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After some of the uneducated, agenda pushing, unthoughtful, unfactual comments following the shooting in California, I just can't help it. Seriously. Are we really people who can be swayed by how someone chose to title themselves? So if I tell you I am a Republican or Democrat, and it matches up with what you are, will you blindly follow me? Really? Please, I hope you are smarter than that. I really hope that you don't agree with everything I say just because of what group I choose to affiliate with.
Of course, I guess we do it all the time don't we? We stand unswayed beside our old alma maters whether they win or loose. We shell out hundreds of dollars to support loosing teams, just because we live in, were raised in, or like the colors of a specific sports team. Why? What is it within our human nature that causes us to do this? Of course, in this circumstance we are called fans. In the other circumstance, we are called "Constituents."
In my humble opinion, which is all this is, an opinion, it seems like the United States would be so much better off if everyone would decide, on their own, what they believe. Take some time to really look at issues and decide within yourself what you believe. Then, ignore what someone labels themselves and support the person who supports you. Did you see that? Our politicians are there to support US – WE don't support them! When did that change? Have you looked up what the definition of 'constituent' is? Take a look at some of the definitions we find:
one who authorizes another to act as agent
having the power to create a government or frame or amend a constitution
someone or something that is necessary in making something else whole
Please people – do not blindly follow a person just because of a party affiliation. Take a good hard look at EVERYTHING they say, especially the things they said before they knew everyone was looking.
Take a look at everything they do – especially the things they did before anyone was looking.
Take time to review facts, true hard facts, before deciding on an issue. Pretend every issue is going to affect your family personally. Don't make a blanket decision about something just because you belong to a certain group of people – you belonged to your family before anything else.
For example. Right now there is all this talk about "gun violence." First off, it isn't 'gun' violence – it's people violence. A gun has never, ever, and never will, kill someone on it's own. A gun can not be violent. Can we agree on that? And other classified it as "another hate crime." Really? Do you think so? What do we call the crimes which aren't hateful?
I'm not going to provide it here, we either want truth and seek it out so it can set us free, or we don't. Take some time to look up statistics in countries with strict gun laws. Does it work? How much, a little or a lot? Maybe it does, maybe it doesn't – you decide.
Here is a quote from someone in regards to the California shootings. I'm not going to say who – if you don't know who and I tell you, that could sway your stance on the quote. I want you to read the quote, digest it, decide how you feel about it with no regard to who said it:
"We have a pattern now of mass shootings in this country that has no parallel anywhere else in the world."
How do you feel about that? You may, given the last few years in America, feel it is a true statement. It sure feels that way. But what if you took time to know more about the subject. Would facts change the way you feel?
Now, I challenge you to see where the United States stands in rankings of firearm homicides. Let me help you out, take a look at the UNODC site. This is information every party has available to them, and so do we! When the US ranks high on any of the charts, the listing shows as "The Americas," which includes Central and South America and all the little islands around us. Take a quick look at pages 25 and 34.
The global average homicide rate stands at 6.2 per 100,000 population, but Southern Africa and Central America have rates over four times higher
than that … – UNODC 2014 Global Homicide Book
Take a look at this map which shows the Mechanism of Homicides – I think you may be just a little shocked. Based on this, not only do we have 'gun violence', we have a pretty large percentage of 'sharp object violence' as well. We aren't safe anywhere – *holds up sign* NO MORE KITCHEN KNIVES! NO MORE PENCILS! Oh wait, we have almost gotten rid of the pencils – hopeful the sharp object violence will go down a little bit now.
If you read this report, you will see a very large number of people committing the crimes are under the influence of drugs and/or alcohol And many times, the victim is as well. They even have proof homicides go down during the alcohol restricted hours in some of the more violent countries. Hmmm, think on this for a moment or two.
Ok people, I am not trying to sway you one way or the other. If I have a family member killed by a firearm – I may be against firearms. If I had a family member saved because someone else had a conceal to carry and saved my family member, I will be for it. But at least these are much better reasons to stand for something rather than which party I have affiliated myself with – isn't it?
It's time to change the meaning of being "politically correct." It's time for us to become politically correct – where we, ourselves, decide what we stand for and what we oppose. It's time for our politicians to support us, and not the other way around. We are not fans, we are constituents. It's time for us to use the internet to seek out the truth, instead of spreading more falsehoods.
It would be nice if more laws would be the easy answers. But don't we already have a major law about killing people? Is one more law going to really make the bad guys less bad? Do we really think by looking at them and saying, you can't have a gun, they will listen?
You know what … for me personally … I don't care what the mechanism was for killing 20+ people. I only care that they have family members who will have an empty seat at their table this Christmas and New Years. I care that there were hurting, sick people using whatever mechanism they used to create that empty seat. Maybe, just maybe, if we concentrated on helping those sick people, the killing rate would go down. Maybe if we concentrated on those who are hurting because they lost loved ones we could make a real difference in someone's life.
I can't help but wonder, what if we did profiles on each and every victim and never mentioned the perpetrator or mechanism? Is it possible one person might make a different choice later? Could one person be changed enough to where they won't pick up a gun and walk into a school, movie theater, bus station? What if the victims had names, faces, and lives stolen from them instead of the perpetrator?
What if our politicians were really working for you, yes YOU, YOU personally? How would things be different? Let's all become Free2Live – Politically Correct as Constituents and not Fans. Become the change we want to see in this world.
For those of us who call our selves Christians, there is a scripture to consider in times like these:
2 First of all, then, I urge that petitions (specific requests), prayers, intercessions (prayers for others) and thanksgivings be offered on behalf of all people, 2 for [a]kings and all who are in [positions of] high authority, so that we may live a peaceful and quiet life in all godliness and dignity. 3 This [kind of praying] is good and acceptable and pleasing in the sight of God our Savior,4 who [b]wishes all people to be saved and to come to the knowledge andrecognition of the [divine] truth. 5 For there is [only] one God, and [only] one Mediator between God and mankind, the Man Christ Jesus, 6 who gave Himself as a ransom [a substitutionary sacrifice to atone] for all, the testimony given at the right and proper time. 7 And for this matter I was appointed a preacher and an apostle—I am telling the truth, I am not lying [when I say this]—a teacher of the Gentiles in faith and truth.
8 Therefore I want the men in every place to pray, lifting up holy hands, without anger and disputing or quarreling or doubt [in their mind]. 1 Timothy 2 (AMP)
Prayers and heart felt thoughts going out to all those lives touched by this tragic event. Prayers going up for all our politicians as they do their best to navigate these times. Prayers going up for those who will cast their votes, let them be for issues and topics and not for people. Pleadings going out to God to continue to keep His hand on this nation.


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subset: commoncrawl


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AVIVA, the global insurance group who have been long-term backers of UK Athletics, are understood to be in talks with the FA about the FA Cup sponsorship E.ON are not renewing at the end of the season, according to the Daily Mail.
Aviva's insurance rivals Prudential are also believed to be interested. Meanwhile, the Football League, looking for new title sponsors to replace Coca- Cola, are quoting two different prices, £7.5m and £9m a year to betting companies.
The news comes in a season of uncertainty regarding sponsorship deals with Blue Square denying that they wanted to end their sponsorship with the Conference. Last week, however, Barclays renewed their title deal with the Premier League.


text:
Author: Emanuel Engel
Directed by: Radu Hăprian
Adaptation by: Aurel Crăciun
Lyrics: Ion Sângereanu
Music: Nicolae Ionescu, George David
Soundtrack: Levente Szöcs
Stage Design: Eustaţiu Gregorian
Cast: Radu Hăprian Dan, Anca Hăprian Dan, Ramona Mincu Benchea,Teodora Popa, Viorica Boda, Mădălin Costea
Stage Director: Elena Madaras
Lights: Ovidiu Tămăşan
Sound: Florin Andrea
Stage Technician: Horia Oarga
Puppet design: Hani Sasu
Tailoring: Mirela Tămăşan
Carpentry: Marin Goia
Minimum age: 4 years
Duration of the performance: 55 minutes
The performance has a strong educational aspect and its based on the central idea that any success or achievement in life cannot be accomplished without personal effort and study. Talent and passion have to be backed by hard work to ensure success. It is a sincere performance, clear-cut, depicting the realities of everyday life and, through theatrical art, trying to connect the child and to spring awareness, regarding this realities. Exhilarating music, which captures the audience from the beginning, accompanies very well the unfolding of the whole performance.
Class Activities
Date : 23 May 2018


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Congolese military forces have opened fire on a crowd of Burundian refugees. The attack occurred last Friday, on September 15. 39 Burundian refugees were killed, 94 injured, with 57 of them critical. The shooting began with what has been reported by Al Jazeera as a peaceful protest over the detention of four Burundian refugees who feared deportation. It escalated when the army tried to disperse the group by firing shots into the air.
There have been various accounts of the event and details are murky, with the UN and Congolese government directly contradicting each other. UN refugee chief Filippo Grandi has labelled it a devastating tragedy and called for a detailed inquiry by the Democratic Republic of Congo (DRC). According to Al Jazeera, the government claims they were attacked by armed persons and not refugees. However, Interior ministry official Josue Boji has said that refugees overran the jail where the four Burundian refugees were being detained, overwhelming soldiers and responding to shots being fired by throwing stones. Maman Sidikou, the head of MONUSCO, said the UN's peacekeeping mission in the country stressed that defence and security forces could resort to force "only as a last resort" and in accordance with international norms, urging "authorities to promptly open criminal investigations."
On September 12, the DRC national army arrested four Burundian refugees because they were reportedly carrying weapons in the town of Kamanyola. Al Jazeera reported that they have since seen footage and claim the refugees were carrying sticks. After being held for two days, the refugees were moved to the general migration directorate, which is responsible for migration and the movements of the national and foreign population. Burundian refugees living in the area feared the detainees would be repatriated and handed over to the Burundian government which, under the 1951 UN Refugee Convention that the DRC has ratified, would be illegal. Burundian refugees then began protesting their detainment outside the office of the National Intelligence Agency. The New York Times and Al Jazeera have both reported that the protesting began peacefully, but took a turn when security forces fired shots to disperse the crowds. Protesters then began to throw rocks, and one refugee reportedly took a gun from a soldier and shot him dead. This is when the security forces opened fire on the crowd. The Congolese government have responded saying they were attacked by armed persons, not refugees, and that an inquiry will be opened into the incident. The UN continues to assert that the people attacked were refugees and asylum seekers who had applied for international protection. They have also called for an inquiry to be opened to establish justice for the victims.
The attack carried out by the Congolese Military Forces on Burundian refugees is disproportionate and should have been handled better. They claim that they were attacked by armed persons, but victims included a ten-year-old girl. However, it is easy to criticize the military and say that they should not have opened fire. While this is true, more preventative measures should have been taken to avoid the situation coming to a head. For a start, military forces should be trained better and have access to more resources so that they are able to peacefully de-escalate tense situations. This would require more support for the Congolese government. This is important considering refugees from around the region seek refuge in the DRC, which puts a strain on their resources.
While it is important to recognize human rights violations like these, we must not lose focus on the root cause of these issues. Having a sole focus on such events distracts from the real problem, which is the reason behind why these people left their country in the first place. We must not forget the root causes behind the conflicts in their own country, and the inability of countries like the DRC to peacefully resolve these situations because they lack the resources to do so.
- Hardened US Military Stance Worsens Situation In Afghanistan - February 5, 2018
- U.S. And Turkey Butting Heads In The Middle East - January 29, 2018
- Nazism Vocabulary Creeps Into Austrian Politician's Rhetoric - January 15, 2018


text:
I'm in one of THOSE moods today, which means I'm probably in a culture shock down swing.
One of the things that hurts me with both languages and sport is that I'm prone to periodic bouts of brain lock. I can practice the basic set plays in basketball and memorize subjunctives and modals and various tenses described as "perfect" but when the actual play starts, and things are moving much faster and more chaotically than in practice, my brain just sort of stops and says "Okay, so now what?" More often than not I'm left standing in one part of the court just sort of blathering to myself. (This, by colossal coincidence, is a pretty apt description of my dating techniques as well.)
It is, I suspect, a form of panic. Rather than living in the moment and reacting to what actually is going on, my brain searches out the perfect thing to do and, not finding it, just takes a little vacation, but I usually pull myself together and move on (albeit, from the far end of the bench whilst I'm holding the stats clipboard trying to keep track of shots and rebounds).
That said, there have only been a few times in my life where I felt completely helpless as a result of brain lock. In one case, it was a the result of ignoring a series of signs, and my own nature, and hoping for the best (which is also a symptom of brain lock).
Back when I was working in Nou-machi, as a rule, I tried to avoid teacher trips. They typically took place in the summer and the staff of the school bussed off to some resort town and had a drunken good time whilst the parents were, for a short time anyway, actually in charge of their children.
I was usually invited to join the trips, but the people issuing the invitations were clearly only doing so out of courtesy. There were a lot reasons for this, part of it having to do with me being a foreigner, and part of it having to do with me working for the school board and not for the school (long, long story that).
One summer, though, in a fit of brain lock, I thought "Well, why not? What's the worst that can happen?" and said yes. The English teachers I worked with, Miss Kato and The Beautiful Miss Takahashi (not her real name–for the record, that is the phrase She Who Must Be Obeyed used to describe her) talked up the trip, which was to a famous lake and ski resort in Fukushima Prefecture. They talked about all the stuff they were planning to do.
Then, at a PTA party, or some such random drinking event, I said I was looking forward to hanging out with all the other teachers and with Miss Kato and the Beautiful Miss Takahashi. They both gave me that look (which is apparently universal to all women all over the world) and explained that "hanging out" was pretty much not going to happen as it was not an English department trip. (Cue ice cold wind sounds, shrieking and horror music theme.)
At this point, the part of my brain that actually, on occasion functions, took the hint (DON'T GO! GET YOUR MONEY BACK! HELL, LET THEM KEEP THE MONEY! DON'T GO!) I also seem to remember complaining about that incident to my friend Charles who said something like "You know, whenever I start to feel like a part of the team, something always happens to remind me that I'm not." In retrospect, this was his way of saying DON'T GO! GET YOUR MONEY BACK! HELL, LET THEM KEEP THE MONEY! DON'T GO!
I, of course, went on the trip because the large part of my brain that was locked up thought things would work out.
Long story short: things didn't work out. We had an expensive, microscopic lunch and rain cancelled the one set of activities everyone was most looking forward to. Therefore, everyone was in a bad mood when we somehow ended up at a glass factory/museum. No one bothered to tell me how long we'd be there or that most of the teaching staff were going to a nearby bar. At that point, I was in the middle of the basketball court two movements behind in the set play, out of position and pretty much already blathering.
What I remember most about this incident was the feeling of panic and not knowing what to do. I was stuck for two days with people who clearly didn't want me around and weren't bothering to keep me informed. After a while I want back to the bus for a short nap. To make matters even more interesting, they'd stuck me off in a room by myself which is something that never, ever happens on a Japanese trip because the group is too important to them.
However, it did give me some time alone to recharge and settle down and get my brain working again (well, so to speak). A long soak in the hot spring bath helped a lot, too (almost as good a form of therapy as a proper haircut).
I ended up having a good time drinking with the principal and vice principal and didn't have to pay for any of the beer, whiskey or sake.
Since then, I've managed to avoid school trips, although I did stumble across someone else's once on a trip to Kyoto. (But that's another odd story.)


text:
- About Company
- Recruitment Process
- Questions Asked in Moonfrog Labs
- Interview Experiences
- Where to Apply ?
About Company :
Moonfrog Labs, is a mobile games based startup headquarted in Banglore, India. MoongFrog labs have developed games like Mafia Wars, CastleVille, Bubble Safari, Cafe World, Yoville, Hidden Chronicles, Empires & Allies. Their flagship hit Teen Patti Gold has been downloaded more than 20 million times and features among the top five highest-grossing games in India on both Android and iOS. In 2017, Moonfrog developed and launched games for two huge Indian IPs — Baahubali: The Game and Alia Bhatt: Star Life.
Know more about Moonfrog Labs
Recruitment Process :
Moonfrog Labs conducts 4-5 rounds to select freshers as Software Engineer in their organisation.
- Online Round
- Technical Round 1
- Technical Round 2
- Technical Round 3
- HR Round
Academic Criteria :
- 70 percent or above in B.Tech, Class X and XII.
- No backlogs at the time of interview
Written Round :
Online round is a programming round. It may be online or offline. To clear this round you must be well prepared with data structures and algorithms.
Technical Round :
The students who clear the written round are called for Technical Interview. To clear this round you should be clear with your basics. You should be prepared with Data structures and Algorithms (tree, graph, string, array). You should be well prepared with system design problems. Students will be expected to write codes in the interview. Be confident about what you have mentioned in the resume. You may be asked puzzles in this round. To be prepared for puzzles you can practice from our Puzzles section.
HR Round
You can expect general HR questions like :
1. Tell me about Yourself
2. Why Moonfrog Labs ?
3. Do you love gaming ?
4. What are your strengths and weaknesses ?
5. Questions form resume
Questions Asked in Moonfrog Labs :
- Jumping Numbers
- Minimum number of jumps
- Reverse an Array
- Topological sort
- Pairwise swap elements of a linked list by swapping data
- monotonically increasing subsequence.
- Tree or not
- Explain a jigsaw puzzle ?
- Print a binary tree in spiral order
- more >>
Interview Experiences
It is always beneficial if you know what it is to be there at that moment. So, to give you an advantage, we provide you Interview Experiences of candidates who have been in your situation earlier. Make the most of it.
Where to Apply ?
Recommended Posts:
- Fab.com Recruitment Process
- eBay Recruitment Process
- Teradata Recruitment Process
- Bloomberg Recruitment Process
- 3DPLM Recruitment Process
- CarWale Recruitment Process
- Practo Recruitment Process
- Dell Recruitment Process
- Quikr Recruitment Process
- OYO Rooms Recruitment Process
- NHAI Recruitment Process
- Arcesium Recruitment Process
- IBM Recruitment Process 2019
- Microsoft Recruitment Process
- Amazon Recruitment Process
- Samsung Recruitment Process
- SanDisk Recruitment Process
- Adobe Recruitment Process
- Flipkart Recruitment Process
- Cisco Recruitment Process
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text:
Advertisingy u no do it?
Advertising (learn more)Advertise virtually anything here, with CPM banner ads, CPM email ads and CPC contextual links. You can target relevant areas of the site and show ads based on geographical location of the user if you wish.
Starts at just $1 per CPM or $0.10 per CPC.
Difference between session and users in segments for content drilldown
Discussion in 'Google Analytics' started by milevkiril, Aug 1, 2018.


text:
Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.
Lactobacillus bacteria belong to the lactic acid bacteria (LAB) and are applied worldwide in the production of a variety of fermented food products. Besides their role in the food industry, some Lactobacillus species are naturally found throughout the human gastrointestinal tract (GIT) in different proportions, with the highest numbers being found in the proximal small intestine (1), yet their most important niche in the human body is probably within the vaginal cavity (2). Several lactobacilli have a documented probiotic function by providing health-promoting effects in humans upon administration. Among them, the well-studied probiotic Lactobacillus rhamnosus GG has been shown to prevent atopic diseases (3–5), decrease the risk of respiratory tract infections (6–8), prevent nosocomial gastrointestinal infections (7), prevent antibiotic-associated diarrhea (9), treat acute infectious diarrhea (10, 11), and be beneficial in the treatment of rotavirus-associated diarrhea (12), although there are always responders and nonresponders. For a more targeted approach, better knowledge of the underlying mechanisms is important. Because of its wide clinical use, L. rhamnosus GG is an important model probiotic strain for use in such targeted approaches.
During the last decade, incredible numbers of studies have focused on the possible immunomodulatory effects of probiotics. Probiotic bacteria are thought to confer such immunomodulatory effects by the microbe-associated molecular patterns (MAMPs) that are present on their cell surface or that are secreted in the environment by them (13). These MAMPs can be highly conserved, such as certain peptidoglycan motifs, or can be more strain specific, such as unique glyco- or lipoproteins. The interaction of a MAMP with its pattern recognition receptor (PRR) on host cells, such as Toll-like receptors (TLRs), results in the induction of a signaling cascade that modulates the expression of various response genes, such as cytokines and defensins (14). Increasing numbers of MAMPs that participate in the host interaction have been identified on the surface of probiotic lactobacilli (13, 15, 16). For instance, lipoteichoic acid (LTA) from L. rhamnosus GG was demonstrated to interact with the PRRs TLR2/6 (17), the S-layer protein SlpA in Lactobacillus acidophilus NCFM interacts with the dendritic cell (DC) receptor DC-SIGN (18), and specific peptidoglycan muropeptides in Lactobacillus salivarius Ls33 are recognized by NOD2 (19).
Recently, pili or fimbriae (i.e., proteinaceous heteropolymeric extracellular appendages) were discovered at the cell surface of L. rhamnosus GG (20). These pili are encoded by the spaCBA gene cluster (LGG_00441 to LGG_00444). The SpaC subunit, located at the tip and along the backbone structure, was shown to be responsible for the high mucus-binding capacity (21). A comparison of the retention times of exogenously applied piliated L. rhamnosus GG and the related nonpiliated strain L. rhamnosus Lc705 also suggests a key role for the pili in promoting residence in the human colon, since piliated L. rhamnosus GG could be detected for 7 days longer in fecal samples from healthy volunteers (21). By mutant analysis of the spaCBA genes, we could show that the loss of pili results in the loss of adhesion to the microvillus-containing Caco-2 intestinal epithelial cell (IEC) line and increased induction of the proinflammatory marker interleukin-8 (IL-8) in these nonphagocytic cells (22). Using recombinant lactococcal constructs, von Ossowski et al. (23) demonstrated that the SpaCBA pilus is a contributory factor in the activation of TLR2-dependent signaling in HEK cells by the recombinant lactococci as well as in the modulation of pro- and anti-inflammatory cytokine (tumor necrosis factor alpha, IL-6, IL-10, and IL-12) production in human monocyte-derived DCs (moDCs). However, a possible immunomodulatory role of the native pili present on the surface of live L. rhamnosus GG has not yet been explored. Interestingly, in the case of the pathogen Streptococcus pneumoniae, interaction of the pilus-associated RrgA adhesin with the complement receptor 3 (CR3), also named integrin CD11b/CD18 or Mac-1, was shown to enhance uptake of the bacteria by murine and human macrophages, on the basis of the results of experiments with a blocking antibody on the wild type and the isogenic mutant lacking RrgA (24). In addition, TLR2 was shown to mediate inflammatory responses to oligomerized RrgA (25).
In this study, we aimed to investigate the role of SpaCBA pili from the probiotic strain L. rhamnosus GG in the interaction with phagocytic innate immune cells. Intestine-resident DCs and macrophages are eager phagocytes capable of sampling bacteria in the intestinal lumen through the extension of transepithelial dendrites (26–28) and are important host cells for probiotic immunomodulatory applications. Here, we chose a simplified model with a murine macrophage-like (RAW 264.7) cell line. Cells of this cell line were coincubated with the L. rhamnosus GG wild-type strain and different pilus mutants of L. rhamnosus GG, so that the role of pili in their natural conformation and organization on live cells could be studied. Such in situ cellular experiments are important because host cellular responses against bacteria are always an integrated sum of the interactions of different MAMPs with different PRRs (13). In addition, we previously showed the importance of the zipper-like and nanospring properties of the pili in promoting close contacts of whole L. rhamnosus GG cells with substrates (29), which cannot be mimicked with isolated pili without their cellular anchor.
Bacterial strains and growth conditions.Wild-type L. rhamnosus GG and its mutant derivatives (Table 1) were routinely grown under nonshaking conditions in Man-Rogosa-Sharpe (MRS) medium (catalog number 288130; Difco, Becton, Dickinson, Erembodegem, Belgium) for 24 h at 37°C, and 1% of this preculture was inoculated into fresh MRS broth and grown overnight at 37°C. Bacteria were washed once with 1× phosphate-buffered saline (PBS; catalog number 14040-091; Invitrogen, Life Technologies, Ghent, Belgium), pH 7.4, and brought to a final concentration of 5 × 107 CFU/ml prior to incubation with macrophages. This concentration was selected on the basis of findings of preliminary tests in which different dose-response relationships (1:1, 1:10, 1:50, and 1:100 cells/bacteria) were evaluated.
Wild-type strain Lactococcus lactis subsp. cremoris MG1363 (30) and the recombinant L. lactis subsp. cremoris clone GRS1185, itself having a nisin-inducible vector that in turn contains the coding region of the spaCBA pilus operon, were grown overnight in M17 broth (catalog number 218561; Becton, Dickinson, Erembodegem, Belgium) supplemented with 0.5% glucose at 30°C. A 1% aliquot taken from this preculture was used to further inoculate fresh M17 broth, and cells were kept overnight at 30°C. In the case of the recombinant lactococcal construct GRS1185, a similar procedure was followed, and the media were supplemented with 7.5 μg/ml chloramphenicol (catalog number C0378-25G; Sigma-Aldrich, St. Louis, MO, USA). After inoculation with 100 μl of preculture, the cultures were allowed to grow until the optical density at 600 nm (OD600) was 0.5. Subsequently, 3 ng/ml of nisin was added to the cultures to induce pilus expression. Nisin (catalog number N5764; Sigma-Aldrich, St. Louis, MO, USA) was prepared as previously described (31). Briefly, a stock solution of nisin (10 mg/ml) was made by suspending 100 mg of nisin per ml in 0.05% acetic acid, followed by 10-fold dilution with dimethyl sulfoxide.
Detection of SpaCBA pili on the L. rhamnosus GG wild type and mutants by immunofluorescence microscopy.To phenotypically characterize the derivative mutants of L. rhamnosus GG, i.e., spaCBA mutant CMPG5357 and the exopolysaccharide (EPS)-deficient mutant CMPG5351, immunofluorescence assays were performed as described previously (32). Briefly, cells were grown to exponential phase and fixed for 1 h in 2.5% formaldehyde (catalog number VELC1374.1000; VWR International, Radnor, PA, USA) and 0.05% glutaraldehyde (catalog number G5882-50ML; Sigma-Aldrich, St. Louis, MO, USA). Cells were then washed and incubated in blocking buffer (0.5%; Boehringer Mannheim, Mannheim, Germany) for 10 min. The samples were further incubated in blocking buffer containing anti-SpaC rabbit polyclonal antiserum (21) or preimmune serum (1:100 and 1:400 dilutions, respectively) for 30 min and subsequently incubated in blocking buffer containing goat anti-rabbit IgG (1:200 dilution) conjugated to Alexa Fluor 488 (catalog number A-11070; Invitrogen, Life Technologies, Ghent, Belgium) for another 30 min. Samples were visualized with a Zeiss Axio Imager Z1 microscope equipped with an AxioCam MRm Rev 3 monochrome digital camera.
GFP labeling of L. rhamnosus GG pilus mutants.The nisRK genes with their own promoter were integrated into the chromosome of the L. rhamnosus GG mutants, as previously described for the wild type (33). Site-specific integration was accomplished by electroporating the suicide plasmid pMEC10 (34) into the L. rhamnosus GG mutants. Integration of pMEC10 at the tRNASer locus was confirmed by PCR as previously described (35). The following primers were used: PRO-567 (5′-GTCGACACAGGATTTGAACC-3′; which corresponds to primer T2 [35]) and PRO-570 (5′-CAAGCCAACAGACGTGCAAGCA-3′; which binds to the 3′ end of the int gene [35]). Subsequently, pMEC45 (36), the expression vector containing gfp under the control of the nisA promoter, was electroporated into CMPG5357/pMEC10 (CMPG1908) and CMPG5351/pMEC10 (CMPG1907). Transformants were verified by PCR analysis. Finally, the expression of green fluorescent protein (GFP) driven by the nisA promoter (pMEC45) in the L. rhamnosus GG derivatives was optimally induced as described previously (33). Briefly, overnight cultures of L. rhamnosus GG FAJ1906/pMEC45, CMPG1908/pMEC45, and CMPG1907/pMEC45 were used to inoculate fresh prewarmed MRS medium diluted 1:50. After 30 min of incubation at 37°C without shaking, 500 ng/ml nisin was added to the cultures, which were then incubated for 2.5 to 3 h. Nisin was used to induce the transcription of the genes under the control of the nisA promoter.
Maintenance and culturing of cell lines.The murine macrophage cell line RAW 264.7 (ATCC TIB-71) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). RAW 264.7 cells were maintained in Dulbecco's modified Eagle medium (DMEM; catalog number 41966-029; Life Technologies, Ghent, Belgium) containing 10% fetal bovine serum (FBS; catalog number SH30071.03; HyClone, USA) and 50 μg/ml gentamicin (catalog number G1397; Sigma-Aldrich, St. Louis, MO, USA) in a 5% CO2 humidified incubator at 37°C. Every 2 or 3 days, the cells were split at a subcultivation ratio of 1:3. For adhesion, phagocytosis, and immunomodulation experiments, RAW 264.7 cells were grown overnight in 12-well culture plates (catalog number 665180; Greiner Bio-One, Ltd., Solingen, Germany) at a concentration of 1 × 106 cells/ml.
Adhesion and phagocytosis assays with murine macrophages.Experiments to assess the adhesion of various L. rhamnosus GG strains to RAW 264.7 cells were carried out as follows on the basis of the methods of Lebeer et al. (22). A 1-ml volume of L. rhamnosus GG and the corresponding mutants (5 × 107 CFU/ml) was added to tissue culture plate wells containing RAW 264.7 cells. The bacteria were allowed to adhere to the cells for 1 h at 37°C. The cells were then rinsed three times with 1× PBS prewarmed to 37°C and resuspended in 1 ml of 1× PBS, pH 7.4. Finally, the cells were detached using a cell scraper, and serial dilutions of bacterial suspensions were prepared and plated out on solid MRS medium for Lactobacillus strains and M17 medium for Lactococcus strains. The percentage of bacterial adhesion was calculated by comparing the total number of bacterial colonies counted after adhesion to the number of cells in the bacterial suspension originally added to the tissue culture plate wells. In addition, the total amount of macrophage-associated bacteria was determined. To this point the aforementioned protocol for adhesion had been followed, but macrophages were then lysed with 0.2% Triton X-100 (catalog number T8787; Sigma-Aldrich, St. Louis, MO, USA) for 15 min prior to preparation of the dilution series in order to liberate internalized bacteria. The percentage of total associated bacteria was then calculated by comparing the total number of bacterial colonies counted after the macrophages were lysed to the number of bacteria originally added. Furthermore, to determine the number of internalized bacteria, phagocytosis assays were performed as described previously (37), with slight modifications. Briefly, RAW 264.7 cells (1 × 106 cells) were incubated for 1 h at 37°C with L. rhamnosus GG or L. lactis subsp. cremoris pilus variants as described above. Bacterial internalization was stopped with the addition of ice-cold 1× PBS, pH 7.4, and macrophages were washed three times to remove nonadhered bacteria. The remaining extracellular bacteria were killed by addition of streptomycin (100 μg/ml) and penicillin (100 U/ml) for another hour. The cells were then washed three times with 1× PBS and lysed with 0.2% Triton X-100 for 15 min. Serial dilutions of bacterial suspensions were prepared and plated out on solid media. The percentage of internalized bacteria was determined by comparing the total number of bacterial colonies counted after the cells were lysed to the number of bacteria originally added. All experiments were repeated at least three times, and all strains were tested in triplicate.
Analysis of bacterium-macrophage interaction by epifluorescence microscopy and flow cytometry.Epifluorescence microscopy was used to visualize the pattern of adhesion of the L. rhamnosus GG strains to RAW 264.7 cells. Macrophages were seeded onto coverslips in 6-well tissue culture plates (catalog number 657160; Greiner Bio-One, Ltd., Solingen, Germany) at a concentration of 1 × 106 cells per well and were grown overnight. An overnight culture of GFP-expressing (GFP+) L. rhamnosus GG (nisin-induced cultures of FAJ1906/pMEC45, CMPG1908/pMEC45, and CMPG1907/pMEC45) was used to inoculate fresh, prewarmed MRS medium diluted 1:50. After 30 min of incubation at 37°C without shaking, 500 ng/ml nisin was added to the culture, which was then incubated for 2.5 to 3 h. Fluorescently labeled L. rhamnosus GG strains were subsequently added to the macrophages at a ratio of 50 to 1 CFU/cell. After 2 h of incubation at 37°C in 5% CO2, the cells were washed three times with 1× PBS, pH 7.4, to remove nonadhered bacteria, and the interaction between the bacteria and the macrophages was examined by epifluorescence microscopy using a Zeiss Axio Imager Z1 microscope equipped with an AxioCam MRm Rev 3 monochrome digital camera (Carl Zeiss Microimaging GmbH, Germany). This assay was repeated at least five times.
For flow cytometric analysis, GFP-expressing L. rhamnosus GG strains were incubated with macrophages for 2 h at 37°C in 5% CO2. The cells were then fixed with 3% paraformaldehyde and resuspended in 1× PBS, and the fluorescence in the macrophages was examined by fluorescence-activated cell sorter (FACS) analysis with a BD Influx cell sorter (Becton, Dickinson, Erembodegem, Belgium). Data were analyzed using FlowJo software, version 7.6.4. The experiment was repeated at least three times, and all strains were tested in triplicate.
Induction of cytokine gene expression in RAW 264.7 cells.Wild-type and mutant L. rhamnosus GG cells were grown overnight in MRS medium and then centrifuged at 2,000 × g for 15 min at 4°C. After washing with 1× PBS, pH 7.4, cells were resuspended in DMEM without FBS and adjusted to a final concentration of 5 × 107 CFU/ml. One milliliter of the bacterial suspension was added to the wells containing RAW 264.7 cells and incubated for 3 h at 37°C in 5% CO2 at 90% humidity. Then, the cells were rinsed three times with prewarmed 1× PBS, pH 7.4. Here, lipopolysaccharide (LPS; 1 μg/ml) from Escherichia coli O111:B4 (catalog number L4391; Sigma-Aldrich, St. Louis, MO, USA) was included as a positive control, and DMEM unsupplemented with FBS was used as a negative control. Additionally, nonpiliated L. lactis subsp. cremoris MG1363 and the recombinant SpaCBA-piliated lactococcal construct (GRS1185) were also included as controls. RNA was extracted from the RAW 264.7 cells by using an RNeasy minikit (Qiagen, MD, USA), following the manufacturer's protocol. The concentration was determined by spectrophotometric measurement of the OD260 (ND-1000; NanoDrop Technologies). In addition, OD260/OD280 and OD260/OD230 ratios were measured and found to range from 2.0 to 2.2, and the RNA quality and integrity were confirmed with an Experion RNA analysis kit (Bio-Rad Laboratories, Hercules, CA, USA). Values of the RNA integrity number (RIN) of between 9.8 and 10 were obtained, with a value of 10 indicating intact RNA (data not shown). Measurement of cytokine gene expression was determined by quantitative real-time PCR (qPCR) as described below. The experiment was repeated at least three times, and all strains were tested in triplicate. To determine whether direct cell-to-cell contact is required for cytokine induction, RAW 264.7 cells were incubated with the L. rhamnosus GG strains in a Transwell culture system (catalog number CLS3401-48EA; Sigma-Aldrich, St. Louis, MO, USA). RAW 264.7 cells were located in the lower chamber, and L. rhamnosus GG cells were located in the upper chamber. The cells were separated by a 0.4-μm-pore-size permeable filter membrane support, thereby avoiding any direct contact between bacterial cells and macrophages.
qPCR analysis.Isolated total RNA (1 μg) was transcribed to cDNA with SuperScript II reverse transcriptase (catalog number 18064-071; Life Technologies, Ghent, Belgium) and oligo(dT) primers (catalog number N8080128; Life Technologies, Ghent, Belgium) according to the manufacturer's protocol. Afterward, nuclease-free water (catalog number AM9935; Life Technologies, Ghent, Belgium) was added to a volume of 100 μl. For each sample, 5 μl cDNA (containing 50 ng of transcribed RNA) was amplified in duplicate with the TaqMan universal PCR master mix (catalog number 4304437; Life Technologies, Ghent, Belgium) in a total reaction volume of 25 μl. We chose to evaluate eight reference genes commonly used as internal controls in expression studies, namely, TATAA box binding protein (TBP), succinate dehydrogenase complex subunit A (SDHA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β2-microglobulin (B2M), peptidyl-prolyl cis-trans-isomerase A (PPIA), ribosomal protein L13a (RPL13A), hypoxanthine phosphoribosyltransferase (HPRT), and β-actin. Interestingly, the TBP, SDHA, and GAPDH genes gave comparable results on the basis of RefFinder analyses (http://www.leonxie.com/referencegene.php) (data not shown). The GAPDH gene was then chosen as a reference gene, so that all results were normalized to those for the GAPDH gene to compensate for differences in the amount of starting material. qPCR was performed for IL-10, IL-6, and GAPDH in a StepOnePlus real-time PCR system (Applied Biosystems, Lennik, Belgium). cDNA plasmid standards, consisting of purified plasmid DNA specific for each cytokine, were used to quantify the target gene in the unknown samples, as described previously (38). For each cytokine, at least one primer or probe was designed to span an intron region of the matching cytokine gene. All primers and probes were designed on the basis of published sequences (39, 40) and chemically synthesized by Integrated DNA Technologies (IDT; Coralville, IA, USA) (Table 2). Each qPCR amplification was performed in duplicate wells in clear 96-well reaction plates (catalog number 4346906; Life Technologies, Ghent, Belgium) under the following conditions: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. qPCR data are presented as a ratio of the amount of cytokine mRNA to the amount of GAPDH mRNA. Nontemplate controls were included for each run.
TLR2 and CD11b blocking assay.The blocking monoclonal anti-human/mouse CD282 TLR2 purified antibody (clones T2.5 and 6C2) and monoclonal anti-mouse CD11b purified antibody (clone M1/70) were obtained from eBioscience (San Diego, CA, USA). RAW 264.7 cells (1 × 106 cells/ml) were incubated with TLR2 (5 to 10 μg/ml) or CD11b antibodies (5 μg/ml) for 30 min at 37°C in 5% CO2. Cells were washed three times with serum-free culture medium to wash off any free antibody. Subsequently, cells were cocultured with bacteria (5 × 107 CFU/ml). After 3 h of incubation, the RAW 264.7 cells were rinsed three times with prewarmed 1× PBS, pH 7.4. RNA was extracted, and measurement of cytokine gene expression was determined by qPCR as described above. Bacterial adhesion to and uptake by macrophages pretreated with CD11b antibodies were also analyzed as described above.
Statistical analysis.Significant differences in the data between wild-type L. rhamnosus GG and its mutant derivatives were evaluated by the unpaired Student's t test. A P value of ≤0.05 was considered statistically significant. All data were also analyzed using SPSS Statistics (version 22) software. The normality of the distribution was assessed using the Shapiro-Wilk test in which significant values greater than 0.05 represented a normal distribution. Graphical tools were also used to test normality by analyzing histograms and quantile-quantile (Q-Q) plots of the sample data.
Detection of pili on the L. rhamnosus GG wild type and isogenic mutants by immunofluorescence microscopy.In addition to the L. rhamnosus GG wild type, the isogenic spaCBA mutant CMPG5357, which lacks the SpaCBA pili (22), and the welE mutant CMPG5351, which is deficient in the production of EPS but shows increased exposure of pili and glucose-rich polysaccharides (20), were the main pilus-expressing variants studied in this work (Fig. 1D). To phenotypically confirm the presence of pili in these derivative mutants of L. rhamnosus GG, indirect immunofluorescence microscopy with anti-SpaC serum was performed. Figure 1B confirms that the pilus mutant CMPG5357 does not contain SpaC monomeric adhesins, as no fluorescence was detected on the mutant cells stained with anti-SpaC serum. In contrast, we were able to visualize SpaC-containing pili on the L. rhamnosus GG wild type (Fig. 1A) and even observed an increased signal with the SpaC antiserum in the EPS-deficient mutant CMPG5351 (Fig. 1C). Staining with preimmune serum as a negative control showed no fluorescence in the wild type or mutants (data not shown).
SpaCBA pili promote L. rhamnosus GG adhesion to RAW 264.7 cells and modulate its internalization.Since intestinal epithelial cells and monocytic cells have different cell surface properties, we subsequently aimed to investigate the role of pili in adherence to macrophages. Compared to the adhesion of the L. rhamnosus GG wild type, the pilus spaCBA-knockout mutant showed a ca. 2.3-fold reduction in adhesion to murine macrophage RAW 264.7 cells, while the EPS-deficient mutant CMPG5351 showed a ca. 2-fold increased adhesion capacity (Fig. 2). Likewise, an increase in the level of adhesion to RAW 264.7 cells of ca. 10-fold was observed for the nisin-induced GRS1185 lactococcal construct producing SpaCBA pili compared to the level observed for the nonpiliated L. lactis subsp. cremoris wild type. To specifically assess the role of SpaCBA pili on macrophage binding, the total amount of macrophage-associated bacteria was also determined, and when RAW 264.7 cells were stimulated with the SpaCBA pilus-deficient mutant CMPG5357, a reduction in the total amount of bacteria of ca. 2.3-fold compared to the total amount found when they were stimulated with L. rhamnosus GG was found. Conversely, the EPS-deficient mutant CMPG5351 showed an increase in the total amount of associated bacteria of ca. 2.1-fold (Fig. 2). Similarly, we could observe an increase in the total number of macrophage-associated bacteria of ca. 9.6-fold for the recombinant SpaCBA-piliated lactococcal cells compared to that for the nonpiliated L. lactis subsp. cremoris wild type.
Furthermore, to examine whether SpaCBA pilus-mediated adhesion affects the uptake of L. rhamnosus GG by macrophages by phagocytosis and related uptake mechanisms, the L. rhamnosus GG wild type and its mutant derivatives were incubated with macrophages, and the number of internalized bacteria was counted. External bacteria were killed by antibiotic treatment consisting of a combination of streptomycin (100 μg/ml) and penicillin (100 U/ml). Previous experiments showed that this treatment resulted in a more than 99% decrease of bacterial survival after 1 h of incubation, so that external bacteria do not significantly bias the results (data not shown). We subsequently observed that the SpaCBA-deficient mutant CMPG5357 was internalized to a ca. 2.7-fold lower degree than the L. rhamnosus GG wild type in RAW 264.7 cells. Conversely, the EPS-deficient mutant CMPG5351 showed a ca. 10.8-fold increased uptake compared to the wild type (Fig. 2). Similarly, the recombinant SpaCBA-piliated lactococcal construct GRS1185 was internalized by RAW264.7 cells ca. 452-fold more than the nonpiliated L. lactis subsp. cremoris wild type.
To further verify our findings, we also performed fluorescence microscopy analyses. First, the pilus mutant CMPG5357 and the EPS-deficient mutant CMPG5351 were transformed with a GFP-expressing plasmid as described in Materials and Methods. RAW 264.7 cells were then incubated with the GFP-expressing bacteria for 2 h, after which the interaction between bacteria and macrophages was analyzed. Notably, in five independent experiments we could barely detect the pilus-knockout mutant (CMPG1908/pMEC45) in the sample (Fig. 3B), while the EPS-deficient mutant (CMPG1907/pMEC45) showed a stronger capacity to interact with RAW 264.7 cells (Fig. 3C) than the L. rhamnosus GG wild type (Fig. 3A). Similarly, our flow cytometric data showed that the spaCBA pilus mutant (CMPG1908/pMEC45) had a lower capacity to interact with macrophages than the wild type, while the EPS-deficient mutant with an increased exposure of pili (CMPG1907/pMEC45) interacted with RAW 264.7 cells more than the L. rhamnosus GG wild type, showing a biphasic interaction curve indicating that the interaction is not synchronized (Fig. 3D). Collectively our data indicate that SpaCBA pili indeed play a crucial role in the adhesion of L. rhamnosus GG to murine macrophages and are also involved in its internalization process, while galactose-rich EPS molecules seem to prevent the adhesion capacity and further phagocytosis of L. rhamnosus GG by macrophages.
Cytokine mRNA induction in RAW 264.7 cells is modulated by the presence of SpaCBA pili on L. rhamnosus GG.Subsequently, we investigated whether the SpaCBA pilus-mediated adhesion influences the induction of the pro- and anti-inflammatory cytokines IL-6 and IL-10, respectively, in RAW 264.7 cells. Macrophages were coincubated with bacteria for 3 h, on the basis of the findings of previous experiments in which different incubation times were tested, and 3 h was found to be the earliest time point with optimal expression of the mRNA of the cytokines under study (data not shown). Our qPCR analysis showed a ca. 4-fold increase in the expression of the anti-inflammatory cytokine IL-10 mRNA in macrophages stimulated with the EPS-deficient mutant CMPG5351 compared to that in macrophages stimulated with the L. rhamnosus GG wild type, whereas the pilus mutant CMPG5357 induced a similar level of IL-10 mRNA (Fig. 4A, left). We also analyzed the role of pili in a recombinantly engineered L. lactis subsp. cremoris strain that produces SpaCBA pili. Interestingly, GRS1185 cells producing SpaCBA pili induced the expression of IL-10 mRNA to levels ca. 4.5-fold greater than those for the nonpiliated L. lactis subsp. cremoris wild type. Taken these findings together, the presence of pili seems to moderately promote IL-10 mRNA induction by L. rhamnosus GG and L. lactis subsp. cremoris, but comparisons of the results for the mutants show that the pili per se are not required for IL-10 mRNA induction. It is also noteworthy that the level of induction of the expression of IL-10 by L. rhamnosus GG or the L. lactis subsp. cremoris wild type is comparable to that by the control, LPS (1 μg/ml) (Fig. 4A, left).
We also tested the expression of IL-6 mRNA as a proinflammatory cytokine, considering its relation with inflammation. As shown in Fig. 4A (right), the expression of IL-6 mRNA was slightly increased by ca. 1.2-fold when macrophages were stimulated with the spaCBA pilus mutant CMPG5357, while the enhanced exposure of pili did not appear to impact IL-6 mRNA induction significantly when the results obtained with EPS-deficient mutant CMPG5351 and the L. rhamnosus GG wild type were compared (P < 0.05). However, stimulation with GRS1185 producing SpaCBA pili resulted in a decreased induction of IL-6 mRNA in RAW 264.7 cells by ca. 2-fold compared to that achieved with stimulation by the nonpiliated L. lactis subsp. cremoris wild type. Taken together, the presence of pili seems to moderately reduce IL-6 mRNA induction by L. rhamnosus GG and L. lactis subsp. cremoris. Importantly, LPS (1 μg/ml) induced IL-6 mRNA to much higher levels than all strains tested.
Subsequently, in order to investigate the role of cell contact in L. rhamnosus GG-mediated cytokine modulation in macrophages, Transwell experiments were performed to prevent direct contact between macrophages and bacteria. IL-10 mRNA expression was examined after 3 h using qPCR. Separation of macrophages from bacteria by a 0.4-μm-pore-size membrane indeed completely suppressed the induction of IL-10 mRNA (Fig. 4B, left). Similar results were found while investigating the expression of IL-6 mRNA (Fig. 4B, right).
Role of the host receptors CR3 and TLR2 in the interaction of L. rhamnosus GG with macrophages.To identify putative receptors on macrophages that are involved in the recognition of L. rhamnosus GG, possibly via the SpaCBA pili, we used previously validated blocking antibodies (24, 41) against complement receptor 3 (CR3; consisting of the two subunits CD11b and CD18) and Toll-like receptor 2 (TLR2). First, we evaluated the effect of CR3 in SpaCBA pilus-mediated adhesion to RAW 264.7 cells by blocking its CD11b component. We observed no significant differences (P < 0.05) in the adhesion of the L. rhamnosus GG wild type, the pilus mutant CMPG5357 and the EPS-deficient mutant CMPG5351 to murine macrophages that had previously been treated with anti-CD11b antibodies (Fig. 5A, left). Even though the difference was not significant, a minor inhibition of the adhesion of the EPS-deficient mutant was found when RAW 264.7 cells were pretreated with the antibody. Furthermore, the level of macrophage ingestion of pilus-expressing strains was slightly but significantly inhibited by ca. 1.2-fold (P < 0.01) by anti-CD11b treatment (Fig. 5A, right) of both the L. rhamnosus GG wild type and the EPS-deficient mutant. However, when analyzing the role of CD11b blocking in the cytokine modulatory capacity of L. rhamnosus GG, we observed that the expression of IL-10 and IL-6 mRNA was not significantly affected by treatment with anti-CD11b antibodies (data not shown). In contrast, antibody blockage of TLR2 showed a significant decrease in the expression of IL-6 mRNA by macrophages after stimulation with all the L. rhamnosus GG strains, while for IL-10 mRNA, this decrease was significant only for the EPS-deficient mutant (P < 0.05) (Fig. 5B).
Adherence is the first step by which components of the bacterial cell surface interact with receptors present on the host cells. Subsequently, surface-bound components and molecules secreted by the bacteria can trigger particular signaling pathways leading to the production of specific host proteins, resulting in specific immune responses. Thus, investigation of the molecular interactions between components on the bacterial cell surface and host cells is of great importance to unravel new mechanisms of regulation of the host immune responses (13). However, the role of bacterial adhesion in immunomodulation, especially in probiotic lactobacilli, remains unclear. Previous studies by our group and others have indicated that there are large differences in the adherence capacities of lactobacilli (reviewed in, e.g., reference 42). We recently showed that the long flexible pili of L. rhamnosus GG promote a strong interaction with substrates by a zipper-like mechanism involving SpaC adhesin subunits present at the tip but also along the pilus fibers (29). In addition, these SpaCBA pili seem to show a remarkable elasticity imparting nanospring properties that might help them to retract closer to substrates even under conditions of shear flow or peristalsis (29). Thus, given its high adherence capacity mediated by pili, L. rhamnosus GG is an interesting model strain with which to investigate the interplay between adhesion and immunomodulation. Here, we aimed to explore the importance of the presence of these pili on native L. rhamnosus GG cells in the interaction with phagocytic innate immune cells, i.e., macrophages. Interest in macrophages as important modulators of the interaction with gut microbiota and probiotics has been renewed, since it has been shown that they can also pass their dendrites through intestinal epithelial cells (26, 43). Moreover, macrophages can differentiate into classical (M1) or alternative (M2) subsets, with the first group promoting T helper 1 (Th1) immune responses and the second one being related to immunosuppression by having an anti-inflammatory phenotype (44, 45).
In this work, we demonstrated that SpaCBA pili of L. rhamnosus GG are crucial for its capacity to adhere to a murine macrophage (RAW 264.7) cell line and that this interaction stimulates bacterial uptake. Additionally, we observed that the pilus-mediated adhesion and enhanced cell-to-cell contact are coupled to an anti-inflammatory response, as monitored by the induction of IL-10 mRNA, since EPS-deficient mutant CMPG5351 with enhanced exposure of the pili and the L. lactis subsp. cremoris mutant with overexpression of the SpaCBA pili showed a markedly increased ability to induce this anti-inflammatory cytokine in RAW 264.7 cells compared to that of their respective wild-type strains. However, we could not observe any significant differences in the expression of IL-10 mRNA when the cells were stimulated with the nonpiliated spaCBA mutant CMPG5357 strain and the wild-type L. rhamnosus GG strain, suggesting that the pili per se are not required for the induction of IL-10 mRNA. IL-10 was selected as a biomarker for immunological activities due to its crucial role in the suppression and prevention of inflammatory responses (46, 47). This cytokine is produced by several immune cells, such as regulatory CD4+ T cells, which are key players in anti-inflammatory immune responses (48), and M2 macrophages, which express an IL-10high phenotype (49, 50). Interestingly, the level of IL-10 mRNA induced was similar to or even greater than that induced by LPS (1 μg/ml), a previously documented bacterial ligand shown by different groups to activate IL-10 gene expression in macrophages (51–53). Our findings after heterologous expression of the SpaCBA pili in L. lactis subsp. cremoris for IL-10 induction in murine macrophages are in agreement with those of previous work of von Ossowski and coworkers using moDCs (23). Given its zipper-like adhesion role, it is highly likely that the pili do not directly induce large amounts of IL-10 mRNA but, rather, mediate close interaction with the macrophages, resulting in an enhanced delivery of anti-inflammatory molecules. The exact IL-10-inducing molecules in L. rhamnosus GG thus remain to be determined, but our Transwell experiments suggest that they are located on the bacterial cell wall. Documented Lactobacillus molecules with anti-inflammatory properties include polysaccharide chains of rhamnose-rich EPSs produced by L. rhamnosus RW-9595M that induce immunosuppression by the production of anti-inflammatory IL-10 in macrophages (54), but they do not seem to be present in L. rhamnosus GG (20). Bernardo and coworkers identified a peptide (STp) secreted by L. plantarum BMCM12 that could induce intracellular IL-10 in human blood and intestinal DCs (67). Likewise, the S-layer protein A (SlpA) from L. acidophilus NCFM was found to mediate the interaction of bacteria with DCs by specifically binding their C-type lectin PRR DC-SIGN, resulting in the induction of IL-10 in the presence of LPS, but IL-10 was not as highly induced by LPS or SlpA alone (18). Furthermore, teichoic acids (TAs) from L. plantarum ATCC 14917 were found to be recognized by TLR2, activate the extracellular signal-regulated kinase pathway, and act synergistically with L. casei Shirota YIT9029 to induce IL-10 production in mouse peritoneal macrophages (55). However, other studies showed that expression of IL-10 was almost entirely unaffected in THP-1 cells after treatment with LTA isolated from L. plantarum KCTC10887BP (56) and that LTA from L. plantarum NCIMB8826 promotes IL-12 rather than IL-10 induction (57). Clearly, multiple Lactobacillus cell wall or secreted molecules could be directly involved in IL-10 mRNA induction.
Furthermore, our results indicate that the SpaCBA pili of L. rhamnosus GG do not directly influence the expression of proinflammatory cytokine IL-6 mRNA. We could observe an increased induction for the SpaCBA pilus mutant CMPG5357 compared to that for the wild type, whereas EPS-deficient mutant CMPG5351, showing an enhanced exposure of the pili, had the same levels of expression of IL-6 mRNA as the wild type. In support of the above-mentioned results, further experiments indicated that the SpaCBA-piliated lactococcus GRS1185 also reduced the level of IL-6 mRNA induction in RAW 264.7 cells compared to that achieved with nonpiliated L. lactis subsp. cremoris. However, these data are not in agreement with those from previous work using human moDCs (23), where the SpaCBA-piliated lactococcal constructs had increased levels of IL-6 production. It is worthy of mention that in the previous study (23), IL-6 production was measured according to the protein levels, while in the present work, gene expression at the mRNA level was evaluated. Moreover, different antigen-presenting cells (i.e., human monocyte-derived DCs and murine macrophages) were used in both studies, so that a variation in the pattern of cytokine production can be expected due to differential receptor expression (58). Taken together, our data suggest that the presence of pili probably indirectly prevents the induction of IL-6 mRNA by other surface molecules. Interestingly, a former study has demonstrated a role for pathogenic pneumococcal pili in the induction of proinflammatory cytokines, as higher levels of IL-6 were found in the serum of mice previously challenged with S. pneumoniae strain T4, which expresses pili, than in the serum of mice treated with its isogenic nonpiliated deletion mutant (59). Although the results from this in vivo study cannot be easily compared with those of our molecular interaction studies with cell lines, it is intriguing that pili from pathogenic bacteria induce proinflammatory immune responses in the host, while our data for the pili of the probiotic strain under study showed instead an indirect antipathogenic role. Nevertheless, future studies will have to investigate the role of pili on probiotic lactobacilli in modulating other anti- and proinflammatory biomarkers in different cell types and hosts with different disease states and genetic backgrounds.
To gain a better insight into the molecular interaction between piliated L. rhamnosus GG and macrophages, we also investigated the role of two PRRs, i.e., CR3 (CD11b/CD18) and TLR2. CR3 is a receptor present on the surface of most immune cells, including monocytes/macrophages, DCs, neutrophils, and natural killer (NK) cells, and plays an essential role in different immunological processes, including cell activation, chemotaxis, cytotoxicity, and phagocytosis (60) and tolerance induction (61). Previous studies demonstrated that pilus-associated adhesin RrgA from S. pneumoniae binds to CR3 and promotes bacterial uptake by murine and human macrophages (24). Using a similar approach with the same anti-CD11b antibodies, our data show that SpaCBA pili of L. rhamnosus GG promote bacterial phagocytosis by RAW 264.7 cells partially through CR3. However, the induction of IL-10 and IL-6 mRNA in these macrophages was not affected by the treatment with anti-CD11b antibodies, suggesting that L. rhamnosus GG-mediated cytokine modulation occurs in a CR3-independent manner. TLR2 has been found to be involved in the recognition of various MAMPs of Gram-positive bacteria. For instance, we recently demonstrated that LTA from L. rhamnosus GG stimulates TLR2/6-dependent activation of NF-κβ signaling in the HEK293T cell line and stimulates proinflammatory cytokines, at least under certain conditions (17). Interestingly, our present results show that induction of IL-6 mRNA by L. rhamnosus GG in macrophages is significantly influenced by the interaction with TLR2. This L. rhamnosus GG-TLR2 interaction is at least partially mediated by L. rhamnosus GG LTA, being a MAMP for TLR2/6, as determined on the basis of our previous work (17). Nevertheless, it is important to emphasize that LPS was still able to induce much higher levels of IL-6 mRNA than all L. rhamnosus GG strains tested, indicating that the induction of IL-6 by L. rhamnosus GG is subtler than that by the more immunostimulatory LPS of E. coli. Blockage of TLR2 also significantly affected the expression of IL-10 mRNA, but only when cells were treated with the EPS-deficient mutant, suggesting that molecules other than SpaCBA pili that are overexposed in this EPS-deficient mutant are signaling through TLR2 and that TLR2 could thus also modulate the anti-inflammatory responses. Interestingly, recent work in Staphylococcus aureus has also shown that its cell surface contains both proinflammatory and IL-10-inducing immunomodulatory molecules. These peptidoglycan-embedded IL-10-inducing molecules also bind to TLR2 on host antigen-presenting cells, activating the PI3K/Akt pathway, resulting in high levels of production of IL-10, which in turn downregulates the adaptive T cell response (62, 63). Thus, the exact receptor(s) by which the SpaCBA pili could mediate cytokine induction remains to be identified. For such studies, sufficient amounts of highly pure pili need to be obtained and preferentially studied in their native configuration. Such experiments are ongoing in our lab but are complicated.
Taken together, our data indicate that L. rhamnosus GG pili play a role in the host interaction with macrophages, mainly by promoting adhesion and internalization. As a result, other MAMPs, both pro- and anti-inflammatory molecules of L. rhamnosus GG, can interact more closely with their respective PRRs. Nevertheless, it remains to be substantiated in more complex models that the pilus-mediated high levels of adhesion of L. rhamnosus GG to host immune cells combined with a low inflammatory potential would be beneficial to humans.
This work was supported in part through BOF program financing (spokesman, Jan Balzarini; PF/10/018) at KU Leuven and in part through the Fund for Scientific Research, Flanders (FWO; grants KaN 150711N and 1520114N), and IOF-SBO financing at the Universiteit Antwerpen. Dominique Bullens is a recipient of a senior researcher fellowship from the Fund for Scientific Research, Flanders (FWO). I.V.O. and A.P. acknowledge project financing from the Academy of Finland-funded Center of Excellence in Microbial Food Safety (CoE-MiFoSa) research program (grant no. 118602 and 141140) and an Academy of Finland general research grant (no. 118165).
Marijke Segers and Hanne Tytgat are acknowledged for many insightful discussions and suggestions. Lieve Coorevits is gratefully thanked for her invaluable help with the culture of RAW 264.7 cells. We also thank Stefanie Roberfroid, Louis Deforche, and David De Coster for their technical assistance with the fluorescence microscope, as well as Geert Schoofs for his assistance with the setup of the flow cytometry experiments.
- Received 3 December 2014.
- Accepted 5 January 2015.
- Accepted manuscript posted online 9 January 2015.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.


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So, the winner of the giveaway is...... Luke & Katie.
I'll be contacting you now, but if you see this let me know too =) Sorry for the delay on posting. I couldn't get on yesterday and today I figured out sitemeter was having some probs, so hooray I got on =)
Check out this new site http://www.momdot.com/ {the name cracks me up} They are launching their new bloggy site featuring lists of blogs, boutiques, & posts of their own. Contests too!
I wish I was at the beach going on this ferris wheel again....but off to buy school supplies for a certain kindergartener-to-be this weekend & visit my parents & Aunt Susie & Uncle in town from Arizona.
Have you seen Wordle? This one is of my blog a couple weeks ago. Just enter your blog address or a list of words you type in here and it generates word art...many colors, many styles.
...here is random happiness


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This is another picture of a Steam Engine on the Ffestiniog railway which was taken in 1978. Unfortunately, I can't make out the name of the engine and although I found another picture of it here, that picture also has the name obscured. The engine is obviously at a station in this picture but the name sign on the side of the picture is unfortunately cut from the frame.
Photo category
TransportationDate Picture Taken:
The Location of this picture is shown below


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What is the new Hormann HST side sectional garage door? A sectional garage door that opens sideways?
That is correct, Hormann have added the long and eagerly awaited HST side sectional door to their range of high quality garage doors. But who would want such a door?
We get asked most days for advice and prices on round the corner garage doors and there are many types now available in the UK all manufactured in a factory environment unlike the RTC doors of old. They were always made of timber, but when timber was well seasoned and not so expensive but they always had to be made into a sliding round the corner system on site, and from scratch - very time consuming and labour intensive. To make such a door these days is still perfectly possible but extremely expensive and you have to start with a high quality joiner and the right timber species to stand a chance of the complete system working.
The Hormann HST side sectional is a garage door designed using the already enormously popular, tried and tested high quality panels from the sectional garage door range, the LPU40. This is a double skinned 42mm thick galvanised steel door made from panel sections varying in depth from around 370 to 500mm and is available up to some very large sizes.
The HST garage door merely uses these superb panels and with some high quality tracking enable these panels to travel sideways and around the corner of the garage aperture providing a garage door with many advantages - minimum headroom required, partial opening for pedestrian access, unique designs and vertical lines for listed or conservation areas and quite honestly and very smooth, quiet and reliable operation with very little moving parts to ever cause a problem.
You also get a good selection of surface finishes either in RAL painted colours or the superb Decograin laminate woodgrain finishes and a choice of optional windows, all double sealed units.
The HST side sectional can be either manually operated or electrically operated using any of the vast range of electric operators already produced by Hormann with different ratings for speed and frequency of operation as well as an enormous range of access controls and security devices.
Take a look at this new side sectional round the corner system for your garage, it may well be the perfect solution and those people who may comment that you lose a wall in your garage for shelving, etc consider the fact that this also forces you to keep your garage tidy!! I know I have one. You cannot lean bikes, garden furniture, old pieces of timber and all those sort of items against the wall so you do not, and with this comes a forced tidiness, perfect!!
Of course if you really want to you can create a false stud wall partition for the door to go behind and get yourself a false wall anyway.
See our information page on the Hormann HST door >


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Kulacom has announced the official launch of its wireless broadband network in Jordan, in partnership with SK Telecom. Eng Basem Rousan, Jordan's Minister of Information and Communications Technology said the launch would help achieve one of the main objectives of the national ICT strategy, namely increasing internet user penetration to 50% by 2012. Kulacom's city-wide network, based on South Korea's homegrown WiBro standard, currently covers over 30% of the population of Greater Amman, providing customers with a full range of voice and data services.
In a separate announcement SK Telecom said that it had signed an additional consulting agreement with Kulacom worth USD4.38 million, as well as a letter of intent (LoI) worth USD2.18 million to cooperate in the latter's WiBro businesses in the Middle East and Eastern Europe. SK Telecom will provide support to Kulacom's Jordanian project, as well as developing new business opportunities in other emerging markets. In particular, SK Telecom plans to send technology and marketing experts to Kulacom headquarters to provide support in business development and management.
Kulacom, registered under the name MetroBeam Wireless Telecommunications Co, was founded in 2006 by Jordanian investors with regional and international experience in the telecoms sector. It was awarded a fixed broadband wireless access licence using the 3.6GHz band in January 2008.


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Yike! ~~ 2NE1,, i love their voice …
ahh~~ I'm surprised Minzy's voice xD so good Live!! ❤
:: Take a Bow ::
Sandra's voice .. is pretty xD >< she give a sweet voice and smoothing …..
ya CL's voice ..no comment ! because def GOOD!!! rocks! so talented
Bommie.. amazing voice !! as we known ..
:: Fire ::
Here's "Fire" Performance^^
Minji.. how come this girl can did like that @0.48 omggg!!!! lol
@YGladies & CodeMonmonSeason2
Filed under: Gossip Star | Tagged: 2NE1 |


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Body Bubbles Body Wash is made from white tea, flax seed, lavender & peppermint and will balance moisture levels while helping to stimulate blood circulation and improve your skin´s defence system. Lavender water gives calming and soothing properties for skin.
99% of the total ingredients are from natural origin.
5% of the total ingredients are from Organic Farming
Natural Cosmetic certified by Ecocert Greenlife according to the Ecocert standard available at www.cosmetics.ecocert.com
INCI: Aqua, Sodium Coco Sulfate, Lavandula Angustifolia (Lavander) Floral Water*, Sodium Cocoamphoacetate, Cocamidopropyl Betaine, Citric Acid, Coco-Glucoside, Glyceryl Oleate, Betaine, Camellia Sinensis (White Tea) Leaf Extract*, Linum Usitatissimum (Flax) Seed Extract*, Benzyl Alcohol, Aroma, Sodium Chloride, Sodium Benzoate, Mentha Pipertia (Peppermint) Essential Oil*, Potassium Sorbate, Limonene**, Linalool**, Geraniol**
** Natural essential oils
It is our guiding principle to always choose the most sustainable solution, and not compromise – even though this stance forces us to exclude certain options, it opens just as many new doors of opportunities, and inspires us to think creatively. It is also extremely important to us to keep our work as transparent as possible, in terms of both production and processes, enabling the consumer to feel sure that they are not being misled – there is a lot of greenwashing going on in many industries, unfortunately.
In terms of iLoveEcoEssentials, our principles have now led to an ECOCERT certified product serie.
Our bottles are manufactured in the UK. The bottles are made with collected consumer plastic that is recyclable.
It´s a jungle out there! …and we wanted to make sure that our products were the very best in every respect. That´s why we chose ECOCERT.
ECOCERT is the world´s leading certification organization, and supports and guides its members to make guaranteed trustworthy products.
ECOCERT sets the standard for natural and organic cosmetics worldwide, ensuring sustainability, environmentally friendly processes and products formulated without GMO, Parabens, Phenoxyethanol, Nanoparticles, Silicon, PEG, synthetic perfumes and dyes or animal-derived ingredients.
It offers the most natural seal of approval, and reassures even the most sustainability-conscious consumer.


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FLEXIMARK® Safety Signs
There are plenty of prohibition signs, such as No Smoking, in our daily lives.
These prohibition, warning or instruction signs can also increasingly be found in industry due to the number of potential hazards. Labour protection is an important issue for every company. When working on machinery, employees are constantly exposed to a variety of hazards such as hot surfaces, electrical voltage or risk of entrapment. Simple and comprehensible safety signs can draw attention to these hazards and thus prevent a lot of accidents.
The ISO 7010 standard for safety signs was introduced to ensure that everyone can interpret the hazards correctly. ISO 7010 defines safety signs that are universally comprehensible. This means that new employees or external visitors can immediately identify any hazards lurking in the environment.
ISO 7010 has been the applicable standard since the beginning of 2013. If your company still has signs complying with the old DIN 4844-2 standard, you are not obligated to replace them. However, in case of damage or accidents, you must demonstrate that the old safety signs meet the same safety standard as the current signs stipulated in the ISO 7010 standard. Because it is very difficult to provide evidence of this, updating the signs to meet the latest standards at the earliest opportunity is advisable from a professional perspective.
In case of damage or accidents, you always have to ask:
"Could this accident have been avoided by better and more comprehensible signs?"
Therefore, we offer you the FLEXIMARK® Safety signs. Those warning, prohibition and safety signs made of laminated polyester provide you an extensive long-term labelling complying with ISO 710.
Points to consider for industrial safety signs:
6. Smooth, laminated surface for easy wiping/cleaning
7. High resistance to alcohols, moisture and oils
8. Powerful adhesive
9. Scratch resistance to ensure long service life
10. If necessary, UV resistance for outdoor use


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Rotarians, Partners and Friends relax on a Sunday afternoonthumbnail view
Chard Rotarians, together with partners and friends spent a relaxing afternoon at the home of Rosemary Ward-Smith, widow of past Rotarian Richard Ward-Smith.
On a warm, sunny afternoon everyone enjoyed a tasty barbecue, prepared by President Nominee Paul Arnold and his wife Anne.
There was an opportunity to enjoy the covered swimmming pool in the garden but most present simply enjoyed the opportunity to talk to friends.


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Remember the servants quarters with the near Oliver Twist-ian beds? Well they've been...improved. Most definitely. At least the maids will be able to sleep now. That's something right?
I got these from Minibeddingshop on Etsy, https://www.etsy.com/shop/minibeddingshop?ref=l2-shopheader-name and I may have to go back for more.
|See? Cute little matresses. I love these.
They fit perfectly on the brass beds.
|I like these mattresses better than the one I sleep on. Why is that?
|Both beds... And one of these days the maid's outfit will be period appropriate. Bare legs and ankles! Oh the scandal!
The blankets they had were kinda flimsy. The Reformists would have kittens. So I got the maids some quilts. Not matching of course...I mean really they're only servants. Pish tosh, any old thing will do.
The first came from MiniBargains on Etsy. https://www.etsy.com/shop/MiniBargains?ref=l2-shopheader-name
|This one is more of a crazy quilt.
This one I got off of Ebay and cannot remember the sellers name for the life of me.
|Pretty nice huh? I need to make them a rag rug or something... that bare floor looks chilly.
These come from Just For You Miniatures on Etsy, https://www.etsy.com/shop/JustForYouMiniatures?ref=l2-shopheader-name I think I've favorited everything in her shop. Gorgeous., absolutely, mind bogglingly, gorgeous.
|Isn't it pretty? I love that its wearable.
|I'm pretty good at tiny sewing but this is beyond me... so delicate!
|The ruffle around the face! OMG gorgeous.
|Fabulous Harry I love the feathers!
|And the tiny little flower details.
|And the fur lining and trim! Have I mentioned I love the feathers?
|Really, her ribbons are on the floor. She needs to take better care of her things.
|The flash washes out the pale green but it looks so pretty in person.


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Surf and immerse yourself in a world of wellness at COMO's newest retreat.
This November, COMO Uma Canggu and COMO Shambhala Estate in Bali will be hosting Sally Fitzgibbons and 16 private clients in an intimate, results-driven eight-day surf and wellness retreat.
"Women's surfing is riding a wave of change, empowerment and respect" says Sally, whose commitment to an empowered lifestyle – in her professional career, her plant-based diet, and love of learning – will inspire everyone, from seasoned surfers to absolute beginners.
To get a sense of her good humour and empathy, COMO caught up with Sally in the midst of the 2019 World Surfing League Championship Tour. She has won 11 World Surfing League Championship events before; this time, winning the World Title would qualify her for the 2020 Olympics, with surfing included for the first time. Yet chasing accolades isn't what motivates Sally: "Surfing allows you to connect to something deeper than just you; it brings something special to your daily life."
During her eight-day dual destination retreat in Bali, Sally aims to help other women make this connection to the world around them by empowering their mind, body and spirit with help from COMO's experts in surf and wellness.
Article c/- COMO Hotels and Resorts.. Read the full article here.
[contact-form][contact-field label="Name" type="name" required="true" /][contact-field label="Email" type="email" required="true" /][contact-field label="Website" type="url" /][contact-field label="Message" type="textarea" /][/contact-form]


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IGNITE 2019: The Inaugural Artivest Alternative Investment SummitApril 10, 2019
For decades, institutional-grade alternatives were within reach of just the largest stewards of capital: giant pension plans, sovereign wealth funds and institutional endowments and foundations. Today, advances in financial technology and industry shifts have sparked seismic change.
What's changed? Everything.
Alternative fund investment minimums stand at a fraction of seven figures. A slow, paper-heavy $13 trillion industry has been set into motion as it moves from analog to digital, and shifts into hypergrowth. Doors have opened for not just Wall Street's weighty few, but a new universe of intermediaries and qualified individual investors. Top-ranked alternative managers are looking downstream to stir up new sources of capital. Frictionless private fund subscriptions take hours instead of months. "Niche" strategies are illuminating new sources of alpha and yield. Operational improvements are enlivening the entire alternatives ecosystem, making it far more accessible. A bright new era of alternative investing has arrived.


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Tag: nematodes
A Suspicious Pattern of Deletions
Winston Ewert recently published a paper in BIO-Complexity suggesting that life is better explained by a dependency graph than by a phylogenetic tree.
Answering Joshua Swamidass on Theistic Evolution: A Religious Agenda?
He defines himself as a Christian who affirms evolution, while also rejecting the name theistic evolutionist.
Nematodes Push Epigenetics to the Limit, as Another "Zombie" Bites the Dust
Isn't DNA the one and only "secret of life"? Neo-Darwinism, a gene-obsessed doctrine, needs it to be.


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Keywords: Tomcat - Amazon Web Services - Technical issue - OtherDescription: Our instances all originally derived from a Bitnami Apache Tomcat AMI. We've since continued to grow our own AMIs off of those servers but the underlying structure is still all original. We have been having a problem recently when running upgrades. It happens after we run an upgrade. It works fine after the upgrade until reboot and then it will fail the instance status check. After it fails the check the instance is unreachable.
I've seen this happen before and then was able to spawn a new image off an earlier snapshot of that instance. I ran upgrade again and watched exactly what it upgraded. I noticed the JDK upgraded. After looking at the JDK directory I found that the bitnami directory that contains the setenv.sh files was missing. I copied it back in and then restarted and then all was good, passed the status checks.
This time however I don't have a recent copy of the problematic instance. So I've disconnected the instance volume and attached it to another one. I'm able to access the volume this way. I saw once again the bitnami folder was missing so I restored it. Then created a new AMI off that volume and launched a new instance. That instance however is also failing the instance status check.
So my question is, is there a better way to identify why these status checks fail? Are there bitnami logs I can find somewhere from the attempted start up to see what went wrong? Is there a tool I can run to analyze the volume?
Any help is greatly appreciated.
Hi @nmb1106,
Thanks for using Bitnami. Can you explain the upgrade process you are applying to the VMs?
Andres R.
Hi @andresrc,
Sorry for the long delay. We're working through a security audit and have been side tracked with that.
I just run the following:
sudo apt-get updatesudo apt-get upgrade
I'll also say though that I upgraded the Open JDK 1.7 to Oracle JDK 1.8 at some point long ago. We needed to switch to Oracle after having some cert problems executing web service requests against some of our customers services. I followed a guide I found some where in the community. It had me install and then update a number of references to the JAVA_HOME and JRE_HOME throughout the instance. Since then the bitnami folder will just disappear after updating sometimes.
My biggest question really though is what tools are available to me that can help me identify why the instance is failing the checks? Are there any logs or anything of that nature to check? Is there anything to look for inside the instance's volume that I detached and attached to another instance? It seems hard to believe that something could just not start and there is no where to look for why. But so far I've found nothing useful.
A colleague will contact you soon to help you with this issue.
Best regards,
Michiel D'Hont
Please, click on if you think my answer was helpful
So, your issue is the "/opt/bitnami" directory getting removed after running "apt-get upgrade"? That's really strange. The only thing that occurs to us you can check, are the system logs at "/var/log", and to save the logs from the "apt-get upgrade" command in case we see anything strange.
If you still have your instance present somehow, and the "/opt/bitnami" directory is present, we have a Support Tool that will gather relevant information for us to debug the issue. Could you please download and execute it on the machine where the stack is running by following the steps described in the guide below? You must click on the platform or cloud that you are using to find the correct instructions.
How to Run the Bitnami Support Tool
Please note that you need to paste the code outputted by the tool in your reply.
If it is possible for you, we would recommend to migrate to a new AMI that does not present this issue, since there might be some kind of logic cleaning /opt after upgrades, although we're unfamiliar with anything similar to this.
Hi @marcos,
No the /opt/bitnami doesn't get removed. A while ago I installed an oracle JDK at /usr/lib/jvm and changed everything in the environment to use the new JDK home instead of the old /opt/bitnami/java home. The /usr/lib/jvm/jre/bitnami directory gets removed which contains the setenv.sh files for setting the java memory arguments.
I don't know if that's the only issue though as I've tried detaching the volume from the instance and attaching it as a secondary to another. Then I put that directory back in place. I then created a snapshot and a new AMI from that volume. Then launched a new instance. That instance spawns with the same problem.
This issue isn't holding us up any longer but my goal of this post was really to find a way to figure out why the instance is failing the status checks. For instance, if my application failed to start up I'd check my logs that are filled with details during the startup. I'm looking for the equivalent to look for here. I certainly am not an expert in linux environments and am really learning as I go out. The answer to this may be very simple and just something I don't know to look for.
There are tons of logs in /var/log, is there a specific log I should look at?
Thank you
Regarding the "/usr/lib/jvm/jre/bitnami" directory, we were not able to locate such a directory in our Tomcat AMIs.
Note that independently of the system's JAVA_HOME value, the Bitnami installation should use the Java installation in "/opt/bitnami/java".
If that is not the case, it is probable the Tomcat scripts have been modified somehow. For instance, check "/opt/bitnami/apache-tomcat/bin/setenv.sh", as it should contain the following line explicitly: "JAVA_HOME=/opt/bitnami/java".
When the directory gets removed after upgrading, what is happening to your application? Is it not starting or are you not able to reproduce it due to the status check fail? Note that when you see the status check not working, it is more probable for it to happen because of SSH settings changes than Tomcat itself not working.
We recommend you checking SSH changes to ensure they are properly configured, and to ensure that SSH is configured to start when booting the VM. You can do this with "chroot" when you mount the volume and run "service", although you will need to mount some directories before for getting the commands to work (/run, /proc, /dev).
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text:
After the breakup of Ricocher, I continued writing new songs. Some of these songs are now ready to be recorded under the name MILLENNIUM | TRILOGY | PROJECT. This first progressive concept album will be titled "part 1: The Trial" and will contain eleven songs with a total playtime of more than 60 minutes. The music style lies somewhere between progressive rock, AOR, symphonic rock.
The story is based on the Millennium Trilogy books and handles about a girl, named Brynn, that is abandoned by society. While everyone sees her as dangerous, suicidal, autistic and schizophrenic, the only thing she tries to do is SURVIVE!!
Now she is facing the judges, the jury and her inner self! It's the crossing that will determine the rest of her life…..
News & updates
Introduction drummer: Igor Koopmans
In the next couple of months we will continue recording the first album of the Millennium Trilogy Project.
Hereby I present you the man behind the drumkit: Igor Koopmans!
He's also known for his drumparts on the last Medea album and off course as the drummer of Casual Silence. Together with the bass parts of Niels Nijssen, he will lay down a solid base for all the songs!!
Introduction bass player: Niels Nijssen
This week we'll start recording the first parts of the album!!
And were better to start than with the bass guitar parts. These bass guitar will be played by one of the best bass players I know: Niels Nijssen!!
He formerly was the bass player in Ricocher and Eleven's Eve and is a guarantee for super solid basslines!! I'm very proud to work with Niels again!
Niels, welcome on board of the MILLENNIUM|TRILOGY|PROJECT
Will you become a part of the MILLENNIUM | TRILOGY | PROJECT?
Thanks for visiting this page!
In May 2019 I will start recording my first solo album, "part 1: The Trial" in the Rock Inc Studios. I won't do this all by myself but with a lot of musicians and vocalists.
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Take a look on this page and find the option that fits you the best!
Thanks for your support!!
John van Heugten