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Using the human brain to understand "omics" in autism
Posted in autism on May 19, 2016| Leave a Comment »
by Shannon Ellis, Ph.D. at Johns Hopkins University
Shannon Ellis, PhD
Autism is a complex neurodevelopmental disorder with a definitively established genetic basis1. However, complete understanding of the genetic etiology remains elusive. While the role for certain CNVs in autism has been definitively established2,3 and exome sequencing studies have begun to uncover rare de novo mutations that play a role in the disorder4–9, we are far from identifying the names of the hundreds of genes likely contributing to the disorder.
While naming the genes that play a role in autism is critical, it has become increasingly clear that changes in the DNA sequence are only a first step toward complete understanding. Additionally, scientists acknowledge that they must know what the genes do, not only what they are. Therefore, our lab has begun to get a handle on what is altered at the level of gene expression and DNA methylation within the primary affected tissue in autism – the brain. This is part of a larger field called "omics": meaning "genomics" (the study of DNA sequence), "transcriptomics" (the study of gene expression), "epigenomics" (the study of how genes are expressed, "proteomics" (how proteins are regulated by DNA expression) and "metabolomics" (the chemical processes of making and breaking down compounds). Our study focused on transcriptomics and epigenomics.
To understand the processes in the brains of people with autism, we had to study the brains of people with autism. Therefore, we worked with the Autism BrainNet (formerly the Autism Tissue Program) to obtain brain samples and extract RNA. We were interested in RNA because it is produced from DNA. Using DNA as a template, RNA is formed as the first step in creating proteins. The proteins are what carry out the function of the cell. The number of copies of RNA is a reflection of the gene expression of the cell: the more copies, the more gene expression. While differences in an individual's DNA are undoubtedly of interest to the study of autism, it is also important to look for differences in gene expression and protein levels to fully understand the disorder. We used the RNA to look at the gene expression of about 14,000 genes. Additionally, in these same individuals, DNA samples were used to estimate methylation levels at cytosines across cytosine-rich regions of the genome.
How DNA is transcribed to RNA then translated to proteins
Last year, we published some results on the transcriptomic part. When we looked at the gene expression, we identified three groups of genes that showed different patterns of expression between autism cases and controls. The first two groups included genes that influence the way neurons work, how they interact with each other, and how they grow and communicate to form the human brain. Interestingly, the genes that showed differences in gene expression were different genes than those identified in previous studies that, rather than looking at gene expression differences, looked to identify DNA differences important to autism. This demonstrates that genes that have differences in their DNA are different from those genes showing downstream differences at the level of gene expression. The third group was made up primarily of M2-microglia genes, suggesting in increased immune response in the brains of autistic individuals. We want to stipulate that this does not mean that alterations in the immune system of the brain cause autism. It could be that abnormal gene expression in the brain triggers an M2 microglia response. Future work is required before we can determine if the increased immune response leads to or is a result of autism. Nevertheless, from a treatment standpoint, this work provides pathways that, despite variable genetic causes, can be targeted for treatment in affected individuals going forward.
We moved this further to compare the gene expression overlap in the brains of people with autism with other neuropsychiatric disorders. Previous studies have shown that there is an overlap in which genes have DNA differences across neuropsychiatric disorders. To establish if this overlap holds up at the level of the transcriptome, we compared gene expression differences across three disorders: autism, schizophrenia, and bipolar disorder. While there was little overlap between autism and bipolar disorder, there was significant overlap between the expression patterns in genes in autism and schizophrenia brains11, with consistent decreased expression at neuronal and synaptic plasticity genes across these two disorders. This work extended the known genetic overlap between neuropsychiatric disorders by establishing a relationship between alterations in gene expression found in both autism and schizophrenia.
Finally, as gene expression is directly regulated by DNA methylation, we looked to determine if DNA methylation differences play a role in autism. Methylation is a process where a methyl group attaches to a part of a DNA sequence and turns down the expression of that gene. To look at DNA methylation, we looked at cytosines. Cytosines are places in our genome where these methyl groups normally attach. Thus far, most work has studied CpG methylation. This refers to when a methyl group attaches to a cytosine (C) that is directly next to a guanine (G) nucleotide. This CpG context is where methylation most frequently occurs in the genome. However, methylation can occur at cytosines next to other DNA nucleotides (C, T, or A), and this is referred to as CpH methylation. As CpH methylation occurs at higher levels in the brain relative to other tissues, we did not want to limit our study to CpG sites alone, but rather wanted to look for differences in CpH methylation as well. We found increased levels of methylation at CpH, but not CpG, sites globally within the autistic brain. We are currently working on why there is this difference and why it is seen in autism, but as CpH methylation is largely specific to the human brain (as compared to blood or other cells), it is a particularly compelling finding.
While we acknowledge we have not answered all the questions, we now have a better understanding of what is going on in the brain of individuals with autism. In particular, in addition to further establishing a relationship between schizophrenia and autism, this work not only highlights a role for increased immune activation within affected individuals, providing a particular pathway to target when considering future therapies, but also, for the first time, suggests a role for increased methylation at cytosines within the CpH context in the brains of autistic individuals.
Gaugler, T. et al. Most genetic risk for autism resides with common variation. Nat. Genet. 46, 881–885 (2014).
Marshall, C. R. & Scherer, S. W. Detection and characterization of copy number variation in autism spectrum disorder. Methods Mol. Biol. Clifton NJ 838, 115–135 (2012).
Sebat, J. et al. Strong Association of De Novo Copy Number Mutations with Autism. Science 316, 445–449 (2007).
Sanders, S. J. et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature (2012).
Yu, T. W. et al. Using Whole-Exome Sequencing to Identify Inherited Causes of Autism. Neuron 77, 259–273 (2013).
Iossifov, I. et al. De Novo Gene Disruptions in Children on the Autistic Spectrum. Neuron 74, 285–299 (2012).
O'Roak, B. J. et al. Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat. Genet. 43, 585–589 (2011).
Neale, B. M. et al. Patterns and rates of exonic de novo mutations in autism spectrum disorders. Nature (2012).
De Rubeis, S. et al. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature 515, 209–215 (2014).
Gupta, S. et al. Transcriptome analysis reveals dysregulation of innate immune response genes and neuronal activity-dependent genes in autism. Nat. Commun. 5, 5748 (2014).
Ellis, S. E., Panitch, R., West, A. & Arking, D. E. Transcriptome Analysis of Cortical Tissue Reveals Shared Sets of Down-Regulated Genes in Autism and Schizophrenia. bioRxiv 29132 (2016).


KPVC Cork, Fascia, Soffit, Gutters, Downpipes, Cladding, Panelling, Window systems and combined wood PVC decking commercial industry in Ireland.
pvc decking suppliers in north ireland. Welcome to Decking NI Ltd Established in 2002, Decking N.I. Ltd has been in business for over ten years.


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Q: Bootstrap web page - centering the whole page I'm setting up a responsive web page with bootstrap, I want the whole page to be centered, with equal width margins on both sides. I've applied the css class to the container div:
 .centered {
    width: 95%;
    margin: 0 auto;
    }

This seems to work fine on lower resolution screens but for a higher resolution laptop,
the whole page still appears aligned to the left.
How do I fix this?

redpajama_set_name: RedPajamaStackExchange


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Q: Move an ImageView in a circular path I need to move an ImageView in a circular path.
The Specifications for the program:
1) Each ImageView has a meteor class that contains the ImageView's

*

*Current coordinates

*Target coordinates

*and a few other various variables and functions

2) When the target coordinates are set the ImageView will move to them at the correct speed
3) The equation for the path of the circle I am using is k+sqrt(-h^2+2*h*x+r^2-x^2) (Top half), k-sqrt(-h^2+2*h*x+r^2-x^2) (bottom half)
below is the code I use the calculate the target coordinates of the top half of the circle.
              if(meteor.getXCoord() == meteor.getTargetCoordsX() && meteor.getXCoord() != meteor.getH() + meteor.getR()) {
                     if (meteor.getYCoord() == meteor.getTargetCoordsY()) {

                         /*
                             b+sqrt(-a^2+2*a*x+r^2-x^2), b-sqrt(-a^2+2*a*x+r^2-x^2)
                          */

                         meteor.setDeltaX(meteor.getSpeedX() + meteor.getXCoord());
                         meteor.setDeltaY(meteor.getSpeedY() + meteor.getYCoord());

                         meteor.setTargetCoordsX(meteor.getDeltaX());

                         //where target coordinate y is set *****
                         meteor.setTargetCoordY((meteor.getK() + (float) Math.sqrt(-1 * meteor.getH() * meteor.getH() + 2 * meteor.getH() * meteor.getDeltaX() + meteor.getR() * meteor.getR() - meteor.getDeltaX() * meteor.getDeltaX())));

                         //bottom half
                     }

               }

My problem is, the target coordinate y becomes NaN according to logcat after the first run through.
Also, according to Log.d every value used to set target coordinate y is set properly.
Additional information:

*

*TargetCoord y & x are floats

*h, k, and r are floats

*delta x & y are floats

*current coordinates x & y are floats

Delta x & y are set above. The values used to set it are speed and current coordinates. speed is the number of pixels moved in 1 millisecond
Also, all meteor.methods have been tested, and work. I think the problem has to do with the calculation of target coordinate y.

A: It might be easier to use a little trigonometry: if you're rotating around (x0,y0) at constant speed, the coordinate updates can be written as
xn = x0 + (x-x0)*cos(u) + (y-y0)*sin(u)
yn = y0 - (x-x0)*sin(u) + (x-x0)*cos(u)

where u is the angular velocity. This avoids the need to take sqrt.


I wrote this review as a part of a Blog Friendly PR outreach campaign for CARD.com, I was compensated for me time but all opinions are my own.
The holiday season is upon us. And we're in search of the perfect gift. But what happens when you cannot find that perfect gift? Or the the person you are buying for is just so hard to shop for? How about Card.com?
Now, how exactly does one load more money onto your Card?
There is a $5.95 monthly fee after the first month. That fee is waived if you do direct deposit amounts over $800. However, if you don't choose to do this, then you can simply just make it a point to go shopping immediately! Your Card.com is good anywhere Visa is accepted. Or you can make cash withdrawals at one of the 22,000 in network ATM's.
I did get to try out Card.com. Sign up only took a few minutes and I was able to choose the design I wanted. It only took a few days to get the card in the mail and then only a a minute or so to activate the card. I chose to call in the activation, which really consisted of punching in the numbers on the card. Very easy.
When it came to shopping, I was nervous. What if it didn't work? I didn't want to be embarrassed that the card would be declined. But, guess what? It worked like a charm. And I was able to pick up some goggles for my son's swimming lessons and some yummy fuel for a race. It was easy.
Now, the question is, would I use Card.com again? Yes. I think the gift giving reason, ease of use, fun designs makes Card.com a great choice.
You can follow Card.com on twitter, Facebook, or pinterest.


As Thanksgiving approaches I see lots of people posting something they are thankful for each day; every single one of these things is a reason to celebrate! We shouldn't wait for a huge anniversary or major milestone to celebrate but enjoy every moment that we are given. Every day is a gift so why not acknowledge this gift with a little celebrating?
Moskato Life is a wine brand that believes in embracing life's milestones in achievable luxury. It offers a sweet, slightly bubbly wine in both Original and Rose.
I was recently given some Moskato Life samples to try and it will definitely become a staple in my wine cooler! Moskato is light and refreshing, I especially enjoy the light carbonation. In addition to tasting delightful, it also has a sweet and fresh scent. I'm not a professional "wine-taster" (although I've tried my fair share), but from what I understand, the scent is a large part of the wine experience in addition to the taste. I think I might even prefer this over standard Champagne bubbly as it has a sweeter taste.
In addition to celebrating every-day moments, Moskato is also great for celebrating large moments. One bottle is perfect for you and five friends to toast each other – be it a birthday, engagement or other amazing news.
Want to learn more about Moskato Life (and are over 21)? Head over to their website to find where to get it for yourself! You can also follow along on their Facebook and Twitter page for updates and promotions!
Thanks to Moskato Life for providing samples of this wine for me to review. All opinions are my own.
Oh this stuff sounds lovely! I'm a fan of bubbly drinks so I'm going to have to try this out. Plus, I love the design on the bottle!
The peacock feather is definitely a fun touch!
I LOVE me some Moscato! It's my favorite– I'll have to keep an eye out for this brand!
Moscato is currently my favorite type of wine (but only when its bubbly)! The Moskato Life website has a product locator so that could also help you find it near you.
Loving that bottle! I heart moscato!
I had a hard time putting the bottle in recycling, kept trying to think of other ways to use it!


River Road Campground - Wag!
Lake George State Forest is home to 21,000 acres of woodlands, swamps, and the River Road Campground. Visitors are welcome at the campgrounds year-round, where you can enjoy all kinds of camping activities such as hiking, birding, and fishing. The area is home to black bears and alligators, so be safe while you are out and about.
This area is surrounded by more than three miles of forest, and the St. Johns River is close by. Tent camping is all that is permitted at River Road Campground. There are restrooms with flushing toilets but no shower facilities. All campsites have a picnic table, fire ring, and grill. Non-potable water and portalets are available on site.
Fishing in the St. Johns River is extremely popular among visitors to the River Road Campground. River fishing and pier fishing are both options for guests. Many visitors like to clean and cook the fish in their campsites.
Walking and horseback riding trails are plentiful near the campgrounds. These hiking trails give guests access to the wilderness of the state of Florida.
Dogs are welcome at the campsite and on the trails surrounding the area. It is unclear as to whether leashes are required for dogs, but while in the campgrounds and the surrounding areas, you will want to keep your dog on a leash for safety reasons. Swamp areas are home to snakes and alligators, and the forests are home to bears and other wild animals. Use bear boxes to prevent bears from coming into your camping area.
Bird watching is common in the area as there are all kinds of beautiful birds that are hard to spot in other areas of the country. Hunting is permitted in the area, so be sure to exercise safety measures while you are in the campgrounds. It is easy for humans and dogs to be mistaken for wildlife from afar.


COLLEGE STATION -- Zach Calzada's first pass in Friday's Texas A&M spring game sailed away from his intended target and ended up somewhere near the bench.
But eventually, the true freshman settled down. By halftime, it was easy to see why Georgia tried to poach Calzada right before national signing day last December.
Luckily for A&M coach Jimbo Fisher, however, Calzada will wear maroon and white for the foreseeable future. And based on his early showing in Friday's intrasquad scrimmage, the quarterback will be one to watch during his time in College Station.
Of the three back-up quarterbacks who took reps during the final practice of the spring, Calzada was arguably the most impressive. The early enrollee was 6-of-15 passing for 103 yards and a touchdown through the first half.
With 9:46 left until halftime, Calzada rolled to his right, stopped and threw a crisp pass to fellow freshman tight end Baylor Cupp, who caught the ball and scored on the 31-yard play. The pass was an example of the velocity and polish Calzada had on his first-half passes.
When he enrolled at A&M out of Buford (Ga.) Lanier, he was considered a 3-star prospect by 247Sports.com's composite ranking. However, he was talented enough that Georgia coach Kirby Smart tried to flip Calzada away from the Aggies at the end of the 2019 recruiting cycle.
Once Calzada officially signed and NCAA rules allowed A&M coach Jimbo Fisher to speak freely about his new signee, Fisher showed praise on his new signee.
"Arm-talent wise, I think he throws as well as anybody in America," Fisher said in February. "He's a tough kid."
Coming into Friday's spring game, the No. 2 quarterback spot on A&M's depth chart was one of the biggest questions of the offseason. Former back-up Nick Starkel, a one-time starter, opted to transfer to Arkansas, leaving a key spot vacant.
In the final scrimmage of the spring, Calzada made a strong case to junior Kellen Mond's key backup. Of the three reserve quarterbacks on scholarship, Calzada had the best first half.
Redshirt sophomore Connor Blumrick, who had been running as the second-team quarterback during the public portion of spring practice, struggled early in the scrimmage. By halftime, he was 3-of-10 passing for 19 yards and one interception. Eighteen of those yards came on one completion.
Redshirt freshman James Foster, a 2018 signee, was efficient when he was on the field. Foster was 6-of-8 passing for 55 yards for the Maroon team.
But Calzada was the one who made the most impressive plays. In addition to making timely throws, he was able to use his legs to roll out of the pocket and evade the pass rush when necessary, a trait necessary to a quarterback's longevity. Even on a few incompletions, Calzada showed the potential Fisher saw when he offered him last year.
In February, the A&M coach said Calzada was only scratching the surface of his potential. Fisher went as far as comparing the 2019 signee with Green Bay Packers legend Brett Favre.
"You got an old-school type [of] guy," Fisher said. "For lack of a better term, a Farve-ish type guy who can just pick the ball up, play, and put guys around him and can make plays and make them win."
A&M hopes that will be the case when the time comes for Calzada to be the top quarterback on the depth chart. With Mond entering his junior year, that certainly won't be this year.
And even after his performance on Friday, Fisher may opt to keep Calzada behind Mond and Blumrick on the depth chart. But that could change as time progresses and Calzada turns into the player Fisher believes he can be.
"He has all the physical talents in the world," Fisher said in February. "He's really blossoming up now, with his size and potential. I think he has a tremendous future ahead of him."


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Les Ouled Jarir (en arabe: أولاد جرير) sont des nomades arabes de la région de Béchar dans le sud-ouest de l'Algérie.

Leur économie était basée traditionnellement sur l'élevage de chameaux avec un peu d'agriculture.

Références

Tribu sahraouie
Groupe ethnique en Algérie

redpajama_set_name: RedPajamaWikipedia


Second Generation WXWorx weather receiver also captures XM radio (audio) and feeds it to my intercom with a small cable. The communication between this receiver and the ATC on the yoke is Bluetooth.
External antennas mounted with velcro to the glareshield. XM radio antenna on the left, GPS external antenna on the right. The ATC will function without an external antenna, but with occasional loss of satellite signal.
Closeup of swivel ball on ProClip yoke mount for the iPaq 4705. Notice the yoke has 4 holes drilled and tapped in a standard AMPS hole pattern, so that it can support a variety of different mounting mechanisms.


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Q: AttributeError: 'PathCollection' object has no property 'hue' I'm trying to get to know seaborn functions. I was trying to execute the jointplot function as it appears on the seaborn website on JupyterLab 2.2.6
https://seaborn.pydata.org/generated/seaborn.jointplot.html#seaborn.jointplot
the code is as follows:
import seaborn as sns
penguins = sns.load_dataset("penguins")
sns.jointplot(data=penguins, x="bill_length_mm", y="bill_depth_mm", hue="species")

trying this I get a blank plot on screen and an error message that reads:
AttributeError: 'PathCollection' object has no property 'hue'

how can I resolve this issue?

A: Just update seaborn in the terminal: $ sudo pip install seaborn -U
To check your version, run this python command:
import seaborn as sns
sns.__version__


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Q: Monitoring rs232 traffic I need to monitor RS232 traffic, I'm communicating with a hardware device and i want to monitor that traffic.
Ideally i would want a Wireshark for serial communication?
Any suggestions ?

it actually pases through a USB, and gives me virtual COM interface.
MS windows...

A: I used this for a couple of days, but I think it is only a 30 day version, so you would have to purchase it for longer use...
However, the different options seemed quite good to me, especially because you have different views depending how deep you want to dig into the communication.

A: You can buy cheap RS-232 data scopes or you can just use an RS-232 breakout box and hook up another computer's Rx data line in tandem with your serial link so that you can watch the data using a terminal program.


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Q: How can I play Dying Light in Germany? The game is not available for sale here, and CD keys apparently will not activate on steam from this region. Could I activate a key on the steam website using Hola Unblocker, or would the gam refuse to download after being added to my account like Wolfenstein the New Order? Is there anything else I can do (that doesn't involve a vpn), or am I just going to have to import the PS4 version?

A: As this game is not prohibited to run in Germany, you can ask someone from a country where it is available to gift or trade it to you. You can then accept the gift and play it normally. This is an intentional loop hole in Steam and works with any Game that does not have the prohibitedToRunInCountries or onlyallowrunincountries flags on it's config, like seen here. This flag prevents you from playing the game while being connected with a German IP.
The advantage of a Gift is that you do not break the Steam Subscriber Agreement, which you do when activating such a game via a Proxy/VPN.
However, be advised that the developer could change his opinion anytime, but I have never actually encountered that and Techland has left the loop hole open for us with Dead Island 1+2 too. with DI1 it was even possible to activate imported Game Keys.
Source of knowledge: I own nearly every Game on Steam I have as uncut version, gifted by an american friend, e.g. South Park.


These signs were created from supplied camera-ready artwork. Our Art Department scanned and digitized the artwork for routing and then cut the material, 1/2" thick Sintra©. We are capable of cutting materials from 1/16" to 3" in thickness.


Most organisms share the same genetic code, based on three nucleotide codons that encode for one amino acid. However, synonymous codons (which specify a single amino acid) are not used at equal frequency by different species. This project assessed the impact of codon usage in protein production in the green alga Chlamydomonas reinhardtii and developed tools for testing gene variants.
Synthetic biology aims to apply principles of engineering to the construction of complex biological systems building up from readily available and well-characterized individual components, also referred as biological parts. Elements that control gene expression such as promoters and transcriptional factors were the first biological parts used in the construction of human-designed genetic circuits. As they are key components of genetic circuits, promoters have been progressively improved in order to drive inducible and tunable gene expression, while at the same time to be insulated from global cell physiology. These parts have been optimized mostly to work in bacteria, which is the favorite chassis in synthetic biology because of its simplicity to grow and amenable genetic manipulation.
The lack of compatibility of parts between organisms is one of the main challenges for the expansion of synthetic biology. It is frequent that parts that work in one organism do not reproduce their behavior in another organism. Although several reasons can lie behind this lack of compatibility, one of the first things to consider is the adequacy of the sequence to be expressed to the codon usage of the host organism. Although most organisms share the same genetic code based on three nucleotide codons that encode for a particular amino acid, synonymous codons that encode for a same amino acid are used in an organism-specific frequency.
Several features make Chlamydomonas reinhardtii an attractive target of synthetic biology: (1) it is a suitable microorganism for biofuel and valuable compound production; (2) cell physiology and metabolism are relatively well studied; (3) it is a haploid organism, and therefore gene manipulation is relatively simple and transformation protocols are well established; (4) it can be used as a platform to develop tools of synthetic biology for land plants, including crops. Several promoter sequences have been used to drive expression of sequences in this alga. However, there is no promoter sequence that allows tunable gene expression, and those which are inducible require conditions that create major changes in cell physiology and metabolism.
We would like to create a tool for gene sequence optimization for Chlamydomonas that refines codon selection by taking into consideration gene expression levels.
Additionally, our tool will allow a more money-efficient gene synthesis, as it will allow the user to control the level of optimization of the sequence. We will use these tools to create an optimized version of the TetON transcriptional factor, which is going to be the base of our synthetic gene expression system. This system has been successfully adapted to mammalian cells but has not been tried to our knowledge in algal systems. We will test different promoter architectures to control the expression of a reporter gene in a TetON-dependent fashion.
Independently of the levels of success of this project, our efforts will contribute to the development of synthetic biology in algae.
Most organisms share the same genetic code, based on three nucleotide codons that encode for one amino acid. However, synonymous codons (which specify a single amino acid) are not used at equal frequency by different species. We are interested in assessing the impact of codon usage in protein production in the green alga Chlamydomonas reinhardtii. We have performed sequence analysis, and developed a platform for measuring the production of a reporter protein, which can be used for testing gene variants. The analysis and materials generated will be useful for transgene design and expression in the alga.
Chlamydomonas reinhardtii is a useful model for plant biotechnology, especially algae. However, the unusual high GC content of Chlamydomonas coding sequences (68%, while Arabidopsis is 44% and human is 52%), strongly biases codon selection towards GC-rich codons, making codon optimization a necessary step for the expression in the alga of genes of other species. Codon optimization consists in replacing infrequent codons of a transgene sequence by synonymous codons used at higher frequency in the host species. This is a necessary step because tRNA molecules abundance is different among species and has co-evolved with codon frequency to optimize translation efficiency. A correlation between codon frequency and the abundance of the isoacceptor tRNA molecules has been reported for a number of organisms, both prokaryotes and eukaryotes (Angov, 2011; Ikemura, 1982). In the case of Chlamydomonas, although tRNA abundance has not been systematically analyzed, there is a good correlation between codon frequency and copy number of the corresponding isoacceptor tRNA gene (Cognat et al., 2008). The majority of codon optimization algorithms use a set of coding sequences of the host to compute codon usage reference values, irrespective of the expression level or translation efficiency of these sequences. This strategy has proven to increase translation efficiency, most likely by the removal of infrequent codons for which the amount of the acceptor tRNA molecule is limiting. However, less is known about the impact in expression level when different sets of genes are used to calculate the reference codon usage, or when the level of codon adaptation varies along the coding sequence. In order to explore codon usage in C. reinhardtii, and to provide the community with a series of tools for codon optimization and protein production, we proposed the following objectives for this OpenPlant fund: 1) to create a web site, with links to tools of codon optimization, describing their utility, and also with links to DNA synthesis companies, including user reviews, 2) to analyze codon usage of different sets of genes, based on expression level, GO term, and pattern of expression, and 3) to assess the impact of codon usage in the production of a protein reporter . During these six months, we have been able to make progress on points 2 and 3, and a detailed description of the work and results is presented below. We think that the website project proposed in point 1 could be useful for synthetic biologists and we would like to start working on it. We ask for the extra 1000 £ funding to cover website creation, diffusion and maintenance. The remaining funding will be used for DNA synthesis to expand the repertoire of Golden Gate-domesticated DNA parts that will be available to the Chlamydomonas community.
1.1. General features of codon usage in Chlamydomonas The high GC content of the Chlamydomonas reinhardtii nuclear genome strongly biases codon usage towards G/C rich codons (Table 1). A close look at the data available in http://www.kazusa.or.jp/codon, which is used by most codon optimization algorithms as reference set, reveals a strong bias towards G or C at the wobble position (3rd letter of codon). Codons of nuclear coding sequences end in a G or a C in 86.2 % of the cases. This contrasts with chloroplast gene codons, which are relatively GC-poor (Table 1), and with STOP codons, for which the codon TAA is used in most cases (1.05‰), followed by TGA (0.54 ‰) and TAG (0.44 ‰) (Table 2, "kazusa table"). In addition to the strong selection of high GC-content codons, a number of amino acids shows bias towards one particular GC-rich synonymous codon (e.g. in the case of Gly, GGC is used in 61.99‰ of the cases, while GGG is used in 9.72‰). This preference correlates with the copy number of the isoaceptor tRNA genes, 17 to 1 copy of the corresponding Gly-tRNAs genes (Cognat et al., 2008).
1.2. Codon usage in different gene datasets The previous data of codon usage comes from the web site http://www.kazusa.or.jp/codon/, and it was calculated from 846 CDSs. The sequences that are used to compute these values are selected only by the quality of the sequence data, and not by their similarity in expression level or translation efficiency. We then were interested in testing whether codon usage changes when it is computed from genes with high translation efficiency exclusively, or from genes with a common pattern of expression. There is no genome-wide data of translation efficiency in C. reinhardtii so far; however, data from other organisms shows a positive correlation between mRNA levels and translation efficiency (Gingold & Pilpel, 2011). Similarly, genes related to translation and ribosome biogenesis / structure are among the most efficiently translated genes (Ingolia, Ghaemmaghami, Newman, & Weissman, 2009). In order to obtain an insight into the differential use of codons in C. reinhardtii, we computed codon usage from the following gene datasets: -Kazusa codon usage table (Nakamura, Gojobori, & Ikemura, 1999). -All CDSs, obtain from primary transcripts only (17,741 sequences) -Highly expressed genes, (see below for their identification) -Genes associated with the GO term "structural constituent of ribosome" -Genes associated with the GO term "ribosome biogenesis" -Genes associated with the GO term "translation" -Genes associated with the GO term "photosynthesis" -Nitrogen starvation up-regulated transcripts (Schmollinger et al., 2014) -Nitrogen starvation up-regulated proteins (Schmollinger et al., 2014) -Nitrogen starvation-down regulated proteins(Schmollinger et al., 2014) -Clusters 1 to 18 of cell cycle regulated transcripts during Chlamydomonas life cycle (Zones, Blaby, Merchant, & Umen, 2015) -P-type gamete specific genes (Lopez et al., 2015) -M-type gamete specific genes (Lopez et al., 2015) -Zygote-specific genes (Lopez et al., 2015) Codon usage was computed for each gene dataset, and the total number and frequency (‰) of each codon is given in Table 2. For each amino acid, the synonymous codon with the highest frequency is highlighted. Codon usage was calculated using C. reinhardtii primary transcripts only (Creinhardtii_281_v5.5.cds_primaryTranscriptOnly.fa, 17,741 sequences), and gene sequences were downloaded from the most recent genome database (v5.5). Differentially expressed genes were obtained from published work, and Phytozome website was used to search for genes associated with specific GO terms. Highly expressed genes were identified in the following way: Tentative Consensus (TC) sequences and constitutive ESTs data were retrieved from the Dana Farber Cancer Institute (ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/Chlamydomonas_reinhardtii/). TCs were blasted against C. reinhardtii primary transcripts and the top hits were kept. Similarly, genes were ranked based on their normalized read counts from an RNA-Seq experiment (Valli et al., 2016). For the latter dataset, read counts from three biological repeats were averaged. Both ESTs and RNA-Seq read counts were normalized by the length of the CDS, and approximately the 1% top genes in each list (167 and 196 genes, respectively) were compared. 116 genes were common to both top lists, and were defined as "highly expressed genes". RNA for the RNA-Seq data was extracted from cells growing in TAP medium, 25°C, continuous light. We do not know about the growth conditions used for the EST experiment, but the high overlap with the RNA-Seq data suggests that similar growth conditions were used. Table 2 shows that the majority of amino acids are encoded by a highly favoured codon among all synonymous codons, and the same codon is preferentially used in all gene datasets analyzed. However, we found four amino acids (Ala, Pro, Ser and Thr) for which the preferred codon changes in different gene datasets. Ala and Pro show a similar pattern: codons GCG (Ala) and CCG (Pro) are the most used codons in "All CDSs" dataset, while in the "Kazusa table", "Highly expressed genes", "Ribosome biogenesis", "Photosynthesis", "Translation" and "Structure constituents of the ribosome" datasets, these amino acids are preferentially encoded by GCC and CCC, respectively. The codon GCC is also preferentially used in the Nitrogen Starvation groups, while the codon CCC is also the most used in one Nitrogen Starvation group and two cell cycle clusters. These alternative preferential codons are recognized by different tRNA molecules, indicating that there is an actual change in the tRNA pool selection among gene groups. Amino acids Ser and Thr use two different preferred codons in different datasets: the "Highly expressed genes" and "Structural constituents of the ribosome" use the codon TCG for Ser more frequently than the codon AGC, which is more frequent in the Kazusa reference table and "All CDSs" datasets. In the case of Thr, only the first cluster of genes of the cell cycle uses slightly more frequently the codon ACG, although the alternative codon ACG, which is dominant in the other gene datasets, has a similar frequency. Based on this analysis, we conclude that codon selection in C. reinhartii is very robust, characterized by a strong preference towards GC-rich codons and with only few alternative preferred codons. Differential codon usage only occurs for the amino acids Ala, Pro, Ser and Thr. Regarding codon optimization of transgene sequences, as most algorithms use Kazusa codon usage as reference, codon selection mirrors the codon selection of genes which potentially are translated with high efficiency ("Highly expressed genes" and ribosome/translation associated genes in our list), except in the case of the amino acid Ser.
1.3. Codon selection along coding sequences Codon optimization tools recode a transgene sequence using only favourite synonymous codons for each amino acid. However, endogenous sequences with such an ideal codon selection are unlikely to occur. We analyzed how distant is codon selection to the most frequent codon usage for each coding sequences of C. reinhardtii by calculating the codon adaptation index (CAI). The CAI of each gene is computed as the geometric mean of the relative adaptiveness () of all the codons in the coding sequence. The relative adaptiveness for codon of kind i that codes for amino acid j is defined as i = Xij / Xjmax , where Xij is the number of occurrences of codon i and Xjmax is the maximum Xij for amino acid j. Then CAI is calculated using the formula: where i(l) is the relative adaptiveness for the l-th codon in the gene and L is the number of codons in the gene (Sharp & Li, 1987). Figure 1A shows the frequency distribution of the codon adaptation index (CAI) for all genes in C. reinhardtii, using the codon usage of "All CDSs" as reference value. No gene has a CAI value of 1 and, therefore, there is no gene that is encoded only by favourite codons. Interestingly, when CAI is calculated only for the "Highly expressed genes" dataset, the distribution is shifted towards 1, indicating that codon usage of this group is closer to the preferential codon selection (Figure 1B). We also looked at a group of genes with no reads in the RNA-Seq experiment ("unmapped gene"), which probably comprises pseudogenes and genes with very low expression. The CAI distribution for this gene group look similar to the distribution of "All CDSs". We then examined how codon usage changes along the coding sequences. Analysis of the "Highly expressed genes" dataset revealed that unfavourable codons are interspersed within stretches of favourite codons, as it is shown in Figure 2A with an example of a coding sequence where the relative adaptiveness value (j) for each codon is indicated. Figure 2B shows a heatmap of j values for the first 200 codons of sequences longer than 600 nts from the "Highly expressed genes" dataset. We noticed that the frequency of unfavourable codons was specially higher in the 5' end of the ORF, which can be seen more clearly by averaging the j value (Figure 2C). We observe that the first codon after the ATG tends to be an unfavourable codon, and then codon adaptiveness increases along the first one hundred codon stretch, creating a sort of "ramp" of codon adaptiveness. This ramp is also observed in the set of "unmapped genes", although it is less pronounced than in the "Highly expressed genes" dataset. The latter observation of a stretch of incremental codon adaptiveness at the beginning of coding sequences could be explained by a mis-annotation of C. reinhardtii ORFs start codons, and these regions being actually part of the 5' UTRs and not ORF. However, similar stretches of low codon adaptiveness have been found in coding sequences of other species, and they have been proposed to act as ramps of translation, regulating ribosome trafficking along the mRNA molecule (Tuller et al., 2010). In this model, the first 30 to 50 residues of a protein are encoded by unfavourable codons that slow down translation initiation, allowing more efficient ribosome binding on the mRNA molecule and avoiding ribosome bottlenecking. Additionally, they could also have a role in coordinating translation with the in vivo folding of the protein.
1.4. Codon optimization of transgenes in C. reinhardtii. Our results show that, despite the high robustness in codon selection observed, there is some variability in codon usage for a number of amino acids. However, Ser is the only amino acid that differs between the set of highly expressed genes and the set of genes used to compute "kazusa" codon usage table, which is the reference set for codon optimization tools. It will be interesting to test the effect of alternative codons for Ser in protein production. A second observation from our analysis is the putative presence of a stretch of low frequently used codons at the beginning of the ORF. It will be also interesting to test its influence in protein production. These factors can be easily tested using the platform that we have developed (see below). Codon usage in C. reinhardtii has attracted recent interest. In a paper from Ralph Bock lab (Barahimipour et al., 2015), the relative contribution of the GC content and codon usage in protein production has been studied. By expressing different YFP gene variants authors concluded that codon usage rather than GC content was a key determinant of translation efficiency. In contrast, GC content was important for gene expression at the level of chromatin structure. It is noteworthy that the YFP gene variants were fully optimized using kazusa codon usage frequency, and none of the factors mentioned before (alternative codons and ramp presence) were tested.
In addition to the analysis of C. reinhardtii gene sequences, we also proposed to build a platform for testing the impact of codon usage in the production of a fluorescent protein reporter. Our experimental design, shown in Figure 3, consisted in the expression of different gene variants of a fluorescent protein (mVenus) that differed in the choice of synonymous codons. These gene variants are expressed using Chlamydomonas transcription regulatory sequences (promoters, terminators and UTRs). The construct also has a gene that confers resistance to an antibiotic, which allows the selection of cells that have incorporated the construct into their genome. The DNA cassette containing these genes can be integrated in any part of the genome, and therefore, its expression is strongly influenced by the transcriptional activity of the neighbouring region. To correct for this positional effect, we also included in the construct a second fluorescent marker (mRuby in the figure) that would allow to normalize the mVenus signal. The sequence of this second reporter is the same in all constructs. Chlamydomonas cell wall-less mutant CC-1883 was our initial choice as recipient strain since it has a high efficiency of transformation. To compensate for the variability between clones, we proposed to analyze a large pool of clones by FACS. The analysis of numerous clones is necessary because we estimate that less than 30% of clones will harbour the complete cassette. During the duration of this grant, we have been able to implement a versatile cloning strategy and test the expression of fluorescent reporters in different strain backgrounds. Although we have not been able to complete the experiment proposed yet, the progress that we have already made has proven to be useful for applications of transgene expression in Chlamydomonas.
2.1. Cloning strategy We decided to adopt a modular cloning strategy based on Weber et al. (Weber, Engler, Gruetzner, Werner, & Marillonnet, 2011). The strategy uses type IIS restriction endonucleases for the assembly of DNA sequences, and provides an inexpensive and fast method for multi-gene construction. Adoption of the syntax suggested by Patron et al (2015) (Patron, 2015) contributes to the standardization of the DNA parts constructed (Figure 4). This syntax consists in 12 fusion sites that enable the assembly and exchange of the different transcriptional units.
2.2. DNA parts A large fraction of the funding given to this project was used to synthesize DNA parts required for the constructs (Figure 5). These DNA parts were transcription regulatory sequences (promoters, UTRs and terminators) and also coding sequences corresponding to fluorescence reporters and drug resistant genes. These sequences were selected from a set of gene expression tools that have been extensively used for gene expression in Chlamydomonas. In all cases, DNA sequences were "domesticated" by removing the restriction enzyme sites that are required for the cloning strategy (BpiI and BsaI). Silent mutations were introduced when these sites fell on coding sequences. Domestication of HSP70-RBCS2 and PSAD promoters, RBCS2 5'UTR, and RBCS2 and PSAD 3'UTR-terminator sequences did not disrupt signals within for strong expression, as it is inferred from the high number of antibiotic resistant clones obtained when these sequences were used to drive the expression of a gene that confer resistance to the antibiotic paromomycin. The selection of fluorescent reporters was based on two recently published articles (Lauersen, Kruse, & Mussgnug, 2015; Rasala et al., 2013)). Rasala et al. 2013 shows that the autofluorescence of C. reinhardtii cells is low in channels 515/550, 554/590 and 575/608 nm (Ex/Em), and therefore, we chose mVenus/YFP, dTomato and mCherry fluorescent proteins. The mVenus gene described in Lauersen et al. (2015) gave a strong signal that could be detected in a fluorescent plate reader. This gene harbours an intron that authors suggest to boost expression. Regarding the second reporters, we have not been able so far to find a suitable fluorescent protein that could be used together with mVenus. We have expressed the red fluorescent protein mRuby2, also described by Lauersen, but we could not detect signal. We are currently testing a number of other red fluorescent proteins (mKate2, mPlum, mStrawberry and mCherry), some of them tested for first time in Chlamydomonas to our knowledge.
2.3. Chassis, transformation and fluorescence measurement The initial strain of choice to express the DNA constructs was the CC-1883. This is a cell wall-less mutant strain which can be easily transformed by the glass bead method (Neupert, Shao, Lu, & Bock, 2012), and it is routinely used in the Baulcombe lab. Transformants that integrated and expressed an antibiotic resistant gene were easily recovered in this background. However, expression levels of a fluorescent reporter were not sufficiently high and failed to be detected in a fluorescent plate reader. This low expression level of transgenes is a general feature of Chlamydomonas strains. Few years ago, Ralph Bock screened for mutant strains that efficiently expressed transgenes, isolating two different mutants, UVM11 and UVM4 (Neupert, Karcher, & Bock, 2009). We requested the UVM11 strain and confirmed that the mVenus fluorescent reporter was produced in this background to detectable levels. Therefore, we have switched to this strain in order to achieve high expression of our reporters. Although our initial plan was to use FACS for the quantification of fluorescence, we eventually decided to use fluorescence microplate readers instead. Plate readers provide a convenient method for quantifying the fluorescence of a large number of clones. We think that the sensitivity of the plate reader will be sufficient to detect differences in translation efficiency of our gene variants, provided a second fluorescent reporter is used for normalization. In the case that this method fails to detect these differences, an alternative method consisting in measuring fluorescence from native polyacrilamide gels can be used (Barahimipour et al., 2015). This method was recently developed by Ralph Bock group to measure differences of translation efficiency of a YFP.
2.4. Conclusions of our "wet lab" work We have presented here the progress developing a platform that can be used to measure the impact of codon usage in the production of a fluorescence protein reporter. Although the initially proposed experiment has not been carried out yet, the tools that we have created and the technology that we have implemented are already useful to the Chlamydomonas community for testing gene expression. With the funding received in this project we have created the first Chlamydomonas-specific MoClo DNA parts following the consensus syntax for gene assembly in Plant Synthetic Biology. Some of these parts are already being used by other researchers of the Plant Sciences department of the University of Cambridge, who will also contribute with new parts to increase the repertoire of DNA parts available for Chlamydomonas research. We think that this will be a major contribution for the development of synthetic biology in Chlamydomonas. Unfortunately, we have not been able to use exclusively open technology in this project. One main component, the efficient transgene expressing strain UVM11, was obtained under MTA signing, and therefore, its distribution is restricted. We think that a potential follow-up of this project could be the creation of alternative open source technology.
Chlamydomonas MoClo DNA parts: These parts will be deposited in the Chlamydomonas Resource Center (http://www.chlamycollection.org/) together with many other parts that are being constructed in the department of Plant Sciences of the University of Cambridge. Plasmid sequences will be available in this website too.
We would like to thank David Baulcombe for helpful discussions and giving his consent to carry out part of this project in his lab; Jim Haseloff and members of his lab for suggestions, reagents and access to equipment; Ralph Bock for sharing Chlamydomonas reinhardtii strains; Matthew Hartley for his support throughout the project. This project was funded by the OpenPlant Fund (July 2015).
Patron, N. J. . . . et al. (2015). Standards for plant synthetic biology : a common syntax for exchange of DNA parts. New Phytologist, 208, 13–19.
Sharp, P. M., & Li, W.-H. (1987). The codon adaptation index - a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Research, 15(3), 1281–1295.
FOLLOW-ON WORK We would like to apply for £1000 funding extension to accomplish the following objectives: -creation of a website dedicated to gene synthesis, with links to tools of codon optimization, resources and DNA synthesis companies, including user reviews, -extension of the repertoire of MoClo DNA parts, including new promoter sequences and other fluorescence reporters.


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Russia: There Will Be a Change
12/12/2016 /in Frontpage Article, Markets /by msv2016
In 2017 the planned ecology duty will become payable. According to the opinion of Russia's president Vladimir Putin, the money could help to construct waste recycling plants, the Russian news agency TASS reported some months ago.
According to the provided information, he has ordered the organization called "All-Russia People's Front"*) to control the situation with recycling, because he is concerned about the quantities of not recycled waste in Russia. In addition, Putin thinks this sphere is "criminalized".
Enormous market potential
On the one hand, Russia is the largest territorial state on earth (about 177 million square kilometers), but on the other hand it is one of the least populated countries (about 144 million inhabitants). However, the Russian amount of industry and household garbage is gigantic compared to other nations. According to the national Court of Auditors, waste in Russia, respectively in the Russian Federation, amounts to 90 billion tons per year. Household garbage has a share of solely 2.3 percent. A current disposal method is the deposition on landfills. According to Greenpeace Russia, the landfill area should cover 41,000 square kilometers. According to specialist media, there are about 1,000 legal landfills as well as 15,000 authorized waste deposits. Furthermore, there shall be about 30,000 illegal landfills. According to the information, 40 percent of industrial waste and 10 percent of household garbage are disposed of in an environmentally friendly manner. For this purpose over 50 large facilities for waste separation and sorting, about 240 facilities for processing of waste and 40 incineration plants were available in 2013. It was only a matter of time until measures have been taken to overcome the waste problem due to the fact that the Russian amount of waste is said to increase annually by around 5 billion tons.
Heretofore there has been a lack of legal conditions. According to Germany Trade and Invest, the economic development agency of the Federal Republic of Germany (GTAI), the Russian law on the protection of the environment from 2007 and the law on production waste and household garbage from 1998 had to be revised. The revision has already taken place in late 2014. Related to the novella of the environmental law, importers and producers of consumer products should set up a fee in the amount of 1.5 to 4.5 percent calculated from the value of the goods or alternatively establish a return and a waste management system or task an external company to perform this function. The implementation of the law was postponed to the beginning of 2017 due to the poor economic situation; the fight against recession and inflation had priority. According to the online-publication "Russia Beyond the Headlines" (RBTH), new amendments to the federal law "On Production and Consumption Waste" took effect on September 26th 2016. "Each region will have to decide how to deal with waste – to sort and recycle it, send it to a landfill, or incinerate it", RBTH reported. "Federal Funding will depend on the implementation of such programs."
Favorable economic situation
So far investments in environmental technology have barely been made due to the economic crisis and especially due to the fact that regions, cities and municipals had to deal with financing problems. However, the situation seems to be improving. According to the Russian Central Bank's opinion, the decrease of the economic growth in 2016 is considerably less severe compared to the previous year. Furthermore, the monetary authorities assume that the import of investment goods will rise again, the inflation will decrease and that the Russian Ruble will be less volatile. The Central Bank expects an increase of imports of investment goods, which is likely to continue in 2017 based on the current trends. This development is supported by the rate of the Ruble, which staggers less meanwhile. The contribution of exports to the growth of the gross domestic product will subside up to and including 2018.
According to experts, the Russian waste management will evolve into a multi-billion dollar business as a few examples show. Therefore, 15 new thermal power plants will be created in the Moscow area, which will use solid waste as fuel. Hereby the deposited volume of waste will shrink by 80 percent. A respective agreement was made among the Moscow area and a joint venture of the state holding company Rostec as well as the Japanese-Swiss company Hitachi Zosen Inova in 2015. There are plans to develop a system, which is adapted to the local conditions, for the garbage collection and transportation for the provision of the generating plant with fuels. The waste management shall be modernized in the Russian Far East as well. A total of 23 sites for the compressing of communal waste with subsequent land-filling are planned to be set up by 2020. Chabarowsk will be the central location for the extraction of secondary raw materials from communal waste, according to GTAI. According to the development and operating company for environmental projects, OAO UK Eco-System, 24.5 million cubic meter waste accrues annually in the Russian Far East.
Projects for plastics recycling
Eco-System intends to build a facility for plastic waste in Chabarowsk (near the border to China) from 2017 until 2019. The project is worth more than 30 million US-Dollar. The profitability of the facility would be given if at least 100 tons plastic waste (particularly from PET) are processed and worked up to secondary raw materials per year. The reason for selecting the regional "Territory of the accelerated social and economical development" (TOR) is to save taxes. The ministry for the development of the Russian Far East establishes via its subordinated development company nine territories in total in the Far East of Russia and one free harbor in Wladiwostok, according to GTAI information. After the realization of the project, Eco-Systems plans further facilities for the processing of aluminum and glass waste as well as recovered paper. However, the problem of selling future arising secondary raw materials is still unresolved. At the moment there are no commercial customers in the Russian Far East, but experts are already monitoring possible markets in the People's Republic of China.
Financing from public and private sources
According to the provided information, the federal law about "Public-Private Partnerships" became applicable in Russia in January 2016. Thus, this financing possibility is also an available option. Additionally, the Russian regions have an own PPP-regulation available. Furthermore, the federal legislation permits awarding concessions by the public authorities at all level of administration, including regions and municipal ties. It will be jointly funded by municipal and regional administrations as well as by the fund for the development of the Far East and the Baikal-region (russ.: FRDW); its sole shareholder is the Vneschekonombank (VEB), a state-owned bank for development and foreign trade. The operating company for environmental projects OAO UK Eco-System and the FRDW are the points of contact regarding projects in this region.
Sanctions Imposed on Russia
The European Union and the United States of America imposed restrictive sanctions on the Russian Federation in 2014 in reaction to Russia's annexation of the Crimea and the deliberate destabilization of an independent neighboring country. In July 2016 the EU extended the sanctions until January, 31st 2017. The sanctions are aiming for parts of the Russian economy. In return Russia imposed sanctions on the EU and the USA.
Sorting Plant to Be Built in Rostov Region
Within the framework of the International Economic Forum in Saint Petersburg, a deal on the construction of a unique environment-friendly unit was signed in June this year. It is intended to build a facility for sorting and processing of solid municipal and industrial wastes in the Volgodonsk district, located in the Rostov region of Russia. The project also includes a landfill site for irreclaimable wastes, optimization of a logistic scheme for wastes collection and transportation, as well as the construction of a waste transfer station, the Russian online journal construction.ru reported in June this year. Investments are estimated at nearly 15 million US-Dollar. Half of the sum will be allocated from the local budget, the rest will be financed by the investor.
Tula: New Landfill
According to media reports, a new solid waste landfill will be constructed near the Russian city of Tula which will be in compliance with all ecological norms and regulations. The wastes from Tula, the Leningrad district and Schekino will be brought there. It is intended that waste treatment facilities will also be built at the landfill which is designed to bury 360,000 tons of wastes. As reported, its lifespan is 16 years.
Integrated Foreign Economic Information Portal http://www.ved.gov.ru/eng/
Russian Investment Agency http://www.investment-in-russia.com/
Invest in Russia http://www.invest-rf.com/
*) This Front is intended to be a coalition between the ruling party and numerous nongovernmental organizations. In June 2013 Putin was elected as its leader.
Tags: GTAI, Hitachi Zosen, landfill, OAO, PET, plastics recycling, recycle it, Recycling, Russia, sort, waste management, waste recycling, waste recycling plant
https://global-recycling.info/wp-content/uploads/2016/12/russia-moscow.jpg 686 1030 msv2016 https://global-recycling.info/wp-content/uploads/2016/08/Global_Recycling_Logo-300-300x96.png msv20162016-12-12 08:22:252016-12-12 08:30:02Russia: There Will Be a Change
USA: Paper Recovery Rate Reached 67.2 Percent in 2016
U.S. Municipal Solid Waste Management Market to exceed $25bn by 2024
Apple's New Robot Disassembles iPhone to Reclaim Precious Materials
Future Market Opportunities for Problematic PET Bottles
Award-Winning Sustainable Recycling
Recycled Glass Market to Exceed $4.4bn by 2025 with 7.6% CAGR
Waste to Energy Market size worth $35.5bn by 2024 Sustainable Phosphorus Use

redpajama_set_name: RedPajamaCommonCrawl


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Vintage Trends and Smart Collecting Tips from a Legendary London Jewelry House
After nearly 150 years in business, the next generation of S.J. Phillips preps for a new chapter in a fresh location…
By Rachel Garrahan
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S.J. Phillips is one of the oldest family-owned antique jewelry stores in London
S.J. Phillips has long attracted the rich and royal to its doors in the heart of Mayfair. Established in 1869 by Solomon Joel Phillips, it is one of the oldest family-owned antique jewelry stores in London.
In addition to exceptional service, knowledge and an inventory that over the years has included jewels and precious objets from the likes of Empress Eugénie and Catherine the Great, it is the store's absolute discretion that has ensured its longtime success. Past clients of whom we are aware include the Queen's grandmother, Queen Mary; Margaret Thatcher; and some of the 20th century's greatest jewelry collectors, including the Rothschild family, Pierpont Morgan, and William Randolph Hearst.
Now, a new chapter of the business has begun following S.J. Phillips' move from its 113 Old Bond Street home of more than 50 years to nearby 26 Bruton Street, once the studio of the Queen's former couturier Norman Hartnell and across the street from where the Queen herself was born.
One thing remains the same: the family and the store's staff remain as passionate and energetic as ever. We asked Nicolas Norton, a member of the business's fourth generation, along with his brother Jonathan and cousin Francis, what the future holds…
Did the move uncover any forgotten treasures in the original store?
"We wish! We have a very efficient record system in place so every piece—however long ago it might have been purchased—is accounted for. There are several pieces, however, that we hadn't looked at for a while, so we enjoyed the trip down memory lane."
What are people looking for in antique jewelry, and what is particularly in demand right now?
"Most of the traditional pieces that we have dealt in over the past 150 years—outstanding examples of early 18th- and 19th-century jewelry, for example—are not as fashionable today as they once were. That said, they are still wonderful jewels and will always command high prices.
It is 20th century jewelry, however, that is more in demand now. So currently, we tend to focus on pieces dating from the first half of the 20th century by leading jewelry houses such as Cartier, Mauboussin, Boucheron, Chaumet, and Bulgari to name but a few. Nonetheless we continue to stock a wide range of jewelry representative of most periods and at all price levels, as it is always important to have something for everybody."
What do you see as the future collectibles, and what is less desirable?
"It's impossible to predict. We have always encouraged our customers to buy what they want, to believe in their own style, taste, and judgment, and not to be dictated to by the vagaries of fashion. Some may wish to purchase a fashion statement, which of course is absolutely fine. Generally, if people buy beautiful, quality pieces, they can't go wrong. Jewelry, from whatever period, is never out of fashion."
How has the business changed in the last decade, and what does the new luxury consumer want?
People have become more discerning. It is more difficult to sell ordinary things but easier to sell the finer items.
The new luxury customer has become ever more demanding in terms of originality and quality in what they are buying. In terms of service, they want greater security and confidentiality.
If the business isn't passed down through the family, what does its future hold?
We are looking forward to celebrating our 150th anniversary in 2019 in our new premises on the second floor of 26 Bruton Street in London. My brother, my cousin, and me are all in our 70s and as S.J. Phillips and the Norton family are synonymous, who can say what will happen beyond that?
What is S.J. Phillips' legacy?
We want to be remembered for the good things. From the beginning, we have been a very personal, family business offering a highly confidential service. Through hard work, persistence and diligence, we have had the privilege to deal in some of the world's most exquisite treasures—many of which are now in major museums around the world or important private collections. Our loyal customers have also been key. Many families have been buying from us for generations. I think our great-grandfather, S.J. Phillips would be very proud of our achievements. (sjphillips.com)
S.J. Phillips
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text:
Govt releases highly redacted COVIDSafe report
The federal government has been forced to release a report on the effectiveness of its controversial contact tracing app COVIDSafe, but has removed all parts relating to this and left only basic information and positive comments.
The report, released this week following a Freedom of Information request, has wholly redacted all of the parts relevant to its effectiveness and comes nearly a year after the government was required to release it.
COVIDSafe uses a smartphone's Bluetooth technology to log close contacts between users and then send these to a national database and state health authorities if a user tests positive for COVID-19. It was launched by the federal government with much fanfare in April last year.
Accompanying legislation enshrining privacy protections around the app in law required the Department of Health to report on the operation and effectiveness of the app and the national database every six months.
This report must be prepared "as soon as practicable after the end of each six-month period", and must then be tabled in Parliament within 15 days of completion.
Credit: Daria Nipot / Shutterstock.com
Despite it now being nearly 18 months since COVIDSafe launched, the government is still yet to produce any full report on the effectiveness of the app.
The Department of Health paid global research firm Abt Associates nearly $250,000 last year to complete the first of these six-monthly reports. The report covering April to October last year was handed to the government in March this year, but is still yet to be publicly released.
Following a Freedom of Information request by The Canberra Times, the government has this week released a highly redacted version of this report, with all analysis of the effectiveness and operation of the act removed.
The only elements left visible in the report relate to the basics of how COVIDSafe works and its launch, and positive elements, such as the quick timeframe it was developed.
The FOI release does reveal that the COVIDSafe report was handed to the Department of Health on 30 March this year. Despite being required to table it in Parliament within 15 sitting days of this happening, there have been 17 sitting days since then and the report still hasn't been released — a contravention of the government's legislative requirements.
Across 11 weeks from September last year, Abt Associates and technical partner Bdna conducted interviews with state and territory health authorities, along with staff from the Department of Health and the Digital Transformation Agency.
"Our evaluation used a mix-methods and multidisciplinary approach blending perspectives from public health evaluation and technology reviews to examine available quantitative and qualitative evidence on the appropriateness, effectiveness and efficiency of the COVIDSafe app and the National COVIDSafe Database," the report said.
"A high level technology review was undertaken according to the request for quotation which excluded COVIDSafe technological alternatives and recommendations for future innovations."
Abt Associates was paid $233,668 to produce this report.
All elements of the report relating to the actual evaluation and analysis of the effectiveness of COVIDSafe were redacted by the government, mostly under s47C. This exemption relates to information involving a "deliberative matter", such as an opinion, advice or recommendation that has been obtained, prepared or recorded.
This exemption is not meant to apply to reports of "scientific or technical experts…including reports expressing the opinions of such experts on scientific or technical matters".
The government did however leave in parts of the report saying it was a "commendable achievement" that COVIDSafe was developed in a short space of time, and another saying that the right model was chosen for the app.
"As our technology review indicates, based on the parameters of knowledge and capabilities at the time of app launch, it is believed that the COVIDSafe app was the correct tool to employ," the report said.
The report said that the key measure of success of the app is its "ability to identify individuals who meet the criteria of close contact".
COVIDSafe has not identified any of these people this year, despite significant outbreaks in New South Wales, Victoria and other areas of the country.
The app is not currently being used at all by NSW health authorities, the only state where it has identified unique close contacts previously.
COVIDSafe has found 17 new close contacts not identified by manual contact tracers in total, while a further 544 people were identified by health authorities after the app found a new exposure time at a NSW venue.
The government paid private contractors nearly $10 million for work on COVIDSafe, while it is still costing $60,000 per month to continue running.
Related: abt associates | COVIDSafe | Department of Health
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Richard Nelson 2 years ago
I don't think this report was written with a full understanding of how the app functions – let alone the nuance of some of the bugs found last year that severely affected its efficacy.
For example: "To further increase privacy around user identity, an update included functionality to randomly generate a new
temporary ID (Temp ID) every few minutes and exchange this in the Bluetooth handshakes instead of a user ID.". The app never exchanged "user IDs", the concept of TempID always came from Singapore's TraceTogether.
Then, "The app's current business rules allow for recording of digital handshakes when other app users are in close proximity (i.e. within 1.5 meters). However, encounters between two individuals are only logged if during the last 21 days there has been at least one single encounter lasting over 15 minutes".
The app itself logs every single exchange, and always did (this was shown by my reverse engineering of the iOS app prior to source code release, and others who reverse engineered the Android version). It has no "business logic" around 15 minutes or 1.5m. This logic is handled server side, and doesn't make sense as written here.
These misunderstandings in the extremely limited (redacted) technical detail cast serious doubts as to the overall correctness of this report. The fact that there is no comparison to efficacy as per the peer reviewed article by the NHS in Nature of their Exposure Notification enabled app is very telling.
Digital Koolaid 2 years ago
JANUARY 1965, Clark Abt opens a two-room office situated above a machine shop in West Cambridge (you already guessed that's Cambridge in the UK). 1966, ABT ASSOCIATES moves to its first designed and owned building at 55 Wheeler Street in Cambridge. 1975, ABT ASSOCIATES Turns 10. 2020, ABT gets paid a lot of money for a review in Australia because it was claimed nobody in this country could possibly do it, but of course we could. 2021, the government refuses to table the report required by legislation and redacts most of the FOI order, demonstrating that freedom of information and the law itself are non-extant in Australia.
Les Buckley 2 years ago
See no point of this ineffective app as it has been shown that new strains are transmitted in less than one minute.
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