#t: brazil wnt

[crystaldeclear]

Jamaica playing so well. 45 minutes left to make history

[the-physicality]

how to pick an nwsl team:

angel city fc (Los Angeles): if you like Christen Press. Very famous ownership group (politically concerning). The team is struggling but has bright young talent. Beckintweed is in her first season as head coach after bringing the team to the playoffs last year as interim.

notable players: christen press, Alyssa Thompson, Gisele Thompson, Messiah bright, Claire Emslie, Sarah Gorden

noteable injuries: Press- extended acl recovery, Jun Endo preseason acl

bay fc (San Jose/Bay Area): if you are an optimist/want to be an early adopter. a lot of exciting internationals, but struggle to win games. First year expansion team so also trying to define themselves. notable players: Rachel Kundananji, Princess, Asistat Oshoala, Deyna Castellanos, Tess Boade, Caprice Dydasco

notable injuries: Alex Loera - acl, Melissa Lowder gk #1 preseason acl

Chicago red stars: if you want to see a redemption story. coming back from a bad season, USA phenom mal Swanson is back to lead attacking talent. Also has USA gk #1 Alyssa naeher. Lorne Donaldson (who coached Jamaica wnt in the 2023 wwc as well as Sophia smith and mal Swanson at a youth level) is in his first year as head coach

notable players: Alyssa Naeher, Mal Swanson, Sam Staab

Houston dash fc: if you like rooting for an underdog and won't get discouraged. a team that has struggled historically but is still fun to watch. A lot of international talent, did very well in the draft. The team has a new head coach Fran Alonso who just came over from the Scottish women’s league. You can watch the team work to implement their new style in real time. Jane Campbell #3 gk for the uswnt as of late won goalkeeper of the year last year. Lost a lot of players to free agency and is rebuilding with rookies, trades, and transfers. somehow both the straightest team and the gayest team at the same time.

notable injuries/abscences: Kiki Van Zanten ankle/ lower leg sei, 3 players on maternity leave

notable players: Jane Campbell, Diana Ordonez, Sophie Schmidt, Paige Nielsen, Tarciane (incoming), Michelle Alozie

Kansas City current: if you like watching bangers. one of the top teams to beat this season. With Malawi sensation Themwa Chawinga this team is difficult to stop and has a lot of attacking prowess. Has some defensive liabilities. Coached by former uswnt head coach vlatko andonoski. Has signed a good number of u18 players

notable players: debinha, lo’eau labonta, themwa chawinga, Vanessa dibernardo, bia zaneratto

New Jersey/New York Gotham fc: if you like the uswnt. recently picked up 4 uswnt players in free agency. Also just got Ann Katrin Berger gk from Chelsea on a transfer. She is very good. Head coach Juan Carlos Amaros won coach of the year last year and the team won the championship. They have struggled with injuries this year and scoring more than one goal in a game.

notable injuries: Midge Purce acl , Abby Smith sei from 2023

notable players: Lynn Williams, Ann Katrin Berger, Crystal Dunn, Rose Lavelle, Tierna Davidson, Esther Gonzalez, Jenna Nighswonger, Emily Sonnett, Midge Purce, Yazmeen Ryan

North Carolina Courage: if you are ok waiting . traditionally a very strong team, has struggled a bit this season without Kerolin who tore her acl on the last regular season game in 2023.

notable injuries: Kerolin acl (Nov 2023)

notable players: Casey Murphy, Brianna Pinto, Tyler Lussi, Manaka, Narumi, Ashley Sanchez, Kerolin

Orlando pride: if you like the brazil women's national team. aka brazil fc. Has gone from a team that struggled to one of the top teams this year, in part due to the players brought in over the offseason, many of whom play for Brazil. Recently brought in Barbra Banda a Zambian striker, who has been very productive.

notable players: Marta, Barbra Banda, rafaelle, ally watt

Portland thorns: if you like soccer dynasties. traditionally one of the most successful teams in the nwsl with a lot of local support, the h th orbs struggled in the first few games with their worst start to the season ever. Following the firing of their head coach, the team has won 6 in a row in interim hc rob gale. Has a lot of strong attacking talent but is vulnerable on defense. The home field is turf.

notable players: Sophia smith, Olivia Moultrie, Christine Sinclair, Janine Beckie, Becky Sauerbrunn

Racing Louisville: if you want to watch a team turn around. a team that has struggled historically has put together a solid team in the offseason under new head coach bev yanez. Got some very good rookies in the draft and is off to a decent start.

notable players: Savannah Demelo, Reilyn Turner, Emma Sears, Uchenna Kanu, Ary Borges

San Diego wave fc: if you like to watch a team underproduce. very successful for a team established in 2022. Has a very strong system but has struggled with injuries as of late. The home field is shared with San Diego state football and is not always in the best condition.

notable injuries: Jaedyn Shaw, Alex Morgan (lower legs out tbd)

notable players: Alex Morgan, Jaedyn Shaw, Kailen Sheridan, Naomi Girma, Abby Dahlkemper, Maria Sanchez

Seattle reign: if you want to watch a team figure out their identity without US national team players. historically successful team that lost a lot of impact players in the offseason to retirement or free agency. Previously owned by the ol group, had to be sold because its owner Michelle Kang also owns the Washington Spirit. Dropping the OL, the brand got a massive upgrade. Is struggling this season. The home field is turf.

notable injuries: Claudia Dickey #1 gk

notable players: Jess Fishlock, Lauren Barnes, Quinn, Bethany Balcer, Alanna Cook, Ji So-Yun, Jordyn Heuitema, Veronica Latsko

Utah royals fc: if you live in utah. a new expansion team that is struggling quite a bit. First time head coach Amy Rodriguez selected ally sentnor as the first draft pick. Has a racist kit and stadium sponsor. Does not have a full roster.

notable injuries: Imani Dorsey Achilles

notable players: Mandy haught, Ifeoma Onumanu, Ally Sentnor

Washington spirit: if you want to watch rookies make magic. owned by Michelle kang, not afraid to make big moves and spend $$$ for a strong team. Has found success recently. Operating under interim hc Adrian Gonzalez until Barcelona head coach Jonathan Giraldez arrives after the end of their season. Croix Bethune is doing very well her rookie season.

notable players: Trinity Rodman, Croix Bethune, Casey Kruger, Andi Sullivan, Ashley Hatch, Hal Hershfelt

[that-soccer-guru]

i so sad about this semis tbh woke up at 5am for the bra x can to cry over it :( the last olympics for marta, formiga, cris wasn't even called up, tamires and erika possibly

-conmebol anon

God I know.

I refuse to wake up for Olympics woso bud, I'm ngl to you. But seeing the posts on the scoreline broke my freaking heart. I was so excited and had so many expectations for Chile and Brazil and things really just fell apart.

Man, and Formiga absolutely deserves a better ride for her last Olympics. That woman has put the midfield on her back for years and they really fell apart in penalties. Marta too, we get to see the golden age of woso and its biggest legend crumbling before Semis is.... painful man, absolutely painful.

Kudos to Canada for pushing through and sticking with their game, I'm glad they're still in it and are showing a lot of grit, but I'm still so freaking bummed out for Brazil.

[the-firebird69]

bja left high and dry his run into chasms.  and he is weak.  so grabbe this just now and prepares to fire on selected targets. and is mean.  means to hit here nearby...and we cant have it. nope and he is off and this is it he dies now.  fully  if we can.  his top generals all there. all will die. and  he begins the countdown...and is counting silently  and he hits death stars and hits ships and curascant and his are there and trumps but mostly trump.  and they agreed no said he was is a blaspheme. and they fire on him shorlty he let loose one great beam and he is not dead did not jump.  and then re arm target that area. and nobody will and he screams it..and they push him into the cravass. he falls and they are fried nope.   says reload they do.  and for real he wishes to target punta gorda and fort meyers and sarasota to clear t and kidnap him.  use him as a hostage.  and he says this round signals it.  we hear ti warm and fire.  he is out. and it is not bg who fired...no it is.  we say this bg knew it was his threat but that is not why he says he is our freind i defend him and moves in shortly..though the controls are fried he wishes to go to manual and yeh to hit nearby  him and they are nuts and lost it. right down the line really macs too try for it.  but no. it is down he fried the controls but....the controls are also in the channels and are secure too. and under the stone.  it is repowering. and yes Gort is there and the Female Gort and more. tons more. of us.  and they are aghast send over fleets cork is up out shortly and bg is fighitng rey his sister and others fight sebastian is ther efighting rey for it.  hue lots of them die and infighting...and it is a sick mess.  he says this is why trump did it..and they agree.and try. and then this no you dont and the empire hits them and blwos up the woods.  and yes makes a cravas no that is other but yeh a huge explosion.  and it is on they yell hey. and are out no the cruiser is macs he hit too cork and others fried them all yu fers suk so badly you hit all our stuff not one target on my list.  and now my fleets.  firing like mad now te star killer is up and aremed hits all targts.  blasts curuscant and takes out huge lasers and hits reflectors it doesnt control tons about five k thusfar  and only a few more thousand and now out none there. and they are slow dimwitted people.  and other planetoids act.  no sit.  and they are now engaging another palnetoud gamemede and it is designed to cut throuhg the layer and it is miels thick and vaboom in and hits and baboom and more and tons of times huge lasers up in smoke tons see it and it hits rapidly.  and tommy a is not out  ok fell sees i t passes out from pain.  then this we get you caa and from here and oh we get ou now ok idiots. and we hit here and we htyou you idiot. oh.  and now we see they are dumb.  rebels targeted nope.  tons of empire targets are hit and yes reflectors move over to curascant. now too and fast and he is empire trump cork is but not that mch and mac wnts trump out but loses death stars.  is in truolbe .  we fire on tons of targets at Gamemede adn the  cool helps immensely we than him he is proud. and boom targets blow.  and more hits now.  andout now you two you sie w empire ok we see.  enough.  and trumps ships not hit yet.  the ground forces were are. and his ships move to attack Hoth and positionnow heat warm and the star Killer hits.  destroys one utterly the others flee.  it is powerful and doped well. tons of doping. and they go now to brazil or near it and collect.  and tons of the ruby red.  and we hit.  they are out soon....and finally they blab there. this is it huge hits and all over..ins pace will b.  and ok that is all no but we see.  mostly planetooids.   adn it is huge though.  massithve hits. and cork is out now. and flies out. and mac fled saw it heat and aim at him. he said adios. adn left adn the fleets move in.  and are terrorized by it goes wide flattens them all and in wide areas out..or on fire depending on armor. bills and wills are not armored as well and were thick many feet.  and they burn. alight lots see them...and rutguer hower says it later..i have seen this stuff and yu say these words to me.....mac its mac.  and he looks i think. and tells you o no and fired on cork and smiles saw it too. huge battles..massive bright balls. impressive yes.  and to him yes.  a culmination of all his lifes work and mine. and it is a sight....huge epiteths no are having fun and a rebel party well ok and we see Hera goes up and starts it off...some empire costumes. and they have drinks adn setup streets and more and says ok and then this is it safe and they say ok  we cahnge it if need be fast.  the streets look like china vendors all over and stores and food and fun tons of rebel stuff and rebel drinks and from far away and we needed this we are in and told  her and yes.  and what is it a huge rebel party and good and we hae them too and it is on we are here. michael Keegan says..and not his real name prob like Hawas yes it is similar like Hawan no lol ahhaah Hawain no damnt and fun costumes sabers and makin them buy sell cosply and tons of realistic ones.  used and new hug elots and garth is htere sells the towable for Sim and hitch mount installs real fast.  bolts on. can do it onsite and setup a shop...tons see it.  has  a dyno too.  we u se it now and garth is pleased sees  he is respected. the star killer fires..and fires and fires and then this we hit now and macs are grouping nearby and the rebels see it and are on alert as they are called away to arty and up now and at them hard real fightersnow and it is mean angry  and the star killer hits death stars. and thre are real rebels at it.  firing. tons cant see who. it is not obvious.   then they land some big ones and hit. the empire does on rebel bases say stop and nope.  and at it again. and then this. we hit youru hometown of missorui and meaninghere and so the middle and we hit you and blam they are hit. out too. other shps move near and try blam and are hit and we move in hit them all and out they go.  giant empire ships hit.  huge ones. and out.  and we fire madly.  now. too. tons try for death stars no good.   we hit now. and trump circled back and he is out   sticks it out and loses. and then this we are not hit by monsters. nope. and then this again who is sauing that and us. ok.  and it fires. hits the ships are out and damaged two done and one escapes.  and trump is slwo forgetshow to fire. and he has a few ships left well about nine.they say oh number nine.  and then this you shut up. and we see. and curascant was his now it iwanes and so was gamemed and hoth no more are out soon. well it has commencced.  fighitng. and tons of it. huge lots in. huge.  tons of them.  gigantic ships hit of empire and rebel and the low lifes  and tons done and trump is less three ships well two adn a half i t limps back he is not happy, breaks radio silence says who. cork suppoedly mac hit him...and they laugh mac did he  is there.  firing on his. and then this who is no im here hes there. and it is skinny the man here tommy f who is not my son anymore. ok.  you oaf. your not letting me succeed he says. we see. tons fire now. huge huge lots.  and at the star killer and we see it will fall shortly, are out momentarily.  but first we use it on a lower setting ad spray them all hits and they are disabled and we hit large groupoings.  firing rapidly and it fires fst and is poerful again. then they retort and hit part of i t. slows it to half speed still fast. adn firing in ten seconds.  and soon...warms an wamo they hit most of th e attackers the rest flee. out now.  warms and fires out.  tons of reloading.    a huge huge fleet about ten nonillion good sized cruisers ten to fifteen miles upt to five  hundred and then trummps three mega ships all beat by us.  and all empire hmm and then this who the hell is firing that thing they are proficient. and we see him nope.  then this.  who.  and nobody can say and it warms now. slowly hums.  finally it is up and runs.  hits the remainingships adn out soon. we fire to exhustion and reload no but close. now reloading on the fly need it.  tons of it in tosn out.  fast too. and between hits  it is on here we fire on you s.  and they balk ok. and fire back and laugh.  not really. and then this we are sitting ducks and they say prove it. seastian will and hit us lol.and ok dummy Sebastian says.  weher  and when.  this planet and you alone with your gear...ok ok i can not alone. and you bring a crew.  so e do this ok.  and we fire tons on them and reload.  tons.  fster adn yes we see Thor Freya Zues Hera mac daddy

https://www.youtube.com/watch?v=-HmWDdmTAE8

[forkickspodcast]

by Daniel Squizzato

If all goes according to plan, Canada���s “bronze-medal celebration” match this past weekend will be the last one they ever play.

For reasons both practical and symbolic, Saturday’s friendly against Mexico — a 3-2 win in front of 22,508 fans at Vancouver’s BC Place — represented the dawn of a new era for the women’s national team.

Canada’s first game since Rio 2016 was meant to celebrate the team’s second straight Olympic bronze medal, while providing a send-off for a trio of retiring veterans: Melissa Tancredi, Rhian Wilkinson and Marie-Eve Nault.

Also honored on the day were Karina LeBlanc, Carmelina Moscato and Emily Zurrer, all part of a core that rebounded from a last-place finish at the 2011 Women’s World Cup to win a surprise bronze at London 2012. That kicked off a half-decade nationwide swoon over this gritty, plucky and, of course, inspirational squad.

As Canada’s long-time core cedes their starring roles to the next generation, expectations are changing. Captain Christine Sinclair and head coach John Herdman have proclaimed that Canada’s target is to hit No. 1 in the FIFA rankings during this quadrennial cycle.

But is that a realistic target, or simply locker-room hype material?

Learning to expect expectations

Prior to the 2015 Women’s World Cup, Herdman claimed his team’s goal was to reach the final, something Canada has never done. Most observers paid the claim little credence, presuming Herdman was simply trying to motivate his squad. Ultimately, the home side went out in the quarterfinals.

But that tournament provided a launching pad for three members of Canada’s new core: Kadeisha Buchanan and Ashley Lawrence (who both recently signed in France; Buchanan with Lyon, Lawrence with PSG), and Jessie Fleming (who has turned many heads in her freshman year at UCLA).

Then, at Rio 2016, the new core welcomed Janine Beckie (who, at 22, is already among Canada’s top 10 all-time scorers), 21-year-old defender Rebecca Quinn and 17-year-old striker Deanne Rose.

The rapid ascent of those players — along with the addition of players like Josee Belanger and Allysha Chapman — are proof that Herdman’s not simply about psychological boosterism. He’s exceptional at evaluating and utilizing talent.

That was surely part of the Canadian Soccer Association’s thought process back in 2013, when they signed the Englishman through 2020. With the benefit of a long-term view, Herdman could gradually integrate new talent, in the hopes of peaking during the 2019 World Cup/2020 Olympic cycle.

And yet, what was supposed to be a transitional squad in Rio ended up punching above its weight, earning a first-ever win over Germany and beating Brazil for the bronze. That vaulted Canada to No. 4 in the world, the team’s highest FIFA ranking yet, and fueled dreams of reaching the global summit.

But if hitting No. 1 is truly the target, then beating Tier-I teams like Germany has to be the norm, not the exception, and having depth all over the field must be the default situation, not unexpected happenstance. The team isn’t there just yet.

Gaining entrance to Tier I

The departure of LeBlanc (retirement) and Erin McLeod (injury) has the goalkeeper position in more flux than it has been in for over a decade. The central defensive triumvirate of Buchanan, Quinn and Shelina Zadorsky has been solid, but there’s no clear option behind those three at the moment.

Lawrence and Belanger have done well as converted fullbacks, but the departure of Wilkinson and Nault has left few ready-made solutions at the position. Leaders like Diana Matheson, Sophie Schmidt and Desiree Scott will keep the midfield humming along, while Canada is actually developing some depth up front, with Beckie, Rose and Nichelle Prince.

Teenagers such as Gabrielle Carle (who was a Rio 2016 alternate), Sarah Stratigakis (who earned her first senior-team start on Saturday) and Marie Levasseur have been easing their way into the senior team, while 16-year-old Julia Grosso and 15-year-old Jordyn Huitema have shown promise.

But Canada’s performances at last year’s U-20 and U-17 Women’s World Cups (going out in the group stage in both tournaments) suggested the pipeline isn’t overflowing with players who are immediately ready to step in for a team hoping to be the world’s best.

Now, with a few more years of development, some of them could play roles in the next cycle. Buchanan, Lawrence, Beckie and Fleming (who’ll all be in their mid-20s) are poised to be standout stars at France 2019. And there is a good chance that Herdman will find more diamonds in the rough, be they from the youth ranks or players joining the national team later in their careers (a la Chapman).

But if you’re holding out hope that the next Christine Sinclair will appear to lead the way, here’s the reality: She’s not coming. Not now, not ever.

A matter of when, not if

The global game has evolved to the point where no single player can unilaterally determine the outcome of games the way Sinclair once did. The Canadian captain, who turns 34 this summer, still brings value to the team, but her years of peak influence are behind her.

But as she leads the team into the next cycle, she provides a living link to the generation that toiled in domestic obscurity for years before breaking through in London. The idea that Canada’s bronze medals “inspired a nation” has been overplayed to the point of self-parody, but for the team’s new core, winning a big tournament isn’t mere fantasy; it’s the next logical step in the team’s progression.

Of course, to be the best, you’ve gotta beat the best (i.e. the U.S.), something Canada’s been traditionally unable to do. The rivalry is staggeringly asymmetrical; the U.S. has won 46 of 55 all-time meetings, with Canada’s last win coming in 2001.

The outcome of their next showdown will give the clearest indication yet of whether reaching No. 1 by 2020 could truly be in the cards for Canada. At this point, the chances of Canada reaching that goal, in that timeframe, look pretty slim.

But that doesn’t mean Herdman’s proclamation is simply smoke and mirrors; rather, it’s a sign that as we close the book on Canada’s bronze generation, it’s time to start thinking about when, not if, the country’s golden generation will emerge.

[abbkineeu]

New Post has been published on Biotech Advisers

New Post has been published on http://www.bioadvisers.com/crispr-cas9-gene-drive-targeting-doublesex-causes-complete-population-suppression-caged-anopheles-gambiae-mosquitoes/

A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

Content introduction:

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

De novo domestication of wild tomato using genome editing

A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

1. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

Here Emily K Schutsky at University of Pennsylvania in Philadelphia, Pennsylvania, USA and his colleagues present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, they found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

Read more, please click https://www.nature.com/articles/nbt.4204

2. De novo domestication of wild tomato using genome editing

Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost. We devised a CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. Agustin Zsögön at Universidade Federal de Viçosa in Viçosa, Brazil and his colleagues report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, their engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Their results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants.

Read more, please click https://www.nature.com/articles/nbt.4272

3. A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. Kyros Kyrou at Imperial College in London, UK and his colleagues found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.

Read more, please click https://www.nature.com/articles/nbt.4245

4. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Base editors (BEs) have been used to create C-to-T substitutions in various organisms. However, editing with rat APOBEC1-based BE3 is limited to a 5-nt sequence editing window and is inefficient in GC contexts. Here, Yuan Zong at Chinese Academy of Sciences in Beijing, China and his colleagues show that a base editor fusion protein composed of Cas9 nickase and human APOBEC3A (A3A-PBE) converts cytidine to thymidine efficiently in wheat, rice and potato with a 17-nucleotide editing window at all examined sites, independent of sequence context.

Read more, please click https://www.nature.com/articles/nbt.4261

5. Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell’s identity and its position in a tissue. Keren Bahar Halpern at Weizmann Institute of Science in Rehovot, Israel and his colleagues recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here they used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs’ tissue coordinates. They applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, they reconstructed LEC zonation and extracted a landmark gene panel that they used to spatially map LEC scRNAseq data. Their approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.

Read more, please click https://www.nature.com/articles/nbt.4231

[bioadvisers]

Bioadvisers shared on Biotech Advisers

A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

Content introduction:

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

De novo domestication of wild tomato using genome editing

A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

1. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

Here Emily K Schutsky at University of Pennsylvania in Philadelphia, Pennsylvania, USA and his colleagues present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, they found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

Read more, please click https://www.nature.com/articles/nbt.4204

2. De novo domestication of wild tomato using genome editing

Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost. We devised a CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. Agustin Zsögön at Universidade Federal de Viçosa in Viçosa, Brazil and his colleagues report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, their engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Their results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants.

Read more, please click https://www.nature.com/articles/nbt.4272

3. A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. Kyros Kyrou at Imperial College in London, UK and his colleagues found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.

Read more, please click https://www.nature.com/articles/nbt.4245

4. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Base editors (BEs) have been used to create C-to-T substitutions in various organisms. However, editing with rat APOBEC1-based BE3 is limited to a 5-nt sequence editing window and is inefficient in GC contexts. Here, Yuan Zong at Chinese Academy of Sciences in Beijing, China and his colleagues show that a base editor fusion protein composed of Cas9 nickase and human APOBEC3A (A3A-PBE) converts cytidine to thymidine efficiently in wheat, rice and potato with a 17-nucleotide editing window at all examined sites, independent of sequence context.

Read more, please click https://www.nature.com/articles/nbt.4261

5. Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell’s identity and its position in a tissue. Keren Bahar Halpern at Weizmann Institute of Science in Rehovot, Israel and his colleagues recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here they used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs’ tissue coordinates. They applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, they reconstructed LEC zonation and extracted a landmark gene panel that they used to spatially map LEC scRNAseq data. Their approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.

Read more, please click https://www.nature.com/articles/nbt.4231

[abbkine]

BioAdvisers said on Biotech Advisers

A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

Content introduction:

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

De novo domestication of wild tomato using genome editing

A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

1. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

Here Emily K Schutsky at University of Pennsylvania in Philadelphia, Pennsylvania, USA and his colleagues present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, they found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

Read more, please click https://www.nature.com/articles/nbt.4204

2. De novo domestication of wild tomato using genome editing

Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost. We devised a CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. Agustin Zsögön at Universidade Federal de Viçosa in Viçosa, Brazil and his colleagues report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, their engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Their results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants.

Read more, please click https://www.nature.com/articles/nbt.4272

3. A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. Kyros Kyrou at Imperial College in London, UK and his colleagues found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.

Read more, please click https://www.nature.com/articles/nbt.4245

4. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Base editors (BEs) have been used to create C-to-T substitutions in various organisms. However, editing with rat APOBEC1-based BE3 is limited to a 5-nt sequence editing window and is inefficient in GC contexts. Here, Yuan Zong at Chinese Academy of Sciences in Beijing, China and his colleagues show that a base editor fusion protein composed of Cas9 nickase and human APOBEC3A (A3A-PBE) converts cytidine to thymidine efficiently in wheat, rice and potato with a 17-nucleotide editing window at all examined sites, independent of sequence context.

Read more, please click https://www.nature.com/articles/nbt.4261

5. Paired-cell sequencing enables spatial gene expression mapping of liver endothelial cells

Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell’s identity and its position in a tissue. Keren Bahar Halpern at Weizmann Institute of Science in Rehovot, Israel and his colleagues recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here they used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs’ tissue coordinates. They applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, they reconstructed LEC zonation and extracted a landmark gene panel that they used to spatially map LEC scRNAseq data. Their approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.

Read more, please click https://www.nature.com/articles/nbt.4231

[that-soccer-guru]

The way the Brazilian goalkeeper j shoved Lindsey hahahahaha

Horan is basically begging to get socked by a Brazilian and I for one vote that the refs let it happen

[that-soccer-guru]

thoughts on the legend herself: formiga

IN THIS HOUSE WE LOVE AND STAN THE QUEEN HERSELF. THE LEGEND. THE BREAKER OF SPIRITS. THE IMMORTAL.

Miraildes Maciel Mota, 7 world cup appearances, every Olympics game since the '96 summer Olympics, 3 time Panamerican gold winner and 6 time Copa América champion, 8 time Brazilian cup winner, one time French cup winner, picked for the FIFA CONMEBOL team of the decade, 200 international caps, known for her nickname Formiga-or "ant" in Brazilian, because she is unselfish and absolutely badass. SHE WAS BORN WHEN BEING A FEMALE SOCCER PLAYER WASNT JUST FROWNED UPON, IT WAS FUCKING ILLEGAL.

DO YOU UNDERSTAND HOW BADASS THIS WOMAN IS. I absolutely love and stan and I know the soccer world will miss her when she decides to retire for good. She embodies the spirit of jogo bonito (the beautiful game) and she's an absolute MASTERCLASS in humanity and resilience.

Yes we absolutely CAN talk about Formiga WHENEVER, WHEREVER MY DEAR ANON.

[that-soccer-guru]

Thoughts on how each player played?

MUCH better game for most of the field! Let me go with the good stuff first.

Naeher—flawless, amazing, we love uncle.

I said last time that Christen and Rose were the best part of the forward core and they went out there and proved me right about my opinions. Amazing chances, good ball handling, great tactical fouling. They went out there and carried those forward chances on their backs.

Broon and Dunn took away every ounce of happiness I had today and I respect them for it. As always, flawless game from them. I saw a tweet that said Dunn's heat map is just a picture of the stadium and its FACTS. She thwarted the Brazilian strikers really well and she put up great chances up top. Broon is the one woman show of the CBs and I can never fault her. In Queen Broon we trust (even today when I was actively rooting against the US, I couldn't help but absolutely SALIVATE at how GOOD she is bro. the one woman defense) Dahlkemper is up there too, although she really fell behind in helping Ertz cover the players finding those gaps in the middle but I'm not gonna discount her good moments, bc she's always reliable.

Ertz had a really hard job to do today, so I won't slam her for the times that Brazil managed to slip through. She moved the line up a lot, played good in recoveries, overall she's a very reliable 6 and I can't fault her for the other players' decisions.

Now, the Bad.

I mean this is hardly bad but I'm never impressed by Alex Morgan and that didn't change today. She's still finding her footing so, you know, we'll see what happens with her. She had some good moments though but overall she got lost in the shuffle a lot which kinda sucks for someone like her. Her replacement, Sophia Smith, also got lost a bit in there, she did have a near goal which was great but I didn't have enough time to really judge her playing.

Lynn Williams.... I'm so tired of saying the same things, I sound like a broken record. She is not made for this team. She's a great player, she has spirit, but fucks sakes this is not the team for her. AND THERES NOTHING WRONG WITH THAT. It's okay to be a great club player. But the missed chances, bad passes, poor covering, it's just not it.

Sonnett had a craaaaaap game. She made some good tackles and good trackbacks but she kept making really bad clearance choices and her passing was NOT with it. I would blame all of Ertz' overextensions on her bad decisions today. Yes, covering Marta is fucking hard. But you're a defender that's your job. And she was going way too far up and abandoning her line which led to her being both a bad defender AND a bad midfielder. Y'all know that despite her personal choices I still think Sonnett is a wonderful player and a defender with great potential. I still think she can be the key to this defense. But she had better get her head on right because this was not her best game. Glimpses of greatness are NOT enough to make up for those costly mistakes.

The "?"

Lindsey Horan had like... a meh? game. She wasn't dazzling, she keeps making really physical choices that are crap. She should've been carded for the free kick interruption, and for bulldozing players, I was just glad she finally got carded for the goalkeeper incident. She had some really brilliant moments on the counter and some glimpses of that playmaking ability she showcases in Portland, but she doesn't look like she's IN the game, which is sucky. At least she didn't drop anyone like she dropped Jflem so, like, yay?

Geriatrics United FC didn't have enough time to fuck anything up so that's great. Although Pinoe did make some reaaaally bad choices in the mid, she had a goal so we can forgive her I suppose. Lloyd as always doesn't impress me and I'm not gonna start saying otherwise today.

Kmew also didn't have enough time to get anything done although she did give that pass for Pinoe's goal I think? so like, wooo! Vlatko hasn't played her enough for me to think that she will be going to the Olympics. Which is unfortunate. The one play I saw her in she has walking and not actively chasing the ball which was just.... odd? bc she's fresh off the bench, she should've been more active. But idk what orders Vlatko gave her on the way in so idk.

For Brazil uh, I was very disappointed with the backline. They went in there on the defensive and I don't get it. Granted, the US have a scary midfield, but so does Brazil. They started on the back foot and hardly got off it. Ludmila didn't look like she was on her A game, Debinha was trying but the passes between all of them were NOT connecting and it sucked. They kept trying for that long ball, which is disappointing, because Brazil's strength is on the short one on one passing. I've never known Brazil to go into a field to play the other team's game, but today they did, and that bummed me out. This should've been a way better game than it was for them.

Overall there's glimpses that the US can self correct. Luckily it looks like the fear of playing the world's #1 still works, even for great teams, so their rivals starting on the back foot is gonna keep being a blessing to count on. Overall there's a lot to improve, I want to see Tierna join Ertz in a double 6 formation for Argentina, just to see if there's a more creative way to have a good containing backline. Press and Rose earned their starting spots for next game. I'd make a case for Horan to be swapped for Kmew, just to see what she's made of.

But knowing the US like we know them, they're gonna come out swinging with the same shit bc WHAT IS CREATIVITY.

[that-soccer-guru]

And I'm gonna say it with my chest

SON OF A BITCH THEY DID LEARN FROM THEIR FUCKUPS

HOWEVER

it's a GOOD FUCKING DAY when Ludmila freaking Da Silva is up top with Debinha. That's a solid fucking midfield and two good scorers that worm their way through holes in the lineup like no ones business.

THIS IS BOUTTA BE A GREAT GAME KIDS!

[that-soccer-guru]

What team do you think will give the US the most problems moving forward. Not necessarily those the Olympics but any team. I loved watching us play Spain in SBC last year. They were the best team at the tournament (in my opinion) but the US just doesn’t know how to quit and always finds a way to win. AKA Julie “the back of yo head is ridiculous” Ertz.

Bonus points if anyone gets that reference!

Oh good question,

As much as it PAINS me to say it'll be a European team, they're the ones dropping the most money and resources on their teams. Barcelona is one of the most dominant teams in Europe behind Lyon, and most of the Spanish team is made up of the Barcelona roster, the same with France and Lyon. So I expect Spain and France to show the most growth and be the toughest opponent. (If France can get its shit together coaching wise that is)

Germany and England are right behind them, with developing leagues and players in international markets getting great experience. I'm half hoping for Italy to get it's shit together at some point soon, if I'm not wrong most of the Juve squad is part of the NT so that'll be a good rally point. China and Japan have really good teams and they have a different way of playing, and a very good way to thwart teams that play the same damn way everytime, so that'll be fun.

Nigeria, South Africa and Cameroon are really interesting NTs from Africa, and the cup of nations is a very good tournament! CONMEBOL ofc has Brazil, but most of the other federations don't pour enough resources into their teams which SUUUUCKS because there's enough talent to give the uswnt a run for their money. But I digress.

So TLDR: Spain and France I expect to give some hard fights, with Germany and England right behind them. I'd love to see latam teams take their women's NTs seriously because I could see Brazil, Colombia, Chile and Argentina rising as high as they always have in the men's side. Also let me not forget the Aussies bc Australia has SO MUCH TALENT.

[that-soccer-guru]

tbh our problem rest mostly in midfield if luana and formiga are not playing, andressinha can't hold it alone and the backline without erika is shit. i mean, rafaelle basically defended alone for the full 90's since kathllen seemed quite lost to me, bruna is not a particularly good defender as we keep sayong and tamires's skill is a waste when not advancing her, she's a fucking beast with corinthians. also we have zero outside backs, it's usually improvised (as camilinha and kathllen were today, even if their position in club is actually that) and tamires should be in mid

overall i feel like we played a bit better that what we expected, holding up quite alright but less then we could've if that makes sense? we had chances that we didn't finished well but we also messed up a lot. i trully think that if luana a d formiga were in the game we could've won or tied

you know I spent all the first half saying "FORMIGA WOULDN'T LET ME DOWN LIKE THIS". So I absolutely feel you. I wish she'd been here because she definitely provides more solid backing for the team, and it showed with how much Marta kept running back.

I give kudos to Rafaelle bc she really fucking played her HEART out. She deserves to bitch slap her teammates for the really stupid mistakes they were making. I really wanted Tamires to shine a little bit more, bc she IS great in Corinthians. But the back foot start was not amazing and I don't think she found her footing.

I respected Bruna only bc of her willingness to crack ankles. Which is entertaining but does not a good defender make.

The chances were just bad bc the passes weren't connecting, but there were moments of brilliance. But when your forwards/mids are spending most of their time in recovery duty, your chances at goal are gonna suffer.

I think Brazil definitely missed its brightest stars today, and I wanted more from them. I know they had it in them. BUT it's time for them to pick themselves up, dust themselves off, and learn from this game.

[that-soccer-guru]

How are we feeling about this game, team CONMEBOL?

[that-soccer-guru]

No sabía que la selección argentina estaba pasando por un mal momento :c Esperemos que dentro de todo logren tener una buena actuación. * La convocatoria de Brasil 👌👌

Pueeeee. Eso es por q la Federación tiene un huevo enorme y prefirieron vetar a las jugadoras que pelearon por lo que les debían (pero vaya dígales que Messi no quiere jugar y venden a la mamá para convencerlo). También me parece muy irresponsable acceder a jugar con la situación del COVID como esta en Argentina y en Estados Unidos. Pero, el caso, será echarles buena vibra.

Esa convocatoria de Brasil está PODEROSA. Va a ser una buena copa para ellas, y eso al menos es algo bueno.

NOW IN ENGLISH

"I didn't know that the Argentina NT was going through a bad time :c let's hope despite everything they have a good tournament. *That Brazil roster 👌👌"

weeeeell, that's because the federation is a piece of shit and they chose to ban the players fighting for the payment they earned rather than just get it over with (but God forbid Messi says he refuses to play for the NT, they'll sell their mother to get him there). I also think it's irresponsible to agree to this when the COVID situation is NOT great in Argentina and the states. But, I'm still gonna be sending them good vibes!

THAT BRAZIL ROSTER IS POWERFUL DUDE! It'll be a good cup for them at least, and that's something good!