thescientist--2020-11-12--Newly Found Proteins Stop Fungal "Bleeding"

Mycelium is the fabric of fungal populations : fungi produce thread-like roots called hyphae , which branch and fuse with one another to form a vast , interconnected network—the mycelium .
It allows fungi to grow rapidly , transport nutrients , and even share information about the local environment over long distances .
The network is also vulnerable ; a wound could lead to catastrophic bleeding of protoplasm that can lead to death .
While some fungal species separate their filaments into compartments with septal walls that can limit leakage , other fungi do not make any walls , and @ @ @ @ @ @ @ respond to an injury .
Now , a team at the National University of Singapore has discovered their secret : large , mechanosensitive proteins called gellins that have not been described before .
When a hyphal filament is injured , the pressurized liquid protoplasm inside the hyphae gushes out .
Immediately , gellins inside the hyphae respond to the shear stress of the protoplasm flow by quickly crosslinking , unfolding , and sticking to each other and to cell membranes .
The crosslinked gellins form a gel plug at the site of injury @ @ @ @ @ @ @ losing all of its protoplasm , evolutionary cell biologist Greg Jedd of the National University of Singapore and colleagues report in Current Biology on November 12 .
“ What ’ s super neat about the gellins is that you ’ re using a physical cue to drive phase separation , ” says cell biologist Amy Gladfelter at the University of North Carolina , Chapel Hill who was not involved in the study .
That is , shear stress of the rapid flow of protoplasm at the injured site triggers the gellins to unfold @ @ @ @ @ @ @ in the protoplasm—liquid and gel .
“ There aren ’ t too many examples of that.
” The project started when Jedd observed that injuries in Phycomyces blakesleeanus and Mucor circinelloides , two fungal species that do not make septal walls , triggered a rapid gelation response that stopped the protoplasm from leaking out .
Suspecting that this response was mediated by a large polymer , the researchers ground up the cells and used protein electrophoresis to identify the large proteins inside the filaments .
They called these proteins gellins—there are two kinds @ @ @ @ @ @ @ the genes that encode them .
Finally , they used CRISPR to eliminate both types of gellins from Mucor circinelloides .
When fungi lacking gellins were injured , they bled out into a protoplasmic puddle and died , confirming that gellins play a key role in sealing up breaks .
“ This was a creative way to discover the proteins that might be responsible for this phenomenon , ” says mycologist Jason Stajich at University of California , Riverside , who was not involved in the research .
He praised the team ’ @ @ @ @ @ @ @ fungal gelation response and “ work backwards to identify the actual molecular basis of that.
” To find out how the gellins actually work , the researchers turned to structural biologist Daiwen Yang also of the National University of Singapore .
Yang purified the gellins and used nuclear magnetic resonance to discover that gellins have 10 “ beta-barrel ” domains .
The first beta barrel domain , which the researchers focused on , had an unusual , very hydrophobic loop that they suspected was involved in binding to cell membranes .
Mechanobiologist Jie @ @ @ @ @ @ @ a gellin by one end and gently pull on the other end , revealing that the protein unfolded at forces consistent with what gellins would experience inside a bursting hypha .
“ It ’ s an elegant set of experiments , ” Stajich says .
The process to stop fungal bleeding has some parallels to mammalian blood clotting .
For example , the von Willebrand factor ( VWF ) that helps blood clot is also mechanically sensitive and responds to the force of flow by changing shape and binding with other VWF proteins @ @ @ @ @ @ @ networks , which are required to overcome the limit of diffusion as an organism gets large , Jedd explains .
“ Each has found ways of dealing with injury , but there is some degree of convergence.
” Spider silk , too , is made of large proteins that are extruded through the spider ’ s silk glands , and the shear stress from that flow induces the formation of intermolecular bonds , thus producing a thread from liquid protein .
Understanding the design principles for these mechanosensitive proteins could have all kinds @ @ @ @ @ @ @ soft robots , the researchers suggest .

Phycomyces blakesleeanus
JUDI THOMAS, MISSOURI MYCOLOGICAL SOCIETY
Mycelium is the fabric of fungal populations: fungi produce thread-like roots called hyphae, which branch and fuse with one another to form a vast, interconnected network—the mycelium. It allows fungi to grow rapidly, transport nutrients, and even share information about the local environment over long distances. The network is also vulnerable; a wound could lead to catastrophic bleeding of protoplasm that can lead to death. While some fungal species separate their filaments into compartments with septal walls that can limit leakage, other fungi do not make any walls, and mycologists haven’t known how they respond to an injury.
Now, a team at the National University of Singapore has discovered their secret: large, mechanosensitive proteins called gellins that have not been described before. When a hyphal filament is injured, the pressurized liquid protoplasm inside the hyphae gushes out. Immediately, gellins inside the hyphae respond to the shear stress of the protoplasm flow by quickly crosslinking, unfolding, and sticking to each other and to cell membranes. The crosslinked gellins form a gel plug at the site of injury almost instantaneously, preventing the fungus from losing all of its protoplasm, evolutionary cell biologist Greg Jedd of the National University of Singapore and colleagues report in Current Biology on November 12.
“What’s super neat about the gellins is that you’re using a physical cue to drive phase separation,” says cell biologist Amy Gladfelter at the University of North Carolina, Chapel Hill who was not involved in the study. That is, shear stress of the rapid flow of protoplasm at the injured site triggers the gellins to unfold and crosslink, creating two distinct phases in the protoplasm—liquid and gel. “There aren’t too many examples of that.”
Protoplasm stops leaking out of an injured hyphal tip in wildtype M. circinelloides. GREG JEDD, TEMASEK LIFE SCIENCES LABORATORY, NATIONAL UNIVERSITY OF SINGAPORE
The project started when Jedd observed that injuries in Phycomyces blakesleeanus and Mucor circinelloides, two fungal species that do not make septal walls, triggered a rapid gelation response that stopped the protoplasm from leaking out. Suspecting that this response was mediated by a large polymer, the researchers ground up the cells and used protein electrophoresis to identify the large proteins inside the filaments. They called these proteins gellins—there are two kinds, gellin A and gellin B—and identified the genes that encode them. Finally, they used CRISPR to eliminate both types of gellins from Mucor circinelloides. When fungi lacking gellins were injured, they bled out into a protoplasmic puddle and died, confirming that gellins play a key role in sealing up breaks.
“This was a creative way to discover the proteins that might be responsible for this phenomenon,” says mycologist Jason Stajich at University of California, Riverside, who was not involved in the research. He praised the team’s ability to make observations of the fungal gelation response and “work backwards to identify the actual molecular basis of that.”
To find out how the gellins actually work, the researchers turned to structural biologist Daiwen Yang also of the National University of Singapore. Yang purified the gellins and used nuclear magnetic resonance to discover that gellins have 10 “beta-barrel” domains. The first beta barrel domain, which the researchers focused on, had an unusual, very hydrophobic loop that they suspected was involved in binding to cell membranes. Mechanobiologist Jie Yan then used magnetic tweezers to grab a gellin by one end and gently pull on the other end, revealing that the protein unfolded at forces consistent with what gellins would experience inside a bursting hypha.
“It’s an elegant set of experiments,” Stajich says.
Using CRISPR, the researchers eliminated the two gellin genes, gellin A and gellin B, from M. circinelloides. In the gellin mutant, an injury causes prolonged protoplasmic bleeding. GREG JEDD, TEMASEK LIFE SCIENCES LABORATORY, NATIONAL UNIVERSITY OF SINGAPORE
The process to stop fungal bleeding has some parallels to mammalian blood clotting. For example, the von Willebrand factor (VWF) that helps blood clot is also mechanically sensitive and responds to the force of flow by changing shape and binding with other VWF proteins.
All multicellular systems have evolved transport networks, which are required to overcome the limit of diffusion as an organism gets large, Jedd explains. “Each has found ways of dealing with injury, but there is some degree of convergence.”
Spider silk, too, is made of large proteins that are extruded through the spider’s silk glands, and the shear stress from that flow induces the formation of intermolecular bonds, thus producing a thread from liquid protein. Understanding the design principles for these mechanosensitive proteins could have all kinds of bioengineering applications, such as self-healing soft robots, the researchers suggest.
T.A. Nguyen et al., “Fungal wound healing through instantaneous protoplasmic gelation,” Current Biology, doi: 10.1016/j.cub.2020.10.016, 2020.


thescientist--2020-11-12--Abcam’s new premium-grade bioactive proteins, for drug discovery, development and manufacturin

Abcam’s new premium-grade bioactive proteins, for drug discovery, development and manufacturing

Sourcing high-quality proteins with batch-to-batch consistency can be a challenge , as endotoxin levels and/or non-native protein conformations and post-translational modifications can affect safety and performance .
Abcam ’ s new premium-grade proteins are manufactured in mammalian cell lines under animal-free conditions limiting endotoxins to ultra-low levels ( < 5 EU/mg ) .
The use of mammalian cell lines produces proteins with the desired conformation and correct post-translational modifications for optimal bioactivity .
They resemble the proteins ’ native state , helping to ensure a safe and consistent outcome during drug discovery , development and manufacturing .
Developed by a @ @ @ @ @ @ @ batch is validated using state-of-the-art technology to ensure consistent quality regarding integrity , purity , concentration , mono-dispersity and correct folding .
Abcam can also design , manufacture and validate proteins to custom needs and specifications .
As a supplier of proteins for a broad range of applications , Abcam provides a comprehensive range of bioactive and research-grade proteins that enable additional cost-effective quality solutions .
• None Batch-to-batch consistency monitored by sophisticated quality control , addressing identity , integrity , purity , concentration , folding and aggregation using methods such as LCMS @ @ @ @ @ @

Sourcing high-quality proteins with batch-to-batch consistency can be a challenge, as endotoxin levels and/or non-native protein conformations and post-translational modifications can affect safety and performance.
Abcam’s new premium-grade proteins are manufactured in mammalian cell lines under animal-free conditions limiting endotoxins to ultra-low levels (< 5 EU/mg). The use of mammalian cell lines produces proteins with the desired conformation and correct post-translational modifications for optimal bioactivity. They resemble the proteins’ native state, helping to ensure a safe and consistent outcome during drug discovery, development and manufacturing.
Developed by a team of industry-expert protein scientists, each batch is validated using state-of-the-art technology to ensure consistent quality regarding integrity, purity, concentration, mono-dispersity and correct folding.
Abcam can also design, manufacture and validate proteins to custom needs and specifications. As a supplier of proteins for a broad range of applications, Abcam provides a comprehensive range of bioactive and research-grade proteins that enable additional cost-effective quality solutions.
Abcam’s premium-grade bioactive proteins provide:


thescientist--2020-11-13--Voters in California Extend Life of Stem Cell Funding Agency

California ’ s Proposition 14 has passed with 51 percent of votes , authorizing the state to issue $ 5.5 billion in bonds to give the California Institute for Regenerative Medicine a second lease on life .
The result was called by the Associated Press yesterday ( November 12 ) .
The agency had last year it was running out of its first $ 3 billion round of funding , which was approved by voters through a previous ballot measure in 2004 .
With the new bonds , the agency will now continue to fill what supporters say is a @ @ @ @ @ @ @ , and fund ongoing clinical trials and training programs for early-career researchers .
Of the new funding , $ 1.5 billion is to go toward projects developing therapies for neurological diseases such as Alzheimer ’ s , Parkinson ’ s , and dementia .
“ We are thrilled ” about Proposition 14 ’ s approval , Jonathan Thomas , the chair of CIRM ’ s governing board , writes in an email statement .
“ We are proud of what we have achieved so far— the cures and therapies we helped develop , @ @ @ @ @ @ @ in additional investments , and the tens of thousands of jobs we created—and we look forward to continuing that work.
” Despite a $ 20 million-strong campaign in support of the measure and the absence of an organized opposition , Proposition 14 passed by just 325,000 votes out of more than 15 million cast , the slimmest margin of victory for any of California ’ s 12 ballot measures this year , and a far smaller margin than the 59 percent approval the 2004 ballot measure received .
The creator of both measures @ @ @ @ @ @ @ patient advocate Robert N. Klein II , attributes the close race to concerns over the cost of the measure amid a time of economic uncertainty , he tells the Associated Press .
See “ California Voters to Decide Future of Stem Cell Funding Agency ” Indeed , it will ultimately cost California taxpayers $ 260 million a year for three decades to pay off the bonds , according to by the nonpartisan federal Legislative Analyst ’ s Office .
That ’ s on top of the annual $ 327 million bill the state @ @ @ @ @ @ @ ballot measure .
In an interview with The Scientist in late October , the CIRM board member Jeff Sheehy criticized the measure for not including wording that would allow the state to recoup a meaningful return on its investment , and for what he said was its potential to worsen perceived conflicts of interest between the agency ’ s board members and the institutions it funds .
Proponents of the measure argued that it would California ’ s economy in the long term .
Proposition 14 ’ s wording encourages the state to @ @ @ @ @ @ @ the cost of stem cell treatments for patients .
“ We are already working on ways to repay [ the trust that voters have placed in us ] and bring stem cell and regenerative therapies to all the people of this great state , particularly for communities that have traditionally been overlooked or underserved , ” Thomas writes in his statement .

ABOVE: © ISTOCK.COM, ADRAGAN
California’s Proposition 14 has passed with 51 percent of votes, authorizing the state to issue $5.5 billion in bonds to give the California Institute for Regenerative Medicine a second lease on life. The result was called by the Associated Press yesterday (November 12).
The agency had announced last year it was running out of its first $3 billion round of funding, which was approved by voters through a previous ballot measure in 2004. With the new bonds, the agency will now continue to fill what supporters say is a critical funding gap in stem cell research, and fund ongoing clinical trials and training programs for early-career researchers. Of the new funding, $1.5 billion is to go toward projects developing therapies for neurological diseases such as Alzheimer’s, Parkinson’s, and dementia.
“We are thrilled” about Proposition 14’s approval, Jonathan Thomas, the chair of CIRM’s governing board, writes in an email statement. “We are proud of what we have achieved so far— the cures and therapies we helped develop, the billions we brought into the state in additional investments, and the tens of thousands of jobs we created—and we look forward to continuing that work.”
Despite a $20 million-strong campaign in support of the measure and the absence of an organized opposition, Proposition 14 passed by just 325,000 votes out of more than 15 million cast, the slimmest margin of victory for any of California’s 12 ballot measures this year, and a far smaller margin than the 59 percent approval the 2004 ballot measure received. The creator of both measures, San Francisco real estate investor and patient advocate Robert N. Klein II, attributes the close race to concerns over the cost of the measure amid a time of economic uncertainty, he tells the Associated Press.
See “California Voters to Decide Future of Stem Cell Funding Agency”
Indeed, it will ultimately cost California taxpayers $260 million a year for three decades to pay off the bonds, according to estimates by the nonpartisan federal Legislative Analyst’s Office. That’s on top of the annual $327 million bill the state is still paying off from the 2004 ballot measure. In an interview with The Scientist in late October, the CIRM board member Jeff Sheehy criticized the measure for not including wording that would allow the state to recoup a meaningful return on its investment, and for what he said was its potential to worsen perceived conflicts of interest between the agency’s board members and the institutions it funds.
Proponents of the measure argued that it would California’s economy in the long term. Proposition 14’s wording encourages the state to use revenue from CIRM-supported research to subsidize the cost of stem cell treatments for patients.
“We are already working on ways to repay [the trust that voters have placed in us] and bring stem cell and regenerative therapies to all the people of this great state, particularly for communities that have traditionally been overlooked or underserved,” Thomas writes in his statement.


thescientist--2020-11-13--SARS-CoV-2 Exits Cells Via Lysosomes

SARS-CoV-2 Exits Cells Via Lysosomes

ABOVE : A pseudocolored electron micrograph shows SARS-CoV-2 ( pink ) inside the lysosomes ( outlined in green ) of an African green monkey kidney cell .
CHRIS BLECK , NIH Scientists know a lot about how SARS-CoV-2 , the virus responsible for the COVID-19 pandemic , gets into cells .
Now , in a study published October 27 in Cell , researchers have determined that mouse hepatitis virus and SARS-CoV-2 , both of which are members of the β-coronavirus family , get out of cells via lysosomes , organelles responsible for taking out cellular garbage , rather than using @ @ @ @ @ @ @ other enveloped viruses .
“ There has n't been a whole lot of cell biology done on coronavirus assembly and release from cells , which is important .
.
.
because we have no antivirals that target these later steps of the virus infection cycle , ” says Carolyn Machamer , a cell biologist at the Johns Hopkins University School of Medicine who was not involved in the study .
This work not only points to new potential targets for inhibiting the later steps of infection , but also provides new understanding of @ @ @ @ @ @ @ she adds .
About five years ago , Teegan Dellibovi-Ragheb , then a postdoc in cell biologist Nihal Altan-Bonnet ’ s lab at the National Heart , Lung , and Blood Institute , started to investigate how β-coronaviruses—the family that includes the pathogens that cause MERS and SARS—get out of cells .
She found that the β-coronavirus mouse hepatitis virus ( MHV ) , which causes severe pneumonia in mice , doesn ’ t use the same secretory pathway that most viruses that have an outer lipid and protein envelope—including γ-coronaviruses , which @ @ @ @ @ @ @ to another job , though , Altan-Bonnet says that no one else in the lab wanted to take up the project to determine how these viruses were getting out of infected cells .
“ Not many people were interested in coronaviruses , ” she says .
“ And then the pandemic hit.
” Altan-Bonnet and Sourish Ghosh , a current postdoc in the lab , got to work , infecting different cell types with either MHV or SARS-CoV-2 .
The researchers found large numbers of the coronaviruses in lysosomes around the time in @ @ @ @ @ @ @ expected to be getting out of the cells to infect new ones .
Furthermore , when the researchers blocked the movement of lysosomes to the plasma membrane in the MHV-infected cells , a key step in lysosome-based exocytosis , they detected much less viral RNA outside the cells .
These findings indicate that β-coronaviruses use lysosomes to exit cells .
Lysosomes are typically quite acidic , with a pH around 4.5 to 5.0 .
This allows specialized enzymes to degrade waste within the lysosome , but not if something went wrong and these @ @ @ @ @ @ @ The research team determined that the lysosomes were somehow being deacidified during MHV infection .
Lysosomes in cells infected with MHV had an average pH of 5.7 , while the uninfected control cells ’ lysosomes had an average pH of 4.7 , leading to reduced lysosomal enzyme activity in infected cells , which would allow the viruses to exit the cells intact .
Based on previous work from other groups and the researchers ’ new evidence that the SARS-CoV-2 protein ORF3a is localized to the lysosomes of infected cells , they hypothesize that @ @ @ @ @ @ @ pH ; they are planning future studies to further investigate the protein ’ s role .
“ We were certainly surprised that it ’ s using this pathway to get out , ” says Altan-Bonnet .
While some viruses without a lipid and protein envelope—such as reovirus , which causes mild gastrointestinal upset in people—that do exit cells via lysosomal trafficking , she adds , “ it ’ s not the typical pathway for enveloped viruses.
” Abnormal immune responses—such as the cytokine storm—can be a feature of COVID-19 , and the process @ @ @ @ @ @ @ to be critical for antigen presentation , which allows immune sentinels to identify and kill infected cells .
Altan-Bonnet shared the findings with her husband , Grégoire Altan-Bonnet , an immunologist at the National Cancer Institute , and they and their colleagues hypothesized that “ maybe the fact that the virus escapes from a different pathway completely messes up the normal immune response , ” he says .
Indeed , the research team found alterations in both antigen presentation and subsequent immune activation in MHV-infected cells , indicating that antigen presentation had been @ @ @ @ @ @ @ is using a different pathway than what we thought previously , ” says Billy Tsai , a cell biologist at the University of Michigan who did not participate in the study .
Some of the open questions center on how the virus deacidifies the lysosomes and then uses the lysosome trafficking pathway , he adds , and it will also be important to see confirmation of these results from other groups .
“ There ’ s going to be a lot of excitement in trying to unravel how these viruses , especially SARS-CoV-2 @ @ @ @ @ @ @ then get out.
” In addition to the basic science questions , the research team is focusing on how their findings relate to COVID-19 ’ s pathology .
“ We follow the literature from the clinical side , and people are making tons of measurements about the cytokine storm and coagulation issues , and there is really not a mechanism which explains how all these things fit together , ” says Grégoire Altan-Bonnet .
He and his colleagues are now exploring whether or not viral egress via lysosomes can explain the immune imbalances @ @ @ @ @ @ @ with MHV , in part because it ’ s safe to work with under biosafety level two conditions , unlike SARS-CoV-2 , which requires biosafety level three .
“ Of course , the mouse strain is not the human strain , ” says Nihal Altan-Bonnet .
“ Since we saw the acidification in the CoV-2 infected cells , we ’ re hopeful that we ’ re going to see the same effects across different coronaviruses.
” S. Ghosh et al.
, β-coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway , @ @ @ @ @ @

ABOVE: A pseudocolored electron micrograph shows SARS-CoV-2 (pink) inside the lysosomes (outlined in green) of an African green monkey kidney cell.
CHRIS BLECK, NIH
Scientists know a lot about how SARS-CoV-2, the virus responsible for the COVID-19 pandemic, gets into cells. Now, in a study published October 27 in Cell, researchers have determined that mouse hepatitis virus and SARS-CoV-2, both of which are members of the β-coronavirus family, get out of cells via lysosomes, organelles responsible for taking out cellular garbage, rather than using the biosynthetic secretory pathway used by most other enveloped viruses.
“There hasn't been a whole lot of cell biology done on coronavirus assembly and release from cells, which is important . . . because we have no antivirals that target these later steps of the virus infection cycle,” says Carolyn Machamer, a cell biologist at the Johns Hopkins University School of Medicine who was not involved in the study. This work not only points to new potential targets for inhibiting the later steps of infection, but also provides new understanding of the basic biology of these viruses, she adds.
About five years ago, Teegan Dellibovi-Ragheb, then a postdoc in cell biologist Nihal Altan-Bonnet’s lab at the National Heart, Lung, and Blood Institute, started to investigate how β-coronaviruses—the family that includes the pathogens that cause MERS and SARS—get out of cells. She found that the β-coronavirus mouse hepatitis virus (MHV), which causes severe pneumonia in mice, doesn’t use the same secretory pathway that most viruses that have an outer lipid and protein envelope—including γ-coronaviruses, which infect birds—use. After Dellibovi-Ragheb moved on to another job, though, Altan-Bonnet says that no one else in the lab wanted to take up the project to determine how these viruses were getting out of infected cells.
“Not many people were interested in coronaviruses,” she says. “And then the pandemic hit.”
Altan-Bonnet and Sourish Ghosh, a current postdoc in the lab, got to work, infecting different cell types with either MHV or SARS-CoV-2. The researchers found large numbers of the coronaviruses in lysosomes around the time in their lifecycles that they’d be expected to be getting out of the cells to infect new ones. Furthermore, when the researchers blocked the movement of lysosomes to the plasma membrane in the MHV-infected cells, a key step in lysosome-based exocytosis, they detected much less viral RNA outside the cells. These findings indicate that β-coronaviruses use lysosomes to exit cells.
Lysosomes are typically quite acidic, with a pH around 4.5 to 5.0. This allows specialized enzymes to degrade waste within the lysosome, but not if something went wrong and these enzymes made it into the cytoplasm. The research team determined that the lysosomes were somehow being deacidified during MHV infection. Lysosomes in cells infected with MHV had an average pH of 5.7, while the uninfected control cells’ lysosomes had an average pH of 4.7, leading to reduced lysosomal enzyme activity in infected cells, which would allow the viruses to exit the cells intact.
A pseudocolored electron micrograph shows SARS-CoV-2 (yellow) inside the lysosomes (purple) of an African green monkey kidney cell. Alan Hoofring, NIH
Based on previous work from other groups and the researchers’ new evidence that the SARS-CoV-2 protein ORF3a is localized to the lysosomes of infected cells, they hypothesize that it may be involved in neutralizing the pH; they are planning future studies to further investigate the protein’s role.
“We were certainly surprised that it’s using this pathway to get out,” says Altan-Bonnet. While some viruses without a lipid and protein envelope—such as reovirus, which causes mild gastrointestinal upset in people—do exit cells via lysosomal trafficking, she adds, “it’s not the typical pathway for enveloped viruses.”
Abnormal immune responses—such as the cytokine storm—can be a feature of COVID-19, and the process of lysosomal degradation had been previously shown to be critical for antigen presentation, which allows immune sentinels to identify and kill infected cells. Altan-Bonnet shared the findings with her husband, Grégoire Altan-Bonnet, an immunologist at the National Cancer Institute, and they and their colleagues hypothesized that “maybe the fact that the virus escapes from a different pathway completely messes up the normal immune response,” he says. Indeed, the research team found alterations in both antigen presentation and subsequent immune activation in MHV-infected cells, indicating that antigen presentation had been disrupted.
“Clearly, this virus is using a different pathway than what we thought previously,” says Billy Tsai, a cell biologist at the University of Michigan who did not participate in the study. Some of the open questions center on how the virus deacidifies the lysosomes and then uses the lysosome trafficking pathway, he adds, and it will also be important to see confirmation of these results from other groups. “There’s going to be a lot of excitement in trying to unravel how these viruses, especially SARS-CoV-2, go through the host cells and then get out.”
In addition to the basic science questions, the research team is focusing on how their findings relate to COVID-19’s pathology. “We follow the literature from the clinical side, and people are making tons of measurements about the cytokine storm and coagulation issues, and there is really not a mechanism which explains how all these things fit together,” says Grégoire Altan-Bonnet. He and his colleagues are now exploring whether or not viral egress via lysosomes can explain the immune imbalances in patients.
The researchers are starting with MHV, in part because it’s safe to work with under biosafety level two conditions, unlike SARS-CoV-2, which requires biosafety level three. “Of course, the mouse strain is not the human strain,” says Nihal Altan-Bonnet. “Since we saw the acidification in the CoV-2 infected cells, we’re hopeful that we’re going to see the same effects across different coronaviruses.”
S. Ghosh et al., β-coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway, Cell, doi:10.1016/j.cell.2020.10.039, 2020.


thescientist--2020-11-13--Q&A: Minks Can Transmit SARS-CoV-2 to Humans, Study Shows

Millions of minks globally have contracted COVID-19 , leading government officials to call for the animals ’ culling to prevent the virus from mutating , potentially reinfecting humans , or thwarting vaccination efforts .
Last week the World Health Organization launched an investigation into virus transmission on mink farms because researchers had identified one mutation labeled D614G in the spike protein of the virus that may increase transmission .
There is no evidence so far that the mutation would increase virulence .
It also “ does not appear , at this point , that that mutation that ’ s been @ @ @ @ @ @ @ have an impact on vaccines and affect a vaccine-induced response , ” noted Anthony Fauci , the director of the US National Institute of Allergy and Infectious Diseases , during a November 12 webinar hosted by the think tank Chatham House .
Humans did initially inadvertently introduce the virus into mink farms , according to an analysis of the viral genomes of minks and of workers from 16 farms in the Netherlands .
There was evidence of human-mink transmission and also transmission from the mink back to humans , researchers reported November 10 @ @ @ @ @ @ @ study coauthor Wim van der Poel , a zoonotic virology expert at Wageningen University in the Netherlands , about his findings .
The Scientist : Tell me the story of how you first started this research project .
Wim van der Poel : The actual research in the mink was started right after we detected the virus in the mink farm .
What happened is that there was a respiratory disease seen in the mink farm .
These farms in the Netherlands have regular visits of specialized veterinarians , and one of these @ @ @ @ @ @ @ normal ” respiratory disease pathogens .
But that was all negative , and then he remembered that the susceptibility of minks was discussed with the [ Dutch ] Ministry [ of Health , Welfare and Sport ] by specialists , [ including van der Poel ] .
We already feared that minks could be susceptible because we knew from animal experiments that ferrets were susceptible , and ferrets and minks are very much related .
See “ Ferret Study Reinforces Role of Aerosols in SARS-CoV-2 Spread ” So then we tested the animals @ @ @ @ @ @ @ appeared to be positive .
We had a meeting with experts in the field right away , and we set up this consortium for further research in the first outbreak farms .
That ’ s the basis of the paper that we published in Science .
TS : In your paper , you use whole genome sequencing to better understand where this virus came from , and how it ’ s being transmitted .
What did this experimental tool tell you about what ’ s going on in these minks ?
WVDP : @ @ @ @ @ @ @ all the mink [ samples ] , but also we tried to obtain samples from all the people working in the farms because it was clear that these people were exposed and also we wanted to know where and how the virus was introduced .
It was traced back to people being infected with SARS-CoV-2 , and these people are likely those who have introduced the virus [ to the farms ] .
We could see that the sequences of the mink and the people working in the farms are related .
From @ @ @ @ @ @ @ see five different clusters of sequences , and the clusters of sequences are single introductions by people .
[ In other words , each farm has a unique subcluster of the virus . ]
We also compared [ the mink farm viral sequences ] to the sequences of the [ virus in the ] general population in the Netherlands , and what we saw is that in the general populations , the sequences are not [ closely related to ] the mink sequences—only the people working in the farms [ carried related viruses @ @ @ @ @ @ @ seems that there is very little transmission from the farms to the general population apart from the people working in farms .
There are sequences that occur far more often in mink than in humans , but in the data that we have , it seems that all the single mutations that we see in minks have also occurred in people .
But we don ’ t see any mutations that we think are really mink-specific and more dangerous to people .
Of course , that needs further investigation because in Denmark , @ @ @ @ @ @ @ of mutations in the spike protein , and that change will result in a change in that protein that they feared would make the virus more aggressive .
TS : The past few months , scientists have been debating whether animals can transmit the virus back to humans .
How does your report add evidence to this debate ?
WVDP : The pandemic is really driven by the circulation in people , but the paper also clearly proves that there is transmission back from the minks to the people .
That ’ s @ @ @ @ @ @ @ the fact that by spreading the virus .
.
.
to other species , you may have a wider epidemic and you may also have the risk of a continuing reservoir in animals .
If we eradicate the virus from people , by controlling it or by vaccination , you would have the risk that you have a remaining reservoir in the mink , for example .
That was also the reason for the Dutch authorities to start culling the mink , because at that time , in June and July , the @ @ @ @ @ @ @ decreasing .
TS : There have been COVID-19 outbreaks in minks all over the world—in Denmark , the Netherlands , and the US .
How should the results of your study inform how public health officials handle SARS-CoV-2 in animals in which the virus can easily spread ?
WVDP : I think that ’ s very important that researchers and public health authorities realize that spreading the virus to other populations of animals may have the risk that you create reservoirs .
Also , from the mink , the virus may spread to @ @ @ @ @ @ @ Mustelidae species like badgers , martens , and otters—these are related to mink , so if the virus spreads from mink to that kind of wildlife , you may end up with a reservoir in these species .
That ’ s something we ’ re also looking into now .
We are testing that kind of wildlife to see whether this occurs .
It ’ s important to look into because if there is a maintained reservoir in wildlife , you may always have the risk of the virus coming back to people @ @ @ @ @ @ @ , “ Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans , ” Science , doi:10.1126/science.abe5901 , 2020 .
Editor ’ s note : The interview was edited for brevity .

ABOVE: © ISTOCK.COM,
GEORGY_GOLOVIN
Millions of minks globally have contracted COVID-19, leading government officials to call for the animals’ culling to prevent the virus from mutating, potentially reinfecting humans, or thwarting vaccination efforts.
Last week the World Health Organization launched an investigation into virus transmission on mink farms because researchers had identified one mutation labeled D614G in the spike protein of the virus that may increase transmission. There is no evidence so far that the mutation would increase virulence. It also “does not appear, at this point, that that mutation that’s been identified in the minks is going to have an impact on vaccines and affect a vaccine-induced response,” noted Anthony Fauci, the director of the US National Institute of Allergy and Infectious Diseases, during a November 12 webinar hosted by the think tank Chatham House.
Humans did initially inadvertently introduce the virus into mink farms, according to an analysis of the viral genomes of minks and of workers from 16 farms in the Netherlands. There was evidence of human-mink transmission and also transmission from the mink back to humans, researchers reported November 10 in Science.
The Scientist spoke with study coauthor Wim van der Poel, a zoonotic virology expert at Wageningen University in the Netherlands, about his findings.
The Scientist: Tell me the story of how you first started this research project.
Wim van der Poel MIRIAN HENDRIKS
Wim van der Poel: The actual research in the mink was started right after we detected the virus in the mink farm. What happened is that there was a respiratory disease seen in the mink farm. These farms in the Netherlands have regular visits of specialized veterinarians, and one of these veterinarians tested the animals for the “normal” respiratory disease pathogens.
But that was all negative, and then he remembered that the susceptibility of minks was discussed with the [Dutch] Ministry [of Health, Welfare and Sport] by specialists, [including van der Poel]. We already feared that minks could be susceptible because we knew from animal experiments that ferrets were susceptible, and ferrets and minks are very much related.
So then we tested the animals by PCR for SARS-CoV-2, and they appeared to be positive. We had a meeting with experts in the field right away, and we set up this consortium for further research in the first outbreak farms. That’s the basis of the paper that we published in Science.
TS: In your paper, you use whole genome sequencing to better understand where this virus came from, and how it’s being transmitted. What did this experimental tool tell you about what’s going on in these minks?
WVDP: What we did is we started sequencing all the mink [samples], but also we tried to obtain samples from all the people working in the farms because it was clear that these people were exposed and also we wanted to know where and how the virus was introduced. It was traced back to people being infected with SARS-CoV-2, and these people are likely those who have introduced the virus [to the farms].
We could see that the sequences of the mink and the people working in the farms are related. From the first 16 farms, you can see five different clusters of sequences, and the clusters of sequences are single introductions by people. [In other words, each farm has a unique subcluster of the virus.]
We also compared [the mink farm viral sequences] to the sequences of the [virus in the] general population in the Netherlands, and what we saw is that in the general populations, the sequences are not [closely related to] the mink sequences—only the people working in the farms [carried related viruses]. In the Netherlands, it seems that there is very little transmission from the farms to the general population apart from the people working in farms.
There are sequences that occur far more often in mink than in humans, but in the data that we have, it seems that all the single mutations that we see in minks have also occurred in people. But we don’t see any mutations that we think are really mink-specific and more dangerous to people. Of course, that needs further investigation because in Denmark, for example, they found a combination of mutations in the spike protein, and that change will result in a change in that protein that they feared would make the virus more aggressive.
TS: The past few months, scientists have been debating whether animals can transmit the virus back to humans. How does your report add evidence to this debate?
WVDP: The pandemic is really driven by the circulation in people, but the paper also clearly proves that there is transmission back from the minks to the people. That’s important here—we have to be aware of the fact that by spreading the virus . . . to other species, you may have a wider epidemic and you may also have the risk of a continuing reservoir in animals. If we eradicate the virus from people, by controlling it or by vaccination, you would have the risk that you have a remaining reservoir in the mink, for example. That was also the reason for the Dutch authorities to start culling the mink, because at that time, in June and July, the number of infections in the Netherlands was decreasing.
TS: There have been COVID-19 outbreaks in minks all over the world—in Denmark, the Netherlands, and the US. How should the results of your study inform how public health officials handle SARS-CoV-2 in animals in which the virus can easily spread?
WVDP: I think that’s very important that researchers and public health authorities realize that spreading the virus to other populations of animals may have the risk that you create reservoirs. Also, from the mink, the virus may spread to other susceptible wildlife. There are other Mustelidae species like badgers, martens, and otters—these are related to mink, so if the virus spreads from mink to that kind of wildlife, you may end up with a reservoir in these species.
That’s something we’re also looking into now. We are testing that kind of wildlife to see whether this occurs. It’s important to look into because if there is a maintained reservoir in wildlife, you may always have the risk of the virus coming back to people.
B.B. Oude Munnink et al., “Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans,” Science, doi:10.1126/science.abe5901, 2020.
Editor’s note: The interview was edited for brevity.


thescientist--2020-11-13--Zebra Finches Recognize the Calls of Over 40 Fellow Finches

ABOVE : Zebra finches are highly social , living in colonies of more than 100 birds .
Their gregariousness may be one reason they are able to recognize dozens of individuals .
© ISTOCK.COM , SAGARMANIS Fast mapping , the ability to rapidly learn an association between two things after very little exposure , is a key tool responsible for the vast repertoire of human language .
It ’ s the reason we can recognize voices from another room and why newborns prefer to listen to their mothers read them The Cat in the Hat over other women .
While @ @ @ @ @ @ @ a human ability , zebra finches can also distinguish dozens of other finches ’ vocalizations , doing so with very little exposure and retaining those memories for at least a month , researchers report today ( November 13 ) in Science Advances .
“ The reason that this study is groundbreaking is because zebra finches are the very first vocally learning species aside from ourselves with any evidence that fast mapping takes place , ” Samantha Carouso-Peck , a behavioral neuroscientist at Cornell University who researches social influences on vocal learning in zebra @ @ @ @ @ @ @ current work , tells The Scientist .
See `` Image of the Day : Watch and Learn '' Prior to the new study , evidence of fast mapping in nonhumans had been suggested for only a single nonhuman animal : Rico the border collie .
Rico is capable of distinguishing the name of more than 200 individual objects and can infer the meaning of a new word after hearing it only once .
Whether this is truly evidence of fast mapping , however , has been debated among scientists who believe that only @ @ @ @ @ @ @ , can be said to truly fast map .
To test zebra finches ’ ability to recognize individual calls , and the number of exposures they need to do so , researchers at the University of California , Berkeley , designed a five-day “ learning ladder.
” Birds were initially trained on a small set of call data that became increasingly larger over several days .
On the first day of testing , each bird in the training arena went through a series of trials during which they heard brief clips of either @ @ @ @ @ @ @ by birds to locate their group when it is out of sight .
These two distinct vocalizations were chosen because they are thought to have the most variation between individuals .
Nineteen birds were trained on songs , while another 19 heard only distance calls .
The vocalizations were parsed , so that one set led to a food reward , while the other set didn ’ t .
In that first test , each finch only needed to discriminate between two individuals ’ vocalizations , one that provided a food reward and @ @ @ @ @ @ @ a key , the finch would start the sound file .
It could then either peck the key again to start a new trial or listen to the entire clip to receive a food reward .
Repeating this process over several trials , the finches learned to associate vocalizations that led to a reward from the ones that did not and to quickly skip vocalizations that weren ’ t rewarding .
Each successive day , sounds from additional individuals were added to the training set .
While finches were originally trained to discriminate @ @ @ @ @ @ @ day two it jumped to four pairs , and then eight on days three through five , for a total of 16 different individuals ’ vocalizations .
The researchers began evaluating the finches ’ performance only on days four and five , when each bird had been exposed at least once to each possible vocalizer .
To be certain the finches weren ’ t simply memorizing the sound files , each bird providing the vocalization was recorded 10 different times .
“ We were pulling randomly from a large library of calls that @ @ @ @ @ @ @ all different every time , ” Frederic Theunissen , an auditory scientist at the University of California , Berkeley , tells The Scientist .
“ Not only are they different vocalizers , but they heard different renditions from the same vocalizer.
” Even with this added layer of complexity , Theunissen says he was “ really impressed ” by how well the finches performed .
All 19 finches trained using song files were able to discriminate between the vocalizers they recognized based on their individual signatures and those they did not , and @ @ @ @ @ @ @ the finches trained using distance calls .
Beyond their ability to recognize other birds based on their calls , the team was also struck by how quickly the finches were able make the distinctions .
The majority of birds could hear as few as 10 exposures to an individual and memorize their signature , while some required fewer than five , strong evidence for fast mapping in the species .
“ We didn ’ t expect the birds to be so good at this , ” Theunissen tells The Scientist .
“ We @ @ @ @ @ @ @ , but the fact that the number of vocalizers is so high , and that they can do this so quickly , was really quite astonishing.
” In the study , the team decided to create what Theunissen calls “ an impossible task ” to test the limits of the finches ’ performance .
He created a new set of stimuli that included both songs and distance calls from 56 distinct vocalizers .
Only four randomly-selected birds went through this mega-task , but on average , they were able to remember 42 individual @ @ @ @ @ @ @ tested the recall of two of the four birds by running them through the same , 56-vocalizer test and found that they were still able to distinguish between individuals—they had retained their memories of the individual calls .
Both the speed of their learning and their retention over time , Theunissen says , “ point towards these really remarkable abilities that these birds have in terms of making auditory memories.
” Because these birds are so social , living gregariously in colonies of more than 100 individuals , it isn ’ t unexpected @ @ @ @ @ @ @ of individuals , says Sarah Woolley , a neuroethologist at McGill University who studies the neurological basis for social behavior in songbirds .
“ We ’ ve always been pretty sure that birds could do that , zebra finches included , ” Woolley says .
“ But no one had really demonstrated it , certainly not to the degree that they do in this paper.
” While the results are “ very interesting evidence ” that zebra finches are capable of fast mapping , Carouso-Peck says that more work on a larger sampling @ @ @ @ @ @ @ can make weightier claims about what they ’ re capable of.
” For example , one of the criticisms of the previous work with Rico the border collie is that fast mapping is a tool for learning language , and therefore only species capable of language truly do it .
While Carouso-Peck says she doesn ’ t necessarily agree , as a next step she would like to see if finches are also capable of fast mapping verbally .
For example , how many times must young zebra finches hear their father ’ @ @ @ @ @ @ @ for themselves ?
See “ Birds Possess an Innate Vocal Signature Based on Silent Gaps ” “ Language is something which we humans tend to hold up as our great crowning achievement , something that we are capable of which absolutely no [ other ] species is capable of , ” Caruso-Peck tells The Scientist .
“ Over the last few decades , we ’ ve had to change the goalposts for what is language in order to maintain that high and mighty position.
” K. Yu et al.
, “ High-capacity auditory @ @ @ @ @ @ @ songbird , ” Science Advances , doi:10.1126/sciadv.abe0440 , 2020 .

ABOVE: Zebra finches are highly social, living in colonies of more than 100 birds. Their gregariousness may be one reason they are able to recognize dozens of individuals.
© ISTOCK.COM, SAGARMANIS
Fast mapping, the ability to rapidly learn an association between two things after very little exposure, is a key tool responsible for the vast repertoire of human language. It’s the reason we can recognize voices from another room and why newborns prefer to listen to their mothers read them The Cat in the Hat over other women. While fast mapping is generally thought of as a human ability, zebra finches can also distinguish dozens of other finches’ vocalizations, doing so with very little exposure and retaining those memories for at least a month, researchers report today (November 13) in Science Advances.
“The reason that this study is groundbreaking is because zebra finches are the very first vocally learning species aside from ourselves with any evidence that fast mapping takes place,” Samantha Carouso-Peck, a behavioral neuroscientist at Cornell University who researches social influences on vocal learning in zebra finches but was not involved in the current work, tells The Scientist.
Prior to the new study, evidence of fast mapping in nonhumans had been suggested for only a single nonhuman species: dogs. Rico the border collie is capable of distinguishing the name of more than 200 individual objects, while another dog named Chaser could discriminate between more than 1,000. Whether this is truly evidence of fast mapping, however, has been debated among scientists who believe that only species capable of language, specifically humans, can be said to truly fast map.
To test zebra finches’ ability to recognize individual calls, and the number of exposures they need to do so, researchers at the University of California, Berkeley, designed a five-day “learning ladder.” Birds were initially trained on a small set of call data that became increasingly larger over several days.
On the first day of testing, each bird in the training arena went through a series of trials during which they heard brief clips of either a song or a distance call used by birds to locate their group when it is out of sight. These two distinct vocalizations were chosen because they are thought to have the most variation between individuals. Nineteen birds were trained on songs, while another 19 heard only distance calls. The vocalizations were parsed, so that one set led to a food reward, while the other set didn’t.
In the team’s experiments, birds in an arena learned to distinguish between vocalizations that came with a reward (Re Vocalizer) and those that did not (NoRe Vocalizer). When birds recognized a call they had heard before, they allowed the clip to play fully, which activated a food reward. When they did not, they hit a button to skip ahead to the next trial. In this way, scientists were able to show that individual finches can remember an average of 42 other vocalizers. Julie Elie, UC Berkeley
In that first test, each finch only needed to discriminate between two individuals’ vocalizations, one that provided a food reward and one that did not. By pecking a key, the finch would start the sound file. It could then either peck the key again to start a new trial or listen to the entire clip to receive a food reward. Repeating this process over several trials, the finches learned to associate vocalizations that led to a reward from the ones that did not and to quickly skip vocalizations that weren’t rewarding.
Each successive day, sounds from additional individuals were added to the training set. While finches were originally trained to discriminate between one pair of individuals, by day two it jumped to four pairs, and then eight on days three through five, for a total of 16 different individuals’ vocalizations. The researchers began evaluating the finches’ performance only on days four and five, when each bird had been exposed at least once to each possible vocalizer.
To be certain the finches weren’t simply memorizing the sound files, each bird providing the vocalization was recorded 10 different times. “We were pulling randomly from a large library of calls that we have, so the calls were all different every time,” Frederic Theunissen, an auditory scientist at the University of California, Berkeley, tells The Scientist. “Not only are they different vocalizers, but they heard different renditions from the same vocalizer.”
Even with this added layer of complexity, Theunissen says he was “really impressed” by how well the finches performed. All 19 finches trained using song files were able to discriminate between the vocalizers they recognized based on their individual signatures and those they did not, and the same was true for 18 of the finches trained using distance calls.
See "Researchers Implant Memories in Zebra Finch Brains"
Beyond their ability to recognize other birds based on their calls, the team was also struck by how quickly the finches were able make the distinctions. The majority of birds could hear as few as 10 exposures to an individual and memorize their signature, while some required fewer than five, strong evidence for fast mapping in the species.
“We didn’t expect the birds to be so good at this,” Theunissen tells The Scientist. “We thought this was going to be interesting, but the fact that the number of vocalizers is so high, and that they can do this so quickly, was really quite astonishing.”
In the study, the team decided to create what Theunissen calls “an impossible task” to test the limits of the finches’ performance. He created a new set of stimuli that included both songs and distance calls from 56 distinct vocalizers. Only four randomly-selected birds went through this mega-task, but on average, they were able to remember 42 individual vocalizers.
A month later, he tested the recall of two of the four birds by running them through the same, 56-vocalizer test and found that they were still able to distinguish between individuals—they had retained their memories of the individual calls. Both the speed of their learning and their retention over time, Theunissen says, “point towards these really remarkable abilities that these birds have in terms of making auditory memories.”
Because these birds are so social, living gregariously in colonies of more than 100 individuals, it isn’t unexpected that zebra finches could discriminate between dozens of individuals, says Sarah Woolley, a neuroethologist at McGill University who studies the neurological basis for social behavior in songbirds. “We’ve always been pretty sure that birds could do that, zebra finches included,” Woolley says. “But no one had really demonstrated it, certainly not to the degree that they do in this paper.”
While the results are “very interesting evidence” that zebra finches are capable of fast mapping, Carouso-Peck says that more work on a larger sampling of birds is needed “before we can make weightier claims about what they’re capable of.” For example, one of the criticisms of the previous work with Rico the border collie is that fast mapping is a tool for learning language, and therefore only species capable of language truly do it. While Carouso-Peck says she doesn’t necessarily agree, as a next step she would like to see if finches are also capable of fast mapping verbally. For example, how many times must young zebra finches hear their father’s song before they can reproduce it for themselves?
See “Birds Possess an Innate Vocal Signature Based on Silent Gaps”
“Language is something which we humans tend to hold up as our great crowning achievement, something that we are capable of which absolutely no [other] species is capable of,” Caruso-Peck tells The Scientist. “Over the last few decades, we’ve had to change the goalposts for what is language in order to maintain that high and mighty position.”
K. Yu et al., “High-capacity auditory memory for vocal communication in a social songbird,” Science Advances, doi:10.1126/sciadv.abe0440, 2020.
Correction (November 17): An earlier version of this story stated that fast mapping had only been shown in one dog, but two border collies have been studied by researchers. The Scientist regrets this error.


thescientist--2020-11-13--The Scientist's LabTalk - Episode 3

The Scientist's LabTalk - Episode 3

Information you provide will be held in confidence and will be shared with the sponsoring vendor ( s ) of @ @ @ @ @ use the information to contact you about your account and to let you know about @ @ @ @ @ you may opt-out at any time .
This allows The Scientist to keep these podcasts @ @ @ @ @ readers .

Information you provide will be held in confidence and will be shared with the sponsoring vendor(s) of this podcast. We may use the information to contact you about your account and to let you know about related programs and products; you may opt-out at any time. This allows The Scientist to keep these podcasts free of charge for our readers.


California ’ s Proposition 14 has passed with 51 percent of votes , authorizing the state to issue $ 5.5 billion in bonds to give the California Institute for Regenerative Medicine a second lease on life .
The result was called by the Associated Press yesterday ( November 12 ) .
The agency had announced last year it was running out of its first $ 3 billion round of funding , which was approved by voters through a previous ballot measure in 2004 .
With the new bonds , the agency will now continue to fill what supporters say is @ @ @ @ @ @ @ research , and fund ongoing clinical trials and training programs for early-career researchers .
Of the new funding , $ 1.5 billion is to go toward projects developing therapies for neurological diseases such as Alzheimer ’ s , Parkinson ’ s , and dementia .
“ We are thrilled ” about Proposition 14 ’ s approval , Jonathan Thomas , the chair of CIRM ’ s governing board , writes in an email statement .
“ We are proud of what we have achieved so far— the cures and therapies we helped develop @ @ @ @ @ @ @ state in additional investments , and the tens of thousands of jobs we created—and we look forward to continuing that work.
” Despite a $ 20 million-strong campaign in support of the measure and the absence of an organized opposition , Proposition 14 passed by just 325,000 votes out of more than 15 million cast , the slimmest margin of victory for any of California ’ s 12 ballot measures this year , and a far smaller margin than the 59 percent approval the 2004 ballot measure received .
The creator of both @ @ @ @ @ @ @ and patient advocate Robert N. Klein II , attributes the close race to concerns over the cost of the measure amid a time of economic uncertainty , he tells the Associated Press .
See “ California Voters to Decide Future of Stem Cell Funding Agency ” Indeed , it will ultimately cost California taxpayers $ 260 million a year for three decades to pay off the bonds , according to estimates by the nonpartisan federal Legislative Analyst ’ s Office .
That ’ s on top of the annual $ 327 million bill @ @ @ @ @ @ @ the 2004 ballot measure .
In an interview with The Scientist in late October , the CIRM board member Jeff Sheehy criticized the measure for not including wording that would allow the state to recoup a meaningful return on its investment , and for what he said was its potential to worsen perceived conflicts of interest between the agency ’ s board members and the institutions it funds .
Proponents of the measure argued that it would California ’ s economy in the long term .
Proposition 14 ’ s wording encourages the @ @ @ @ @ @ @ to subsidize the cost of stem cell treatments for patients .
“ We are already working on ways to repay [ the trust that voters have placed in us ] and bring stem cell and regenerative therapies to all the people of this great state , particularly for communities that have traditionally been overlooked or underserved , ” Thomas writes in his statement .


thescientist--2020-11-16--Moderna's COVID-19 Vaccine 94 Percent Effective: Initial Data

Moderna announced that its COVID-19 vaccine candidate is 94.5 percent effective at protecting people from infection , compared to patients who received a placebo saline shot , according to an interim analysis of 95 participants the company released today ( November 16 ) .
“ These are obviously very exciting results , ” Anthony Fauci , the director of the US National Institute of Allergy and Infectious Diseases , tells CNN , noting he expects the first vaccinations to begin toward the end of December .
“ It ’ s just as good as it gets—94.5 percent is truly outstanding.
@ @ @ @ @ @ @ vaccine on par with competitor Pfizer ’ s COVID-19 vaccine , which was found to be 90 percent effective in a preliminary analysis of 94 participants .
Both companies are expected to seek authorization for emergency use in the US and from other global regulators in the coming weeks .
The European Medicines Agency announced today that it has begun a rolling review of the Moderna vaccine .
Both vaccines use mRNA to instruct cells to create many copies of a piece of the SARS-CoV-2 virus , causing the immune system to be @ @ @ @ @ @ @ try to invade .
Although there are other vaccines in development that use this technology , none have been approved yet .
As mRNA can quickly degrade in warm temperatures , vaccine storage presents a serious practical challenge .
While Pfizer ’ s vaccine must be stored at –70 °C , Moderna ’ s can be kept at –20 °C .
Moderna also said today that once thawed , its vaccine can remain stable for a month at 2–8 °C .
“ The fact that two different vaccines made by two different companies @ @ @ @ @ @ @ in a new messenger RNA concept , both worked so effectively confirms the concept once and for all that this is a viable strategy not only for COVID but for future infectious disease threats , ” Barry Bloom , a public health professor at Harvard University , tells The New York Times .
It ’ s unclear exactly how many of the trial ’ s 30,000 participants , who received either two doses of the vaccine or the placebo , have contracted COVID-19 .
The interim analysis includes 95 confirmed cases—five who had @ @ @ @ @ @ @ received the placebo .
Among this group , 11—all in the placebo group—developed severe COVID-19 .
“ It was one of the greatest moments in my life and my career .
It is absolutely amazing to be able to develop this vaccine and see the ability to prevent symptomatic disease with such high efficacy , ” Tal Zaks , Moderna ’ s chief medical officer , tells CNN .
Like the Pfizer vaccine , Moderna ’ s reportedly does not have any serious side effects , but a small percentage of participants in @ @ @ @ @ @ @ aches and headaches .
Pfizer did not release data on the vaccine ’ s performance to prevent severe cases .
Both companies announced their findings in press releases , not in peer-reviewed journals , and neither company provided the detailed data that would allow outside experts to evaluate their claims .
As the studies continue , the figures on the vaccines ’ efficacy may change .
It is also unclear how long the immunity provided by the vaccines will last .
Moderna and Pfizer conducted their trials using slightly different protocols .
While @ @ @ @ @ @ @ display at least two symptoms of disease in addition to a positive test for the virus to be officially counted as a COVID-19 case , the Pfizer trial required only one symptom and a positive test .
Pfizer started counting cases seven days after the second injection of the vaccine , and Moderna waited 14 days .
Moderna has a $ 1.525 billion contract to provide the US with 100 million doses of the vaccine through Operation Warp Speed , with an option to purchase an additional 400 million doses .

ABOVE: © ISTOCK.COM,
DEJAN_DUNDJERSKI
Moderna announced that its COVID-19 vaccine candidate is 94.5 percent effective at protecting people from infection, compared to patients who received a placebo saline shot, according to an interim analysis of 95 participants the company released today (November 16).
“These are obviously very exciting results,” Anthony Fauci, the director of the US National Institute of Allergy and Infectious Diseases, tells CNN, noting he expects the first vaccinations to begin toward the end of December. “It’s just as good as it gets—94.5 percent is truly outstanding.”
The results put Moderna’s vaccine on par with competitor Pfizer’s COVID-19 vaccine, which was found to be 90 percent effective in a preliminary analysis of 94 participants. Both companies are expected to seek authorization for emergency use in the US and from other global regulators in the coming weeks. The European Medicines Agency announced today that it has begun a rolling review of the Moderna vaccine.
Both vaccines use mRNA to instruct cells to create many copies of a piece of the SARS-CoV-2 virus, causing the immune system to be prepared to attack the virus should it try to invade. Although there are other vaccines in development that use this technology, none have been approved yet.
As mRNA can quickly degrade in warm temperatures, vaccine storage presents a serious practical challenge. While Pfizer’s vaccine must be stored at –70 °C, Moderna’s can be kept at –20 °C. Moderna also said today that once thawed, its vaccine can remain stable for a month at 2–8 °C.
“The fact that two different vaccines made by two different companies with two different kinds of structures, in a new messenger RNA concept, both worked so effectively confirms the concept once and for all that this is a viable strategy not only for COVID but for future infectious disease threats,” Barry Bloom, a public health professor at Harvard University, tells The New York Times.
It’s unclear exactly how many of the trial’s 30,000 participants, who received either two doses of the vaccine or the placebo, have contracted COVID-19. The interim analysis includes 95 confirmed cases—five who had been given the vaccine and 90 who received the placebo. Among this group, 11—all in the placebo group—developed severe COVID-19.
“It was one of the greatest moments in my life and my career. It is absolutely amazing to be able to develop this vaccine and see the ability to prevent symptomatic disease with such high efficacy,” Tal Zaks, Moderna’s chief medical officer, tells CNN.
Like the Pfizer vaccine, Moderna’s reportedly does not have any serious side effects, but a small percentage of participants in the study experienced symptoms such as body aches and headaches. Pfizer did not release data on the vaccine’s performance to prevent severe cases.
Both companies announced their findings in press releases, not in peer-reviewed journals, and neither company provided the detailed data that would allow outside experts to evaluate their claims. As the studies continue, the figures on the vaccines’ efficacy may change. It is also unclear how long the immunity provided by the vaccines will last. Moderna and Pfizer conducted their trials using slightly different protocols. While participants in the Moderna trial needed to display at least two symptoms of disease in addition to a positive test for the virus to be officially counted as a COVID-19 case, the Pfizer trial required only one symptom and a positive test. Pfizer started counting cases seven days after the second injection of the vaccine, and Moderna waited 14 days.
Moderna has a $1.525 billion contract to provide the US with 100 million doses of the vaccine through Operation Warp Speed, with an option to purchase an additional 400 million doses.


thescientist--2020-11-16--Cell Culture Advances Fuel Disease Research

Novel cell culture technologies have a large impact on how scientists study human disease .
Researchers use three-dimensional culture techniques @ @ @ @ @ to recapitulate three-dimensional physiological structures and shed light on cell behavior during tissue and organ @ @ @ @ @ cell culture vital for understanding degenerative pathologies and potentially restoring lost organ function .
Download @ @ @ @ @ 's Creative Services team to learn how researchers use the latest cell culture techniques to

Novel cell culture technologies have a large impact on how scientists study human disease. Researchers use three-dimensional culture techniques, microfluidics, and bioprinting to recapitulate three-dimensional physiological structures and shed light on cell behavior during tissue and organ development. These advancements make cell culture vital for understanding degenerative pathologies and potentially restoring lost organ function.
Download this ebook from The Scientist's Creative Services team to learn how researchers use the latest cell culture techniques to


thescientist--2020-11-16--Optimizing Library Preparation for Tumor Samples

Comprehensive tumor profiling using next generation sequencing ( NGS ) technologies is key to discovering new , targetable biomarkers for cancer research .
However , converting tumor samples into NGS libraries can be challenging .
Tumor samples may be formalin-fixed and paraffin-embedded ( FFPE ) to preserve tissue structure , which reduces tumor DNA quality .
Similarly , cell-free DNA ( cfDNA ) samples collected from liquid biopsies often yield low quantities of tumor-derived DNA .
Careful library conversion of low-quality , low-quantity DNA samples is critical to sequencing success .
Download this application note from IDT to discover • @ @ @ @ @ @ @ DNA Library Prep Kit with IDT xGen hybridization reagents for the sensitive and accurate detection of low-frequency tumor variants • How researchers successfully detect tumor-associated variants in matched formalin-fixed , paraffin-embedded , and cell-free DNA samples .
For research use only .
Unless otherwise agreed to in writing , IDT does not intend these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use .
Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or @ @ @

Comprehensive tumor profiling using next generation sequencing (NGS) technologies is key to discovering new, targetable biomarkers for cancer research. However, converting tumor samples into NGS libraries can be challenging. Tumor samples may be formalin-fixed and paraffin-embedded (FFPE) to preserve tissue structure, which reduces tumor DNA quality. Similarly, cell-free DNA (cfDNA) samples collected from liquid biopsies often yield low quantities of tumor-derived DNA. Careful library conversion of low-quality, low-quantity DNA samples is critical to sequencing success.
Download this application note from IDT to discover
The benefits of combining the xGen TM Prism DNA Library Prep Kit with IDT xGen hybridization reagents for the sensitive and accurate detection of low-frequency tumor variants
Prism DNA Library Prep Kit with IDT xGen hybridization reagents for the sensitive and accurate detection of low-frequency tumor variants How researchers successfully detect tumor-associated variants in matched formalin-fixed, paraffin-embedded, and cell-free DNA samples.
For research use only. Unless otherwise agreed to in writing, IDT does not intend these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.


thescientist--2020-11-16--Black in X Addresses Long-Standing Inequity in STEM

On May 25 , Christian Cooper was bird-watching in New York City ’ s Central Park when he asked a woman to put her dog on a leash , as was mandated for the area .
The woman responded to the request by saying she was going to call the cops and she was “ going to tell them there is an African-American man threatening [ her ] life.
” As evidenced by the video taken by Cooper during the exchange , she did just that and urged the police to come immediately .
While the incident was unique in @ @ @ @ @ @ @ , scores of Black birders , naturalists , and ecologists recounted experiencing white onlookers calling the police on them while in outdoor spaces .
One birder , for instance , tweeted that after having dealt with many police calls , he now displays a sign to explain what he ’ s doing while in natural areas .
In June , a geoscientist shared a 10-minute-long video describing how survey work yields daily stares and sometimes tense encounters with people displaying neo-Nazi or other white supremacy symbols .
“ Unfortunately , the safety of @ @ @ @ @ @ @ , ” Angelica Patterson , a PhD candidate at Columbia University , tells The Scientist in an email .
Cooper ’ s encounter—combined with uproar over the deaths of George Floyd , Breonna Taylor , and other Black Americans at the hands of law enforcement—have propelled a number of initiatives to raise the profile of Black people in STEM and awareness of the continuing effects of racism .
In the immediate aftermath of the incident in Central Park , economist Anna Gifty Opoku-Agyeman had the idea of celebrating Cooper and other Black birders @ @ @ @ @ @ @ with a goal of providing a space for Black naturalists to get to know one another and show that interest in birding and other outdoor activities is not exclusive to white people .
Opoku-Agyeman , a cofounder of The Sadie Collective , an organization dedicated to helping Black women break into the world of finance and economics , shared her idea on Twitter and had conversations with others in @ BlackAFInSTEM , an organization for Black scientists .
She realized that Cooper ’ s experience wasn ’ t a one-off incident , but @ @ @ @ @ @ @ in the field , she tells The Scientist .
Enthusiasm for Black Birders Day spread on social media , and what was meant to be a day stretched into the first-ever Black Birders Week , which ran from May 31 through June 5 .
Each day featured a theme and encouraged Black birders to share pictures of themselves in nature or of their favorite bird species .
Thousands of tweets went out with the hashtag # BlackBirdersWeek , and the event attracted attention from some high-profile organizations , including the National Audubon Society @ @ @ @ @ @ @ to the creation of # BlackInNature , a social media space where Black ecologists and naturalists of all kinds , not just birders , can connect .
From there , Twitter exploded with Black scientists celebrating their disciplines .
Soon , # BlackInAstro , # BlackInNeuro , # BlackInCardio , # BlackInMicro , and others had their own awareness weeks and were garnering international participation .
As momentum grew , the awareness weeks progressed beyond hashtags and began integrating live events with expert speakers and sessions focused on career development .
Researchers who @ @ @ @ @ @ @ also created their own accounts and hashtags , such as @ LatinxinSTEM and # LGBTQinSTEM .
“ If you had asked me a month ago , ‘ Who are the other Black microbiologists ?
’ .
.
.
I would have had one , maybe two people that I would have known offhand , ” Black in Micro co-organizer Ariangela Kozik , a postdoc at the University of Michigan , tells The Scientist .
Now , because of Black in Micro Week , “ I know at least 30 very well , and @ @ @ @ @ @ @ re all one cohesive group that needs each other in order to move forward .
We ’ re not an island or a silo , ” Kozik explains .
All of the different groups , collectively known as “ Black in X , ” connect via Slack , where they discuss the logistical challenges of pulling off an awareness week and share advice .
“ It kind of solidifies the fact that we are not out here on our own , ” Kozik says .
“ Now that we can finally see each @ @ @ @ @ @ @ other.
” The topics of the events , which are still ongoing , vary from week to week , and increasingly include career development sessions .
Black in Chem , for instance , held a live “ elevator pitch ” contest , encouraging participants to present their research and interests succinctly .
The microbiologists discussed the science around the COVID-19 pandemic , and the neurologists addressed the need for mentorship .
Moving forward , each group is taking a slightly different approach so that their organizations become lasting entities , not ephemeral Twitter @ @ @ @ @ @ @ their awareness weeks for next year and have ongoing digital meetups to stay in touch with one another .
Black in Neuro held a digital mini-conference for October 31 through November 4 .
Because of how interdisciplinary modern science is , some scientists are able to participate in multiple weeks , growing their networks and learning about other areas they might not be well-versed in .
This isn ’ t good just for Black scientists , it ’ s good for science , says Ayanna Jones , a leader within Black in Chem @ @ @ @ @ @ @ .
It hasn ’ t been long enough to say what the lasting effects of this movement will be , but several of the organizers who spoke to The Scientist shared that as a result of growing awareness of the issues faced by academics who are members of underrepresented groups , their institutions have been undertaking conversations about acknowledging racism and assessing what needs to be done to create lasting change .
This isn ’ t a simple fix , as most of the problems are thought to be rooted in implicit bias @ @ @ @ @ @ @ Black students are less likely to begin STEM degrees and drop out of them far more often than their white peers are .
Entrance into graduate school creates another barrier due to factors such as the cost of the GRE and the fact that Black students are less likely to have attended prestigious undergraduate institutions or worked at unpaid internships .
In their careers , Black scientists are less likely to receive funding or get their research published .
Even with equal resumes , people with African -American–sounding names are less likely to @ @ @ @ @ @ @ .
“ Systemic bias in the workplace , unwelcoming spaces and cultures in science departments , and outright harassment of people of color venturing outdoors have prevented opportunities for all of these spaces and visible roles to be diversified , ” says Patterson .
“ When doing science work or recreating in the outdoors alone , [ Black ] individuals will have to be more cognizant of their surroundings , be mindful of what time of day they are going outside , and be prepared with a way to communicate with friends , @ @ @ @ @ @ @ a park or forest if one finds themselves in a hostile situation.
” Several organizers also pointed to the prevalence of microaggressions , acts that don ’ t constitute outright discrimination , but lead to feelings of discomfort and exclusion .
This can include giving compliments with a racial caveat , such as , “ You ’ re so articulate for a Black woman , ” negating lived experiences by saying “ I don ’ t see color , ” or questioning a person ’ s qualifications .
“ Especially with Black women @ @ @ @ @ @ @ when we say things , ” explains Devin Swiner , a graduate student at the Ohio State University and one of the organizers of Black in Chem .
“ People are always trying to check my credentials .
If I say something in a group meeting , then people might not take me seriously .
But if my advisor or another man in my lab is like , ‘ Oh , no , Devin had a really great point , ’ then it ’ s like , ‘ Oh , maybe Devin knows @ @ @ @ @ @ @ That happens pretty often.
” By themselves , these behaviors are irritating , says Jones , but they can snowball into an unwelcoming , unhealthy culture .
“ So it ’ s all these microaggressions building up and the next thing you know , you have a macroaggression , ” Jones explains .
“ There ’ s all these statistics about the lack of Black people in STEM , but let ’ s break that down .
Where did that begin ?
And what is the root cause ?
”

ABOVE: © ISTOCK.COM, CASTIGATIO
On May 25, Christian Cooper was bird-watching in New York City’s Central Park when he asked a woman to put her dog on a leash, as was mandated for the area. The woman responded to the request by saying she was going to call the cops and she was “going to tell them there is an African-American man threatening [her] life.” As evidenced by the video taken by Cooper during the exchange, she did just that and urged the police to come immediately.
While the incident was unique in making national news, in its aftermath, scores of Black birders, naturalists, and ecologists recounted experiencing white onlookers calling the police on them while in outdoor spaces. One birder, for instance, tweeted that after having dealt with many police calls, he now displays a sign to explain what he’s doing while in natural areas. In June, a geoscientist shared a 10-minute-long video describing how survey work yields daily stares and sometimes tense encounters with people displaying neo-Nazi or other white supremacy symbols.
“Unfortunately, the safety of Black naturalists continues to be an issue,” Angelica Patterson, a PhD candidate at Columbia University, tells The Scientist in an email.
Cooper’s encounter—combined with uproar over the deaths of George Floyd, Breonna Taylor, and other Black Americans at the hands of law enforcement—have propelled a number of initiatives to raise the profile of Black people in STEM and awareness of the continuing effects of racism.
Black in X
In the immediate aftermath of the incident in Central Park, economist Anna Gifty Opoku-Agyeman had the idea of celebrating Cooper and other Black birders. Black Birders Day was born, with a goal of providing a space for Black naturalists to get to know one another and show that interest in birding and other outdoor activities is not exclusive to white people.
Opoku-Agyeman, a cofounder of The Sadie Collective, an organization dedicated to helping Black women break into the world of finance and economics, shared her idea on Twitter and had conversations with others in @BlackAFInSTEM, an organization for Black scientists. She realized that Cooper’s experience wasn’t a one-off incident, but that racist encounters were common while out in the field, she tells The Scientist.
Enthusiasm for Black Birders Day spread on social media, and what was meant to be a day stretched into the first-ever Black Birders Week, which ran from May 31 through June 5. Each day featured a theme and encouraged Black birders to share pictures of themselves in nature or of their favorite bird species. Thousands of tweets went out with the hashtag #BlackBirdersWeek, and the event attracted attention from some high-profile organizations, including the National Audubon Society.
The success of the event led to the creation of #BlackInNature, a social media space where Black ecologists and naturalists of all kinds, not just birders, can connect.
From there, Twitter exploded with Black scientists celebrating their disciplines. Soon, #BlackInAstro, #BlackInNeuro, #BlackInCardio, #BlackInMicro, and others had their own awareness weeks and were garnering international participation. As momentum grew, the awareness weeks progressed beyond hashtags and began integrating live events with expert speakers and sessions focused on career development. Researchers who are members of other underrepresented minority groups also created their own accounts and hashtags, such as @LatinxinSTEM and #LGBTQinSTEM.
“If you had asked me a month ago, ‘Who are the other Black microbiologists?’ . . . I would have had one, maybe two people that I would have known offhand,” Black in Micro co-organizer Ariangela Kozik, a postdoc at the University of Michigan, tells The Scientist. Now, because of Black in Micro Week, “I know at least 30 very well, and about 100 more.”
Ariangela Kozik, co-organizer of Black in Micro COURTESY OF CALVIN UNIVERSITY
“We’re all one cohesive group that needs each other in order to move forward. We’re not an island or a silo,” Kozik explains. All of the different groups, collectively known as “Black in X,” connect via Slack, where they discuss the logistical challenges of pulling off an awareness week and share advice. “It kind of solidifies the fact that we are not out here on our own,” Kozik says. “Now that we can finally see each other, we can now support each other.”
The topics of the events, which are still ongoing, vary from week to week, and increasingly include career development sessions. Black in Chem, for instance, held a live “elevator pitch” contest, encouraging participants to present their research and interests succinctly. The microbiologists discussed the science around the COVID-19 pandemic, and the neurologists addressed the need for mentorship.
Moving forward, each group is taking a slightly different approach so that their organizations become lasting entities, not ephemeral Twitter trends. Some groups have already planned their awareness weeks for next year and have ongoing digital meetups to stay in touch with one another. Black in Neuro held a digital mini-conference for October 31 through November 4.
Because of how interdisciplinary modern science is, some scientists are able to participate in multiple weeks, growing their networks and learning about other areas they might not be well-versed in. This isn’t good just for Black scientists, it’s good for science, says Ayanna Jones, a leader within Black in Chem and a PhD student at Emory University.
It hasn’t been long enough to say what the lasting effects of this movement will be, but several of the organizers who spoke to The Scientist shared that as a result of growing awareness of the issues faced by academics who are members of underrepresented groups, their institutions have been undertaking conversations about acknowledging racism and assessing what needs to be done to create lasting change.
This isn’t a simple fix, as most of the problems are thought to be rooted in implicit bias and have been reinforced over generations. Black students are less likely to begin STEM degrees and drop out of them far more often than their white peers are. Entrance into graduate school creates another barrier due to factors such as the cost of the GRE and the fact that Black students are less likely to have attended prestigious undergraduate institutions or worked at unpaid internships. In their careers, Black scientists are less likely to receive funding or get their research published. Even with equal resumes, people with African -American–sounding names are less likely to be called to interview for a job.
“Systemic bias in the workplace, unwelcoming spaces and cultures in science departments, and outright harassment of people of color venturing outdoors have prevented opportunities for all of these spaces and visible roles to be diversified,” says Patterson. “When doing science work or recreating in the outdoors alone, [Black] individuals will have to be more cognizant of their surroundings, be mindful of what time of day they are going outside, and be prepared with a way to communicate with friends, family members, or authorized person of a park or forest if one finds themselves in a hostile situation.”
Several organizers also pointed to the prevalence of microaggressions, acts that don’t constitute outright discrimination, but lead to feelings of discomfort and exclusion. This can include giving compliments with a racial caveat, such as, “You’re so articulate for a Black woman,” negating lived experiences by saying “I don’t see color,” or questioning a person’s qualifications.
“Especially with Black women, people don’t believe us when we say things,” explains Devin Swiner, a graduate student at the Ohio State University and one of the organizers of Black in Chem. “People are always trying to check my credentials. If I say something in a group meeting, then people might not take me seriously. But if my advisor or another man in my lab is like, ‘Oh, no, Devin had a really great point,’ then it’s like, ‘Oh, maybe Devin knows what she’s talking about.’ That happens pretty often.”
By themselves, these behaviors are irritating, says Jones, but they can snowball into an unwelcoming, unhealthy culture. “So it’s all these microaggressions building up and the next thing you know, you have a macroaggression,” Jones explains. “There’s all these statistics about the lack of Black people in STEM, but let’s break that down. Where did that begin? And what is the root cause?”