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On August 22, 2016 August 31, 2016 By ViolincatIn DIFT, Generation 2, Our Different Paths, Sims 316 Comments
I was woken up by a loud conversation the next morning. The voices were those of my family members and of two other people. One voice was familiar, the other one, not so much.
Throwing on some clothes, I headed out to see what all the noise was about.
In the living room were two men- one was my mother's old friend, Sebastian Vanderburg, who has worked with the Hidden Springs police department for as long as I could remember. The other man was younger and unfamiliar. Despite his younger age, he had a much stricter appearance and I got the feeling he wasn't here just to chit-chat.
"Irene, there you are!" my mother called to me. "Captain Vanderburg and deputy Westen wanted to speak with you and David."
I noticed how she used Sebastian's title and not his name. The other man's presence must have been the reason for that- she didn't want him to think Sebastian had any kind of impartiality to our family. For someone who wasn't really in the know of recent events, my mother had good gut instincts.
"Good morning, mom, dad, Gerald. Good morning Captain Vanderburg and Deputy Westen. How may I help you?" I greeted the two men. Considering the recent events, there were few good reasons for them to be here, but it still wouldn't hurt to be polite.
"Ms. Meadows, I assume you are aware of the recent passing of your CEO. And of the leak of some very sensitive information regarding the company you are currently on the payroll of," the deputy didn't waste time.
"I am aware, it was pretty much all over the news yesterday," I admitted. "We were all pretty shocked—no one expected Mr. Collier to die so young. But I assume you wanted to talk with David about this? He's still asleep." I bit my lip. How would David fare talking to the police right now? He still wasn't dealing with yesterday's events too well. "Should I wake him up…?"
"That won't be necessary yet, Ms. Meadows," the deputy shook his head. "We would very much like to talk with you first."
This isn't going too well, I began to think. There was something about the way that deputy looked at me that didn't sit well with me. It was almost like he was trying to trip me up.
"Alright, then. I'm here." I sat down with the people in my living room. Throughout the exchange, Sebastian Vanderburg remained quiet, but I had the weirdest feeling he was here on our side. But why would he need to be on our side, unless…
"Ms. Meadows, where were you this Tuesday night, from 5 to 12 pm?" he asked without breaking eye contact.
Oh well, we always knew this was a possibility, I thought. They were on to us.
"I worked late at the office that day. I didn't leave the building until late that night," I told him. It wasn't a lie, really. I was at the office, after all.
"I'm sorry, but shouldn't she have a lawyer for questioning?" my father chimed in. He was a farmer, yes, but that didn't mean he didn't know quite a bit about the legal system.
"Agreed," my brother chimed in. "You are violating the law by questioning her without a lawyer."
The deputy gave a sad smile and turned to Sebastian, who in turn calmed us down "This is not an interrogation per se. And if anything, I'm here to make sure Deputy Westen doesn't do anything outside his jurisdiction."
Well, that was just weird.
"Ms. Meadows, I understand how my presence here, so early in the morning after the events of yesterday, can seem very accusatory. I assure you, this is not why I'm here. However, I would like to know whether Mr. Collier Jr. was with you on the evening in question."
"Me?" David asked, coming into the room. "What do you mean?"
"Hello, Mr. Collier. I'm glad to see you are doing so well, despite your loss. My sincerest condolences to you regarding your father's passing," the deputy nodded at him. "I have to ask, were you alone on this Tuesday evening? Or were you, perhaps, in someone's company?"
"I was alone, until I met with Irene back at the office. We came here after work." David just entered the room, but he also quickly caught on that something was weird here. "But why does it matter? Am I under suspicion of anything? Or is she?" He pointed at me.
"No," the deputy gave a weary sigh. "Neither of you is under official suspicion of anything. However, I'm sorry to inform you, Mr. Collier, but your mother is our main suspect in the death of your father, Lionel Colier. Autopsy was able to find traces of succinylcholine in his system—his death was not natural and due to certain information, we have good reason to suspect Selma Collier is responsible. I'm here on official visit from Twinbrook to check her statement. When we questioned her, she has told us that she spent the times in question with her son, David. However, upon questioning, both her neighbors and yours informed me that the house lights were off for the entire night, as if no one was home. So, of course, I thought her story wasn't quite sound. It turns out, I was right."
"Wait, why would you think my mother committed murder just because she lied about being with me that night? Maybe she had good reason to say that?" David defended and I didn't blame him. This thing the deputy did? It was a trick- he knew perfectly well that Selma Collier wasn't with David that night. But how?
"He's right," I added. "Sometimes women lie about these things, you know. Sorry David, but maybe she was meeting a 'male friend' and wasn't comfortable admitting that to the police?"
"In Twinbrook?" the deputy shook his head. "Look, the thing is, even if you and Mr. Collier Jr. held up her alibi, it wouldn't have worked. You see, several people have spotted her in Twinbrook, including the workers of the motel she has checked into while in the city under a fake name. Now tell me, don't you think it's suspicious that the wife of a CEO of a huge pharmaceutical company arrives into a city under a fake name only a day before the man himself dies of a 'heart attack'? And there is information pointing to her having purchased the poison?"
"Yes, it is very suspicious," I admitted. "But it still doesn't mean she actually did it. The Colliers have a lot of enemies. It wouldn't be out of character for someone to try and frame her in the midst of all this." I didn't know why I was defending Selma Collier, but something about the whole situation just didn't sit well with me.
"Someone like, say, a young woman whose family was robbed by the company and who has been working at said company since high school?" the deputy looked at me.
Ah, so now he was bringing out the big guns. Even Captain Vanderburg looked shocked at this. "Listen, Deputy Westen, I realize that now, after all the information about Collier Enterprises' illegal activities came to light, it really makes sense for me to want to hurt the people involved, but tell me, if I was really responsible for Lionel Collier's passing would I really murder him and try to frame his wife for it, only to make up defenses for her afterwards? Would that make any sense to you? Besides, anyone can tell you that I was in Hidden Springs for this whole last week."
"I know you were here," the man smiled. "And I don't think you did it. However, if she is really innocent of murder, why is she lying about her true whereabouts and intentions for being in Twinbrook? Wouldn't that only make her situation worse?"
He had a good point. Why would she lie about this?
Gerald chimed in again "Look, if you got the 'proof' of her purchasing the poison from a huge server leak, doesn't it mean someone is out for their family for real? How do you know that information is legit? I mean, did you find any more of the poison in Selma's possession?"
"Well, no," the deputy admitted.
"In fact, from what he tells me, the woman looked completely baffled when presented with the information that she was a suspect for poisoning," Captain Vanderburg added.
"I have never seen my mother baffled by anything in her life," David admitted. "If she was, then she really didn't do it. Now I really think someone is trying to frame her."
"And they're doing a pretty good job of it," my mother chimed in. "You guys need to think big. Who would have wanted to have both Colliers out of the way, one in jail, the other dead? Find that person and you have your killer."
The deputy listened to all of us carefully, taking in as much information as he could. Finally, he nodded and got up.
"Thank you for your time, all of you. You definitely cleared some information up for the Twinbrook police department. I think we can go now, Captain Vanderburg."
"Alright, then, I'll go start up the car. Simon, Celeste, it's been good seeing you two. And Deputy Westen, let me tell you, no one in this family would commit anything illegal. That I can vouch for." He headed out for his car.
"Wait," David yelled out. "So, is my mother still under suspicion?"
"Well," Deputy Westen shrugged, "Considering how we came by the information that accused her, her own silence and lies on the matter are her worst enemies right now. As soon as she explains her side of things, her lawyer will be able to help her."
"So, she's still in trouble…" David frowned.
"I'm surprised you are so worried, Mr. Collier. I thought you were dead set on exposing your family's activities?"
My jaw dropped to the floor. How could he know about this? Was someone tailing us the whole time?!
The man noticed my expression and smiled "You see, in this age of nonstop surveillance, many go out of their way to disguise bugs and hidden cameras in the strangest objects, like, say, a statue, a necklace, or even a painting. And sometimes painted dragons really are watching over you. At least you had the common sense to wear gloves while breaking and entering, Ms. Meadows."
"You know about this?! How are you not arresting us?" I blew up.
"For what? Technically, Mr. Collier here wasn't violating any laws—he was in his father's office. And you were with him. And since you weren't even the ones who leaked the information… it was done from somewhere in Twinbrook… Anyways, take care, you two. Next time, you might not get so lucky."
And with that, he left, leaving both of us and my family just standing there dumbstruck.
"So," my dad found his speech first. "You two want to fill me in on what was happening lately?"
Well, considering how it was his discovery that started off this whole mess, he had a right to know.
"Yeah, dad. You guys might want to get comfortable. It's quite a story."
Naturally, neither of my parents was happy to hear that their kids were leading an illegal investigation and that their daughter broke into her boss's office while he was gone. However, they understood why we felt like we needed to do those things.
"So now, someone else leaked the information you guys have been trying for years to access," my father pondered. "From what you're saying, I'm guessing there was a second server key that David's father always had on him. Whoever leaked that information must have gotten pretty close to Lionel to access it."
"Well, there isn't much we can figure out sitting here, in Hidden Springs," mom brought up. "And I don't know how much this police deputy is willing to do to get to the truth. I mean, the Colliers are a pretty hated family. Sorry, David. But it's kind of true. So, I don't know how much chance your mother will have now, even if she's innocent."
"It's ok, Mrs. Meadows. I know how things stand at the moment. I just don't understand why she's lying to them," David threw his hands up. "The deputy is right- she's making things worse for herself."
"I think I need to call Roman," Gerald shook his head. "If he's finished catching up with his wife, I think he'll be able to learn more through his channels. And honestly, I'm just curious about who it was that leaked the information before us."
As if on cue, his phone rang—Roman called him first.
"Gerald, are you able to talk right now? I have some information that may interest you on this?
"Hold on, can I put you on speaker? My whole family and David are here and they're interested in this too," Gerald explained.
The hacker clearly agreed, and Gerald set the phone on the table for us all to hear his news.
"You guys! So, do you know Selma Collier has already been arrested?"
"Yes," Gerald told him. "We've just been visited by a detective from Twinbrook who was here to verify whether her alibi of being with David that night was holding up."
"And it totally didn't, right? I mean, she must have been in Twinbrook already. Here's the deal—it seems that someone may have lured her out there with information that Lionel was cheating on her there. Which would totally be why she headed there incognito—imagine how much money she could get out of a divorce after catching Lionel in the act?"
"It must have been a trap," David lit up. "They must have been trying to get her to the location so that she would be conveniently present when the crime happened."
"Yes, that is very possible!" Roman sounded really excited. "You guys! This is getting more interesting by the day—I don't think Selma Collier is our culprit at all! I've been going through her online information all day, and not only are there no traces of any pre-murder research at all, but there seems to be a very interesting kind of spyware on her machine. It was really hard to detect and it's totally that could, upon prompting from the person running it, create fake internet searches or even purchase information."
"Who could make something like this? I know I can't," Gerald admitted.
"I don't know if I could have made it either. I remember there was this one person working in our department during that life plant research project, who would have had the skill to create something like that… She probably would've been able to point us in the direction of this thing's creator if she was here still."
"Maybe we can find her?" I perked up. This whole fake crime situation was getting out of hand: it was one thing to get the Colliers in jail for something they actually did. But to frame the wife for the husband's murder was a little too much.
"I don't know. She wasn't that young when she worked for us. She's probably way retired by now and really hard to find, but I can take a look at what happened to her after the project," Roman agreed.
"How do we know she isn't the person who engineered the spyware herself?" David asked. "I mean, if she worked on your project, which got canned because of my father's intervention, she must have also been affected? You and Gerald utilizing the internet to hack into my father—who's to say she isn't still alive and doing the same?"
"Well, he has a point there," my father chimed in. "After all, revenge seems to be the fad of the day."
"What was the woman's name?" my mother asked. "Maybe we can help look for her as well."
"Let me remember," Roman paused. "Alma? No, wait it wasn't Alma. It was an "A" name though, for sure. Wait, I had some official records from the days of that research. Let me see if Tia kept them. Wait a bit."
He left the phone for a while and we could hear him asking Tia for his old research papers.
"This is insane, you know," my mother shook her head. "To go to so much trouble just to frame someone for a crime she didn't commit, when there is probably enough in the server leak to connect her to things she must have been involved in…"
"You think Selma was involved in Lionel's illegal activities?" I was curious.
"Well, the server leak didn't really have her name involved in anything, did it? But, it is safe to assume she knew some things about her husband schemes," Gerald supposed.
"No. I don't think she would've known as much as you think, actually," David shook his head, slumping back in his seat. "I mean, my mother likes living a rich lifestyle, that's a fact. And I'm sure she has done her best to ignore as many of my father's legal violations as she could if it means she gets to continue living this kind of a lifestyle. But I don't think Dad would have willingly involved her in anything that required as much trust as hiding a crime, to be honest. They didn't have the best opinion of one another, really. So, she was likely not officially involved in any of his violations."
"Well, that definitely gives whoever engineered this whole situation more incentive to frame her, then," my father mused.
"I'm back!" Roman came back on the line. "Hey, guys, here's the name of that woman who worked with us: Aileen Johnson. It wasn't really common knowledge that she was really good with computers, since you know, people assume that the elderly are tech-illiterate, but she definitely had a way with programs. Not that she knew I knew it about her. I kind of stumbled upon her coding like crazy one day- it was a work of art."
"Why would she hide that kind of skill? Isn't such computer literacy really useful for scientific research?" David was incredulous.
"No idea. But everyone just mostly considered her this nice old lady, who got left penniless by her husband- I guess she wanted to maintain her image for some reason. Let's see, maybe she went back to her hometown? Appaloosa Plains, was it?"
"Appaloosa Plains!?" my parents both jumped a little.
"And you say her name was Aileen?" my mother was on the edge of her seat.
"Umm, yes. Why, do you know her?" Roman was confused.
"Can you look up some information on her past?" my father added. "Like, past employment, marriages…"
"Yeah, shouldn't be too hard."
"What's happening? How do you know this person?" David looked around confused.
"My maternal grandfather was married to and embezzled by a woman named Aileen, years ago. And they lived in Appaloosa Plains. I mean, as soon as they discovered her scheme, that Aileen fled town, so since this woman was from Appaloosa Plains too and trying so hard to maintain her 'nice old lady' image, maybe she was that same person," I explained.
"Wow… your family seems to have this kind of luck with people, doesn't it?" he was amazed.
"You could say that again," my mother chimed in. "This witch preyed on my father when he was mourning my late mother and stole from him for years. Almost ruined my relationship with him, too."
"It's in the past now, dear," Dad patted her. "She's out of our lives."
"Maybe not," Roman's voice sounded through the phone. "It looks like my Aileen was indeed your Aileen. What was your father's name, Mrs. Meadows?"
"Brandon Lewis. He owned a ranch in Appaloosa Plains until his passing several years earlier," Mom explained.
"Yep, we've got the same Aileen. This one's records, which were strangely hard to break into, for someone her age, show that she is, indeed your former step-mother."
Everyone paused, mulling over the information.
"Well…" Mom exhaled.
"That was unexpected," agreed Dad.
"Looks like she could be our culprit, then," Gerald scratched his head. "People like that, they don't really change, do they?"
"So, she's targeting my family? Where is she anyway?" David requested.
"There isn't really much information about her after the project's end, to be honest. I think she moved away from Hidden Springs, though. I can look into this further, but hunting down her current location will take a couple of days," Roman explained.
"That would be most welcome. Thanks for all your research, Roman," I thanked the hacker.
"You guys are most welcome! Take care. I'm going to go for now—Tia's making her famous lasagna!" the guy was clearly happy to be back home and I kind of felt bad for our family distracting him from spending all the time with his family.
"Enjoy!" Gerald hung up the phone. "Well, what do we do now?"
"We wait for Roman's information on Aileen's whereabouts," my mother offered.
"And see where that leads," Dad added.
"This could take a while though," I felt impatient. I wanted to know what was going on and I wanted to know now!
"Anyway… why don't all of you get ready for breakfast? Or rather, lunch, by now," mother got up and headed toward the kitchen.
"Yes, those detectives woke us all up rather early, didn't they?" Dad headed off to get properly dressed.
Gerald just went back to his room, to read some article he's already been reading before the detective's arrival.
I watched David with anticipation and some worry. No doubt, he wasn't taking all this information about his family too well.
For his part, however, he just started playing with Gerald's cat. This in itself surprised me- Murka was a former stray and she usually clung to my brother, chasing away others. But here she was, happily purring in David's hands, after approaching him herself. He looked relaxed, if anything.
"What are you thinking?" I asked.
"I'm thinking that I want to prove my mother didn't kill Dad," he replied. "I couldn't come to terms with the fact that she could have done it. But she didn't. And for whatever reason, she is afraid to talk to the cops truthfully…"
"Yeah, it doesn't sound right."
I looked out the window, which looked out on the Hidden Springs lake. Hmm, we couldn't do much from here, could we?
"You know, the detectives didn't require us to stay here, in this city…"
David looked at me with interest "Are you suggesting what I think you're suggesting?"
"Well, it would be easier to figure out what's going on if we could talk to Selma, wouldn't it?"
"I do want to talk to her. I can't imagine what she is feeling now, if she is in jail. I mean… not like her normal lifestyle, is it?" David frowned. "And I don't like the idea of someone being so good at framing her. Even if she hasn't ever been the best mother."
"I understand. We should go to Twinbrook. As soon as possible. I doubt our absence would do much at the company now, everything is in chaos already."
"Yeah. It probably would have been similar if our original plan worked," he admitted.
"But our plan didn't involve any murders and framing. And anyway, I think we need to figure out who has targeted your family all the way from Twinbrook and why."
What I didn't say was, if Selma wasn't involved in Lionel's criminal activity, someone was just going after the Colliers as a family. Which meant that, eventually, they'll come after David too. And I wanted to make sure the police caught them before they got the chance to do it.
"Tomorrow? I can pack really fast," David agreed. "We can leave as soon we have the tickets."
"It's decided then. We're going."
The Sunshine Blogger Award (Updated)
On August 16, 2016 By ViolincatIn UncategorizedLeave a comment
I added in the questions from Dandylion240 and Stormysims!
Huge thanks to both RosemaryMarie and RaymondSanti who nominated me for this! They both have really cool stories on their respective blogs- if you aren't reading them, check them out!
Also, I was nominated by Dandylion240 at her Bring Me Back To Life legacy and by StormySims at The Life and Times of the Willows. They both have amazing stories going on, which are very captivating.
What is the Sunshine Blogger Award?
Well, it is of course an award for bloggers from other bloggers. It is a beautiful way to share all the amazing things other bloggers do and write about. To recognize their efforts in inspiring others to do what they love.
Thank the person who nominated you and add a link to their blog.
Answer the 11 questions sent by the person who nominated you.
Nominate 11 bloggers and add their links.
Notify the bloggers you…
On August 11, 2016 August 16, 2016 By ViolincatIn Uncategorized21 Comments
Notify the bloggers you included.
Keep the rules in your post.
Questions from RosemaryMarie:
What was the last picture you took with your phone?
It's actually a picture of the lizard that decided to "visit" my apartment!
If you could have personally witnessed anything in history, what would it be?
Hopefully, something that people still wonder about, like how the pyramids were built, or any of those "alien encounters". Just to quell my own curiosity.
If you could be any age for a month, what age would you choose?
If I could also live the way I lived at that age, I'd love to go back to being 6. No school, my best friend lived across the street, I had fun with my neighbor's grandkids who came to visit for the summer…. it was a good time.
What is the strangest thing you've ever eaten?
I'd have to go with this dish called Ghormesabzi from a local Mediterranean restaurant near me. It was pretty good, but a bit too heavy on the spices for me.
Probably Karlsson-on-the-Roof, by Astrid Lindgren. It's so funny!
Yes, I do. Never saw any, but that doesn't mean they don't exist.
What is the longest you've gone without sleep?
Probably around 60 hours? Back in high school, I had to go on this overnight trip to the local uni. Due to some nerves, I ended up not sleeping the night before the trip. Once at the uni, I tried to compensate the lack of sleep by consuming way too much coffee, which in turn led to more insomnia… Let's just say, I was lucky none of the counselors there thought I was under the influence, because I was practically a zombie!
Coke, Pepsi or neither?
Honestly, I usually can't taste the difference between them. I prefer Cherry Coke or Dr. Pepper, usually.
What did you do for your last birthday?
I went to a live concert with my family. And afterwards, to a restaurant!
New Years Eve! Mostly it's because in Russia, which I'm originally from, it was The Holiday! It still is in my family, we get together, watch holiday concerts, eat, drink. It's a fun old tradition.
What is on your desk?
My laptop, mouse, jar with pencils and table lamp.
Questions from RaymondSanti:
What inspires you to write?
These characters basically come to live in my head and they want their stories told! The least I can do is write them.
One of your bad habits?
My terrible sleeping schedule. You guys probably noticed how I end up reading and liking your posts at the weirdest time of day or night…
What do you want the most right now?
A job. A real money-earning job. Preferably one that I can do fairly well and where I get along with my coworkers and boss.
Something you never leave home without?
My phone. If I ever leave my phone at home, I feel lost without it.
If you could ask any question to anyone and get a truthful answer, what would you ask?
That's a tough one. I might ask my former high school crush whether it was ever possible for anything to happen between us relationship-wise. But then again, I was definitely not mature enough for a relationship back then.
Baths or showers?
Showers. They just take less time.
What's your favorite number?
Either 4 or 7. I can't really explain why.
If you had the chance to torture your worst enemy, would you?
Nope. Even if I totally hated them, the most I'd do is get into an actual fight with them. But torture is immoral and, as many studies show, it's pretty useless.
What do you admire in your friends?
They give me a new perspective on life. I mean, the other day, my closest friend drove a really long distance to come see me. And my online friends are always improving my mood when I'm down. You're all wonderful.
If you could invent anything, what would it be?
Something that makes people less prone to hate one another? If that were possible. Imagine how much humanity could achieve if there was no hatred, racism, bigotry?
Given the chance to rule the world… would you?
No! That is way too much responsibility for one person. And I'm definitely not someone who could do such a difficult job.
Questions from Dandylion240:
Anything but orange and pink.
Do you like to travel?
Not too much. It's pretty exhausting.
If you could go anywhere, where would it be?
Someplace cold, like Sweden, maybe. Or someplace in the UK.
How long have been playing the sims?
Since the summer of 2013.
What motivated you to start writing?
Seeing other people's stories and wanting to try a hand at creating a tale myself.
If you could meet anyone in from the past, who would it be?
Perhaps, my great grandmother Sophia. She was a very wise and amazing woman, but she passed away when I was only four.
What's your favorite song?
I don't really have a favorite song. I have been pretty obsessed with The Cat Empire lately though.
Day or Night?
Night. I feel much more productive at night.
Do you plan out every detail of the stories you write or do you just let the story take you to unexpected places?
I try to plan, but in the end it still changes. So, most of the time, the end result of the story is way different from what I originally had in mind.
Do you like to write first or third person?
I generally find 1st person easier, since you get to see things from the character's perspective. Although, lately I've been trying a hand at 3rd person narrative.
If you could do anything right now, what would it be?
I actually really want to go to the beach right now. Because it's so hot today and the beach is always windy and cool.
Questions from StormySims:
If you could be anywhere in the world (or fictional world) right now, where would you be?
Just anyplace really calm and cool. Preferably, not in California. Although, the Shire from LOTR would be a pretty neat place to be.
What drew you to start playing The Sims, TS2, TS3 or TS4?
I started playing the mobile Sims Freeplay. And then, for my birthday, I got the actual Starter Pack from the Sims 3.
What is your favorite genre to write about?
I definitely love mysteries and adventures. My least "favorite" is romance, since I find it really difficult to write believable romance scenes.
If you could make any one of your characters in any of your stories come to life, who would it be, and why?
From this story, Celeste, since she is very caring and understanding, no matter what. From my Tumblr story, The Roommies, I'd totally love to make Bernardo Vida real, since he seems to be someone I'd really get along with.
When writing your story on a blog, do you prefer to write on a white background, a black background or a colored background? Why?
I almost never use the WordPress blog for actual writing, I just use Microsoft Word. And lately I've been using this pale green background while writing, since it's supposed to be better for eyes.
When you are writing or taking pictures in game for your story, what do you have going on in the background? (TV, music, etc.)
I prefer silence while writing. I can have a lot of noise going while taking the pictures though.
If you were a Sim, would you choose to be a PlantSim, Zombie, Alien, Werewolf or a witch? Why?
I'd love to be a Witch. So many possibilities there.
If you could be any animal, what would it be, and why?
A cat. They have really cool physical abilities and everyone loves them.
What is your favorite season?
IRL, it's winter. In game, Summer or Spring.
What is your favorite Sims world?
Appearance-wise, Isla Paradiso is the most beautiful, even though it lags so much. Otherwise, I like Monte Vista quite a bit.
What has been your favorite Sims challenge that you either have done before or like to read?
I like the Bring Me To Life challenges, where you have to resurrect an EA ghost and let them live a full life.
My nominees are:
Forever Darkness
The Chrysantemum Tango
Camp Wildberry
The Van Winkle Legacy
Dustland Fairytale
The Mayfields
My questions for my nominees:
Do you have any favorite characters in your own stories? Who?
What are your favorite genres to read or watch?
Which fictional character do you wish you could be real life friends with?
Would you rather land the perfect job or find true love?
Do you cook or bake? If not, would you want to learn how to?
What is your dream career?
What is the one thing you absolutely can't live without?
Which country do you most want to visit?
Are you a day or a night person?
Would you want to become famous?
What do you like the most about gaming?


text:
Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol
Chao Sun1,2,
Xianjuan Dong2,
Rubing Zhang2 &
Congxia Xie1
BMC Biotechnology volume 21, Article number: 3 (2021) Cite this article
(R)-(+)-perillyl alcohol is a naturally oxygenated monoterpene widely used as the natural flavor additives, insecticides, jet fuels and anti-cancer therapies. It was also readily available monoterpene precursors. However, this natural product is present at low concentrations from plant sources which are not economically viable. Therefore, alternative microbial production methods are rapidly emerging as an attractive alternative to make (R)-(+)-perillyl alcohol production more sustainable and environmentally friendly.
We engineered Escherichia coli to possess a heterologous mevalonate (MVA) pathway, including limonene synthase, P-cymene monoxygenase hydroxylase and P-cymene monoxygenase reductase for the production of (R)-(+)-perillyl alcohol. The concentration of (R)-(+)-limonene (the monoterpene precursor to (R)-(+)-perillyl alcohol) reached 45 mg/L from glucose. Enhanced (R)-(+)-perillyl alcohol production was therefore achieved. The strain produced (R)-(+)-perillyl alcohol at a titer of 87 mg/L and a yield of 1.5 mg/g glucose in a 5 L bioreactor fed batch system.
These datas highlight the efficient production of (R)-(+)-perillyl alcohol through the mevalonate pathway from glucose. This method serves as a platform for the future production of other monoterpenes.
Perillyl alcohol is a natural monoterpene that exists in two optical forms. (R)-(+)-perillyl alcohol is produced in perilla leaves, citrus, lemon and lavender [1,2,3] and has extensive applications. It serves as a natural flavor additive for food, as an insecticide in agricultural fields, as a jet fuel in aviation fields, and as a healing agent for anti-cancer therapeutics [4,5,6,7]. The bioconversion of readily available monoterpene precursors (such as (R)-(+)-limonene) are recognized as valuable oxygenated derivatives [8]. Up to 70–97% of (R)-(+)-limonene is present in citrus oils [9], produced to levels that exceed 60,000 t per year [10]. Many terpenes are synthesized from limonene, such as perillyl alcohol, carvone and α-terpineol. The enantiomers of carvone cost US $ 30–60 per kg; whilst (S)-(−)- and (R)-(+)-perillyl alcohol cost US $4500/kg [7, 11]. As the chemical synthesis of perillyl alcohol is of high cost and leads to environmental pollution, the biosynthesis of perillyl alcohol from renewable carbon sources is regarded as an economically feasible industrial process. New and more effective synthesis procedures are however required.
(R)-(+)-perillyl alcohol can be synthesized in Escherichia coli [12], Pseudomonas putida [5, 13,14,15,16], Mortierella minutissima [17], Fusarium verticilloides [18], Aspergillus strain [19, 20] from the biotransformation of (R)-(+)-limonene to (R)-(+)-perillyl alcohol (Table. 1). The recombinant Pseudomonas taiwanensis VLB120 harboring cymAa and cymAb from Pseudomonas putida can convert (R)-(+)-limonene to (R)-(+)- perillic alcohol [15] (Table 1). The maximum yield of (R)-(+)-perillyl alcohol was 258.1 mg/L for 3 days using Mortierella minutissima cultivation containing 0.5% (R)-(+)-limonene at 15 °C [17]. This produced the highest levels of (R)-(+)-perillyl alcohol currently reported. It should be as a substrate for the above microbial transformation reaction, but the raw materials were not easily obtained [22]. Metabolic engineering for natural compound production can be enhanced through gene modifications that increase enzyme activity. Many terpenoids have been produced at high titers by metabolic engineering, including monoterpenes, sesquiterpenes, diterpenes and tetraterpenes [23,24,25,26,27,28,29].
Table 1 Biosynthesis of (R)-(+)-perillyl alcohol l by microbial transformation
Isopentenyldiphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) form the building blocks of monoterpenes, sesquiterpenes and diterpenes during synthesis, the precursors of which include geranyl pyrophosphate (GPP), farnesylpyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP), respectively. These precursors are derived from mevalonate (MVA) and are catalyzed by geranyl pyrophosphate diphosphate synthase (GPPS) (Fig. 1). However, the microbial mediated production of monoterpenes is limited by toxicity [29] and poor GPPS expression [30, 31]. In this study, we initially engineered E. coli to produce (R)-(+)-perillyl alcohol from glucose by the heterologous expression of P-cymene monoxygenase hydroxylase (CymAa) and a P-cymene monoxygenase reductase (CymAb) (Fig. 1). Genetic modifications to enhance the production of (R)-(+)-perillyl alcohol were also performed. These included the codon optimization of cymAa, cymAb and ClLS and exogenous GPPS expression. The highest performing strain SC04 was then cultured under fed-batch conditions for the assessment of its potential for large-scale production.
(R)-(+)-perillyl alcohol production via the MVA pathway. Enzymes are depicted in the legend
Perillyl alcohol production from limonene
It is well established that during (S)-(−)-perillyl alcohol production, titers can be increased by 100 mg/L using Escherichia coli [32]. Many terpenoids have been produced to high titers through metabolic engineering, including monoterpenes, sesquiterpenes, diterpenes and tetraterpenes. Limonene hydroxylase from Bacillus stearothermophilus BR 388 (LHBS), was proved to produce (R)-(+)- perillyl alcohol 0.51 mg/L with the substrate (R)-(+)-limonene [12, 21]. LHBS was ligated into pET28a(+) to create pSC00, meanwhile, cymAa and cymAb were created pSC01 in Table S1. Limonene is toxic to most microorganisms and its concentrations regulate the growth of BL21 (DE3). It was therefore important to assess the optimal concentrations of limonene for fed-batch production. Different (R)-(+)-limonene concentrations were assessed (0.2 mM to 3.0 mM) in growth assays. With the increasing of (R)-(+)-limonene concentration, cell growth was inhibited. At (R)-(+)-limonene concentrations of 1.0 mM, the OD600 decreased from 2.5 to 2.0 for SC01 (Fig. 2a). When (R)-(+)-limonene was used at 2 mM, the maximum production was 86.9 mg/L and the conversion yield of perillyl alcohol was 30% for SC01 (Fig. 2b). For another strain SC00, it produced the low concentration of 1.5 mg/L, and cell growth was suppressed (Fig. 2b). In the later study, the strain SC01 was used as the research basis.
Effects of limonene concentration on the activity of SC00 and SC01. When the OD600 reached 0.6, samples were induced at 30 °C for 24 h using 0.2 mM IPTG and different contents of (R)-(+)-limonene (0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2.0 mM). a OD600 and (b) titres (mg/L) over different concentration of limonenes for 2 strains. c standard substance perillyl alcohol. d the fermentation product with 2 mM (R)-(+)-limonene for SC01. Error bars: SD from three independent cultivations
Products were identified by GC-MS after organic extraction by ethyl acetate. As shown in Fig. 2c and d, control assays with extracts of E. coli lacking a cDNA clone did not yield (R)-(+)-perillyl alcohol, whilst E. coli carrying cymAa and cymAb from Pseudomonas putida produced (R)-(+)-perillyl alcohol to detectable quantities. The biosynthetic pathway for (R)-(+)-perillyl alcohol production was successfully constructed using (R)-(+)-limonene as a substrate.
Limonene production from glucose
We optimized the yields of limonene to improve the production of perillyl alcohol. pTrcHis2B and pET-28a (+) were assessed. pYJM14 contains ERG8, ERG19, IDI from Saccharomyces cerevisiae [33]. pSC02 contains geranyl pyrophosphate synthase GPPS from Abies grandis that converts IPP/DMAPP to GPP, and (R)-(+)-limonene synthase CILS from Citrus limon that converts GPP to limonene [34] (Table S1). SC02 was cultured in fermentation medium under sealed shake-flask conditions.
The levels of (R)-(+) limonene in the culture media were plotted against known concentrations of (R)-(+)-limonene. The (R)-(+)-limonene concentration of SC02 reached 38 mg/L and 45 mg/L (mg limonene per liter of culture, the same as follows) with 0.2 mM IPTG and 10% n-dodecane overlay at 24 h and 48 h of induction. When 10% DINP (diisononyl phthalate) was used instead of the n-dodecane overlay, the limonene titers were 29 mg/L and 40 mg/L respectively at 24 h and 48 h (Fig. 3c). The enantiomer forms produced by the strain cultures were analyzed using GC with a Cyclosil-B column, and the production of (R)-(+)-limonene by SC02 was confirmed (Fig. 3a and b).
Limonene production using the SC02 strain. a The enantiomeric distribution of limonene was analyzed using the Agilent Technologies 7890B GC System on a Cyclodex B column (30 m × 0.25 mm internal diameter; film thickness = 0.25 μm). GC conditions were as follows: 50 °C hold, 2 °C/min ramping to 160 °C; carrier: high-purity helium, linear velocity: 1 ml/min; temperature of the injector: 250 °C; split ratio: 1:20. Compounds of interest: (S)-(−)-limonene at 21.2 min and (R)-(+)-limonene at 22.02 min. b The fermentation broth was mixed and centrifuged, and the organic layer was analyzed. c The total production of Limonene was measured after 24 h and 48 h of IPTG induction and 10% n-dodecane or DINP induction
Microbial perillyl alcohol production using the MVA pathway
CymAa and CymAb catalyze the biosynthesis of (R)-(+) perillyl alcohol using (R)-(+) limonene as a direct substrate through a hydroxylated reaction at the 7 position. We cloned cymAa and cymAb from Pseudomonas putida into pSC04&pSC05. Terpenoid synthase is expressed to low levels in E. coli [35], so cymAa and cymAb were codon optimized (Table S1). According to the analysis of chirality of the intermediate limonene produced by SC02, we concluded that the configuration of perillyl alcohol was R-type (Fig. 3b). Expression levels in the perillyl alcohol pathway for SC04 were investigated by qRT-PCR. The results indicated that ERG8, ERG19 ERG12, and IDI significantly increase compared to the control. Moreover, changes in the expression of mvaE, mvaS, CILS, GPPS, cymAa and cymAb were comparable to the control (Fig. 4a). We used n-dodecane or DINP as the extraction solvent and the organic phase was collected and centrifuged to remove cell contaminants. Samples were then subjected to GC-MS analysis and trace levels of perillyl alcohol were detected. pYJM14 and pSC04&pSC05 were co-transformed into E. coli BL21 (DE3), resulting in recombinant strain SC04&SC05. SC04 that was cultured in sealed shake flasks to assess perillyl alcohol production. The fermentation broth of the strain SC04 was extracted by ethyl acetate, TLC, product refined and optical rotation detection. The specific rotation value of the product was [α]D20 = + 82 ± 1 (C 1.8, CHCl3), the standard was [α]D20 = + 84 (C 1.8, CHCl3) [36]. From the above, the fermentation product can be identified as (R)-(+)-perillyl alcohol.
(R)-(+)-Perillyl alcohol production using SC04 and SC05 strains. a relative expression levels in SC04 and SC05 engineered for (R)-(+)-perillyl alcohol production. Data was for 24 h in LB medium with 0.2 mM IPTG and 20 g/L glucose. Gene expression was calculated using the 2-△△CT. system and are shown as the mean ± SD (n ≥ 3). Identical letters indicate no significant differences (p < 0.05). (5) Total production of (R)-(+)-perillyl alcohol was measured at 48 h after induction with 0.2 mM IPTG and10% n-dodecane or 10% DINP, Data are the means of 3 repetitions ± SD
The (R)-(+)-perillyl alcohol levels for SC04 reached 12.5 mg/L after induction by 0.2 mM IPTG and 10% DINP overlay for 48 h, which was 2.4-fold higher than SC05 (7.2 mg/L for 48 h) (Fig. 4b). In the absence of DINP, SC04 and SC05 produced 3.1 mg/L and 1.5 mg/L of (R)-(+)-perillyl alcohol. We included n-dodecane overlays in the cultures, and thus a small amount of perillyl alcohol was detected [32]. The production of (R)-(+)-perillyl alcohol was 9.3 mg/L in SC04 and 5.6 mg/L in SC05, mainly due to the rapid extraction of limonene to the organic phase, and as n-dodecane overlay could not be used for perillyl alcohol fermentation. In the SC04, the production of (R)-(+)- perillyl alcohol and mevalonate was 12.5 mg/L and 102 mg/L after 48 h of induction (BL21 (DE3)/pET-28a(+) not detected, Fig. S1). So, the production of (R)-(+)-perillyl alcohol was through the mevalonate pathway from glucose.
Fed-batch culture of engineered E.coli strains
High cell densities for (R)-(+)-perillyl alcohol production were achieved through fed-batch fermentation using SC04 in a 5 L bioreactor. According to the residual glucose concentrations, feeding rates were maintained at ≤1 g/L. Figure 5 shows the time-course of (R)-(+)-perillyl alcohol production and cell density during fermentation. (R)-(+)-perillyl alcohol biosynthesis was initiated from 5 h of induction and 10% DINP was added during the fed-batch process. After 36 h of induction, the production of (R)-(+)-perillyl alcohol peaked at 87 mg/L with DINP overlay and the yield was 1.5 mg/g glucose. The theoretical conversion yield of (R)-(+)-perillyl alcohol from glucose is 28.1% (1.5 glucose~ 0.5 (R)-(+)-perillyl alcohol) However, the engineered strain achieved a cell-density OD600 of 108 for two-phase fermentation. In the late fermentation stage, the content of perillyl aldehyde and acetic acid increased to 45 mg/L and 8 g/L respectively, which hindered the production of (R)-(+)-perillyl alcohol [37]. The accumulation of IPP and DMAPP may have influenced the growth of the engineered strain [25, 38, 39]. However, the two-phase fermentation of (R)-(+)-perillyl alcohol fermentation eliminated toxic intermediates. (R)-(+)-perillyl alcohol biosynthesis requires many overexpressed genes, which influence host cell viability and reduce productivity [40].
Time-course of (R)-(+)-perillyl alcohol production during fed-batch fermentation of SC04
(R)-(+)-perillyl alcohol is produced in perilla leaves, citrus, lemon and lavender and has a range of applications. Many terpenes are synthesized from limonene, such as perillyl alcohol, carvone and α-terpineol. As the chemical synthesis of perillyl alcohol is of high cost and is environmentally unfriendly, the biosynthesis of perillyl alcohol from renewable carbon sources is regarded as an economically feasible industrial process. Indeed, the development of (R)-(+)-perillyl alcohol as an anti-cancer drug has been limited by the costs associated with extracting the compound from its natural oil. Terpenes are produced by microorganisms and can be scaled-up to increase drug production [41]. We used E.coli as a host and confirmed the feasibility of (R)-(+)-perillyl alcohol production. The levels produced were however, too low for industrial requirements.
Through comparison of strain SC00 and SC01, we found that SC00 by expressed LHBS from Bacillus stearothermophilus BR 388 produced a little, 1.5 mg/L (Fig. 2b), It may be that cofactors (NADPH, ATP) are involved in the fermentation process, which reported the production of 0.51 mg/Lwith (R)-(+)-limonene substrate [21]. so we directly selected the cymAa and cymAb which convert limonene derived from the metabolism of p-cymene [15]. Enzyme activity was lower than other terpene synthases, that reduce perillaldehyde or perillic acid [15]. We initially engineered SC02 and showed successful (R)-(+)-perillyl alcohol production using GC-MS (Fig. 2d). The maximum production of (R)-(+)-perillyl alcohol composed with (R)-(+)-limonene as substrate was 86.9 mg/L and lethal concentrations of limonene to E. coli were 2 mM (Fig. 2b). (R)-(+)-Limonene accumulates in biological membranes leading to damage. The compound is also insoluble in water [42]. It has been reported that limonene is inhibitory to multiple microorganisms [43]. A recent report revealed that 0.025% (v/v) limonene inhibited the growth of E. coli [44]. Thus, the tolerance and solubility of limonene requires improvement to increase both the concentrations and yields of (R)-(+)-perillyl alcohol.
In previous studies, terpenoids have been studied using the MVA pathway [23, 25, 26, 45]. Our research group constructed engineered E.coli which synthesized isoprene by the mevalonate (MVA) pathway, and produced isoprene to 665.2 mg/L under flask conditions [46, 47]. GPPS and CILS were engineered to improve (R)-(+)-limonene production, and SC03 produced no detectable limonene (data not shown), highlighting the need for terpenes. When no extracts were added during limonene fermentation, no detectable SC02 was observed (data not known), indicating that limonene is rapidly lost from the culture medium. As limonene from the fermentation system was highly volatile and anti-microbial [48, 49] other collection methods have been reported including culture extraction, solvent overlay, solid-phase micro-extraction, gas shipping to a cold trap, and adsorbent polydimethlsiloxane bars [48, 50, 51]. Whilst not all of these methods are appropriate, they do prevent product inhibition and toxicity, avoiding evaporative loss of the limonene product using the two-phase system. Davies et al. [52] showed that an overlay of n-dodecane enhanced limonene recovery. N-dodecane was selected as a favorable solvent for terpenoid extraction experiments due to its low volatility [52]. In engineered E.coli, DINP fermentation prolongs both the growth and production phases, leading to (S)-(−)-limonene concentrations of 1.35 g/L [53]. (R)-(+)-limonene obtained by n-dodecane extraction was 45 mg/L for 48 h (Fig. 3c). It may be that the distribution coefficient of (R)-(+)-limonene in n-octane is larger than that of the DINP phase. The strain fermented by the DINP overlay also inhibited cell growth, and the OD600 decreased from 3.4 to 2.5 (Fig. 3c). Jongedijk et al. [48] engineered Saccharomyces cerevisiae to express (R)-(+)-limonene synthase from Citrus limon. Trapping of the headspace in limonene synthase expressing strains resulted in 0.12 mg/L (R)-(+)-limonene. Pang et al. [54] engineered Yarrowia lipolytica to achieve (R)-(+)-limonene at 11.705 mg/L, through the overexpression of HMGR and the optimization of the fermentation conditions. Cao et al. [55] engineered Yarrowia lipolytica to increase limonene titers to 23.56 mg/L by encoding neryl diphosphate synthase1 (NDPS1) and limonene synthase (LS). Here, we engineered E. coli to produce (R)-(+)-limonene to 45 mg/L.
We confirmed that the chirality of perillyl alcohol was (R)-type in SC04, which was performed with (R)-(+)-limonene as an intermediate (Fig. 3b). We then engineered SC04 & SC05 and investigated the effects of copy numbers on (R)-(+)-perillyl alcohol production. We hypothesized that cymAa and cymAb genes produced by the lower copies of SC05 could not repress all promoters. In contrast, SC04 had 40 copies encoding the repressor from medium and middle-copy plasmids. The genes contributed to higher levels in SC04 than in SC05 (Fig. 4a). This hypothesis was supported by the production of perillyl alcohol, which was 1.7-fold in SC04 and SC05 (from 7.2 mg/L to 12.5 mg/L) (Fig. 4b). In addition, the levels of ERG12, ERG8, and ERG19 in the MVA pathway were higher than those of the control strain, whilst the levels of GPPS, ClLS, cymAa and cymAb showed no obvious changes compared to controls (Fig. 4a). Most intermediates can be efficiently converted to IPP, but the production from IPP to perillyl alcohol was weak. This suggests the enhanced production of (R)-(+)-perillyl alcohol through regulating metabolic imbalances.
(R)-(+)-perillyl alcohol production was achieved by assembling biosynthetic genes encoding a heterologous MVA pathway. The highest-performing strain (SC04) accumulated up to 87 mg/L of (R)-(+)-perillyl alcohol under fed-batch fermentation conditions. The synthesis of (R)-(+)-perillyl alcohol reported in this study has not been systematically optimized. From the perspective of fermentation process (Fig. 5), the low yield of (R)-(+)-perillyl alcohol depends on two aspects. On the one hand, the by-product perillaaldehyde leads to the decrease of (R)-(+)-perillyl alcohol content. It may be due to the presence of alcohol dehydrogenase [56] (such as yigB) which can convert geraniol to geranial in E. coli. Therefore, the key gene cymAa and cymAb were overexpressed and optimized. On the other hand, the fermentation control was optimized to reduce the content of acetic acid [57] and promote cell growth.
We have engineered E. coli to produce (R)-(+)-perillyl alcohol from glucose through the MVA pathway. In this study, it was firstly reported that (R)-(+)-perillyl alcohol was synthesized from glucose in recombinant Escherichia coli, which produced 87 mg/L of (R)-(+)-perillyl alcohol. It provides new methods for the synthesis of chiral perillyl alcohol. Efforts should now be directed towards the optimization of the MVA pathway.
Strains and culture conditions
Metabolic engineering for natural compound production can be enhanced through gene modifications that increase enzyme activity. All experimental materials used in this study are listed in Table S1. E.coli were grown in LB (tryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L). During the production of (R)-(+)-limonene and (R)-(+)-perillyl alcohol, strains were cultivated in shake-flasks in medium containing glucose 10 g/L, MgSO4 1 mM and riboflavin 0.05 mM during fed-batch fermentation in glucose 20 g/L, K2HPO4 9.8 g/L, ferric ammonium citrate 0.3 g/L, citric acidmonohydrate 2.1 g/L, MgSO4 1 mM, riboflavin 0.05 mM and 1 mL trace element solution, including (NH4)6Mo7O24·4H2O 0.37 g/L, ZnSO4·7H2O 0.29 g/L, H3BO4 2.47 g/L, CuSO4·5H2O 0.25 g/L, and MnCl2·4H2O 1.58 g/L. As required, ampicillin (100 μg/ml) and kanamycin (50 μg/ml) were added for selection.
Plasmid construction
Limonene hydroxylase (pOT435) gene (LHBS, GenBank Accession No. AF039527.1) from Bacillus stearothermophilus BR 388, were codon optimized and cloned into pUC57. LHBS were PCR amplified and cloned into pET28a (+) with EcoI/XhoI restriction sites, creating pSC00 (pET28a-LHBS). P-cymene monoxygenase hydroxylase (cymAa, GenBank Accession No. AAB62299.1) and P-cymene monoxygenase reductase (cymAb, GenBank Accession No.:AAB62300.1) from Pseudomonas putida were codon optimized by BGI, and cloned into pUC57. cymAa and cymAb were PCR amplified and subcloned into pET28a (+) with BamHI/SacI restriction sites, creating pSC01 (pET28a-cymAa-cymAb). ClLS (GenBank Accession No.:AF514287.1) of Citrus limon and GPPS (GenBank Accession No.:AF513112.1) of Abies grandis were optimized by BGI and synthesized by GeneWiz (Suzhou, China), producing pUC57-ClLS&pUC57-GPPS. MvaE-mvaS was then excised from pYJM20 [46] and ligated into pET-28a (+) to create pET28a-mvaE-mvaS. GPPS and ClLS which were truncated in the N-terminus, were cloned and assembled into pET28a-mvaE-mvaS at the SacI/AatII sites to generate pSC02 (Table S1). CymAa-cymAb fragments were obtained through Pseudomonas putida using AatII and PacI and ligated into pSC02 to create pSC03 (Table S1). MvaE, mvaS, and GPPS were cloned from pSC02 into pCOLADuet-1 at the BamHI/XhoI sites, generating pcolaDuet-mvaE-mvaS-GPPS-ClLS. CymAa, cymAb were cloned from pSC03 into pcolaDuet-mvaE-mvaS-GPPS-ClLS at the XhoI/PacI sites to produce pSC05 (Table S1).
pYJM14 was constructed from pTrcHis2B through the introduction of ERG8, ERG12, ERG19 and IDI from S. cerevisiae [33, 58]. All plasmids and primers are shown in Table S2.
RT-PCR analysis
Total RNA was isolated from 24 h cultures using commercially available SPAKeasy RNA kits. RNA was reverse transcribed using TaKaRa Primer Script RT reagent Kit and RT-PCRs were performed. Each reaction contained 1 μL cDNA, 5 μL TB green Premix Ex TaqII, 0.2 μL 50 × ROX Reference Dye, 0.2 μM for/rev primer, and ddH2O up to 10 μl. RT-PCR conditions were as follows: 30 s at 95 °C, 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Gene expression was normalized to the absolute transcript levels of rpoD. qRT-PCRs were performed on a Primer 5.0 program (Table S3). Relative gene expression was calculated using the 2ΔΔCt method for each treatment. Reactions were repeated a minimum of 3 times.
Shake-flask fermentation
Cultures were produced in in 25 ml of LB. E. coli strains with each recombinant plasmid inoculated in a gyratory shaker at 37 °C and 180 rpm. IPTG (0.2 mM) was added to induce recombinant protein expression upon an OD600 of 0.6. Cultures were incubated at 30 °C with 2-phase fermentation [59] used for limonene and perillyl alcohol extraction from the aqueous broth due to the toxicity of terpenes. We added 10% (v/v) n-dodecane or DINP (disononyl phthalate) following IPTG induction and cultures were incubated for 48 h. Cell densities, glucose levels, limonene levels and perillyl alcohol production were then assessed.
Fed-batch fermentation
For (R)-(+)-perillyl alcohol production on a larger scale, fed-batch cultivations were performed in a 5 L bioreactor system (Biostat B plus MO 5 L) using 2 L of fermentation fluid. Seed cultures (100 ml, 10 g NaCl, 5 g yeast extract, and 10 g of tryptone per 1 L) were added to shake flasks overnight at 37 °C and Sparger aeration was performed to maintain high dissolved oxygen (DO) levels. Post-fermentation, the pH of the broth was maintained at 7.0 through ammonia addition. Fermentation was performed during the growth stages under the following conditions: 37 °C, agitation 400 rpm and airflow at 1 L/min. Antifoam 204 was added as required. DO was maintained at 20% saturation through the control of air flow and stirrer speed (1–2 L/min and 400–900 rpm, respectively). When cells reached an OD600 of ~ 20, the temperature was switched to 30 °C and 0.2 mM IPTG and 0.05 mM riboflavin were added. DINP (10%) was added after 4 h when the initial glucose levels were exhausted, as indicated by the increase in DO. The fed batch mode was initiated through the feeding of 60% glucose at appropriate rates. Residual glucose levels were maintained to low levels through the addition of acetic acid. Samples were periodically collected and OD600 values were determined prior to centrifugation for the separation of the organic and aqueous phases. Organic layers were removed for all GC–MS analysis.
E. coli growth was determined through OD600 measurements on a spectrophotometer (Cary 50 UV-vis, Varian). The Shimadzu GC-MS system (TQ8050) was used for Limonene and perillyl alcohol identification. GC-MS conditions were as follows: 30 m DB-5MS column (internal diameter 0.32 mm, film thickness 0.25 μm); temperature: 50 °C hold, ramped up 10 °C/min to 250 °C with a final hold at 250 °C for 10 min. Highly pure helium was used as a carrier at a linear velocity of 1 ml/min; an injector temperature of 250 °C; a split ratio of 1:10; an ion source temperature of 230 °C and mass range of m/z 40–500. Limonene and perillyl alcohol and perillaldehyde peaks were identified through the retention times of external standards and MS comparisons via the National Institute of Standards and Technology (NIST) database. Fermentation broths were mixed, centrifuged, and the organic layer was taken for GC-MS analysis.
The enantiomeric distribution of limonene was analyzed using the Agilent Technologies 7890B GC System on a Cyclodex B column (30 m × 0.25 mm internal diameter; film thickness = 0.25 μm). GC conditions were as follows: 50 °C hold, 2 °C/min ramping to 160 °C; carrier: high-purity helium, linear velocity: 1 ml/min; temperature of the injector: 250 °C; split ratio: 1:20. Compounds of interest: (S)-(−)-limonene at 21.2 min and (R)-(+)-limonene at 22.02 min. The fermentation broth was mixed and centrifuged, and the organic layer was analyzed.
The concentration of mevalonate was determined by gas chromatography (GC) analysis on a Aglient19091J-413 HP-5 column (30 m × 0.32 mm internal diameter, film thickness = 0.25 μm). The broth was acidified to pH 2.0 with 3 M HCl and mixed with anhydrous Na2SO4. The mixture was extracted twice with ethylacetate. Mevalonate in the solvent layers was then analyzed by GC. GC conditions were as follows: 50 °C hold, 10 °C/min ramping to 250 °C, hold 5 min. Carrier: high-purity helium, linear velocity: 1 ml/min; temperature of the injector: 260 °C.
Specific rotation of (R)-(+)-perillyl alcohol. The fermentation products were subjected to preparative TLC (PTLC) to isolate pure product [60]. Specific rotation was detected in the automatic polarimeter (JIAHANG). Test conditions: wavelength of sodium light 589 nm, room temperature, length 10 cm, 1.8% (W/V) of CHCl3. Three times for each detection.
The data supporting the conclusions of this article are included within the article.
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Alonso-Gutierrez J, Chan R, Batth TS, Adams PD, Keasling JD, Petzold CJ, Lee TS. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab Eng. 2013;19(Complete):33–41.
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Zheng G, Dan X, Abdallah II, Dijkshoorn L, Setroikromo R, Lv G, Quax WJ. Metabolic engineering of Bacillus subtilis for terpenoid production. Appl Microbiol Biotechnol. 2015;99(22):9395–406.
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Chang HC, Oriel P. Bioproduction of Perillyl alcohol and related Monoterpenes by isolates of Bacillus stearothermophilus. J Food Sci. 2010;59(3):660–2.
Chubukov V, Mingardon F, Schackwitz W, Baidoo EE, Alonsogutierrez J, Hu Q, Lee TS, Keasling JD, Mukhopadhyay A. Acute limonene toxicity in Escherichia coli is caused by limonene-hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase (AhpC). Appl Environ Microbiol. 2015;81(14):4690–6.
Ma SM, Garcia DE, Redding-Johanson AM, Friedland GD, Rossana C, Batth TS, Haliburton JR, Dylan C, Keasling JD, Petzold CJ. Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases. Metab Eng. 2011;13(5):588–97.
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Chatterjee T, Bhattacharyya D. Biotransformation of limonene by Pseudomonas putida. Appl Microbiol Biotechnol. 2001;55(5):541–6.
The authors appreciate the contribution of the Dr. Yujin Cao for his advice on strain design. The authors also acknowledge Cong Wang for the assistance on the conduction of GC-MS.
This work was supported by the National Natural Science of China (grant number 21878166), and the Taishan Scholar Project of Shandong Province, China (grant number ts201511033).
A State Key Laboratory Base of Eco-Chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, 53 Zhengzhou Rd., Qingdao, 266042, China
Chao Sun & Congxia Xie
CAS Key Laboratory of Bio-based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Rd., Qingdao, 266101, China
Chao Sun, Xianjuan Dong & Rubing Zhang
Chao Sun
Xianjuan Dong
Rubing Zhang
Congxia Xie
CS, RZ and CX conceived and designed research. XD contributed analytical tools. CS performed experiments and analyzed data. CS wrote the manuscript. All authors read and approved the manuscript.
Correspondence to Rubing Zhang or Congxia Xie.
Strains and plasmids used in this study. Table S2. Primers used in this study for plasmids construction. The restriction sites in the primers were underlined. Table S3. Primers for RT-PCR used in this study. Fig. S1. Mevalonate and (R)-(+)-perillyl alcohol production engineered MVA pathway. The total production of MVA and (R)-(+)-perillyl alcohol was measured after 48 h of IPTG induction.
Sun, C., Dong, X., Zhang, R. et al. Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol. BMC Biotechnol 21, 3 (2021). https://doi.org/10.1186/s12896-020-00662-7
Perillyl alcohol
Mevalonate pathway
Microbial production

redpajama_set_name: RedPajamaCommonCrawl


> DAC> concerning a colon in a filename -- valid in *nix, not in Windows.
> DAC> "aux%??" (I don't recall the encoding of colon)?
(via windows update if necessary) have support for the �.
currancy, rather it is to discuss the merits of an unused symbol.


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Reaching every corner of the world. Arirang TV is to present an unbiased view and reliable information of Korea to the world through the wide range of programs such as Korean news, cultural, educational and entertainment show, dramas, documentaries and more.
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128 2017-02-08 'S. Korea-China Ties 2017', by Arirang TV and CGTN
'S. Korea-China Ties 2017', by Arirang TV and CGTN
On January 26, leading global TV stations in Korea and China hosted a discussion while the relationship between Korea and China still remains strained. Arirang TV, Korea's leading global broadcast network, collaborated with CGTN (formerly CCTV World), a Chinese network launched to enhance China's influence in the world by broadcasting news from the Chinese perspective.
Through the program, the participants held a heated discussion on issues such as THAAD and North Korean Nuclear weapon problems, on which the two countries have opposing views. The discussion was hosted by anchors Moon Conn-young from Arirang News Center and Zou Yue from CGTN. Each station also invited experts to participate in the dialogue.
Arirang TV invited Lee Doo-won, Prof. of Economics at Yonsei University while CGTN's guest was Teng Jianqun, Senior Researcher from CIIS (China Institute of International Studies).
Mr. Teng stated on the program that THAAD has negatively impacted the Korea-China relationship and even the economy because businessmen are feeling the tension. He also emphasized that the problem of THAAD is between China and the US. However, Prof. Lee commented that China's efforts to switch the focus of its economy from exports to domestic trade are the cause of the decrease in trade volume and that it is too early to determine whether that decrease is a long-term trend.
Prof. Lee also emphasized that China holds the key to solving the North Korean nuclear problem in terms of economy, politics and security. Mr. Teng refuted this by saying that China no longer wields such a strong influence over North Korea as it did in the past. CGTN's anchor, Mr. Zou commented that Korea, China and the US all oppose North Korea's nuclear program while Ms. Moon mentioned that many countries expect China to play a larger role in resolving the North Korean nuclear problem although they are not asking China to be solely responsible.
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136 [Bizline] The Bye-Buy Sensation 2017-03-09 10440
135 [Arirang Special] Significance of Kim Jong-nam's Death 2017-03-09 3814
134 Arirang TV, Cooperation Agreement on Broadcasting with Vietnam & Mexico 2017-03-09 4315
133 아리랑국제방송, 멕시코 의회방송 Canal del Congreso와 방송교류협정 체결 2017-02-17 4346
132 [Upfront] Assessment of the Rise of Medical Tourism in Korea 2017-02-08 4625
131 History behind Korea-Japan Conflict on Dokdo Island 2017-02-08 3892
130 Arirang Radio's Sound K dal.komm Concert 2017-02-08 4386
129 Realities of North Korea by Former North Korean Diplomat Thae Yong-ho 2017-02-08 3788
128 'S. Korea-China Ties 2017', by Arirang TV and CGTN 2017-02-08 4900
127 아리랑라디오, Sound K 달콤콘서트 2017-01-19 4810
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Business Telegraph > industry > Sunak to meet Tory MPs to head off unrest over cost of living crisis
Sunak to meet Tory MPs to head off unrest over cost of living crisis
Rishi Sunak will attempt to quell growing Conservative unrest over the cost of living as delegations of MPs press him to cancel a planned tax rise and ditch VAT on energy bills in exchange for savings on tackling the pandemic.
The chancellor will convene MPs for several evening summits this week as tensions grow over pressures faced by households from April, with a planned 1.25-percentage-point rise in employee national insurance contributions (NICs) and soaring energy bills once the price cap is reviewed.
Labour will hope to pile pressure on Sunak and Boris Johnson on Tuesday with a binding vote in parliament on cutting VAT on home energy bills.
The motion, put forward by Labour in its opposition day debate, guarantees parliamentary time for a bill to enact the cut, a device that means Tory MPs must actively oppose it rather than abstain.
MPs' demands are wide-ranging but the cut to VAT, which Sunak and Johnson have previously ruled out in favour of more targeted action, has "the most widespread support", according to one veteran Tory.
"It doesn't hurt that it's something we can now do because of Brexit," said another senior Tory plugged into several key parliamentary pressure groups.
Many are set to urge the chancellor to go further and delay the NICs rise from April, expected to bring in £12bn that Sunak wants to use to cut NHS waiting lists and fund the new social care system in the medium term.
David Jones, a former Brexit minister, said the pressure on many households would be immense from April.
"To have this tax increase at the same time the cap is removed from energy prices is very difficult. That is a couple of thousand pounds increase for many families," he said.
One senior backbencher, John Redwood, warned Sunak on Monday that his own ambitions depended on his standing in the party on these issues.
"Those who brief that Mr Sunak should be the next PM should tell him imposing an avalanche of unpopular and unwise tax rises will undermine that ambition," he tweeted.
Another MP said they would urge Sunak to ditch the rise and focus on making savings on pandemic spending, including NHS test and trace. "I will urge him to cut the NICs rise – £12bn is not a lot of money compared to the current Covid spend. There is a great deal of support in the parliamentary party for that."
Though not all MPs said they would back revoking the NICs rise, one minister warned it had left Sunak exposed.
"The party hates the NICs rise. Sunak himself wouldn't have done it apart from to head off other spending demands with warnings of more tax rises. So no wonder he comes under pressure to ditch it when there's a hook to do it. The chancellor has to realise that the party and the country are just not where he is on tax and spend."
Johnson said he had discussed cost of living pressures with Sunak on Sunday night. "I understand how difficult it is for people, I understand the pressures that people are facing on household finances," he said on Monday.
"It's making life very tough. And we've got to make sure that people are aware of all the things that they can do, all the money that we're putting in via local councils to help people in hardship, the cold weather payments, the warm homes discount, the money for pensioners."
Cabinet ministers have all but rejected a windfall tax on North Sea oil and gas profits, proposed by Labour. MPs said that was less of a priority. "Most colleagues think it's counterproductive at a time when we want to encourage inward investment," a former minister said.
Johnson's spokesman said a windfall tax had "not been ruled out" but hinted again that a targeted approach would be taken. "We want an approach that helps those most in need," the spokesman said.
The communities secretary, Michael Gove, underlined that new support measures were likely to be targeted to the most vulnerable. "I think that the prudent and the responsible thing to do is to recognise that we need to take a balanced approach, and a balanced approach means that when we can support, we provide support most for those in the most difficult circumstances," he told Sky News.
Ministers are understood to have discussed extending the warm homes discount on bills for the most vulnerable but Tory MPs argue it does not help enough households.
Treasury sources said they were reluctant to raise the tax burden on the oil and gas sector, pointing to additional taxes on the extraction rate of oil and gas and headline tax rates on profits more than double other businesses.
Labour has criticised the education secretary, Nadhim Zahawi, who told LBC that "oil and gas companies … are already struggling", though the companies are expected to report "near record" income in 2021-22.
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Tag: North Brunswick Environmental Commission
Elmwood Cemetery's 1st Annual BioBlitz
June 12, 2019 Heather Fenyk Comment
Article by Howard Swerdloff, New Brunswick Environmental Commission
On Saturday June 8, 2019, the Elmwood Cemetery hosted their first annual "BioBlitz." (A"BioBlitz" is an event that focuses on finding and identifying as many species as possible in a specific area over a short period of time.) The event was sponsored by the Lower Raritan Watershed Partnership (LRWP), the Americorps Watershed Ambassadors Program, New Brunswick Environmental Commission, North Brunswick Environmental Commission, and the Elmwood Cemetery.
Photo credit: Roger Dreyling
Over 4 dozen area "citizen scientists" and experts scoured the 50 acre site identifying and cataloging the fish, mammals, insects, aquatic invertebrates, fungi, plants, and birds. They identified 8 species of fish; 8 species of mammals; 47 species of insects; 15 species of aquatic invertebrates; 20 species of fungi; 37 species of plants; and 42 species of birds (the latter are catalogued on E-bird: https://ebird.org/nj/view/checklist/S57201308 ) — a total of 177 different species.
Photo credit: Johnny Malpica
The event inspired many two-way conversations between our community participants and the volunteer scientists. Instead of a didactic "top down" learning experience, both groups shared their knowledge and understanding of the local environment in a way that enhanced the specialized knowledge of the expert scientists, and, in turn, the expert scientists helped community volunteers develop a deeper knowledge and appreciation for the natural world and local environment. The experts' final reports will be ready in a month; their findings will be shared with the volunteers. Elmwood Cemetery plans to make a Bioblitz an annual event.
Elmwood Cemetery, a Special Forested Habitat Refuge
The cemetery is nestled between the New and North Brunswick communities. It was established in 1868 as a "Victorian Garden Cemetery" during the rural cemetery movement, and to this day all of Elmwood's lanes and paths are lined with evergreens and flowering native trees. Cemetery managers are building on this legacy of careful planning and land protections to secure Arboretum accreditation, which will allow them to further advance the planting, study, and conservation of woody plants and trees in the area.
June 8 BioBlitz in New/North Brunswick
June 8, 2019 Heather Fenyk Comment
A BioBlitz is an event that focuses on finding and identifying as many species as possible in a specific area over a short period of time. At a BioBlitz, scientists, families, students, teachers, and other community members work together to get a snapshot of an area's biodiversity.
On June 8 the LRWP and partners including the Americorps Watershed Ambassadors Program, New Brunswick Environmental Commission, North Brunswick Environmental Commission, and Elmwood Cemetery will host our inaugural BioBlitz of Elmwood Cemetery in North Brunswick!
Elmwood Cemetery is a special forested habitat refuge nestled between the urban New and North Brunswick communities. The Cemetery was established in 1868 as a "Victorian Garden Cemetery" during the rural cemetery movement, and to this day all of Elmwood's lanes and paths are lined with evergreens and flowering native trees. Cemetery managers are building on this legacy of careful planning and land protections to secure Arboretum accreditation, which will allow them to further advance the planting, study, and conservation of woody plants and trees in the area. Elmwood is located at 425 Georges Rd, North Brunswick Township, NJ 08902.
Our BioBlitz will run from 5:30AM-2PM and include public talks by expert naturalists about local natural history, and a chance for the public to work with these experts in an active survey of mammals, fish, plants, insects, aquatic invertebrates, birds and fungi.
5:30AM – Check-in opens
6:00AM – Birds
7:00AM – Mammals
8AM – Plants
9AM – Aquatic Invertebrates, Insects
10AM – Fish, Fungi
11:30AM – Procession of Species to Elmwood Office lawn
12:00PM – Data reports, lunch, conversation
2PM – Wrap up
Our Expert Scientists:
Aquatic Invertebrates: Von Scully, Erin Dempsey, Jen Helminski with NJ Watershed Ambassadors
Birds: Laurie Gneiding, NJDEP Ornithologist/Ecologist for Environmentally Sensitive Areas
Fish: Chuck Sedor with NJ Division of Fish & Wildlife, Bureau of Freshwater Fisheries
Fungi: Randy Hemminghaus and Nancy Addotta with the NJ Mycological Association
Insects: Trisha Nichols with the Philadelphia Insectarium Butterfly Pavilion
Mammals: Brionna Primiani with NJ Wildlife Services
Plants/Trees: Michele Bakacs with Middlesex County EARTH Center – Rutgers Cooperative Extension
Please register to be part of one of these species teams!


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Black Sherif settles medical bills of new mothers in Ghanaian hospital
Deborah Akpobome
Ghanaian rapper, Black Sherif has demonstrated a kind gesture towards women that recently gave birth to children.
The 'Kwaku The Traveller' hit-maker paid the medical bills of mothers who were stuck due to inability to pay what the hospital charged them.
He visited Ridge Hospital in Accra, Ghana and cleared the expenses preventing the women from being discharged with their babies.
Black Sherif also provided the mothers with food and toiletries during his visit to the hospital on 18th of January 2023.
In related news, a lecturer at the Nnamdi Azikiwe University, Awka, Anambra State, who gave birth to a set of septuplets (seven babies) cried out for financial assistance.
Mrs Ngozi Uzodike delivered two boys and five girls, at Obijackson Women and Children Hospital, Okija, however, one of the girls died.
She was charged N19million by the hospital, a bill she was asked to pay before she can be discharged with the remaining six babies.
The woman who works at the Department of Business Administration, was torn between tears of joy and sorrow as she lamented that she is unable to pay the medical bill.
She explained that she had not been paid salaries since she started working at the institution in December 2020.
Mr. Celestine Uzodike, corroborated her story while speaking with pressmen on Wednesday, saying that that his wife was properly employed as a fulltime a lecturer at the institution, but unfortunately, had not been paid since.
According to her husband, she had been promoted from Assistant Lecturer to Lecturer II, yet she had never earned a kobo as salary.
READ: BBNaija's Uriel expresses shock as her boyfriend posts his wedding photo after she refused to get down with him
The distraught man appealed to the Federal Government to pay the two years salary arrears owned to his wife, and also come to their aid.
Mr Uzodike lamented that they are not able to foot the hospital bill and take care of the surviving two boys and four girls, all by themselves.
He said; "The babies came out prematurely at 26th week gestation period and the hospital said they will need to be placed under intensive care unit and each of them will cost N3million. One of them has died, remaining six babies, that is why they billed us like that."
The new mum, who was very weak to speak after the delivery, said; "All I want is for philanthropists both in Anambra State and Nigeria at large to come to our aid in other to offset the hospital bill."
Related Topics:black sherifhospitalmothers
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I've transitioned to passing message with rap music – Black Sherif


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How Much Space Should a Carpet Runner Have on Each Side?
By Leslie Rose Updated September 26, 2017
A carpet runner is a carpet that's much longer than it is wide. Carpet runners are most commonly used in hallways. There are no hard and fast rules that dictate the width of a carpet runner relative to the space it occupies. You must decide what looks best in your hallway.
Center the carpet to leave an equal amount of space between the carpet and each wall.
Consider the purpose of your runner. This may sound like common sense, but if you're purchasing a runner to cover unsightly stains or scratches in a hallway, the dimensions of the runner must be larger than the area that you aim to cover. If you're trying to hide unsightly flooring, the runner should be as large as possible -- from wall to wall, or close to it. There's no right or wrong dimensions for a carpet runner, so the function of the runner should take precedence.
Normal Dimensions
In general, 6 inches on each side is an acceptable distance between the wall and the runner. If your hallway is 3 feet wide, for example, then a 2-foot-wide runner should be adequate. For wider hallways found in more public areas, you may consider a runner that leaves more space between its edges and the wall. Look for a runner that's at least two-thirds of the width of the hall. If possible, the space around the carpet runner should be equal on all four sides. If this isn't possible, be sure to center the carpet runner so the spaces found on opposite sides of the runner are equal.
You can experiment to see which dimensions work for you by purchasing a roll of butcher's paper. Lay the roll down in your hall and trim it to the size that looks best. Measure the dimensions and make a note to keep with you when you shop for your runner.
If you happen to find a carpet runner that's larger than the ideal size, it may be possible to have it altered. Some businesses that sell rugs and carpets will do in-house alterations. Ask the clerk at the store for details. If your area runner is purchased used or from a home improvement store that doesn't specialize in carpet sales, you may just have to live with imperfect dimensions, or keep shopping.
Leslie Rose has been a freelance writer publishing with Demand Studios since 2008. In addition to her work as a writer, she is an accomplished painter and experienced art teacher. She has a Bachelor of Arts degree in art with a minor in English.
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Diagnosing pulmonary tuberculosis (TB) may be delayed until culture results become available.
We ascertained the accuracy of a stepwise diagnostic algorithm for the rapid diagnosis of pulmonary TB by GeneXpert from sputum and/or bronchoalveolar lavage (BAL) followed by a Mycobacterium tuberculosis-specific BAL ELISPOT assay in patients with a suspected diagnosis of pulmonary TB at a clinical referral centre in Germany.
Among 166 patients with a presumptive diagnosis of pulmonary TB, 81 cases were confirmed by M. tuberculosis culture from sputum and/or BAL. In 66 out of 81 (81.5%) cases, patients initially had M. tuberculosis detected by GeneXpert from sputum; in addition, six out of 81 (7.4%) cases were diagnosed by GeneXpert on BAL fluid (together 72 out of 81 (88.9%) patients). Out of the remaining nine patients with negative GeneXpert results from sputum and BAL, BAL ELISPOT identified eight patients with culture-confirmed TB correctly (median time to culture positivity 26 days). At a cut-off of >4000 early secretory antigenic target-6- or culture filtrate protein-10-specific interferon-γ-producing lymphocytes per 1 000 0000 lymphocytes, the specificity of the BAL ELISPOT for active TB was 97%.
In low TB incidence countries, nearly all patients with active pulmonary TB can be identified within the first few days of clinical presentation using a stepwise strategy with GeneXpert and BAL ELISPOT.
Conflict of interest: J. Heyckendorf has received personal fees for sponsor-independent lectures at sponsored symposia from Chiesi, Gilead, Janssen, Lucane and Hain, outside the submitted work. C. Lange has received personal fees for sponsor-independent lectures at sponsored symposia from Chiesi, Gilead, Abbvie, MSD, Becton Dickinson, Janssen, Lucane, Novartis and Thermo Fisher, outside the submitted work. B. Kalsdorf has received personal fees for sponsor-independent lectures at sponsored symposia from Oxford Immunotec and Lucane, outside the submitted work.
Support statement: C. Lange and J. Heyckendorf are supported by the German Center for Infection Research (DZIF). Funding information for this article has been deposited with the Crossref Funder Registry.


text:
package ecologylab.bigsemantics.generated.library;

import ecologylab.bigsemantics.generated.library.SlashdotItem;
import ecologylab.bigsemantics.metadata.builtins.MetadataBuiltinsTypesScope;
import ecologylab.bigsemantics.metadata.builtins.RichDocument;
import ecologylab.bigsemantics.metadata.mm_name;
import ecologylab.bigsemantics.metametadata.MetaMetadataCompositeField;
import ecologylab.bigsemantics.namesandnums.SemanticsNames;
import ecologylab.serialization.annotations.simpl_collection;
import ecologylab.serialization.annotations.simpl_inherit;
import ecologylab.serialization.annotations.simpl_nowrap;
import java.util.ArrayList;
import java.util.List;
import java.util.Map;

@simpl_inherit
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		return items;
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redpajama_set_name: RedPajamaGithub