For an early test of Swanson's protocol, Kindergarten teacher Danielle Brown recorded herself leading a small group of students in playing a game to learn addition and subtraction. It was her first time using the game, and before trying it again she wanted feedback on how she communicated the game's purpose and instructions.
Comment on the video, within one week. Using their agreed-upon norms for feedback, the other members of the group review the video and use YouTube's commenting feature to address the teachers' question. Comments should include timestamps that connect them to the specific moments in the video related to the viewers' observations. To be helpful, feedback should be provided within a few days of the lesson featured. Says Swanson: "Any more than that and a teacher is in a different unit, they've already figured it out, or decided to put it aside." The teacher whose video is featured can reply to specific comments for clarifications and additional information. In Swanson's model, a group meets again at the end of a cycle—in-person or virtually—to discuss the feedback and plan the next cycle.
Repeat steps 3 and 4 for the other group members, until everyone gets feedback. Then begin a new cycle.

subset: commoncrawl


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The Media and Local Communities - Supplementary Submission
Written response to a question coming out of FRIENDS' presentation to the House of Commons Standing Committee on Canadian Heritage on the future of local TV in Canada.
Mr. Jean-François Lafleur
Standing Committee on Canadian Heritage
House of Commons
131 Queen Street, 6th Floor
Ottawa, ON
Dear Mr. Lafleur:
During FRIENDS' appearance before your Committee on April 12, we undertook to respond to a question posed by Mr. Maguire in writing, as there was no time available for an oral response on that occasion.
Mr. Maguire asked: "Some of the local television news is in pretty good shape south of the border, and I'm just wondering if there's anything we can learn from the Americans to strengthen local TV news that we have here?"
FRIENDS' response:
Support Measures for Local TV in the United States
Over the last seven decades, the U.S. has adopted a robust set of mechanisms that support local TV broadcasters. The extent of support far exceeds anything adopted by Canada. These measures include:
Protection of program rights purchased by local broadcasters
Both cable and satellite companies are required to "black out" programming imported into local markets that has been purchased by the local broadcaster. In Canada, simultaneous substitution or "simulcast" offers only limited protection against identical programming imported into a local market at the same time.
"Distant signals" forbidden
DTH satellite companies in the U.S. are all but forbidden to import distant signals due to the negative impact on local broadcasters.1 In Canada, DTH and cable providers are free, for example, to beam Vancouver TV station signals into the homes of Toronto viewers, thereby fragmenting the available audience for local broadcasters even further, and without compensation.
Paying for local signals
"Retransmission consent" allows local TV stations in the U.S. to negotiate compensation for their carriage by cable operators. Over two decades, this has evolved into cash payments to local broadcasters estimated currently at $2.5 billion per annum.2 In Canada, a variation on this approach was proposed by the CRTC in 2010, but found to be ultra vires by the Federal Court of Appeal,3 which suggests that a Broadcasting Act amendment would be required for implementation.
Compensation for expropriation
Local broadcasters in the U.S. are to be reimbursed from a $1.75 billion fund established by Congress for the cost of relocating their signals4 when 600 MHz broadcast spectrum is repurposed for mobile services starting in 2017.5 In Canada, no compensation has thus far been offered to local TV broadcasters, who face similar, if not greater incremental costs than their American counterparts.
U.S. local TV also benefits from a much higher share of total TV advertising (43% of the TV ad 'pie' vs. 20% in Canada). U.S. local TV stations have continued to grow advertising revenues – from $15.8 billion in 2009 to $20.8 billion in 2014 – and are forecast to continue to grow advertising revenues through to 2019 (to $23.2 billion).6
Because of this much more supportive public policy framework, U.S. local TV stations are in far better financial health, and better equipped than their Canadian counterparts to compete with new Internet-based media.
• • •
National Content – International Comparisons
We also wish to provide further information in response to a question from Mr. O'Regan: *"**Let me ask you and give you an opportunity maybe to address best practices. When you talk about Canadian content and the promotion or protection of Canadian content what are some of the better practices that you've seen in comparable economies, obviously outside of the United States, but I'm thinking more of European countries and Australia in terms of the amount of national content and how they go about their cultural industries through demanding carriers, whether they be foreign carriers that broadcast within or domestic carriers, what percentages do they ask be of local content or national content, but domestic content?*
There was insufficient time available to respond fully to his question last week. We would like to share with the Committee a graphic originally produced by CBC/SRC two years ago, based on 2012 data. It is possible that CBC/SRC has more up-to-date information but, in our view, these data respond in broad terms to Mr. O'Regan's question:
If we can be helpful to the Committee in other ways during your media and local communities study, please do not hesitate to contact us.
Yours sincerely,
Ian Morrison
1 See, For example, http://www.fcc.gov/guides/television-broadcast-stations-satellite
2 Industry estimate.
3 Reference Re Broadcasting Act, 2011 FCA 64, [2012] 4 F.C.R. 231. http://reports.fja-cmf.gc.ca/eng/2012/2011fca64.html
4 Authorized under the 2012 Spectrum Act
5 See Decision on Repurposing the 600 MHz Band, August 2015, http://www.ic.gc.ca/eic/site/smt-gst.nsf/eng/sf11049.html
The U.S. also implemented an incentive auction, designed to facilitate the repurposing of spectrum by encouraging OTA TV broadcasters to voluntarily relinquish some or all of their spectrum usage rights in exchange for proceeds from an auction of new mobile licences. Broadcast Incentive Auction 101, Federal Communications Commission,
6 Source: BIA/Kelsey Consulting
Apr 12, 2016 — Policy Brief: The Media and Local Communities FRIENDS presentation to the House of Commons Standing Committee on Canadian Heritage on the future of local TV in Canada.


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Dear participants of the 42nd Chess Olympiad and the 87th FIDE Congress!
The organizers have made it possible for you to obtain electronic visas to enter Azerbaijan.
The procedure is very convenient for participants and organizers recommend that you follow it strictly.
Participants are asked to send to the organizers:
- a color scan of their passport
- an identity picture, in color, on white background and clearly visible face
- a filled-out visa application form: the form is available in the Olympiad's registration system; the participants also have the possibility to send it directly to the visas department: firstname.lastname@example.com The application form was sent to all Federations by e-mail.
If you have difficulties with the form or any questions related to visas, please address firstname.lastname@example.com
Ready electronic visas will be sent to Federations and participants via mail.
Participants have to show electronic visas at the time of registration for the flight to Baku and then at Passport Control Points at the Baku Airport.


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2Pint Software – The Home Of Microsoft P2P Extensions › Forums › Public Forums › All About Peer To Peer › Is IPv6 required to be enabled for BR Reporter?
Todd MillerGuestAugust 26, 2016 at 1:43 pmPost count: 65#16386 |
I have installed BC Reporter on one client and deployed a BC enabled advertisement to that client. Upon receipt of the package at the client, there was an error displayed on the machine involving BITSBCReporter.exe. The error was recoreded in the system's application event log. In my org, we have IPv6 disabked on the clients by setting TCPIP6/parameters/Disabledcomponents set to 0xffffffff Is IPv6 required to be enabled for the reporting tool to work without error?
The text of the entry for BITSBCReporter.exe is below.
Application: BITSBCReporter.exe
Framework Version: v4.0.30319
Description: The process was terminated due to an unhandled exception.
Exception Info: System.Net.NetworkInformation.NetworkInformationException
at System.Net.NetworkInformation.SystemIPInterfaceProperties.GetIPv6Properties()
Phil WilcockKeymasterAugust 26, 2016 at 1:53 pmPost count: 25
hi Todd,
NO need for IP6.
it's a known issue, fixed in the next release which is due any day now.
The workaround is to create a reg key – HKLM:\Software\2Pint Software\BCReporter
and a DWORD value named IncludePolicyStats which should be set to 0
Todd MillerGuestAugust 26, 2016 at 4:46 pmPost count: 65
When I looked, that value was already present in the registry and set to 0. Maybe the code sets it this way on its own if an error is encountered?
I have a second problem where the logs are hanging while attempting to capture the MAC address, but I will open another thread as it is a separate issue I think.
Andreas HammarskjöldKeymasterAugust 26, 2016 at 5:58 pmPost count: 64
I think a debug log would be a good start. That version of the Reporter had a few known issues. Or hold off until tomorrow (or whenever Phil can get his updated reports finalized).
- You must be logged in to reply to this topic.


text:
The development of data centres is a key part of the government's strategy for attracting foreign direct investment to Ireland.
They believe they raise our "visibility" internationally as a tech hub while also bringing economic benefits in the form of jobs, both in construction and after completion.
There are drawbacks, however, with data centres creating enormous pressures on the power grid at a time when Ireland is already struggling to meet targets on climate change and greenhouse gas emissions.
The scale of power usage can be extraordinary - a proposed development by Amazon at Mulhuddart in Dublin will consume 4.4% of the state's entire energy capacity.
The focus on creating more construction jobs may also be a double-edged sword as building inflation contributes to enormous cost overruns on projects like the National Children's Hospital.
In other countries, conditions for building data centres are more onerous and in the United States, some cities that fought hard for these developments have come to realise the economic dividends are not quite so extensive as they believed.
We want to look deeper into an issue that divides opinions sharply.
On one side, opponents of data centres point to the meager amount of jobs left once construction is finished, the drain on energy supply, and Ireland's already significant difficulties in meeting climate change targets.
On the other, supporters believe data centres bring not only jobs involved in their development, but also wider job creation from technology giants.
We want to look behind the scenes at government level and the IDA to see exactly what Ireland's strategy is and whether it is changing amid environmental concerns.
We also want to examine the experience of other countries - particularly Denmark and the United States - when it comes to the development of data centres.
Are there lessons to be learned from their experiences?
Have you any information that you think would help this investigation, contact us at [email protected]
If you want to know how your contribution is used, or anything else about how Noteworthy works, you can find out more here.


text:
As bold and loud as cold water to the face, Socket-to-Me speaks with absolute conviction. A cascade of confident lines, each socket is right out there to tell the world you know your stuff.
Hand-forged recycled steel. Hand-painted finish.
Sku: 219B02BL
Finish: Black
Dimensions: 14.375 in. W x 5 in. H x 4.813 in. Ext
Item Weight: 2.5 lb.
Lamping: 2 x 100W Medium, Not Included
Click here to view/download the Specification Sheet
Collections: Mid-Century Modern, Socket-to-Me, Wall Lights
Type: Vanity
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702-792-6900 | Contact Us | 4445 S Valley View Blvd, Ste 6, Las Vegas, NV 89103


text:
"Lord of the Rings" star Elijah Wood was among the attendees at the DIO/ANTHRAX show at the Greek Theater in Los Angeles, CA on Saturday (Oct. 30). Check out pictures from Launch: Dio, Anthrax, Fireball Ministry.
'Lord Of The Rings' Star Attends DIO/ANTHRAX Show In L.A.: Photos Available
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text:
Home > Classes & Events
At Cottage Health, we're dedicated to teaching our patients how to care for themselves through every stage of their lives. As a teaching hospital, we are devoted to training the best healthcare professionals in the nation and advancing medicine and medical research.
For patients, education is a key to prevention. For caregivers, education is a cornerstone of excellent patient care. At Cottage, we're committed to both.
As we plan for the coming weeks and the evolving situation with COVID-19, Cottage Health has decided to cancel all public events through the end of December. We understand this may be disruptive to schedules and wanted to provide as much advance notice as possible. Registrations fees will be refunded. We appreciate your understanding.
Como parte de nuestro plan para las próximas semanas para la situación cambiante causada por el COVID-19, Cottage Health decidió cancelar todos los eventos públicos hasta que termine diciembre. Entendemos que esto podría alterar sus planes y queríamos avisarle lo antes posible. Reembolsaremos los cargos de inscripción. Gracias por su comprensión.


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The Life, Blood and Rhythm of Randy Castillo
Date Added: 14th September 2018
From Mariachi to Ozzy, from Native roots to Mötley Crüe, Randy Castillo became one of the greatest drummer in rock. His personal story was even more magical.
Year: 2018
Duration: 87 min
Director: Wynn Ponder
Cast: Zakk Wylde, Vince Neil, Lita Ford, Mike Inez, Bill Ward
Studio: Freestyle Digital Media
IMDB: Discover more...
IMDB Rating: Not yet rated
Amazon Rating:
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text:
Within a few days of United Airlines announcing a new policy for transporting pets, more than 56,000 people have signed a petition asking for an exemption for military families with dogs.
United Airlines said this week that starting June 18 it would no longer transport dozens of breeds with short or snub noses, including bulldogs, boxers and pugs, in the cargo hold, because studies have shown that they have difficulty breathing in planes.
Passengers can still bring small pets, including many of the breeds banned in cargo, into the cabin if the animal's carrier fits under the seat without obstructing passengers from exiting.
But the policy, which doesn't address service and emotional-support animals, also bans the transport of animals in crates bigger than 30 inches tall anywhere on planes.
The petition on Change.org says United has been the most affordable airline for transported animals, especially large breed dogs such as Labradors, golden retrievers and huskies — breeds that can only be transported in crates more than 30 inches tall.
"I have a German Shepherd and we are living in the UK as a military family," Ashley Swearingin wrote on the petition site. "I can't imagine how we will get our baby girl home now."
Another military family member, Lynette Layne, wrote on the petition site: "SEMPER FI, Always Faithful!!!! We should as a country never leave a man, woman or animal behind!!!"
The new policy may cause some hardship with military families based in Guam, where United Airlines is the only major U.S.-based carrier serving routes to the mainland.
The carrier said Thursday that it will allow military families and State Department and foreign affairs personnel to book new reservations to transport their pets in and out of Guam under the previous regulations — but only until the new policy takes effect June 18.
"Unfortunately, we are unable to provide any other exceptions," said United spokesman Charles Hobart. "We know this can present challenges, but we hope our customers understand that we are making these improvements to help safeguard the health and safety of pets in our care."
In most cases, travelers could book flights on other major U.S. airlines, some of which allow large breeds but tend to charge higher fares.
The changes were prompted by the death of a 10-month-old French bulldog on a United Airlines flight in March from Houston to New York — a story that went viral on social media. United also flew two other dogs to the wrong destination in the days after the bulldog scandal.
After the incident, United stopped transporting all animals in the cargo compartment, the service that it will relaunch starting June 18.
The chances of your dog dying in flight are slim, but loading your dog on a plane like a piece of cargo still is a bad idea and I'd never do it.
To read more about the travel and tourism industries, follow @hugomartin on Twitter.


text:
Backstage with Al Di Meola and Friends Photo by Siimons
- April 3rd, 2012
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text:
Our correspondent from the region reported that the Turkish occupation army and its mercenaries stationed in the Azzaz area returned Monday evening to shell the village of Şewarge in Sherawa district in Afrin canton, using heavy weapons and missiles, some of which fell on the houses of the village.
The shelling continues.
On May 6, the Turkish occupation army and its mercenaries shelled the villages of Malikiya, Şewarge and Murnaz in the Shera district of Afrin canton with shells and heavy weapons. The shelling caused material damage to the houses and property of the residents.


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Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza
Contributed by Peter Palese
The NS1 protein of influenza A virus contributes to viral pathogenesis, primarily by enabling the virus to disarm the host cell type IFN defense system. We examined the downstream effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels by measuring expression of over 13,000 cellular genes in response to infection with wild-type and mutant viruses in human lung epithelial cells. Influenza A/PR/8/34 virus infection resulted in a significant induction of genes involved in the IFN pathway. Deletion of the viral NS1 gene increased the number and magnitude of expression of cellular genes implicated in the IFN, NF-κB, and other antiviral pathways. Interestingly, different IFN-induced genes showed different sensitivities to NS1-mediated inhibition of their expression. A recombinant virus with a C-terminal deletion in its NS1 gene induced an intermediate cellular mRNA expression pattern between wild-type and NS1 knockout viruses. Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.
Influenza viruses are responsible for an average of 20,000 deaths and 114,000 hospitalizations per year (1). Highly pathogenic strains of influenza A virus have emerged occasionally in recent history, producing pandemics such as the one in 1918, which resulted in the death of 20–40 million people worldwide (2, 3). Although the mechanism of increased pathogenicity has been genetically traced for a few unusually virulent strains [e.g., the PB2 and hemagglutinin genes of the Hong Kong H5N1 viruses appear to contribute to their virulence in mammals (4)], the cause of severe pandemics, such as the one in 1918–1919, and the contribution of individual influenza virus genes to pathogenicity remain largely unknown.
Influenza A virus has a negative-strand RNA genome that encodes on 8 RNA segments 10 or 11 proteins, depending on the strain. Segment 8 encodes an mRNA that is alternatively spliced to express the nonstructural protein-1 (NS1) and the nuclear export protein, NEP (5). The NS1 protein, which binds double-stranded RNA and forms dimers in vivo, has been suggested to perform several important accessory functions for the optimal replication of the virus in its host (5). Importantly, the NS1 protein represses the host cell antiviral response by multiple mechanisms. These mechanisms include the inhibition of the IFN-inducible double-stranded RNA activated kinase PKR (protein kinase RNA-regulated) (6–8) and the blocking of IFN-β production by preventing NF-κB (9), IFN-regulatory factor (IRF) 3 (10), and IRF7 activation (11).
DNA microarray technology is increasingly being used to examine the effects of viral infection on host cell gene expression (12). The advantages of this discovery-based approach is that one can monitor the expression of thousands of genes simultaneously, perhaps identifying coexpressed genes from a single pathway or those that function in multiple pathways. Here, we measured the global pattern of cellular gene expression in cells infected with the wild-type (wt) influenza A/PR/8/34 virus and compared it with those in cells infected with NS1 mutant viruses lacking all or part of the NS1 gene. In addition, we have examined the effect on cell gene expression of a recombinant virus in which the NS gene of influenza A/WSN/33 virus was substituted with the NS gene of the 1918 pandemic influenza A virus. The cellular genes that were differentially regulated by these viruses and the degree of overlap between them were identified by using the resolver Expression Data Analysis System. We conclude from this analysis that NS1 expression is necessary but most likely not sufficient for evasion of the host innate defenses. Finally, we identified multiple cellular pathways and new potential antiviral genes whose expression is influenced by the presence or absence of NS1.
Materials and Methods
Viruses and Cells.
Wild-type influenza A/PR/8/34, delNS1, and NS1 (1–126) viruses were generated, propagated in 7-day-old eggs, and titrated by plaque assay as described (9, 13, 14). Influenza A/WSN/33 virus and 1918 NS WSN recombinant virus were propagated and maintained as described (15). The lung epithelial cell line A549 was purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM containing 10% FBS, 2 mM glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin at 37°C.
Viral Infections, RNA Extraction, and Immunofluorescence Assays.
Infections with wt and recombinant influenza A viruses were performed in A549 cells grown in 10-cm2 dishes or T-150 flasks. In all cases, the multiplicity of infection (moi) was adjusted before each independent experiment to ensure that at least 80% of the cells were infected 8 h postinfection as determined by immunofluorescence using a viral nucleoprotein (NP)-specific antibody. Total RNA extraction and mRNA selection procedure is described (ref. 16; http://ra.microslu.washington.edu/Website/protocol/archive_protocol.html).
Northern Blots and Electrophoretic Mobility-Shift Assay (EMSA) Analysis.
EMSAs were done to determine the activation of NF-κB as described (9), by using nuclear extracts from infected A549 cells and a DNA probe containing a mouse H2 κB binding site. Northern blot analysis of total RNA isolated from A549-infected cells using specific [32P]ATP-labeled probes was performed as previously described (9).
Microarray Conditions and Image Analysis.
Microarrays were constructed by the University of Washington's Center for Expression Array Technology with PCR products generated from the 15,000 set of sequence-verified IMAGE consortium clones (http://ra.microslu.washington.edu/Website/genelist/genelist.html). For microarray conditions, see supplemental text, which is published as supporting information on the PNAS web site, www.pnas.org. In accordance with proposed MIAME (minimum information about a microarray experiment) standards (17), raw data, including sample information, intensity measurements, error analysis, microarray content, and slide hybridization conditions will be made available in the public domain (http://expression.microslu.washington.edu).
Influenza PR8 Virus Lacking the NS1 Gene Induces High Levels of IFN-β mRNA and NF-κB Activation in Human Lung Epithelial Cells.
Recent evidence in cell lines and in mice suggests that the influenza A NS1 protein antagonizes the cellular IFN response (13) in part by blocking NF-κB, IRF3, and IRF7 activation (9–11). We examined whether the same was true in A549 cells, a human lung epithelial cell line that may more accurately represent the infected cells found at the physiological sites of influenza virus infection. Both NF-κB activity (Fig. 1A) and IFN-β mRNA levels (Fig. 1B) increased dramatically by 9 h postinfection with a recombinant influenza A virus lacking the NS1 gene (delNS1 virus). In contrast, the activation of NF-κB and production of IFN-β mRNA were considerably lower in cells infected with wt PR8 virus. We confirmed that expression of IFN and NF-κB regulated genes were induced in delNS1 virus-infected cells by examining a selected set of IFN and/or NF-κB stimulated genes by Northern blot (Fig. 1B). The results demonstrate that the NS1 protein of influenza A virus down-regulates the IFN response in a human lung epithelial cell system and support the hypothesis that this viral protein has an inhibitory effect on IFN and NF-κB pathways.
Induction of Antiviral Gene Expression in Response to Influenza PR8 Virus Infection of Lung Epithelial Cells.
We next examined global cellular gene expression levels in cells infected with viruses containing mutations in the NS1 gene and compared them with those in cells infected with the parental wt PR8 virus. Two recombinant viruses were tested, delNS1 virus (13), and NS1 (1–126) virus lacking the C-terminal 104 aa of the NS1 protein (9). A549 lung epithelial cells in monolayers were infected at an moi resulting in approximately 80% of cell infection (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org.). We chose this moi to infect most cells with 1–2 virus particles. At 8 h postinfection, total RNA was extracted to be analyzed by microarray. Differentially expressed genes were selected based on ratio and statistical criteria from combined replica experiments. Because approximately 20% of cells are not infected, down-regulated genes in the array represent substantially down-regulated genes in infected cells or genes that are also down-regulated in noninfected neighboring cells. PR8 wt virus infection perturbed the expression of the fewest number of cellular genes (84 genes) whereas delNS1 virus infection resulted in the largest regulation of cellular genes (115 genes). The NS1 (1–126) virus regulated an intermediate number of cellular genes (93 genes). Thus, influenza A virus infection resulted in ≈0.5–1% of all of the cellular genes on the array being differentially regulated. A summary of the number of genes that are significantly regulated by each virus and the degree of overlap between gene sets is shown [Fig. 2; and see Table 1 (which is published as supporting information on the PNAS web site) for data on individual experiments]. Genes that were regulated by each virus can be classified in three primary groups: genes whose expression was similar between wt and mutant NS1 virus infections (Fig. 2, solid black and diagonal hatched), genes whose expression was similar between mutant NS1 viruses but not wt virus infections (Fig. 2, horizontal hatched), and genes whose expression is more significantly regulated by the individual viruses alone (Fig. 2, solid gray).
To confirm that induction of NF-κB and IFN-β regulated genes could be detected by microarray, we examined the mRNA levels for the cellular genes tested by Northern blot. The NFKBIA, IL-8, TNFRSF6, and HLA-C genes were all induced at 8 h postinfection with delNS1 virus but not by wt PR8 virus (Fig. 1B and Fig. 7, which is published as supporting information on the PNAS web site). In contrast, the levels of actin-α were unaffected (actin-β not present). It is important to stress that the Cy3/Cy5 ratio obtained by repeated microarray experiments underestimates the relative change in mRNA expression. Thus, a 2-fold change in expression by microarray is most likely a conservative cutoff for a differentially regulated gene, because the levels of the same mRNA measured by Northern blot appear to be more dramatically changed. The discrepancy between Northern and microarray results has previously been observed by using the same array platform (18).
There were 24 genes that were induced more than 2-fold with greater than 99% confidence (P ≤ 0.01) during infection with both wt and mutant PR8 viruses (Fig. 2, solid black, and Fig. 3A). There were 10 more genes whose expression was coregulated by all 3 viruses but with reduced confidence levels (P value > 0.01) in one set of experiments (Fig. 2, diagonal hatched, and Fig. 3A). Sixteen genes in this group were previously known to be stimulated by IFN and 4 more are potential antiviral genes. The IFN-stimulated genes (ISGs) up-regulated by viral infection include the influenza virus inhibitory IFN-induced gene MX1/MXA, in addition to IFI75, IFI41, IRF1, ISGF3-γ/IRF9 and others (Fig. 3A, asterisk). Note that, even though these genes were differentially regulated during infection with both wt and mutant viruses, some genes (e.g., GBP and IRF1) were induced to higher levels in delNS1 virus-infected cells (Table 2, which is published as supporting information on the PNAS web site). Other potential antiviral genes of interest that were consistently induced by PR8 virus infection include TNF-related apoptosis inducing ligand (TRAIL/TNFSF10), complement C1S component, and MYD88. The latter is an adapter molecule that participates in Toll-like receptor signaling (19).
To determine whether the sequence of differentially regulated expressed sequence tags (ESTs) aligned with any known or predicted gene, a blast search of the human genome database was performed on the available sequences for each uncharacterized clone. Three ESTs aligned with regions containing known or predicted genes with potential antiviral activity (Fig. 9, which is published as supporting information on the PNAS web site). These ESTs align with an exon of cig5 similar to inflammatory response protein 6 (IMAGE ID 120600), an exon of LOC129607 which shares similarity with thymidylate kinase (IMAGE ID 207838), and the 3′ noncoding region of LOC143274 that is similar to IFIT2/ISG54 (IMAGE ID 289496).
Mutations in the NS1 Gene Lead to Enhanced Antiviral Gene Expression of IFN, Cytokine, and NF-κB-Regulated Genes During Influenza Virus Infection.
We would predict from biochemical analysis in cell culture (Fig. 1B and refs. 9 and 10) and phenotypic analysis in mice (13, 14) that the mutant NS1 viruses would induce a more pronounced antiviral response involving genes regulated by IFN and/or NF-κB. Indeed, delNS1 and NS1 (1–126) viruses induce a generally higher increase in ISGs and in NF-κB mediated gene expression than wt virus [Fig. 3A, Fig. 10 (which is published as supporting information on the PNAS web site), and Table 2]. In addition, both mutant viruses induce the expression of an additional 24 genes (Fig. 3B), 5 of which were known to be associated with the IFN pathway (including 2 ESTs revealed by blast; see Fig. 9) and 6 were potential antiviral genes based on available annotation. Furthermore, if one also considers the ISGs that are not exclusively coregulated by both mutants but enhanced during either NS1 (1–126) (1 ISG and 7 genes with potential antiviral activity, Fig. 3C) or delNS1 virus (3 ISGs and 5 genes with potential antiviral activity, Fig. 3D) infection, it is evident that the antiviral response, including IFN and NF-κB regulated genes, is more robust in the absence of the NS1 protein (Fig. 3 and Table 2). Interestingly, two genes that are induced to high levels in delNS1 virus-infected cells (PMAIP1 and GBP1) also appear to be induced by overexpression of a constitutively active form of IRF3 (20). The higher activation of IRF3 observed during infection with delNS1 virus as compared with wt PR8 virus (10) may explain these results.
Collectively, the mutant NS1 viruses appear to have a significant impact on genes in the signal transducer and activator of transcription (STAT)-signaling pathway. For instance, the expression levels of STAT1 (Fig. 3D) and STAT3 (Table 2) were greater in cells infected with NS1 mutant viruses relative to wt A/PR/8/34 virus. In addition, the expression of two members of the suppressor of cytokine signaling (SOCS) family of proteins, STATI2/SOCS2 and SSI-3/SOCS3, whose transcription is regulated by STAT proteins (21–23), was affected in cells infected with delNS1 virus relative to wt (Table 2 and Fig. 4A). These proteins function to modulate cytokine and growth factor signaling in a negative feedback loop. Interestingly, SOCS2/STATI2 is down-regulated in delNS1 virus-infected cells (Fig. 3D), whereas SSI-3/SOCS3 is up-regulated (Fig. 3B), suggesting that the two genes may have opposing functions in modulating cytokine response, at least during influenza virus infection. The expression of another SOCS family member (SOCS1/SSI-1, Fig. 4A) and of the N-Myc interactor (NMI) (Table 2), which has also been shown to interact with STAT proteins (24), was also increased by PR8 virus infection. We examined expression of all SOCS-box-containing proteins, along with its associated cytokines and receptors that were present on the microarray (Fig. 4A). We found that, in addition to the STAT and SOCS genes discussed above, the leukemia inhibitory factor (LIF) and its receptor (LIFR), whose signaling is modulated by SOCS proteins (23, 25), were induced in PR8 virus-infected cells. The SOCS genes modulate cytokine response via different mechanisms. SOCS1/SSI-1 binds directly to Janus kinases (JAKs) and inhibits their catalytic activity (26), SOCS2/SSI-2/CIS binds to the cytoplasmic domain of cytokine receptors (27), and SOCS3/SSI-3 functions via binding to receptors in which JAKs are already bound (28).
Other intriguing evidence that the STAT pathway may be regulated during influenza virus infection is an increase in dual-specificity phosphatases, DUSP4 and DUSP5, mRNAs during delNS1 virus infection (Fig. 3D). DUSP6 mRNA was also increased but its P value was 0.02 (Fig. 11, which is published as supporting information on the PNAS web site). Although the human DUSP genes, which are homologous to vaccinia virus H1 phosphatase (VH1), are known to primarily act on mitogen-activated protein (MAP) kinases [such as extracellular signal-regulated kinase 1 (ERK1)], the viral homolog VH1 has been shown to dephosphorylate and inhibit STAT proteins (29). The mRNA for several members of the DUSP gene family (e.g., DUSP1, -2, and -5) were also found to be induced in other large-scale gene expression studies of both bacterial (30) and viral infections, including influenza virus infection of dendritic cells (31). Another gene critical to IFN signaling that may also be involved in STAT signaling is the IFN-inducible gene PKR. Levels of PKR mRNA increased in cells infected with NS1 mutants relative to wt virus. We presume that increased levels of type I IFN (Fig. 1B) in the absence of NS1 result in increased PKR mRNA levels. Finally, PKR itself has been suggested to be involved in the serine phosphorylation of STAT1 (32) and therefore may also contribute to the overall regulation of STAT signaling.
Proteins containing tripartite motifs are a growing family of known and predicted proteins that have been proposed to function by forming specific subcellular compartments, for example, promyelocytic leukemia (PML) nuclear bodies (33). These genes are predicted to contain, a RING finger (3 zinc binding domains), a B-box (type 1 and/or 2), and a coiled-coil region. Tripartite motif (TRIM) genes regulated during influenza virus infections are shown in Fig. 4B (see also Fig. 12, which is published as supporting information on the PNAS web site). We propose that this set of TRIM genes may represent a new class of antiviral proteins that are coordinately regulated.
The NS Gene from Pandemic 1918 Influenza Virus Is More Effective than the NS Gene from WSN Virus at Blocking Expression of ISGs in Human Lung Cells.
To further explore the potential contribution of the NS1 protein in influenza virus virulence, we used a recombinant WSN virus containing the NS gene from the 1918 pandemic strain (15). Because this recombinant virus was generated in the influenza A/WSN/33 virus strain background, it was also necessary to measure the effect of WSN virus infection on host cell gene expression in A549 lung cells so that a direct comparison could be made. Introduction of the 1918 NS sequence into the WSN background resulted in an overall expression pattern that was very similar to the parental WSN virus (Fig. 5). However, differences were observed for specific ISGs. Although both WSN wt and 1918 NS recombinant viruses appeared to induce lower levels of expression of IFN-regulated genes than the PR8 wt virus (Fig. 13, which is published as supporting information on the PNAS web site), the wt WSN virus did induce expression of some ISGs to high levels [MXA/MX1, ISG15, IFITM1, and ISGF3-g (IRF9)]. In contrast, infection with 1918 NS WSN virus failed to significantly induce any ISG (Figs. 5 and 13). We also noted that NS 1918 virus (and to a lesser extent wt WSN virus) had the opposite effect of PR8 virus infection on the expression of SSI-1/SOCS1, STAT1, and leukemia inhibitory factor (Fig. 4A), as well as of some NF-κB-regulated genes (e.g., SAA1 and BIRC2 and -3, Fig. 10). The compare plot in Fig. 5 also reveals that several cellular genes, in addition to ISGs, are differentially expressed between cells infected with wt or 1918 NS WSN viruses. For example, protein kinase inhibitor-β (PKIB) and Toll-like receptor 5 (TLR5) were up- and down-regulated, respectively, by 1918 NS virus but not by wt WSN. Conversely, CSPG6 is down-regulated during infection with wt WSN virus but not by 1918 NS WSN. We also found several ESTs that exhibit different expression levels between the two viruses. These results emphasize the advantage of combining recombinant virus and microarray technology to identify a few candidate genes for future study. Whether these differences in gene expression are partially responsible for the high virulence associated with the 1918 virus remains to be investigated.
We have shown that the influenza A virus NS1 gene has a significant impact on host cell gene expression in human lung epithelial cells. These data confirm that the NS1 protein plays a central role in inhibiting IFN, cytokine, and NF-κB pathways. Although we cannot rule out that cytokines, especially IFN, present in the egg-grown virus preparations played some role in the observed differences, chicken IFN is unlikely to signal in human cells. Interestingly, the expression of some ISGs was not affected by the presence of the NS1 gene, whereas the induction of other ISGs was reduced in wt PR8 virus-infected cells. A mutant virus containing a truncated version of the NS1 gene, lacking the C-terminal domain, exhibited an intermediate phenotype, suggesting that this region plays a significant role in NS1 function. In a different study, we have found that the C-terminal NS1 region plays mainly a structural role on NS1 function by enhancing its dimerization (X.W. and A.G.-S., unpublished results). The differential cellular response after infection with wt and mutant NS1 influenza viruses correlates well with their known virulence in mice. For instance, wt virus grows to high titers in lungs of infected mice that eventually die from infection. In contrast, delNS1 virus is attenuated in wt mice, but is lethal in mice with deficient IFN signaling (13). This finding suggests that the inability of the virus to block the initial cellular antiviral response allows the immune system to rapidly clear the delNS1 virus infection. The NS1 (1–126) virus, again, exhibits an intermediate phenotype in mice (unpublished observations).
Our experimental approach allowed us to identify genes associated with antiviral pathways. Multiple members of the SOCS and TRIM gene families are coregulated during PR8 virus infection (Fig. 4), and several genes associated with apoptosis are influenced by NS1 protein expression (Fig. 14, which is published as supporting information on the PNAS web site). blast searches against the draft human genome with EST sequences are consistent with some of these ESTs representing exons, alternatively spliced exons, or untranslated regions of expressed mRNAs (Fig. 9). However, some other ESTs appear to align with DNA sequences outside areas corresponding to predicted mRNAs (Fig. 9). In this context, two recent large-scale expression studies (34, 35) found that many cytosolic RNA species hybridize to regions outside known or predicted exon regions. We have also observed that some influenza virus coregulated genes are located in close proximity in chromosomal DNA, e.g., RI58, MPHOSPH, IFIT1, LOC143274 on chromosome 10, and TRIMs 22, 21, and 34 on chromosome 11 (Fig. 15, which is published as supporting information on the PNAS web site).
Large-scale gene expression studies are increasingly being used to compare the host cell response to infection with different pathogens. Remarkably, a comparison between host cell genes that are regulated by influenza PR8 virus infection in epithelial lung cells (this manuscript) and those regulated by a variety of pathogens (including PR8 virus, Escherichia coli, and Candida albicans) in dendritic cells (31) revealed that 27 cellular genes not only were regulated by PR8 virus infections in both cell types but also were regulated by E. coli and C. albicans in dendritic cells. This finding suggests that these genes, which consist largely of IFN-inducible genes as well as DUSP and TRIM family members, represent the core of the innate cellular immune response that is conserved in multiple cell types. In contrast, some genes do not exhibit similar trends in gene expression after PR8 virus infection in dendritic and lung cells, which presumably reflects the specialized functions of these two cell types (or other parameters that differ in the two experiments). However, because influenza virus infection is known to decrease the levels of cellular protein translation, future experiments are planned to assess the combined effects of cellular RNA changes with translational attenuation in the global pattern of protein expression in influenza virus-infected cells.
In this study, we have also explored the possibility of using microarray analysis to examine the role of the 1918 NS gene in virulence. A virus containing the NS gene from the 1918 pandemic influenza virus was more effective at inhibiting a subset of ISGs in human lung epithelial cells than the parental WSN virus strain, even though the overall cellular response was similar. These differences were most likely due to their slightly different NS1 sequences, although we cannot formally rule out the possibility that the NEP protein, whose coding sequence partially overlaps with the NS1 gene, also plays a role. Although it is tempting to speculate that the negative effect of 1918 NS on ISG expression may be responsible for its unusually high pathogenesis in humans, additional experiments will be required to assess the contributions of the NS1 and other 1918 viral proteins.
Significant differences in the IFN response to influenza WSN and PR8 infections in A549 cells were observed (Fig. 13). Although it is possible that this difference is due to their NS1 genes, which differ by 7 aa (see Fig. 16, which is published as supporting information on the PNAS web site), it is likely that additional influenza virus genes contribute to the WSN virus-mediated block of the antiviral response. Because the cellular antiviral response depends on a balance between negative and positive regulators of gene expression, it is possible that positive viral regulators are more effective in wt PR8 than in WSN virus infection. For example, PR8 virus infections may generate more double-stranded RNA than WSN virus infections. Interestingly, both viral strains represent examples of human viruses that were adapted to mice. PR8 virus, as a result of mouse adaptation, became highly attenuated in humans (36), which is consistent with the cellular gene expression pattern induced on infection of human lung cells. As of this moment, the virulence of WSN virus in humans is unknown. It will also be interesting to compare the cellular gene expression patterns induced on infection with influenza A, B, and C viruses. Although these three types of influenza viruses have common strategies of replication, influenza C viruses are believed to cause mild or asymptomatic infections in humans, and influenza B viruses have developed specific strategies to counteract the activity of some IFN-inducible genes, such as ISG15 (37).
In summary, the combination of gene profiling analysis of infected human lung epithelial cells with reverse genetics techniques has given new insights into the cell antiviral response against influenza virus and its regulation, and on the role of the NS1 gene in preventing this response. In addition, clear differences were found in cellular gene expression induced by different influenza virus strains. These differences may have a significant role in virulence. The generation of databases allowing the comparison of different viruses and viral strains with respect to their impact on cellular gene expression is likely to help to rapidly predict the pathogenicity of new or past virus strains by using high throughput gene profiling techniques.
We acknowledge Estanislao Nistal-Villan and Zhihong Wang for their excellent technical assistance, Ted Holzman and Bart Kwieciszewski for their bioinformatic support, and Jeff Furlong for developing Expression Array Management Database. We thank the University of Washington Center for Expression Arrays for providing microarray services. This work was funded in part by National Institutes of Health grants (to M.G.K., P.P., and A.G.-S.). T.M.T. was supported by U.S. Department of Agriculture, Agricultural Research Service, Current Research Information System (CRIS) Project 6612-32000-022-93.
ISG, IFN-stimulated gene
moi, multiplicity of infection
wt, wild type
IRF, IFN-regulatory factor
EST, expressed sequence tag
STAT, signal transducer and activator of transcription
DUSP, dual-specificity phosphatase
PKR, protein kinase RNA-regulated
NS1, nonstructural protein-1
TRIM, tripartite motif
SOCS, suppressor of cytokine signaling
- Accepted June 5, 2002.
- Copyright © 2002, The National Academy of Sciences
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Title: Magnerobo Ga-Keen
English: Magnerobo Ga-Keen
Synonyms: Magne Robo Gakeen, Gakeen Magnetic Robot, Robotman Gakeen, Magnetic Robot Ga Keen, Magnos Robot
Japanese: マグネロボ・ガ・キーン
Type: Tv
Total: 39
Status: Ongoing
Aired: Sep 5, 1976 to Jun 26, 1977
Premiered: Fall 1976
Genres: Action, Mecha, Sci-Fi
Duration: 24 min. per ep.
Rating: PG-13 - Teens 13 or older
Summary: Dr. Kazuki, who perceived of an invasion of Earth by the Izaru people builds a robot based on the science of magnetism and sphere joint theory. Undergoing a dangerous augmentation process, Dr. Kazuki's daughter Mai becomes the pilot of "Mighty" or "Magnetman Minus". Takeshi Houjou becomes the pilot of "Puraiza" or "Magnetman Plus". The pilots would hold each other and then physically trasform their joint bodies in a metallic plate locking itself on the Gakeen (Short for "Gathering Keen") robot's frame thus enabling the super robot to move and fight. This sequence was notable, to an adult's eye, for its almost-sexual connotation. (Source: Wikipedia)


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September 27 is the annual observance of the National Gay Men's HIV/AIDS Awareness Day. First observed in 2008, the purpose of this day is to: raise HIV/AIDS awareness among gay men, encourage testing and advocate for greater treatment and prevention services for gay men. It is also a day to recognize the disproportionate impact of HIV/AIDS on gay men.
According to the Centers for Disease Control and Prevention (CDC), gay and bisexual men are the population most affected by HIV.
In 2014:
- Gay and bisexual men accounted for an estimated 83 percent (29,418) of HIV diagnoses among males and 67 percent of all diagnoses.
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Nearly 1 in 7 gay and bisexual men living with HIV are undiagnosed. Getting tested is the critical first step toward protecting yourself and your partner from HIV. Sept. 27 is a great day to do it.
Many facilities have extended testing hours. The CDC recommends that everyone between the ages of 13 and 64 be tested for HIV at least once as part of routine health care. Some gay and bisexual men may benefit from more frequent testing (e.g., every 3-6 months), as well as from the use of PrEP to prevent HIV in those at high risk.
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"There are 14 National HIVAIDS Awareness Days that we observe throughout the year that promote awareness in different communities including Youth, Black, Latino, and Transgender individuals," said Lorenzo Lowe, Director of HIV Prevention for Compass, the Gay and Lesbian Community Center of Palm Beach County. "The reason why it's so important to recognize these days in these different communities speaks to the challenges we all continue to face in the fight against HIV/AIDS. Truth is, the responsibility to get tested, use condoms, and adhere to treatment falls on everyone regardless of your status. This is a responsibility that needs to be exercised everyday by everyone. I would hope that conversations about prevention become secondary. Remember it was just in the early 80's we were in a race between education and catastrophe. This same race continues today and National Gay Men's HIV/AIDS Awareness Day is one day among many in which we realize there is still work to do."
You can find a list of testing sites throughout South Florida by visiting http://srdappsdoh35.doh.state.fl.us/ClinicSearch/ClinicSearch.aspx.


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I am 18, and as I grasp this issue of homosexuality, I realize that it is something I always knew, sort of a silent understanding with myself, but life went on, and growing up I never made it a big issue. I felt different from a very early age, but than again, I never knew what it was like to be very normal anyway, so I figure that I didn't miss much. Having an older brother guaranteed me to an adolescence of tomboy activities. Hanging out with his friends was much cooler than hanging out with dumb girls who only cared about what the boys thought anyway. The teenage years struck, with a startling result, and all of my brothers friends began to be interested in me. Not friend interested, but girlfriend interested. I had already had my slew of boyfriends… always looking for something that I suppose would never be there. Then I had my first lesbian experience, and I finally admitted to myself that I was gay. You would never know it from looking at me, which is better I suppose, because I want people to know me for who I am, not my sexual orientation. Anyway, attending a magnet Art High School, where allot of the guys were openly gay, led me to finally coming out to my friends. Some of them said they had their suspicions because I never had boyfriends around, and others said they had no clue. They all took it well, and the point is, they accepted ME, That segued into me telling my brother. He accepted it in disbelief, because he always thought that everything about me was perfect. I told him that I don't see my sexuality as a curse, and that if he wanted to think everything around my life was still perfect, he could. I have yet to tell my parents. I figure I will wait until I am out of the house, either in college, or after that. Part of me thinks they already know, but that they really don't want to admit it. My parents are Catholic Republicans, very proud of my grades and talent, and constantly trying to fix me up with the "perfect" guy. I let them think that I don't have a steady boyfriend because I go to a high school where allot of the guys are gay, and that I truly love going to movies with their co-worker's sons. I do this because at this time in my life, it's easier than hurting them. I know that time will end soon enough, and that they will love me nonetheless, but until then, I keep it to myself. I am who I am, and that is enough for me.
It is early in the morning, in the other room I can hear the soft sleeping sounds of my partner, Terry. I am concerned that she will wake and find me in here on the computer, instead of studying for exams. She is 11 years older than I, and sometimes can be rather paternalistic, (notice I didn't say maternalistic). I also refer to this as her wearing the "pants of crank". Nevertheless, since I came across this site and read a lot of the sad, happy, inspiring and just plain brave stories of so many men and women, I have wanted to add my own insights and share my "coming out story".
I like to say that in a sense, I never actually "came out" because I had never known I was "in". It took me 'til I was in my later twenties to realize that I was a lesbian, bisexual, gay, whatever you want to label it. Was I so blind and stupid I lived in a closet for 25 odd years and didn't even know it? (Actually, I was feeling a little cramped, oh, and the mothballs….). Gay friends and acquaintances said later "We knew it all the time". Maybe they are just trying to prove their strong "gaydar" skills or maybe they're right, I don't know.
My mother was not wholly surprised that day I told her I was dating another woman. I think she half suspected I would not let her drift into middle age without pulling some rabbits out of the hat. She even asked if I was "doing this just to spite her". (Funny how she always thinks everything is about her). She should have expected it from me, the child who always fought for the underdog, who could never leave well enough alone, who lived with her heart on her sleeve. If our family had a black sheep (and you didn't include my alcoholic father, who was really more of a "black-out"), I was it.
The first time I had sex with a woman I was more than a little drunk, a lot lonely, and a little bit in love with Meg, this smart funny, troubled, self proclaimed "dyke" I had met through a close friend of mine who was gay. ( Hey, we were friends for years, and I thought for the longest time her partner was just her roomate. Yes, they slept in the same bed, did everything together, but so???)
Loving another woman, touching the landscape of her body that was so like my own, kissing her, holding her, was a little strange and yet beautiful experience for a woman who thought she was straight and just hadn't met that special guy. It was almost like the ultimate way of loving myself. The thing was, I didn't feel ashamed, embarrassed, or that I had done anything "wrong". Instead, I felt like announcing to the clerk at the gas station and anyone else who would listen about this wonderful experience I had just had. (I didn't).
When I left her apartment that morning I knew that something very meaningful had just happened, but I thought it meant I was "open" in my sexuality, that I was curious, bisexual, whatever. But I still told myself it didn't mean I was gay.
I had always been attracted to men, had my share of boyfriends and sexual experiences as a teenager but never long term, and then as I got older and gained a lot of weight I chose to be uninvolved because I didn't feel I could be intimate with anyone looking and feeling about my body as I did. Plus it wasn't like a lot of guys were chasing fat girls. So I went off to university and then spent the first five years of my twenties celibate, lonely, busy with school, friends, hobbies, my career, and fantasies of losing weight, meeting that perfect man, and having the white picket fence.
Eventually I did lose quite a bit of weight and was feeling more comfortable with myself and started dating and actively meeting men. It felt hollow. It also felt like I just wanted sex. I didn't feel like I connected at all with men on an emotional level. I didn't understand them the way I did women. And then there was Meg.
It took a few months after that (and also a few more nights with Meg, though we decided to eventually be just friends), for me to realize I really was gay. Of all things it was a porno movie that finally made me sure.(Who knew they could be educational or enlightening?) As I watched the man and woman on the screen I thought, that's not what I want anymore. I was sexually attracted to men, yes, but I didn't want to live with one, spend my life with one, now that I knew there were other possibilities. To a lot of gays, I know, it was never a choice, but I think in a way, it was for me. I could choose to do what gave me the most happiness and joy, and who wouldn't choose that? Still it was difficult for me to grasp that the "American Dream" of husband, wife, kids, and picket fence would never be for me. And I grieved for that. It had been all I had ever wanted since I was a little girl.
So I was confused, stressed out, coming to terms emotionally with my realization that I wanted to love a woman. And like others in my situation I called up the GLBT group in my city and spoke to the woman who was then the president of the group for some advice and reassurance. It is her that I hear soflty snoring in the next room.
She is the love of my life, I feel that I have been so blessed to have a best friend, life partner and lover all rolled into one human being. Terry is more transgendered than gay and I suppose I am more bisexual than lesbian so we are kind of the classic femme/ dyke couple. If there were a husband in this relationship, she'd be it, and I the wife. But its different because she has just enough felinity so that she thinks like a woman, loves like a woman, even though she'd rather be shopping for power tools than shoes. Over the years she has considered a sex reversal operation, and maybe one day she will. It won't matter to me. I have discovered that its not male or female that I love but a person, a soul. And in her I have the best of both worlds.
We have been together four years now, and live our lives very openly. I talk to strangers, friends, family and coworkers about my partner the way they talk about their husbands or wives. I am very matter of fact about it. In the beginning, I told family and those close to me "Guess what, I am seeing someone new. Only, its not a guy." Then I would just stand there, with a small smile on my face and wait for their reaction. For some, like my brother, it was easy, he just said, "Ok, if your happy, that's great". For others, like my mother, it is an ongoing road to acceptance. (The other day we were talking and I told her that I was happy. She said "No you're not. You just think you are". I wonder, isn't that the same thing?).
Oh, and the American Dream thing? I didn't have to give it up after all. My partner builds a great white picket fence., and we're planning our first baby next year.
Next blog post: Allen's Story https://wordpress.com/post/lovejudgenot.wordpress.com/611


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Title: Going Beyond Mentoring to Help Others Advance in Their Careers
Description: Learn about a new movement that forward-thinking people are adopting which is focused on sponsoring versus mentoring to elevate - in particular, under represented individuals - in the workplace.
Presenter: Lori Gold Patterson
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I was saddened, but not surprised, at Jorge Posada's retirement from MLB.
The all-star catcher spent his entire career with the New York Yankees, my favorite team of all sports. My loyalty is due in part to growing up relatively close to Yankee Stadium. My years as a fan have spanned from the tail end of Yogi Berra's career to Posada's….and will continue.
He will always be a favorite among the Yankee Nation, but will he eventually end up in the Hall of Fame?
A Yankee fan site has expressed some doubt, but I disagree.
His career stats fall comfortably within those posted by other Hall of Fame catchers. There is no doubt he was a key component in the Yankee's core quintet – which included Jeter, Rivera, Williams and Pettite – that produced a string of World Series Championships in the late nineties.
In an era that produced scandals regarding the use of performance enhancement drugs, Posada was a model exception.
I hope I can write a post about his entry in the Hall of Fame in five years.
I also hope Rivera sticks it out for at least another ten years.


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Description: Cycnoches chlorochilon is an unusual orchid from South America characterized by large green and white, highly fragrant and waxy flowers on pendulous flower spikes. Cycnoches are unique among most orchids in that they produce separate male and female flowers, usually on different inflorescences. The genus gets its name from the elongated column on the male flowers, which resembles the arched neck of a swan; some sources attribute the name to the shape of the flower, which resembles a swan when viewed from a certain angle.
Cycnoches have cylindrical pseudobulbs with thin, slightly pleated deciduous leaves.
Propagation: Propagated commercially from seed or by tissue culture. Also can be propagated by separating the pseudobulbs.
Cultivation: Easily grown indoors under medium to low light levels (800-1,000 foot-candles) and at temperatures from 55 to 85 degrees. They are best grown in fine fir bark or New Zealand sphagnum moss. Repot every year when the new growth begins.
In winter, after they lose their leaves, cycnoches require a pronounced dry period (no water). Once new growth begins in the spring, plants should be kept moist and fertilized heavily with a 20-20-20 fertilizer.
Bright light (1,000 foot-candles or more) promotes the formation of female flowers, while subdued light (less than 1,000 foot-candles) encourages the formation of male flowers.
Pests: Cycnoches' soft, thin leaves are vulnerable to spider mites. Slugs and snails will eat new roots and flower buds.
Comments: Cycnoches chlorochilon has the largest flowers (more than 5 inches across) of the genus. The spicy vanilla fragrance alone makes this orchid worth growing. Don't let the cultural requirements (not watering the plant for three months) scare you. This is one orchid with a flower both beautiful and bizarre and a fragrance you'll never forget.
Cycnoches chlorochilon
This is a work in progressw so please ignore the mess....
Common Name The Green-Lipped Cycnoches - in Venezuela "El Pelicano"
Flower Size 7" or less [17.5 cm]
This medium sized, epiphytic, hot to warm growing species is found from southeastern Panama, Colombia and Venezuela in semi-dense wet forests at elevations around 400 to 850 meters with fusiform pseudobulbs enveloped by non-foliaceous sheaths and carrying 4 to 7, plicate, elliptic-lanceolate leaves that blooms in the late summer and fall on an axillary, pendant, 3" [7.5 cm] long, racemose, few to several flowered inflorescence arising from the leaf nodes just below the apex of the newest, mature, large, fusiform to conical pseudobulb.
Synonyms Cycnoches ventricosum Bateman var chlorochilon [Klotsch] P.H.Allen 1952
Photo by © Danny Lentz, plant grown by the Atlanta Botanical Garden
Cycnoches Chlorochilon at 700 meters nms state aragua
Panama, Central America, Southern America French Guiana, Southern America Guyana, Southern America Suriname, Southern America Venezuela, Southern America Colombia, Southern America Brazil North, Southern America
|Large, impressive green flowers that have the fragrance of banana popsicles! This South American species wants plenty of water and fertilizer in Spring and Summer months but in Winter after the leaves start turning brown and eventually fall off keep the mix dry only watering if the pseudobulbs begin to shrivel. This plant typically produces 1-4 flowers on an inflorescence.
"green swan orchid. related to catasetum, cycnoches has an intense smell that i find pleasant and haunting."
Presenting : Cycnoches chlorochilon 'Green' x 'Yellow'
Cycnoches are epiphytic lowland orchids in the Tribe Cymbidieae, Subtribe Catasetinae. They are closely allied to Catasetum, Mormodes, Clowesia and Dressleria. The name Cycnoches is derived from the Greek words 'kyknos' meaning swan and 'auchen' meaning neck and is in reference to the thin arching column of the male flowers. They are found from Mexico down through Central America into the Amazon Basin of South America. They have cigar shaped fleshy pseudobulbs of several internodes topped by a few thin heavily veined large leaves. The pseudobulbs themselves can be from approximately 8 inches tall to more than 20 + inches tall. As a general rule, the taller the plant, the thicker the pseudobulb. The flowers which are produced on the apical portion of the pseudobulb can be single or up to as many as 30 + on an inflorescence. But sadly, I had only one bloom on the first time I have posessed this plant. The other buds have blasted. They can range from about an inch in size to over 6 inches. The inflorescence can be erect to arching to pendulous. Each pseudobulb, depending on the species, can produce one to several spikes with some producing up to 6 or more. The flowers are fairly long lived especially when compared to Catasetums and many are extremely fragrant. You will need to smell them yourself to decide on a description. My flower smell sweetly spicy to me. ( Bud; 07-22-2012 )
Cycnoches chlorochilon care and culture
Cycnoches chlorochilon is native to Panama, Colombia and Venezuela. In Panama, they meet in the province of Chiriquí and along the river La Maestra at altitudes from sea level up to 25 m...
Cycnoches chlorochilon also called as The Green-Lipped Cycnoches, Cycnoches ventricosum var chlorochilon, is a species of the genus Cycnoches. This species was described by Johann Friedrich Klotzsch in 1838.
Cycnoches chlorochilon is native to Panama, Colombia and Venezuela. In Panama, they meet in the province of Chiriquí and along the river La Maestra at altitudes from sea level up to 25 m. In Venezuela, they grow on lonely, little-foliage trees near Caracas, at heights of 900-1000 m and along the old road between Caracas and Maracay. There are reports of encountering them in the states of Aragua and Miranda at an altitude of 850 m, in the state of Yaracuy at an altitude of 500 m and in the state of Zulia. In Colombia, it is the most common species of this genus. They grow on hot lowlands below 1000 m.
It is a medium sized, epiphytic, hot to warm growing species, which reaching 30-50 cm in height, with fusiform, up to 20 cm long pseudobulbs enveloped by non-foliaceous sheaths and carrying 4 to 7, plicate, elliptic-lanceolate, 12-30 cm long and 2.5-5.0 cm wide leaves.
The Green-Lipped Cycnoches blooms in the late summer and fall on an axillary, pendant, 7.5 cm long, racemose, few to several flowered inflorescence arising from the leaf nodes just below the apex of the newest, mature, large, fusiform to conical pseudobulb. The flowers on each inflorescence are usually either male or female, but most often male flowers. In any case, however, they are large, fleshy and differ only in the construction of the spine. The petals of both whorls are yellowish-green. The lip is white and has a dark green, egg-shaped cavity below the fleshy, pointed, triangular, dark green thickening. The spine is green and its top is greenish-white. Female flowers are seen less frequently than male ones, but they are identical to male ones, with the exception of the construction of the spine.
Cultural information should only be used as a guide, and should be to be adapted to suit you. Your physical location; where you grow your plants, how much time you have to devote to their care, and many other factors, will need to be taken into account. Only then can you decide on the cultural methods that best suit you and your plants.
Cycnoches chlorochilon needs a light level of 10000-45000 lux. This species react in different ways to growing at different levels of light. The amount of light at which they grow determines the sex of emerging flowers. A low level of light results in the formation of female flowers, and at high light levels, male flowers develop. Sometimes the flowers are both male and female flowers, but usually on one inflorescence, all flowers are of the same sex. Strong air movement must be ensured all the time, regardless of the level of light.
It is a thermophilic plant. The average summer temperature during the day reaches 31 ° C, at night 22 ° C, with a daily difference of 9 ° C. In winter, the average day temperature is 33-34 ° C, at night 19-20 ° C, with a daily amplitude of 12-14 ° C.
he Green-Lipped Cycnoches needs the humidity of 85-90% for most of the year, only for the period of 2-3 winter months drops to 65-70%. The high humidity is very important.
Substrate, growing media and repotting:
Cycnoches chlorochilon can be grown in containers with good drainage or on cork or tree fern rootstock. In the case of mounted plants, there are practically no problems with decay caused by plant flooding, but many people are not able to provide such plants with sufficiently high humidity in the summer. Therefore, plants are usually grown in clay or plastic pots filled with soil, which provides excellent drainage and aeration of the roots. When the plants are dormant, it is usually better to remove them from the pots, cut the roots with a sterile tool and place the pseudobulb group in the empty pot until the end of winter.
When new growths occur in spring, the plants should be repotted to slightly larger pots, in order to keep the pseudobulb group and that there would only be room for one-year new increments. The pots are filled with loose medium and quickly draining excess water. You can use a medium-sized fir bark or chopped tree-fern fiber with pearlite, which helps keep the substrate cool, but also retains some of the moisture. The addition of charcoal also improves the permeability of the substrate, and also protects against acidification. They appear to grow better and have fewer problems with decay if it is repotted every year.
In the period from late spring to autumn rainfall is very abundant. Then their number decreases quite quickly and a 2-month dry season follows at the end of winter and at the beginning of spring. The cultivated plants should be abundantly watered during active growth, but the roots must always dry quite quickly after watering. The substrate around the roots can never be desiccated or soggy.
This species require strong fertilization, so during active growth they should be fertilized with a full dose of balanced fertilizer every week. You can use high-nitrogen fertilizer from spring to mid-summer, then switching to high-phosphorus in late summer and autumn.
Rest period:
This orchid requires a dry winter rest, and it is very susceptible to overdose during this period. If during the rest period the Cycnoches chlorochilon is taken out of the pot, after we remove the old roots, we can place a group of pseudobulbs in an empty clay or plastic pot until new increments appear in the spring. The plants mounted and those in empty pots should be misted or very lightly watered once a week. No substrate around the roots facilitates faster drying and protects against rotting. When new growths appear in spring, the plants should be planted in a fresh substrate, but the amount of water when watering can be increased only when new roots begin to grow. Fertilization should be eliminated until new growths appear and more watering begins in the spring. Special attention should be paid to the fact that water does not accumulate on growing new increments because they are particularly prone to rotting.
Native to:
Brazil North, Colombia, French Guiana, Panamá, Venezuela


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Please leave your information on our page. Brag about your kids, your grandkids, your fur babies, your latest endevours. Let us know what you have been doing for the last 40 years!
Alice Baker (Mitchell)
Divorced 3Divorced
2 beautiful daughters
4 wonderful grandsons
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Becki McKey (Kanada )
Married 2Becki McKey,Kanada
Keith And I have been Married 39 Years And have been Blessed with two Beautiful Daughters
That Have Blessed Us With 7 Grandchildren'in which our Precious "Jessie " left this world too soon is in the Arms of God Our Father! We Never Forget Our Blessing!
We now reside in Mississippi, our camp For Over 25 Years is now our Home, We Are So Excited for
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Keith is in his final years of working with Local 53 and looking forward to retirement and for myself I
am a Bus driver in Mississippi! I truly enjoy my job and Look forward to what God Has in store for our next chapter in Our Life!
We look forward to our Reunion and Reuniting our Special Class And Forever Friendships ❤
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MarriedMarried Lynn Duhon 3 years ago. Live on False River. We own a convenient store on the North end of the Island....Island Oasis. Can't wait to see everyone.
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Married 4Billy and I will be married 39 years in April. We have eight grown kids (thank you God) including spouses, eleven grandchildren so far. Yes so far. I work for the state and I can see the faint light of retirement. LOL
Jeannine Simoneaux (Cockerham)
A reset highlight, and my life, was to visit at home as well as add a reception with my favorite teacher of all, Mrs. Dolores Day. She taught me fifth grade at Tanglewood. She is just as beautiful and sharp as ever, at 90 years old. Also, I was able to make a girls trip to California, with PaulaWilliams Zumo. A friend since fifth grade, two weeks ago.
His Peace to All!
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Married 2I have been married to Mike for 35 years. We have 2 sons, both still single so, no grandchildren yet. Our boys live in Austin TX and Shreveport LA. I am self employed, doing contract work from home as a project coordinator. We have lived in Central all of our married life.
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Susan is retired now after 30+ years in the medical field. I have been working for ExxonMobil Chemical for 29 years and look forward to retiring soon.
We would like to spend more time traveling around the country as well as visiting Scotland, Ireland and many other great places.


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On the snowy turf of Waldo Stadium, Northern Illinois cooled off a hot Broncos squad with a second half comeback on Friday, Nov. 28, that included six WMU turnovers. The Huskies strong second half offense topped the Broncos, 31-21, which would have given Western an appearance in the MAC championship.
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