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Opened 3 years ago
Closed 3 years ago
#6955 closed defect (invalid)
Build error with media-autobuild_suite - decklink / pthread?
|Reported by:
|Owned by:
|build system
|decklink pthread
|Blocked By:
|Reproduced by developer:
|Analyzed by developer:
Building of FFmpeg is currently impossible.
The build process of the current master stops with the following error messages:
Likely error: from E:/AB-Suite/build/ffmpeg-git/libavdevice/decklink_common.h:28, from E:/AB-Suite/build/ffmpeg-git/libavdevice/decklink_dec.cpp:49: E:/AB-Suite/build/ffmpeg-git/compat/w32pthreads.h:101:19: note: candidate: int pthread_mutex_init(void**, void*) static inline int pthread_mutex_init(pthread_mutex_t *m, void* attr) ^~~~~~~~~~~~~~~~~~ E:/AB-Suite/build/ffmpeg-git/libavdevice/decklink_dec.cpp: In member function 'virtual ULONG decklink_input_callback::Release()': E:/AB-Suite/build/ffmpeg-git/libavdevice/decklink_dec.cpp:578:16: warning: deleting object of polymorphic class type 'decklink_input_callback' which has non-virtual destructor might cause undefined behavior [-Wdelete-non-virtual-dtor] delete this; ^~~~ make: *** [/build/ffmpeg-git/ffbuild/common.mak:63: libavdevice/decklink_dec.o] Error 1
You can look at the log files here:
[logs.zip] (https://0x0.st/sZCA.zip)
Attachments (1)
Change History (4)
comment:1 Changed 3 years ago by cehoyos
To make this a valid ticket, please at least provide the configure line you tested and run make several times, provide (only) the complete, uncut console output of the last make run.
Changed 3 years ago by BlackScreen
FFmpeg - make logfile
comment:2 Changed 3 years ago by BlackScreen
I have attached the log of make from the ffmpeg-git dir of the suite.
comment:3 Changed 3 years ago by BlackScreen
- Resolution set to invalid
- Status changed from new to closed
Sorry for the bug report. It was an error in the suite.
Note: See TracTickets for help on using tickets.

subset: commoncrawl


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Because shit like this needs to fill the screen every time a prospective employer, prospective client, or prospective romantic partner types the name "Megan Thode" into a search engine:
Entitled much?
...for the rest of her life.
Wednesday, February 13, 2013
For Megan Thode
3 comments:
Ugh...I had to deal with this SO much in grad school. If you can't/don't want to do the work that's required, then don't get into the program.ReplyDelete
- AnonymousFebruary 14, 2013 at 3:17 PM
What a stupid, selfish, immature reaction that this bitch has done. Glad to hear Judge rejected it.ReplyDelete
She'll never get a job again, that's for sure.
Yep! Stupid is as stupid does, and it looks like this dingbat is going to learn the hard way.ReplyDelete
Subscribe to: Post Comments (Atom)


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I've struggled a great deal in finding and setting priorities in my work life and in managing the many side projects that I have going on. Some people naturally have that ability to self-organize, and some don't. For those that don't, they need a Shelly in their lives.
Shelly has a natural ability to listen to someone's organizational challenges, and make them seem like mole-hills instead of mountains. It's because she helps you break those down into small and logical steps, where all you could see is a giant mass of panic.
More than just helping someone get organized, Shelly helps you discover the panic-free, completely organized part of you that got buried under a pile of mental tasks. Shelly helped me realize that there are things in my life that I do that help me retain focus and accomplish tasks. I just had to discover them and learn to do them intentionally.
With Shelly's amazing help, I was able to balance work, a side project, preparation for an overseas lecture, and a hackathon. Without her, none would have been successful, but with her, work was happy, the lecture was a hit, and I won first place.
Five out of five stars. Would definitely recommend.
Jennifer has a very calm manner that both motivates and instills confidence. She has loads of practical strategies that truly boost your efficiency. I feel very lucky to have the opportunity to learn from her- I highly recommend her.
When I hired Shelly to help me get my office organized, one of my goals was to be able to more easily locate things I had stashed in the 20′ x 4′ storage closet in my office over the past ten years. The other day I was jumping up and down when I needed an important document and I knew exactly where to find it. Shelly made this daunting task almost fun. The pain of when I first started this project is ABSOLUTELY gone!
When I met Jennifer, I was a new startup business in desperate need of administrative assistance. I needed someone to help create systems, procedures and facilitate in creating order where there wasn't any. I decided that Microsoft Outlook would be the right solution for me. Although I could navigate some of the features, I was barely utilizing it to its full capacity. Needless to say I was overwhelmed and so was my email, contact database and calendar. Jennifer took the time to understand my business, listened to what I hoped to accomplish, helped identify the best starting point and my most immediate needs and very patiently and methodically walked me through the steps to setup a system that fit me and my business. Jennifer is knowledgeable, methodical, patient. We sat side-by-side through every step until I was comfortable with moving forward alone. What seemed overwhelming to me was simplified and very comprehensive thanks to Jennifer's professionalism and PATIENCE.
Shelly – You have made a very BIG & Wonderful difference in my home-office life!
Prior to hiring you, I never would have believed that I "Needed You" – a Professional Organizer –after all I am a tenured and Extremely Organized Mortgage Loan Officer, having current systems in place that allow me to be a Step-Ahead of my clients' needs to always Exceed their Expectations!
However, my home-office was beginning to look more like an office – not a pretty sight. But with your Listening skills and Expertise, you patiently worked WITH me over several sessions, to come up with additional organizational systems customized to my need. You transformed my office back into a home-office.
My new home-office now reflects the rest of my home's décor very nicely. And I have TONS of room to work in. My "paper claustrophobia" is all gone. My wonderful husband of 40 years was also very pleased with Shelly's results and the fact that "Shelly is much more economical than a Marriage Counselor!!"
Even though I am pretty organized, my computer was a mess! I was deleting emails because I couldn't stand looking at them and I was missing important documents. In addition, I was wasting precious time looking for documents and emails. It was frustrating and time-consuming! After working with Jennifer, like everything in our house having its place, my email and documents all have a place to land and to live. I have more focus now and I save tons of time searching for documents.
Jennifer is not only knowledgeable, she is also so patient and a master at helping you customize a system that will work way after she leaves your computer. Jennifer has such integrity – she follows through on what she promises and has a great way of getting to the heart of what is really going on with email and how to solve the problems quickly. I love her so!
Thanks for helping me out and showing me the way! Things are already running quite a bit more smoothly!
I hired Jennifer because I felt so out of control with my computer files, which was making me very frustrated and unproductive. She helped me get my file cabinets to match up within my emails, word documents, and my hard copies. She also helped get my computer and phone to "talk" to each other.
Jennifer has made my life less stressful! I can actually find the information that I'm looking for. She is patient and had a way of simplifying my problems. I highly recommend Jennifer to help organize your messes!!
Shelly did a great job working with me to bring order to the chaos my basement had been since moving in! It was transformed from a scary and depressing place to a place I actually enjoy working and relaxing in! We then went on to attack closets, cabinets and drawers with equally impressive results! The best thing was the changes have all been sustainable and have saved me endless time hunting for things!
Even though I consider myself relatively tech-savvy, I knew I was not maximizing the benefit of technology in my business. I hired Jennifer to help me streamline how I use technology to make my business more efficient and effective. As I explained what I was frustrated with, she instantly understood and was able to explain easy to implement changes. I highly recommend Jennifer for her patience, her insight and her vast knowledge of computer software.
After our last meeting, I really went to town and started digging in. Honestly, Shelly, I feel confident that I've got a handle on the "mountain of projects" that I faced when we first met. Thank you so much for all of your help. You were certainly a breath of fresh air and the inspiration for my accomplishments! I will always highly recommend your services!
You had some great tips on managing email at the Self-Employed St. Louis lunch today. Thank you for sharing. Your stories made me feel pretty good since I only had about 50 emails in my business inbox. You inspired me to cut it down to six this afternoon–all things I need to deal with Monday.
Shelly has been an answer to many prayers! Got my phone up and running… Shelly shortened my learning curve by a year… at least!
I've been working with Jennifer for almost three years now. I first met Jennifer when she gave a presentation to a networking group of professional women.
Her presentation addressed email overload and how best to manage the daily email traffic and save time. Some of the tips she gave were so enlightening and helped me so much so that I asked her to give the same presentation to the staff members at the office. They too benefited and immediately implemented some of her ideas and recommendations.
Since that time I have asked Jennifer to speak to several other networking groups with the same results. If you are struggling with some computer issues and need help, I would highly recommend you give Jennifer a call. Jennifer makes navigating our daily computer tasks so much easier and faster, and she is just a delight to work with.
Jennifer becomes your co-partner and will carefully assess your unique situation. After understanding the scope of my needs, she got right to work helping me sort and organize in a sensible fashion that helped me reach my end goal. Since I'm easily distracted, I particularly appreciated that Jennifer patiently yet firmly kept my project moving forward, never judging when I got off task. I highly recommend working with Jennifer!


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The New Ramen Burger Los Angeles Location!
239 S. Vermont Ave / at 3rd · Neighborhood: Koreatown
There hasn't been a restaurant opening that's gotten me this excited in a long while. Ever since I tried Keizo Shimamoto's famed Ramen Burger at the Japan America Kite Festival last year, I've been keeping alert to any word of an actual Ramen Burger brick and mortar shop to open in Los Angeles.
Come Sept 5th, I'll be jumping for joy and so will my tastebuds.
New Yorkers have been lucky with a few Ramen Burger shops from Keizo which have opened in the past months, but this new LA location, a teensy tiny little stall in Koreatown on the corner of 3rd and Vermont, will be the first of its kind for us burger fans on the Westcoast.
The facts:
In addition to the original ramen burger which started the whole craze, there will be a variety of new equally delicious menu items including a salmon ramen burger, a "works burger" (with crispy bacon and a fried egg) a veggie option and a burger featuring the spicy Lincoln Lee's La Sriracha Macha hot sauce. Sides will include garlic fries, seaweed and sea salt fries and fries with red miso cheese. Coming soon: Possibly ramen fries?? Stay tuned…
And if you feel so inclined to show off your love for the burger-with-the-noodle-bun, there's a variety of merch available including tees, iPhone charms and cute made-in-Hawaii ramen burger keychains which I couldn't resist purchasing.
If somehow you happen to have never heard of the Ramen Burger, allow me to explain. It is an original creation from Keizo Shimamoto who spent years in Japan perfecting this trademarked burger. It's a handmade bun, made from fried ramen noodles and inside is a 100% ground beef patty with baby arugula, scallions and a secret shoyu glaze. It's chewy, soft and exploding with flavor. Careful there, you might get some dripping down your arm. Mmmmm. Just lick it off and enjoy.
Standing outside the stand discussing how delicious the burgers were and licking our fingers, my foodie friends (@KristieHang, @Chef_Jay, @TammyLaLaLand) and I suggested a surf and turf burger jokingly to Keizo's brother Jeff… voilá! A masterpiece was created! Perhaps if we're lucky, it just may just end up on the menu!
The Ramen Burger will be at the up and coming 626 Night Market (July 18 + 19) and there's word that Keizo will try to make it in town (possibly) August 2nd.
Come hungry and be prepared to stand in line! This sh•t is about to go down!
Click here to read about my first ever taste of the ramen burger! And click here to Watch!
Tip: If the line is too long outside. Go inside for a drink. You'll be able to order off the Ramen Burger menu from the neighboring Lock and Key Bar which shares the same kitchen. I must say, my idea of a great weekend afternoon is a burger, a cocktail and some bumpin' music. They have a pretty hip #IceCreamSundays shindig which features some really tasty cocktail drinks. New drink menu is set to launch very soon.
We got a sneak peak and a taste…
If you made it this far in the post, thank you kindly for reading! Your reward is… getting to see a pic of the newest Ramen Burger creation coming soon…
A ramen churro ice cream sandwich. OMG. Heavenly cinnamon, crunchy sweetness. A must get. Trust me.


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Democratic frontrunner Joe Biden and close rivals Elizabeth Warren and Bernie Sanders have feuded over healthcare at the party's third debate.
The event in Houston, Texas, was the first time the three presidential hopefuls faced off in a debate.
Mr Biden attacked the feasibility of Mr Sanders' universal healthcare plan, Medicare for All, while Ms Warren and Mr Sanders defended the measure.
Only the 10 highest-polling Democrats in the field of 20 were on stage.
A former Vice-President, Mr Biden is leading current polls by a considerable margin, with Massachusetts Senator Ms Warren and Vermont Senator Mr Sanders in second and third. The rest of the field are polling in single figures.
A broad range of issues from climate change to gun control were debated, but it was healthcare which sparked the fieriest clashes.
Why is healthcare controversial?
The issue of how to reform the US healthcare system has become a point of disagreement between moderate and more progressive Democrats.
Mr Sanders and Ms Warren both back Medicare for All, which would expand an existing federally run programme for the elderly to cover all Americans in a single-payer insurance system.
Mr Biden criticised Mr Sanders' Medicare for All plan as being too expensive to work and said the country should focus on improving President Barack Obama's Affordable Care Act instead.
"I know the senator [Warren] says she's for Bernie. Well, I'm for Barack. I think Obamacare worked," Mr Biden said. "My plan costs a lot of money…but it doesn't cost $30tn (£24tn)."
Ms Warren defended the policy, saying families have to deal with exorbitant healthcare bills now, and only wealthier individuals and corporations would see increased costs from the plan.
Some of the lower polling candidates also attacked Mr Sanders and Ms Warren, with Minnesota Senator Amy Klobuchar saying that Medicare for All: "I don't think that's a bold idea. It's a bad idea."
How about gun control?
Former Texas congressman Beto O'Rourke, whose hometown El Paso was the site of a mass shooting that left 20 dead, was asked whether he supports confiscating assault-style rifles.
"Hell yes we're going to take your AR-15, your AK-47," Mr O'Rourke said to applause. "We're not going to allow it to be used against your fellow Americans anymore."
On gun control California Senator Kamala Harris drew President Trump into the debate, saying: "Obviously he didn't pull the trigger, but he's certainly been tweeting out the ammunition."
In their opening remarks most of the candidates attacked President Trump, blasting him for being a divisive leader and accusing him of racism.
What else happened?
Though some sparks flew there was also a lot of common ground between the candidates.
Here are some of the other memorable moments:
- Businessman Andrew Yang used his opening speech to announce he would be using donations to his campaign to start a "freedom dividend", giving $1,000 (£810) a month to 10 American families, prompting laughter from other candidates.
- New Jersey Senator Cory Booker, who is a vegan, was asked by a Spanish-speaking moderator whether he wanted Americans to stop eRead More – Source


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Over its evolutionary history, Saccharomyces cerevisiae has evolved to be well-adapted to fluctuating nutrient availability. In the presence of sufficient nutrients, yeast cells continue to proliferate, but upon starvation haploid yeast cells enter stationary phase and differentiate into nonquiescent (NQ) and quiescent (Q) cells. Q cells survive stress better than NQ cells and show greater viability when nutrient-rich conditions are restored. To investigate the genes that may be involved in the differentiation of Q and NQ cells, we serially propagated yeast populations that were enriched for either only Q or only NQ cell types over many repeated growth–starvation cycles. After 30 cycles (equivalent to 300 generations), each enriched population produced a higher proportion of the enriched cell type compared to the starting population, suggestive of adaptive change. We also observed differences in each population's fitness suggesting possible tradeoffs: clones from NQ lines were better adapted to logarithmic growth, while clones from Q lines were better adapted to starvation. Whole-genome sequencing of clones from Q- and NQ-enriched lines revealed mutations in genes involved in the stress response and survival in limiting nutrients (ECM21, RSP5, MSN1, SIR4, and IRA2) in both Q and NQ lines, but also differences between the two lines: NQ line clones had recurrent independent mutations affecting the Ssy1p-Ptr3p-Ssy5p (SPS) amino acid sensing pathway, while Q line clones had recurrent, independent mutations in SIR3 and FAS1. Our results suggest that both sets of enriched-cell type lines responded to common, as well as distinct, selective pressures.
Organisms live in changing environments, and thus evolutionary adaptation to varying conditions is crucial for an organism's long-term survival. Some environmental stressors may be rare while others may occur more frequently, even on a regular basis, such as circadian cycles. Such changes include fluctuation in temperature or nutrient levels (Tagkopoulos et al. 2008; Van der Linden et al. 2010), and seasonality is an ubiquitous driver of fluctuating selection in organisms with generation times of a month or less (Messer et al. 2016). A few mechanisms have been identified that can help organisms to prepare for recurring stressors (Dhar et al. 2013), for example, bet hedging, which results in expression of different sets of genes (or different levels of gene expression) within different subgroups of cells in the population, thereby generating phenotypic heterogeneity within an otherwise isogenic population. This strategy has been shown to increase the long-term fitness of yeast grown with variable application of either heat shock, "diauxic lag" phase duration, or utilization of different carbon sources (Levy et al. 2012; New et al. 2014; Wang et al. 2015). Another mechanism is adaptive anticipation, where an organism uses the information of the present environment to preadapt in the anticipation of the forthcoming changes. Physiological adaptive anticipation in single-cell organisms (including yeast) is well-documented and is becoming a new paradigm for microbiology (Mitchell et al. 2009, 2015; Brunke and Hube 2014; Siegal 2015; Yona et al. 2015).
In response to starvation for one or more nutrients, a fraction of the cells in a stationary yeast population exit the mitotic cycle and become Q, a state physiologically similar to that seen in higher eukaryotic G0 cells (Gray et al. 2004; Valcourt et al. 2012). The organization of a Q cell's internal structures and genome is very different from that of a proliferating, NQ cell's; there is an increase in storage carbohydrates and stress protectants such as glycogen and trehalose, increased width of the cell wall (Aragon et al. 2008), sequestration of proteins (Suresh et al. 2015), telomere clustering (Guidi et al. 2015; Rutledge et al. 2015; Laporte et al. 2016), and global transcriptional shutoff (McKnight et al. 2015; Young et al. 2017). These changes are programmed, energy dependent, and are considered physiologically adaptive during stress (Smets et al. 2010; Klosinska et al. 2011; De Virgilio 2012). Most of the transcriptional changes associated with transition to Q state are simply correlated with slower growth during starvation, and as such are not specific for the Q state (Valcourt et al. 2012). However, a set of Q-specific core genes, showing altered transcription rates during starvation that are independent of any growth rate-associated patterns of expression, has been previously identified. For example, increased transcription was detected for genes involved in membrane lipid biosynthesis, protein modification, response to toxins, and metal ion transport, while decreased transcription was found for genes related to cytokinesis, chromosome organization and biogenesis, and organization of the nuclear pore complex (Klosinska et al. 2011). Still, the most significant property of yeast Q cells, relative to proliferating NQ cells, is their ability to maintain viability over long periods of time during the growth-arrested phase, and to resume mitotic growth once growth-promoting conditions are restored (Gray et al. 2004). Transition to Q cells may be triggered by early signals of nutrient depletion, so that the population contains individuals in different physiological states with associated transcriptional profiles. Production of a stable fraction of Q cells is characteristic for yeast strains. A population-level bet hedging strategy could result in subgroups of Q cells and NQ cells, which may increase the chances of that population's survival in variable environmental conditions (Wloch-Salamon et al. 2013; Wloch-Salamon 2014; Honigberg 2016).
The extracellular concentration of nutrients available for free-living fungi can change by many orders of magnitude during the growth of a population over time, and even during the lifespan of a single cell. Micro-organisms cope with such changes in part by controlling the rates of uptake of these molecules, by tuning their ability to sense them, and by transcriptional induction of relevant permease genes. In Saccharomyces cerevisiae, distinct mechanisms have evolved to sense the extracellular presence of carbon sources (such as glucose) and nitrogen sources (including amino acids) (Ozcan et al. 1996; Didion et al. 1998). S. cerevisiae appears to have adapted to a wide range of nutrient availability by maintaining at least 15 different amino acid permease genes and at least 17 sugar transporter genes (Gaber et al. 2003). An extreme adaptation to scarcity of nutrients is manifested in diploids by the production of spores, and in haploids by the production of Q cells.
In this study, we set out to identify genetic mechanisms involved in the regulation of Q cell formation. To this end, we repeatedly subjected populations of haploid S. cerevisiae cells to two sequential environmental conditions: (1) feast, under which adapted cells are expected to divide as quickly and efficiently as possible, and (2) famine, where some cells in the population transition to nondividing Q cells, which are able to survive long periods of starvation and thus are crucial to the long-term survival of the population. After each cycle, we enriched our experimental lines for either Q or NQ cells, exploiting the fact that Q cells are more dense than NQ cells (Allen et al. 2004), and repeated the process for 30 such cycles, which we determined was equivalent to ∼300 generations (cell doublings). For one set of lines, only Q cells were repeatedly propagated, while for the other set of lines, only NQ cells were propagated. The NQ line populations are expected to be enriched in cells that do better in the nutrient-rich condition, as they ignore environmental signals of nutrient depletion and do not enter the Q program. By contrast, the Q line populations are expected to be enriched in cells that do better under famine, detecting and/or anticipating upcoming nutrient scarcity, since repeated harvesting of only Q cells should reward cells that enter the Q cell program. For the Q-enriched population, we presume that fitness advantages during logarithmic growth become less important overall, because we assume that only Q cells make it to the next cycle. Because the populations were grown and then starved on the same rich medium (YPD plates) the change in environment was not acute, but instead was gradual. This may allow cells, when reaching early stationary phase, to naturally anticipate signals of the consecutive changes, and respond by differentiating into Q or NQ cells. After the 30 successive cycles of cell type enrichment, growth, starvation, and separation, we isolated several single colonies from the final populations for whole-genome sequencing. These 30-cycle clones (C30) were also used to investigate possible trade-offs in their fitness under conditions differing from those in which they were selectively enriched.
During the course of the experiment, we observed that populations produced a significantly increased fraction of the cell type (Q or NQ) for which they were enriched, which was also true for the C30 clones. Analysis of whole-genome sequences of the C30 clones allowed us to identify potentially Q- or NQ-promoting mutations. Interestingly, NQ cell enrichment in our conditions may be enhanced by disabling the SPS signaling pathway, as SSY1 was frequently mutated in NQ lines but not in Q lines. In Q lines, we found repeated SIR3 and FAS1 mutations. We also found genes independently mutated in both lines, including ECM21, RSP5, MSN1, SIR4, and IRA2. Our results reveal distinct patterns of molecular adaptation for increased production of either Q or NQ cells.
Materials and Methods
Strains, media, and cycles of growth and starvation (G-S)
We used a derivative of the laboratory haploid strain s288C (Mat α, ura3::KanMX4, his3Δ1 leu2Δ lys2Δ) [modified according to Cubillos et al. (2009)]. A single colony from a YPD plate was grown to stationary phase in 10 ml liquid YPD at 30° for 2 d (density of ∼2 × 108 cell/ml). The culture was diluted 1:100 and three 100 μl aliquots were used to start replicate populations for the experiment by plating them as patches on 10 ml YPD small Petri plates (50 mm diameter), such that the initial density of the entire plate was ∼2 × 105/ml. After 4 d of incubation at 28°, the entire patch was washed from the plate using 10 ml of sterile water, 4 ml of which was then fractionated (see below) in a Percoll gradient, to give two fractions: a more dense, lower fraction (Q) and a less dense, upper fraction (NQ). Each of the six resulting fractions (an NQ and a Q from each of the three replicates) was then used to found experimental lines NQ I–III, and Q I–III, respectively (Figure 1A). We used calibration curves, based on combined OD reads and Burker chamber cell counting for the NQ and Q lines, to plate 1 × 106 cells onto a 10 ml YPD plate (for a final media density of 1 × 105/ml), which was incubated for 3.5 d at 28°, at which point the cells had reached stationary phase (density ∼2 × 108 cells/ml). The fractionation, followed by the YPD plating, was then repeated for 30 cycles. For all of the NQ experimental lines, we used only cells from the upper fractions (NQ cells) for each subsequent cycle, and likewise for the Q lines we selected only the lower fractions after each centrifugation (Q cells). Starting at cycle 10, we sampled aliquots of the populations from each fraction every five cycles, and stored them as frozen glycerol stocks at −80°. The total quantity of the Q and NQ cells in the populations was estimated by optical density.
Fractionation and isolation of Q and NQ cells
We used a previously described method (Allen et al. 2006) to obtain the gradient for density fractionation. In brief, we mixed Percoll and 1.5 M NaCl, 9:1 v/v, and 7.5 ml of this solution was centrifuged in a swinging bucket rotor at 10,078 × g for 20 min. Then 4 ml of the culture washed from the plates (in 50 mM Tris, pH 7.5) were placed on top of the gradient and were centrifuged for 60 min at 417 × g. We carefully removed the upper (NQ) and lower (Q) cell fractions into separate tubes using pipettes. Based on OD, ∼106 cells were transferred to a YPD plate of 10 ml, for an initial density of 105 cells/ml (for the entire volume of the small Petri plate). In addition, at the beginning of the experiment, at the 10th and then every 5th cycle (that is 15th, 20th, 25th, and 30th), the quantity of each of the Q and NQ fractions in all experimental lines was assessed. All fractions were collected to separate tubes and then serially diluted. Based on specific density curves (separate for Q and NQ fraction) and spectrophotometric OD measurement, the population density was estimated.
During the experiment we observed an increase in the quantity of all selected fractions in each of the experimental populations. That is, the NQ fractions from the NQ lines became thicker as did the Q fractions from the Q lines (Figure 1B). To determine if this phenotypic effect was heritable and still present in the populations derived from the evolved clones, we checked the quantity of NQ and Q fractions of the ancestral strain and 11 evolved clones. Latin numbers refer to the experiment (QI 1, 13; QII 5, 6; QIII 8, 9; NQI 3, 11; NQII 15; and NQIII 6, 7). Each of these clones and the ancestral clones were subjected to a single cycle of G-S prior to fractionation, similarly to as in the main experiment. It included growth on YPD plate for 3.5 d and single fractionation in a Percoll gradient. The number of cells in each of the isolated fractions was estimated based on OD measurements. This procedure was repeated three independent times for each of the clones.
Phenotypic changes of evolved clones: maximum growth rate (MGR)
MGR of each C30 clone was estimated in liquid YPD medium culture. A frozen sample was transferred by a metal pin to fresh liquid YPD medium dispensed into 500 μl aliquots in 96-deep well plates and incubated without shaking for 3 d at 30°. Then, 2 μl of this stationary phase culture were transferred to 100 μl of fresh YPD liquid medium in a 96-well plate, which was then sealed. Plates were then placed into a Tecan instrument, incubated at 30° with agitation at 1250 rpm, and wide-band optical density (OD) readouts were acquired every 10 min with the Infinite m200 Microplate Reader (Tecan). The OD readouts were corrected for background effects, log-transformed, and used to determine the range of exponential growth. This was performed by finding Q and NQ limits of OD, between which the Pearson coefficient r was highest. Regressions were based on no less than 10 time points and had an r of 0.99. MGR (1/hr) was then estimated from the slope of the linear regression of the log OD vs. time. This experiment was repeated in triplicate for each C30 clone. One-way ANOVA and post hoc Tukey test were used for statistical analysis.
Phenotypic changes of evolved clones: relative growth (RG)
Initially, reference and query strains were mixed in 1:1 proportions. The conditions used were exactly the same as for the cycles of G-S that were used during the experiment, except that instead of fractionation on a Percoll gradient we kept the populations in Percoll solution for 2 hr with no centrifugation or fractionation (the same amount of time that was required for centrifugation and sample collection during the experimental conditions), and then washed the cells using standard Tris buffer (as during the experimental procedure). The initial mixed populations were diluted to a density of 2 × 105/ml to: (1) calculate their initial proportions and (2) start the actual G-S cycle. We applied two cycles of G-S, and then counted the fraction of cells of each competing strain by counting the number of fluorescent and nonfluorescent colonies on a YPD plate. Fitness as the RG rate on plates in competition with ancestral marked with YFP was measured assuming transitivity of fitness interactions. The YFP marker was amplified from genomic DNA from the BY4741-YFP.natR strain and integrated into the ancestral strain according to previously described protocol (DeLuna et al. 2008). The reference ancestral YFP-marked strain, 16 evolved experimental clones from NQ and Q lines (QI 1, 13; QII 5, 6, 12; QIII 8, 9, 11; NQI 11, 14; NQII 3, 15; and NQIII 6, 8, 11) and the unmarked, original ancestral strain, were separately grown overnight to stationary phase in liquid cultures. The reference strain was mixed with each of the query strains isostoichiometrically. Mixed cultures were diluted and ∼105 cells were used to inoculate a plate (10 ml YPD) for the first round of G-S. After 3.5 d, the resulting large patches were harvested and incubated for 2 hr in Percoll solution. Then, ∼105 cells were used to inoculate another plate (10 ml YPD) for the second round of G-S. After each round of G-S, the frequency of competing strains was estimated by plating dilutions to count CFU (using a transilluminator to distinguish colonies that were florescent). The fitness of each of the clones relative to the reference strain was computed as the ratio of the Malthusian parameters of both strains (Lenski 1991). At least three replicate competition experiments per strain were performed. Ancestral clones grew 6% (average RG = 1.06) faster than the YFP-marked derivative. We corrected for this difference by dividing growth of each evolved query clone by this number. General Linear Models: Nested ANOVA model was used: repetitions nested in clones that were nested in line. Post hoc Tukey test was used for statistical analysis.
Phenotypic changes of evolved clones: regrowth after starvation
Clones derived from the final experimental cycle (C30) (Q I 1, 13; Q II 5, 6; Q III 8; NQ I 11; NQ II 3, 15; and NQ III 6, 11) were transferred from the frozen culture to fresh YPD agar medium using a metal pin. The colonies were incubated for 3 d. Then, a small quantity of a colony was transferred to liquid culture for overnight growth on a rotating wheel at 30°. The next day, five drops of 50 μl were put onto 25 ml fresh YPD plates such that the grown patches did not touch each other; this was done in triplicate. After 2, 4, 8, 15, and 22 d, the population density and viability were estimated. Each time, one patch from the plate was washed out using water. Cell density was estimated based on Coulter Counter results. Then we serially diluted the cultures and plated them on a fresh YPD plate. After 3 d of incubation at 30°, we counted CFU and estimated the fraction of the cells able to undertake mitotic divisions and grow to form a colony on the plate. Viability is expressed as the cell fraction of cells that were able to undertake mitotic division and form a colony in the fresh YPD plate.
Plate-washing assay
The plate-washing assay was performed as described (Cullen and Sprague 2000). Ten microliters of the stored population derived from each of the experimental and ancestral clones were put on a YPD plate. Invasion was allowed to proceed for 3 d at 30°. Plates were washed with water to remove cells that did not invade the agar. Care was taken not to break the agar surface. Photos were taken right before and immediately after performing the washing assay. Colonies for which we observed visible intrusion into the agar surface were marked X when only part of the area of the original colony was visible, and XX when the whole area of the colony was visible (Supplemental Material, Figure S1).
Whole-genome sequencing of evolved clones
We struck out the experimental populations from the three NQ (NQ I, NQ II, and NQ III) and three Q (Q I, Q II, and Q III) lines from the final cycle (C30) to single cells on YPD plates. We isolated two ancestral clones and a total of 47 clones from the NQ lines (16, 15, and 16 from NQ I, NQ II, and NQ III, respectively) and 47 clones from the Q lines (16, 16, and 15 from Q I, Q II, and Q III, respectively). Clones were grown separately in fresh liquid YPD in a 96-deep well plate. After 2 d of growth at 30°, we used the YeaStar Genomic DNA Kit (Zymoresearch) according to the provided protocol for isolation of total genomic DNA from each clone. We measured DNA concentrations using a Qubit fluorometer and the Quant-iT dsDNA HS kit (Invitrogen) (according to provided protocols), and prepared Nextera-based genomic libraries as previously described (Kryazhimskiy et al. 2014). These 96 (94 evolved and two ancestral clones) DNA libraries were mixed together and sequenced on a single flow-cell lane on an Illumina HiSequation 2000 to generate paired-end, 100 bp reads.
Sequencing data analysis: counting SNV in whole-genome sequences
We used two independent methods to analyze whole-genome sequences. For the first method, obtained sequences were mapped to the reference genome (R64-1-1; RefSeq assembly accession: GCF 000146045.2) using bowtie2 software (Langmead and Salzberg 2012) with option very sensitive. Obtained SAM files were transformed to BAM, sorted, and indexed using samtools (for more information go to http://www.htslib.org/doc/samtools-0.1.19.html). Optical duplicates were deleted using samtools rmdup. Samtools mpileup was used to look for the SNV (with the following settings: -u, and –q20). The resulting file was transformed to VCF format (using bcftools view). Variant filtering was done with a custom Python script. Variants with the following setting were qualified for further analysis: not present in the ancestral (t02) sequence, QUAL parameter (in VCF file) was ≥30, coverage was >9, present in ≥85% of all good quality reads. Coverage and percent of variant confirming reads were extracted from the DP4 field of the VCF file. For annotation, we used software annotate-0.1 (http://depts.washington.edu/sfields/software/annotate/). For the second method, genomic analysis was done using CLC-Genomic Workbench 7.5 software. Unmapped postfilter, paired-end reads as FASTQ files provided by the Stanford Sequencing Centre were downloaded directly to the CLC GW software. We mapped two of the ancestral clones (t01 and t02) and 94 evolved, C30 clones to the previously downloaded reference sequence: S. cerevisiae. EF4. 68. dna. toplevel (Genome). No masking was applied to the reference genome. Mapping was random with the following parameters set: mismatch cost 2, insertion and deletion costs 3, length of fraction 0.5, and similarity of the fraction 0.8. The mapped sequences of clone t01 were filtered against known variation in the mapped sequences of the t02 clone, which was treated as the reference. We only considered variation supported by >85% of the mapping reads. We mapped whole-genome sequences of the 94 evolved clones to the reference genome. Then, we checked basic variant detection using the following parameters: ignore broken pairs, ploidy: 2; minimum coverage: 5; minimum count: 2; minimum frequency: 50%. Detected single nucleotide polymorphisms (SNPs) were filtered against previously found variations in clone t02. Variations found in each of the clones (ignoring broken pairs and nonspecific matches) were checked for amino acid change in the annotated genes. Each of the SNPs was confirmed by visual inspection in ∼50% of the cases of the aligned reads, and then annotated as to whether they were in coding or noncoding regions.
Construction of the mutation phylogenetic tree
A fasta file was generated for all clones, and each clone was represented by a sequence, made out of all the variant positions present across all clones. The file also had an entry for the reference sequence. The fasta sequences were aligned using the MUSCLE package (Edgar 2004). The aligned file was then converted to a format that can be read by programs in the PHYLIP software distributed by the author (Felsenstein 2005). The tree was generated by the programs dnapars, retree, and drawgram from the PHYLIP software and imported in illustrator CS6.
Data availability
Strains and isolated clones are available upon request. Genome sequences have been deposited in SRA (NCBI) as BioProject: PRJNA383491, with accession number SRP104610. File S1 contains descriptions of all Supplemental files. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.
Experimental design
Experiments were performed using a derivative of the S. cerevisiae S288C laboratory strain (Matα, ura3::KanMX4, his3Δ, leu2Δ, lys2Δ) (Cubillos et al. 2009). A single colony of this strain was grown in liquid YPD media to stationary phase and the resulting population was separated into Q and NQ cells by density centrifugation on a Percoll gradient, with the upper (less dense) fraction consisting of NQ cells and the lower (more dense) fraction consisting of Q cells (Figure 1A) (Allen et al. 2006). We then serially propagated cells from only the Q cell layer, or from only the NQ layer, through 30 G-S cycles. Each cycle started by plating 100 µl of the culture, containing ∼106 cells, as a "patch" onto a small Petri plate containing 10 ml of YPD agar medium (2% glucose), yielding an initial population density of 105/ml for the entire plate of medium. After 3.5 d of growth at 28°, after the point where the cells had already reached stationary phase and were experiencing nutrient deprivation (density of ∼2 × 108/ml for the whole plate) (Allen et al. 2006; Davidson et al. 2011), Q and NQ cells were separated by centrifugation, and the relevant fraction (Q or NQ) was diluted and ∼106 cells plated again to give 105 cells/ml (for the 10 ml YPD agar plate) to start the next cycle. In three of the experimental lines (named NQ I, NQ II, and NQ III) only NQ cells were serially propagated, while in the other three lines (Q I, Q II, and Q III) only the Q cells were serially propagated (Figure 1B). Each of the six experimental lines were subjected to 30 of these 3.5-d G-S cycles (Figure 1B). After the 30th cycle (∼120 d), 15–16 clones were isolated from each of the experimental lines (a total of 47 from NQ lines and 47 from Q lines) and were whole-genome sequenced to identify mutations that might have been selected by the one selection regime or the other. These clones (all or only chosen ones) were in addition checked for phenotypic traits: MGR, survival during G-S cycles, survival in starvation, and invasive growth.
Increase of the enriched cell type proportion in the experimental populations
To determine whether serial enrichment of a particular cell fraction resulted in a stably increased production of such cells within the population, we measured the relative proportions of each cell type (on a population basis for each of the enriched lines) throughout the experiment. We observed significant increases in the relative proportion of each serially enriched cell type (Figure 2, A and B). The ancestral population, prior to the serial cell type enrichment cycles, had a NQ cell proportion typical of the S288C strain of 25% ± 1% and a corresponding Q cell proportion of 75% ± 1%. After 30 G-S cycles with serial enrichment for NQ cells, the proportion of NQ cells (taking the average of all three NQ lines) increased to 82% ± 2%, and the proportion of Q cells correspondingly decreased to 18% (Figure 2A). The percentage of NQ cells for the individual NQ I, NQ II, and NQ III lines, after 30 cycles, was 87, 79, and 80%, respectively. Likewise, after 30 G-S cycles, but with serial enrichment for Q cells, the percentage of Q cells (average of all Q lines) increased to 87% ± 5%, while NQ cells correspondingly decreased to 13% (Figure 2B). The 30-cycle Q percentages of the individual Q I, Q II, and Q III lines were 91, 76, and 95%, respectively. The Q II line, which acquired a mutation in MMS2 and a mutator phenotype (explained below), showed no significant change in cell type proportions during the 30-cycle experiment.
To determine whether the changes in cell type proportions displayed by the enriched populations were also reflected by individual cells taken from the populations, we chose 11 single colonies from the six final (30th cycle) populations, and one colony from the ancestral preenrichment parent strain, and measured the fractions of each cell type that they generate under the experimental conditions. We performed one cycle of growth, starvation, and fractionation, identical to the experimental serial G-S enrichment cycles (Figure 1B) in triplicate for each of the chosen clones. The average proportion of NQ cells for all NQ lines was 84% ± 4%; the average for each line was: NQ I 88.9% ± 2.2%; NQ II 82.9% ± 2.2%; and NQ III 89.0% ± 8.9%. By contrast, the average proportion of Q cells in all Q lines was 96% ± 6%, and the average for each line was: Q I 98.59% ±7.9%; Q II 89.54% ± 6.6%; and Q III 99.61% ± 4% (Figure 2C).
Adaptations to a specific environment results in trade-offs in different environments
We hypothesized that there may be fitness trade-offs due to adaptation to the specific conditions (Kassen 2002; Bono et al. 2016) that were used in our experiment, similar to previous findings (Wenger et al. 2011). To test this hypothesis, we determined the fitness of the ancestral clone and clones from the NQ and Q experimental lines in three different conditions: (1) Growth in YPD medium, with the presence of sufficient glucose (2%), i.e., a condition to which clones producing increased numbers of constantly dividing NQ cells may be better adapted; (2) a fluctuating environment, consisting of two alternating G-S cycles, without separation and selection of cells (in this changing environment, the ancestral, "generalist" cells might be better adapted than either of the enriched, evolved "specialist" populations from Q or NQ lines); and (3) viability after long-term starvation and regrowth ability, a condition under which clones producing increased numbers of Q cells might be better adapted.
Condition (1): We used MGR, measuring each evolved clones' individual growth rate during logarithmic growth, as our assay. Division time (1/hr) measured in liquid YPD culture is an approximation for the early stage of each experimental G-S cycle, when cells are actively growing on solid YPD medium (Figure 1B). Under these conditions, we observed some differences between the lines (Figure 3A): one-way ANOVA showed differences in MGR between lines (F = 17.61, d.f. = 6, P < 0.0001), yet there was no significant difference between the NQ (I–III) and the Q (I–III) lines. Instead, a significant difference was observed between the ancestral and the evolved clones (post hoc Tukey test). All evolved populations statistically increased their MGR, with NQ lines having the highest MGR (Figure 3A). The MGR of the average of all (15–16) experimental clones from each experimental line (with ancestral MGR set as (1) was as follows: NQ cells: MGRNQ I = 0.89 ± 0.05; MGRNQ II = 0.90 ± 0.05; and MGRNQ III = 0.89 ± 0.06; and Q cells: MGRQ I = 0.79 ± 0.08; MGRQ II = 0.80 ± 0.1; and MGRQ III = 0.75 ± 0.06.
Condition (2): To assess the evolved clones' adaptation to a changing environment, we measured the RG rate of 16 selected evolved experimental clones from NQ and Q lines and ancestral clones ("query" strains) in competition with a YFP-tagged version of the ancestral strain (the "reference" strain) on plates during two G-S cycles (Figure 3B). Nested ANOVA showed significant difference between lines (ancestral, NQ, and Q): Flines = 9.57; d.f. = 6; P = 0.003; however, there was no difference between the clones within each line and within their replicates: Fclones = 1.25, d.f. = 12, P = 0.39, Frepetition = 0.89, d.f. = 34, P = 0.63). A post hoc Tukey test showed a significant difference in RG between the ancestral line (RGancestral = 1.00 ± SD = 0.01) and all NQ and Q II–III lines; however, a post hoc Tukey test showed that average RG from clones from the Q I line (RGQ I = 1.02 ± 0.02) did not differ from the ancestral clone's RG rate (Figure 3B). Though not significantly different, the average RG rate of Q clones showed higher fitness by 2% compared to the average of all the NQ line clones (RGallNQlines = 0.99 ± 0.03 and RGallQlines = 1.01 ± 0.02, respectively) (Figure 3B).
Condition (3): We examined the clonal population's ability to regrow after starvation on YPD plates. We used two clones from the ancestral population, t01, and t02 (derived from the same single colony population that was used to start the whole experiment), and 10 clones from the experimental lines. From each clone, we derived populations that were checked for ability to regrow after 2, 8, 15, and 22 d of starvation on YPD plates. Analysis of GLM nested ANOVA showed significant differences between days (not nested) and lines (ancestral, Q, and NQ), but no difference between clones within lines (Fdays = 166.210, d.f. = 3, P < 0.001; Flines = 16.22, d.f. = 6, P < 0.001; and Fclones = 0.479, d.f. = 4, P = 0.75). Differences in viability, measured as the percentage of the entire population that was still able to reenter mitosis, divide, and produce a colony, became statistically significantly different among lines after 8 d of starvation with the Q lines showing a significant advantage. In this condition, clones derived from Q lines were fitter than either the ancestral or NQ line-derived clones (Figure 3C). The difference became more pronounced with increasing time of starvation, as measured on days 15 and 22.
Strains may have acquired adaptive mutations during the experiment
To determine whether selection for Q or NQ cells resulted in the enrichment of particular mutations that might predispose cells to form those particular cell types, we whole-genome sequenced t01, t02, and 94 evolved clones (15–16 from each of the three Q and three NQ lines). In total, across all the sequenced genomes of all clones, we found 138 (including 13 frameshifts) nonunique events of nonsynonymous SNV within gene coding regions (Table S1). The phylogenetic relationships among clones are shown in the (Figure S1). The presence of multiple mutations shared across different experimental lines is an unexpected result. We can hypothesize that: (1) it is possible that some of the shared mutations arose early in the experiment, before splitting to independent lines, and as such were spread within the experimental lines; (2) that some of these mutations were present in the initial culture because although we used what we believed was a single colony, there may have been more than one colony that was picked (if that was the case, then low frequency mutations would not have been detected when we sequenced just two clones from the ancestral population); and (3) there could have been unintended mis-labeling during the experiments [if so, we believe it is most probable (although we cannot be sure) that there was not any switching between Q and NQ lines]. Overall, we feel that because our choice of the presumably adaptive mutations is based on the fact that multiple mutations in a gene are present in several experimental clones and not that the clones belong to any specific line (except what is shared between NQ or Q line), that if mis-labeling occurred, it did not influence the choice of which mutations were adaptive.
One of the identified frameshift mutations was in the MMS2 gene (Thr44fs, middle branch in the Figure S1). The MMS2 gene is involved in error-free postreplicative repair (Broomfield et al. 1998) and, when inactivated, confers a mutator phenotype, significantly increasing the spontaneous mutation rate (note that mms2 mutants do not display any characteristic mutational spectrum) (Xiao et al. 1999). As expected, the 22 clones that carry this particular MMS2 mutation have acquired substantially more SNV than the rest of the clones, with an average of 15.7 mutations per clone as compared to only 4.9 mutations per clone for the remaining 72 clones. These 22 clones are excluded from the following analysis and discussion; however, their associated datasets are included in Table S1. After excluding clones with the MMS2 mutation, we identified 50 SNV in 40 genes (nuclear genome only), including one dubious ORF, and one gene of unknown function (Table S1) from a total of 72 sequenced clones. In 32 of these genes, only a single mutation was observed, with these mutations being present in 1–16 clones. Because of the experimental set-up where we transferred a fraction of the populations to the next G-S cycle, we do expect that clones isolated from each line at the end of the experiment (C30) would share some of the SNV that arose during the experiment. That is indeed the case, and SNV was often shared among clones with common ancestry from the same experimental line (Figure S1 and Table S1).
To identify mutations that may have been selected by the experimental conditions, we focused on genes in which we observed multiple different mutations, either specific to only Q or NQ lines, or found among both Q and NQ lines. In NQ lines only, we observed multiple mutations in SSY1, while in Q lines only, we observed multiple mutations in SIR3. We also observed genes that were independently mutated multiple times in both Q and NQ lines (MSN1, ECM21, SIR4, and RSP5). We also focused on single mutations that were observed in many Q clones but no NQ clones (FAS1) or in all NQ clones and only three Q clones (IRA2). We interpret the mutations that arose in this group of eight genes as possibly adaptive to the growth and enrichment environments we performed (Figure 4).
Adaptive mutations shared between NQ and Q lines
There are four genes in which we identified widespread multiple mutations across both the NQ and Q experimental lines (Figure S1 and Table S1). These are MSN1, ECM21, RSP5 and SIR4 (Figure 4). Two different missense mutations in MSN1, both affecting the same codon, are present among all non-MMS2 clones: Lys86Thr is found in QI and QIII clones, and Lys86Glu is found in clones from all six lines (Q I–III and NQ I–III). These results suggest that these mutations arose in the beginning of the experiment before splitting of the lines. MSN1 encodes a transcriptional activator of invertase and glucoamylase expression. MSN1-null mutants show either a decrease or complete absence of invasive growth, typical for haploids growing on solid agar in response to nitrogen and carbon limitation (Gagiano et al. 1999; Cullen and Sprague 2000). Because our experimental protocol involves a step where we collect cells for the next cycle by washing the cells off an agar plate, we performed a plate-washing test (see Materials and Methods) to determine the invasive growth phenotype for all 94 sequenced clones. Despite having a wild-type MSN1 gene, the ancestor clones did not show an invasive growth phenotype; this was expected as the S288C strain background does not show invasive growth. The invasive growth phenotype was also absent from all 36 Q clones, even though they all had one of the two MSN1 missense mutations, suggesting that these mutations do not increase invasive growth. However, we found that 33 NQ clones (out of a total of 36, all of which have the MSN1 Lys86Glu mutation) do show an invasive growth phenotype (Figure S1 and Table S1). Thus, apparently, the MSN1 mutations are not responsible for any changes in the invasive growth phenotype, and instead, the presence of other NQ line-specific mutations such as the SPS pathway genes (see Discussion below) may be conferring the invasive growth phenotype to the NQ clones. Interestingly, the three NQ clones that lack an invasive growth phenotype are the only clones (out of all sequenced clones) that contain a presumed inactivating frameshift mutation in the RIM8 gene; null mutations of RIM8 are known to decrease or abolish invasive growth (Madhani and Fink 1998), and thus this mutation may be responsible for the lack of invasive growth in these NQ clones. Overall, invasive growth among NQ cells suggests an expected phenotypic trait for cells in search of nutrients, as opposed to cells more prone to enter quiescence.
We also observed multiple mutations in SIR4 and ECM21 across both Q and NQ cell lines. We observed five mutations (Ser57*, Asn89fs, Ser154*, Gln595fs, and Pro1124fs) in the SIR4 gene, which regulates de novo assembly of heterochromatin by scaffolding, nucleation, and anchoring (Kueng et al. 2012, 2013). Mutated sites are widespread along the entirety of this long gene (1358 aa). The first three mutations, all presumably inactivating since they are nonsense or frameshift, occur within the first domain (aa 1–270), which is responsible for yKu80 and DNA binding. The Gly595fs frameshift mutation leaves the above yKu80/DNA binding domain intact as well as a second Rap1 binding domain (aa 142–591) intact, although it is not known if a partial protein would be stable. The frameshift mutation Pro1124fs resides in the last domain, which is responsible for Sir4p dimerization and Sir3p and YKu70 binding (Kueng et al. 2012, 2013; Gartenberg and Smith 2016). Found among all lines, and shared by 67 clones, were two mutations in the ECM21 gene. Both mutations introduce premature stop (positions Arg106* and Glu282*) before the PY-motif, which is necessary for interaction with Rsp5p (Lin et al. 2008).
IRA2 mutation found in all NQ clones
An IRA2 stop mutation, Gln1365*, is present in 39 clones that were derived mainly from NQ cell lines (the mutation is present in all 36 NQ clones, but in only three Q clones). It is possible that this mutation also arose at the beginning of the experiment, before the culture was split between the NQ and Q selection lines. Ira1 and Ira2 are Ras GTPase-activating proteins (GAP) that act as negative regulators of the Ras-cAMP signaling pathway by increasing the rate Ras proteins (encoded by RAS1 and RAS2) hydrolyze GTP to GDP (Tanaka et al. 1990). Such mutations in the Ras/cAMP signaling pathway have been previously identified as adaptive under glucose-limited conditions (Lang et al. 2013; Chiotti et al. 2014). These mutations result in elevated glucose signaling, resulting in the cell's misperception that glucose is present even when it is not (Smets et al. 2010).
The amino acid signaling pathway (SPS) and endocytosis may play a role in the enrichment of NQ cell frequency
There are 22 mutated genes among the 36 non-MMS2 NQ clones (Table S1). YeastMine GO analysis of this gene group reveals enrichment for the biological process of response to amino acid (GOID 0043200, Bonferroni corrected P-value = 0.05) based on the presence of three genes in the group, SSY1, PTR3, and SSY5, which together constitute the SPS amino acid sensing pathway (Didion et al. 1998). Another enriched process is ubiquitin-dependent endocytosis (GOID 0070086, Bonferroni corrected P-value = 0.04) based on the genes ECM21, RSP5, and RIM8. The most significantly enriched cellular components are the plasma membrane part (GOID 0044459, Bonferroni corrected P-value = 0.006), based on genes PHO89, SSY1, RSP5, PTR3, RIM8, SSY5, and IRA2; the plasma membrane extrinsic component (GOID 0019897, Bonferroni corrected P-value = 0.03) due to genes RSP5, PTR3, and SSY5; and the plasma cytoplasmic side (GOID 0009898, Bonferroni corrected P-value = 0.04) due to genes RSP5, RIM8, and IRA2.
We identified multiple mutations in the SSY1 gene, all of which occurred only in clones derived from NQ lines (Figure 4, Table S1). The SSY1 gene encodes a nontransporting nutrient sensor, and acts as the first step in the SPS pathway (Didion et al. 1998; Klasson et al. 1999) that senses extracellular amino acids (the following steps involve the PTR3 and SSY5 gene products, see above). All four of our observed SSY1 mutations, Tyr747*, *853Tyr, Glu526fs, and Gly492Arg, presumably leave an intact cytoplasmic domain (Conrad et al. 2014). The other members of the SPS amino acid signaling pathway are mutated only in the NQ clones (PTR3: D263fs and SSY5: E155fs). Quite interestingly, we observed mutations related to SPS signaling and membrane receptor catabolism across all Q-enriched lines. In particular, point mutations in ASI1, BRO1, DOA1, and RSP5 arise exclusively in Q lines (apart from a single instance in NQ lines, involving RSP5). It was shown that mutations affecting these genes restore amino acid uptake in SPS sensor-deficient strains (ssy1 leu2). This allows for growth of cells that are otherwise unable to respond to extracellular amino acid concentrations, by compromising degradation pathways responsible for membrane receptor turnover (Forsberg et al. 2001). The expectation then would be that these mutants, which arose in clones from the Q lines, should result in upregulated degradation pathways. This mechanism is also suggested by studies linking degradation pathways to chronological lifespan in yeasts and other systems (Alvers et al. 2009; Takeda et al. 2010; Kruegel et al. 2011).
Chromatin silencing role in populations with an elevated Q cell fraction
We found 20 genes mutated in the 36 non-MMS2 clones from Q lines (Table S1). Yeast Mine GO analysis of this gene group showed enrichment in the cellular components of transcriptional repressor complex (GOID 0090568; 0017053, Bonferroni corrected P-value, respectively, = 0.001 and 0.003), due to the presence of genes SIR3, SIR4, and BUR6 in the group, and enrichment in cytoplasmic side of membrane (GOID 0009898; Bonferroni corrected P-value = 0.02, genes: RSP5, BUD2, and IRA2).
We identified two mutations in the SIR3 gene present in a total of 16 Q clones (Figure 4, Figure S1 and Table S1). Mutation E317* is present in clones from all three Q lines, suggesting that it might have been preexisting before splitting into experimental lines, or that it might be the result of unintentional mislabeling of clones (as explained before). An additional mutation in the SIR3 gene is present within the QII line clones that carry the MMS2 mutation. Both SIR3 mutations (E317* and I246fs) leave the first SIR3 domain, BAH (bromo-adjacent homology, aa 1–214), intact, which is crucial for chromatin repression. All 15 of the clones with SIR3 E317* mutations have a preexisting FAS1 missense mutation (G588A) that may have a synergistic effect. It has been suggested that the Fas1 "housekeeping" gene product (a subunit of fatty acid synthetase), in addition to its catalytic function, is required for the biosynthetic control of the yeast FAS complex (Wenz et al. 2001).
Mutations in SIR2 and other possible adaptive mutations among the clones with MMS2 mutation (mutator lineage)
We cannot entirely exclude the possibility of additional adaptive mutations in the clones with the MMS2 mutation (mutator clones). The presence of additional, including multiple, SNVs in the genes that we classified as possibly adaptive—such as MSN1 (Ser200Phe and Arg186Trp) found in Q and NQ mutator clones, SIR3 (Val653Phe) found only in Q mutator clones, and SSY1 (*853Tyr) present only in NQ mutator clones—supports this hypothesis. There are two genes that acquired multiple SNVs, at least one of which is present within the mutator clones: SIR2 (Leu332Pro, Cys469*, and Ser420*) and KEL1 (Arg237Thr and Gln814*). All of the SIR2 mutations are spread within NQ clones. This may reflect the unique role of Sir2p among sir complex genes in the asymmetric partitioning of oxidation-damaged proteins between mother and daughter cells (Aguilaniu et al. 2003). Furthermore, Sir2p is responsible for rDNA silencing (Smith et al. 1998), an activity that supports entry to quiescence. Mutations in SIR2 and SSY1 cooccur as in the cases of C469* in SIR2/G492R in SSY1 and L332P in SIR2/*853Y in SSY1 (the latter pair of mutations appears within the mutator line). This might suggest an epistatic relationship between these genes that confers fitness advantage among NQ cells.
Finally, there are five genes with multiple SNVs present only within mutator clones; HSL7 (Val198Glu and Thr347fs), EFM4 (Ser96Tyr and Ser21Pro), SSK2 (Tyr852Ser and Leu853*), WHI2 (Gly141* and Leu76fs), and BRO1 (Asp228Tyr and Leu216Ile).
Two mutations, G141* and Leu76fs, that deactivate the WHI2 gene are present within 26 mutator clones. The mutations leave intact the first out of five protein domains, similar to the mutations found previously in yeast evolving under nutrient-scarce conditions (Wenger et al. 2011; Lang et al. 2013). Whi2p is required for full activation of the general stress response and regulates growth during the diauxic shift. Null mutants have increased competitive fitness in YPD media. We also found two mutations in the SSK2 gene (L853* and Y852S) in the mutator strains. Ssk2 is a kinase (MAPK) of the Hog1 mitogen activated pathway, activated in yeast by high osmolarity stress (Hersen et al. 2008). Although it is difficult to presume the adaptive nature of these mutations in strains that contain many other mutations simultaneously, the presence of these mutations (the mutations in the same genes as seen in non-MMS2 clones, as well as the WHI2 and SSK2 mutations seen only in MMS2 clones) is suggestive of an adaptive nature.
In this study, we sought to elucidate genetic mechanisms involved in the regulation of Q cell formation. Haploid yeast cells were sequentially subjected to two opposite environmental conditions of (1) feast, when cells should divide as quickly and efficiently as possible, and (2) famine, when transition to quiescence is crucial for survival. After successive enrichment for only one type of cells (either NQ or Q) that were used to found subsequent generations, we observed changes that indicated cell survival strategies differing from the ancestor. The enriched cell lines showed persistent and significant differences from the ancestor in proportions of Q and NQ cells, and also showed fitness trade-offs when grown in different environments. We found that the ancestral clone, a laboratory strain that presumably has been long adapted to a changing environment due to cyclic growth (serial transfer) on rich YPD-type medium, has higher fitness than either the NQ- or Q-enriched cell line clones in an assay monitoring growth through two feast–famine cycles; the ancestor thus retained its ability to both grow fast when nutrients are abundant, but also to be able to physiologically adapt to periods of starvation while nutrients are scarce. By contrast, clones from the NQ lines, which we found to be much more highly enriched in NQ cells than the ancestral strain, grow faster than either the ancestor or Q line cells in conditions when logarithmic growth rate is supported and there is no need to produce Q cells. Finally, clones from the Q experimental lines, which produce a slightly (but significantly) higher percentage of Q cells than the ancestor, outcompete the ancestral as well as NQ line clones in viability assays after prolonged starvation.
There are no easy-to-use markers for Q and NQ cells developed so far (Čáp et al. 2009; Rutledge et al. 2015; Laporte et al. 2016); however, separation of Q and NQ cells from the population can be achieved based on differences between the densities of NQ and Q cells (Allen et al. 2006; Davidson et al. 2011; Klosinska et al. 2011; Čáp et al. 2012), which is the method we used for our serial enrichment strategy. However, it is possible that to some extent we also selected or enriched for cells of various density or size characteristics not necessarily linked to their Q or NQ status. Although none of the genes we identified as possibly adaptive (Figure 4) were listed in the yeast GO annotation as cell size control regulator (GO:0008361), we cannot unequivocally exclude such a possibility, since the effects of gene mutations on cell size and density are not usually assessed, which also takes into account the fact that most clones acquired several mutations during the experiment.
Increase of the NQ cell fraction may be a consequence of a dysregulated SPS signaling pathway
An increase in the production of NQ cells in the NQ lines was already visible after 10 cycles of G-S and did not change much after further cycles (Figure 2A). The most striking result from the whole-genome sequencing of our C30 serially enriched clones was the fact that multiple mutations in the SSY1 gene (Tyr747*, Glu526fs, and Gly492Arg) were found in 32 of the 36 NQ clones (note that this does not include the MMS2 mutator NQ clones that also have mutations in the SSY1 gene, see Supplemental Material). The SSY1 gene encodes a nontransporting nutrient sensor (Didion et al. 1998; Klasson et al. 1999), acting as the first step in the SPS pathway, during which the extracellular amino acid concentration results in conformational changes of the Ssy1p cytoplasmic domain, which signals to downstream targets (Conrad et al. 2014). In the presence of extracellular amino acids, the primary amino acid sensor Ssy1p is stabilized in a conformation that triggers intracellular signaling (Ljungdahl and Daignan-Fornier 2012). It has been previously observed that mutation of SSY1 codon 382 from threonine to lysine (or arginine) results in constitutive signaling, showing that mutant Ssy1p can initiate signaling in the absence of extracellular signaling (Gaber et al. 2003; Poulsen et al. 2005, 2008). All the mutations we observed (Gly492Arg, Glu526fs, and Tyr747*, plus *853Tyr in the mutator clones) presumably leave an intact cytoplasmic domain (Conrad et al. 2014). This, similarly to previous discoveries (Gaber et al. 2003; Poulsen et al. 2005, 2008), could possibly result in constitutively active signaling in the absence of extracellular amino acids. In all the clones with SSY1 mutations, the other genes from the SPS pathway were not mutated, which is also the condition for constitutive signaling found in previous research (Gaber et al. 2003). PTR3 and SSY5 were also mutated but in NQ clones with no mutation in SSY1 (Table S1). In our experiments, an altered activity of the SPS pathway could be a mechanism that led to increased NQ cell production. In this rationale, constitutive activity of Ssy1p, the extracellular amino acid sensor, could lead cells to overestimate the amount of extracellular nutrients, which should hinder entry to quiescence. Alternatively, if the mutations are inactivating, downregulation of the pathway, e.g., by inactivation of the downstream effector Ssy5p, increases replicative lifespan (Tsang et al. 2015); again, this is a trait that is expected to confer a fitness advantage among NQ cells.
Another gene that, when mutated, may confer advantage to NQ cells is IRA2; we observed a single frameshift (and thus possibly inactivating) mutation that was present in all 36 NQ clones, but in only three Q clones (Table S1). We presume that this mutation is more adaptive for the NQ than the Q cells. Inactivating mutations in IRA2 (and its close homolog IRA1) are known to provide a fitness benefit in multiple contexts in glucose-limiting conditions (Wenger et al. 2011; Kvitek and Sherlock 2013; Chiotti et al. 2014; Venkataram et al. 2016) and can result in cells continuing to divide even in conditions of low glucose. Interestingly, IRA2 had an elevated transcriptome level during glucose quiescence (Klosinska et al. 2011). Taken together, it seems quite plausible that the IRA2 frameshift mutation may provide a fitness benefit for NQ-enriched lines, where uncontrolled growth during early starvation conditions may confer an advantage.
Q cell advantage may arise from a possible role of SIR3 mutations
We observed a significant increase in the fraction of Q cells in two Q lines (Q I and Q III lines). Ultimately, Q line cells survive to the next experimental cycle only when they efficiently convert to the Q state. However, these cells also need to be able to reestablish cell division when the conditions improve. Both of these needs have to be taken into account when considering the possible role of the selected mutations. Present in all Q lines, two mutations in the SIR3 gene (Ile246fs and E317*) introduce truncation after the BAH domain. Remarkably, the BAH domain alone has the binding properties of full-length Sir3p, suggesting that the domain represents an independent nucleosome-binding module (Connelly et al. 2006; Onishi et al. 2007). We hypothesize that the SIR3 mutant product is still available for selective binding, which attracts the SIR2-SIR4 complex. Thus, the chromatin silencing function is sustained. However, if the protein is truncated, it would seem that Sir3p dimerization through its extreme C-terminal wH (winged helix) domain, necessary for SIR-complex spreading along nucleosomes, might be impaired (Kueng et al. 2013). SIR3 also has an important role in telomere hypercluster formation that extends chronological life span during quiescence. However, this is a rather slow process that reaches its plateau at ∼6–7 d of culture at 30° (Guidi et al. 2015). In addition, recent research shows that telomere hyperclustering is not required for cell survival in early quiescence (Laporte et al. 2016). Thus, for our experiments, where cells were starved for >3 d, we speculate that telomere hyperclusters may not have yet formed, and that this function of Sir3p may not be not adaptive during our short-term starvation conditions, although it is possible that it plays an early role that is adaptive in our conditions. Sir3p also plays a role in the epigenetic inheritance of silent chromatin in yeast (Motwani et al. 2012). Differentiation into distinct cell types involves establishing unique patterns of gene expression. Once established, epigenetic mechanisms operate to maintain these patterns as cells divide.
Mutations shared among Q and NQ lines are important for the general stress response and nutrient acquisition
Invasive growth is believed to allow budding yeast to search for nutrients, as the elongated cells remain associated and in communication with each other, but also to anchor yeast colonies to the stable surface (Honigberg 2016). Thus, as the population proliferates, cells can invade solid agar media or move through static liquid media in search of carbon or nitrogen. The invasive growth test revealed an obvious distinction between NQ cells, in which the invasive phenotype was present, and Q cells, where this phenotype was completely absent (Figure S1). This was the case even for Q cells that were carrying the MMS2 mutation, suggesting that other factors influence this phenotype. Interestingly, rim8 mutation within a NQ lineage appeared to reduce invasiveness (Figure S1), consistent with a role of RIM8 in invasive growth (Madhani and Fink 1998). Mutations, such as in IRA2 (despite the predominant partitioning in NQ lines) and MSN1 may directly reflect additional modes of adaptation in the growth media, nutrient depletion, and/or yeast harvesting from the agar plate.
With this experimental set-up we wanted to begin to explore the regulation of the yeast Q state (Klosinska et al. 2011; Čáp et al. 2012; Honigberg 2016) and the growing interest in nonnatural, oscillating environment experimental design (Mitchell et al. 2015). Nutrient depletion causes yeast populations to produce Q cells at a certain frequency. By introducing selection and serial enrichment of Q or NQ cells, we uncovered pathways that appear to facilitate or hinder entry to quiescence, respectively. In the case of NQ-enriched cells, the presumed adaptive mutations we observed in the SPS signaling pathway may result in dysregulation of their adaptive anticipation mechanism and thus allow them to bypass transition to quiescence. In the case of Q-enriched cell populations, we speculate that because they were repeatedly subjected to >3.5 d of starvation, the deep reorganization of the internal cell structures such as (1) spatial confinement of enzymes, (2) mitochondrial and ER protein reorganization, and (3) loss of organelle contact sites might be not necessary or even a "waste of energy" (Suresh et al. 2015). In such a case it could be advantageous for cells to avoid it. All of the adaptations we observed for NQ- and Q-enriched populations seem to be costly and incur trade-offs in the other conditions. Our findings demonstrate the value of nonnatural dynamic perturbations in exposing hidden sensitivities of cellular regulatory networks.
We thank the reviewers for helpful comments; Ryszard Korona, Gavin Sherlock, Joanna Rutkowska, and Sandeep Venkataram for help, remarks, and comments; Katarzyna Pawlik and Joanna Bobula for technical assistance; and Sever Salamon for assisting with realization of the figures. The work was funded by the National Science Centre via a HARMONIA grant to D.M.W.-S. (DEC-2011/01/M/NZ8/01031), grant number 2014/13/B/NZ8/04668 awarded to K.T., and grant number DS/INoS/762/2017.
Supplemental material is available online at www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.041749/-/DC1.
Communicating editor: B. J. Andrews
- Received March 4, 2017.
- Accepted April 11, 2017.
- Copyright © 2017 Wloch-Salamon et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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For some years the artist has spent his summers in the United States, working in a studio he maintains in upstate New York. In 2007 Hume had a solo show at White Cube, London entitled American Tan. The exhibition combined a group of paintings on aluminium board and sculptures all inspired by an American icon – the cheerleader. The artist decided to bring together a selection of motifs from his most recent work to form a portfolio of screenprints. Hume opted for a collage process where the prints are laid down on a brushed aluminium card. The Sister Troop is a technical tour de force and Hume's most intricate print series to date. Limited availability.
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|First Name
|Last Name
|Artist Description
|Gary Hume (born Kent, UK, 1962) studied at Goldsmiths College of Art, London, emerging to become part of the Young British Artists group in the early 1990s. His work is characterized by a bright palette of flat colours, often using household gloss paints on polished aluminium panels with imagery drawn from popular contemporary culture in a manner recalling the Pop Art movements of the 1960s and 1970s. Hume refers to three main strands in his paintings as 'flora, fauna and portraiture'. A major exhibition pairing Hume with the late Patrick Caulfield at Tate Britain in 2013 has been followed by regular appearances in the RA Summer Exhibition and a 2017 solo show at Sprüth Magers, London.
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|Screenprint on brushed aluminium card
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|Collaged screenprint on brushed aluminium card. Printed on 410gsm Somerset Satin paper, from a portfolio of 10 screen prints titled The Sister Troop published by the Paragon Press in 2009.
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|Also known as silkscreen. In its simplest form, this is a technique by which the artist blocks out a section of a fine, woven screen (formerly made of silk), which is stretched over a frame. With a squeegee, ink is pressed evenly through the screen on to a sheet of paper beneath. Only the areas of the screen not blocked out will be printed. The artist will use as many of these transparent sheets (separations) as the numbers of colours required in the print, and each sheet must be aligned very carefully with the others. The films are transferred on to the silkscreens via a light-sensitive process: only the areas which are painted by the artist will be blocked out and will not allow ink through.
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Some people from this present era may not be so familiar with the tragic events of Holocaust during the World War II. Some may have tried to forget it and don't even want to remember it but there is an official day to honour those who have perished in this event. Holocaust Remembrance Day is one of the World's histories that the Nation of Israel and other Jewish communities around the world are commemorating and remembering every year.
When is Holocaust Remembrance Day 2015
Holocaust Remembrance Day 2015 in the Gregorian calendar falls on the dawn of Wednesday, April 15 and ends in the nightfall of Thursday, April 16.
Holocaust Remembrance Day in Hebrew calendar is observed as a National Memorial Day and public holiday on the 27th day of the month of Nissan. If the actual day falls on a Friday, Israel observes the Holocaust Remembrance Day on the foregoing Thursday. When it falls on a Sunday, they observe it on the following Monday to avoid adjacency with the Shabbat. It always falls on the months of April or May.
Since Holocaust Remembrance Day this calendar year 5775, is not falling on a Sunday, it will be stay on Wednesday, 27th day of Nissan.
Read also INTERNATIONAL HOLOCAUST REMEMBRANCE DAY
How the day was established
It has been more than 60 years since that catastrophic event of Holocaust has occurred but for all who have survived this tragic happening including the families of those who survived, the memoirs are still real and alive though there are some who have not been involved consider it to be part of the primeval history.
Before the day was established as a remembrance for all the victims of Holocaust, including the six million European Jews and million others who are homosexuals, mentally and physically disabled, Ukranian, Russian civilians, and Gypsies, there are various days suggested to commemorate the event. It was debated for two years until in 1950 they have chosen the 27th day of Nissan which also marks the anniversary of the Warsaw Ghetto uprising.
Finally in April 12, 1951, the Israel's Parliament (Knesset) proclaimed the date of remembrance on the 27th day of Nissan and it was formalized and established by the Knesset on August 19, 1953. It was named as "Yom HaShoah Ve-Hagevurah" in Hebrew which means the "Day of Remembrance of Holocaust and Heroism" and later shortened into Yom HaShoah. Although it was established in Israel, other Jewish communities around the world are also commemorating the day.
Remembering the Holocaust Victims
Since Holocaust Remembrance Day is a newly set national holiday, there are no set of rules and customs established yet for the day. But generally, it is a day observed with candle lighting, singing, poems, prayers and ceremonies which are held in cemeteries or in a memorial landmark of Holocaust for reciting the names of the victims. There are often six candles lit to represent the six million Jews.
In the early 1960's, the sound of the siren on the Holocaust Remembrance Day stops all the motorist and striders for a two-minute silence of remembering. The siren blows twice at sundown and at 11:00 in the morning. All the radio and television broadcasting networks align their programs in parallel with the holiday. The public entertainment businesses such as theatres and cinemas are closed during the holiday.
The memories of the victims of the Holocaust will live on, in any manner this day is being remembered.