Daily Archives: June 1, 2020

Research from the Max Planck Institute for the Science of Human History has demonstrated that when young birds are nurtured by their parents for extended time periods, they become more intelligent and better equipped for survival.

The study places new emphasis on the critical role of parents in the evolution of intelligence. Like birds, children need a supportive environment that helps brain development reach its fullest potential.

While observing two species of birds in their natural habitats, the team investigated the influence of parenting on learning in adolescence and survival in adulthood.

At a study site in Sweden, the researchers observed Siberian jays. The young jays live in family groups for up to four years under the care of a breeding pair.

Using a field test, the experts discovered that birds who stayed with their parents longer were able to learn faster. As a result, they were more intelligent, better equipped for long-term survival, and more likely to start their own family.

The experts also observed New Caledonian crows in the wild to understand how the young birds learn to make tools for food retrieval, which takes about a year.

The researchers found that Caledonian crows can stay with their parents for up to three years a childhood that is much longer than that of most other crows.

The team noted that parents and other adults were extremely patient with young crows. While adults used a tool to get food, they fed the juveniles and let them watch closely. The crows even tolerated tool theft and physical contact by juveniles.

According to the researchers, this tolerant learning environment is responsible for the large brain size and intelligence of New Caledonian crows.

The study authors argue that the influence of parenting on the evolution of cognition has been overlooked. Parental care gives children the time and opportunity to make mistakes and learn from them.

Extended parenting has profound consequences for learning and intelligence, explained study co-author Michael Griesser.

Learning opportunities arise from the interplay between extended childhood and extended parenting. The safe haven provided by extended parenting is critical for learning opportunities. It creates extended developmental periods that feed back into the extended childhood.

The study is published in the journal Philosophical Transactions of the Royal Society B.

By Chrissy Sexton, Earth.com Staff Writer

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Parents play a crucial role in the evolution of intelligence Earth.com - Earth.com

And has seemed to embrace the last few months.

A great event and a great cause ... winners all around! It was so fun having millions watch me shank shots, ask for lost ball rulings, and rip my pants wide open! Brady said online.

Brady has been in the public eye for a long time and does just fine as the center of attention on football Sundays. Hes often, though, seemed uncomfortable in that same glare off the field. He has cited a desire for privacy often and avoided politics. Some of his most telling interviews were those he walked out of as means of declining to engage in their subject matter.

That has changed. Whether Brady wanted to keep his life to himself or didnt feel he was allowed to be as open as hed have liked to be before this offseason, the quarterback is suddenly willing to put himself out there in ways he hasnt been in a long time.

For example: Hes starting a production company. Hes doing a nine-part documentary with ESPN about himself. He was mostly micd up for an entire Sunday of golf that was seen by a record-setting average 5.8 million homes reaching even more viewers once the pants incident went viral.

The only thing I saw from TB12 and them was he split his pants at one point and he sunk a hole-in-one, said Jason McCourty, errantly referring to the birdie Brady made from the fairway. So, I was happy to see he split his pants. The next time I see him, Ive got to give him a hard time.

Brady has also recently shared that his wife, Gisele Bundchen, at times, was unhappy with their marriage as a result of his time commitment to football. He has also told us (on TikTok) that he exercises more than she does but also spends more money, has more clothes, and is more of a whiner when sick. Hes posted selfies with goofy filters and photoshopped a Caddyshack reference. He is, as the kids say, very online.

More significantly, Brady has delved into the political. He has requested, via an Instagram post, justice for George Floyd, the Minnesota man who died last Monday after a Minneapolis police officer knelt on his neck while arresting him. Earlier in May, Brady signed a letter from the Players Coalition to the Justice Department demanding a federal investigation into the February killing of another Black man, Ahmaud Arbery.

He has screenshot a TMZ post about his accidental barging-in on a Tampa man whose home he mistook for that of Buccaneers offensive coordinator Byron Leftwich. He addressed a mini-controversy on social media, screenshooting a report claiming tension between he and Patriots offensive coordinator Josh McDaniels and captioning it please stop this nonsense! Please be more responsible with reporting.

Beyond the confines of the Internet, hes also selling his custom Escalade, with its blacked-out windows and privacy curtains. Hes currently living in a home (Derek Jeters, if you havent heard) in Tampa that he told Howard Stern is far less private than the Brookline manse his family inhabited for the last few years.

Where I lived in Chestnut Hill, I was pretty private for a long time, Brady said. So I forgot, in a way, like people could drive up to your house. You couldnt drive up to my house where I lived in Chestnut Hill. Here, they could pull right up to the back of the house.

This is a little different because when you go out to the backyard, theres a lot of boats that have pulled up and people out the front.

Oh, also, he did multiple hours with Stern.

Maybe this was part of the plan. Its smart business Bradys Twitter-follower count has grown from 977,730 on March 11 to more than 1.2 million now, the new followers roped in by the 37 tweets (out of a lifetime total of 206) hes sent since then.

Maybe it just makes him happy.

He wanted to go somewhere and enjoy himself, have more fun than he was having, Bradys friend Jay Feely said during an guest spot on NFL Networks Good Morning Football, speaking of Bradys change of scenery. He earned that right. He was a free agent and well see how that ends up.

The degree to which Patriots players are discouraged from having active presences online, and otherwise with the public, is usually overstated but still a factor.

Maybe it took the confluence of a global pandemic that requires the public to stay inside and become round-the-clock content consumers and the biggest change of his professional life to get Brady on TikTok, but there he is.

This has not all gone over well in New England. While Brady stories continue to generate tons of interest, plenty of Patriots fans including those still rooting for the quarterback, as long as hes not playing New England have voiced the opinion that its getting tiring hearing about his every move. (Anyone who has ever social media-stalked an ex knows that these things can both be true.)

Beyond the specific dynamics of this offseason, though, its fascinating to watch one of the NFLs defining players change this way. Bradys always done most of his talking on the field, but hes starting to get chatty in other arenas, as well.

Nora Princiotti can be reached at nora.princiotti@globe.com. Follow her on Twitter at @NoraPrinciotti.

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The social evolution of Tom Brady: From Foxborough to TikTok - The Boston Globe

For as long as living things have been building proteins based on the code carried by messenger RNA molecules, aminoacyl-tRNA synthetases have been there. These enzymes, AARSs for short, link transfer RNAs (tRNAs) to the corresponding amino acids. That would seem to be a big enough job for one class of enzymesand when protein-based life began, it was. But as organisms became more complex, AARSs picked up additional domains that allow them to do much more.

By the time you get to humans, the synthetase has become highly decorated with those additional domains, says Paul Schimmel, a Scripps Research Institute biochemist who studies these add-on jobs.

Living things possess at least one type of AARS molecule for each of the 20 proteinogenic amino acids. For some amino acids, there are two varieties, with a separate enzyme for use in protein translation that takes place in the mitochondrion. All of these synthetases have a core segment that is involved in linking tRNAs and amino acids, and all but one harbor one or more additional accessory domains. Plus, by alternatively splicing their mRNAs or fragmenting the proteins post-translationally, cells can make more than 300 different protein variants from AARS genes. Some of these variants moonlight as inflammatory cytokines. Others orchestrate the formation of blood vessels. The AARSs for glutamic acid and proline are merged into a two-part protein; the linker between them seems to control immune activity and fat metabolism, and may even influence life span. Many AARSs have been linked to human diseases caused by defects not in protein assembly, but in these other, noncanonical functions.

I heard how skeptical the field was about those discoveries. I dont blame them. I would be confused too.

Xiang-Lei Yang, Scripps Research Institute

Some researchers now view the enzymes as drug targets for cancer, immune disease, and other conditions. The company Schimmel cofounded, aTyr Pharma in San Diego, envisions the AARS proteins themselves as an entirely new class of drugs, distinct from small molecules or other biologics. The firm is currently running a clinical trialtesting a piece of the histidine enzyme, HisRS, for treating inflammatory lung disease.

Alternative AARS functions have been known in lower organisms such as bacteria since the 1980s, but their activities arent extensive, says Schimmel. Then, starting in the 90s, Schimmel and others began to uncover noncanonical functions in higher eukaryotes, starting with unexpected roles in angiogenesis. The discovery of new functions for these ancient proteins was a big surprise, says David Dignam, a biochemist at the University of Toledo. But given the diverse functions that researchers studying AARSs have uncovered, many of which touch on crucial disease pathways, Dignam says he thinks aTyrs approach makes sense. Arguing that you can make medicines based on this, I think, is very logical.

While other proteins have adopted secondary functions, the quantity and variety of side gigs found in the AARSs is remarkable, says Schimmel. And he doesnt think its a coincidence. These particular synthetases have been present and available for evolution to modify since protein-based life began. Given their essential role in protein synthesis, theyre consistently produced, and unlikely to disappear from any viable genome. That makes them a stable substrate for new functional domains. Moreover, they possess specific amino acid binding sites, ready to interact with other proteins.

Its lock and key, says Schimmel. Any protein that sticks out a nice side chain that matches a synthetase could eventually become a partner.

Schimmel says hes long been fascinated with AARSs original function: interpreting the genetic code. Back in the 90s, Schimmels lab, then at MIT, was sequencing the AARS genes. We were interested in developing small molecules that would target them and kill their activities in specific ways, he says. For example, if the AARS of a pathogen was different enough from that in people, he reasoned, one could develop an antibiotic that shuts off protein synthesis in the infectious agent.

Schimmels then-postdoc Keisuke Wakasugi got curious about the sequence of the gene encoding TyrRS, the AARS for tyrosine. In humans, TyrRS includes an extra segment at the carboxyl end of the enzyme, a feature that isnt present in prokaryotes or lower eukaryotes. The amino acid sequence for this part of the protein was similar to that for a human cytokine, EMAP II, which recruits circulating immune cells into tissues to promote inflammation. Wakasugi decided to test that carboxyl domain for cytokine-like activity.

Thats a silly idea, Schimmel recalls thinking. But Wakasugi went ahead, and sure enough, the TyrRS carboxyl domain acted just like EMAP II, inducing cultured phagocytes and leukocytes to migrate and release inflammatory signals. The full-length TyrRS, in contrast, didnt influence the cells behavior. That hinted at the possibility that the carboxyl domain could be broken off the TyrRS for immune functions. No one in the lab would believe the finding at first, so Wakasugi repeated the experiments, with the same results.

Although it would take more than a decade to show that such AARS fragments were truly present and relevant in a living animal, Wakasugi knew he was onto something. Paul and I were very excited to discover a novel and unexpected function of human TyrRS, recalls Wakasugi, now a biochemist at the University of Tokyo. Throughout this project, I felt that we opened the door to a whole new research field.

As part of the same study, Wakasugi also investigated the amino-terminal, catalytic domain of TyrRS, wondering if it might also influence cell migration. It behaved in a manner reminiscent of the cytokine interleukin-8 (IL-8). Both the TyrRS amino-terminal fragment and IL-8 bind to the IL-8 receptor on certain leukocytes, causing them to migrate in culture.

Aminoacyl tRNA synthetases are crucial players in protein synthesis, linking tRNAs to the amino acids dictated by the codon sequence. All AARSs have also been found, in diverse in vitro and in vivo systems, to play non-protein synthesis roles in a number of body systems. This table includes a sampling of the more well-studied examples.

Schimmel recruited Xiang-Lei Yang, a postdoc with expertise in structural biology, to join his lab at Scripps in La Jolla, California, to investigate how TyrRS might manage alternative functions. Yang zeroed in on a particular sequence of amino acids, glutamic acidleucinearginine, required for the synthetase fragments cytokine activity. The same sequence was also found in IL-8 and related cytokines. In crystal structures, she found that full-length TyrRS buried this motif, but it was exposed in the cytokine-like fragment.

IL-8 was known to promote the formation and growth of blood vessels, so Wakasugi also tested his TyrRS amino-terminal fragment for angiogenic activity. When he injected a bit of gel containing the fragment into mice, blood vessels grew and suffused the gel. To explore that action further, Schimmel phoned his Scripps colleague Martin Friedlander, an ophthalmologist and cell and developmental biologist, and asked him to test the TyrRS fragment in his mouse models of eye vascularization. Friedlander agreed, but also asked for a control. So along with the human TyrRS fragment, Wakasugi provided a natural splice variant of the tryptophan enzyme, TrpRS, that lacks the glutamic acidleucinearginine motif.

The results, Friedlander recalls, werent exactly what he expected. TrpRS, the supposed control, had a much more potent effect, says Friedlander, who is also president of the Lowy Medical Research Institute in La Jolla. But that effect was the opposite of TyrRS action: rather than promote angiogenesis, as Wakasugi had seen in the gel, the TrpRS fragment blocked it in mammalian cell culture, chicken embryos, and mouse eyes. TyrRS and TrpRS may have evolved as opposing regulators of angiogenesis, says Wakasugi.

Scientists were initially resistant to the idea that an AARS could have functions beyond protein synthesis. Yang recalls attending a conference, shortly after Wakasugi published his work on angiogenesis, where others were unaware that she was a Schimmel acolyte. Thus incognito, I heard how skeptical the field was about those discoveries, she recalls. I dont blame them. I would be confused too.

Aminoacyl-tRNA synthetases play a fundamental role in protein translation, linking transfer RNAs to their cognate amino acids. But in the hundreds of millions of years that theyve existed, these synthetases (AARSs) have picked up several side jobs. One of these is to manage the development of vertebrate vasculature.

Multiple AARSs play roles in the development of the vertebrate circulatory system. During development, the serine enzyme SerRS downregulates the expression of vascularendothelial growth factor A (VEGF-A), preventing over-vascularization.

In addition, a combo synthetase for glutamic acid and proline, GluProRS, links up with other proteins to form the interferon- activated inhibitor of translation (GAIT) complex to block VEGF-A translation.

A piece of the tryptophan synthetase TrpRS also contributes to dampening angiogenesis by binding and blocking VE-cadherin receptors on endothelial cells so they cant link together to form blood vessel lining.

Meanwhile, a fragment of the tyrosine synthetase TyrRS appears to promote the growth of blood vessels by stimulating migration of those endothelial cells.

According to Scripps Research Institute biochemist Paul Schimmel, the addition of accessory domains that perform such tasks parallels major events in the evolution of circulation. The first blood vascular system, which lacked the endothelium present in modern vertebrates, probably arose in a common ancestor of vertebrates and arthropods around 700 million to 600 million years ago. Around this same time, TyrRS acquired a glutamic acidlysinearginine motif that today is thought to promote angiogenesis. Then, around 540 million to 510 million years ago, an ancestral vertebrate evolved a closed vascular system, with blood pumping through vessels lined by endothelium. At some point around that same time period half a billion years ago, the TrpRS picked up a WHEP domain, which today regulates its ability to block angiogenesis. In addition, SerRS acquired a domain unique to this enzyme, which now prevents over-vascularization in developing zebrafish, and likely other vertebrates.

GluProRSs role in angiogenesis, on the other hand, doesnt seem to be so precisely timed to the evolution of vasculature. A linker protein tied together the AARSs for glutamic acid and proline enzymes around 800 million years ago, before circulatory systems existed.

While the TyrRS and TrpRS functions Wakasugi and colleagues had discovered were interesting, it wasnt clear that the enzyme fragments genuinely performed these functions in vivo. Yang realized that to give herself and other scientists confidence about noncanonical functions of AARSs, shed have to find evidence that they were present in animals.

The team still hasnt done so for TrpRS or TyrRS, but Wakasugi found her opportunity with the serine enzyme, SerRS. Multiple published genetic screens in zebrafish had identified defects in vascular development when SerRS was mutated. But mutations that knocked out the enzymes ability to link tRNAs and amino acids did not cause such defects, indicating that something else was going on.

To figure out what, Yang turned to a sequence, christened UNE-S, that is found in vertebrate, but not invertebrate, SerRS. Yangs teamshe joined the Scripps faculty in 2005, and now shares a lab with Schimmelquickly identified a nuclear localization sequence within UNE-S, and determined that mutations altering this signal caused the vascular defects in zebrafish embryos. In the nucleus, they found, SerRS seems to minimize the expression of vascular endothelial growth factor A (VEGFA). The study, published in 2012, was the first to illustrate an essential, natural role for an AARS accessory domain in a living animal. Shortly thereafter, the team reported that nuclear SerRS blocks VEGFA by competing and interfering with c-Myc, a transcription factor that normally promotes the genes expression.

Meanwhile, Schimmels and Yangs groups continued to try to explain the noncanonical functions of TrpRS and TyrRS, even as they found more side gigs for these enzymes. Yang led studies on the TrpRS fragments structure and mechanism. She discovered that full-length TrpRS doesnt influence angiogenesis because its capped by a WHEP domainso called because this domain appears in aminoacyl tRNA synthetases for tryptophan (W), histidine (H), glutamic acid (E), and proline (P), as well as in the glycine and methionine enzymes. Yangs team found that when uncapped by proteases in the extracellular space, TrpRS binds to a cellular receptor called VE-cadherin. Specifically, tryptophans in the receptor seemed to enter the TrpRSs active site to create the bond. Thats why Wakasugi saw that only the fragment, not the full TrpRS, blocked angiogenesis.

More recently, Schimmel has also been interested in plant-based amino acidlike compounds, such as resveratrol, the stuff in red wine thats thought to counter oxidative stress. Resveratrol and tyrosine are similar in that both contain a phenolic ring, and thats important for resveratrols ability to influence the expression of pro- and anti-oxidative genes. In 2015, Schimmels team reported that under conditions of stress, TyrRS moves into the nucleus of human cultured cells or living mice, where any resveratrol present fits neatly into TyrRSs active site. This turns off the normal TyrRS catalytic activity to connect tyrosine molecules with the appropriate tRNAs. Instead, TyrRS stimulates the activation of PARP-1, an enzyme involved in DNA repair.

A few years later, the team found that an alternatively spliced version of TyrRS stimulates platelet proliferation in mice and cultured cells, and could potentially be used to treat people with a low platelet count.

Schimmel expects noncanonical AARS functions will keep the group busy for a long time. We are barely scratching the surface of what is to be learned, he says. I am as excited, or even more excited, about these enzymes as I was when I started out decades ago.

As evidence of noncanonical functions for AARSs was trickling out of Schimmels lab, Paul Fox, a biochemist at the Cleveland Clinics Lerner Research Institute, was studying the control of inflammation in macrophages. Specifically, his team was investigating a complex generated when the cells were exposed to the cytokine interferon-. A protein complex called GAIT (for interferon- activated inhibitor of translation), generated within macrophages, binds to and blocks mRNAs related to inflammation. Inside the complex, the researchers found GluProRS, an enzyme that includes the AARSs for glutamic acid and proline.

We ran into it just absolutely by accident, Fox recalls. I didnt think it was an interesting enzyme. But he knew of Schimmels work, and he picked up the phone to call Scripps.

One minute into the call, Schimmel interrupted to welcome Fox to what Schimmel called the most exciting area of AARS research: the noncanonical functions. Schimmel also promised his assistance, Fox says. Hes been a big supporter and a friend ever since. With tools supplied by Sunghoon Kim, a former Schimmel lab postdoc now at Yonsei University in South Korea, Foxs team discovered that interferon- causes GluProRS to become phosphorylated, abandon its post in translation, and join up with the other GAIT members to halt the production of inflammatory proteins.

Its not clear why the glutamic acid and proline synthetases paired up approximately 800 million years ago, but Fox has a hypothesis, which he published in 2018. Proline is synthesized from glutamic acid, and at that period in evolution, emerging animal proteins began to include more proline. That may have led to a rise in the production of ProRS that sopped up all the available proline, requiring more to be made from glutamic acid. That might have resulted in a deficit in glutamic acid levels, impairing protein synthesis. The solution to that was to fuse the two synthetases into a single gene, so they have to be made in the same amounts, says Fox. No ones stealing from the other.

The linker between the two synthetases is crucial for GAIT complex activity; its made of three WHEP domains that bind to target RNAs. Fox speculates that sometime after the linker appeared, the cell coopted it to regulate inflammation.

More recently, Foxs team wondered if the GAIT pathway might function in cells besides macrophages. When the researchers looked at fat cells, they saw that insulin treatment caused GluProRS to become phosphorylated and leave the protein-synthesis machinery. But it didnt join the other GAIT partners. Instead, it paired with a normally cytosolic protein called fatty acid transport protein 1 (FATP1). Together, the molecular duo went to the fat cells membrane, where the transporter brought fatty acids into the cell.

I am as excited, or even more excited, about these enzymes as I was when I started out decades ago.

Paul Schimmel, Scripps Research Institute

The researchers engineered a mouse lacking the phosphorylation site needed to free GluProRS to find FATP1. With less fatty acidstorage ability, the mice were lean, weighing about 15 percent to 20 percent less than control animals. Moreover, they lived nearly four months longer, giving them a lifespan that was increased by about 15 percent. A similar gain in people would correspond to a decade or more. If we could target that phosphorylation site, maybe we could increase life-span, says Fox. His lab is in the very early stages of looking for a small molecule to inhibit that phosphorylation event.

In the various jobs that AARSs have taken on above and beyond their traditional role, Schimmel and colleagues see a theme: they keep cells and bodies stable. They seem to be something thats playing a modulatory role, restoring more of a homeostasis, says Leslie Nangle, a former Schimmel lab grad student who is now senior director for research at aTyr Pharma. Many researchers think its risky to mess with such essential enzymes, says Kim, but he and Schimmel see potential in targeting AARSs for treating disease. Schimmels company aTyr, of which Kim and Yang are also cofounders, hopes to turn the enzymes themselves into biologic therapeutics. In addition, in Seoul, Kim directs the nonprofit drug discovery organization Biocon, where researchers are developing several small molecules that interact with AARSs, as well as biologics based on natural AARS variants.

Biocon is currently testing molecules to treat cardiac fibrosis, alopecia areata (an autoimmune disease that causes hair loss), and inflammation. A fibrosis treatment now under Phase 1 study targets the site on the proline synthetase that links the amino acid to its tRNA. Fibrosis results from an accumulation of collagen, which is two-thirds proline. Biocon researchers have found that a drug can go after that active site, knocking down the canonical function by more than 90 percent in healthy cultured cells without greatly affecting the synthesis of other proteins or cell proliferation, says Kim. At first, he and his colleagues didnt believe their results, but hes come to see sense in them. A normal cell is not necessarily doing high level protein synthesis all the time, he says. As long as it has a certain degree of residual activity going on, then a normal cell can be perfectly happy.

For cancer and other conditions, Biocon is developing small molecule candidates that avoid the tRNAamino acid linking site or target the extracellular activities of secreted AARSs, meaning that protein synthesis should not be affected. Similarly, aTyr researchers expect that the firms therapeutics, based on AARS derivatives themselves, to be relatively safe. Coming from a world of natural physiology, you start to feel better about it, says aTyr CEO Sanjay Shukla.

In the developing mouse retina, fragments of the tryptophan synthetase, TrpRS, that are missing a restrictive protein cap (B, C) prevent vascularization of a secondary tissue layer (right). (The right images include the shadows of the adjoining primary layers, which are shown at left.)

PNAS, 99:17883, 2002, 2002 NATIONAL ACADEMY OF SCIENCES

Nangle and colleagues, alongside aTyrs subsidiary Pangu Biopharma in Hong Kong, began by cataloging natural AARS splice variants and then screening them for interesting biological activitiesin a variety of human cellbased assays. They looked for effects on cell proliferation and protection, immunomodulation and inflammation, cell differentiation, transcriptional regulation, and cholesterol transport. We figured theres got to be some therapeutic benefit there, says Schimmel.

Currently, aTyr is pursuing an immuno-modulator based on the WHEP domain of the histidine enzyme HisRS. In human T cell cultures, full-length HisRS quieted activated cells and reduced cytokine production. In further experiments, aTyr researchers found that the WHEP domain hooks up with receptors on those immune cells to dampen activity. The company hopes that its modified version of the HisRS WHEP peptide, attached to a bit of antibody to help it last longer in the bloodstream, will have the same quieting effect in an inflammatory disease called sarcoidosis. This disease affects a variety of organs, most often the lungs, and can sometimes require lifelong treatment with immune-suppressing steroids. Those medications come with a list of misery-inducing and dangerous side effects ranging from insomnia to glaucoma to infection.

aTyr presented results from several animal models of inflammatory lung disease at the American Thoracic Society meeting in 2017, 2018, and 2019, and those findings suggest the companys candidate 1923 looks promising. For example, the cancer drug bleomycin can cause lung damage, but HisRS or its WHEP domain reduced inflammation and fibrosis.19 Rats treated with bleomycin breathe quickly to compensate for their damaged lungs, but those treated with 1923 recovered normal respiratory rates.

aTyrs 1923 has already been through a Phase 1 trial for safety in healthy people without any red flags. Now, the company is running a Phase 1/2 study in people with sarcoidosis, looking to confirm safety, find the right dosage, and perhaps even see signs of efficacy. Patients enter the trial while taking steroids, and the aim is to taper down the steroid dosage during the study. Those receiving 1923, its hoped, will see their symptoms stay the same or improve, while those on placebo should see them worsen as the steroid doses are lowered.

Its a testament to the need for a new treatment that volunteers are willing to risk having their symptoms intensify if they end up in the placebo arm, says participating physician Daniel Culver, a pulmonologist at the Cleveland Clinic. [Steroids are] very toxic, says Culver, who notes that one of his patients calls his steroid prescription the Devils drug because it does almost as much harm as good. People are very, very motivated to find something different.

The study plans to enroll 36 participants, but has been delayed by the COVID-19 crisis. With such a small sample size, Culver doesnt expect a home run, but he says he hopes the data will be good enough to embark on a larger, Phase 3 study. aTyr is also planning a Phase 2, 30-person trial of 1923 for respiratory complications associated with COVID-19.

If aTyr succeeds, it will be the first instance of a therapeutic built from an AARSbut probably not the last. As Kim sees it, AARSs are ready and waiting to respond to anything that challenges homeostasis, from cancer to the novel coronavirus. I rename the synthetases Molecular 911.

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Protein Synthesis Enzymes Have Evolved Additional Jobs - The Scientist

Dr Yu Jie, associate at LSE IDEAS and senior research fellow at Chatham House, dissects the possible evolution of the China-UK trade relationship

COVID-19 acts as a catalyst to provoke many domestic and foreign affairs debate in British politics, including its future relationship with China, the world second-largest economy. While many of the Tory and Labour grandees have called for reckoning with China, there are some which have insisted on the need for continued positive engagements with China on global issues. Foreign Secretary Dominic Raab ambivalently suggested there could not be a return to business as usual with China-UK trade relations after COVID-19. The defining emotion for Downing Street is confusion, the direction nebulous.

The defining emotion for Downing Street is confusion, the direction nebulous.

This discombobulated mood highlights the difficult trade-offs the UK faces in its relationship with the Middle Kingdom in a period of frequent changes in Brexit foreign policy coupled with post-COVID 19 world order.

For the Chinese government, British trade officials efforts to boost economic ties cannot happily coexist with the UK House of Commons Foreign Affairs Committee remarks about the Chinese governments cover-up and disinformation campaign during the outbreak.

On top of the pressure, the UK is under from the United States and other Five Eyes intelligence-sharing partners to revise the decision to allow Huaweis participation in critical infrastructure, such a controversy could further damage a relationship that was already turbulent.

Seen from Beijing, China has been very clear about what it wants from a relationship with Britain, but Britain appears unable to decide what it wants from China.

After hailing a so-called golden era of bilateral ties during a state visit by President Xi Jinping in 2015, Chinese leaders now see the shock of the Brexit vote steering the relationship in a totally new direction without, apparently, a clear agenda. All of the suspicions that the Communist Partys leaders had about the dangers of Western democracy have been confirmed in spades. They may have also learned a more pointed lesson from the Conservative party about the dangers of externalising internal party conflicts.

In contrast, China has a very clear set of economic priorities for the China-UK trade relationship irrespective of Brexit or COVID-19.

Firstly, Chinese companies continue to look to the UK to provide a secure home for investment, providing opportunities to enhance their global brand value or to make new acquisitions without the fierce resistance encountered in continental Europe and the US.

Secondly, as China grapples with a shuttered economy and international isolation led by the United States, it is looking to calm trade ties with Western powers committed to the principles of free trade and economic globalisation, like the UK, though not at the expense of relations with the EU.

Thirdly, Beijing remains eager to be recognised by established economies and has high hopes of the UK acting as a supporter for Chinas global ambitions as George Osborne did with the UKs membership of Asia Infrastructure Investment Bank. All three could theoretically be one of Chinas prized win-win outcomes, and perhaps also benefit the British economy to some degree.

It is also important to remember why China has prioritised its relationship with the UK over other major European economies. While British media often focus on Chinas interest in education and tourism connections, Beijing has long admired British expertise in financial and corporate governance. As far back as the 1950s, Mao Zedong was championing the desire to become more like Britains developed economy.

Today, while France and Germany have been important for providing manufacturing technologies, the UK is a more useful partner for the next stage of Chinese development: a shift to a consumption-led and service-based economy; the move to currency convertibility via Chinese yuan trading in the City of London; and a welcoming and absorbing of Chinese investment abroad. And in fact, the direct investments from China to the UK remains at a very low level compared with continental Europe. And most of the direct investments focus on real estates which well trumped over other sectors in terms of volume.

Yet, an inconsistent message from London jeopardises these economic possibilities by challenging a key element of the Chinese Communist Partys legitimacy: a resurgent Chinese nationalism. The party has strongly promoted the idea that its leadership has ended over 150 years of foreign bullying of China and helped the country regain its rightful place at the center of world affairs.

The UK is traditionally viewed by the Chinese as a former colonial power which inflicted humiliation on China in the Opium Wars.

So it is no wonder comments on Hong Kong by British Parliamentarians have sparked rage in Beijing. The UK is traditionally viewed by the Chinese as a former colonial power which inflicted humiliation on China in the Opium Wars. Seen in this light, the government must show its people that it can stand up to strong words from Britain.

Some parts of the world do not understand the sense of humiliation todays Chinese feel when they look back on the past, at least in the version that is popularly presented domestically.

Judging by the growing anti-China rhetoric from many parts of the world, Chinas relations with the West have soured significantly. This trend could not have come at a worse time, which emboldens China hawks in the United States to promote decoupling amid deteriorating relations between Beijing and Washington. This deterioration also poses a tremendous challenge to post-Brexit Global Britain.

To reconsider its relationship with China, the British government must better understand how economic and security strands of the relationship are connected and become more skilful at interacting along both lines. To nurture that partnership, London will have to balance mitigating its security concerns with realism about how much it can change the Chinese governments outlook and political choices. And it will have to accept that it cannot divorce its security stance from its hopes for a profitable economic relationship.

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The expected evolution of the China-UK trade relationship - Open Access Government

More News02 Jun 2020 | 3:15 AM

Nashik, Jun 1 (UNI) With 28 more people testing positive for the deadly novel coronavirus here on Monday, the tally of infected patients in the district rose to 1,250.

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INTRODUCTION

Whole-genome duplication (WGD) or polyploidy provides genomic opportunities for evolutionary innovations and adaptation (14). Polyploidy is rare in animals, possibly because of barriers to sex determination, and physiological and developmental constraints, especially nuclear-cytoplasmic interactions and related factors (5, 6). Further, polyploid animals appear to be incapable of coping with genomic and developmental chaos resulting from the merging of two genomes because of changes in structural variation, regulatory imbalance, gene expression bias, and activation of transposable elements (TEs), as documented in many other allopolyploids (1, 3, 710). A newly formed allopolyploid line of fishes (11) experienced more severe chaotic changes than polyploid plants (7, 8, 10, 12, 13). These rapid and dynamic changes have genetic and epigenetic bases (7, 14). Biased subgenomic changes may help alleviate chaos from genome mergers (15), and subsequent coordination may help stabilize the subgenomic functions in newly synthesized allotetraploid plants (7, 12, 16) after the initial genome shock (17) between divergent subgenomes that coexist in the same cell nucleus. However, the long-term consequences for polyploid animals in rediploidization remain elusive.

Goldfish (Carassius auratus red var.) belongs to family Cyprinidae in the most specious order of fishes (Cypriniformes), which contains many polyploid species (1820). The tetraploid carp was domesticated hundreds of years ago in China and Europe, and it is the economically most important fish in freshwater aquaculture (21). Goldfish (C. auratus red var.) is the most commonly kept pet globally, and it constitutes a model system for studying neurobiology and physiology. This allotetraploid (2n = 4x = 100) species was formed by the interspecific hybridization of two diploids (2n = 2x = 50); subsequent chromosome-doubling restored meiotic paring and disomic inheritance (18, 19). Goldfish has nearly twice as many chromosomes as zebrafish and most of other cyprinids (22, 23). Its numerous small chromosomes pose a great challenge to assembling and annotating both subgenomes at the chromosomal level. Because no extant diploid progenitors are available for study, the evolution of this complex polyploid genome remains poorly understood (21, 24, 25), despite the recently published draft genome (26).

Here, we report a reference-quality, chromosome-scale assembly of goldfish including identification of both subgenomes and analysis of the variation and expression changes between them. We also evaluate the subgenomic evolution of goldfish and common carp. Our results indicate that allotetraploid goldfish and common carp have diverse strategies for balancing dynamic subgenomic diversification during continuous rediploidization. The diverse and continuous evolutionary processes broaden our understanding of the evolution and function of genomes in allopolyploid vertebrates and may explain why most polyploid animals fail to survive.

Allopolyploid genomes are much more complicated than diploid ones due to their polynomic inheritance and gradual random decay of progenitors genomes during rediploidization (2, 3). A high-quality genome assembly is required to discriminate changes from related species in allopolyploids. Our high-quality genome of a gynogenetic goldfish (C. auratus red var., n = 100) was assembled using data from a combination of three technologies (Fig. 1), including 325.34 gigabases (Gb; 203) of Illumina sequence data (Illumina GAII, HiSeq 2000), 128.51 Gb (80) of single-molecule real-time (SMRT) long reads (PacBio RS II and Sequel), and 231.50 Gb of clean BioNano mapping data (Bionano Genomics Irys). The final assembly consisted of 5477 scaffolds, with a scaffold N50 of 2.94 megabases (Mb) after gap filling (Table 1 and data S1_14), which resulted in a genome size of 1.64 Gb. This was similar to the size estimated by flow cytometry (1.71 Gb; Fig. 1) and slightly higher than the k-mer analysis (1.43 Gb; Fig. 1). In addition, using 307.46 Gb of data generated by high-throughput chromosome conformation capture sequencing (Hi-C seq) technology (Annoroad Gene Technology Co. Ltd., Beijing; data S1_5, 6), 1.59-Gb (96.95%) genome-level sequences were aligned and ordered into 50 scaffolds that potentially matched the chromosomes (Table 1, fig. S1A, and data S2_1). A genetic map consisting of 50 linkage groups was constructed in 79 F2 offspring using 7466 single-nucleotide polymorphism markers developed from 147.10 Gb of RNA sequencing (RNA-seq) data (27). Next, the genome scaffolds (1.07 Gb, 65.24%) were anchored on the genetic map (data S2_2). Sequence assemblies on the pseudochromosomes matched perfectly to the anchored 50 linkage groups, confirming the high-quality assembly of the allotetraploid goldfish genome (fig. S1, B and C, and data S2_35). BUSCO and CEGMA showed the assemblies to be complete (data S2_6). Our assembly conformed to Vertebrate Genome Project standards for reference genomes (https://vertebrategenomesproject.org/) and covered more sequences than the published goldfish genome by Chen et al. (26), which consisted of 1.24 Gb with an anchor ratio of ~66% from 1.82 Gb contigs (Table 1). Subsequently, we compared the completeness and collinearity between our assembly and the published genome using MUMmer (v3.23), based on the minimum clustering unit of 50 kilobases (kb), which is the smallest unit that we could check, and consistent with 50-kb resolution of Hi-C clustering. Within the total length of 1.06-Gb collinearities from 2380 clustering groups, we found general consistency, while some structural differences existed between the two. In our study, among 277 inversions and 1338 translocations identified, 63.95% were validated by PacBio long reads, optical map, and/or Hi-C data (Fig. 2A, fig.S1D, and data S2_7, 8). These efforts obtained a general improvement of quality scores in our genome assembly relative to the published data. For example, the optical map and Hi-C analyses obtained improvement at an inversion boundary (Fig. 2A).

(A) Genome size and karyotype of goldfish. (a) Image of a gynogenetic goldfish (C. auratus var.). Photo credit: Shaojun Liu, Hunan Normal University, China. (b) Diagram of C value. The X axis presents the fluorescence index, and the Y axis presents the frequency of cells. Sample/calibration ratio equals the peak X value of the calibration sample divided by X value at the peak of the target sample. The first sharp peak with green dashed line displays the X axis and cell frequency of chicken blood, and the second one with red dashed line represents the X axis and cell frequencies of goldfish. C value of sample is sample/calibration ratio calibration samples C value. (c) Goldfish have 100 chromosomes and 100 signals after the chromosomes are stained with DNA probe (probe A) [9468-bp fragment of 36 copies of a repetitive 263-bp fragment; adopted from Liu et al. (11)]. (B) Sequencing technologies for primary assembly. (C) Genome assembly, Hi-C cluster, and genetic map construction. Genome size assessment by k-mer analysis is performed by 40 Illumina paired-end reads after the primary assembly. Next, scaffolds are clustered into 50 pseudochromosomes by using Hi-C data obtained by chromosomes; the genetic map was constructed by using the data of Kuang et al. (27) (D) Annotation and chromosome-scale organization. Annotation of scaffolds was performed using a combination of ab initio prediction, transcript evidence gathered from RNA-seq of embryos and eight kinds of adult tissues (gonads, brain, liver, spleen, kidney, eye, epithelium, and fin), and homologous genes information from five fish genomes, by using EVidence Modeler (EVM). Final set of 50 pseudochromosomes was generated after pairwise validation among Hi-C clustering results, genetic map, and collinearity analyses. (E) Subgenome identification. After extracting the homologous genes of goldfish and other species, the species tree is constructed by using single-copy genes from 10 genomes. Gene trees were constructed by defining homologous gene clusters using whole-genome sequences/transcripts from 10 cyprinid species of Cyprininae (C. auratus, Cyprinus carpio), Labeoninae (Labeo rohita), Poropuntiinae (Poropuntius huangchuchieni), Schizothoracinae (Schizothorax oconnori, Schizothorax waltoni, Schizothorax macropogon, and Schizothorax kozlovi), Danio rerio, and Ctenopharyngodon idellus. After comparing the species tree and nucleic gene trees, the matrilineal (clustered with Schizothorax) and patrilineal markers from the gene trees were labeled back to 25 pairs of pseudochromosomes. The origin of pseudo-chromosomes was identified by most of the supported markers.

NGS, next-generation sequencing; LG, linkage group.

(A) Diagrams displaying the rearrangement identified in the syntenic blocks between our assembly and the goldfish genome published by Chen et al. (26). The syntenic block between two assemblies was identified with an inversion event (a), in which the rearranged boundary on our genome was continuously covered by the long reads of the optical map (b), and supported by the strong clustering signal from the heat map (c) constructed by high-depth sequencing data from chromosome conformation capture technology. (B) Allotetraploid goldfish and common carp genome synteny. Blocks represent the genomic overview of assembled chromosomes of subgenome M and subgenome P from goldfish and subgenome B and subgenome A from common carp (Hebao red carp) (29). Colored lines indicate the orthologous sites of gene blocks and their colinear relationships between subgenomes M and B and subgenomes P and A, respectively. Numbers M01 to M25, P01 to P25, B01 to B25, and A01 to A25 indicate the homologous chromosomes of M and P subgenomes from goldfish, and B and A subgenomes from common carp, respectively, numbering in order according to the collinearity relationships to zebrafish genome (fig. S3). The numbers beside M and P chromosomes indicate the supporting rate from homoeologous gene makers with clear origin of parental ancestor determined by the gene trees. (C) Phylogenetic relationships and timing of WGD/polyploidization events in Cyprininae, with nodes based on protein-coding genes of goldfish, common carp, golden-line barbel, grass carp, and zebrafish. Dated divergence time of grass carp and the ancestor of Cyprininae was 20.9 Ma ago, and the putative matrilineal and patrilineal progenitors were 15.1 Ma ago (T1), after the WGD event (T2). Divergence of the polyploid Cyprininae radiation was dated at 13.8 Ma ago (T3), and the divergence between goldfish and common carp was dated at 10.0 Ma ago (T4). Orange and blue branches indicate the putative M and P progenitors, respectively. Photo credit: Goldfish and common carp by Shaojun Liu, Hunan Normal University, China; golden-line barbel and grass carp from FishBase; zebrafish from Wikimedia. (D) Phylogenetic tree based on protein-coding genes from single-copy orthologs, rooted with human and chicken. Alignments were performed by MUSCLE, and the maximum likelihood tree was reconstructed by PhyML.

The annotation predicted 43,144 genes with an average length of 17,025 base pairs (bp) and 9.78 exons per gene (data S1_7 and data S2_9). Of the genes, 39,205 (90.87%) were functionally annotated (data S2_9). Accuracy and completeness of the annotation were validated further through 97.78% coverage of annotated genes by RNA-seq data. The annotation included 6788 transfer RNA (tRNA), 1380 ribosomal RNA (rRNA), 1324 small nuclear RNA (snRNA), and 3385 microRNA genes (data S2_10). Repeat annotation indicated an overall repeat content of 39.49% (data S2_11), which was less than the 52.2% of zebrafish (28) and comparable to ~40% of African clawed frog (9). The most abundant TEs in the goldfish genome were type II, which represented 21.19% of the whole-genome sequence. Other annotated TEs included 174 superfamilies.

To identify the goldfishs subgenomes from two progenitors, we used phylogenetic information of the Cyprininae (18, 19). Several species in the genera Carassius (including goldfish) and Cyprinus were reported to have undergone the same allopolyploidization approximately 10 to 12 million years (Ma) ago (19). The matrilineal copy of several nuclear genes was grouped with genus Schizothorax (18, 19), and they diverged from the patrilineal ones 17 to 19 Ma ago (18, 19). By comparing phylogenies between the mitogenomes and homoeologous gene pairs of whole-genome sequences/transcripts from 10 cyprinid species, analyses confirmed that Schizothorax was monophyletic; it shared a matrilineal ancestor with all allotetraploid species of Cyprininae (Fig. 1E and data S1_8). The analysis identified 1274 homoeologous gene pairs of goldfish. Mapping these homoeologous gene pairs onto the 50 pseudochromosomes identified matrilineal (M) and patrilineal (P) subgenomes, with an average support rate of 95.34% (Fig. 2B and table S1). To clarify the relationship between each subgenome pair of goldfish and common carp (Hebao red carp) with subgenomes of A and B of Xu et al. (29), we first constructed the collinearity relationship between the two species. Analyses using MCScanX found that 9266 orthologous gene pairs had unambiguous one-to-one relationships between subgenome M (from goldfish) and subgenome B (from common carp), and 6991 orthologous gene pairs had clear one-to-one relationships between subgenome P (from goldfish) and subgenome A (from common carp; Fig. 2B). The results showed a consistency of 74.58% between subgenomes M and B and 66.97% between the subgenomes P and A. This suggests that subgenomes M and B are from the matrilineal genome of the Cyprininae ancestor, while subgenomes P and A are patrilineal ones (Fig. 2B and data S2_12, 13).

The ancient WGD within Cyprininae was identified via phylogenetic analyses of genome-wide markers, which integrated subgenomes M and P. Analyses compared both subgenomes of goldfish, common carp, and golden-line barbel (Sinocyclocheilus grahami) to zebrafish and grass carp. A MCMCTree analysis in the Phylogenetic Analysis by Maximum Likelihood (PAML) package (v4.8) (30) with four calibration points suggested that the progenitor-like genomes diverged approximately 15.09 Ma ago (T1; Fig. 2C). Following allopolyploidization (T2), S. grahami originated about ~11 Ma ago (T3), followed by goldfish and common carp at ~9 Ma ago (T4). These dates were more recent than those (13.75 and 9.95 Ma ago, respectively) estimated by using 568 single-copy genes (Fig. 2, C and D, and fig. S1E). The new estimate (13.75 to 15.09 Ma) of timing for the Cyprininae WGD (T2; Fig. 2C) was earlier than those (10 to 12 Ma ago) based on gene markers (19). The expansion and contraction of gene families were estimated to infer the evolutionary history after the Cyprininae-specific WGD event relative to the teleost-specific WGD event in zebrafish and grass carp (fig. S1, F and G, and data S2_14). Within 4453 expanded gene families, there were 313 transcription factors, indicating a significant abundance (P < 0.01, Fishers exact test). Most of them were known to be involved in embryonic development, especially organogenesis during differentiation of germinal layers (fig. S1H).

Evolution of subgenomes has been reported in allopolyploid angiosperms (8, 10) and vertebrates (9). They typically showed conservation of one progenitor-like genome and diversification of the other by large structural variation in collinearities compared to the ancestral genome. In contrast, comparison of allotetraploid goldfish annotated genes, repeats, and noncoding RNAs (ncRNAs) showed approximately equal representations between the two subgenomes. Approximately 1.5 Gb of chromosome-scale sequences were partitioned into subgenomes M and P, which was consistent with the number of genes in M (20,913, 52.07%) and P (19,248, 47.83%). The proportions of repeat sequences and ncRNAs were also similar between the two subgenomes (data S3_1). Thus, the two subgenomes had similar gene densities and distributions in most chromosomes, except for significantly higher densities in three M (chr1, chr20, and chr43) and one P (chr27) chromosomes (P < 0.05, two-tailed paired t test; data S3_2). The two subgenomes also contributed similar proportions of all TE families with annotation against public database (fig. S2, A and B, and data S3_3). Further, the comparison between two subgenomes of common carp in genomic contents showed nearly equal representations between subgenomes B and A (29).

To further investigate the evolution of two subgenomes after allopolyploidization in carp-like fishes, we compared subgenomes M and P of goldfish and subgenomes B and A of common carp with zebrafish to define the changes in synteny and genomic divergence. To integrate the collinearities between goldfish and common carp, we aligned 43,144 high-confidence gene models to 50 goldfish and the 25 zebrafish chromosomes. The results indicated that 12,450 genes of subgenome M and 11,042 genes of subgenome P were located on syntenic blocks (P = 1.09 105), and 7568 orthologous gene pairs had a clear two-to-one relationships to zebrafish (Fig. 3A and fig. S3). Collinearities between homologous goldfish M and common carp B chromosomes identified 15.12% inversions and 10.30% translocations, and the ones between goldfish P and common carp A chromosomes showed 22.29% inversions and 10.74% translocations, which indicated more rearrangement in the patrilineal subgenomes (data S2_12). We also validated the boundaries of all rearranged regions identified by both collinearities against goldfish from Chen et al. (26) and common carp; 69.01% of regions on subgenome M and 67.64% on subgenome P were assembled continuously in our genome by sequencing data (data S3_4). With respect to GC (guanine-cytosine) content and repeat densities, we used a sliding window of 50 kb for comparative analysis. We found that the patrilineal subgenomes P and A had greater GC content than matrilineal subgenomes M and B (Fig. 3B, a, and data S3_5), indicating subgenome-specific variation in these two species. However, the distributions of repeat densities yielded an opposite pattern; more repeats occurred in subgenome P than M in the goldfish, and in the common carp, more repeats were in B than in A. These differences may have owed to species divergence (Fig. 3B, b, and data S3_5). The distributions of GC content and repeat density might contribute to different kinds of divergence (data S3_5). In addition, we obtained 28 and 16 specific blocks with significantly biased repeat densities between subgenomes in goldfish and common carp, respectively, and labeled those potential sources of divergence between the two species on each chromosome (P < 0.05, Wilcoxon rank sum test; Fig. 3A, fig. S3, and data S3_6).

(A) Syntenic blocks between goldfish and common carp and syntenic blocks between goldfish and zebrafish with color-labeled rearrangements in two examples. The other 23 groups of syntenic analysis are shown in fig. S3. All 50 pseudochromosomes of goldfish show clear two-to-one orthologous relationships to the 25 chromosomes of zebrafish. Orange bars and numbers mark the chromosomes from matrilineal (M and B) subgenomes, while blue denotes the patrilineal (P and A) ones. Gray bars and numbers mark the chromosomes of zebrafish. Light gray lines indicate the syntenic blocks, light orange lines indicate the rearrangements between goldfish and common carp, and the pink lines indicate the rearrangements between goldfish and zebrafish. The red and black bars on each chromosome indicate the biased repeat densities, in which the red ones indicate the syntenies with significantly higher repeat densities relative to the black ones (P < 0.05, Wilcoxon rank sum test). (B) Boxplots of distributions of (a) GC content and (b) repeat density in consistent syntenies, rearranged regions, and the boundaries of rearranged regions. Orange and blue boxes mark the values from matrilineal (M and B) and patrilineal (P and A) subgenomes, respectively. Boxes with black lines indicate the distributions of goldfish, and the ones with dashed lines indicate the common carp. (C) Distributions of consecutive gene retentions in subgenomes M and P, which do not differ from each other significantly (Fishers exact test, P = 0.35). (D) Dating the time of pseudogene formation in subgenomes M and P of goldfish, subgenomes B and A of common carp, as described in Supplementary Methods and Analysis 6. X axis represents an estimated time of pseudogene formation; Y axis represents the frequencies of pseudogenes in every 0.5-Ma unit. Orange lines display the frequencies of pseudogenes on subgenomes M or B along time (X axis), and blue lines display the frequencies of pseudogenes on subgenomes P or A. Gray bars indicate the timing of allopolyploidization (13.8 to 15.1 Ma ago). (a) and (b) display the distributions of all pseudogenes frequencies from each subgenome along time in goldfish and common carp; (c) and (d) display pseudogenization events specific to each subgenome in goldfish and common carp. (E) Boxplot displays the distributions of Ka/Ks ratios for (a) 7568 homoeologous gene pairs between M and P subgenomes calculated against zebrafish and grass carp and (b) distributions of Ka/Ks ratios displayed in boxplot for (a) 7568 homoeologous gene pairs between subgenomes B and A calculated against zebrafish and grass carp. Central line in each boxplot indicates the median value. Top and bottom edges of the box indicate the 25th and 75th percentiles, and the dashed lines extend 1.5 times the interquartile range beyond the edges of the box.

Most genes (>88%) were retained in both subgenomes, and the distribution of consecutive (more than two contiguous genes) retentions of syntenic blocks was not biased between them (P > 0.05; Fig. 3C and fig. S2C). The tracing of the gene loss by deletion showed 1737/2727/1677 M/P/shared losses in goldfish and indicated that subgenome P experienced more small-scale deletions of genes (11.53% of 23,652) than subgenome M (7.14% of 24,327; P < 0.01). In common carp, 1009/1409/1574 B/A/shared losses indicated more small-scale deletions in subgenome A (6.98% of 20,172) than B (4.81% of 20,977). Subgenome P tended to lose more genes related to pathways of amino acid metabolism, oxidative phosphorylation, base repair, and homologous recombination (fig. S2C and data S4_1) than subgenome M. Genes lost across all subgenomes occurred in no more than two consecutive genes in all syntenic blocks of zebrafish (data S3_7). Analyses identified fewer pseudogenes in subgenome M (2.90%, 705/24,327) than P (4.33%, 1023/23,652; P < 0.01) and also fewer pseudogenes in B (2.33%, 486/20,815) than A (3.86%, 754/19,509).

According to the dating of pseudogene formation, pseudogenes accumulated continuously in both goldfish and common carp (Fig. 3D). In goldfish, pseudogenes formed asymmetrically and continuously after allotetraploidization, while in common carp, both subgenomes experienced the accelerated accumulation of pseudogenes after allotetraploidization (Fig. 3D, a and b). To test whether the functional loss continued after the divergence between the two species, we grouped the pseudogenization events into the one shared by two species, either MB or PA, and the other occurred specifically in each subgenome. Subgenomes M and B shared 79 pseudogenes, while P and A shared 156. These shared pseudogenes in the patrilineal subgenomes of goldfish and common carp were involved in pathways of base excision repair and homologous recombination (data S4_2). We found 626/867/407/598 pseudogenes specific to M/P/B/A subgenomes, respectively, which suggested that more gene loss events occurred independently in each subgenome than the ones they shared. The dating of pseudogenes specific to each subgenome showed the same distributions after the divergence of both species: continuous accumulations in goldfish (Fig. 3D, c) and accelerated accumulation of pseudogenes in common carp (Fig. 3D, d). Notably, the pseudogenes specific to subgenome P were also predicted to be involved in DNA repair and homologous recombination (data S4_3). Together, these analyses supported a bias in gene loss between goldfish and common carp.

Nonsynonymous mutation (Ka) values, synonymous mutations (Ks) values, and the ratios of these values (Ka/Ks) between the two subgenomes were compared against the reference genomes of zebrafish and grass carp to identify alterations in evolutionary rates. In goldfish, all homoeologs in subgenome M (zebrafish median Ka/Ks = 0.12; grass carp = 0.18) had a significantly lower Ka/Ks ratio than those in subgenome P (median Ka/Ks = 0.13 and 0.19; P < 0.01 for both, Wilcoxon rank sum test; Fig. 3E), while single-copy genes and all genes showed no significant bias between the two subgenomes (P > 0.05; data S3_8, 9). In common carp, both homoeologous and all genes in subgenome B had significantly lower Ka/Ks ratio than in subgenome A (P < 0.01), while single-copy genes showed no significant bias between the two subgenomes (P > 0.05; data S3_8, 9). Ka and Ks values and Ka/Ks ratios in syntenic blocks indicated that no significant correlation existed with structural changes (fig. S2, D to F). The distributions of Ka/Ks ratios between each paired M and P, or paired B and A, chromosomes also showed no difference (fig. S2G); only three syntenic blocks showed a significant biased Ka/Ks ratio between subgenomes M and P, while only seven syntenic blocks were biased significantly between subgenomes B and A (P < 0.05, Wilcoxon rank sum test). These results indicated species-specific gene fates. Signatures for positive selection occurred in 128 homoeologous genes in goldfish, symmetrically including 0.31% (65/20,913) of genes in M and 0.33% (63/19,248) in P. Statistical comparisons of both overall and pairwise homoeologous chromosomes detected significant differences between matrilineal and patrilineal subgenomes, as well as species-specific changes.

More species-specific alterations occurred between parental genomes than asymmetrical changes. This might had led to the diversities of expression in homoeologous gene pairs. To test for this, we compared transcriptome changes between the subgenomes in six adult tissues and 15 developmental stages using the homoeologous genes that were confirmed with high correlations between biological duplicates and among developmental stages (fig. S4, data S1_9, and data S3_10, 11). In goldfish, expressions of homoeologous gene pairs did not show a bias between the homoeologs among all six adult tissues or at eight developmental stages. This pattern held except in seven specific stages around the reprogramming of embryogenesis (31, 32), where M subgenomic homoeologs were expressed 4.8% higher than the P ones (Fig. 4, A and B). In common carp, expression of homoeologous gene pairs showed B-biased expression in five stages around the reprogramming of embryogenesis, one stage in pharyngula period, and two stages in hatching period (Fig. 4C).

(A) Boxplot of log10(TPMM/TPMP) for homoeologous gene pairs showing medians in six adult tissues of goldfish. Red dashed line shows the equal ratio of log10(1). All adult tissues show no bias of expression between genes stemming from the subgenome M or P. (B) Boxplot of log10 [(TPMM + 0.1)/(TPMP + 0.1)] for homoeologous gene pairs showing medians in 15 developmental stages of goldfish [16 cell, 32 cell, 64 cell, 128 cell, 1000 cell; 8, 12, 16, 18, 24, 30, 46, 64, 71, and 84 hours post-fertilization (hpf)]. Red dash shows the equal ratio of log10(1). Time points from 64-cell to 22-somite stages biased expression of M homoeologs, which average 4.8% more than P genes within homoeologous gene pairs; early embryos (16- and 32-cell stages), pharyngula, and hatching period embryos show no bias of expression. (C) Boxplot of log10 [(TPMB + 0.1)/(TPMA + 0.1)] for homoeologous gene pairs from common carp shows medians in zygotically controlled developmental time points. Red dashed line shows equal ratios of log10(1). Time points from 32-cell to germ ring, 25% otic vesicle closure (OVC), long pec, and pec fin stages indicate biased B-homoeolog expression; other stages show no expression bias. (D) Expression patterns of 9090 homoeologous gene pairs from goldfish where the trend displays expression of either biased toward M or P homoeologs (EBM or EBP; 6223 genes in total, 68.46%) when gene pairs are coexpressed in at least one development stage. (E) Expression trend of 9090 homoeologous gene pairs from goldfish displaying an expression shift between two homoeologs (ES; 1644 genes in total, 18.09%) where one copy is expressed higher than the other at earlier time points, then the other copy surpasses it in later development stages. (F) Expression patterns of 4241 homoeologous gene pairs from common carp where the pattern displays either biased B- or A-homoeolog expression (2811 gene in total, 66.28%) when homoeologous gene pairs coexpressed in at least one development stage. (G) Expression patterns of 4241 homoeologous gene pairs that indicate expressional shift between two homoeologous gene pairs (414 gene in total, 9.76%); one homoeologous gene copy expressed higher than the other at an earlier time point, then the other copy surpasses it later in development. Patterns include two groups: genes (185, 4.36%) with ES before germ ring stage and genes (229, 5.40%) with ES post-germ ring stage, which accounts for most genes. Among the ES genes, 32 (0.75%) have more than two time shifts. (H) Comparison of DNA methylation levels between the two subgenomes in brain and liver tissues. (I) Comparison of DNA methylation levels between the two subgenomes in embryos of 12 developmental stages.

In goldfish, the number of differentially expressed homoeologs differed among developmental stages (fig. S5), and their expression levels exhibited spatial and temporal variation throughout development. Expression of 9090 homoeologous gene pairs showed three patterns. First, most gene pairs (68.46%, 6223/9090) displayed an expression bias toward either M (39.69%, 3608) or P (28.77%, 2615) homoeologs (Fig. 4D and Table 2). Second, expression shifted between two homoeologs (18.09%, 1644/9090) during different developmental stages (Fig. 4E and Table 2). Among them, 11.88% (1080) displayed a shift after the reprogramming of embryogenesis, while 184 genes shifted more than once through various developmental stages. Third, 13.45% of homoeologs (1223/9090) were expressed equally throughout all developmental stages. Approximately 39.69% homoeologous pairs displayed biased expression toward the M homoeologs throughout all development stages with a slightly higher expression levels in stages around germ ring, while 18.09% homoeologous gene pairs were equally expressed. These trends were consistent with those in the common carp using the same analysis with 4241 homoeologous pairs (Fig. 4, F and G, and Table 2). The expression-bias shift of both M/B and P/A genes occurred most frequently in the germ ring of the gastrula stage, which is crucial for germ-layer development in both goldfish (395 shifts) and common carp (117 shifts; data S3_12).

EBM, expression bias toward matrilineal (M and B) homoeologs; EBP, expression bias toward patrilineal (P and A) homoeologs; ES, expression shift between two homoeologs.

In goldfish, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses found overrepresentation of the M- and P-preferentially expressed genes of goldfish in 91 pathways (data S4_4, 5) and of expression-bias shifted genes in 61 pathways (data S4_6). Most of these preferentially expressed homoeologs shared enrichment of 13 pathways, while some M- and P-preferred genes were overrepresented in DNA replication and repair and in spliceosome and phototransduction, respectively. The results indicated that homoeologous genes from both subgenomes contributed similarly to biological pathways (fig. S6, A to D).

DNA methylation could have occurred during polyploidization, and the methylation-associated genes could have been inherited as epialleles (33). In goldfish, the subgenomes had indistinguishable levels of DNA methylation (difference less than 20%) in both gene body and promotor regions (fig. S7) in brain and liver tissues and among 12 developmental stages (Fig. 4, H and I, and data S1_10). A high level of DNA methylation in early stages of embryos was inherited from the sperm rather than eggs and decreased over time during development (fig. S7, A and B). Expression levels of homoeologous gene pairs correlated negatively with DNA methylation patterns, especially in the proximal promotor regions (figs. S7, C and D, and S8). Thus, DNA methylation may have played a role in the regulation of homoeologous gene expression. Analyses of co-DNA methylation by weighted gene coexpression network analyses (WGCNA) (34) identified 182 homoeologs (12.5%, 182/1454), with the DNA methylationlevel shifts between M and P homoeologs corresponding to their expression shifts but with no significant difference in either promoter or gene body regions among embryos of 12 time points (P > 0.05, Fishers exact test; fig. S7, E and F, and data S3_13).

Polyploid vertebrates such as goldfish and common carp might have experienced chaotic changes in their early stages of polyploidization (15 Ma ago), as reported in newly formed allopolyploid fishes (11) and in other newly synthesized polyploid plants (7, 12, 13). Our analyses of goldfish and common carp indicate similarities and differences in structural change. Significant bias (P < 0.05, Wilcoxon rank sum test) occurs in Ka/Ks ratios in homoeologous gene pairs between subgenomes M and P in the goldfish (M < P), and yet no significant bias occurs in Ka/Ks in single-copy genes. This suggests that the goldfish genome was prone to retain functionally constrained genes after its WGD (35). Specifically, the goldfish genome usually retains only one copy of homologous DNA repairrelated genes, which is consistent with the pattern in plants (36). Further, goldfish subgenome P continues to lose these genes via pseudogenization, yet this does not occur in common carp subgenome A, suggesting variable strategies in different species. This provides functional flexibility for both subgenomes during evolution and adaptation. Likewise, biases in post-polyploidization gene loss have been studied in plants (37, 38), and more work is necessary to elucidate this both in plants and animals. Our analyses reveal that short fragment loss only involved one or two consecutive genes of subgenomes M and P; this differs from flowering plants and African clawed frog, which have lost more and longer fragments (9, 10, 38). Considering gene expression, a few surviving animals could have evolved a balanced strategy to maintain genome stability. This would limit structural changes and genomic diversification by reprogramming homoeologous gene expression during embryonic development, which is critical for survival in both natural and controlled environments. Cyprinines can serve as models for investigating the evolution of vertebrate polyploidization, and they may explain why polyploidization events are far less common in animals than in plants. The dosage balance hypothesis is an attractive explanation for the patterns of post-polyploidization gene retention and loss (39), and future functional work is necessary to completely paint the picture.

Three gynogenetic goldfish from the same inbred line were collected to extract genomic DNA (data S1). DNA from one fish was used in whole-genome sequencing by Illumina and SMRT (Pacific Biosciences) sequencing platforms. We used wtdbg (v1.1.006) (40) to assemble the long reads and polished the resulting contigs with short reads. Another goldfish was sampled for optical mapping (BioNano Genomics Irys) and Hi-C library construction, which produced chromosome-level scaffolds. For the RNA-seq, eight adult tissues were sampled from one male and one female goldfish. Two groups of mature eggs and embryos were taken from 15 developmental stages of goldfish [16 cell, 32 cell, 64 cell, 128 cell, 1000 cell, 8 hours post-fertilization (hpf), 12 hpf, 16 hpf, 18 hpf, 24 hpf, 30 hpf, 46 hpf, 64 hpf, 71 hpf, and 84 hpf] and 14 developmental stages of common carp for RNA-seq. Further, brain and liver tissues and embryos from 12 developmental stages of goldfish were sampled for whole-genome bisulfite sequencing (WGBS). All experiments were approved by Animal Care Committee of Hunan Normal University (2014278) and followed guidelines of the Administration of Affairs Concerning Experimental Animals of China.

On the basis of the scaffolds linked by the Irys optical map, 50 pseudochromosomes were clustered with the Hi-C data. Next, a genetic map of goldfish based on genotyping was constructed by adopting the pooling-sequenced transcriptomic data of Kuang et al. (27), which were based on an inbred line of two parents and 79 F2 individuals. Subsequently, the two-to-one colinear relationships between goldfish and zebrafish were identified by using MCScanX (41). Last, 25 homologous chromosome pairs were generated after pairwise validation among Hi-C clustering results, genetic map, and collinearity analyses.

Protein-coding genes were annotated using a combination of ab initio prediction, transcript evidence gathered from RNA-seq of embryos and 16 adult tissues (ovary/testis, brain, liver, spleen, kidney, eye, epithelium, and fin for both female and male), and homologous genes prediction from five fish genomes (Ctenopharyngoden idellus, Danio rerio, Gasterosteus aculeatus, Tetraodon nigroviridi, and Sinocyclocheilus anshuiensis), with EVidence Modeler (v1.1.1). Functional annotations mainly included the following methods: (i) searching against known sequence data (Swiss-Prot/Gene Ontology) by BLASTP with E value at 1 105 and online comparison against the KEGG database by KEGG Automatic Annotation Server (KAAS) and (ii) InterProScan (v5.21-60.0) predicted conservative motifs and domains.

OrthoMCL (42) was used to cluster gene families for zebrafish, grass carp, golden-line barbel, common carp, goldfish, and species of Schizothorax. PhyML (v3.1) (43) was then used to build the phylogenetic trees for each gene family. A species tree was also constructed by using single-copy genes from the above 10 genomes. In the topology of gene trees, homoeologs located in the same clade with Schizothorax were considered to be M markers, while the remaining P copies constituted the hypothetical P species. The M/P markers were labeled back to 25 pairs of pseudochromosomes. The origins of pseudochromosomes were thereby identified by most of the M/P markers.

Times of speciation and progenitors divergence were estimated by the divergence time using MCMCTree in the PAML package (v4.8) (30). The general time reversible (GTR) nucleotide substitution model was used with a relaxed clock analysis. The multiple calibration points based on literature and fossil records were listed in detail in Supplementary Methods and Analysis 5. We used the divergence time of putative M and P progenitors in Cyprininae, and synonymous substitution levels between putative maternal and paternal homoeologs in goldfish, common carp, and S. grahami, respectively, to estimate an absolute substitution rate. The Ks values were measured with the method as implemented by using the yn00 program in PAML (v4.8) (30).

Putative gene-loss events were traced from the syntenic blocks between zebrafish and the two subgenomes of goldfish. In the triples of consecutive genes within syntenic blocks from the zebrafish genome and two goldfish subgenomes, missing genes were considered as deletions or pseudogenes. Sequences of potential missing genes were confirmed with The BLAST-Like Alignment Tool (BLAT) alignment and mapping coverage of Illumina short reads. Deletions had little support, and pseudogenes contained various defects including premature stop codons, frameshifts, disrupted splicing, and/or partial coding deletions. More details were provided in Supplementary Methods and Analysis 6.

RNA-seq data of six adult tissues, mature eggs, and all embryos with two biological duplicates were mapped to reference genome using Tophat (v2.1.1) (44). Gene expressions in each sample were estimated by RSEM (v1.2.19) (45) and quantified as values of transcripts per million (TPM). Gene expressions with TPM > 0.5 were considered to be detectable. Then, we analyzed expression variation among homoeologous genes in 15 developmental stages by developing coexpression networks with WGCNA (v1.63) (34), following the workflow of Session et al. (9).

We analyzed DNA methylation level of brain and liver tissues from WGBS data using Bismark (v0.19.0) (46) with three steps. More details about the methylation differences in functional elements between two subgenomes are provided in Supplementary Methods and Analysis 8.

Originally posted here:

From asymmetrical to balanced genomic diversification during rediploidization: Subgenomic evolution in allotetraploid fish - Science Advances

Throughout several movies in the Marvel Cinematic Universe, the relationship between Tony Stark and Steve Rogers took center stage. To Chris Evans, the evolution of the two characters that occurred through 2019s Avengers: Endgame was one of his favorite parts of the MCU.

In an interview with theAwards Chatter Podcastby The Hollywood Reporter, Evans discussed his recent role in Apple TV+s miniseries Defending Jacob and his time in the MCU playingSteve Rogers. On the podcast, Evans said that some of his favorite moments in the MCU took place in Avengers: Endgame and involved the scenes between Steve Rogers and Robert Downey Jr.s Tony Stark.

everything from Endgame was really special to me because my headspace was very much in the reflective, grateful part of it. You almost feel like youre living in a memory; you feel like its almost like the moments already passed, so youre really just trying to soak it in and just appreciate what this journey has been like, Evans said.

He continued, Like I said, in Endgame, there are just so many great moments. I love scenes with Downey. I love seeing the evolution of those two characters. They usually give Cap great motivational speeches and things like that. Any of those scenes where theres all of us together, and it just is a real reflective and special moment.

RELATED: The Phone Call From Robert Downey Jr. That Changed Chris Evans Life Forever

While Evans loved working on Avengers: Endgame and the evolution between Steve Rogers and Tony Stark, he told the Awards Chatter Podcast that his favorite scene in the MCU actually occurs in Captain America: The Winter Soldier.

The Russos are real, real cinephiles, and they have such knowledge and love for certain scenes in certain movies. And you can tell when they get excited about certain moments that they want to make iconic; they were really excited about that first elevatorfight scene inWinter Soldier, Evans said on the Awards Chatter Podcast. You could tell that they wanted it to be special, and as a result, thats one of my favorite fight sequences.

When Evans was offered the role of Captain America in the MCU, he was hesitant to accept. In fact, he turned down the role multiple times.

The problem was initially, it was a nine-movie contract. And they said, if these movies take off and do very well, and my life changes and I dont respond well, I dont have the opportunity to say, listen, I need a f*cking break. That just scared me, Evans told Variety in 2014. They called back and they tweaked the deal. It went from nine (films) to six. I said no again.

To help try and convince him to take the role, Downey Jr. called Evans.

I remember getting on the phone with him and strongly suggesting that he not shrink away from the offer, Downey said. I said, Look man, you might not like the fact that youve played one of these guys before (in Fantastic Four), but you know, the thing is this can afford you all sorts of other freedoms I also thought he was the perfect guy for the job.

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Chris Evans Loved 'the Evolution' Between Iron Man and Captain America in the MCU - Showbiz Cheat Sheet

Kareena Kapoor Khans flashback photos prove that the diva hasnt changed a bit and ages like fine wine.

Age is just a number when it comes to the Kapoor sisters. They truly age like fine wine and when it comes to Bebo, she looks like the finest bottle of wine there is. In this new Then vs Now segment, we often look back at the celebritys style and how it has evolved over the years. These throwbacks are generally for us to see how far the leading ladies of B-Town have come.

However, things changed when we took a look back at the Begum of Bollywoods photos and to our surprise, we found no stark difference.

THEN

The ever-glamorous Bebo has always been glowing. The diva has managed to maintain the same even after years passing by. Her signature smudged kohl look has still managed to make the same impact it did years ago. Adding to it, she often resorted to metallic lips which if we look back now seems a bit icky. However, then, she was definitely on-trend.

This one award function look in 2004, however, seems off as she stepped out in an embellished kurta and left her highlighted waves open in a haphazard manner.

Coming to her clothes, mini skirts and bodycon dresses were definitely her go-to. She was often seen in different versions and colours of it. Casual tees and jeans were something she seemed to be most comfortable in.

NOW

Her style now has definitely matured. Fashion pieces and creations are strategically selected so she looks her best. While solid colours still remain to be her go-to, Kareena is still seen in her casual jeans and t-shirt. What has evolved is her ability to experiment with her attires. From different hairdos to clothing, she is often seen bringing new things to the table.

What are your thoughts about her style? Let us know in the comments section below.

For more Fashion & Beauty updates: Follow @pinkvillafashion

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Then Vs Now: Kareena Kapoor Khan's evolution proves her GLAM is on point no matter the decade - PINKVILLA

Number nine shirts are among the most iconic and coveted numbers in football.

And since the dawn of the 21st century, Huddersfield Town have had a wide variety of players to have donned the jersey.

Some only wore it for just a season, while others had it for campaign after campaign, while there's even one player to have had it across a couple of spells at the John Smith's Stadium.

Here's our list of those players to have worn Town's number nine since 2000:

Huddersfield entered the new century with Stewart wearing the club's number nine shirt.

However, his time at Town donning the coveted jersey in the new millennium lasted only a month as, with 15 goals to his name that season by the end of January, he was controversially sold to Division One rivals Ipswich Town for 2.5m.

He helped the Tractor Boys reach the top flight via the play-offs as Town missed out by finishing eighth that season.

He later moved to Sunderland in 2002 before being transferred to Bristol City in 2005.

During that time, he had loan spells at Preston North End and Yeovil Town, before joining the Glovers permanently in 2007.

He stayed there until 2008 and ended his playing days with three seasons at Exeter City before retiring in 2011. He is currently assistant manager to Darrell Clarke at Walsall.

Smith inherited the number from Stewart after his controversial sale to Ipswich Town.

He joined Town from Sheffield United and stayed until 2003, when he joined Northampton Town after the Terriers suffered relegation.

Smith held the number nine shirt from 2000 until the end of the 2001/02 season.

After leaving the Cobblers, Smith also had spells at Darlington, Blyth Spartans and Kettering Town before retiring.

Booth came through the ranks at his hometown club and made his debut in March 1992.

His exploits in front of goal led to him being bought by fellow Yorkshire side Sheffield Wednesday in the summer of 1996 for some 2.7m.

As well as a brief spell on loan at Tottenham Hotspur in 2001, he made the return to Town from Hillsborough in March of that year, though his efforts could not stop Huddersfield being relegated to English football's fourth tier.

The following season, Booth took up the number nine shirt this century for the 2002/03 campaign, in which he found the back of the net six times in more than 30 games as Town were promoted.

After this season, the number nine shirt passed to Pawel Abbott, but Booth remained at Town until he retired in 2009. He remains at the club as an ambassador.

Abbott initially joined Town on loan from Preston North End in 2004 to replace Jon Stead after he was sold to Premier League Blackburn Rovers.

He scored four goals in six games during his loan spell, and was bought by Town on a permanent basis on the back of his good form.

Overall he scored more than 40 times in over 100 appearances for Huddersfield.

He wore the number nine shirt during this time before a move to Swansea City where he was sold in the January transfer window in 2007.

The Jamaica international joined Town on a three-year-deal from Watford in 2009.

He took up the number nine shirt and had a productive campaign as he scored 16 times in 43 games in all competitions.

Robinson made only a handful of appearances for Huddersfield in the 2010/11 season and moved to Milwall on loan in September 2010, later joining the London side permanently.

He had a brief return to Town in 2013 on loan from Derby County and his past clubs include Doncaster Rovers, Scunthorpe United, Motherwell, Port Vale and Swindon Town.

Robinson is currently contracted to Southend United but has been on loan at Colchester United in League Two this campaign.

Two spells with Town this decade for the forward and on both occasions he wore the number nine.

Cadamarteri first signed for Huddersfield in 2007 and joined after being released by Leicester City.

He left the Terriers after two seasons and scoring six times in 51 games, heading north of the border to join Dundee United in the SPL.

Two seasons later, he rejoined Town in January 2011 and ended up staying for a further year-and-a-half in West Yorkshire, before leaving for good when he was not offered a new deal in the summer of 2012 by then-Terriers boss Simon Grayson.

After two seasons at Carlisle United, Cadamarteri hung up his boots in 2014 due to a knee injury.

After joining Town in 2009 from non-league Gateshead, Novak would have to wait until his final season with the Terriers until he donned the number nine shirt.

He was Huddersfield's number nine in the 2012/13 campaign, and in total scored six times in nearly 40 appearances across all competitions that season.

He departed the Yorkshire side in the summer of 2013 when his contract expired and linked up with former Town boss Lee Clark once more at Birmingham City.

He joined Chesterfield on loan during his time at St Andrew's and has since played for Charlton Athletic and Scunthorpe United. He is currently at League Two side Bradford City.

Vaughan joined Huddersfield initially on loan from Norwich City in 2012 and was initially handed the number 18 shirt and scored 14 times.

It was only when he joined the club permanently during the following summer transfer window from the Canaries that he took up the number nine.

He stayed at the John's Smith's Stadium for more than two further seasons by which time he had scored 33 goals in 95 games.

After joining hometown club Birmingham City on loan in November 2015, he joined the Blues permanently in 2016.

Since then, the now 31-year-old has had a host of clubs, including Bury, Sunderland, Wigan Athletic, Portsmouth and Bradford City, and is currently on loan at League One side Tranmere Rovers.

The current wearer of Town's number nine has done so since he joined the club in 2016.

It had been vacant during much of the 2015/16 season following the departure of Vaughan from the Terriers to hometown club Birmingham City.

DR Congo international Kachunga first became a Town player in 2016 when he was signed by David Wagner on a season-long loan from FC Ingolstadt in Germany.

His 13 strikes in all competitions that season helped Huddersfield win promotion to the Premier League via the play-off final victory against Reading, also ending the campaign as Town's top scorer.

Kachunga spent two seasons in the Premier League with Town but only has one goal in the top flight to his name against Watford. This season, the forward has found the back of the net just twice so far.

See original here:

Who wore it best? - The evolution of Huddersfield Town's number nine shirt this century - YorkshireLive

Shares in Evolution Petroleum (ASQ:EPM) are currently trading at 2.44, but a key question for investors is how much the current economic uncertainty will affect the price.

One way of making that assessment is to look at the profile of the stock to see where its strengths are. Evolution Petroleum is a player in the Oil & Gas sector. The good news is that it scores well against some important financial and technical measures. In particular, it has strong exposure to two influential drivers of investment returns: high quality and a relatively cheap valuation.

To understand where that shows up, here's a closer look:

GET MORE DATA-DRIVEN INSIGHTS INTO ASQ:EPM

Good quality stocks are loved by the market because they're more likely to be solid, dependable businesses. Profitability is important, but so is the firm's financial strength. A track record of improving finances is essential.

One of the stand out quality metrics for Evolution Petroleum is that it passes 5 of the 9 financial tests in the Piotroski F-Score. The F-Score is a world-class accounting-based checklist for finding stocks with an improving financial health trend. A good F-Score suggests that the company has strong signs of quality.

While quality is important, no-one wants to overpay for a stock, so an appealing valuation is vital too. With a weaker economy, earnings forecasts are unclear right across the market. But there are some valuation measures that can help, and one of them is the Earnings Yield.

Earnings Yield compares a company's profit with its market valuation (worked out by dividing its operating profit by its enterprise value). It gives you a total value of the stock (including its cash and debt), which makes it easier to compare different stocks. As a percentage, the higher the Earnings Yield, the better value the share.

A rule of thumb for a reasonable Earnings Yield might be 5%, and the Earnings Yield for Evolution Petroleum is currently 17.5%.

In summary, good quality and relatively cheap valuations are pointers to those stocks that are some of the most appealing to contrarian value investors. It's among these shares that genuine mis-pricing can be found. Once the market recognises that these quality firms are on sale, those prices often rebound.

Finding good quality stocks at attractive prices is a strategy used by some of the world's most successful investors. If you want to find more shares that meet these rules, you can see a comprehensive list on Stockopedia's StockRanks page.

Read more:

Two factors that could drive the Evolution Petroleum share price higher - Yahoo Finance UK