NVIDIA GeForce RTX 3080 12GB GPU ‘production resumed’, makes no sense
NVIDIA GA102 GPUs sitting around, so production has reportedly resumed with AIBs making new GeForce RTX 3080 12GB graphics cards, before next-gen is here.
NVIDIA is reportedly resuming production of its GeForce RTX 3080 12GB SKU, after production stopped some two months ago now.
The company released the newer GeForce RTX 3080 12GB graphics card earlier this year in January, an upgraded version over the GeForce RTX 3080 10GB graphics card. The biggest two differences between the GeForce RTX 3080 10GB and GeForce RTX 3080 12GB is that firstly, the obvious difference in 12GB of GDDR6X memory over 10GB of GDDR6X memory. Secondly, the RTX 3080 12GB is an LHR “Lite Hash Rate” model, while the GeForce RTX 3080 10GB has full-speed crypto mining performance.
But now, NVIDIA faces warehouses of current-gen Ampere-based GeForce RTX 30 series graphics cards, in the months leading up to its next-gen Ada Lovelace-powered GeForce RTX 40 series graphics cards. This is why we’re hearing that NVIDIA will reportedly launch the flagship GeForce RTX 4090 graphics card first, and the GeForce RTX 4080 and GeForce RTX 4080 later… because there are too many higher-end GeForce RTX 30 series graphics cards left over in warehouses, and now more GeForce RTX 3080 12GB graphics cards are being made.
In previous news, we heard from leaker Moore’s Law is Dead and his sources that AIB partners told NVIDIA to go “pound sand” over leftover Ampere GPUs leading into next-gen Ada Lovelace. NVIDIA is working with AIB partners to get rid of existing GeForce RTX 30 series GPUs, with price drops for graphics cards across the board, and flagship GPUs like the EVGA GeForce RTX 3090 Ti FTW3 dropping $1000+ in the last couple of weeks alone.
NVIDIA is actively helping AIBs purge their old graphics cards, as a “compromise” of sorts in the “NVIDIA AIB Revolt” that I reported on last week. In that article, I covered that MLID said that his sources have said that AIBs are being helped by NVIDIA to clear their reserve stockpiles of GA102 (GeForce RTX 3090 Ti, GeForce RTX 3090, GeForce RTX 3080 Ti, GeForce RTX 3080 12GB, GeForce RTX 3080). His sources state that this was “the compromise” of the “NVIDIA AIB Revolt” which is when AIBs told NVIDIA to “go pound sand” over their warehouses of older-gen GPUs before the next-gen Ada Lovelace cards arrive.
Source 1: NVIDIA has directly stepped in to help AIBs clear their reserve stockpiles of GA102 in exchange for big pre-purchases of Enthusiast Lovelace. This was the compromise for the “NVIDIA AIB Revolt”. But even with help, it’s still going to take months for AIBs to sell through what they have, and they’re on their own when it comes to s telling off GA104 and lower”. Source 2: The channel is already starting to run dry of RTX 3090s, and the RTX 3090 Ti is starting to show signs of depleting stock as well. After that, we’ll see how aggressive we need to get for the RTX 3080 Ti and 3080 12GB to sell through”. Source 3: Late August is when the more aggressive actions will kick in from both AMD and NVIDIA if there’s still a ton of inventory left over. NVIDIA can still stomach going a bit lower on pricing if needed, but AMD won’t get much cheaper”.
Now, as for the AIB partners that told NVIDIA to “go pound sand” that had AIB sources explaining: “This month we told NVIDIA to go pound sound. We don’t want to buy anymore Ampere or Turing stock until we liquidate our existing warehouses. At this point we’re even willing to risk Lovelace allocation if they threaten us”.
The AIB source continued: “NVIDIA underestimated how desperate we were getting, but no more. They’re finally working with us on solutions to clear as much stock as possible before Lovelace launches in October. BTW – the overwhelming majority of Used mining stock hasn’t even hit the west yet. We need to get rid of these New cards NOW”.
Another source explained: “Well, over here (ASIA) miners are definitely selling most of their stock off. I’d expect those cards to start flooding the west within weeks”.
And now… now we have GeForce RTX 3080 12GB SKU going back into production. Hmmm.
MSI Gaming GeForce RTX 3080 LHR 12GB (RTX 3080 Gaming Z Trio 12G LHR)
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Lilliput Library no 84 can be found at 237 Highcliff Road. Its guardian is Debbie McEwan. The fantastic artwork is courtesy of Roz Wood. Go take a look and a book.
Lilliput Library no 85 is at ABC Balclutha Central which is located at 83 Charlotte St, Balclutha. It’s the first in the area and is dedicated to children’s books. Alice Muir from Milton painted this wonderful Lilliput.

domain: library


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HOCKEY legend Rhona Simpson, of Paisley, has challenged her Scotland Under-21 squad to show her what they’re made of when they take on the Scotland A team in two games this weekend.
The former Great Britain Olympian hopes to see her charges compete well against their opponents as the Under-21s crank up their preparations for the European Nations Championship later this summer.
And although results may not be the prime objective for the two games, future selection is certainly on the agenda this weekend.
Simpson, herself a former Olympic striker, is beginning to exert a bit of pressure on her young charges.
She said: “These games give me the chance to look at my players and for them to put into practice what they have been doing on the training ground.
“It is a final selection weekend, so there will be a bit of added pressure which the girls need to start being able to handle.”
Simpson is in the throes of her build-up programme for the European Nations Championships in Lille at the end of July, where Germany, Belarus and Spain are the pool opposition.
Simpson admitted: “We still have a fair way to go, but we do have a good run of games coming up which will hopefully increase the intensity of the programme.”
Before Lille, Simpson has several opportunities to hone her youngsters into the finished article as they have friendly encounters with England, France and Belgium, as well as the Junior Celtic Cup.
But the young Scots have already tasted success against quality opposition as they won two and drew one in six encounters with current European champions Germany.
“Our strengths are our pace, particularly in attack,” said Simpson.
“We’ve a good understanding of our individual roles and responsibilities, also we’ve a number of players who can score from open play and penalty corners.”
The CALA Edinburgh pair of Emily Bain and Amy Brodie, along with Grove’s Daniele Johnston, have scored goals at international level, but it was Morag McLellan and Ali Howie who found the net in the most recent German test matches.
In contrast, Gordon Shepherd’s Scotland A side has more of a hybrid composition about it.
He explained: “The idea here is to give players who are not in the under 21 set-up a pathway to be seen for possible selection for Scotland.
“I have supplemented the squad with some players who were not selected for the recent World Cup Qualifier.”
That will allow the likes of Nikki Kidd, Clair Scott, Susan McGilveray, Becky Ward, Amy Rowan, Becky Merchant and Kelburne’s Julie Bryce to catch Shepherd’s eye for possible Commonwealth Games selection.
And Shepherd has certainly left the carrot dangling, adding: “I hope to find that we have some really good youngsters to add to the depth of talent already playing at the top level.”
The sides play at Forthbank this afternoon and again at Bellahouston tomorrow.

domain: news


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KERALA: THE two day national seminar on the ‘World of EMS’ was held to commemorate the birth centenary of Comrade EMS Namboodiripad at his birth place, Perinthalmanna in Malappuram district. Inaugurating the seminar CPI(M) general secretary Prakash Karat recalled his experience with EMS Namboodiripad, who was the first chief minister of a democratically elected communist government in Kerala and the general secretary of the Party for nearly one and a half decade.
Prakash reiterated that the Party would continue its struggle against neo-liberal economic policies and the pro- imperialist stand of the UPA government. He also emphasised the importance of a self-critical analysis on the recent electoral setbacks and assured that the Party would examine whether it had distanced away from certain sections of the society. He also added that the electoral defeat is only temporary and the Party will continue to be the leading force in resisting the pro-rich, anti-people policies. The consolidated attacks of the right wing political parties, and other sectarian forces backed by imperialism can never hamper our struggle to resist neo-liberal economic policies and UPA government’s servile attitude towards imperialism, he said.
It is none but EMS Namboodiripad who identified the threat of majority communalism against democratic ethos and designed our strategy against the fascist forces. Even in the early eighties, he warned about the strengthening of the right wing Hindutva forces and their penetration into our secular society. Talking about the contributions of Comrade EMS Namboodiripad, Prakash described him as the most original and outstanding Marxist produced by the developing world in the 20th century. No other Marxist thinker theorised about the class and caste associations of our society as EMS did. He evaluated Indian national movement from a working class perspective and played a pioneering role in developing communist perspective for parliamentary forums.
The minister for local administration, Paloli Muhammedkutty presided over the seminar. Presenting a paper on democratic perspective of development, Prabhat Patnaik, vice chairman of the State Planning Board said that Kerala could never duplicate other states in development. If we imitate other states, much of our freedom would be negated and indiscriminate encouragement of capitalist investment would be dangerous to Kerala, he said.
The Finance minister T M Thomas Issac recalled the role played by EMS Namboodiripad in the successful implementation of decentralisation of power in Kerala.
The evening session kindled nostalgic revolutionary memories of Comrade EMS when Prakash Karat laid the foundation stone for EMS Memorial Complex on the banks of river Nila in a ceremony charged with emotions in which thousands of comrades and sympathisers participated. A Vijayaraghavan, MP presided over the function and the daughters of EMS, Dr Malathi, EM Radha and son in law Dr A D Damodaran attended. Dr Sumangala, the grand daughter of EMS sang melodious revolutionary songs belonging to the resistance music genre.
On the second day, the session started with CPI(M) Polit Bureau member S Ramachandran Pillai’s paper on ‘EMS and Coalition Politics in Kerala’. He urged the activists to follow the model of EMS in waging restless and sustained struggles against bourgeois social system and reactionary ideas. He also cautioned the Party activists not to confuse between the third front and the third alternative, which the Party seeks to build. He warned the Party activists to be vigilant against the influence the existing bourgeoisie society is trying to exert on them. Instead of being influenced by the unequal society, the communists should try to change the society, he said and called upon the activists to be prepared to face any challenges.
Many other learned scholars and practicing politicians presented their views on various facets of Comrade EMS’s life and related these to the contemporary task of the communist movement. Madhavankutty, famous journalist criticised the media for targeting CPI(M) adversely. Speaking on ‘Coalition Experiments and Media,’ he accused the media of giving shape to a ‘mega media rainbow coalition’ against the CPI(M). KKN Kuruppu, historian presented a paper on coalition politics in pre independent days. T N Seema, AIDWA state president spoke on Left perspective on women’s interventions.
The last session which dealt with EMS’s relationship with the cultural front was inaugurated by M A Baby, minister for Culture and Education. The session was presided over by Prabha Varma, the poet and was enriched by the presence of luminaries from the cultural front. Veteran poet Akkitham and Dr K G Poulose, vice chancellor of Kerala Kalamandalam participated. Baby described EMS as a leader who recognised the importance of cultural activities for the movements which work for social change.
The two days of discussion on EMS’s life, politics and contributions recharged the comrades with inspiration and confidence with the light radiating from the brilliance of the ‘Genius of the epoch’.
Kolkata Observes EMS Centenary
ADDRESSING a large indoor rally to remember E M S Namboodiripad, the legendary communist leader, in his centenary year, CPI(M) general secretary Prakash Karat said that the party would go to the people as in the past, learn from them and battle ahead, defeating all adversities.
The function was organised at the Calcutta University Centenary Hall on July 31 evening, with Biman Basu in the chair and CPI(M) state secretariat members on the dais.
Prakash Karat said EMS was not merely a theoretician, he was also of that rare quality that allowed one to put theory into practice. He would analyse the evolving situation and draw correct lessons from them. He placed a remarkable address on the problems plaguing the Malabar kisans when he was speaking at a legislative session in Delhi in the pre-independence years. This was the address that later served to inspire the land reforms movement in the country and gave birth much later to the movement for land reforms in places like Kerala and West Bengal.
EMS’s was also a correct analysis of the nationalities question in Kerala and, apart from calling for an integrated concept of what was then called a province, EMS also spoke firmly in favour of democratic decentralisation of power, financial as well as administrative.
Prakash Karat said the CPI(M) was at present under assault from the forces of reaction, indigenous and foreign. The attacks assumed a sharp dimension during the run-up to the 15th Lok Sabha elections and has continued since in Bengal.
From the date of the announcement of election schedule, 70 comrades have been martyred here in West Bengal, said Prakash Karat. In Kerala, the attack was open and overt. In Bengal the attack is covert and in the guise of ‘Maoism.’ Prakash Karat was bitterly critical of the politics of ‘Maoism’ being practiced in some parts of the country.
Analysing the election results of the 15th Lok Sabha elections, Prakash Karat pointed out that the results were an “exception in Bengal where the LF has been in office for 32 long years.” The CPI(M) has full confidence, and it would learn from the people the correct lessons to drive forward in the days to come. The life and achievements of EMS would serve as source on inspiration in this task.

domain: sociology


text:
Egg production in Jan-Aug 5% higher YoY, Kyiv region in lead
Egg production in January-August 2020 amounted to 5.938,8 mln units, the data of the State Statistics Service show. This indicator is 5% lower from the comparative period of 2019.
In 8M 2020, birds' eggs production is dominated by five regions:
- Kyiv: 1.738,9 bln units
- Khmelnytsky: 533.9 mln units
- Donetsk: 448.1 mln units
- Dnipropetrovsk: 344.6 mln units
- Cherkasy: 307.8 mln units
As a reminder, since the start of 2020, egg exports from Ukraine totalled 80.86 thou. units, 15% lower YoY.
In January-July 2020, birds' eggs production in Ukraine totalled 10.09 bln units, 1.2% less from the comparative period of 2019 (10.21 bln units).

domain: agronomy


Asked by: Sidibe Baldofskihome and garden home appliances
What is the smallest permissible pipe size for vents?
Last Updated: 15th May, 2020
Click to see full answer.
Herein, what is the minimum size of a vent pipe for kitchen sink?
The minimum diameter of the vent pipe is 1 1/4 inches in both cases.
Furthermore, how many fixture units can a 2 inch vent handle? 24 fixture units
Likewise, can you reduce the size of a vent pipe?
The line can be reduce in size and be kept small if there are enough additional vents to give the required aggregate area out through the roof. 2. The pipe size cannot be reduced but has to be full size from the house sewer all the way to the roof.
Can you vent a toilet with a 2 inch pipe?
The Dry Venting Toilet If the toilet has a 3-inch waste pipe, the vent must be located within 6 feet of the toilet trap; the distance is 10 feet for 4-inch pipe. Plumbers usually use 2-inch pipe for this, which is larger than the plumbing code requires.


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About 20 passengers are in critical condition while 26 other persons are mildly injured and receiving treatment at the Winneba Trauma and Specialist Hospital in two separate accidents which occurred on the Kasoa -Cape Coast Highway.
The first accident happened at Gomoa Mpota while the second happened at Gomoa Mankesim .
Police at Winneba say three vehicles that were involved in the first accident, a Sprinter Benz, a Kia Rhino and a DAF trailer have been towed away from the accident scene.
Winneba Municipal Fire Service Commander, D03 Kwesi Hughes, confirmed this to Adom News.
He said an unsuccessful overtaking by the Kia Rhino vehicle resulted in a head-on-collision.
In the second accident, an eyewitness revealed that, a Sprinter Benz Bus with the Registration number GN 2572- 10 From Cape Coast to Accra burst a tyre, and as a resulted somersaulted leaving the occupants in a critical condition .
The Winneba Municipal Fire Service Commander, D03 Kwesi Hughes, said the second accident was caused by the overloading of passengers.
He said, instead of the 23 passenger capacity, there were 36 passengers on board the vehicle that had food stuff as well.
All the 46 passengers are receiving treatment at Winneba Trauma and Specialist Hospital.


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Plays with I Am Another You (Sun, Nov 5 / 4 pm)
Directed by Rhayne Vermette
2017, Canada, 15 min
This is the story of the godlike architect, Carlo Mollino, animated within the desk space of failed architect, Rhayne Vermette. Made, with love on 16mm, 35mm and Super 8, this classic Pygmalionesque tale investigates intersections between cinema and architecture.

domain: design


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This year, fans of JoJo’s Bizarre Adventure have lost some beloved talent. Earlier this summer, news broke of Unsho Ishizuka’s death, and a brand-new piece has confirmed Nobuo Tanaka passed away as well just recently.
According to Nikkan Sports, Tanaka passed away in Japan on October 17. The well-known voice actor was 83 years old and died after a battle with esophageal cancer.
For those unaware of Tanaka by name, you will surely know several of the series he lent his voice to. Famously, the actor played Dio Brando in the original JoJo’s Bizarre Adventure OVA back in November 1993. Tanaka took up the role one more in JoJo’s Bizarre Adventure 2000 OVA series, bringing Dio to life in a supporting role.
Outside of JoJo’s Bizarre Adventure, Tanaka enjoyed success with his work on Gatchaman, Lupin III, and Detective Conan. The actor voiced many supporting anime roles over his tenure, and he provided narration for a few variety shows in Japan as well.
While Tanaka has not played an active role in JoJo’s Bizarre Adventure for some years, his legacy with Dio lives on. Currently, the title’s actual TV series is exploring its ‘Golden Wind’ arc featuring Giorno Giovanna. The blond-haired lead just so happens to be the illegitimate son of Dio Brando, so there is still plenty of story to explore with the infamous villain even today.
Which of Tanaka's roles will you remember him by? Let me know in the comments or hit me up on Twitter @MeganPetersCB to talk all things comics and anime!
JoJo's Bizarre Adventure was originally created by Hirohiko Araki for Shueisha's Weekly Shonen Jump in 1987. Currently the second longest running series in the magazine with over 100 volumes collected, the series tells the story of the Joestar family, who are each entwined in a destiny battling outrageous foes. The series has been split up into eight parts, with each part following a different generation of the Joestar family.
JoJo's Bizarre Adventure: Golden Wind is the fifth part of Araki's series and follows Giorno Giovanna, the son of former series villain Dio Brando, as he joins the mafia group Passione in order to change and reform them into reputable thieves and crooks from the inside. The series officially began October 5 in Japan, and can currently be found streaming on Crunchyroll.

domain: entertainment


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An NRA ad released on March 4 called out “every Hollywood phony” and the “lying media” to put them on notice that their “time is running out.”
The ad was released just hours before the 90th Oscars began. It promotes NRA spokeswoman Dana Loesch’s new show and is clearly intended to send the signal that the claims of leftists in Hollywood, the media, and pro-athletes will not go unchallenged.
Loesch opens the ad by saying, “We’ve had enough of the lies, the sanctimony, the arrogance, the hatred, the pettiness, the fake news. We are done with your agenda to undermine voters’ will and individual liberty in America.”
She continues, “So to every lying member of the media, to every Hollywood phony, to the role model athletes who use their free speech to alter and undermine what our flag represents, to the politicians who would rather watch America burn than lose one ounce of their personal power … your time is running out”:
“To every lying member of the media, to every Hollywood phony, to the role model athletes who use their free speech to alter and undermine what our flag represents…Your Time is running out. The clock starts now."
–@DLoesch #Oscars90 pic.twitter.com/SDVjeTxyfB
— NRATV (@NRATV) March 4, 2018
In the latter part of the ad, Loesch gets specific, referencing certain media and news personalities by name, putting them on notice as well.
She said, “To the Joy-Ann Reids, the Morning Joes, the Mikas, to those who stain honest reporting with partisanship, to those who bring bias and propaganda, to CNN, the Washington Post, the New York Times, your time is running out.”
AWR Hawkins is an award-winning Second Amendment columnist for Breitbart News, the host of the Breitbart podcast Bullets with AWR Hawkins, and the writer/curator of Down Range with AWR Hawkins, a weekly newsletter focused on all things Second Amendment, also for Breitbart News. He is the political analyst for Armed American Radio. Follow him on Twitter: @AWRHawkins. Reach him directly at firstname.lastname@example.org. Sign up to get Down Range at breitbart.com/downrange.


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Approx .40 acres on Main Street in Philadelphia, MS. Land is currently zoned R-1 residential sellers will work with buyers and city to get zoned to Commercial with acceptable contract. Main lot is divided into four city lots within the city. Corner lot with lots of city street frontage.
- Site Link
- Property SubType Other
45 +/- acres in the Sandtown Community of Neshoba County, Mississippi just East of Philadelphia. Multiple house sites with natural timber regrowth.
MLS ID 319742
Courtesy of: BHHS Gateway Real Estate
Office Phone: 6018530414
Listing Agent: Johnnie Rea
Updated: 19th February, 2020 5:16 AM.
348 MAIN ST Philadelphia, MS

domain: geography


text:
Research Article | Open Access
Roberta Lane de Oliveira Silva, Manassés Daniel Silva, José Ribamar Costa Ferreira Neto, Claudia Huerta de Nardi, Sabrina Moutinho Chabregas, William Lee Burnquist, Günter Kahl, Ana Maria Benko-Iseppon, Ederson Akio Kido, "Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane", The Scientific World Journal, vol. 2014, Article ID 357052, 12 pages, 2014. https://doi.org/10.1155/2014/357052
Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane
One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.
Sugarcane (Saccharum spp.) is a major crop with vegetative propagation and capacity to accumulate high levels of sucrose in the culms [1–3]. World production of this crop in 2011 resulted in about two billion tons of raw material, which corresponded to a gross income of more than US$ 52 billion . Despite the economic importance of sugarcane, the knowledge of relevant genetic mechanisms remains challenging, due to the fact that this crop presents one of the largest and most intricate genomes of the plant kingdom, with diploid numbers ranging from 100 to 130 chromosomes, indicating a high ploidy level, as well as regular aneuploidy events [5–7]. Because of this complexity, the use of molecular tools represents an attractive approach to the improvement of sugarcane breeding programs. Moreover, transcriptomic studies have been prioritized, allowing identification of candidate genes involved in developmental processes and plant responses to environmental cues, which have eventually led to the discovery of functional molecular markers . Reverse transcription quantitative real-time PCR (RT-qPCR) is based on a high specific polymerase chain reaction associated with sensitive fluorescence, allowing the detection of variations in gene expression, including discreetly transcribed genes [9, 10]. This technology has been used as a diagnostic tool for identification of plant pathogens, transgene expression , and human diseases [11, 12] and confirmation of transcriptional profiles generated by different methodologies, such as EST libraries , Microarray , HT-SuperSAGE , and RNAseq . The reliability of RT-qPCR data based on relative quantification is depending upon comparative transcription of target genes to stable reference genes [17, 18]. The use of reference genes that undergo changes in transcription across experimental groups can dramatically alter the conclusions on targeted gene expression . In order to test for normalization of reference gene expression, several statistical algorithms, such as GeNorm , NormFinder , and BestKeeper , have recently been developed. Despite the importance of proper reference gene selection for reliable and accurate RT-qPCR assays, most reports involving sugarcane have not described, or compared, methods in order to determine the efficiency of reference genes [21–23], suggesting arbitrary criteria for this selection. To our knowledge, only one systematic study aimed to assess, standardize, and validate reference genes (GAPDH, β-tubulin, β-actin, and 25S rRNA) for tissue and genotype-specific gene expression analysis in sugarcane . Additionally, this kind of study has not been carried out before under stress conditions which often alter the behavior of some genes. Thus, a rigorous selection of reference genes for expression profiling validation in sugarcane under biotic or abiotic stress was yet to be described. In the present work, screening and validation of new reliable reference genes for expression analysis in sugarcane roots were carried out. Besides, additional resources for target validation were evaluated, especially considering comprehensive transcription profiling, like those provided by HT-SuperSAGE , in sugarcane, revealing hundreds of candidate genes responsive against drought stress, requiring subsequent validation.
2. Materials and Methods
2.1. Plant Material and Treatments
Sugarcane drought assay: accessions were previously evaluated in a glasshouse trial conducted by the Center for Sugarcane Technology (CTC) in Piracicaba, Brazil (22° 41′ S; 47° 33′ W), aiming to identify drought-tolerant and drought-sensitive ones. Four accessions were selected as drought-tolerant (CTC6, CTC15, SP83-2847, and SP83-5073) and another four as drought-sensitive (CTC9, CTC13, SP90-3414, and SP90-1638). Some of them were previously reported as tolerant and sensitive to drought based on chlorophyll and water content measurements together with field observations . Briefly, for the drought stress assay, plants of each accession were grown under glasshouse conditions (°C (maximum), °C (average), and °C (minimum) and % (average) relative humidity under natural photoperiod) in 40 L pods in a randomized experimental design (comprising six repetitions) under daily irrigation (4 L·h−1) until reaching three months of age. Plants were submitted to drought conditions by continuous dehydration caused by the interruption of irrigation during 24 hours. Roots of both, stressed and unstressed plants, were collected, immediately frozen in liquid nitrogen, and stored at −80°C until total RNA extraction. On the other hand, the salinity stress assay was performed as follows: acclimated plants of the RB931011 clone in vitro cultivated (referred to as salt tolerant by the Brazilian RIDESA program of sugarcane breeding) were grown in a greenhouse (UFPE, Recife, PE, Brazil; 8° 04′S, 34° 55′W) in pots containing washed sand (washed) and they were watered daily with Hoagland solution, throughout three months. Later, NaCl (100 mM) was added to the nutritive solution as the salt stress. Roots from both stressed and nonstressed (negative control) plants were collected after stress induction (30 and 90 min) and immediately frozen in liquid nitrogen until a total RNA extraction was performed.
2.2. Total RNA Isolation, Purification, cDNA Synthesis, and HT-SuperSAGE Libraries
Total RNA was extracted with RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions, treated with DNAse (Qiagen), and purified with RNeasy Mini kit (Qiagen). RNA samples were quantified using Quant-iT RNA assay kit (Invitrogen) with the Qubit fluorometer (Invitrogen). RNA integrity was verified in 1.5% agarose gel electrophoresis with blue-green loading dye (LGC Biotechnology) staining. The purified RNA (1 μg) of each sugarcane accession was reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions and resuspended in a final volume of 20 μL. The cDNA synthesis reaction was incubated at 42°C for 2 minutes (genomic DNA digestion), 42°C for 15 minutes (reverse transcription), and 95°C for 3 minutes (enzyme inactivation) and stored at −20°C. The procedures for HT-SuperSAGE library generation followed Matsumura et al. , including the attachment of library-specific adaptors carried out by GenXPro GmbH (Frankfurt, Germany), allowing the identification of library-specific reads after SOLEXA sequencing. Concerning the drought stress, four libraries were generated as described by Kido et al. : the bulk of tolerant accessions under stress and the respective negative control and the bulk of sensitive accessions after stress and the respective negative control. The bulks were composed by equimolar amounts of poly-A+ mRNA from all accessions comprising the respective library. In relation to the salt stress, equimolar amounts of total RNA from each sample/time were assembled to compose the two bulks used to generate the SuperSAGE libraries (stressed and control).
2.3. Primer Design, Amplification Efficiency, and RT-qPCR Analysis
Sugarcane ESTs (Table 1) from dbEST database (http://www.ncbi.nlm.nih.gov/nucest), related to independent pathways as an attempt to minimize the effects of coregulation, were used for primers design with the Primer 3 software  with minor modifications: the amplicon length range was set to 70–200 bp, the melting temperatures were between 40°C (minimum), 50°C (optimum), and 60°C (maximum), and the CG content ranged from 45 to 55% (optimum of 50%). In relation to H1 and SAMDC, the primers sequences were obtained from the literature . All primer pairs were synthesized by Bioneer Corporation (South Korea) and some details of these primers are given in Table 1. An initial standard PCR was performed with the potential reference genes, using the sugarcane cDNA samples (bulk of tolerant and sensitive accessions), in order to investigate the PCR products. Amplicons were analyzed on 1.5% agarose gel electrophoresis followed by blue-green loading dye staining (LGC Biotechnology). Additionally, a dissociation curve analysis was made, in RT-qPCR assay, to confirm the specificity of the amplification by the candidate genes. Calibration curves using a dilution series of the cDNA pool (1, 10−1, 10−2, 10−3, and 10−4) were made to calculate the PCR amplification efficiencies  for each quantified candidate gene, their respective correlation coefficients , and interceptors. The RT-qPCR amplifications were performed on LineGene 9660 model FQD-96A (Bioer), using SYBR Green detection. Each reaction mixture comprised 1 μL of template cDNA (diluted 5-fold), 5 μL of HotStart-IT SYBR Green qPCR Master Mix 2x (USB), 0.05 μL of ROX (normalization dye), 1.95 μL of ultrapure water, and 1 μL of each primer (0.05 μM), forming a final volume of 10 μL. Three biological and three technical replicates were used in each run for RT-qPCR analysis, and a no template control (NTC) was also included. The reactions were subjected to an initial denaturation step of 95°C for 2 min, followed by 40 cycles at 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s in 96-well reaction plates. The dissociation curves were analyzed at 65–95°C for 20 min after 40 cycles. The baseline and quantification cycle (Cq) were determined using the LineGene 9600 software (version 1.1.10).
|Databases: dbEST (NCBI; http://www.ncbi.nlm.nih.gov/) and Gene Index (SoGI; http://compbio.dfci.harvard.edu/tgi/); Rodrigues et al. . TUB: alpha-tubulin; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; H1: histone H1; SAMDC: S-adenosylmethionine decarboxylase; UBQ: ubiquitin; 25S rRNA: 25S ribosomal RNA; PFP1: pyrophosphate fructose-6-phosphate 1-phosphotransferase alpha subunit (EC 184.108.40.206); AS: glutamine-dependent asparagine synthetase (EC 220.127.116.11); PIP1-1: plasma membrane intrinsic protein; ACC oxidase: 1-aminocyclopropane-1-carboxylate oxidase (EC 18.104.22.168).|
2.4. Data Analysis
The potential reference genes were ranked, and the number of candidate genes required for an optimal normalization was indicated according to their gene expression stability using sugarcane cDNA samples, after being analyzed by the GeNorm (version 3.5) , NormFinder (version 0.953) , and BestKeeper (version 1)  software. The GeNorm and NormFinder input data were based on relative quantities applying the ΔΔCq method . The GeNorm software determines the reference gene stability measurement as the average pairwise variation of each reference gene with all the other reference genes and enables the elimination of the least stable gene and the recalculation of the values, resulting in the ranking of the most stable genes. The average expression stability value (-value) was a parameter for quantification of stable reference gene candidates, in which a low M-value indicated a more stable expression . The NormFinder tool was applied to identify and rank the most suitable genes for RT-qPCR normalization from the set of candidates, considering intragroup and intergroup variations, in a model-based approach of mixed linear effect modeling . The BestKeeper software, using raw Cq values as input, was applied to identify the most stable expressed genes by a Pearson correlation coefficient (geometric mean of Cq values of candidate genes), calculating the standard deviation (SD) of Cq values among the entire data set. The relative gene expression levels (based on the relative quantities after the ΔΔCq method) were evaluated with the tool (Relative Expression Software Tool, version 2.0.13), which bases its performance on pairwise comparisons using randomization and bootstrapping techniques (Pairwise Fixed Reallocation Randomization ) . The normalization of the RT-qPCR was performed by taking the geometric averages of the combined reference genes, using the negative control to normalize this relative expression, and testing the hypothesis of significant differences between the control and treatment. With the input of multiple target and reference genes and based on the normalized values of the target genes, the software indicates the direction of the difference between the groups, as well as their value. Also, the MIQE guidelines (The Minimum Information for Publication of Quantitative Real-Time PCR Experiments)  were followed in order to increase transparency and reliability of the results obtained.
3.1. RNA Integrity, Specificity, and Efficiency Amplifications
All the reference candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) amplified the cDNAs generated from the RNAs samples (Supplemental Figure 1(a) in Supplementary Material available online at http://dx.doi.org/10.1155/2014/357052) using the proposed primers. Based on standard PCR amplifications, only a single product was observed with a specific primer pair (Supplemental Figure 1(b)) and these results were supported by the dissociation curve analysis (Supplemental Figure 1(c)). Based on the standard curves using a serial dilution of the cDNA pool (Supplemental Figure 2), the real-time PCR amplification efficiency , considering the selected six candidate genes, ranged from 98.34% to 100.89%, with correlation coefficients varying from 0.984 to 0.999, while the slopes ranged from −3.50 to −3.21 (Table 2). Considering the efficiency of 100%, the value of the expected slope should be −3.32, while slopes ranging from −3.10 to −3.58 would represent efficiency comprising 90% to 110%, thereby characterizing acceptable reactions. These parameters derived from the RT-qPCR analysis, and others in accordance with the MIQE Guidelines, are shown in Tables 2 and . The results showed favorable conditions for amplification, efficiency in successive dilutions, and acceptable variations in gene expression across samples, representing potential for choosing a suitable reference gene. Thus, in gene expression studies, fluctuations due to pipetting errors, variations in the quantification of samples, or the concentration of reagents could be normalized with the aid of these suitable reference genes .
|Based on MIQE Guidelines . Tm: melting temperature (°C); N.D.: not detected; NTC: no template control; TUB: alpha-tubulin; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; H1: histone H1; UBQ: ubiquitin; SAMDC: S-adenosylmethionine decarboxylase; 25S rRNA: 25S ribosomal RNA; PFP1: pyrophosphate fructose-6-phosphate 1-phosphotransferase alpha subunit (EC 22.214.171.124); AS: glutamine-dependent asparagine synthetase (EC 126.96.36.199); PIP1-1: plasma membrane intrinsic protein; ACC oxidase: 1-aminocyclopropane-1-carboxylate oxidase (EC 188.8.131.52).|
3.2. Gene Expression Stability of the Reference Gene Candidates
The six candidate genes selected for normalization (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) in RT-qPCR tests showed Cq values ranging from 13.06 to 28.00 (Supplemental Table ). Most of the candidate genes presented Cq values with slight variations (below one cycle), except UBQ and SAMDC. Based on these values, 25S rRNA was the most abundantly transcribed gene (average Cq = 14.00), while αTUB was the least abundant (average Cq = 27.48). These data, in order to assess the gene expression stability of the reference gene candidates, were used in GeNorm , NormFinder , and BestKeeper  analysis.
3.2.1. GeNorm Analysis
Considering the average expression stability values (M-value), αTUB ( = 0.61), GAPDH ( = 0.62), and histone H1 ( = 0.63) were the most stable genes while SAMDC represented the most variable ( = 0.87) gene. However, all of them showed an expressive high stability with M-values below 1 (Figure 1), suggesting that all the six candidates may be adequate for normalizing gene expression data under the conditions used in the present work. Besides, based on the pairwise variation data (Figure 2), it was possible to determine the optimal number of reference genes required for the relative quantification analysis and to investigate the effect of gene addition in this normalization. The data suggested that the addition to the two most stable genes (αTUB and GAPDH) considering a third gene (; Figure 2), a fourth (), or even more genes ( and ; Figure 2) still exhibited desired values (below 0.15 as proposed by Vandesompele et al. ). To normalize the gene expression in the above mentioned sugarcane samples, αTUB, GAPDH, and H1 seem to be sufficient (Figure 1).
3.2.2. NormFinder and BestKeeper Analysis
Basically, the gene expression stability ranking provided by the NormFinder and BestKeeper software exhibited the same order, with only the first two candidates switching places comparing the ranking (Table 3). The two software programs identified histone H1 ( = 0.28; CV ± SD = ) and αTUB ( = 0.32; CV ± SD = ) as the most stable genes, followed by GAPDH, 25S rRNA, UBQ, and SAMDC (see respective values in Table 3). Again, SAMDC showed the highest instability, in agreement with the GeNorm results. All the three software programs presented fairly consistent results showing the first three (H1, αTUB, and GAPDH) as the most stable and reliable genes for RT-qPCR data normalization. Two of them (H1 and αTUB) are reported as RT-qPCR normalizing genes suitable for sugarcane roots under abiotic stress for the first time.
|: average expression stability value; CV: coefficient of variance; SD: standard deviation; TUB: alpha-tubulin; H1: histone H1; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; 25S rRNA: 25S ribosomal RNA; UBQ: ubiquitin; SAMDC: S-adenosylmethionine decarboxylase.|
3.3. Normalization of Target Genes
In order to assess the applicability of the recommended histone H1, αTUB, and GAPDH as reference genes in relative expression studies using RT-qPCR, four targets based on sugarcane SuperSAGE unitags annotated as glutamine-dependent asparagine synthetase (AS, EC 184.108.40.206), pyrophosphate fructose-6-phosphate 1-phosphotransferase alpha subunit (PFPα1, EC 220.127.116.11), plasma membrane intrinsic protein (PIP1-1), and 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase, EC 18.104.22.168) were evaluated (Table 4). HT-SuperSAGE survey pointed SD282748 unitag as a potential AS being upregulated 1.92 times in the drought-tolerant bulk after the stress (24 h of continuous dehydration) as compared to the unstressed control while no relevant unitag frequency change was observed () with the sensitive contrast (Table 4). The RT-qPCR relative quantification results confirmed the overexpressed status in relation to both cDNA bulks with 1.473-fold change for the tolerant bulk compared to its negative control (Figure 3(a)) and no significant change (1.038 times) considering the sensitive bulk in the comparative contrast (Figure 3(b)). In turn, SD179780 unitag (annotated as PFPα1) did not respond () to water deficit stimulus in any contrast analyzed involving sugarcane drought-tolerant or -sensitive accessions, in agreement with the RT-qPCR results, showing constitutive expression of this gene (Figures 3(a) and 3(b), resp.). In an attempt to explore the use of the proposed reference genes, a target relative to the ASS122537 unitag (ACC oxidase) from the salt HT-SuperSAGE libraries, which was induced (UR) by the salt-tolerant accession (1.95 times after the salt stress exposition, NaCl 100 mM), showed overexpression after 30 min according to RT-qPCR results (Figure 3(c) and Table 4). Another target relative to the ASS140030 unitag (PIP1-1) from sugarcane after salt stress presented RT-qPCR results confirming the constitutive expression observed in silico (), even after the bulk has been opened in two times of salt exposition (30 and 90 min, Figures 3(c) and 3(d) and Table 4). The same PIP1-1 had been validated by RT-qPCR with cDNAs from the drought-tolerant and -sensitive sugarcane accessions (24 h of continuous dehydration) using GAPDH and 25S rRNA as the reference genes, in a previous work, showing differential expressions as expected by the SuperSAGE analysis .
|AS: glutamine-dependent asparagine synthetase (EC 22.214.171.124); PFP1: pyrophosphate fructose-6-phosphate 1-phosphotransferase alpha subunit (EC 126.96.36.199); ACC oxidase: 1-aminocyclopropane-1-carboxylate oxidase (EC 188.8.131.52); PIP1-1: plasma membrane intrinsic protein. #Bulk with four accessions by each library; FC: fold change [ratio of the frequencies (normalized to 1,000,000) observed in the stressed library in relation to the control library]; &relative expression level by REST software (v.2.0.13) after the ΔΔCq method, * ; UR: upregulated; ns: not significant at . The time in the parentesis represents the salt stress exposition (min).|
The understanding of sugarcane physiology under environmental stress remains under intensive research, due to the socioeconomic importance of this crop and the increasing unpredictability of environmental conditions worldwide. In this regard, gene expression analysis is an attractive approach to dissect plant physiological response to stress conditions. Nevertheless, reference gene selection has received limited attention in sugarcane. RT-qPCR is currently one of the most used techniques for gene expression analysis, due to its rapid, specific, and highly sensitive parameters. However, problems with RNA samples variations, standardization, and protocols efficiency (RNA extraction, RT, and qPCR) have routinely been observed . Furthermore, the choice of normalizing genes remains one of the most time consuming and difficult steps in RT-qPCR. It requires reference genes to be constitutively expressed under external stimuli. Additionally, it needs to exhibit little or no behavior change in different cell types or tissues, as well as in specific developmental stages and experimental conditions [36–38]. Stal Papini-Terzi et al.  described transcriptional profile of signal transduction events in different sugarcane tissues, using reference genes selected based upon the literature (tubulin and actin), microarray data, and ESTs (polyubiquitin and 14-3-3 proteins). To individually normalize gene expression in sugarcane under certain conditions, Rocha et al.  relied upon four reference genes (14-3-3, polyubiquitin, GAPDH, and 25S rRNA). Moreover, Rodrigues et al.  used β-tubulin as the reference gene based on previous data . To our knowledge, Iskandar et al.  represented an attempt to prospect stable sugarcane reference genes by checking the reliability of four genes (β-actin, β-tubulin, GAPDH, and 25S rRNA) in leaf, root, and internode tissues of some sugarcane cultivars and representatives of Saccharum genus, but none of them under abiotic stress. According to the authors, GAPDH was the most stable gene (CV = 51%) comparing different tissues, followed by β-actin and β-tubulin (CV = 81% and 68%, resp.); regarding species, β-actin showed the lowest coefficient of variation (31%) followed by GAPDH (33%). Although these methods are useful for prospecting candidate reference genes  or addressing gene expression using validated reference genes for target tissues [22, 23], selection using more appropriate statistical approaches should be the method of choice for identification of new reliable reference genes. In this way, software programs like GeNorm, NormFinder, and BestKeeper have assisted researchers by indicating reference genes suitable for expression profiling normalization studies . In the present study, a group of potential reference genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were evaluated by the three software programs, in order to evaluate their reliability for expression profiles normalization in sugarcane roots under abiotic stress (24 h of continuous dehydration). Basically, all three software programs pointed histone H1, αTUB, and GAPDH as the most reliable reference genes, with some of them switching places in the ranking. This set of genes was employed here as reference genes to validate sugarcane cDNAs relative to unitags from SuperSAGE libraries composed of roots of plants after stress exposition. Thus, the gene expression stability ranking provided by NormFinder and BestKeeper software showed the same order after the third place. Besides, based on the GeNorm analysis and M-values, all the six candidates may be suitable for normalizing gene expression data as presented here. But, combining the two best candidates (αTUB and H1) with the most variable one (SAMDC, = 0.87), as reference genes normalizing the target AS (induced in SuperSAGE analysis), the REST software did not detect the expected overexpression (comparison 1, Table 5) due to the SAMDC largest standard deviation (SD) influencing the value calculated, consequently leading to a false negative and possible misinterpretation of data. Alias, any other combination including SAMDC as reference gene did not reveal the alleged overexpression (comparisons 2–5, Table 5), indicating that this gene is not suitable for gene expression normalization in roots of sugarcane accessions under the evaluated stress. However, Hong et al.  reported SAMDC as the most reliable reference gene in grass Brachypodium distachyon when evaluated under four abiotic stress conditions (high salt (300 mM), cold (4°C for 5 h), heat (42°C for 2 h), and drought (400 mM mannitol)). In addition, Li and Chen , when describing SAMDC as a target gene, verified that this gene was induced in roots of rice seedlings at three leaf stages (after application of 171 mM salt for 24 h and 20 mM exogenous abscisic acid (ABA) and dehydration using 15% PEG6000). These results highlight the need to choose appropriate reference genes for each experiment, especially under stress conditions.
|REST software analysis after the ΔΔCq method. UR: upregulated; ns: not significant at ; TUB: alpha-tubulin; H1: histone H1; SAMDC: S-adenosylmethionine decarboxylase; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; 25S rRNA: 25S ribosomal RNA; UBQ: ubiquitin.|
On the other hand, the other five genes (αTUB, GAPDH, H1, UBQ, and 25S rRNA) could be successfully employed in the normalization analyses, composing different combinations of reference genes (comparisons 6–11, Table 5), with similar results to that observed for the proposed set (H1, αTUB, and GAPDH). In relation to the 25S rRNA gene, it was the most abundant transcript (Cq 13.06), in agreement with results previously obtained from rice (Cq values of 15 ) and sugarcane (Cq values of 16.6 ). This can be explained by the fact that rRNA comprises the majority of total RNA present in a cell and, thus, further dilution for its use in RT-qPCR approaches  would be required. In the present work, only a 1 : 5 dilution was applied. Furthermore, the abundance of transcripts can affect the stability and, therefore, the normalizing results for the reference gene candidates . In turn, GAPDH was also one of the most stable genes, confirming it to be an appropriate reference gene for experiments involving sugarcane roots under water deficit conditions. Concerning αTUB, it was indicated by GeNorm as the most stable gene using the bulks of accessions, both under regular irrigation and after 24 h of continuous dehydration. By the NormFinder and BestKeeper analysis, this gene was the second most appropriate reference gene. However, Fan et al.  assessing the reliability of reference genes in 14 different tissues and developmental stages of Phyllostachys edulis observed that αTUB showed a larger variation ( = 1.94) among all candidates. Similar results were reported by Zhong et al.  with litchi (Litchi chinensis Sonn.) under several experimental conditions (tissues, organs, developmental stages, and varieties), showing αTUB as the most variable gene among 10 candidates. Under biotic and abiotic stresses αTUB also displayed instability, as demonstrated by Die et al. , Hong et al. , and Zhu et al. . Thus, the selection of suitable reference genes to normalize gene expression in sugarcane and other plant species seems to be essential because reference genes may be differently regulated in different species, displaying particular gene expression patterns . The proposed reference genes (H1, αTUB, and GAPDH) validated the gene expression of sugarcane cDNAs related to SuperSAGE unitags, showing upregulation or even constitutive basis, in the mentioned drought assay. Among those targets, AS is a crucial component of the asparagine synthesis, acting as a key member in nitrogen assimilation, recycling, and storage in higher plants . The overexpression results observed in the present study supported those found in a previous microarray  showing AS induction in wheat accession considered tolerant to drought after 36 h of irrigation suppression. AS were also detected by RT-PCR, using mRNA samples from roots and shoots (two-week-old plantlets), significantly induced after salinity (250 mM), osmotic stress (using mannitol 5.0% (w/v)), and exogenous abscisic acid (ABA) application (20 mM) . Altogether, these results indicate the involvement of the AS gene in response to several stresses. Considering PFPα1, the constitutive expression observed in the present assay has been reported previously and was in agreement with Lim et al.  who demonstrated by semiquantitative RT-PCR that the expression of PFPα1 in wild-type Arabidopsis was also constitutive in different tissues (roots, leaves, and flowers) and also in distinct developmental stages (15, 25, and 45 days after planting). PFPα1 is responsible for the addition of phosphate to the second D-fructose 6-phosphate in the glycolysis pathway  and is essential for maintenance of carbohydrate metabolism and other processes in plant cells . In sugarcane, PFPs are known to play a prominent role in sucrose accumulation, especially in immature and metabolically active tissues, taking part in glycolysis and in carbon compartmentalization .
The effort to explore the use of the proposed reference genes (H1, αTUB, and GAPDH) normalizing the expressions of sugarcane cDNAs (associated with SuperSAGE unitags) also from roots of plants under salt stress (100 mM) was effective in a preliminary study. This way, the induction of ASS122537 unitag (annotated as ACC oxidase, enzyme responsible to convert the ethylene precursor ACC to ethylene, ), as revealed by in silico SuperSAGE analysis, was confirmed by RT-qPCR after 30 minutes of salt exposition (tolerant accession), suggesting that rapid ethylene production is an adaptive response to the new conditions imposed by the salt stress to the evaluated genotype. Unfortunately this overexpression was not detected during the 90 min of stress exposition. Nevertheless, there is evidence that a variety of stressful conditions trigger the synthesis of ethylene . Regarding salt stress, it has been observed that this hormone signaling may be required for triggering the tolerance process. Yang et al. , when evaluating Arabidopsis mutants (ein2-5, ein3-1, and ctr1-1) and wild plants ecotype Col-0, found that mutants insensitive to ethylene (ein2-5 or ein3-1) were more sensitive to saline stress when compared to their wild counterpart. The opposite was found in the mutant sensitive to ethylene (ctr1-1), which showed significant tolerance to salt stress. Concerning the PIP1-1, the RT-qPCR results showed no significant differences in any of the two sampled times, confirming the SuperSAGE results with the bulk comprising both sampling times. The same target in RT-qPCR assay confirmed differential expressions expected by the SuperSAGE analysis, with root cDNAs from the drought-tolerant and -sensitive sugarcane accessions (24 h of continuous dehydration) and GAPDH and 25S rRNA as the reference genes . An explanation could be that the time intervals used for stress exposition were not enough for PIP1-1 expression. In rice, Guo et al.  reported PIP1-1 expression in response to salt stress (250 mM NaCl) after 2 h of stress exposition.
The potential of the six proposed reference genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) was confirmed after they were tested with cDNAs from sugarcane roots under drought stress (24 h of continuous dehydration) and analyzed by three different software programs (GeNorm, NormFinder, and BestKeeper). With the exception of SAMDC, all the other candidate genes seem to be suitable for sugarcane expression profiling normalization, but three of them (αTUB, H1, and GAPDH) were considered as the best reference genes. In this study, two new reference genes were reported for the first time for sugarcane (αTUB and H1), to undergo a RT-qPCR validation study involving expression in roots under abiotic stresses. Also, the present work pointed GAPDH and 25S rRNA genes, both indicated by Iskandar et al. , as reference genes in a previous study, also suitable for use with sugarcane root under abiotic stress. Using the proposed set of reference genes (αTUB, H1, and GAPDH), it was confirmed that the relative expression profile, with the aid of the REST software, of cDNAs was associated with unitags (26 bp) and annotated as AS and PFPα1, using a bulk of cDNAs relative to the drought-tolerant sugarcane accessions (four accessions, 24 h of continuous dehydration), in agreement with the HT-SuperSAGE data. Another two unitags (associated with ACC oxidase and PIP1-1) had their expression profiles validated by RT-qPCR, using cDNAs from sugarcane roots after salt stress exposition (100 mM NaCl), in an attempt to explore other possibilities using these reference genes. In conclusion, this set of reference genes will be useful for dissecting gene expression in sugarcane roots, especially in advanced transcriptomic studies using SuperSAGE or RNAseq approaches covering abiotic stresses.
Conflict of Interests
The authors have declared that they have no conflict of interests.
The authors acknowledge the Brazilian institutions FINEP (Financiadora de Estudos e Projetos), FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco), and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support and fellowships. They also thank Dr. Björn Rotter, Ruth Jungmann, Dr. Peter Winter (GenXPro GmbH, Germany), and Eng. Ernestino Ciambarella (Labtrade do Brasil) for scientific and technical advice.
Supplementary Material showing the quality of the total RNAs from sugarcane roots under drought stress (FIGURE S1 - A), the amplicons of expected size for each gene used in the study, displaying primer specificity as required for RT-qPCR amplification (FIGURE S1 - B), and the melting curves showing a single peak for six potential reference genes (αTUB; GAPDH; H1; SAMDC; UBQ and 25S rRNA) and four targets (PFPα1; AS; PIP1-1 and ACC oxidase).
Supplementary Material (FIGURE S2) showing standard curves using a dilution series (100, 10-1, 10-2, 10-3 e 10-4) of six potential reference genes (αTUB; GAPDH; H1; SAMDC; UBQ and 25S rRNA) for sugarcane roots under drought stress and the respective curves for four targets (PFPα1; AS; PIP1-1 and ACC oxidase).
Supplementary Material (TABLE S2) based on MIQE checklist for authors, reviewers, and editors.
Supplementary Material (TABLE S1) showing Cq values of potential reference genes for gene expression normalization (RT-qPCR) with cDNAs from sugarcane roots under drought stress (24 h of continuous dehydration).
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Which Best Explains the High Surface Tension of Water.
Chapter 2: Introduction to the Chemistry of Life
By the end of this section, yous will be able to:
- Draw the backdrop of water that are critical to maintaining life
Scout a video about why we demand oxygen and how it causes problems for living things.
Practice y’all ever wonder why scientists spend time looking for h2o on other planets? Information technology is considering water is essential to life; even minute traces of it on another planet tin bespeak that life could or did exist on that planet. H2o is one of the more than abundant molecules in living cells and the ane most disquisitional to life as we know it. Approximately 60–seventy percent of your torso is made upwardly of water. Without it, life simply would not exist.
Water Is Polar
The hydrogen and oxygen atoms inside h2o molecules grade polar covalent bonds. The shared electrons spend more than time associated with the oxygen cantlet than they do with hydrogen atoms. In that location is no overall charge to a water molecule, but in that location is a slight positive charge on each hydrogen cantlet and a slight negative charge on the oxygen atom. Because of these charges, the slightly positive hydrogen atoms repel each other and form the unique shape. Each water molecule attracts other h2o molecules because of the positive and negative charges in the dissimilar parts of the molecule. H2o also attracts other polar molecules (such as sugars), forming hydrogen bonds. When a substance readily forms hydrogen bonds with water, it can dissolve in water and is referred to as
(“water-loving”). Hydrogen bonds are not readily formed with nonpolar substances like oils and fats . These nonpolar compounds are
(“water-fearing”) and volition non deliquesce in h2o.
Water Stabilizes Temperature
The hydrogen bonds in water allow it to absorb and release heat energy more slowly than many other substances.
is a measure of the movement (kinetic energy) of molecules. As the move increases, free energy is higher and thus temperature is higher. H2o absorbs a nifty deal of energy before its temperature rises. Increased energy disrupts the hydrogen bonds between water molecules. Because these bonds can be created and disrupted rapidly, water absorbs an increase in energy and temperature changes only minimally. This means that h2o moderates temperature changes inside organisms and in their environments. Equally free energy input continues, the balance between hydrogen-bond formation and destruction swings toward the destruction side. More bonds are broken than are formed. This process results in the release of individual h2o molecules at the surface of the liquid (such as a body of water, the leaves of a plant, or the skin of an organism) in a process called
evaporation. Evaporation of sweat, which is xc percent h2o, allows for cooling of an organism, because breaking hydrogen bonds requires an input of energy and takes heat away from the body.
Conversely, equally molecular move decreases and temperatures drop, less energy is present to break the hydrogen bonds betwixt water molecules. These bonds remain intact and begin to form a rigid, lattice-like structure (e.thou., ice) (Effigy 2.8
a). When frozen, ice is less dumbo than liquid water (the molecules are farther autonomously). This ways that ice floats on the surface of a body of water (Effigy two.8
b). In lakes, ponds, and oceans, ice volition grade on the surface of the water, creating an insulating barrier to protect the animal and institute life below from freezing in the water. If this did not happen, plants and animals living in water would freeze in a cake of ice and could not move freely, making life in cold temperatures difficult or impossible.
Water Is an Excellent Solvent
Considering water is polar, with slight positive and negative charges, ionic compounds and polar molecules can readily dissolve in it. H2o is, therefore, what is referred to as a
solvent—a substance capable of dissolving another substance. The charged particles will grade hydrogen bonds with a surrounding layer of water molecules. This is referred to as a sphere of hydration and serves to keep the particles separated or dispersed in the water. In the instance of table salt (NaCl) mixed in water, the sodium and chloride ions split, or dissociate, in the water, and spheres of hydration are formed effectually the ions. A positively charged sodium ion is surrounded past the partially negative charges of oxygen atoms in h2o molecules. A negatively charged chloride ion is surrounded by the partially positive charges of hydrogen atoms in water molecules. These spheres of hydration are as well referred to as hydration shells. The polarity of the water molecule makes it an effective solvent and is important in its many roles in living systems.
Water Is Cohesive
Accept you ever filled upwards a glass of water to the very summit and so slowly added a few more drops? Earlier it overflows, the h2o actually forms a dome-similar shape above the rim of the glass. This water tin can stay to a higher place the drinking glass because of the property of
cohesion. In cohesion, water molecules are attracted to each other (considering of hydrogen bonding), keeping the molecules together at the liquid-air (gas) interface, although at that place is no more room in the glass. Cohesion gives rise to
surface tension, the chapters of a substance to withstand rupture when placed under tension or stress. When yous drop a small scrap of paper onto a droplet of water, the paper floats on summit of the water droplet, although the object is denser (heavier) than the water. This occurs because of the surface tension that is created by the water molecules. Cohesion and surface tension proceed the water molecules intact and the item floating on the acme. It is even possible to “float” a steel needle on summit of a glass of water if you lot identify information technology gently, without breaking the surface tension.
These cohesive forces are besides related to the water’southward property of
adhesion, or the attraction betwixt water molecules and other molecules. This is observed when water “climbs” upwards a harbinger placed in a glass of water. You will notice that the water appears to be college on the sides of the straw than in the middle. This is because the water molecules are attracted to the harbinger and therefore adhere to it.
Cohesive and adhesive forces are important for sustaining life. For instance, considering of these forces, water tin can flow up from the roots to the tops of plants to feed the plant.
Concept in Action
To larn more about h2o, visit the U.S. Geological Survey Water Science for Schools: All About Water! website.
Buffers, pH, Acids, and Bases
The pH of a solution is a measure of its acidity or alkalinity. You accept probably used
litmus paper, paper that has been treated with a natural water-soluble dye so information technology can be used as a pH indicator, to test how much acid or base (alkalinity) exists in a solution. You might accept even used some to make sure the water in an outdoor swimming pool is properly treated. In both cases, this pH test measures the amount of hydrogen ions that exists in a given solution. Loftier concentrations of hydrogen ions yield a low pH, whereas low levels of hydrogen ions result in a high pH. The overall concentration of hydrogen ions is inversely related to its pH and can be measured on the
(Figure 2.xi). Therefore, the more than hydrogen ions nowadays, the lower the pH; conversely, the fewer hydrogen ions, the higher the pH.
The pH calibration ranges from 0 to 14. A change of one unit on the pH scale represents a alter in the concentration of hydrogen ions by a factor of 10, a change in ii units represents a modify in the concentration of hydrogen ions by a factor of 100. Thus, small changes in pH stand for large changes in the concentrations of hydrogen ions. Pure water is neutral. It is neither acidic nor basic, and has a pH of 7.0. Anything beneath 7.0 (ranging from 0.0 to vi.ix) is acidic, and annihilation in a higher place vii.0 (from 7.1 to 14.0) is alkaline. The blood in your veins is slightly element of group i (pH = seven.4). The environment in your stomach is highly acidic (pH = 1 to 2). Orange juice is mildly acidic (pH = approximately 3.5), whereas baking soda is basic (pH = 9.0).
are substances that provide hydrogen ions (H+) and lower pH, whereas
provide hydroxide ions (OH–) and raise pH. The stronger the acrid, the more than readily it donates H+. For example, hydrochloric acid and lemon juice are very acidic and readily give up H+
when added to water. Conversely, bases are those substances that readily donate OH–. The OH–
ions combine with H+
to produce water, which raises a substance’s pH. Sodium hydroxide and many household cleaners are very element of group i and give up OH–
rapidly when placed in water, thereby raising the pH.
Near cells in our bodies operate within a very narrow window of the pH calibration, typically ranging simply from 7.2 to 7.6. If the pH of the body is outside of this range, the respiratory arrangement malfunctions, equally do other organs in the body. Cells no longer function properly, and proteins will interruption downwards. Deviation outside of the pH range tin induce coma or even cause expiry.
Then how is information technology that we tin ingest or inhale acidic or bones substances and not die? Buffers are the cardinal.
readily absorb backlog H+
or OH–, keeping the pH of the body carefully maintained in the same narrow range. Carbon dioxide is part of a prominent buffer system in the homo body; it keeps the pH inside the proper range. This buffer organisation involves carbonic acrid (HiiCO3) and bicarbonate (HCO3
–) anion. If also much H+
enters the body, bicarbonate volition combine with the H+
to create carbonic acid and limit the decrease in pH. Too, if too much OH–
is introduced into the arrangement, carbonic acid will apace dissociate into bicarbonate and H+
ions. The H+
ions can combine with the OH–
ions, limiting the increase in pH. While carbonic acid is an important production in this reaction, its presence is fleeting because the carbonic acid is released from the trunk as carbon dioxide gas each time we breathe. Without this buffer system, the pH in our bodies would fluctuate besides much and we would neglect to survive.
Water has many properties that are critical to maintaining life. It is polar, allowing for the formation of hydrogen bonds, which allow ions and other polar molecules to deliquesce in water. Therefore, water is an fantabulous solvent. The hydrogen bonds betwixt water molecules requite water the power to hold heat ameliorate than many other substances. As the temperature rises, the hydrogen bonds between water continually break and reform, allowing for the overall temperature to remain stable, although increased energy is added to the system. Water’s cohesive forces let for the property of surface tension. All of these unique properties of water are important in the chemistry of living organisms.
The pH of a solution is a measure of the concentration of hydrogen ions in the solution. A solution with a high number of hydrogen ions is acidic and has a depression pH value. A solution with a high number of hydroxide ions is basic and has a loftier pH value. The pH scale ranges from 0 to 14, with a pH of 7 beingness neutral. Buffers are solutions that moderate pH changes when an acid or base is added to the buffer system. Buffers are important in biological systems because of their ability to maintain constant pH weather condition.
a substance that donates hydrogen ions and therefore lowers pH
the attraction betwixt water molecules and molecules of a different substance
a substance that absorbs hydrogen ions and therefore raises pH
a solution that resists a change in pH by arresting or releasing hydrogen or hydroxide ions
the intermolecular forces between water molecules caused by the polar nature of water; creates surface tension
the release of water molecules from liquid water to form h2o vapor
describes a substance that dissolves in water; water-loving
describes a substance that does not dissolve in water; h2o-fearing
filter paper that has been treated with a natural water-soluble dye and so it can exist used as a pH indicator
a scale ranging from 0 to 14 that measures the approximate concentration of hydrogen ions of a substance
a substance capable of dissolving another substance
the cohesive forcefulness at the surface of a body of liquid that prevents the molecules from separating
a mensurate of molecular move
Humphrey, W., Dalke, A. and Schulten, Yard., “VMD—Visual Molecular Dynamics”,
J. Molec. Graphics, 1996, vol. 14, pp. 33-38. http://www.ks.uiuc.edu/Research/vmd/
- Figure 2.7 by Gautam Dogra
- Figure 2.viii
- water ice lattice past Jane Whitney
- (b) by Carlos Ponte
- Figure two.10 by Cory Zanker
- Figure ii.11 past Edward Stevens
Which Best Explains the High Surface Tension of Water


text:
Hours after raping a gay man, the attacker was killed in a shootout with police "You can go home in peace. It's all over," a detective told the victim.
The right is looking for a victory in liberal Massachusetts so that they can export their campaign to other states.
The town has some other creative ways they'll be using to let him know he isn't welcome.
After looking at this picture, maybe the "Concerned Women for America" should be more friendly to gender nonconforming folks.
Here is a list of our top three female campaign managers making all their own rules in the 2018 midterms.
"We don’t know where we’re going to be in a few months time. I’m afraid."
"You can go home in peace. It's all over," a detective told the victim.
Protesters who used bullhorns at a pride festival for hate speech just won a court victory.
And, he says, the "Crown of England and Parliament" are in on the scheme to use LGBTQ rights as the weapon to make it happen.
"Every opinion is valid, but some opinions can be really harsh," she said about the discrimination she's faced.
He's filed multiple nutty lawsuits nationwide - like suing for the right to marry his laptop.
Moore may be disgraced, but his argument that the Supreme Court nominee is the victim of a liberal plot is shared by many fellow conservatives.
And it looks incredible.
"Adherence to a series of internal policies that do not contemplate the existence of intersex people is not good reason" to deny someone a passport.
There has been an explosion of children detained by the government, and the money to house them has to come from somewhere.
The book will tell the story of the riots from the point of view of the Stonewall Inn itself.
The 2018 election may be one of the most important midterms for transgender people.
He overheard a conversation between two young gay men that brought back a lot of painful memories.
He hates Pride because people "frolic unashamedly" on "rainbow floats."
If Democrats wanted support from organized labor, Wohlforth wanted support for LGBTQ rights. And she was the woman in the position to make it happen.
Mark Judge is a conservative writer who thinks gays are "dangerously promiscuous" and women should be beaten "like a gong."
A fan of the opposing team was trying to get under his skin and turned to slurs.
But their "proof" is so laughable, it's not helping them one bit.
A far-right website thought it was insulting the 11-year-old gay boy, but really....
Australia's Therapeutic Goods Administration has recommended banning "poppers," angering the country's gay community.
She was one of four women killed by the deranged Border Patrol agent.
Sesame Workshop continues to say Muppets don't have a sexual orientation, but they obviously do.
He said we are living in "a sequel to the story of Sodom and Gomorrah."
President Miguel Díaz-Canel announced his support for "marriage between people without any restrictions" as the country prepares for a new constitution.
The top leaders of hate groups will mingle with Republican politicians at the Value Voters Summit this weekend.


A “supervolcano” in Italy is closer to erupting than previously thought, scientists have warned.
Campi Flegrei, situated just nine miles west of Naples – the other side of the city from Vesuvius – “may be approaching a critical stage,” according to scientists from University College London and the Vesuvius Observatory.
Christopher Kilburn, Director of the UCL Hazard Centre, said in a paper written for journal Nature Communications: “Further unrest will increase the possibility of an eruption… It’s imperative that the authorities are prepared for this.”
Campi Flegrei - which means "Flaming Fields" in a blend of Italian and ancient Greek - is not as well known internationally as Vesuvius, but could be just as devastating for the city of Naples and Pozzuoli, where it’s located, if it erupts.
The paper makes clear that an eruption is not imminent, but a critical phase is on the horizon. Since 2012, the alert level for Campi Flegrei has been at amber – previously it was green.
The experts, who’ve been studying the supervolcano’s activity over the past 500 years, say the eight-mile-wide caldera has been “restless” for nearly seven decades, with small earthquakes throughout the 1950s, 1970s and 1980s. Its periods of inflation and deflation, placing the crust under stress while the magma is trapped below, were previously thought to diffuse energy – but according to the team’s new analysis, this is not the case.
The port of Pozzuoli, says the data, has risen over 4m since 1950, and there have been 20,000 shocks in the intervening years. Before 1950, ground levels had been gently sinking. The movement is caused by magma below ground.
The last time Campi Flegrei was so active was in 1538, when, after 100 years of activity, the volcano erupted.
There is no word on when an eruption is due, but Kilburn notes that it’s part of a “trend” that has been noted in other supervolcanoes such as El Hierro in the Canary Islands and Soufriere Hills on Montserrat, which killed 19 people when it erupted in 1997.
Nearly a million people live in Naples, and there are 360,000 living across Campi Flegrei’s caldera.
An eruption would also disrupt air traffic, as seen in 2010, when Iceland’s Eyjafjallajökull volcano erupted, stopping all transatlantic flights due to ash clouds.
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text:
Before there was Clover...
Before there was Clover...
before there was Huey Lewis and the News, the Doobie Brothers, Southern Pacific, Elvis Costello singing “Alison,” or Tommy Tutone’s “867-5309/Jenny”...
there were three teenagers who met around the wild blooming of the Hippie era on Haight Street and Muir Beach, the Avalon Ballroom and the Fillmore Auditorium.
Alex Call and Mitch Howie were childhood friends who grew up in the Homestead Valley area of Mill Valley, California, across the Golden Gate Bridge north of San Francisco. Homestead Valley was a special place that seems almost like a California Zen Mayberry in retrospect, where white-collar, blue-collar, and nascent hipness were comfortably at home with each other. In 1966 Alex was just out of high school. He and Mitch, who was in his junior year, had been playing around on guitar and drums for the last few years, and they had a few other players coming and going, but as lifelong pals they were the core.
John McFee was a fifteen year old escapee of a difficult background in Orange County. His father was a too tough Bakersfield oilfield guy; John had been raised on Merle Travis, Joe Maphis, and Buck Owens. John’s brother Bob had introduced Alex and Mitch to John, and a bond had been forged among the three. They called their band “The Tiny Hearing Aid Company”, from a newspaper ad that said, “fits entirely in the ear.”
Their home gig was the old Tavern at Muir Beach, a prohibition era speakeasy that featured a dance hall room with a deck that led to the wind-swept beach. It became a hippie haven, away from the civilizing and restricting influences of weed-busting cops and straight people in general. The gigs on the deck attracted lithe hippie chicks from Tam High in Mill Valley and elsewhere: a good place for young musicians to be free, love-wise and otherwise.
Playing various shows around the Bay Area, The Tiny Hearing Aid Company began getting some attention, including that of one Johnny Ciambotti, a bass player from a San Francisco band called the Outfit. Ciambotti was a handsome, talented, debonair guy with both college sophistication and Los Angeles street smarts. He was older by a few years and had played in bluegrass bands in LA before ending up in San Francisco along with so many others of that day. He saw the potential of THAC and left the Outfit - and he picked the new name: Clover.
Why Clover? Because the music that the four gravitated towards had a strong country, bluegrass, even western swing influence, yet was still R&B based rock and roll. They wore cowboy shirts, overalls, and shit-kicker boots along with Salvation Army suits. Clover was a “country” band in that forays into the City, while common, were more like raids, whereas their favored habitat was the wide-open green hills of west Marin County.
Eventually, Clover signed a recording contract with Fantasy Records - to some extent through the influence of the Fantasy recording group Creedence Clearwater Revival, by way of recommendation of CCR members - and made two albums for the label. Subjectively, the Clover members were unfortunately not very satisfied with their recordings, primarily because they considered the finished product to fall short of the band’s potential.
Teamed up by Fantasy with a talented classical musician, Ed Bogas, as producer, the process of “record making” presented some real challenges. The band felt that in spite of Ed’s brilliance, and despite feeling that they as a group had some good material and some good musical ideas, their collective lack of studio savvy and recording experience led to the final results falling short of their hopes and expectations (Mitch, Alex, and John were still teenagers and Ed, his many talents notwithstanding, had never produced a record before).
Their disappointment was sadly validated by the spectacular lack of success for the two albums, and when the contract with Fantasy had run its course, the band moved on, and in fact not long after the end of the “Fantasy years,” Clover added two new members to the lineup.
Hugh Cregg, now better known as Huey Lewis, joined the group on harmonica and lead vocals. Alex had been the band’s main lead singer - Ciambotti would occasionally sing one of his compositions, but Alex really was pretty much “the” lead singer - and the addition of Huey added another element of focus for the band’s vocal sound. And he is truly a fantastic harmonica player…
Also enhancing and expanding the band’s musical capabilities was the addition of Sean Hopper on keyboards. In addition to his great keyboard skills, Sean also added another voice - he is an excellent singer - to contribute to the band’s vocal harmonies.
Although they joined after the Fantasy contract had ended, Huey and Sean are solidly connected to the band’s music from the Fantasy albums. As is well documented, eventually the band re-located to England and made two albums for Phonogram Records, produced by Robert John “Mutt” Lange, and Mitch Howie had left the group. Nevertheless, Huey and Sean performed much of the material from the Fantasy albums live with Clover many times both before and after Mitch’s departure, and of course they contributed greatly to the ongoing music of Clover’s catalog moving forward.

domain: artistic


text: Missing Dog Found Stuck in Drain Is One Tough Cookie The pup had been missing for more than a year By Newser Editors and Wire Services Posted Nov 28, 2015 3:59 PM CST 14 comments Comments A dog named Cookie is rescued from a storm drain more than a year after it went missing. (Twitter) (Newser) – Officials in Maryland say a 30-pound dog rescued from a storm drain had been missing for over a year. Rescue crews got the dog out of the drain on Friday after a man walking his own dog spotted the other pup in the storm drain. It took crews two hours to get the Wheaten Terrier out of the drain. The 1-year-old dog named Cookie had reportedly been missing since October 2014. Montgomery County Fire and Rescue Service spokesperson Pete Piringer says the dog's owner has been located and will pick the dog up. Piringer says it's believed Cookie got into the drain through a pipe at a nearby construction site.

domain: pet