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BWSTL lewis and clark trail 2.20.11.wmv
If this doesn't stream well, reduce to 240p resolution
Posted by Craig on 9:53 AM Links to this post
BWSTL- Lewis and Clark Trail February 20, 2011
The first hike of the spring BWSTL Hiking Club was at the Lewis and Clark trail in St.Charles County. The temperature was 70 degrees for a February weekend. Vicky and I have hiked this 5.3 mile trail dozens of times and the turn out for the BWSTL walk was again fantastic. 30 people came out for the walk.
The whole group started at once
Looking up at the first big uphill climb
Looking Southwest
Looking Northeast
Rob ( Backwoods sales person) and his friends move along the trail
Turkey tail fungus
A: It is an Oriole feather ( I can't say I have ever seen an Oriole before)
Rick and Janine. Thanks so much for organizing these great days out in the woods
Animal Sightings:
humans, humans and more humans
Posted by Craig on 6:58 PM Links to this post
Afternoon Hike in the Snow at Broemmelsiek Park February 13, 2011
The temperature soared to 50 degrees and finally both Vicky and I were over our flu season and could go for a walk. We went over to Broemmelsiek Park, which we know well, but decided to walk it in opposite direction.
It was a nice day to be out
The snow was still deep in some spots all through the woods
The creek water was running well under the ice
Animal Tracks in the snow:
Deer, Racoon, Opposum, Coyote, ( Bear or Bobcat?)
4 miles in 1 hr 45 minutes
Vicky braking trail and crossing a creek at Broemmelsiek Park
Posted by Craig on 3:02 PM Links to this post
Milestone- 250 miles hiked in Great Smoky Mountain National Park
Official confirmation that I have hiked 250 miles of trails in the Great Smoky Mountain National Park
I didn't think that it would be that big a deal to actually have a momento of the event, after all I have the memories of all those hikes, but I may not get back to that park for some time, given the other hikes I want to do, so it does represent a very good feeling of accomplishment and lots of fond memories.
Trail Total
256.4 miles
Posted by Craig on 8:14 AM No comments: Links to this post
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text: Home Insights Back to Insights Flying with Bluefin 12 December 2017 Tweet Share Tom Bridge Digital Marketing Manager It's time to fasten your seatbelts and get ready for a drive through the digital world of SAP. Tom Bridge explains what attracted him to Bluefin and how they're helping him fly. I knew a career change was imminent. Career wise I was comfortable but I had an itch to be back in the Big Smoke; to be at the forefront of innovation, new technologies and the latest ways of working. I wanted to be back on Slack (yes we use it!), use terms like 'Digital transformation' and be able to accurately explain what it means (please don't ask!)…and that's when I landed at Bluefin. But as an obsessive marketer, I had some 'wants' for taking on my next role: I want to work in a collaborative environment. I want to learn – to get under the bonnet of the SAP world. I want to be the best – I want to be a Usain Bolt I want visibility – I need to know the direction of the business and how I'm supporting it . I need flexibility – the opportunity to work in a way that suits my style. I'm a bit fussy, aren't I? So, does this ring true? Let's explore… A truly great bunch of bananas (I mean people!) Cliché alert: I can honestly say that the people here are genuinely a great bunch; friendly, conscientious and willing to help you grow. Although we're a busy bunch, people will give you the time to work on your development – if you ask, you get. No matter whether you're at the office, working on-site or from home, the team is always online. It's an environment where you can comfortably bounce ideas around, test new ways of working and, yes, learn a thing or two. We're proud of what we've achieved but we're also perfectionists, always looking to do things better or more efficiently. For the consultants especially, who are often working across timezones and multiple locations, this way of working is critical for our success. Thought leadership The emphasis here at Bluefin is to asisst you in building your own personal brand, enabling you to establish yourself as a thought leader in your specialist field. We're all (healthily) obsessed with being the best at what we do. Right from the start, the emphasis is on growing our teams and not just with the techy stuff. We encourage our consultants to help us curate thought leadership, through our blogs, articles and industry publications. We know how brilliant they are, so we raise awareness of their expertise in the digital world. The flexibility to work at your best It's the 21st Century, or at least that's what the calendar says! At Bluefin, they really get that the old-fashioned approach to working simply doesn't cut it. Most of the team, consultants mainly, work remotely. We work across some incredible locations across Europe, Asia and America. Sunny Madrid is the next location on the horizon. Remote working is a part of the culture here and finding the right balance is key. I personally, acknowledge that I need days where I block the world out and get things done. Being able to have the flexibility to work in a different space if needed is important - especially having worked for myself in the past. Join the training train A flexible approach is not the only thing that matters. As with any career, the trick is to be agile; nimble enough to react to the latest trends, so you're not left as a dinosaur licking your wounds. SAP is no different. In fact, it's possibly one of the most rapidly changing technology companies in terms of new product development. So, we must to stay on top of the latest trends. Golden Fridays are Bluefin's way of getting all the teams together in one space and to share new thinking, watch presentations on the latest developments and a chance to eat pizza. You can't learn without pizza! I've found that with training, if you ask you usually get. Since joining I've already started working towards professional marketing qualifications supported by the company. I mentioned visibility earlier and this is a good time to bring it up. Each department, or Centre of Excellence (COE) as we call it, have regular 'town hall' meetings – a chance to catch up with the team and check on collective progress. Again, this is a chance to voice opinions about the direction of the company and to contribute to the conversation. From my experience, that's not something you usually get a chance to be involved in, so it's nice to have a chance to have my say and be listened to. Your oasis in the big city Before you consider Bluefin, I recommend you take the trip to Chiswick Park, run by the team at Enjoy Work. Their philosophy is simple – work should be a fun and stimulating place to be. Chiswick Park is set away from the city, with its very own river /stream thing, waterfall and green space. The summers here are amazing – they even lay out deck chairs for us! The Enjoy Work team run a jam-packed series of activities and events; everything from exercise classes, mindfulness courses, fireworks and even pumpkin carving. As I write, the team are currently setting up our very own winter wonderland complete with a Ferris wheel and mulled wine. What more do you need? See what you're missing out on. Why do they bother? As part of Mindtree, one of our core values is to support employee wellbeing; happier, healthier employees get the best results. Therefore, the team actively encourage engagement in the park's activities and support personal development. Being a member of the social club is yet another chance to get involved with life here at Bluefin. Each month the team organises activities and we've done everything from ice skating, sushi making, candle stick making (that's still on the agenda). It's a great opportunity to regroup and let your hair down and enjoy a beer if that's your thing. Life through blue tinted glasses Going back to my checklist, I'm pretty sure I've ticked a lot of those boxes with a whopping great big permanent marker. I was looking for a space to be provocative and to be supported by a passionate and driven team. For others, like our consultants, they are driven by innovation, changing and improving the processes of international businesses like Jaguar Landrover, Noble Industries and Surrey County Council. If you're motivated to catapult your career, here's your opportunity. Get in touch with the recruitment team today. About the author Tom Bridge Digital Marketing Manager

subset: commoncrawl


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Varian streamlines customer service with remote monitoring software
Varian Medical Systems is using Axeda's remote monitoring software to quickly and efficiently service customers all over the world.
Running a global manufacturing operation is no small task, and when the finished product can literally mean the difference between life and death, the stakes for customer satisfaction are at their highest. So when Varian Medical Systems Inc. wanted to improve its product monitoring and customer service, it turned to remote monitoring software from Axeda -- and found new ways to connect to customers around the world.
Varian, based in Palo Alto, Calif., claims to be the largest manufacturer of medical devices and software used to treat cancer. It operates plants in North America, Europe and China, selling products that are involved directly in diagnosing and treating cancer, as well as back-office processing of patient data.
When Varian began searching for a remote monitoring service 12 years ago, the company wanted a way to better connect staff and customers to experts to quickly solve problems. Prior to Axeda, Varian was using pcAnywhere for remote access. The march of progress away from analog connections to digital networks, however, rendered Varian's pcAnywhere system outdated, according to Dan DuBeau, program manager of product support engineering at Varian.
"This became more and more of a problem as our customers were needing faster and faster service," DuBeau said. "We went out looking for a solution to pcAnywhere for desktop sharing. Axeda was brand new at the game; we were their second customer."
Desktop sharing expands Varian's problem solving abilities
While the Axeda remote monitoring suite did not originally include desktop sharing, the vendor added that feature at Varian's request. "Our relationship with Axeda began with the promise of desktop sharing. It was what we really needed," DuBeau said. "If, for example, an executive is sitting at his desk in California and needs to solve a problem at one of our facilities across the world, we need to be able to share that desktop in order to solve the problem. Back-and-forth communications will break down eventually, so you really need to see what they see."
Over the last 12 years, Axeda's remote monitoring has evolved quite a bit, according to DuBeau. When Varian first adopted Axeda, the system required a customer on one end and an expert at the other sharing information to make a connection. That process has now mostly been automated, he said.
Software monitoring is another large part of how Varian is using Axeda, DuBeau noted. "Some of the most intense monitoring that we do is on our patient information side, in our SQL database," he said. "If there is an error in the database, we need to get that error at the same time the customer does, if not before."
With Axeda's help, Varian started to branch out into more "proactive" activities, such as monitoring the disk usage of databases and being able to send out a warning message when they hit 95% capacity, DuBeau explained. "Our needs have changed over the last 12 years, but what Axeda can do has also changed," he said.
Remote monitoring software cuts time, expenses for customer calls
Varian also uses Axeda remote monitoring to walk customers through product installation without the need for on-site assistance. Even in instances where hardware installations require a Varian rep to make a site visit, the post-installation monitoring of that hardware -- for service after sale and contract and warranty support -- is done remotely through Axeda, according to DuBeau.
"This has always been a customer satisfaction initiative from the beginning," he said. "A side benefit of that is that we're saving possibly $10 million a year. It's difficult to measure what we're not doing, but we know that what we're saving is significant and effective."
The savings are largely travel-based, DuBeau explained, noting that on average, a typical service call includes two hours to travel to the site, two hours at the site and two hours back. With the Axeda remote response functionality, four hours of travel time is eliminated, allowing service reps to complete three customer calls in the time it once took to complete one -- tripling productivity.
Perfecting remote customer service takes time, innovation
While the remote monitoring software is accomplishing the majority of what Varian wants, the two companies are working together to develop new features to suit Varian's changing needs, according to DuBeau. One of these works-in-progress is an ad hoc solution that Varian refers to as "zero footprint." "A customer can have a workstation that we've never seen before and call us because of trouble," said DuBeau. "We need to be able to install an agent remotely, complete the service call remotely and then wrap it up by uninstalling the remote system -- leaving their workstation exactly as it was before we came in. We only want to install the software for the purpose of the call." DuBeau said that Varian expects the zero footprint capability to be ready by the end of January next year.
One of the most significant challenges Varian has experienced, he added, is that prior to concentrating on remote service, the company had no software-embedded serialization of its products. Each installation of the patient management database looked similar save for a serial number sticker on each computer, in compliance with FDA regulations. Without embedded serialization, Varian remote reps had no way of checking the software to confirm they were servicing the correct hospital. "That was a shortcoming in the implementation of serialization of our products," DuBeau noted. "When you're doing remote support with a customer on the phone, they never know the serial number of their product, and we can't expect them to."
DuBeau explained that Varian has been dealing with this hurdle by working with its marketing and engineering departments to implement a connection to its SAP database, reaching out to its ERP system to find out where its installed base is and taking out the guesswork and human error that often occurs with manual entry of serial numbers. "Everything that goes out the door has to have some sort of software identifier so we know who we're dealing with when we connect to a customer," he said.
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text:
MADISON (WORT) — No charges will be filed against Madison Police Officer Matt Kenny who shot and killed 19-year-old Tony Robinson. Dane County District Attorney Ismael Ozanne made the much anticipated announcement Tuesday and gave details of the moments leading up to the police shooting. Dylan Brogan was there.
About Dylan Brogan
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WORT News Department


text:
Apple iOS 6 Maps vs. Google Maps 2.0 for iOS: labels & local searchAs Apple prepares its latest enhancements to Maps with iOS 7, Google has released its own major update for Google Maps on iOS. Here's how the two compare in location data and local search, our fourth segment looking at mobile maps.
Source: Apple
Introducing iOS 6.0 Maps
Using Maps Offline
2: Maps and visualizations
3: Transit directions
4: Map labels & local search
5: Routing & traffic
As previous segments in this series have detailed, Apple's new iOS 6 Maps introduced cutting edge technology related to efficient 2D and perspective vector maps, as well as 3D visualizations of satellite maps, a feature Apple calls Flyover. In these areas, Apple is well ahead of the mobile apps from Google and Nokia, the existing market leaders.
Apple's mobile iOS 6 Maps is even well ahead of Google and Nokia in the arena for 3D mapping visualizations using a full powered desktop.
Google Maps 3D still a work in progress, even on full power desktops
On top, Apple will be bringing its own desktop class Maps app to OS X Mavericks, providing larger map views without the performance constraints imposed by mobile devices.
Apple's Maps also handle offline use much more efficiently. If you do any traveling where you work outside of the network service area, being able to efficiently work offline is a major feature.
For example, after going off the mobile grid on the way to the airport in Hamburg, Germany, Google Maps for iOS gave me nothing but pixelated mess of jagged lines well before I got there, while Apple Maps properly cached enough vectors show where I was, navigate the highways and read the street labels of the major roads.
Google Maps fail rapidly, while Apple Maps continue to work for miles offline
At the same time, Apple's iOS 6 Maps doesn't provide its own transit information, relying instead on third party partner apps to deliver this. And in many reviews, Apple's new Maps have been criticized over misplaced map labels and inferior search results.
To see how well (or poorly) Apple's map labeling and location search stands up to competitors (and companion iOS 6 alternatives in the App Store, including Google Maps for iOS and Nokia Here), we conducted a series of tests to see how well each performs at finding and locating points of interest and (as will be detailed separately in a future segment) in providing driving directions and traffic information. You can skip the historical context of iOS 6 Maps below and jump to our comparison of iOS map apps on page two.
iOS 6 Maps was not Apple's first location project
Last fall's release of iOS 6 Maps wasn't Apple's first move into the business of maps and location-oriented technology. The company began laying the groundwork for location awareness and finding and labeling locations at least five years earlier. Within six months of launching the original iPhone, Apple added the beginnings of a location system to its Maps app, originally based on WiFi base station data compiled by Skyhook Wireless (because the original iPhone lacked hardware support for "real" GPS in the form of the satellite-based Global Positioning System).
In iOS 2, alongside the release of the App Store, Apple began recognizing the potential for misuse of location data and the need for protecting users' security from intentional or inadvertent mishandling by third party apps. It gave users the ability to turn location data off before it even launched the App Store, and forced app developers to ask the user for permission before accessing the device's location for any reason.
In iOS 3.0, Apple added remote location capabilities for MobileMe's new "Find my iPhone" feature, similarly protected via a user-secured account. It also introduced Core Location, a framework that enabled third party apps to develop turn by turn navigation apps.
In parallel, Apple also launched a new version of iPhoto in early 2009 that added geolocation awareness to photos on the desktop, a feature it named Places. Beyond just reading the geotag metadata of a photo and plotting it on a map, iPhoto also performed reverse lookup of place names from the latitude and longitude data, as Apple's Phil Schiller demonstrated at Macworld Expo. While Apple continued to use Google's map data, it was beginning to develop its own location database for Places.
At the same time, Apple also began acquiring location based mapping companies, including Placebase in July 2009. That startup's employees had been working on maps product before Google brought its own Maps to the web for free; Placebase then changed direction to offer a more customizable product to layer "private and public data sets" together. Former Placebase employees subsequently joined the "Geo Team" at Apple to work on a series of location-based initiatives in iOS and OS X, and in apps like iPhoto.
Apple continued hiring staff to work on maps, with one job posting in late 2009 noting, "we want to take Maps to the next level, rethink how users use Maps and change the way people find things. We want to do this in a seamless, highly interactive and enjoyable way. We've only just started."
By 2010, most of Apple's iPhone installed base now had GPS, but Apple's other products, including the iPod touch, all WiFi-only iPads and Macs, still relied on WiFi location. That spring, Apple began providing its own location information in place of data from either Skyhook or Google. It also began supporting iPhoto Places on iOS 3.2 for iPad.
And that summer, Apple released iOS 4 with additional security features related to location services, which it also enhanced to "define geographic regions and monitor when the user crosses the boundaries of those regions," a technology referred to as geofencing.
Apple also enhanced its Map Kit framework in iOS 4, adding support for map overlays to "provide a way to create more complex types of annotations. Instead of being pinned to a single point, an overlay can represent a path or shape that spans a wider region." Apple told developers, "you can use overlays to layer information such as bus routes, election maps, park boundaries, and weather maps on top of the map."
The company also acquired Poly9, a Canadian company that produced mapping products used by companies including Skype, which was later purchased by Microsoft. And through the end of 2010, Apple continued to hire new engineers related to mapping services, asking for candidates with "deep knowledge of Computational Geometry or Graph Theory" and posting listings related to "distributed image processing on a server cluster."
In 2011, the company announced in April that it was "collecting anonymous traffic data to build a crowd-sourced traffic database." The collected data sparked a controversy after users discovered caches of large amounts of data that were being unnecessarily backed up to iTunes, issues that Apple addressed with an iOS update in May.
Apple subsequently introduced iOS 5, which added new support for geofencing in location-based Reminders, a feature that later made its way to the Mac. This feature was an application of the Location Services and Map Kit enhancements Apple had first introduced in iOS 4.
Maps-gate, right out of the gate
When iOS 6 arrived, it should not have been huge surprise that Apple was now serving its own maps within the app, rather than Google's. At the same time, however, a variety of features that users commonly associated with Google Maps were actually Apple's own work.
This included the original iOS Maps user interface (which Apple patented) and the Map Kit frameworks developers use to interact with it and to locate places and draw points of interest, bus routes, arbitrary regions and other annotations on a map. Developers were fully aware of this, but many users did not seem to be.
However, after years of internal work, the new iOS 6 Maps was released last fall without full and official recognition by Apple that some elements of the new product were brand new and still being completed, particularly its Flyover 3D satellite renderings, which were only finished in a variety of U.S. cities.
Critics, unaware of Apple's years of work invested in Maps (many of whom apparently thought that that iOS 5 Maps was entirely a Google product) virtually ignored all of the new product's innovations to focus on four things: Flyover glitches in areas that hadn't been fixed (such as the Brooklyn Bridge), the lack of Google's Street View, the lack of integrated transit data, and a variety of improperly located or otherwise incorrect points of interest.
Maps, a primary strategic product for Apple in iOS 6, rapidly became one of the most ridiculed and dismissed products in recent history. Pundits' contempt for the new Maps grew so loud and scathing that Apple's chief executive Tim Cook issued an apology for any inconvenience to users and recommended a variety of alternative mapping tools in the App Store and on the web for iOS 6 users.
It has since been widely reported that Scott Forstall, then acting as the head of iOS development, refused to make the public apology himself. That was said to be the last straw in what has been characterized to be a series of contentious management issues, and resulted in Forstall being stripped of his responsibilities. Management of the Maps team has now been delegated to Eddy Cue, who runs the rest of Apple's online services including iCloud, iTunes and the App Store, and the Siri service integrated into Maps.


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Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS) toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN) Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.
Citation: Johnson C, Kannan TR, Baseman JB (2011) Cellular Vacuoles Induced by Mycoplasma pneumoniae CARDS Toxin Originate from Rab9-Associated Compartments. PLoS ONE 6(7): e22877. https://doi.org/10.1371/journal.pone.0022877
Editor: Stefan Bereswill, Charité-University Medicine Berlin, Germany
Received: May 11, 2011; Accepted: July 6, 2011; Published: July 29, 2011
Copyright: © 2011 Johnson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by an award number U19AI070412 from the National Institute of Allergy and Infectious Diseases and The Kleberg Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Mycoplasma pneumoniae, the etiologic agent of primary atypical pneumonia, is associated with a wide variety of respiratory infections ranging from tracheobronchitis and pharyngitis to extrapulmonary infections, including joint and central nervous system disorders [1]. It is believed to be responsible for 20–30% of all community-acquired pneumonia and is also linked to acute and chronic airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease [2], [3], [4], [5]. M. pneumoniae is generally considered an extracellular pathogen that adheres to mucosal epithelial surfaces, although it has the capability to replicate and survive intracellularly for extended periods [6], [7]. Cytadherence of M. pneumoniae to host cells is a prerequisite for colonization and infection, which is primarily achieved through an assemblage of proteins localized at its tip organelle [8], [9], [10]. However, alternate mechanisms of adherence to host cells have been reported by us and are likely to be physiologically relevant [11], [12]. Following mycoplasma infection, the cytopathology that M. pneumoniae elicits has historically been attributed to several factors, including hydrogen peroxide and superoxide radicals generated by mycoplasma metabolism, nutritional parasitism, and the host inflammatory responses [13], [14], [15], [16]. Earlier, we proposed that only metabolically active mycoplasmas could introduce toxic factors into respiratory cells resulting in tissue injury [14].
Recently, we described a unique M. pneumoniae ADP-ribosylating and vacuolating cytotoxin designated CARDS toxin [17] that shares sequence similarity to the known ADP-ribosylating toxin, Bordetella pertussis pertussis toxin S1 subunit, and exhibits an ADP-ribosyltransferase activity in the presence of host cell proteins [12], [17]. Interestingly, rCARDS toxin elicited a distinct vacuolating phenotype in cultured mammalian cell lines, which was not exclusively dependent upon ADP-ribosylating activity [17]. CARDS toxin is the first ADP-ribosyltransferase described that exhibits both ADP-ribosyltransferase and vacuolating activities. rCARDS toxin-induced vacuolation appears particularly relevant to the pathogenic process, as histopathology associated with M. pneumoniae infection includes cellular vacuolation, marked deterioration of ciliary movement, and ultimately cell death [15], [18], [19]. Furthermore, baboon tracheal rings exposed to rCARDS toxin exhibited a vacuolated appearance, in addition to a reduction of ciliary movement and loss of cellular integrity of the respiratory epithelium [17]. Importantly, we observed characteristic vacuolation of the bronchial epithelium in rCARDS toxin-treated mice [20].
Toxin-mediated vacuole formation has been extensively examined in Helicobacter pylori [21]. Its vacuolating cytotoxin, designated VacA, induces vacuole formation upon acid activation and in the presence of weak bases, like ammonium chloride [22], [23], [24]. Cellular uptake of the acidotropic dye, neutral red, revealed that the intralumenal environment of VacA-induced vacuoles is acidic, and further studies determined the endosomal origin of these vacuoles [25], [26], [27], [28]. The vacuolar membranes of VacA-induced vacuoles were enriched in markers for late endocytic compartments, including lysosome associated membrane protein 1 (LAMP1) and the GTPase, Rab7, but not markers for early endocytic compartments [28]. Rab7 GTPase enrichment onto vacuolar membranes was further linked to its role in vacuole biogenesis [29]. Rab GTPases are key regulators of vesicle traffic in eukaryotic cells and also play an important role in membrane fusion events by recruiting, tethering and docking cellular factors to target membranes. Rab GTPases delineate membrane composition and identity, as they recruit specific effectors to restricted membrane microdomains [30], [31]. Rab5, a protein associated with the plasma membrane and early endosomes, regulates endocytosis at early stages [32]. Rab7 regulates transport from early endosomes to late endosomes [33] or from late endosomes to lysosomes [34] and is associated with vesicular-tubular structures that develop from the perinuclear area to the cellular periphery. Rab9 regulates membrane cycling between late endosomes and the trans-Golgi network (TGN) and is mostly localized to the perinuclear region [35], [36]. While the literature regarding the role of Rab GTPases in membrane trafficking is continually evolving, it has been proposed that VacA-induced vacuolation alters membrane traffic at the late endosomal stage, or inhibits transport, directly or indirectly, between late endosomes and lysosomes [29].
Vacuolation appears to be closely linked to M. pneumoniae infection and the presence of CARDS toxin [17], [20]. As CARDS toxin does not share significant sequence similarity with known vacuolating toxins, including VacA, we decided to investigate the formation and composition of vacuoles generated by rCARDS toxin. Using a panel of markers that distinguish vacuole properties, we confirmed the association of a specific, unexpected Rab GTPase with rCARDS toxin-induced vacuoles. Based on these results we propose a novel mechanism of rCARDS toxin-mediated, late endosomal vacuole biogenesis.
Characterization of vacuole formation in rCARDS toxin-treated mammalian cells
As rCARDS toxin-induced vacuolation of CHO cells was previously demonstrated [17], we analyzed other mammalian cell lines in order to compare the specificity and degree of vacuolization. First, we examined human lung carcinoma epithelial A549 cells, as M. pneumoniae is a respiratory pathogen, and rCARDS toxin-induced vacuolation was readily evident (Fig. 1). Other epithelial cell lines examined included kidney (Vero) and cervical (HeLa) cell lines, which exhibited similar vacuolization patterns. rCARDS toxin-mediated vacuolization was dose and time-dependent in all cell lines tested with vacuole formation occurring between 8 to 10 hours at rCARDS toxin concentrations ranging from 5 to 75 µg ml−1. Nascent vacuoles first appeared around the perinuclear region, followed by increased numbers and sizes of vacuoles. Within 24 h smaller vacuoles coalesced into larger vacuoles which could occupy the entire cell. As the vacuoles grew in size, they retained more neutral red, indicating their acidic nature and providing a method to quantify rCARDS toxin-induced vacuolation (Fig. 1). When we examined rCARDS toxin-induced vacuole formation in the presence of the V-ATPase inhibitor, bafilomycin A1, decreased neutral red uptake (NRU) correlated with increased concentrations of bafilomycin A1 (Fig. 1). This observation indicated that rCARDS toxin-mediated vacuolation was dependent on V-ATPase enzymes, which are found on the membranes of intracellular organelles, like endosomes and lysosomes. Similarly, decreased NRU was observed in the presence of the ionophore, monensin, reinforcing the acidic intracellular pH of the vacuoles and suggesting an endocytic origin of vacuole formation (Fig. 1).
(A) A549, HeLa, CHO, and Vero cell monolayers were treated with rCARDS toxin for 24 h at 37°C and images captured Neutral Red Uptake (NRU) by rCARDS toxin-induced HeLa cell vacuoles was observed. Original magnification: ×400. (B) HeLa cells were treated with rCARDS toxin for 24 h at 37°C, and representative images of neutral red-treated cells were compared. Note the diversity of vacuole sizes. (C) HeLa cells pretreated with varying concentrations of bafilomycin A1 or monensin were incubated with rCARDS toxin for 24 h at 37°C. Neutral red in vacuoles was quantified by OD550 nm and expressed as percentage of control cells. All values are the mean of two different experiments run in triplicate ± SD (DeltaGraph).
rCARDS toxin-induced vacuoles originate along the endocytic pathway
rCARDS toxin-induced vacuoles in HeLa cells accumulate the extracellular fluid-phase marker, Lucifer yellow, indicating that these vacuoles derive from the endocytic pathway (Fig. 2). Fluorescence intensity varied among rCARDS toxin-induced vacuoles, with the majority of smaller vacuoles (98%) exhibiting the most intense fluorescence. In the presence of the cytoskeletal inhibitors, nocodazole and colchicine, toxin-induced vacuolation was markedly reduced at 24 h, as NRU measurements were 4% and 12% of toxin-treated 'control' cells, respectively.
HeLa cells were intoxicated with rCARDS toxin in the presence of 500 µg ml−1 Lucifer yellow in MEM medium for 24 h at 37°C. Cells were fixed and visualized by visible light and fluorescence microscopy. (A) DIC; (B) Fluorescence; and (C) Merged image. Lucifer yellow is clearly discernible in a subpopulation of vacuoles (Panel C; see arrows). Original magnification: ×400.
Enrichment of the late endosomal Rab9 GTPase around rCARDS toxin-induced vacuoles
As rCARDS toxin-induced vacuoles appear to derive from the endocytic pathway, we examined individual vacuoles for the presence of endosomal and lysosomal markers including Rab5, EEA1, Rab7, Rab9, cathepsin D, LAMP1 and LAMP2 using indirect immunofluorescence. The early endocytic specific markers, Rab5 GTPase and EEA1, were distributed throughout the vacuolated cells in a dispersed pattern with no evidence of selective association with rCARDS toxin-induced vacuoles (Fig. 3). Also, vacuolar membranes did not exhibit discernible selective labeling with the late endosomal GTPase, Rab7. Interestingly, we observed intense enrichment of the late endosomal Rab9 GTPase, as well as LAMP1 and LAMP2, around rCARDS toxin-induced vacuolar membranes (Fig. 3). Since LAMP1 and LAMP2 are known to shuttle between lysosomes, endosomes and the plasma membrane, and since we did not observe an association of rCARDS toxin-induced vacuolar membranes with the lysosomal hydrolase cathepsin D (data not shown), we focused on the late endosomal-specific Rab9 GTPase. We examined whether the enriched Rab9 GTPase around rCARDS toxin-induced vacuoles was due to its overexpression or redistribution by comparing it with another late endosomal marker, Rab7. rCARDS toxin-treated and untreated HeLa cells were analyzed by immunoblot using anti-Rab9 antibodies (Fig. 4). We did not observe increased expression of Rab9 (or Rab7 as negative control) in vacuolated cells at different time points, indicating that the accumulation of Rab9 around rCARDS toxin-induced vacuoles was likely due to redistribution, rather than its increased synthesis during intoxication and vacuole development.
Untreated and rCARDS toxin-treated HeLa cells were incubated for 24 h at 37°C and processed as described in Materials and Methods. Representative DIC and fluorescence images of untreated and intoxicated cells are shown. Note that Rab9, LAMP1 and LAMP2 antibodies react with perinuclear region-associated vesicles in untreated cells (left panels) while in intoxicated cells Rab9, LAMP1 and LAMP2 localize and concentrate around specific vacuoles (right panels). Original magnification: ×400.
HeLa cells were intoxicated with rCARDS toxin and incubated for 12 or 24 h at 37°C. rCARDS toxin-treated and untreated cell extracts were separated on NuPAGE Bis-Tris 4–12% gels and transferred to nitrocellulose membranes. A) Immunoblot analysis was performed with primary antibodies against Rab9 (mouse) and Rab7 (rabbit) and secondary goat anti-mouse and goat anti-rabbit antibodies conjugated to alkaline phosphatase. B) Quantification of Rab7 and Rab9 band intensities was performed by scanning immunostained bands and analyzing images with KODAK Image software.
Distribution and localization of Rab9 at rCARDS toxin-induced vacuolar membranes
Early rCARDS toxin-induced vacuoles were generally delineated by a single bounding membrane, which supported their endocytic origin. Vacuolar fusion events were also observed, accounting for the large size of several vacuoles (5 µm) and leading to their occasional occupation of the entire cell. Consistent with the indirect immunofluoresence results, immunogold-labeled Rab9 was clearly embedded around the margins of CARDS toxin-induced vacuoles (Fig. 5), while immunogold-labeled Rab7 was rarely associated with the vacuolar membranes (Fig. 5). This observation reinforced the specificity of Rab9 binding to toxin-induced vacuoles. Also, gold-conjugated secondary antibodies did not bind to vacuolar membranes, which further supported the unique vacuolar association of Rab9 (data not shown).
CARDS toxin-treated and untreated HeLa cells were incubated for 24 h at 37°C, then labeled with primary antibodies reactive against Rab9 (mouse) or Rab7 (rabbit), followed by goat anti-mouse or goat anti-rabbit IgG gold complex (10 nm), respectively. Representative circles indicate heavy concentration of immunogold labeled Rab9 particles localized to vacuolar membranes in rCARDS toxin-treated cells (A), which are absent in untreated controls. In contrast to Rab9, Rab7 is observed very rarely around the vacuoles in toxin treated vesicles (B). Bar, 100 nm. Original magnification: ×80,000.
Rab9 is required for rCARDS toxin-induced vacuole formation
To further confirm the association of rCARDS toxin-induced vacuoles with Rab9, HeLa cells were transiently transfected with either green fluorescent-tagged Rab9 or Rab7 GTPases. We observed the clustering of GFP-Rab9 around vacuolar membranes in intoxicated cells, in contrast to non-intoxicated HeLa cells where GFP-Rab9 was distributed evenly throughout the cytoplasm (Fig. 6). To further delineate the role of Rab9 in vacuole formation, HeLa cells were transiently transfected with mCherry-tagged DN-Rab9. The majority of cells expressing DN-Rab9 lacked visible vacuole development (Fig. 6) supporting the role of Rab9 in rCARDS toxin-mediated vacuole formation. Conversely, HeLa cells expressing DN-Rab7 behaved similarly to toxin-treated, non-transfected cells. In other words, DN-Rab7 transfected cells exhibited rCARDS toxin-induced vacuolation (Fig. 6), which is consistent with the minor or indeterminate role of Rab7 in toxin-induced vacuoles.
HeLa cells were transiently transfected with wild-type pEGFP-Rab9 or pDsRed DN-Rab9 or pDsRed DN-Rab7 and incubated for 24 h at 37°C before addition of rCARDS toxin for an additional 24 h. rCARDS toxin-treated and untreated HeLa cells were analyzed by light and fluorescence microscopy. (A, B) Representative DIC and fluorescence images of untreated HeLa cells transfected with WT pEGFP-Rab9 (C, D) Representative DIC and fluorescence images of rCARDS toxin-treated HeLa cells transfected with WT pEGFP-Rab9. (E, F) Representative fluorescence and fluorescence+DIC merged images of rCARDS toxin-treated HeLa cells transfected with pDsRed DN-Rab9. Note the absence of vacuole formation in DN-Rab9 transfected cells (white arrows and dispersed fluorescence). Also, note distinct vacuoles in non-transfected cells (black arrows and no fluorescence; F). (G, H) Representative fluorescence and fluorescence+DIC merged images of rCARDS toxin-treated HeLa cells transfected with pDsRed DN-Rab7. Note cells transfected with pDsRed DN-Rab7 exhibit distinct vacuoles (H). Original magnification: ×400.
M. pneumoniae infection is associated with cellular vacuolation, marked deterioration of ciliary movement and ultimately cell death [4], [15]. Interestingly, rCARDS toxin can substitute for live M. pneumoniae organisms in eliciting the characteristic airway histopathology in intoxicated mice [20] and tracheal organ cultures [17]. Similarly, rCARDS toxin induces extensive vacuole formation in vitro in cultured mammalian cell lines (Fig. 1). These vacuoles initially form around the perinuclear region, fuse, and grow in size until much of the cytoplasm is occupied by a single vacuole or several large vacuoles (Fig. 1). This process would require the progressive addition of membranes, which is consistent with the alteration of membrane trafficking along the endocytic-endosomal pathway. As bacterial toxins have been extremely useful in the study of different aspects of cell biology, the aim of this study was to characterize and elucidate the ontogeny of rCARDS toxin-induced vacuoles.
We observed rCARDS toxin-mediated vacuole formation in multiple cell lines and in a concentration-dependent manner. Similar to VacA, rCARDS toxin-induced vacuoles accumulate neutral red, indicating the acidic nature of the vacuolar lumen [27]. rCARDS toxin distinguishes itself from VacA, as it does not require acid activation or weak bases to induce vacuolation. However, rCARDS toxin-induced vacuolization is prevented by the vacuolar ATPase inhibitor, bafilomycin A1 [37], similar to VacA (Fig. 1). Both bafilomycin A1 and monensin (Fig. 1) are reported to alter intracellular trafficking by preventing early endosome acidification along the endocytic pathway [38], [39] The mechanisms by which both bafilomycin A1 and monensin inhibit rCARDS toxin-mediated vacuole formation are unclear, although similar observations were reported with Escherichia coli subtilase cytotoxin [40].
Endosomal origin of rCARDS toxin-induced vacuoles
The impermeable fluorescent dye, Lucifer yellow, has been used as a marker for fluid phase endocytosis in mammalian cells and Saccharomyces cerevisiae [41]. The accumulation of Lucifer yellow within rCARDS toxin-induced vacuoles, especially with the smaller vacuoles (Fig. 2) and the retention of neutral red (Fig. 1) is similar to the H. pylori VacA vacuoles [28], revealing their endosomal origin. As cytoskeletal structures are essential for a number of cellular functions, including intracellular transport of endosomes and lysosomes, we analyzed the effect of nocodazole and colchicine on CARDS toxin-induced vacuole formation. These agents prevent polymerization of microtubules and, therefore, depress membrane traffic from early endosomes to late endosomes. As noted earlier, rCARDS toxin-induced vacuolization was markedly reduced by these drugs, further supporting the endocytic nature of these vacuoles [42], [43], [44].
In both untreated and rCARDS toxin-treated cells EEA1 localized to multiple small early endosomal vesicles distributed throughout the cytoplasm with no obvious selective enrichment to toxin-induced vacuoles (Fig. 3). In contrast, we demonstrated that rCARDS toxin-induced vacuolar membranes concentrated Rab9, a small GTPase primarily associated with late endosomes (Fig. 3) [45]. Moreover, we showed an association of LAMP1 and LAMP2 with rCARDS toxin-induced vacuolar membranes. Although LAMP1 and LAMP2 proteins are mainly associated with lysosomes, they also shuttle between endosomes and the plasma membrane [46]. These data favor the possibility that rCARDS toxin-induced vacuoles are derived from late endosomes and that rCARDS toxin may be altering membrane trafficking at this stage. As seen by the neutral red uptake and light microscopy, it is clear that rCARDS toxin vacuoles originate from the perinuclear region and are enriched with Rab9, LAMP1 and LAMP2.
Rab9 plays an essential role in rCARDS toxin-induced vacuole formation
Interestingly, we did not observe the clustering of another small GTPase associated with late endosomes, Rab7. This finding is in contrast to VacA-induced vacuoles, which contained Rab7 [28], [47]. The functional involvement of Rab9 on M. pneumoniae rCARDS toxin vacuole formation was tested by overexpression of DN-Rab9 mutants, which resulted in a significant decrease in rCARDS toxin-induced vacuole development (Fig. 6). These data suggest that the extensive fusion of Rab9-containing late endosomes appears to be crucial for the formation of rCARDS toxin-induced vacuoles. As DN-Rab7 did not interfere with the ability of rCARDS toxin to form vacuoles, in contrast to overexpressed DN-Rab9 (Fig. 6), Rab7 does not appear to play a meaningful role in rCARDS toxin-induced vacuolation. Notably, the late endosomal Rabs, Rab7 and Rab9, have different biological functions and are not always present on the same endosome [45]. Since rCARDS toxin selectively associates with the late endosomal Rab9 GTPase, but not the Rab7 GTPase, we suggest that rCARDS toxin may affect selective subpopulations of late endosomes that are Rab9 positive and Rab7 negative.
In this study we clearly observed the association of Rab9 with rCARDS toxin-induced vacuoles. There is increasing information that viruses and intracellular bacterial pathogens hijack Rab GTPases in order to enter cells through the endocytic pathway, or recruit membranes from the ER or the Golgi apparatus to form replicative intracellular niches [31]. The functions of the Rab GTPases can be modulated by these pathogens to allow their intracellular growth. Numerous studies have shown that selective inclusion or retention of Rab GTPases on vacuolar membranes regulates the biogenesis of phagosomes inhabited by several bacteria. H. pylori inhibits phagosome maturation by localizing Rab7 to the bacterium-containing phagosome, while mycobacteria secrete factors that prevent the acquisition of Rab7 by the phagosome, leading to enhanced intracellular survival [30], [48], [49]. Salmonella typhimurium acquires multiple Rabs within Salmonella-containing vacuoles and Salmonella-induced filaments, including Rab7 and Rab9, in order to block fusion with lysosomes and survive intracellularly [50]. Further, it has been found that Rab9 GTPases are key components for replication of several unrelated enveloped viruses, including HIV1, Ebola, Marburg and measles [51]. Since viral assembly initiates at late endosomes for enveloped viruses and viral budding is completed at the plasma membrane, it is hypothesized that these viruses depend on Rab9 to transport viral proteins to the TGN and out to the plasma membrane [51]. As previously mentioned, M. pneumoniae is able to establish an intracellular residence, thereby contributing to persistence and chronic airway infections. Whether M. pneumoniae utilizes Rab9 in order to achieve this intracellular residence requires further clarification. Alternatively, the endogenous proteins, TGN38 and mannose-6 phosphate-receptor, shuttle between the late endosomes and the TGN, and bacterial toxins, including Pseudomonas aeruginosa exotoxin A, have also been shown to utilize the retrograde transport pathway through Rab9 to access the ER [35], [52]. rCARDS toxin may utilize a similar pathway by recruiting Rab9.
With over 70 Rabs identified in the human genome, much remains to be understood regarding the role of these regulatory GTPases in intracellular trafficking, endosomal and phagosomal organelles and microbial pathogenesis. Interestingly, toxins have been helpful in the study of endocytosis and intracellular transport [53]. Future studies with rCARDS toxin may define the molecular mechanisms by which this unique toxin exploits Rab9 vesicles for vacuole formation and delineate how Rab9 connects to various components of the transport machinery, such as cargo adaptors, molecular motors, vesicle fusion protein and other Rab GTPases.
As mentioned earlier, hallmarks of M. pneumoniae infection are characteristic and programmed injury to respiratory cells with accompanying histopathology and inflammatory responses. As it became evident that rCARDS toxin was sufficient in replacing live M. pneumoniae in the production of vacuolization, we focused on understanding toxin-mediated vacuolating pathways. Here, we demonstrate that rCARDS toxin-induced vacuoles originate from late endosomal compartments and recruit the small GTP-binding protein, Rab9, which distinguishes CARDS toxin from VacA. Further investigation into the origin and mechanisms of CARDS toxin-induced vacuolation may clarify the role of M. pneumoniae and its ADP-ribosylating and vacuolating CARDS toxin in infection and disease progression.
Materials and Methods
Chemical reagents and antibodies
Neutral red, bafilomycin A1, colchicine, nocodazole, and monensin were purchased from Sigma and Lucifer yellow from Molecular Probes (Invitrogen). Antibodies reactive against EEA1, Rab7, cathepsin D, LAMP1, and LAMP2 were obtained from Abcam. Rab 5 antibody was purchased from Cell Signaling Technology and Rab 9 antibody from Novus Biologicals. Alexa Fluor 488 goat anti-mouse and goat anti-rabbit affinity-purified antibodies were purchased from Invitrogen.
Expression and purification of rCARDS toxin
rCARDS toxin was expressed in BL21 DE3 (LpxM) [54], purified using nickel column chromatography (Qiagen), and desalted using PD10 desalting columns (GE Healthcare) as previously described by us [17]. Similarly expressed and purified M. pneumoniae recombinant EF-Tu and recombinant pyruvate dehydrogenase ß subunit proteins were used as negative controls [17].
Mammalian cell growth conditions
HeLa (CCL-2), Vero (CCL-81), CHO-K1 (CCL-61), and A549 (CCL-185) cells were obtained from the American Type Culture Collection (ATCC). HeLa and Vero cells were passaged in Minimal Essential Medium (MEM) (ATCC) with 10% fetal bovine serum (FBS) (Atlas Biologicals). CHO-K1 (CCL-61) and A549 (CCL-185) cells were passaged in F-12K medium (ATCC) with 10% FBS. All cell lines were maintained at 37°C in 5% CO2 in 75 cm2 flasks (Corning).
rCARDS toxin-induced vacuolation of mammalian cell lines
Mammalian cell lines were grown in T25 cm2 flasks (Corning) and incubated in the presence or absence of rCARDS toxin at varying concentrations and time intervals at 37°C. Cell vacuolation was visualized using inverted light (Olympus CK 40) or differential interference contrast (DIC) microscopy (Carl-Zeiss Cell Observer).
Quantification of rCARDS toxin-induced vacuolation by NRU
The NRU assay was performed as described with slight modification [55]. Briefly, HeLa cells were seeded in 96-well plates at a concentration of 4×104 ml−1 for 24 h, before addition of rCARDS toxin at varying concentrations (5 µg to 75 µg ml−1) for different time points. For specific experiments, HeLa cells were pre-incubated with nocodazole (250 ng ml−1) and colchicine (10 µM) for 1 h before addition of 50 µg ml−1 rCARDS toxin for 24 h at 37°C. Then, MEM culture medium was aspirated from HeLa cells, and 100 µl of neutral red solution (40 mg ml−1 neutral red prepared in phosphate buffered saline [PBS pH 7.4], filter sterilized and diluted 1∶100 in MEM medium) were added to each well. Incubation was continued for 2 h at 37°C; cells were washed with PBS; neutral red was extracted with 150 µl of destain solution (50% ethanol, 49% distilled water, and 1% glacial acetic acid); and OD550 nm was determined using a microtiter plate reader spectrophotometer (Opsys MR, Dynex Technologies Inc.). NRU was also measured in the presence and absence of monensin and bafilomycin A1.
Cytochemical staining of rCARDS toxin-induced vacuolation
Movement and localization of Lucifer yellow carbohydrazide into vacuoles were assessed by incubating HeLa cells in the presence of 75 µg ml−1 rCARDS toxin and 500 µg ml−1 Lucifer yellow in MEM medium for 24 h at 37°C. Cells were washed with PBS, fixed in 2% paraformaldehyde (methanol free, Electron Microscopy Sciences) in PBS, and visualized with DIC and 488 filter sets using a Carl Zeiss Cell Observer Z.1 microscope operated by a multichannel acquisition system (AXIO Vision ver.4.7.2). Small and large vacuoles enriched with Lucifer yellow were counted (>500 vacuoles counted) to analyze the percentage of vacuoles associated with Lucifer yellow.
Immunofluorescence analysis of endosomal membrane markers localized to rCARDS toxin-induced vacuoles
HeLa cells were seeded in 4-chamber glass slides (Lab-Tek II Chambered #1.5 Borosilicate Coverglass, Fisher) at a concentration of 5×104 cells/chamber 24 h before intoxication. Then, cells were incubated in the presence or absence of 75 µg ml−1 rCARDS toxin for 24 h at 37°C. Cells were washed once with PBS, fixed in 2% paraformaldehyde (methanol free) in PBS for 15 min at room temperature (RT), washed with PBS; and permeabilized in 0.2% Triton X-100 in PBS for 5 min. Cells were again washed in PBS with 1% normal goat serum (NGS, GIBCO) and incubated with primary antibodies diluted 1∶100 (EEA1, Rab 5, Rab 7, Rab 9, Cathepsin D, LAMP1, and LAMP2) in PBS with 0.2% NGS for 1 h. Cells were washed with PBS containing 0.2% NGS and incubated with secondary antibodies diluted 1∶500 (Alexa Fluor 488 goat anti-mouse or goat anti-rabbit) in PBS with 0.2% NGS for 1 h at RT. Cells were washed again with PBS and visualized with DIC and 488 filter sets using a Carl-Zeiss Cell Observer Z.1 microscope.
Comparative immunoblot analysis of endosomal membrane markers in rCARDS toxin-treated and untreated HeLa cells
HeLa cells grown in T25 cm2 flasks were incubated in the presence or absence of 75 µg ml−1 rCARDS toxin for 24 h at 37°C. After visual detection of vacuolation by light microscopy, HeLa cells were washed and scraped into PBS, and protein concentrations were determined by bicinchoninic acid (BCA) quantitation (Pierce). Intoxicated and non-intoxicated HeLa cells were boiled in SDS-PAGE loading buffer, and total cell protein (2 µg/well) was separated by gel electrophoresis on 4–12% NuPAGE gels and transferred to nitrocellulose membranes. Membranes were blocked in 5% blotto in Tris-Buffered Saline-Tween-20 (TBST) for 1 h at RT. Primary antibodies were diluted 1∶100 in 3% blotto in TBST and incubated with membranes for 1 h. Secondary goat anti-rabbit (Zymed) and goat anti-mouse antibodies (Zymed) were diluted in TBST at 1∶3000.
Immunoelectron microscopic analysis of rCARDS toxin-treated HeLa cells
HeLa cells grown in T25 cm2 flasks were intoxicated with 75 µg ml−1 rCARDS toxin or buffer alone and incubated overnight at 37°C. HeLa cells were washed with PBS and fixed with 1% glutaraldehyde/4% formaldehyde for 2 h at RT. Then, cells were scraped into fixative and sections prepared and mounted on Formvar-coated nickel grids. Individual grids were blocked with 100 mM Tris-HCl containing 1% BSA and 1% NGS and incubated with antibodies reactive against Rab7 (rabbit polyclonal; 1∶10 dilution) or Rab9 (mouse monoclonal; 1∶10 dilution) in PBS containing 1% BSA. After three washes in PBS with 1% BSA, grids were incubated with secondary goat anti-rabbit 20 nm gold particles (Rab7) and goat anti-mouse 10 nm gold particles (Rab9) antibodies diluted 1∶10 in PBS with 1% BSA. Individual grids were analyzed with a JEOL 1230 transmission electron microscope at an 80-kV accelerating voltage after staining with 7% uranyl acetate followed by Reynold's lead citrate.
Transient transfection of wild-type (WT) and dominant-negative (DN) Rab7 and Rab9 in HeLa cells
HeLa cells were seeded in 4-chamber glass slides at a concentration of 5×104 cells/chamber for 24 h at 37°C prior to transfection. Cells were either transfected with pEGFP-rab7 WT (Addgene plasmid 12605), pEGFP-rab9 WT (Addgene plasmid 12663), DsRed-rab9 DN (Addgene plasmid 12676), or DsRed-rab7 DN (Addgene plasmid 12662) [56] using FuGENE 6 transfection reagent (Roche) according to manufacturer's instructions. Twenty-four h after transfection, cells were intoxicated with 75 µg ml−1 rCARDS toxin, incubated for another 24 h, and washed with PBS. Cells were visualized with DIC and 488 or 633 filter sets using a Carl Zeiss Cell Observer Z.1 microscope.
Author Contributions
Conceived and designed the experiments: CJ TRK JBB. Performed the experiments: CJ. Analyzed the data: CJ TRK JBB. Wrote the paper: CJ TRK JBB.
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- 56. Choudhury A, Dominguez M, Puri V, Sharma DK, Narita K, et al. (2002) Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J Clin Invest 109: 1541–1550.


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Should I choose a custom website or should I choose an open source CMS - that is the question. Customers must make a decision between building a custom website and using of CMS, before they come to us.
There are many benefits to both. Only if you know your needs you can really establish which one will work best for you. For most businesses, budget is the key factor in deciding between the two, but let's see what are the advantages and disadvantages of both approaches.
Content Management Systems (CMS) such as WordPress, Drupal or Joomla! have become very popular in recent times. Content Management Systems give more power to the regular people, who don't understand programming languages. They are almost free to use and this means that the final price for the website building will be quite low. You don't have a big budget for different modules and extensions, because they are already developed. Community development takes care about providing new features, options, plug-ins, themes and most updates are free.
That sounds great, isn't it?
But let's imagine that you have today your BIG idea for your website, you choose one of the best working content management systems, find a theme that meets your needs and your new website is ready. You get the job done with a smaller budget based on your needs. You will decide tomorrow you want something new on your website, but it turns out that what you are looking for is new to the internet and you can't integrate it into your site. It turns out that your CMS don't support similar extension or it doesn't work the way you want. You may have to pay more to someone, who must research and customize, and that will cost even more time. In this case you want your website to meet your requirements, you need to do some customization, but your developer may not be able to change everything.
Open source CMS are not unlimited, as the built from scratch websites are. If you choose a CMS you will have to follow the existing protocols and architecture when building your website which takes more time.
All this comes to show that the custom solution may be a better solution in the long term.
Custom Website
Custom designed and developed websites are preferred because of their uniqueness. Your business deserves a unique online presence and the custom website will always represent who you are and what you do.
The most important things are the powerful administrative panel with quick and intuitive control over the information on the website and the freedom that the customer has when he choose the website design.
All the features and functionality of the custom websites turn your website into an asset of your business. You will have a website that will set you apart from your competition.
When you choose a custom solution you build something without architectural limitations. The custom websites allow the developer to build the website around the content you have. All the customer wants is possible.
New functions or abilities are possible any time, but you will have to pay your developer to update the website. This means that the website will be more expensive and the development process will take a little longer.
If your website needs some custom functionality, need to be competitive and unique, building a custom website might be a better choice.


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Final Fantasy VII, Final Fantasy VIII, Final Fantasy IX, Final Fantasy XII
Final Fantasy VII
Afterwards (665 words)
It was quiet, afterwards. PG
All's Fair in Love and Strife (210 words)
A terribly bad pun written in honour of Squeenix giving Zack a last name. G
As Easy As This (1740 words)
Insert super happy ending here. PG
TIMESTAMP: Time Goes Ever On and On (440 words) For them, change is relative. G
Blood Brothers (AU 36,155 words)
Zack and Cloud are blood brothers - two souls bonded by one fate. But what happens when one brother is lost and the other has to sacrifice everything he is to try and save him? NC-17
(Zack/Cloud; Cid/Vincent)
Contrary to Appearances (crossover with Kingdom Hearts 445 words)
Even when one of them's tied up, it's hard to tell who's on top. PG
Death Penalty (125 words)
It's almost worse to remember than to have forgotten in the first place. G
(gen - Cloud)
Guided Tour (125 words)
Either the trip or the destination's going to make him throw up. G
Handbasket (1330 words)
Sephiroth was starting to think Zack was making up holidays for the sake of dressing them up in strange clothes. G (warning for crossdressing)
Heart and Home (1600 words)
It was the perfect solution to the situation. No, really. G
TIMESTAMP: Edible Chef (275 words) The perks of domesticity. PG
Helping Hands (125 words)
Advent Children. Because really, how else did Cloud get back to the church? G
(Zack/Cloud/Aeris undertones)
If at First (3050 words)
Sometimes help comes from entirely unexpected sources. And makes you question your sanity. G
(gen - Zack, Cloud)
In the Closet (1450 words)
Closet sex. Seriously. NC-17
In the Country (1280 words)
Because sometimes I do write porn. NC-17
In Service of the King (AU 3075 words)
How far would you go to uphold a corrupt regime? PG
(gen - Turks, Rufus)
It Runs in the Family (non-AU AU 1250 words)
All things considered, Zack was mostly just surprised he hadn't found out sooner. G
TIMESTAMP: Family Matters (540 words) Cloud makes SOLDIER. Zack takes advantage. PG
Life Becomes Him (500 words)
On SOLDIER, boyfriends and where facial hair fits into the mix. G
Long Distance Shot (670 words)
Phone calls are always an adventure with Reno on the other end of the line. G
(gen - Reno, Rude)
Luxuries (125 words)
Because you don't need an excuse for hot spring sex. NC-17
A Matter of Contrast (1190 words)
There are all sorts of side effects to living. PG
Of Solace (840 words)
Patience. Friendship. Quiet. What more do you need to chase the bad dreams away? G
(gen - Sephiroth, Zack)
Out of Place (125 words)
This might be one of the more awkward moments in his life. G
Over Drinks (crossover with Trigun 3100 words)
You always hear about the pain of those who are left behind, but what about those who have to go on ahead? G
(Zack/Cloud/Aeris; Vash/Wolfwood undertones)
Riding Out the Storm (AU crossover with Final Fantasy IX 1625 words)
For the brave and reckless, lightning diving can be a very lucrative career. It's also one hell of an adrenaline rush. PG
Running Silent (AU 1290 words)
A demon has possessed the prince. The Baron of Gongaga has gone to try and find a solution. A lowly stable boy is finding things difficult without him. G (with one instance of violence)
Smooth Shuffle (AU 3200 words)
1927, Chicago. One of Big Man Maud's men goes out to dig up some dirt on the Shinra gang. PG
(gen - Turks; suggested Tseng/Elena)
Sticky Notes (AU 2920 words)
Cloud hates Mondays. Luckily, Zack is creative. PG
Stone Crazy (AU crossover with Cowboy Bebop 1730 words)
The Bebop goes after an escaped convict from Midgar Prison - only Jet thinks they could use some extra help bringing this one in. PG
(gen - Spike, Jet, Sephiroth, Zack, Cloud)
Talk Is (640 words)
When Reno's not talking, Rude starts looking out for armageddon. G
(gen - Rude, Reno)
Talking Round Toothbrushes (crossover with Final Fantasy VIII 520 words)
Seifer Almasy, SOLDIER First Class, is having a bug problem. And by bug, he means Zack Fair. G
Tease (790 words)
Tequilla shots and foreplay. Yes, at the same time. R
That one ficbit about anime conventions (500 words)
if a picture of him dressed like this shows up on the cover of the interdepartmental newsletter he is going to kill Zack. G
That one ficbit about cop!Cloud (AU 260 words)
Zack was teetering at the top of a ladder with a power drill in hand when the knock came. G
(gen - Zack/Cloud pre-slash)
Touching (1690 words)
The whole situation is decidedly unappealing, and to top it off he's hearing things again. PG
(Attempted Don Corneo/Cloud; past Zack/Cloud)
TURK Narcotic Division (AU crossover with Final Fantasy XII 1485 words)
Sure, the NYPD's good and all, but TURKs get the job done with a helluva lot more style. PG
(gen - Larsa, Reno)
Unshakeable (1310 words)
The first time it happened, Zack figured he had to be dreaming. G
(gen - Zack, Cloud)
Verses and Sequels - Final Fantasy VII
-Idiots in Hades- (crossover with Kingdom Hearts)
Guided Tour (2165 words)
DiZ sends Riku to retrieve something from the Underworld. He meets a very unusual ghost along the way. G
(gen - Riku, Zack; Zack/Cloud undertones)
An Idiot in Hades (1635 words)
Cloud comes back for his idiot. G
Domestic Disturbance (500 words)
In which Cloud gets kissed and several people have absolutely no idea what's going on. PG
Dreamings (400 words)
He had to reassure himself that it was real. R
-The New Black-
Situation (725 words)
Occurs immediately prior to The New Black. Sephiroth and his SOLDIERs arrive to deal with the situation. G
The New Black (1620 words)
There are some mornings even coffee can't fix. G
Different Strokes (1690 words)
The weirdest thing is how it isn't really as weird as it should be. PG
(Zack/Cloud; Sephiroth/Zack/Cloud pre-slash)
-Wild Ones verse (AU)-
Playing With Fire (2225 words)
Prequel. Zack decides that Sephiroth's spending too much time cooped up at the castle. G
(Zack/Cloud; Sephiroth/Zack/Cloud pre-slash)
Sword Master (500 words)
Prequel. To say it had been a shock, that first time, would have been an understatement. PG (warning for gore)
When Summers End (500 words)
Prequel. It was summer again. G
Wild Ones (abandoned WIP - 16,300 words)
Irrevocably tied and holding nothing back, two men search the world for what's been lost. This is but a small part of their story. Overall PG
(currently Zack/Cloud)
-Zack's verse (AU)-
Zack's (2960 words)
The gang's out for an evening on the town - Cloud just wishes they could have left him at home instead. PG
(Zack/Cloud preslash)
Traditional (4800 words)
Clearly no one has told Zack that first dates are supposed to be awkward. Cloud's pretty sure he's awkward enough for the both of them. G
Christmas at Zack's (6050 words)
Zack invites Cloud to a party at his bar - on Christmas Eve. G
Pearly Whites (2215 words)
Reno needs a favour. Cloud needs to be somewhere else. PGish
Festival (125 words)
Zack and Cloud on the town. G
Final Fantasy VIII
The Blowup (crossover with Final Fantasy IX 825 words)
Zell and Zidane play a game. It's not exactly as inappropriate as it sounds. PG
(gen - Zell, Zidane, Squall, ensemble)
Firestarter (AU crossover with Kingdom Hearts 1280 words).
The first thing Axel learned about Enforcer Almasy was to stay the fuck out of his way. PG
Holding the High Ground (AU crossover with Final Fantasy FFXII 880 words)
The plan to draw the Galbadians into close quartered fighting around the citadel was going perfectly - right up until the point when the defenders started losing. G
(gen - Seifer, Balthier)
Satisfying (100 words)
Seifer likes the way the SeeD cadet uniform looks. NC-17
Suits and Smiles (635 words)
There are some things the SEED exams just don't prepare you for. G
Talking Round Toothbrushes (crossover with Final Fantasy VII 520 words)
Seifer Almasy, SOLDIER First Class, is having a bug problem. And by bug, he means Zack Fair. G
Final Fantasy IX
The Blowup (crossover with Final Fantasy VIII 825 words)
Zell and Zidane play a game. It's not exactly as inappropriate as it sounds. PG
(gen - Zell, Zidane, Squall, ensemble)
In the Moment (775 words)
For an actor, life is made of moments. For a thief, even more so. PG
Riding Out the Storm (AU crossover with Final Fantasy VII 1625 words)
For the brave and reckless, lightning diving can be a very lucrative career. It's also one hell of an adrenaline rush. PG
Final Fantasy XII
Anticipate (125 words)
Partners in sky piracy. G
(gen - Vaan, Penelo)
Desperate Times (1780 words)
Fight, flee or... what was our third option again? PG
Fighting Words (1875 words)
Pre game. Swordplay as foreplay for politics - because what else would you expect in Arcadia? G
(gen - Vayne, Drace)
Holding the High Ground (AU crossover with Final Fantasy FFVIII 880 words)
The plan to draw the Galbadians into close quartered fighting around the citadel was going perfectly - right up until the point when the defenders started losing. G
(gen - Seifer, Balthier)
Moments (1830 words)
The Arcadian Emperor has a party. Luckily an uninvited sky pirate shows up to make things more interesting. G
A Question of Trust (crossover with Pirates of the Caribbean 1470 words)
The problem with being one of the good guys is that it often conflicts with the basic nature of being a pirate. G-PG
(Jack Sparrow/Balthier/Vaan)
Sun, Moon and Stars (1520 words)
Well, what do you do when you've got some time to yourself? NC-17
(Balthier solo; references to Balthier/Basch, Balthier/Vaan, Balthier/Fran)
Tempting When Wet (435 words)
The motly crew stops for a rest on the Phon coast. Some party members are not as worn out as others. G
(one-sided Basch/Vaan)
To Catch a Thief (3550 words)
Post-game. Vaan's done a lot of growing up since he became a sky pirate. Basch isn't entirely certain why he's started to notice, but he's definitely not the only one. PG
TURK Narcotic Division (AU crossover with Final Fantasy VII 1485 words)
Sure, the NYPD's good and all, but TURKs get the job done with a helluva lot more style. PG
(gen - Larsa, Reno)


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Shipping Policy
We work to ship your items as fast as possible. We realize that quick shipping and receiving is as important to you as price and customer service. Most in stock orders will be shipped the same day if you get your order in before our daily pickup. Holidays and off days will create more time needed for shipping.


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Apple iPad Update May Debut in June 2011 With Mini USB Drive
The iPad, which debuted earlier this year and sold over 3 million in the first 80 days, is expected to undergo an update in the second quarter, the report notes. Some of the other changes expected to come include a thinner model that is lighter to carry.
Other Wall Street soothsayers expect a new version of Apple's iPad may hit as early as the first quarter and possibly include a smaller seven-inch screen, compared with its current 9.7-inch display, according to an earlier Bloomberg report.
As talk of an iPad upgrade begins to filter out ahead of the busy holiday selling season, the effect could create a slowdown in iPad sales as shoppers hold off buying the current version in anticipation of one with a few more bells and whistles. Apple may rely on the old ace in the hole and slash prices to drive sales. That strategy, however, does well for revenues but hurts the bottom line.
Apple shares were down 2.63% to $283.51 in morning trading, compared with its close Monday.


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Sunday, August 24, 2008
Ebb and Flow
3 comments:
Hi Sarah, these are lovely.
By chance I had just been looking at Kurt Jackson's paintings as I'd wanted to re-view a beetroot painting that
Katherine put up a while back (London Art Fair?), and found by accident his two rivers project. I don't know if you've seen it but you can download a pdf of it from Messums:
download link.
Hope you're enjoying your 'new' home and you don't miss France to much, at least you still have the sea!
- 8:59 am
- Katherine Tyrrell said...
Oh - I come along to say how lovely this look and Julian beat me to it and mentions me!
I'm loving this mauvey colour you've got going in this and the last painting - what are you using?
- 4:13 pm
- vivien said...
utterly beautiful and totally catching the atmosphere and colour and light of those estuaries :>)
KJ also has a book on the Cornish estuaries
You are near enough to see his work often at the Lemon Street Gallery in Truro - lucky you < sigh >
- 2:09 pm


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It's understood that the electronic cigarette market has always moved at a daunting pace, where new products surface what seems to be each week. As a result, vendors carrying these products and the manufacturers who make them are constantly upping the anty with the next 'hit item' to rock the scene. This steady flow of competitiveness has influenced some to meet the demand of these vendors and manufacturers by offering ecig advertising services to help build that next company or brand.
And so… the Ecig Advertising Group has emerged.
With analysts estimating that the electronic vaporizer market is at an outstanding $2.5 billion with projections of continuous growth, entrepreneurs are quickly making their way to the thriving industry. The figures and spark of opportunity has inspired a slew of ecig companies to rise in the heat of the moment, placing their attention on the marketing of their company, brand and products. In fact, last year ecig marketing budgets for the largest ecig companies grew by more than 100% year over year. There were six of the largest companies that spent nearly $60 million combined on advertising and promotions. As a result of this high demand for ecig advertising, a group of highly recognized and experienced figures within the electronic cigarette industry came together to create an industry group that offers advertising solutions specifically designed for the ecig industry. The Ecig Advertising Group (ECAG) is comprised of a group of experienced individuals that know the importance of ecig advertising. Through various community and industry ties, ECAG is able to offer ecig advertising specific solutions and pin-point proven strategies that provide industry leading results.
Ecig advertising is a hot topic these days because the competition within the industry has become so thick. The trouble these companies face is that electronic cigarette advertising or anything related is restricted on many networks, leaving ecig companies disgruntled from limited options of exposing their company and brand to the public. This is why it is vital for a company such as the Ecig Advertising Group to provide services that do not have restrictions and allow ecig advertisers to get their brand seen by vapers at a low-cost.
To learn more about the Ecig Advertising Group, feel free to visit www.EcigAdvertisers.com.


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While at EY over the past 9+ years in M&A and Strategy domain, I have grown through ranks from Associate to an Engagement Manager and now a Director with the firm. I have interviewed nearly 150+ candidates through on and off campus program with extremely diverse profile/experiences and for many different EY offices (such as NY, Chicago, Dubai, Singapore, China, India and many European countries). Also I currently assume leadership role in driving onboarding and internal case training efforts for new graduate and experienced hires for the firm. Therefore I very well understand 'BIG skill gaps' students encounter which require professional counsel.
Whether you are an undergrad or a prospective MBA student or an experienced job-seeker, I will work with you to come up with an approach to storyboard your original answer in a dramatically different way that can persuade the interviewer and tell them why you matter!. Whoever structures and articulates a better story eventually wins!


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Mrs. Faigy Prupas, Youth Director
The Lower Merion Synagogue Youth Department has a full range of programming for children from toddlers through college. We look forward to seeing your children at our Shabbat groups, holiday programs, and special events. For questions about the Youth Department, please contact Mrs. Faigy Prupas at firstname.lastname@example.com
"Shevet Achim" Shabbat Children's Programs
Meet from 10:00 until 11:30 a.m.
"Achdut" Ages 3-5/6
Leader: Leta Borck & the LMS teens.
Upstairs double classroom, snacks provided
"Achva" 1st-2nd Grade Children
Leaders: ZC Schwartz
Upstairs classroom, snacks provided
"Batya" 3rd-5th Grade Girls
Leader: Aviva Hassan
Lower Level classroom, snacks provided
"Chaverim" 3rd-5th Grade Boys
Leader: Josh Hassan
Upstairs classroom, snacks provided
Babies Room
Opens at 10 am, upper level, for parent supervised play.
Teen Minyan is led by Joshua Friedman and the LMS Teens on the lower level beginning at 9:15 a.m.
The Parsha Forum for Middle School Girls is led by Miriam Coren and begins at 9:15 with Teen Minyan. The group then meets in the Social Hall.
Shabbat Afternoons
Bnos: Shabbat afternoons on the upper level for girls ages 3through Grade 6. Contact Meira Friedman at 610-617-9529 with any questions.
Please check the weekly announcements for meeting times.
Pirchei–Torah Youth: Shabbat afternoons in the social hall for boys in Grades K-8. Contact Rabbi Silver or Rabbi Sruli Schwartz with any questions.
Please check the weekly announcements for meeting times.
Bnei Akiva: Snif meets one hour and 15 minutes before Mincha on the lower level. For children in grades 3-8. For information, please contact Miriam and Asher Coren at firstname.lastname@example.com. Please check the weekly announcements for meeting times.
Mommy & Me: Wednesdays from 9:30-10:45 am on the lower level. Mothers and female caregivers are invited to participate with children up to 3 years old. Mommy & Me is facilitated by Ayala Aidman.
Mishmar & Cholent: Everyone is invited on Thursday nights from 9-10 pm, to an open Beit Midrash followed by cholent.
For questions about any of our youth programs, please contact Mrs. Faigy Prupas at firstname.lastname@example.com


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U.S. Attorney General Loretta Lynch announced at the United Nations on Wednesday that the Department of Justice would be working with several major metropolitan police forces, starting with New York, to create a globe-girdling police force targeting "violent extremism."
It's on like Donkey Kong.
New York Mayor Bill de Blasio made it clear that his city has joined 25 other cities worldwide because the global police force will target "intolerance."
"The Strong Cities network will be a message to all families who have lost loved ones because of extremism that something is being done in a new and powerful way," de Blasio said.
I'm guessing the "something" might look a bit like this:
It's not that I don't trust our government; I just don't trust the people who are in our government.
Especially not when they say they are going after "homegrown" terrorism and using a global police force to fight "intolerance."
See, the real problem is that the people in charge of this new statist terror are liberals, the same people who brought us the old statist terrors, from the KKK to the Nazis and Communists.
This particular iteration of liberal fascism is being run by people who, since the start of the Obama Administration, have classified anyone who is conservative, a Bible believer or a veteran as a potential terrorist, while virtually ignoring the very real threats from Islamist groups and drug cartels that are sending their agents across our open borders.
The New York Daily News wrote: "(De Blasio) also stressed that the alliance would not focus on any one type of extremism, and pointedly referenced past violent attacks like the 1994 shootings on greater Boston Planned Parenthood clinics and the racially motivated Charleston, S.C., shooting in June that killed nine African-American churchgoers."
So right there, de Blasio's already implying that pro-lifers are a target because of something from 1994, and since the media have spun the Charleston church shootings into a tale of an allegedly conservative-inspired massacre of blacks by Confederate flag owners, the "Global Military Police" will no doubt have a list of conservatives who need to be put behind bars for public safety.
This represents probably the greatest surrender of American rights and authority since, oh, a couple of days ago when the Obama Administration basically got run down by Russia, which is building itself a nice little nest there in the Middle East.
"We will be vigilant against all forms of violent extremism, whether it's based in religious, or racial, or nationalistic or ideological intolerance," deBlasio said.
Unless they're Muslim.


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There's just one problem here -- The Dems are about as hawkish as the Republicans!! From Sen. John Kerry (D-MA) to Sen. Maria Cantwell (D-WA), Dems voted en masse to support the wars in Afghanistan and Iraq. Many, such as Hillary Clinton & Cantwell, seem hell bent on supporting a similar incursion into Iran.
In fact, if you look at the history of the US, the art of invading foreign countries has been perpetrated by Presidents on both sides of the aisle. We entered WWI under President Woodrow Wilson. WWII came about on FDR's watch. Nuclear bombs were dropped on Japan under Harry Truman's watchful eyes. We became bogged down in Vietnam under the Kennedy and Johnson administrations.
All of the aforementioned US presidents were Democrats!!
More importantly, most of the wars the US becomes involved (ensnared?) in have little to do with specific military objectives. While our invasion of Iraq offers a current example, a recent revelation shows that the war against North Vietnam fits the same bill.
According to Canada.com,
Former U.S. secretary of state Henry Kissinger quietly acknowledged to China in 1972 that Washington could accept a communist takeover of South Vietnam if that evolved after a withdrawal of U.S. troops - even as the war to drive back the Communists dragged on with mounting deaths.Of course, such a statement seems to fly in the face of the "we need to protect the world against Communism" argument foisted time and again on the American public. If we could accept a Communist government in Vietnam, then why in the hell were we there in the first place?
The late U.S. president Richard Nixon's envoy told Chinese Premier Zhou Enlai: "If we can live with a communist government in China, we ought to be able to accept it in Indochina."
The sad answer to that question is that imperialistic powers -- and the US certainly fits the definition -- need to have an enemy. Enemies allow the hegemonic powers to funnel millions and billions of dollars to the military-industrial complex which, in turn, funnel money back to insure those same political leaders stay in power. Enemies also divert public attention away from all the shortcomings of any given administration -- things like poverty, woeful health care, the theft of basic civil rights and lackadaisical education.
It turns out that Vietnam, like Iraq, wasn't anything like our leaders told us it was. So, if you think electing a slew of Democrats this November will change the tenor and direction of this nation, you haven't been paying close attention to history.


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Seattle mayor bans city-funded travel to Indiana following passage of anti-gay legislation
The mayor of Seattle has banned all city-funded travel to Indiana in response to the recently passed anti-gay Religious Freedom Restoration Act, signed by Indiana Governor Mike Pence (R), reports KOMO.
Mayor Ed Murray (D) addressed the ban on Saturday saying, "Laws that say you can discriminate have no place in this country."
Indiana is reeling from threats of boycotts, with corporations dialing back conducting business in the state over a law considered discriminatory to the LGBT community.
Murray joins San Francisco Mayor Ed Lee in instituting a ban, adding that, as Seattle's first openly gay mayor, he's not doing it because of his sexual orientation.
"I'm not doing this because I'm a gay man," he explained. "You cannot say, 'You can't come into my restaurant because you're black and it's my religious belief not to serve you.' That is a settled question."
"Indiana's S.B. 101 doesn't reflect the values of our City. Seattle has been a leader in the fight to protect civil rights and ensure equality for all people – no matter who you are, or who you love," Murray said. "This is why I am ordering that none of our taxpayer dollars should go toward supporting this discriminatory law. To those in Indiana today who are working hard in the fight for equality – know that Seattle stands with you as you continue your efforts to end discrimination and protect civil rights for everyone."
Murray noted that the U.S. Conference of Mayors is scheduled to be held in Indiana next year, and said he will request a change of venue if the RFRA is still in place.