Analysis of DNA repair gene polymorphisms and survival in low-grade and anaplastic gliomas.
In this dataset, eight gene variants mapping to five different DNA repair genes (ATM, NEIL1, NEIL2, ERCC6 and RPA4) which were associated with survival.
Functions of alternative replication protein A in initiation and elongation.
aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2.
An alternative form of replication protein a expressed in normal human tissues supports DNA repair.
Findings provide the first direct evidence for the function of alternative form of RPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.
A naturally occurring human RPA subunit homolog does not support DNA replication or cell-cycle progression.
Results suggest that RPA4 cannot support cell proliferation but can support processes that maintain the genomic integrity of the cell.
Eukaryotic single-stranded DNA binding proteins: central factors in genome stability.
Recently a human homologue to the RPA2 subunit, called RPA4, was discovered and RPA4 can substitute for RPA2 in the RPA complex resulting in an "alternative" RPA (aRPA), which can bind to ssDNA with similar affinity as canonical RPA.
Typhus and other rickettsioses: emerging infections in Germany.
RpA4, has not yet been associated with any human disease.
An alternative form of replication protein a prevents viral replication in vitro.
The "canonical" RPA is composed of three subunits (RPA1, RPA2, and RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4, that can substitute for RPA2 in complex formation.
Prevalence of tick-borne zoonotic bacteria in questing adult ticks from northern Spain.
RpA4/DnS14 by sequencing of the citrate synthase (gltA) gene.
Rickettsia sp. strain RpA4 detected in Portuguese Dermacentor marginatus ticks.
Recently, a new rickettsia genotype, RpA4, was detected in several Dermacentor sp.
The role of wild boar (Sus scrofa) in the eco-epidemiology of R. slovaca in Northeastern Spain.
RpA4 was identified in 4 specimens of D. marginatus removed from wild boar.
Ticks and associated pathogens collected from domestic animals in the Netherlands.
DnS14/RpA4 (14.0%), and Borrelia burgdorferi sensu lato (0.3%) were detected in the D. reticulatus ticks, which appeared to be free from B. canis infection.
Prevalence data of Rickettsia slovaca and other SFG Rickettsiae species in Dermacentor marginatus in the southeastern Iberian peninsula.
SFG rickettsia has been detected in 85.15% of ticks studied (26.7% of positives have been to R. slovaca, the causative agent of TIBOLA-DEBONEL, and 73.3% to SFG rickettsia closely related to strains RpA4-JL-02-DnS14-DnS28).
Prevalence of spotted fever group Rickettsia species detected in ticks in La Rioja, Spain.
strains RpA4, DnS14, DnS28 and JL-02 (59.3%) were found within this tick species.
Detection of members of the genera Rickettsia, Anaplasma, and Ehrlichia in ticks collected in the Asiatic part of Russia.
Spotted fever group rickettsiae in ticks feeding on humans in northwestern Spain: is Rickettsia conorii vanishing?
RpA4/DnS14, R. helvetica, Rickettsia sp.
Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage.
Hyperphosphorylated replication protein A is preferentially localized to double-stranded break repair and the DNA damage checkpoint complexes in response to DNA damage.


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text:
National offers the smallest buck regulator ICs
by Vince Biancomano
Santa Clara, Calif.&#8212National Semiconductor says its two new step-down switching regulators for handheld devices and automotive power adapters, the LM34917A and LM34919, are the industry's smallest non-synchronous buck regulators to feature a supply voltage rating up to 50 volts, 1.25-amp continuous output, intelligent current-limiting, and over-voltage protection. These chips, in the company's PowerWise family, are suited to applications that require high-input voltage regulation such as MP3 players utilizing Firewire connectivity (IEEE1394), and MP3 players and auxiliary power adapters with direct connection to a 12-volt (universal) automotive power outlet where transient protection requirements can exceed 40 volts.
The chips include all of the functions needed to implement a low-cost, efficient buck bias regulator. The LM34917A (12-bump, 1.97-by-2.3 mm micro SMD package) features programmable switching up to 2 MHz, thus facilitating the use of ultra-small inductors and capacitors for very small solution size. The chip utilizes a constant on-time (COT) control scheme, which requires no loop compensation. It has a peak efficiency of at least 90 percent.
The chip's COT feedback regulation scheme results in fast load transient response and simplifies circuit implementation. The operating frequency remains constant with line and load variation, owing to the inverse relationship between the input voltage and the on-time. The valley current-limit results in a smooth transition from constant-voltage to constant-current mode without foldback when the output is overloaded. Additional features include VCC under-voltage lockout, thermal shutdown, gate drive under-voltage lockout, and maximum duty-cycle limiting. The LM34917A can deliver up to 1.25 amps to the load.
The LM34919 (10-bump, 1.51-by-1.97 mm package) can deliver up to 600 mA. It features the same COT feedback regulation scheme used in the LM34917A.
Datasheets : For more information on the LM34917A and LM34919 or to order samples and an evaluation board, click here and here.
Pricing : Available now, the LM34917A is priced at $1.35 each and the LM34919 is priced at $1.05 each, both for 1k quantities.
National Semiconductor , 1-800-272-9959, www.national.com
0 comments on "National offers the smallest buck regulator ICs"

redpajama_set_name: RedPajamaCommonCrawl


text:
In 2012, the dramatic and unique Cinema Suite was unveiled
Europe's first Jiva Spa at 51 Buckingham Gate in London offers a blissful vitality pool
Once the home of lords and ladies of the Royal Court, Taj 51 Buckingham Gate is a five-star luxury hotel right next to Buckingham Palace and just a stroll to Mayfair shops and London's famous landmarks
Nothing speaks luxury and safety like being a neighbour to the reigning queen of England, and that is exactly what Taj 51 Buckingham Gate offers in abundance: a home away from home in likely one of the most prestigious and safe neighbourhoods in London.
With a history dating back to Tudor England and the court of Queen Elizabeth I, Taj 51 Buckingham Gate now includes three palatial townhouses overlooking a private courtyard, with each offering a wide choice of suites and residences.
Between the three buildings, the property provides guests with more space and flexibility to accommodate families, state delegations or top business executives.
The beautifully designed suites and residences feature between one and six bedrooms, spacious living areas and fully equipped kitchens, designed to provide unforgettable home-away-from-home experiences, right in the heart of London.
For the ultimate five-star luxury London experience, the four signature suites are individually designed and expertly crafted to combine elegance and space with refined comfort. Each offers a unique experience to match one's taste and mood.
The Golden Keys concierges and acclaimed butler service attend to all personal requirements, ensuring bespoke experiences are easily and effortlessly arranged.
This admired address has played host to truly dazzling weddings, private events and parties and milestone celebrations. It is discreetly positioned in SW1 – at the heart of central London - a gemstone's throw from Buckingham Palace, St. James's Park and close to Westminster, Whitehall, The Houses of Parliament and Victoria station. Many of London's most illustrious landmarks are also within walking distance, including Westminster Abbey, Big Ben, the Tate Gallery, National Gallery and Trafalgar Square, not to mention the enticing shops and restaurants of St. James, Mayfair and Knightsbridge and the world-renowned medical centres of Kensington.
Taj 51 Buckingham Gate Suites and Residences offers an exceptional plethora of dining options. Kona serves some of London's most innovative afternoon teas (all with bespoke halal versions), while Bistro has something for everyone with its international favourites. Journey to the south west coast of India at Michelin-starred Quilon or, if visiting in the summer, enjoy the beauty of St. James' Courtyard - a tranquil escape away from the hustle and bustle of central London. Nestled in its idyllic gardens is the hotel's shisha lounge, serving a variety of flavours including blueberry, mint, apple and watermelon alongside a selection of comforting mezze favourites and desserts.
Europe's first Jiva Spa at 51 Buckingham Gate in London celebrates the marriage of two award-winning brands – the traditional Indian healing rituals of Taj's own heritage brand Jiva and the advanced derma-science of the British-born Temple Spa. The unique treatment menu celebrates ancient and modern healing therapies, along with Eastern and Western wellness rituals.
Jiva's natural products and signature essential oils are renowned for their healing properties, while Temple Spa products are rich in natural botanicals, crafted with advanced derma-science, gorgeous Mediterranean aromatics and feel-good textures that feel like cashmere.
A specially appointed relaxation area includes a vitality pool, steam room, sauna and an emotional shower. It is the perfect couples' retreat or mother/daughter treat, that can be combined with a glamorous makeover by a celebrity stylist before indulging in one of the most iconic London experiences – afternoon tea, served in theatrical elegance, just minutes away from Buckingham Palace.
IN December 2012, Taj 51 Buckingham Gate Suites and Residences, London unveiled the Cinema Suite, created by acclaimed Indian designer Sabyasachi Mukherjee. At once a journey through world cinema and an ode to tradition, the 1,832 square foot, two-bedroom suite, with dining room, study, powder room and kitchen, was designed in tribute to the history of film and takes inspiration from motion pictures across the globe. Aptly, the Cinema Suite opened just ahead of celebrations marking 100 years of Indian film in 1913.
The suite is inspired most notably by 1940s and 1950s Hollywood as well as the Indian Cinema era of the 1950's. The suite is a celebration of personalised detail and global culture. Visitors will marvel at breathtakingly rare Bollywood film prints, remarkable history books, vintage mirrors and a myriad of portraits and drawings. From the leather Chesterfield to precious glassware, intriguing art and antique rugs, world arts and crafts are displayed throughout. Each wall is a work of art in itself, with striking Nina Campbell wallpapers in burnt orange, black and red, laden with curios collected from over 32 different cultures. Sumptuous silk drapery meets magnificent oak flooring, while priceless antique furniture displays ornaments such as rare Chinese porcelain, Bhutanese frames and 1920s embroidered Indian lamps.


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We strive our best to answer all questions and concerns within 24 hours of receiving the email. If customers have any defects or issues with their products please email us at cherryluo@buddyvape.com with a photo or video of the malfunction so we may further assist you. We will answer all your questions and inquires in a prompt manner. For any other questions or concerns, contact us at cherryluo@buddyvape.com.


The School of International Studies at Renmin University of China (RUC) was founded in April 2000, based on the reorganization and integration of the teaching and research faculty. With comprehensive courses in International Studies and a first-rate faculty, the School gained nationwide recognition for political science, for cultivating talent in international affairs, and for related teaching and research in International Studies. The program places special emphasis on American politics and American foreign policy with the objective of providing students with a deep understanding of American political systems and foreign policy. The school continues to strengthen this program through cooperation with domestic and international and universities.
The School of International Studies has a large library, but only a small collection of English language resources.


text:
Q: Erroneous use of auto type specifier with Eigen objects I have a main function which looks like:
#include <iostream>
#include <vector>
#include <Eigen/Dense>

using Eigen::Vector3d;
using namespace std;

void ext_func(vector<Vector3d> &a, vector<Vector3d> &b, vector<Vector3d> &c);
int main()
{
    vector<Vector3d> a = {};
    a.push_back(Vector3d(1.,1.,0.));
    a.push_back(Vector3d(2.,1.,0.));

    vector<Vector3d> b = {};
    b.push_back(Vector3d(.5,.7,2.));
    b.push_back(Vector3d(0.,.2,-1.2));

    vector<Vector3d> c = {};
    c.push_back(Vector3d::Zero());
    c.push_back(Vector3d::Zero());

    ext_func(a,b,c);

}

From main, I call an external function:
void ext_func(vector<Vector3d> &a, vector<Vector3d> &b, vector<Vector3d> &c)
{
    double fac = 0.01;
    for (size_t i = 0; i < a.size(); i++) {
        auto a_temp = a[i] + fac * b[i] / 50.;
        cout << "Before" << endl << fac*a_temp << endl;
        a[i] = a_temp;
        c[i] += fac*a_temp;
        cout << "After" << endl << fac*a_temp << endl << endl;
    }
}

The output is:
Before
 0.010001
0.0100014
    4e-06
After
 0.010002
0.0100028
    8e-06

Before
     0.02
0.0100004
 -2.4e-06
After
     0.02
0.0100008
 -4.8e-06

Which is not what I want to get (I want a_temp to stay constant once it's assigned). I understand that the problem is that I assign a new value to &a and so when I'm calling a_temp I'm basically incrementing a second time my initial a[i]. The problem is solved by replacing auto with Vector3d or Vector3d&&.
What I'm not sure about is what is auto really doing here.

A: When calculating with matrices in the eigen library, the overloaded operators return an object representing the calculation expression. This expression is not yet evaluated (ie. the actual calculation is not yet performed).
In your example, the type derived by auto is Eigen::CwiseBinaryOp<Eigen::internal::scalar_sum_op<double>, const Eigen::Matrix<double, 3, 1>, const Eigen::CwiseUnaryOp<Eigen::internal::scalar_quotient1_op<double>, const Eigen::CwiseUnaryOp<Eigen::internal::scalar_multiple_op<double>, const Eigen::Matrix<double, 3, 1>>>>.
The actual evaluation of the expression is done when the result is needed (eg. when assigning to a variable, printing, etc.).
Since a_temp in your case is the expression (not the result), the expression is evaluated multiple times :

*

*when printing it as part of the Before line

*when assigning to a[i]

*when assigning to c[i]

*when printing it as part of the After line

The second of these ends up modifying one of the operands of the expression, so subsequent evaluations of the expression will have a different result.

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text:
Scottish Interests
Special Events_Burns Dinner
Scottish Golf Day
New! Events Calendar (07/24/2016)
Follow us on Facebook at Celtic News Bytes for a look at what we're doing and what's coming up!
Looking for other information? Try these links! If there's one you think should be listed, use hte Contact Us form to let us know!
Scottish Clans: http://www.scotclans.com/scottish-clans
Blackhawk Pipes and Drums: http://blackhawkpipesanddrums.org/
Celtic Festival and Highland Games of the Quad Cities: http://celtichighlandgames.org/
Quad City Piper: http://www.quadcitypiper.com/
Scottish Heritage Society of Iowa: http://www.iowascots.org/
Hawkeye Area Grand Gaelic Society of Iowa (HAGGIS): http://haggis-iowa.com/
St. Patrick Society of the Quad Cities: http://www.stpatsqc.com/
Courses and Castles/Prints by Bill Hawthorne http://coursesandcastles.com/
Note: The painting in the website banner is Castle Eiliean Donan, and is a painting by Bill.
(Please note: links were valid as of time listed. Website may change. The Scottish American Society is not responsible for out-of-date information on third-party sites.)
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text:
Hepatic reference gene selection in adult and juvenile female Atlantic salmon at normal and elevated temperatures
Kelli C Anderson1,2 &
Abigail Elizur1
The use of quantitative real-time polymerase chain reaction (qPCR) has become widespread due to its specificity, sensitivity and apparent ease of use. However, experimental error can be introduced at many stages during sample processing and analysis, and for this reason qPCR data are often normalised to an internal reference gene. The present study used three freely available algorithms (GeNorm, NormFinder and BestKeeper) to assess the stability of hepatically expressed candidate reference genes (Hprt1, Tbp, Ef1α and β-tubulin) in two experiments. In the first, female Atlantic salmon (Salmo salar) broodstock of different ages were reared at either 14 or 22°C for an entire reproductive season, therefore a reference gene that does not respond to thermal challenge or reproductive condition was sought. In the second, estrogen treated juvenile salmon were maintained at the same temperatures for 14 days and a reference gene that does not respond to temperature or estrogen was required. Additionally, we performed independent statistic analysis to validate the outputs obtained from the program based analysis.
Based on the independent statistical analysis performed the stability of the genes tested was Tbp > Ef1α > Hprt1 > β-tubulin for the temperature/reproductive development experiment and Ef1α > Hprt1 > Tbp for the estrogen administration experiment (β-tubulin was not analysed). Results from the algorithms tested were quite ambiguous for both experiments; however all programs consistently identified the least stable candidate gene. BestKeeper provided rankings that were consistent with the independent analysis for both experiments. When an inappropriate candidate reference gene was used to normalise the expression of a hepatically expressed target gene, the ability to detect treatment-dependent changes in target gene expression was lost for multiple groups in both experiments.
We have highlighted the need to independently validate the results of reference gene selection programs. In addition, we have provided a reference point for those wishing to study the effects of thermal challenge and/or hormonal treatment on gene stability in Atlantic salmon and other teleost species.
The use of quantitative real-time polymerase chain reaction (qPCR) has become widespread due to its specificity, sensitivity, broad dynamic range, cost effectiveness, high throughput capability and the need to measure exact levels of gene transcription [1, 2]. Despite its apparent ease of use, there are many stages in between initial sampling and performance of the qPCR that can introduce variability, and essentially affect the quality and reliability of the data produced.
There are a few common standardisation techniques that are used to compensate for introduced variability; for example, data can be normalised to sample size (tissue weight or number of cells), quantity of RNA extracted, or a stably expressed reference or 'housekeeping' gene [1]. However, there are potentially significant drawbacks associated with each of these methods. RNA extractions are routinely carried out on tissue samples that may contain various cell types. For this reason, ensuring that the cellular make-up of dissected tissue is consistent between animals of different disease or developmental state can be quite difficult [1], and the specific contribution of each cell type to the total amount of RNA extracted could be disproportionate between samples. Subsequently, qPCR results acquired may reflect changes in cell composition and not a response to experimental conditions. While ensuring that the input amount of total RNA is the same between samples for cDNA synthesis is essential, this should not be used as a complete standardisation strategy. Standard RNA quantification methods mostly measure the ribosomal RNA (rRNA) fraction that can account for ~80% of total RNA. Therefore, this method relies on the assumption that the ratio of messenger RNA (mRNA) to rRNA does not change as a result of the experimental treatment or condition [3, 4]. However, previous studies have demonstrated that stability of the rRNA fraction cannot be taken for granted [5, 6]. Additionally, this method of standardisation does not control for pipetting error during cDNA synthesis, inhibitory factors contained in the tissue, or error introduced during the reverse transcription and qPCR phases of sample processing and analysis respectively [1]. Normalisation to one or several internal control (reference) genes is by far the most common method used to manage technical or other variation when estimating gene expression levels. Its inclusion in qPCR studies is preferred over other methods of normalisation since the reference mRNA template is present at all stages of processing and analysis, and will therefore reflect the cumulative change in sample dynamics [7]. However, inappropriate use of a single or multiple reference genes for normalisation can limit one's ability to detect small changes in mRNA abundance, or alter the fundamental findings and conclusions of a study [8].
In the past, many studies have assumed the stability of a small group of 'classic' reference genes without proper validation [4]. However, guidelines for reference gene selection are becoming more stringent, and there are now various prerequisites that a candidate reference gene must fulfill before it can be considered appropriate for normalising experimental error. For instance, the reference gene transcript level should not change as a result of experimental treatment [4], it should be expressed at a level that is similar to that of the target gene [1, 9] and amplification should be RNA specific with the absence of pseudogenes and contaminating genomic DNA (gDNA) [10]. In fish species, it is now apparent that the expression of many commonly used reference genes may vary on the basis of gender [11], tissue type [12], developmental stage [13] and experimental conditions such as type and length of exposure to exogenous chemicals [14]. Therefore, indiscriminate use of reference genes without stringent testing and validation could lead to incorrect expression profiling and interpretation of results [8, 11].
For many studies, a question remains as to what is the best way to determine whether candidate reference genes are appropriate for normalising non-biological sample variation. At the present time, there are three popular algorithms which have been specifically designed to determine the most suitable reference gene or combination of genes from a panel of candidates, namely BestKeeper [7], GeNorm [4] and NormFinder [15]. Recently it has been demonstrated that discrepancy can sometimes occur between results obtained from these software packages due to differences between algorithms, and it has been suggested that additional external evaluation is necessary to independently confirm the validity of genes selected by computer programs [16–18].
Salmonids are the most widely studied group of teleosts. However, studies investigating the usefulness of reference genes in qPCR have mainly focused on immature salmon [14, 19, 20], and no data are available for the expression of candidate reference genes for adult fish over an entire reproductive season. In addition to this, there are fundamental knowledge gaps concerning whether the age of an adult fish, or rearing temperatures outside of the optimum range modulate the expression of reference genes as demonstrated for other target genes during reproductive development [21]. In order to be able to assess the effects of temperature on reproduction, a reference gene is required whose expression is stable throughout reproductive development and across temperature ranges. In the present study, we have assessed the expression stability of elongation factor 1 alpha (Ef1α), hypoxanthine phosphoribosyltransferase 1 (Hprt1), TATA-box-binding protein (Tbp) and β-tubulin, all of which are routinely used reference genes in the field of teleost reproductive physiology. Gene expression was measured in hepatic tissue from 2+ (maiden) and 3+ (repeat) year old female Atlantic salmon reared at either 14 or 22°C during a reproductive season. Reference gene stability was then determined using the freely available BestKeeper, GeNorm and NormFinder algorithms, and methods recommended elsewhere in the literature [17]. Then using the same method, we determined whether the same reference gene candidates (except β-tubulin) were suitable normalising genes in a study where juvenile Atlantic salmon were given either a blank or 17β-estradiol (E2) implant, and maintained at either 14 or 22°C for 14 days. Studies such as these could become even more critical in years to come as wild and farmed fish species are affected by climate change, and we explore novel methods such as hormonal therapy to improve reproductive performance under thermal challenge.
qPCR assay validations
The efficiency and R2 value for all primer pairs were above 0.96 and 0.95 respectively (Table 1). For each reaction a single amplicon was produced as determined via melt curve analysis and agarose gel electrophoresis (data not shown); amplicon identity was confirmed through sequencing. No detectable qPCR product was produced for no-template controls and negative reverse transcription controls which confirms the absence of contamination by gDNA or other sources.
Table 1 qPCR primers used to amplify fragments of candidate reference genes
Stability analysis of candidate reference genes
In the adult broodstock experiment, Ef1α consistently had the highest transcript abundance, Hprt1 and β-tubulin had intermediate transcript abundance, and Tbp had the lowest (Figure 1, raw data are available at http://doi.pangaea.de/10.1594/PANGAEA.772212) Additional file 1: Table S1. When considering the standard error of the average quantification cycle (Cq) for all groups of fish studied, Tbp had the lowest transcript abundance variance followed by Ef1α, Hprt1 and β-tubulin (Figure 1). When Kruskal-Wallis analysis coupled with Bonferroni's correction was performed on a month-by-month basis, no significant differences in transcript abundance were found for Tbp during the reproductive season (Figure 1). For Ef1α, significant differences in transcript abundance were found for two sets of statistical comparison, for Hprt1 four sets of comparison were statistically significant and for β-tubulin five sets of comparison were statistically significant (Figure 1). When Kendall's tau correlation analysis was performed between Cq value and sample point, significant Cq-time relationships were found for all genes at p ≤ 0.01 except β-tubulin. For Tbp, sample point accounted for 7.2% of Cq variation, for Ef1α time of sampling accounted for 9.3% of Cq and for Hprt1 time accounted for 29.37% of Cq variation.
Abundance of hepatic mRNA transcripts for Tbp, Hprt1 and Ef1α and β-tubulin in female maiden Atlantic salmon reared during the reproductive season. The average quantification cycle (Cq) of all groups analysed, and the standard error is shown at the top left of the graph. Bars show the average Cq (+SEM) for each group of fish (n = 7). Different superscripts denote statistical significance between groups at a given sample point at p ≤ 0.05.
The BestKeeper algorithm ranked Tbp as the most appropriate reference gene while GeNorm ranked Tbp and Hprt1 as the most suitable pair after the step-wise elimination of the least stable genes (Table 2). This partly agreed with the results from NormFinder where Tbp was ranked third and Hprt1 was ranked first. Hprt1 was given a rank of three by BestKeeper which was not in agreement with any other algorithm. Ef1α was given a rank of two by BestKeeper and NormFinder and three by GeNorm. However, all three algorithms were in agreement when β-tubulin was assigned a rank of four, and was named as the least appropriate candidate gene for use in qPCR normalisation. When the hepatic expression of the target gene vitellogenin (egg yolk protein) was normalised using β-tubulin, then compared to the data normalised by Tbp, the ability to detect treatment-dependent changes in target gene expression was lost during January and March (Figure 2, raw data are available at http://doi.pangaea.de/10.1594/PANGAEA.772212) Additional file 2: Table S2.
Table 2 Ranking of candidate reference genes
Vitellogenin gene expression normalised to either Tbp or β-tubulin in female maiden Atlantic salmon reared at either 14 or 22°C and repeat salmon reared during the reproductive season. The mean (+ SEM) gene expression levels for each group of fish (n = 7) are displayed. The asterisk is placed above sample points where different target gene expression results were obtained after normalization.
In the juvenile salmon experiment, Ef1α had the highest transcript abundance followed by Hprt1 then Tbp (Figure 3, raw data are available at http://doi.pangaea.de/10.1594/PANGAEA.772213) Additional file 3: Table S3. This order is consistent with results from the adult experiment and in fact, the average Cq values for each gene between the different studies were very similar. Ef1α had the lowest transcript abundance variance as demonstrated by the low standard error for this gene, Hprt1 had an intermediate level of variance and Tbp had the highest (Figure 3). The standard error for the three genes examined in this experiment ranged between 0.68 and 1.4 cycles which is lower than 1.44 and 2.04 cycles that was observed for the same genes (not including β-tubulin) in the adult experiment. Kruskal-Wallis analysis followed by Bonferroni's correction revealed that no significant differences in Cq existed between experimental groups within a given sample point for EF1α (Figure 3). The same could not be said for Hprt1 where one significantly different comparison was found or for Tbp where six sets of comparisons were statistically significant. Kendall's tau correlation analysis revealed a small but significant relationship between Ef1α Cq and sample point, where sample point accounted for 3.61% of Cq variation (p ≤ 0.05). No significant relationship between time and expression of Tbp or Hprt1 was found.
Abundance of hepatic mRNA transcripts for Tbp, Hprt1 and Ef1α in juvenile Atlantic salmon given a blank silastic implant. The average quantification cycle (Cq) of all groups analysed, and the standard error is shown at the top left of the graph. Bars show the average Cq (+ SEM) for each group of fish (n = 7). Different superscripts denote statistical significance between groups at a given sample point at p ≤ 0.05.
Unlike the adult experiment where two out of three programs listed Tbp as an acceptable reference gene, all three algorithms ranked Tbp as the least stable reference gene candidate in the juvenile experiment (Table 2). GeNorm selected Ef1α and Hprt1 as the most appropriate pair of candidates, BestKeeper selected Ef1α and NormFinder selected Hprt1 as the best candidate. It is interesting that NormFinder listed Hprt1 as the most stable single gene, though listed Ef1α and Tbp as the best combination of two genes to use for target gene normalisation. Similarly, addition of the least stably expressed gene (Tbp) into a panel of Hprt1 and Ef1α made a significant contribution (0.24) to the normalisation factor calculated by GeNorm despite its lower ranking. Based on the results from GeNorm, all three reference genes should be used for accurate normalisation of qPCR data.
We have reported on the stability of candidate reference genes for the entire reproductive season of maiden and repeat spawning female Atlantic salmon reared under cool (14°C) and warm (22°C) conditions in Tasmania. Statistical analysis of Tbp Cq value revealed that no significant differences were present between experimental groups of fish within a given sampling point for the entire reproductive season. Tbp also had the lowest transcript abundance variance over the entire eight month period which suggests that it is the most stably expressed gene of the candidate panel tested. However, correlation analysis revealed that 7.2% of the variation in transcript abundance could be accounted for by sampling point. This may indicate that Tbp gene expression is down-regulated (has a higher Cq) to some extent over time because it is linked to reproductive status. For this reason, a degree of caution should be exercised when directly comparing the normalised expression levels of target genes from fish sampled in different months, particularly between the start and end of the reproductive season (i.e. August'07 versus April'08) as normalisation could introduce some error.
Two statistically significant comparisons were found for the Cq value of Ef1α during March; in November and March a total of four significant comparisons were found for Hprt1 and five were found for β-tubulin. Therefore, it is not surprising that EF1α had the second lowest level of Cq variance followed by Hprt1 then β-tubulin. The presence of statistically significant differences in Cq value indicates that these genes may not be suitable candidates for target gene normalisation during certain months of the year. In fact, normalisation of a hepatically expressed target gene (vitellogenin) to β-tubulin resulted in a loss of the ability to detect treatment-dependent changes in target gene expression during January and March when compared to target gene expression data normalised by Tbp (Figure 2). These results confirm the work of previous authors where inappropriate use of a reference gene significantly altered the interpretation of qPCR results [8, 22].
In a similar fashion to Tbp, a significant correlation between sample point and Cq was found for Ef1α that accounted for 9.3% of Cq variation. Again, it may be at the discretion of the researcher to decide whether comparisons of qPCR data are of critical importance between months at the start and end of the reproductive season, or whether within month assessment of gene expression levels will suffice. However, the strongest relationship between sample point and transcript abundance was found for Hprt1 as ~30% of Cq variance could be attributed to the month of sampling. The apparent connection between gene expression and developmental state, the number of statistically significant comparisons found and high standard error compared to other candidate genes made Hprt1 a poor choice for qPCR data normalisation in the present study. Even though no significant correlation was found between β-tubulin and sample point, the use of a β-tubulin to normalise target gene expression variance resulted in experimental bias due to a combination of its high standard error and high within-month Cq variation (Figure 2). Based on the statistical analysis performed, the usefulness of candidate reference genes for normalisation is as follows: Tbp > Ef1α > Hprt1 > β-tubulin.
The BestKeeper algorithm provided rankings that were in agreement with the ranking obtained (above) via independent statistical analysis. GeNorm also selected Tbp as an ideal reference gene, however this gene was chosen in combination with Hprt1 which showed the largest relationship between Cq and sample point, and four statistically significant comparisons. However, GeNorm successfully identified β-tubulin as the least stably expressed candidate gene. NormFinder also ranked β-tubulin as the least suitable gene for target gene normalisation. However, unlike the other algorithms, NormFinder assigned a rank of three to Tbp which is surprising given the lower standard error, within month variance and Cq-time correlation of Tbp compared to the rest of the candidate panel. However, ranking of the remaining genes between algorithms was ambiguous which indicates that the output of such programs should not be used complacently.
Disagreement between programs concerning the suitability of candidate reference genes presumably occurred as a result of the different statistical principles and assumptions underlying each algorithm. For example, the BestKeeper program assumes that if candidate reference genes are stably expressed, then gene expression levels should be highly correlated. Thus, pair-wise correlations for every possible gene pair combination are performed, and the geometric means of highly correlated genes are used to calculate an index. The level of correlation between each individual gene and the index is then determined, and a higher coefficient of correlation is indicative of a more stable gene. Unlike BestKeeper, GeNorm uses relative gene expression levels (calculated using the ΔCq method) and not raw Cq values to calculate the average pair-wise variation between candidate genes. A stability value (M) calculated as a result of this analysis can then be used to eliminate the least stable gene in a step-wise fashion until only the two genes remain. Lastly, NormFinder allows the user to define experimental treatments through the use of group identifiers which is a feature unique to this software. As a result, NormFinder takes into account both inter- and intra-group variability when using relative gene expression levels to calculate a stability value then subsequently rank candidate genes. Based on our data, it appears that all three reference gene selection programs are useful for eliminating the least stably expressed gene from a panel of candidates. For this reason external validation of program outputs is warranted to account for conflict between algorithms, program assumptions and limitations when complex experimental designs are employed.
In addition to the adult experiment, we also determined the suitability of the same candidate reference genes (except β-tubulin) for use in a study where juvenile salmon were given a blank or E2 pellet and reared at either 14°C or 22°C for 14 days. In the juvenile study, the gene expression of Ef1α did not significantly change as a result of experimental treatment within each sampling point. However, a significant Cq- sample point correlation was found for this gene that accounted for 3.61% of Cq variance. While this is a statistically significant relationship (p ≤ 0.05), it is probably not of major concern due to its magnitude. One significant comparison was detected for Hprt1 gene expression at day 14, and a total of six sets of comparison were significant for Tbp. Ef1α also had the lowest transcript abundance variance, followed by Hprt1 then Tbp. This strongly suggests that stability of candidate reference genes in this experiment is as follows: Ef1α > Hprt1 > Tbp.
All three reference gene programs were in agreement when assigning a rank of three to Tbp which was the same result achieved through external statistical analysis (above). Through this analysis it has become apparent that freely available algorithms are consistently able to identify the least stable gene from a panel of candidates. BestKeeper found Ef1α to be the most stable candidate followed by Hprt1 while the opposite was found using NormFinder. In a similar fashion to the adult experiment, the BestKeeper algorithm gave the same rankings to the reference gene candidates as independent statistical analysis and therefore has been the most reliable algorithm in our studies. GeNorm selected Ef1α and Hprt1 as the most suitable pair of candidates; although the addition of Tbp to the gene panel significantly improved overall stability value (by 0.24) despite its apparent lower stability. When the expression of a hepatically expressed gene was normalised to Tbp alone or in combination with Hprt1 and Ef1α, significantly different results were achieved for two groups of fish (data not shown). Additionally, variance in the target gene expression data, and therefore noise, was reduced when all three genes were used for normalisation instead of Tbp alone (data not shown).
In a broad sense, our data agree with previous studies that outline the need to validate reference genes on an experiment-to-experiment basis [11, 14]. For example, in the adult experiment there is strong evidence to suggest that Tbp was the best candidate for target gene normalisation, while in the juvenile experiment Tbp was ranked third but may still prove useful as a reference gene in combination with other candidates. Based on our data, we recommend Tbp and Ef1α as a starting point when selecting candidate reference gene for research using female Atlantic salmon broodstock where hepatic gene expression will be measured during reproductive development. Furthermore, Ef1α and Hprt1 appear to be quite stable in juvenile fish treated with E2 under thermal challenge. As a final point, all three algorithms correctly identified the least stable candidates in both of our experiments and can therefore prove useful to initially screen data and eliminate the most undesirable genes.
In recent years it has become clear that no single gene is stably expressed under all experimental conditions for any given tissue or species. Our study further highlights the need to evaluate reference gene stability separately for every experiment as it is likely that no one gene will be consistently stable across experiments. Freely available stability assessment programs are user friendly and can provide valuable information in gene expression studies. However, we recommend that independent statistical validation be carried out as an additional safe guard against inappropriate adjustment of target gene expression. Furthermore, full justification for the reference genes selected should be provided in any publications containing relative qPCR data.
For the adult experiment, maiden and repeat cultured adult females were held at the Salmon Enterprises of Tasmania (SALTAS) Wayatinah Hatchery (Tasmania, Australia) at ambient temperature and photoperiod in either 200 (maidens) or 50 (repeats) m3 circular tanks at stocking densities of 12-18, and 24-36 kg m-3 for maidens and repeats, respectively until early January 2008. In January, fish were transferred to temperature-controlled 4 m3 tanks (14 fish per tank) under simulated ambient photoperiod according to the following experimental groups (28 fish per group): maidens reared at 14°C, repeats reared at 14°C, maidens reared at 22°C and repeats reared at 22°C. Fish were not fed from the time of transfer to the temperature controlled systems in January which is consistent with hatchery practice for management of this experimental stock of fish. All fish were maintained at the nominated temperature (14 or 22°C) until early April when all fish were exposed to a temperature ramp down over 11 days to 8°C to induce final oocyte maturation and ovulation as described in King and Pankhurst [23].
Fish from both maiden and repeat groups were sampled on the 31st August and 2nd November 2007, and 7th January 2008 and after introduction to the controlled temperature regimes on the 14th February 2008, 28th March and 25th April. For sampling, fish were netted from the holding tanks, terminally anaesthetised in Aqui-S ™ (Crop & Food, New Zealand) and sections of liver were transferred to 1-2 mL of RNA Later ™ (Qiagen, Germany) to stabilise mRNA for later measurement of gene expression. Samples were held overnight at 4°C, then stored at -20°C. This research activity was undertaken with approval from the Animal Ethics Committees of the University of the Sunshine Coast and Griffith University (approval numbers AN/A/07/35 and EAS/02/07/AEC respectively).
For the juvenile experiment, 84 juvenile female Atlantic salmon (217.2 g ± 4.68 g) were housed at the SALTAS, in 4 separate 1000 L tanks (21 fish per tank) with independent recirculating fresh water for each temperature treatment. At day 0, all fish were anesthetised with Aqui-S™ (25 ppm), weighed, implanted with a blank or E2-containing pellet (10 mg.kg-1) and placed in thermo-regulated tanks in one of four experimental groups: E2 pellet at 14°C, E2 pellet at 22°C, blank silastic pellet at 14°C and blank silastic pellet at 22°C (n = 21 per group). At 3, 7 and 14 days post implantation, 7 fish were sacrificed from each group using a lethal dose of Aqui-S™ (50 ppm). Liver sections were dissected and stored as described above. This experiment was conducted under approval from the Animal Ethics Committee of the University of the Sunshine Coast (approval number AN/A/07/35).
RNA isolation and cDNA synthesis
Total RNA was isolated from 15 mg of hepatic tissue using the Illustra RNAspin Mini kit (GE Healthcare) according to the manufacturer's protocol. RNA yield and 260/280 purity ratio were determined using the NanoDrop 2000 (Thermo Scientific). An RNA integrity number (RIN) was determined for a random sample of hepatic RNA using a 2100 Bioanalyzer (Agilent) to establish RNA quality. All RNA was stored at -80°C until use.
Four hundred nanograms of liver-derived RNA were used to synthesise cDNA for use in qPCR using the QuantiTect® reverse transcription kit (Qiagen). This kit includes a DNA elimination step to remove potential contamination of PCRs by genomic DNA. Following synthesis, cDNA was stored at -20°C until use.
Primer design and standard curve construction
Gene specific primers for Hprt1, Ef1α, β-tubulin and Tbp were designed from species-specific mRNA sequences (freely available on GenBank) using Primer3 software (http://frodo.wi.mit.edu/primer3/, Table 1) by Pankhurst et al., (2011). All primers were designed to have an optimum annealing temperature of 60°C and were supplied by GeneWorks (Australia). Validation curves containing at least five points were carried out in triplicate using serially diluted cDNA as the template. Following amplification, the size of all qPCR products was determined by running 4 μl of the product on a 2% agarose gel; gene identity was then confirmed through sequencing. Reaction efficiencies (Table 1) were automatically calculated by Rotor-gene software version 1.7.87 using the equation: E =[10(-1/M)]-1, where E is equal to efficiency and M is equal to slope.
qPCR cycling conditions
qPCRs were conducted on a Rotor-gene 6000 series thermal cycler (Qiagen) using the following reaction mix: 5 μl Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen), 200 nM each primer, 3.6 μl PCR grade water and 1 μl cDNA template (total 10 μl). Cycling conditions were as follows: 50°C for 2 min; 95°C for 2 min; 40 cycles of 95°C for 15 sec; 60°C for 15 sec, and 72°C for 20 sec (acquiring). At the end of cycle 40, a melt curve analysis was performed to confirm amplification of a single product as follows: 90 sec preconditioning step at 72°C, followed by a temperature gradient up to 95°C at 1°C per 5 sec. For every gene analysed no-template controls (NTCs) were included to detect possible contamination; negative reverse transcriptase controls were also analysed to detect contaminating DNA if present.
Candidate reference gene analysis
Raw Cq values were transformed to relative expression levels using the 'ΔCq' method and the equation Q = E(minCq - sampleCq) where Q = relative quantities, E = efficiency (+ 1) and minCq refers to the Cq of the sample with the highest expression level (lowest Cq) in the data set for any given gene. Input for GeNorm and NormFinder were data transformed to relative expression levels using the ΔCq method, stability was assessed according to the program's instructions. Sub-group identifiers were included for NormFinder analysis. For BestKeeper, data were entered without modification as raw Cq according to the program's instructions.
Previous authors have recommended that statistical differences in qPCR be detected non-parametrically [17, 24]. Therefore, statistical differences between groups on a monthly basis were analysed using the Kruskal-Wallis test coupled with Bonferroni's Correction to reduce the risk of type 1 error using SPSS (version 17.0). The P value for all analyses was initially set at 0.05 before adjustment by Bonferroni's correction. Kendall's tau non-parametric correlation analysis was performed to assess whether a significant relationship existed between raw Cq value and sampling point for each gene in both experiments. The R2 method was used for interpretation of the correlation results where the correlation coefficient is squared, then multiplied by 100 to give the percent of variation in Cq accounted for by time. Target gene (vitellogenin) expression was calculated using Relative Expression Software Tool (REST©) [25].
The data sets supporting the results of this article are available in the PANGAEA® repository (http://doi.pangaea.de/10.1594/PANGAEA.772212 and http://doi.pangaea.de/10.1594/PANGAEA.772213 for the adult and juvenile experiments respectively).
Huggett JF, Dheda K, Bustin SA, Zumla A: Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005, 6: 279-284. 10.1038/sj.gene.6364190.
Bustin SA, Vandesompele J, Pfaffl MW: Standardization of qPCR and RT-qPCR: new guidelines seek to promote accurate interpretation of data and reliable results. Genetic Engineering & Biotechnology News. 2009, 29: [http://genengnews.com]
Suzuki T, Higgins PJ, Crawford DR: Control selection for RNA quantitation. Biotechniques. 2000, 29: 332-337.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology. 2002, 3: research 0034.0031-0034.0011.
Hansen MC, Nielsen AK, Molin S, Hammer K, Kilstrup M: Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels. J Bacteriol. 2001, 183 (16): 4747-4751. 10.1128/JB.183.16.4747-4751.2001.
Solanas M, Moral R, Escrich E: Unsuitability of using ribosomal RNA as loading control for Northern blot analyses related to the imbalance between messenger and ribosomal RNA content in rat mammary tumors. Anal Biochem. 2001, 288: 99-102. 10.1006/abio.2000.4889.
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004, 26: 509-515.
Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GAW, Zumla A: The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem. 2005, 344: 141-143. 10.1016/j.ab.2005.05.022.
Bustin SA, Nolan T: Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004, 15: 155-166.
Kreuzer K-A, Lass U, Landt O, Nitsche A, Laser J, Ellerbrok H, Pauli G, Huhn D, Schmidt CA: Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of ß-actin transcripts as quantitative reference. Clin Chem. 1999, 45: 297-300.
Filby AL, Tyler CR: Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish. BMC Mol Biol. 2007, 8 (10): doi: 10.1186/1471-2199-8-10
Olsvik PA, Lie KK, Jordal A-EO, Nilsen TO, Hordvik I: Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol Biol. 2005, 6 (21): doi: 10.1186/1471-2199-6-21
Tang R, Dodd A, Lai D, McNabb WC, Love DR: Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin. 2007, 39 (5): 384-390. 10.1111/j.1745-7270.2007.00283.x.
Arukwe A: Toxicological housekeeping genes: do they really keep the house?. Environ Sci Technol. 2006, 40: 7944-7949. 10.1021/es0615223.
Andersen CL, Jensen JL, Ørntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004, 64: 5245-5250. 10.1158/0008-5472.CAN-04-0496.
Hibbeler S, Scharsack JP, Becker S: Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus. BMC Mol Biol. 2008, 9 (18): doi: 10.1186/1471-2199-9-18
Setiawan AN, Lokman PM: The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale. BMC Mol Biol 11. doi:10.1186/1471-2199-1111-1175
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Ingerslev H-C, Pettersen EF, Jakobsen RA, Petersen CB, Wergeland HI: Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.). Mol Immunol. 2006, 43: 1194-1201. 10.1016/j.molimm.2005.07.009.
Jorgensen SM, Kleveland EJ, Grimholt U, Gjoen T: Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon. Marine Biotechnol. 2006, 8: 398-408. 10.1007/s10126-005-5164-4.
Pankhurst NW, King HR, Anderson KC, Elizur A, Pankhurst PM, Ruff N: Thermal impairment is differentially expressed in maiden and repeat spawning Atlantic salmon. Aquaculture. 2011, 316: 77-87. 10.1016/j.aquaculture.2011.03.009.
Radoni A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A: Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004, 313: 856-862. 10.1016/j.bbrc.2003.11.177.
King HR, Pankhurst NW: Ovulation of Tasmanian Atlantic salmon maintained at elevated temperatures: implications of climate change for sustainable industry development. 6th International Symposium on the Reproductive Physiology of Fish. Edited by: Norberg B, Kjesbu OS, Taranger GL, Andersson E, Stefansson SO. 2000, Bergen, 396-398.
Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E: Housekeeping genes as internal standards: use and limits. J Biotechnol. 1999, 75: 291-295. 10.1016/S0168-1656(99)00163-7.
Pfaffl MW, Horgan GW, Dempfle L: Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002, 30 (9): 36-10.1093/nar/30.9.e36.
This work was supported by Salmon Enterprises of Tasmania Pty. Ltd. (Saltas), the Australian Government through the Fisheries Research & Development Corporation and the Australian Seafood Cooperative Research Centre. K. Anderson was supported by an Australian Post Graduate Award through the University of the Sunshine Coast. Thanks are extended to Ned Pankhurst for designing the salmon experiments, critically reviewing the manuscript and expert supervision, to Harry King for experimental design, fish maintenance and expert guidance, and to SALTAS staff for assistance with fish husbandry and management.
Faculty of Science, Health and Education, University of the Sunshine Coast, Locked Bag 4, Maroochydore DC, Queensland, 4558, Australia
Kelli C Anderson & Abigail Elizur
Australian Seafood Cooperative Research Centre, Box 26, Mark Oliphant Building, Science 10 Park Adelaide, Laffer Drive, Bedford Park, Adelaide SA, 5042, Australia
Kelli C Anderson
Abigail Elizur
Correspondence to Abigail Elizur.
KA took part in sampling, performed all laboratory work/data analysis and drafted the manuscript. AE acquired funding, took part in sampling, critically reviewed the manuscript and provided principal supervision for KA.
Additional file 1: Table S1. Abundance of hepatic mRNA transcripts for Tbp, Hprt1 and Ef1α and β-tubulin in female maiden and repeat Atlantic salmon reared at either 14 or 22°C during the reproductive season. (DOCX 13 KB)
Additional file 2: Table S2. Vitellogenin gene expression normalised to either Tbp or β-tubulin in female maiden and repeat Atlantic salmon reared at either 14 or 22°C during the reproductive season. (DOCX 14 KB)
Addtional file 3: Table S3. Abundance of hepatic mRNA transcripts for Tbp, Hprt1 and Ef1α in juvenile Atlantic salmon given a blank or E2 silastic implant at 14 or 22°C. (DOCX 12 KB)
Anderson, K.C., Elizur, A. Hepatic reference gene selection in adult and juvenile female Atlantic salmon at normal and elevated temperatures. BMC Res Notes 5, 21 (2012). https://doi.org/10.1186/1756-0500-5-21
Reference Gene
Candidate Reference Gene
Juvenile Salmon
Adult Experiment
Thermal Challenge


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Q: Estilização do input Date
Bom, queria saber como faço para estilizar um input do tipo date

A: O padrões estéticos do input date vão depender dos navegadores que a suportam. Se quiser uma opção cross-browser e personalizável, terá que usar um plugin, ou algo do tipo. Aqui vai um exemplo com o datePicker do Jquery:

$(function() {
  $("#datepicker").datepicker({
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    buttonText: "Select date"
  });
});
<link rel="stylesheet" href="//code.jquery.com/ui/1.12.0/themes/base/jquery-ui.css">
<script src="https://code.jquery.com/jquery-1.12.4.js"></script>
<script src="https://code.jquery.com/ui/1.12.0/jquery-ui.js"></script>

<input type="text" id="datepicker">

Você pode estilizar esse dataPicker com as classes do Jquery-ui.css, exemplo:

$(function() {
  $("#datepicker").datepicker({
    showOn: "button",
    buttonImageOnly: true,
    buttonText: "Select date"
  });
});
.ui-datepicker {
 background-color: #666;
 border: 1px solid #66AFE9;
 border-radius: 4px;
 box-shadow: 0 0 8px rgba(102,175,233,.6);
 display: none;
 margin-top: 4px;
 padding: 10px;
 width: 240px;
  font-family: sans-serif;
}
.ui-datepicker a{
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}
.ui-datepicker a,
.ui-datepicker a:hover {
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}
.ui-datepicker a:hover,
.ui-datepicker td:hover a {
 color: #2A6496;
 -webkit-transition: color 0.1s ease-in-out;
    -moz-transition: color 0.1s ease-in-out;
      -o-transition: color 0.1s ease-in-out;
         transition: color 0.1s ease-in-out;
}
.ui-datepicker .ui-datepicker-header {
    margin: 0;
 margin-bottom: 4px;
 text-align: center;
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Guia completo de estilizações


Quadriga was formed to serve clients interested in unique design solutions for complex projects. From day one, we've focused on our clients and their objectives, rather than a particular project. We have been fortunate to develop a varied base of repeat clientele with whom we share basic values and frequent collaboration.
Since our beginning in 1997, we have been committed to advancing our clients' interests, with careful attention to budget and constructability. To this we weave principles of stewardship, respect for cultural context, and spirit of place in pursuit of uniquely successful solutions.
Quadriga is organized as a collaborative studio composed of passionate and talented designers committed to design excellence and meeting the needs of our clients. Arranged in teams that are composed according to the needs of each project, we employ a horizontal framework that encourages involvement and broadens staff knowledge, while fostering individual interests and expertise.


For Christopher Knowles, time is a landscape, a visual pattern. The poet and artist collects alarm clocks, he carries at least one wristwatch, and when he meets an acquaintance he might tell her: It's been 3,782 hours since I last saw you. If she counts the days and hours she will find that he is correct. If you tell Knowles your birthday, he will tell you what day of week you were born, and for any historical date he can tell you exactly what phase the moon was in. Like a person on a hilltop surveying the landscape, Knowles seems to visualize the years and days and hours spreading out before him.
Across five decades, Knowles has explored these structures through a multidisciplinary practice of performance, poetry and art-making. His typings—geometric patterns of words and letters printed on a typewriter using colored inks—have been exhibited at major museums and made their way into the collections of MoMA and the Museum Ludwig. At first glance Knowles's work can seem confusing, naïve and arbitrary, but when you pay attention precise patterns emerge from his poetry, his typings, his use of alarm clocks.
In 1973, when he was just thirteen years old, Knowles recorded an audio tape that wound up in the hands of Robert Wilson. On the tape, Knowles recites a poem about his sister, chanting a series of variations of the three phrases "Emily likes the TV," "because she watches the TV," and "because she likes it," over and over again in different constellations, until the sentences dissolve into pure sound and all meaning is wiped away, only to be put back together again like Russian dolls being reassembled.
Wilson was instantly intrigued. He transcribed Knowles's words and found that the seemingly random variations were not random at all. What at first had sounded arbitrary was in fact extremely structured and precise and constituted a pattern, establishing a world of its own. Wilson invited Knowles to see his play The Life and Times of Josef Stalin—a wordless, twelve-hour long piece with more than 120 performers—and he traveled to visit Knowles at his school in Schenectady, New York. Wilson didn't like the way the teachers were treating Knowles, correcting his actions and his speech, dogmatically imposing their reality onto his, and he convinced Knowles's parents to pull him out of school. Soon Knowles was living with Wilson, the beginning of a long and unusual collaboration.
In the '60s and early '70s, Wilson's works had been silent—no text, no libretto, no words. He had been creating plays with the deaf-mute Raymond Andrews, but now Knowles's formal and plastic approach to language started to enter Wilson's pieces. One day, Knowles was speaking in an elaborate Victorian English, apparently reciting a text he had read and memorized. This soliloquy became the central piece of Wilson's 1974 play A Letter for Queen Victoria.
It was as if Knowles were the embodiment of the postmodern ideas Wilson, Glass, Ashbery and Robbe-Grillet were wrestling with: how to free language from meaning, how to embrace contradictions, how to dissolve plot and structure to give way to something freer and more fractured. What was so difficult to achieve for Wilson and Glass and their baffled audiences—getting rid of narrative, not always looking for meaning—was second nature to Knowles.
WIlson relates how on a nine-hour flight from Amsterdam to New York, Knowles had secretly placed some of his forty-eight alarm clocks in overhead bins, under the seats and in restrooms. He had set them to go off at different intervals, creating a sonic a structure, a kind of symphony for the nine-hour flight. Wilson had fallen asleep, but was woken up when the alarms began going off.
For most people time has a flow. It runs like a river—from the past, through the present, towards the future. There are deadlines to deliver on, appointments to remember, and so we divide the plane of time into years and days and hours, and attempt to adhere to these schemes through an arsenal of alarm clocks, calendars, diaries and watches.
For some physicists, however, time is not a flow but a dimension much like a landscape. Just like Manhattan still exists while you and your consciousness are in Brooklyn, October 1976 still exists while your consciousness is in January 2017. According to this idea of the block universe, all moments exist simultaneously on the plane of time: your birth, your first kiss, last Saturday's party and Sunday's hangover too. All of it exists at once, and the sense that the present is somehow more real and alive than the past is just a trick of the consciousness, our limited minds trying to make sense of it all.
Christopher Knowles seems able to visualize the block universe more clearly than most people. In his calendars he skips between decade-old memories and seemingly random birthdays, in and out of the present day. He seems amused with our feeble attempts at measuring time, the alarm clocks, the calendars, the watches. By toying with the crushing conformity of these time-keeping devices, Knowles is creating his own patterns, a private kingdom. Or maybe a kingdom for two.


Mokuren Dojo: Ju don't know Ju!
So, they say that this judo thing that we do is based on the principle of "ju", which is supposed to mean something like "gentle," but westerners tend to assign some screwed-up connotations to the word, "gentle" - connotations like "flaccid" or "weak" or "wimpy."
Sensei Strange (pictured above, working his ju magic) has discussed the kanji for ju at his blog a couple of times here and here. And I'm no Japanese language scholar, so I thought I'd dig a bit into the English denotations for the words we frequently use for ju.
I think the first 3 definitions for the adjective GENTLE are interesting, and I especially like the first couple of definitions for the verb TO GENTLE. Brings to mind "The Uke Whisperer!" There are several interesting definitions in the entry on SOFT, but overall, those ideas don't fit my understanding of ju.
So, how do y'all think judo or ju is like or unlike those denotations for SOFT or GENTLE?


Many people already know that eBay listings with embedded video convert more sales than listings without video. But most people still don't take advantage of this.
It is very easy to embed video into an eBay listing. You don't need a website or your own hosting account. All you need is an account with YouTube if you don't already have one.
Just go to YouTube and follow the directions to set up an account, and then upload your chosen video from 9anime. Make sure you give it a good title and enter the keywords you think people will use to search for the video. You then will have the option to embed the video. Copy the "embed" code supplied, then go to your auction listings description, open the "html" tab and paste in the code. It's really that simple.
Once your auction listing is complete, copy the URL from the address bar. Go back to the description for your uploaded video on YouTube and paste the URL. You may want to write a plug for visitors to purchase your product. For example: "Click here for more information".
By adding videos to your listings, you give potential buyers more information about the products you are selling. Buyers like to have a lot of information before they commit to making a purchase, so the more information you provide, the better it will work out for you in the long run.
When you then link your YouTube video back to your eBay listing, you are generating a method to bring more visitors to your listing. More visitors equal more sales. Just be sure to go back and remove the YouTube links when your auction ends.
As you can clearly see, embedding YouTube videos in your eBay listings is a simple and effective way to increase sales. Just make sure you own the rights to the videos, do not plagiarize and always be sure your videos are of high quality. If they are not, some buyers may think the quality of your products may be similar to the quality of your videos and if your videos are poor, then prospective buyers may be turned away.
So what kinds of videos can you use?
You could have someone make a video review of your product. Video reviews are extremely powerful marketing tools and if they are done right they could significantly increase your sales. If you don't know anyone who will do a video review, you may want to check if you can have it done cheap on a site like fiverr.com.
You could also make a video of someone demonstrating your product. Maybe emphasize on how easy your product is to use and how much it would benefit your customers if they purchased it.
If you are selling a video tutorial or video course you could embed a small clip of the tutorial so your visitors can get an idea of what they are about to purchase.
As you can see, embedding YouTube videos into your eBay auction listings is really a no-brainer. It's simple, effective, and just good marketing practice. Just remember to make sure the videos are high quality and begin raking in the sales.


text:
Vols Promote Tight Ends Coach Larry Scott to Offensive Coordinator
(utsports.com) KNOXVILLE, Tenn. — University of Tennessee football head coach Butch Jones announced today the promotion of Larry Scott to offensive coordinator. Additionally, Mike Canales has agreed to terms to become the quarterbacks coach and defensive backs coach Charlton Warren was given additional duties as special teams coordinator.
Scott, Canales and Charlton Warren will be available to the media today at 5 p.m. in the Ray and Lucy Hand Studio.
"We spoke to a lot of very quality candidates," Jones said. "Our goal was to gather as much information as possible in a timely manner but also go through a detailed process, which we did. We feel strongly about our offensive staff and that Larry Scott is the best fit moving forward to lead the unit.
"Larry played an important role in the success we had offensively last year and was heavily involved in all aspects of our game plan, both during the week and on game day. We felt it was vital to maintain our continuity on offense and keep building on what we have established the past four seasons.
"I'm also really excited about adding Mike Canales to our staff. Mike has recruited, coached and developed numerous quarterbacks at the collegiate level. He will be of great benefit to our players and staff with his extensive experience and knowledge of the quarterback position."
Scott, who has 16 years of coaching experience, joined UT in 2016 as tight ends/special teams coordinator and was part of an offensive unit that set a school-record with 63 touchdowns. The Vols averaged 47.8 points over the last five games and scored 30-plus points in nine of 13 games in 2016. Offensively, the Vols ranked second in the SEC in scoring offense and fifth with 449.2 yards per game.
The Vols averaged 443.7 yards of total offense and produced 400 yards of total offense in eight of their 13 games, and 450-plus yards in seven of 13 outings. With a red zone scoring percentage of 88.0 percent, the Vols ranked second in the SEC and 28th in the NCAA.
A native of Sebring, Florida, Scott spent three seasons (2013-15) as the Miami Hurricanes' tight ends coach and served as interim coach for the final six games of the 2015 season. As an interim head coach, Scott led the Hurricanes to a 4-2 mark and a berth in the Sun Bowl. Scott spent eight seasons at the University of South Florida where he served a variety of roles, including tight ends, offensive line and running backs coach. He was part of a staff that helped USF to its fifth-straight eight-win season. He also spent time at Wharton High School, Freedom High School and Sebring High in Florida.
Canales arrives in Knoxville with 32 years of coaching experience, including 23 years as an offensive coordinator. He has coached five different quarterbacks that have earned All-America recognitions and been a part of teams that have appeared in eight bowl games. He spent the 2016 season at his alma mater, Utah State, where he served as the assistant head coach/running backs/tight ends coach.
From 2010-15, Canales served as the associate head coach/offensive coordinator/quarterbacks coach at North Texas from 2010-15. He also spent portions of the 2010 and 2015 seasons as an interim head coach for the Mean Green. During his six seasons at North Texas, he mentored 20 offensive players who earned all-conference honors, while helping the program win just their third bowl game in school history in 2013. He coordinated a unit in 2014 that ranked tops in the nation with a 94.3 percent red zone scoring percentage. In 2013, he directed an offense that scored 414 points, the second-most in school history and the most since 1951.
Under Canales' guidance in 2013, quarterback Derek Thompson ranked second-best in school history for single-season passing yards (2,896), completions (251), completion percentage (64.4) and total offense (3,053). During the 2012 season, Thompson threw for a career-high 2,649 yards and set the school single-game record for completion percentage (89.3 pct.) against Kansas State when he completed 25 of 28 passes. Thompson finished his career with 7,457 passing yards and 42 touchdown passes. Canales spent a portion of the 2010 season as interim head coach at North Texas and led the team to a 2-3 mark.
Prior to joining North Texas, Canales served two stints at South Florida where he was the offensive coordinator from 2007-09 and the offensive coordinator/quarterbacks coach from 1996-2000. He directed a unit in 2008 that led the Big East Conference in scoring and finished second in total offense. He helped his team reach a national ranking as high as No. 2 during the 2007 season and No. 10 during the 2008 season. Under his leadership, South Florida quarterback Matt Grothe became the Big East Conference career leader in total offense.
Canales spent three seasons (2004-06) as the offensive coordinator at the University of Arizona. He was a part of Herm Edwards' staff with the New York Jets in 2003 where he served as wide receivers coach. He tutored Santana Moss, who finished among the top 15 NFL receivers that season with 74 receptions for 1,105 yards and 10 touchdowns.
From 2001-02, Canales was passing game coordinator and quarterbacks coach at North Carolina State, where he helped develop NFL All-Pro quarterback Philip Rivers of the San Diego Chargers. Rivers was a two-time All-ACC honoree and a Heisman Trophy candidate. North Carolina State finished 11-3 and ranked 12th in the final AP Poll in 2002, averaging 392.0 yards on offense. The offense featured a 3,000-yard passer in Rivers, a 1,000-yard receiver in Jerricho Cotchery and a 1,000-yard rusher in T.A. McLendon. Canales also spent time at Pacific (1995), Snow College (1987-94) and Brigham Young (1985-86). During his time at Snow College, the team ranked in the top 10 nationally in seven of his eight seasons. He also coached the softball team for seven years, leading the team to six region titles.
Categories: College, Sports, Sports – Local Sports News, Sports – UT Vols


It is a universal social security programme for all Indians.
APY will replace the Swavalamban scheme.
The 50% or 1000 Rs share of Government can only be enjoyed by the individuals who are not income taxpayers.
decrease in photosynthetic productivity of plants.
Increase in number of insects and other pests.
Which of the following specie is endemic to India?
Which of the following Environmentalists first gave the concept of Biodiversity 'hotspots' ?
Red Panda is not closely related to the Giant Panda, rather it belongs to the skunk and raccoon family.
In India it is found along the western Himalayas in parts of Himachal Pradesh and Uttarakhand.
The red panda (Ailurus fulgens), also called the lesser panda, the red bear-cat, and the red cat-bear, is a mammal native to the eastern Himalayas and southwestern China.
The red panda is the only living species of the genus Ailurus and the family Ailuridae. It has been previously placed in the raccoon and bear families, but the results of phylogenetic analysis provide strong support for its taxonomic classification in its own family Ailuridae, which, along with the weasel, raccoon and skunk families is part of the superfamily Musteloidea] Two subspecies are recognized. It is not closely related to the giant panda.


Pictured: The property at at 3900 Buck Road in Huntingdon Valley, Pa.
HUNTINGDON VALLEY, Pa. -- Now's your chance to own a piece of movie history in Montgomery County!
A more than 16,000 square foot estate in Huntingdon Valley, that was seen in the Rocky spin-off movie 'Creed,' is up for auction.
And the listing from Maxx Spann Real Estate and Auction Company says you can name your price!
The foyer, with its double staircase, was used as a setting in the movie. The brochure for the auction calls the staircase the "other famous Rocky steps."
PHOTOS: LA premiere of "Creed"
The property has its own movie theater, arcade game room, and a wet bar. Outside you'll find an in-ground pool, hot tub, and decorative statues.
It has six bedrooms, six full bathrooms five half bathrooms. The master bedroom features a fireplace, vaulted ceiling, and marble bath and dressing area.