SARS-CoV-2 Antibody Durability

COVID-19 vaccine development depends on identifying potent neutralizing antibodies .
Recent research studies suggest that neutralizing antibodies found in the @ @ @ @ @ decay rapidly and the level of antibody protection may correlate with infection severity .
In @ @ @ @ @ by The Scientist , scientists studying the SARS-CoV-2 immune response will discuss their research on @ @ @ @ @ memory in recovered COVID-19 patients and the implications for protective immunity .
• Assessing antibodies @ @ @ @ @ of COVID-19 patients • Examining immune memory formation and persistence in individuals that recovered from @ @ @ @ @ PhD Professor , Department of Immunology Canada Research Chair in Tissue Specific Immunity Associate Chair @ @ @ @ @ of Immunology University of Toronto

FREE Webinar
Monday, October 26, 2020
2:30 - 4:00 PM, Eastern Time
Register Now
COVID-19 vaccine development depends on identifying potent neutralizing antibodies. Recent research studies suggest that neutralizing antibodies found in the blood of recovered COVID-19 patients decay rapidly and the level of antibody protection may correlate with infection severity. In this webinar brought to you by The Scientist, scientists studying the SARS-CoV-2 immune response will discuss their research on SARS-CoV-2 antibody persistence and immune memory in recovered COVID-19 patients and the implications for protective immunity.
Topics to be covered
Assessing antibodies to SARS-CoV-2 in the saliva of COVID-19 patients
Examining immune memory formation and persistence in individuals that recovered from mild COVID-19
Register Now
Meet the Speakers:
Jennifer Gommerman, PhD
Professor, Department of Immunology
Canada Research Chair in Tissue Specific Immunity
Associate Chair, Graduate Studies, Department of Immunology
University of Toronto
Marion Pepper, PhD
Associate Professor, Department of Immunology
University of Washington


thescientist--2020-10-06--Study Points to Novel Role for Microglia in Down Syndrome

ABOVE : Confocal images showing activated microglia in hippocampal brain slices of a mouse with a murine version of Down syndrome GIOVANNI MORELLI Overactivation of the brain ’ s immune cells , called microglia , may play a role in cognitive impairments associated with Down syndrome , according to research published today ( October 6 ) in Neuron .
Researchers in Italy identified elevated numbers of the cells in an inflammation-promoting state in the brains of mice with a murine version of the syndrome as well as in postmortem brain tissue from people with the condition .
The team additionally @ @ @ @ @ @ @ of activated microglia in juvenile mice could boost the animals ’ performance on cognitive tests .
“ This is a fabulous study that gives a lot of proof of principle to pursuing some clinical trials in people , ” says Elizabeth Head , a neuroscientist at the University of California , Irvine , who was not involved in the work .
“ The focus on microglial activation , I thought , was very novel and exciting , ” she adds , noting that more research will be needed to see how the effects @ @ @ @ @ @ @ translate from mice to humans .
Down syndrome is caused by an extra copy of part or all of human chromosome 21 , and is the most commonly occurring chromosomal condition in the US .
Children with Down syndrome often experience cognitive delays compared to typically developing children , although there ’ s substantial variation and the effects are usually mild or moderate .
People with the syndrome also have a higher risk of certain medical conditions , including Alzheimer ’ s disease .
A number of studies have identified elevated levels of @ @ @ @ @ @ @ while separate research has also connected inflammation to cognitive delay or decline in people and research animals .
In their study , neuroscientists Laura Cancedda , Giovanni Morelli , and Bruno Pinto of the Italian Institute of Technology in Genoa and colleagues set out to investigate a role for microglia—which , when in a so-called activated state , release cytokines known to promote neuroinflammation .
The team focused first on a mouse model of the condition , in which part of chromosome 16—the murine equivalent of human chromosome 21—is triplicated .
These so-called @ @ @ @ @ @ @ of the traits seen in people with Down syndrome , including delayed development and difficulties with motor and cognitive skills , but not the dysfunctional neurogenesis or other abnormalities in brain development characteristic of some other mouse models of the condition and people with Down syndrome .
Comparing the brains of juvenile Dp ( 16 ) mice with those of control animals , the researchers didn ’ t find any differences in the overall numbers of microglia .
However , Dp ( 16 ) mice had higher numbers of microglia in an activated @ @ @ @ @ @ @ and protein expression patterns associated with neuroinflammation .
The researchers next knocked out some of these activated microglia in the brains of Dp ( 16 ) mice—either by feeding animals a drug that reduces the overall number of microglia , or by injecting the animals with acetaminophen , an anti-inflammatory drug that helps inhibit microglial activation , once a day for three days .
Both sets of treated mice performed better on lab measures of cognition—such as discrimination between familiar and unfamiliar objects—than Dp ( 16 ) mice that hadn ’ t been @ @ @ @ @ @ @ mouse model of Down syndrome produced similar results .
The effect of acetaminophen wore off relatively quickly , Morelli notes : mice tested a couple weeks after their last injection showed microglial morphology and cognitive performance similar to that of untreated mice .
Additional experiments showed that the drug didn ’ t have a significant effect in adult animals , suggesting that microglial activation is particularly relevant during earlier stages of brain development , Cancedda adds .
To connect their findings to humans , the researchers examined the hippocampi of postmortem brains from @ @ @ @ @ @ @ they reached 40 .
An analysis of gene and protein expression patterns and cell morphology revealed the same tell-tale signs of microglia activation that the team had found in mice .
Tarik Haydar , a neuroscientist at Children ’ s National Hospital in Washington , DC , who was not involved in the work , says that he was impressed by the study ’ s thoroughness .
How microglia influence the developing brain , particularly as it relates to Down syndrome , has been largely unknown , he adds .
The study authors @ @ @ @ @ @ @ they answered it quite comprehensively.
” Both Haydar and Head praise the team ’ s use of two separate mouse models and inclusion of postmortem human brain tissue , noting that similar findings across all three provide good evidence for microglial cells ’ relevance in Down syndrome .
“ The fact that they ’ re getting the same signatures in their young mice and they ’ re seeing something similar in younger human brains , I thought was really convincing , ” says Head .
In an email to The Scientist , Victoria @ @ @ @ @ @ @ del Mar Medical Research Institute in Barcelona , calls the study a “ tour de force ” and praises its use of multiple techniques .
She adds that while the findings provide evidence “ that abnormal microglia may be a possible cause ” of cognitive delays , Down syndrome “ is a complex syndrome that combines alterations not only in the brain immune system but also in other systems , ” including the cardiovascular system and wider patterns of gene expression .
“ Chronic treatment with anti-inflammatory drugs may not be sufficient ” @ @ @ @ @ @ @ she writes , although it ’ s worth scientists investigating to see if it could help .
Cancedda says that researchers will need to learn more about how and over what timescale acetaminophen , a widely available and relatively safe drug , acts to reduce cognitive problems in the juvenile mice they studied , with an eye to possible clinical trials in the future .
She cautions that there ’ s still a lot left to understand about acetaminophen ’ s effects on cognition and that people should not experiment with taking the @ @ @ @ @ @ @ agrees that the findings provide good justification for studies in humans , but should be interpreted with caution until carefully controlled trials have provided more information—not least because of potentially harmful side-effects or interactions between acetaminophen and other medications .
“ The proper way to do this , just as the authors suggest , is to do a controlled clinical trial , ” says Head .
“ I would love to see a clinical trial do that.
” B. Pinto et al.
, “ Rescuing over-activated microglia restores cognitive performance in juvenile animals @ @ @ @ @ @ @ model of Down syndrome , ” Neuron , doi:10.1016/j.neuron.2020.09.010 , 2020 .

ABOVE: Confocal images showing activated microglia in hippocampal brain slices of a mouse with a murine version of Down syndrome
GIOVANNI MORELLI
Overactivation of the brain’s immune cells, called microglia, may play a role in cognitive impairments associated with Down syndrome, according to research published today (October 6) in Neuron. Researchers in Italy identified elevated numbers of the cells in an inflammation-promoting state in the brains of mice with a murine version of the syndrome as well as in postmortem brain tissue from people with the condition. The team additionally showed that drugs that reduce the number of activated microglia in juvenile mice could boost the animals’ performance on cognitive tests.
“This is a fabulous study that gives a lot of proof of principle to pursuing some clinical trials in people,” says Elizabeth Head, a neuroscientist at the University of California, Irvine, who was not involved in the work. “The focus on microglial activation, I thought, was very novel and exciting,” she adds, noting that more research will be needed to see how the effects of drugs used in the study might translate from mice to humans.
Down syndrome is caused by an extra copy of part or all of human chromosome 21, and is the most commonly occurring chromosomal condition in the US. Children with Down syndrome often experience cognitive delays compared to typically developing children, although there’s substantial variation and the effects are usually mild or moderate. People with the syndrome also have a higher risk of certain medical conditions, including Alzheimer’s disease.
A number of studies have identified elevated levels of inflammation in people with Down syndrome, while separate research has also connected inflammation to cognitive delay or decline in people and research animals. In their study, neuroscientists Laura Cancedda, Laura Perlini, Giovanni Morelli, and Bruno Pinto of the Italian Institute of Technology in Genoa and colleagues set out to investigate a role for microglia—which, when in a so-called activated state, release cytokines known to promote neuroinflammation.
The team focused first on a mouse model of the condition, in which part of chromosome 16—the murine equivalent of human chromosome 21—is triplicated. These so-called Dp(16) mice show some of the traits seen in people with Down syndrome, including delayed development and difficulties with motor and cognitive skills, but not the dysfunctional neurogenesis or other abnormalities in brain development characteristic of some other mouse models of the condition and people with Down syndrome.
Comparing the brains of juvenile Dp(16) mice with those of control animals, the researchers didn’t find any differences in the overall numbers of microglia. However, Dp(16) mice had higher numbers of microglia in an activated state—they showed cell morphology, electrophysiology, and protein expression patterns associated with neuroinflammation.
The fact that they’re getting the same signatures in their young mice and they’re seeing something similar in younger human brains, I thought was really convincing. —Elizabeth Head, University of California, Irvine
The researchers next knocked out some of these activated microglia in the brains of Dp(16) mice—either by feeding animals a drug that reduces the overall number of microglia, or by injecting the animals with acetaminophen, an anti-inflammatory drug that helps inhibit microglial activation, once a day for three days. Both sets of treated mice performed better on lab measures of cognition—such as discrimination between familiar and unfamiliar objects—than Dp(16) mice that hadn’t been treated. Testing acetaminophen on a different mouse model of Down syndrome produced similar results.
The effect of acetaminophen wore off relatively quickly, Morelli notes: mice tested a couple weeks after their last injection showed microglial morphology and cognitive performance similar to that of untreated mice. Additional experiments showed that the drug didn’t have a significant effect in adult animals, suggesting that microglial activation is particularly relevant during earlier stages of brain development, Cancedda adds.
To connect their findings to humans, the researchers examined the hippocampi of postmortem brains from people with Down syndrome who died before they reached 40. An analysis of gene and protein expression patterns and cell morphology revealed the same tell-tale signs of microglia activation that the team had found in mice.
Tarik Haydar, a neuroscientist at Children’s National Hospital in Washington, DC, who was not involved in the work, says that he was impressed by the study’s thoroughness. How microglia influence the developing brain, particularly as it relates to Down syndrome, has been largely unknown, he adds. The study authors “not only asked that question, they answered it quite comprehensively.”
Both Haydar and Head praise the team’s use of two separate mouse models and inclusion of postmortem human brain tissue, noting that similar findings across all three provide good evidence for microglial cells’ relevance in Down syndrome. “The fact that they’re getting the same signatures in their young mice and they’re seeing something similar in younger human brains, I thought was really convincing,” says Head.
In an email to The Scientist, Victoria Puig, a neuroscientist at the Hospital del Mar Medical Research Institute in Barcelona, calls the study a “tour de force” and praises its use of multiple techniques. She adds that while the findings provide evidence “that abnormal microglia may be a possible cause” of cognitive delays, Down syndrome “is a complex syndrome that combines alterations not only in the brain immune system but also in other systems,” including the cardiovascular system and wider patterns of gene expression. “Chronic treatment with anti-inflammatory drugs may not be sufficient” to treat cognitive problems in people, she writes, although it’s worth scientists investigating to see if it could help.
Cancedda says that researchers will need to learn more about how and over what timescale acetaminophen, a widely available and relatively safe drug, acts to reduce cognitive problems in the juvenile mice they studied, with an eye to possible clinical trials in the future. She cautions that there’s still a lot left to understand about acetaminophen’s effects on cognition and that people should not experiment with taking the drug outside a clinical setting.
Head agrees that the findings provide good justification for studies in humans, but should be interpreted with caution until carefully controlled trials have provided more information—not least because of potentially harmful side-effects or interactions between acetaminophen and other medications.
“The proper way to do this, just as the authors suggest, is to do a controlled clinical trial,” says Head. “I would love to see a clinical trial do that.”
B. Pinto et al., “Rescuing over-activated microglia restores cognitive performance in juvenile animals of the Dp(16) mouse model of Down syndrome,” Neuron, doi:10.1016/j.neuron.2020.09.010, 2020.
Clarification (October 7): The fourth paragraph of this article has been updated to add the name of study coauthor Laura Perlini.


thescientist--2020-10-06--How Ice Shelf Loss Will Change the Antarctic Ecosystem

ABOVE : An iceberg fracturing off the Antarctic Peninsula JEROEN INGELS This month , Arctic sea ice shrank to its second lowest level on record , while the Antarctic Peninsula is one of the most rapidly warming places on the planet .
The calving of the world ’ s largest iceberg—a 1-trillion-ton mass called A-68 that split from the Larsen C ice shelf in 2017—is one of almost a dozen significant ice shelf loss events at the southern pole in the past few decades .
Under a “ business as usual ” climate scenario , in which carbon emissions continue @ @ @ @ @ @ @ melting of Antarctic ice will cause global sea level to rise up to three times as much as it did in the last century .
To better understand how climate change is shaping Antarctic ecosystems , the National Science Foundation sponsored a workshop in November 2017 , bringing together almost 40 interdisciplinary researchers to assess the current state of knowledge regarding ice shelf loss and identify critical information gaps .
Their review , published yesterday ( October 5 ) in WIREs Climate Change , pulls from decades of research that have already noted @ @ @ @ @ @ @ shelf thinning and retreat .
The Scientist spoke with Jeroen Ingels , a marine ecologist at Florida State University and the lead author of the new study , about the most pressing takeaways from the team ’ s findings .
The Scientist : When people think about Antarctica , they 're thinking about penguins , arguably the most iconic residents .
But who are the other major players in the Antarctic food web ?
Jeroen Ingels : It ’ s mainly the large organisms associated with the ice , [ such as ] @ @ @ @ @ @ @ krill , which have cycles that are associated with the appearance and disappearance of sea ice .
Then you have phytoplankton blooms , and increasingly more salps [ gelatinous invertebrates ] as well .
So there ’ s this food web that ’ s situated in the open sea .
But of course , all of that is also connected to the bottom , benthic environment .
You have lots of different benthic organisms , from microbes to meiofaunal , macrofaunal , and megafaunal species .
The seafloor beneath ice sheets is analogous @ @ @ @ @ @ @ it was considered azoic [ lifeless ] until people started looking .
But a few decades ago , they were able to drill a hole through the ice and peer down , and you see there ’ s actually quite a bit of life there .
TS : What is going to happen to that food web as climate change takes place and ice melts ?
JI : I think one of the important things to realize is that it 's not going to be uniform .
We like to think of climate @ @ @ @ @ @ @ , but we know very well now that it ’ s going to be different in different places .
The same is true for the Antarctic .
Some places ice is disappearing and other places it ’ s growing .
In areas where the ice disappears , the whole system on the seafloor is going to change .
The distribution of species that are dependent on sea ice production will shift closer to shore in the place where the ice shelf used to be .
Phytoplankton blooms can come into these areas .
@ @ @ @ @ @ @ food down to the sea floor .
Species that are adapted to the dark depths are going to be invaded by species that are more adapted to exploiting the new food sources .
All these ecological matches that were once standard suddenly turn into mismatches , and trying to figure out what the consequences will be is quite difficult to do .
TS : What is happening to Antarctic ice sheets in the Anthropocene and how is that different from what has happened in the past ?
JI : The ice sheets are @ @ @ @ @ @ @ the rapidity [ are unprecedented ] .
It happens , but it happens over tens of thousands of years .
It does n't happen in a few decades .
But the warming itself is changing oceanographic patterns , causing melting at the base of the ice shelves .
All these feedback loops are increasing the rate at which [ ice shelves ] are breaking off and collapsing , and it seems like they are poised to make this place disappear as fast as possible .
TS : In the review , you called @ @ @ @ @ @ @ unpredictable.
” What makes it so difficult to predict ?
JI : It ’ s the interactions between the atmosphere and the ocean , and the variable climate patterns in different regions of the Antarctic , that make it difficult for models to speak specifically to how much ice is thinning .
It ’ s a lot for any model to calculate what exactly is going to happen and how long it ’ s going to take .
I think that ’ s something that scientists are working really hard on , but @ @ @ @ @ @ @ resolution needed to address those particular questions .
TS : What is it going to take to establish the long-term , collaborative , interdisciplinary initiatives needed to address the challenges of studying the Antarctic ?
JI : I think it has become quite clear that research investment [ is needed ] in infrastructure , in science , and in time to tackle the big questions in the Antarctic .
Look at space programs , you ask one huge question and you get hundreds of millions [ of dollars ] in funding for [ @ @ @ @ @ @ @ obligation to start understanding what is happening to our planet that .
.
.
we are actually dependent on .
I think it ’ s reasonable to say that we should increase our efforts to try and understand it , and to make it perhaps a bit more livable and manageable .
Editor ’ s note : The interview was edited for brevity .

ABOVE: An iceberg fracturing off the Antarctic Peninsula
JEROEN INGELS
This month, Arctic sea ice shrank to its second lowest level on record, while the Antarctic Peninsula is one of the most rapidly warming places on the planet. The calving of the world’s largest iceberg—a 1-trillion-ton mass called A-68 that split from the Larsen C ice shelf in 2017—is one of almost a dozen significant ice shelf loss events at the southern pole in the past few decades. Under a “business as usual” climate scenario, in which carbon emissions continue unabated, it is estimated that the melting of Antarctic ice will cause global sea level to rise up to three times as much as it did in the last century.
To better understand how climate change is shaping Antarctic ecosystems, the National Science Foundation sponsored a workshop in November 2017, bringing together almost 40 interdisciplinary researchers to assess the current state of knowledge regarding ice shelf loss and identify critical information gaps. Their review, published yesterday (October 5) in WIREs Climate Change, pulls from decades of research that have already noted changes to Antarctic ecosystems due to ice shelf thinning and retreat.
The Scientist spoke with Jeroen Ingels, a marine ecologist at Florida State University and the lead author of the new study, about the most pressing takeaways from the team’s findings.
Jeroen ingels
The Scientist: When people think about Antarctica, they're thinking about penguins, arguably the most iconic residents. But who are the other major players in the Antarctic food web?
Jeroen Ingels: It’s mainly the large organisms associated with the ice, [such as] penguins and whales. Both feed on krill, which have cycles that are associated with the appearance and disappearance of sea ice. Then you have phytoplankton blooms, and increasingly more salps [gelatinous invertebrates] as well. So there’s this food web that’s situated in the open sea. But of course, all of that is also connected to the bottom, benthic environment. You have lots of different benthic organisms, from microbes to meiofaunal, macrofaunal, and megafaunal species.
The seafloor beneath ice sheets is analogous to the deep sea, where originally it was considered azoic [lifeless] until people started looking. But a few decades ago, they were able to drill a hole through the ice and peer down, and you see there’s actually quite a bit of life there.
TS: What is going to happen to that food web as climate change takes place and ice melts?
JI: I think one of the important things to realize is that it's not going to be uniform. We like to think of climate change as a warming climate all over, but we know very well now that it’s going to be different in different places. The same is true for the Antarctic. Some places ice is disappearing and other places it’s growing.
In areas where the ice disappears, the whole system on the seafloor is going to change. The distribution of species that are dependent on sea ice production will shift closer to shore in the place where the ice shelf used to be. Phytoplankton blooms can come into these areas . . . that are able to send food down to the sea floor. Species that are adapted to the dark depths are going to be invaded by species that are more adapted to exploiting the new food sources. All these ecological matches that were once standard suddenly turn into mismatches, and trying to figure out what the consequences will be is quite difficult to do.
All these feedback loops are increasing the rate at which [ice shelves] are breaking off and collapsing, and it seems like they are poised to make this place disappear as fast as possible. —Jeroen Ingels, Florida State University
TS: What is happening to Antarctic ice sheets in the Anthropocene and how is that different from what has happened in the past?
JI: The ice sheets are clearly melting, and the timescales and the rapidity [are unprecedented]. It happens, but it happens over tens of thousands of years. It doesn't happen in a few decades. But the warming itself is changing oceanographic patterns, causing melting at the base of the ice shelves. All these feedback loops are increasing the rate at which [ice shelves] are breaking off and collapsing, and it seems like they are poised to make this place disappear as fast as possible.
TS: In the review, you called the thinning of the ice shelves “unpredictable.” What makes it so difficult to predict?
JI: It’s the interactions between the atmosphere and the ocean, and the variable climate patterns in different regions of the Antarctic, that make it difficult for models to speak specifically to how much ice is thinning. It’s a lot for any model to calculate what exactly is going to happen and how long it’s going to take. I think that’s something that scientists are working really hard on, but they haven’t quite solved the resolution needed to address those particular questions.
TS: What is it going to take to establish the long-term, collaborative, interdisciplinary initiatives needed to address the challenges of studying the Antarctic?
JI: I think it has become quite clear that research investment [is needed] in infrastructure, in science, and in time to tackle the big questions in the Antarctic. Look at space programs, you ask one huge question and you get hundreds of millions [of dollars] in funding for [decades]. We have an ethical obligation to start understanding what is happening to our planet that . . . we are actually dependent on. I think it’s reasonable to say that we should increase our efforts to try and understand it, and to make it perhaps a bit more livable and manageable.
Editor’s note: The interview was edited for brevity.


thescientist--2020-10-07--CRISPR's Adaptation to Genome Editing Earns Chemistry Nobel

This year ’ s Nobel Prize in Chemistry has been awarded jointly to Emmanuelle Charpentier of the Max Planck Institute for Infection Biology and Jennifer Doudna of the University of California , Berkeley , the Royal Swedish Academy of Sciences announced today ( October 7 ) .
They are recognized for their pioneering work in developing CRISPR technology , which has revolutionized the field of gene editing .
“ Today ’ s Nobel Prize in Chemistry recognizes CRISPR-Cas9 , a super-selective and precise gene-editing tool where chemistry plays an incredibly important role , ” Luis Echegoyen , the president of @ @ @ @ @ @ @ both women are members , says in a statement .
“ This discovery , originally derived from a natural defense mechanism in bacteria against viruses , will have untold applications in treating and curing genetic diseases and fighting cancer , as well as impacts on agricultural and other areas .
The future for this technique is indeed bright and promising.
” “ I don ’ t know quite what to say .
I ’ m about as shocked as I ’ ve ever been , ” Doudna tells The Scientist , reacting to @ @ @ @ @ @ @ I ’ m really so grateful.
” CRISPR-based technology allows scientists to precisely manipulate DNA , a desirable ability for many fields .
CRISPR has contributed to discoveries across disciplines , conferring mold and pest resistance to important crops , leading to new cancer therapies , and , more recently , repairing damage within mitochondrial DNA .
During the COVID-19 pandemic , CRISPR has also been used in new diagnostic tools for detecting the virus .
“ This type of research will always lead potentially to a way to discover new pathways that @ @ @ @ @ @ @ bacteria , ” Charpentier said during a call with the Nobel Prize committee , “ but also as a way to find new mechanisms to target genes and their expression.
” Charpentier completed both her PhD and her first postdoc in the lab of Patrice Courvalin , a microbiologist at the Pasteur Institute in Paris .
“ She ’ s a very obstinate scientist .
She will not quit the problem , and will work on it until the end , ” Courvalin tells The Scientist .
“ Science is all her life.
@ @ @ @ @ @ @ Streptococcus pyogenes , Charpentier discovered a new immune molecule , called tracrRNA , that neutralized viruses by breaking apart their DNA .
After publishing her findings in 2011 in Nature , she initiated a collaboration with Doudna in which the pair isolated and refined the molecular underpinnings of these genetic scissors .
Their landmark paper , published in Science in 2012 , tracked closely with work being undertaken by other researchers at the time .
Virginijus Šikšnys , a biochemist at Vilnius University in Lithuania , published a paper in PNAS in September @ @ @ @ @ @ @ team further characterized the bacteria ’ s immune mechanism .
While Charpentier and Doudna ’ s paper published first , in June , it was submitted later .
Their results reaffirmed earlier findings that CRISPR could be used to cut double-stranded DNA at specific , predetermined sites along the genome , and further simplified the tool by showing that crRNA and tracrRNA could be fused to create a single , synthetic guide to direct the cutting mechanism to a site of interest .
The next year , in 2013 , Doudna ’ s @ @ @ @ @ @ @ cells .
The adoption of CRISPR and the subsequent iterations that endowed the tool with new abilities have been global .
At the same time , there have been ongoing disputes over the ownership of the intellectual property .
The first patent for the use of CRISPR gene editing technology in eukaryotes was granted to the Broad Institute of MIT and Harvard , based on the work of Feng Zhang ’ s group , back in April 2014 .
While the University of California has sought to have its own patents , based @ @ @ @ @ @ @ , take precedence , in February 2017 , the US Patent Trial and Appeal Board sided with the Broad—a decision later upheld by US Court of Appeals for the Federal Circuit .
The battle is far from over in the US , and another patent battle rages in Europe , where the European Patent Office has already revoked at least one Broad patent .
See “ A Brief Guide to the Current CRISPR Landscape ” Charpentier and Doudna join 53 women who have previously won a Nobel Prize , including only five who @ @ @ @ @ @ @ last prize in chemistry went to Frances Arnold in 2018 .
“ It ’ s stunning , it ’ s appropriate , and it brings joy to those of us who are women in science , ” Lila Gierasch , the editor in chief of the Journal of Biological Chemistry , tells The Scientist .
The pair will share the prize of 10 million Swedish kronor , or about $ 1.12 million .

ABOVE: © NOBEL MEDIA 2020 ILLUSTRATION: NIKLAS ELMEHED
This year’s Nobel Prize in Chemistry has been awarded jointly to Emmanuelle Charpentier of the Max Planck Institute for Infection Biology and Jennifer Doudna of the University of California, Berkeley, the Royal Swedish Academy of Sciences announced today (October 7). They are recognized for their pioneering work in developing CRISPR technology, which has revolutionized the field of gene editing.
“Today’s Nobel Prize in Chemistry recognizes CRISPR-Cas9, a super-selective and precise gene-editing tool where chemistry plays an incredibly important role,” Luis Echegoyen, the president of the American Chemical Society, of which Doudna is a member, says in a statement. “This discovery, originally derived from a natural defense mechanism in bacteria against viruses, will have untold applications in treating and curing genetic diseases and fighting cancer, as well as impacts on agricultural and other areas. The future for this technique is indeed bright and promising.”
“I don’t know quite what to say. I’m about as shocked as I’ve ever been,” Doudna tells The Scientist, reacting to the news of her award. “I’m really so grateful.”
CRISPR-based technology allows scientists to precisely manipulate DNA, a desirable ability for many fields. CRISPR has contributed to discoveries across disciplines, conferring mold and pest resistance to important crops, leading to new cancer therapies, and, more recently, repairing damage within mitochondrial DNA. During the COVID-19 pandemic, CRISPR has also been used in new diagnostic tools for detecting the virus.
“This type of research will always lead potentially to a way to discover new pathways that could be useful for developing therapeutics against bacteria,” Charpentier said during a call with the Nobel Prize committee, “but also as a way to find new mechanisms to target genes and their expression.”
A CRISPR CUT: The guide RNA (gRNA) forms a complex with Cas and directs the enzyme to cleave the target DNA to which the gRNA binds through complementary sequence. The cell tries to repair the DNA break, which often results in the insertion (as shown) or deletion of nucleotides that changes the reading frame of the gene and a creates premature stop codon. The Scientist staff
Charpentier completed both her PhD and her first postdoc in the lab of Patrice Courvalin, a microbiologist at the Pasteur Institute in Paris. “She’s a very obstinate scientist. She will not quit the problem, and will work on it until the end,” Courvalin tells The Scientist. “Science is all her life.”
Through her study of the bacteria Streptococcus pyogenes, Charpentier discovered a new immune molecule, called tracrRNA, that neutralized viruses by breaking apart their DNA. After publishing her findings in 2011 in Nature, she initiated a collaboration with Doudna in which the pair isolated and refined the molecular underpinnings of these genetic scissors.
Their landmark paper, published in Science in 2012, tracked closely with work being undertaken by other researchers at the time. Virginijus Šikšnys, a biochemist at Vilnius University in Lithuania, published a paper in PNAS in September that year in which he and his team further characterized the bacteria’s immune mechanism. While Charpentier and Doudna’s paper published first, in June, it was submitted later. Their results reaffirmed earlier findings that CRISPR could be used to cut double-stranded DNA at specific, predetermined sites along the genome, and further simplified the tool by showing that crRNA and tracrRNA could be fused to create a single, synthetic guide to direct the cutting mechanism to a site of interest. The next year, in 2013, Doudna’s team applied her new technology to human cells.
The adoption of CRISPR and the subsequent iterations that endowed the tool with new abilities have been global. At the same time, there have been ongoing disputes over the ownership of the intellectual property. The first patent for the use of CRISPR gene editing technology in eukaryotes was granted to the Broad Institute of MIT and Harvard, based on the work of Feng Zhang’s group, back in April 2014. While the University of California has sought to have its own patents, based on Charpentier and Doudna’s work, take precedence, in February 2017, the US Patent Trial and Appeal Board sided with the Broad—a decision later upheld by US Court of Appeals for the Federal Circuit. The battle is far from over in the US, and another patent battle rages in Europe, where the European Patent Office has already revoked at least one Broad patent.
See “A Brief Guide to the Current CRISPR Landscape”
Charpentier and Doudna join 53 women who have previously won a Nobel Prize, including only five who have been awarded in chemistry. The last prize in chemistry went to Frances Arnold in 2018. “It’s stunning, it’s appropriate, and it brings joy to those of us who are women in science,” Lila Gierasch, the editor in chief of the Journal of Biological Chemistry, tells The Scientist.
The pair will share the prize of 10 million Swedish kronor, or about $1.12 million.


thescientist--2020-10-07--Stomach Acid & Heartburn Drugs Linked with COVID-19 Outcomes

The uncertainty of the COVID-19 pandemic has made our stomachs churn , and now , evidence suggests that intense heartburn may be linked with worse symptoms of the disease .
Some drugs that neutralize stomach acid , such as famotidine , which President Donald Trump is taking , are associated with reduced severity , but others , such as Prilosec , correlate with higher infection rates and risk of death , at least in patients hospitalized with SARS-CoV-2 infections .
“ Everyone has some level of acid reflux , ” says Helder Nakaya , a systems biologist at the University @ @ @ @ @ @ @ been studying the link between stomach acid and SARS-CoV-2 infection .
“ But I want to be clear .
.
.
we can not claim that this increased risk of death would apply to everyone with reflux.
” The link does suggest that stomach acid might be a factor that ’ s often overlooked when it comes to COVID-19 , Nakaya says .
In a retrospective analysis posted on medRxiv of roughly 1,300 hospitalized COVID-19 patients , Nakaya and colleagues found that individuals taking proton-pump inhibitors , including Prilosec , had a two- @ @ @ @ @ @ @ with hospitalized patients not taking those drugs .
This observation falls in line with a study published in late August in the American Journal of Gastroenterology by doctors in the US that also found people taking a proton-pump inhibitor twice a day for acid reflux had higher odds of testing positive for SARS-CoV-2 compared with individuals taking that type of drug once a day or individuals who took a histamine-2 receptor blocker such as Pepcid AC .
Proton-pump inhibitors “ may undermine the gastric barrier to SARS-CoV-2 entry and reduce microbial diversity in @ @ @ @ @ @ @ risk getting COVID-19 , the US-based team writes in its report .
Another retrospective analysis published online in Gastroenterology in May found that in a cohort of 1,620 hospitalized patients , proton-pump inhibitors had no relationship to the patients ’ outcomes .
Meanwhile , famotidine , which blocks the histamine-2 receptor on cells , correlated with a reduced risk of patients being intubated or dying from COVID-19 .
Prior to analyzing that larger dataset , the authors had heard anecdotal evidence that famotidine might make COVID-19 symptoms less severe , and they ’ @ @ @ @ @ @ @ experiments also backed up the idea , study coauthor Joseph Conigliaro , the division chief of general internal medicine in the Department of Medicine at Northwell Health in Manhasset , New York , tells The Scientist in an email .
In April , Northwell partnered with Alchem Laboratories and launched a randomized clinical trial to test whether giving up to 360 milligrams of famotidine intravenously to COVID-19 patients would improve their health outcomes compared with patients who received standard of care treatment .
The trial later came under fire with a government whistleblower @ @ @ @ @ @ @ evidence and for the high doses of the drug being used , according to the Associated Press .
Still , Northwell ’ s research on famotidine continued .
“ Neither the whistleblower complaint or the AP story had much of an effect on us .
However , they are part of an overall trend to politicize Covid-19 clinical trials and prospective therapies that is deeply unfortunate , ” Matthew Libassi , a spokesperson for the Feinstein Institutes for Medical Research , the research arm of Northwell , writes in an email to The @ @ @ @ @ @ @ as it makes it harder to conduct medical science research.
” Despite the pushback , additional evidence for famotidine ’ s role in fighting viruses has surfaced .
A past study published in 1996 had shown that famotidine could reduce viral replication of HIV , and a more recent computational analysis published in May also suggested that the histamine blocker could have some antiviral properties—specifically , that it might inhibit a protease that processes proteins vital for SARS-CoV-2 replication .
The computational results offered “ some biological plausibility ” to the hypothesis that @ @ @ @ @ @ @ , but “ in the end I think the computational models do not tell the whole picture , ” Conigliaro says .
That ’ s why he and his colleagues conducted their retrospective study on patients admitted to the hospital between February 25 and April 13 ( these patients were not part of Northwell ’ s clinical trial testing famotidine ) .
Analysis of the data revealed a distinct difference in the overall health outcomes of the patients taking famotidine compared with patients not given the drug .
( Why the patients were @ @ @ @ @ @ @ some had a history of gastric reflux and were taking the medication prior to being admitted to the hospital . )
“ I expected a difference , but not so pronounced , ” Conigliaro says .
“ I was also surprised that doses as low as what was used is the retrospective study would work that well .
We had anticipated that bigger doses would be needed.
” In the study , some patients received up to 20 milligrams of famotidine per day for five days intravenously , while others received it orally @ @ @ @ @ @ @ patients can take as much as 160 milligrams four times a day .
Those who got the drug in the hospital fared better than those who did not , the study found .
Julian Abrams , a gastroenterologist at Columbia University Irving Medical Center-New York Presbyterian Hospital and a collaborator of Conigliaro , notes that the link between famotidine and reduced severity of COVID-19 is still only correlational at this point , but nevertheless warrants more attention .
Nakaya ’ s work offers a bit more insight into potential mechanisms .
He and @ @ @ @ @ @ @ to look at the relationship between antacids and COVID-19 at all .
Their project began as an offshoot of another one that Nakaya ’ s graduate student , Leandro Jimenez , had started .
Jimenez had been analyzing the transcriptome data of patients with Barrett ’ s esophagus , a condition in which acid reflux causes damage to the lining of the esophagus .
The data from biopsies and a gene expression repository revealed increased expression of the gene ACE2 , which encodes a cell surface protein that SARS-CoV-2 uses to enter human @ @ @ @ @ @ @ s esophagus compared with individuals who did not have the condition .
That increased gene activity was tied to regulation of intracellular pH pathways , specifically , their enrichment .
That connection suggests increased ACE2 expression is linked with lower pH inside cells , possibly a result of exposure to stomach acid .
“ And that raised a flag , ” Nakaya says , which led his team to hypothesize that Barrett ’ s esophagus , as well as gastroesophageal reflux disease ( GERD ) , may be a comorbidities of COVID-19 that @ @ @ @ @ @ @ To test the link between stomach acid and SARS-CoV-2 infectivity , Nakaya ’ s team exposed human monocytes in cell culture to different pH conditions and then to SARS-CoV-2 .
The researchers used these immune cells because they are known to be susceptible to infection by the coronavirus , Nakaya says .
Under normal oxygen levels , the cells in culture with a pH lower than 7.4 had a higher expression of ACE2 and also a higher viral load .
The result indicates that intracellular pH may influence the ability of SARS-CoV-2 to @ @ @ @ @ @ @ But it wasn ’ t clear , says Nakaya , that the finding would have any clinical relevance , so the team dug into the medical records of patients in Manaus and São Paulo , Brazil , who had been hospitalized for COVID-19 and found that proton-pump inhibitors correlated with an increased risk of death .
That association , Nakaya says , suggests that it might not be the drugs themselves that lead to worsening COVID-19 symptoms , but instead that the proton-pump inhibitors are , the team writes , “ important markers @ @ @ @ @ @ @ caused by the excess stomach acid in GI tissues.
” In other words , the low pH that cells are dealing with—and that patients are trying to treat with a proton-pump inhibitor—might make the cells more vulnerable to SARS-CoV-2 infection .
The results , however , don ’ t explain why famotidine correlated with better outcomes in Conigliaro and Abrams ’ s study , supposing the drug is also used to suppress stomach acid .
“ We don ’ t think that stomach acid is the explanation for the findings , ” says @ @ @ @ @ @ @ t know why we found what we did.
” The past work on HIV and the computational analysis suggest that famotidine works as an antiviral , and a study published today ( October 7 ) also supports that idea .
The research shows that ranitidine bismuth citrate , another histamine antagonist and antibiotic combo used to treat stomach acid , suppresses SARS-CoV-2 replication in infected golden Syrian hamsters and improves their virus-related pneumonia symptoms .
But famotidine , Conigliaro says , may have benefits other than being antiviral ; it may actually prevent @ @ @ @ @ @ @ a SARS-CoV-2 infection and causing a life-threatening cytokine storm .
There ’ s some evidence for this from an observational study in which patients severely ill with COVID-19 were given a cocktail of histamine blockers , one of which was famotidine , and had better outcomes than did patients receiving the standard of care whose cases were reported elsewhere .
Those data and other research “ seem to suggest that the anti-histamine effect is what prevents patients from getting the cytokine storm , ” Conigliaro explains , noting that cells with the histamine-2 @ @ @ @ @ @ @ as the stomach .
The results from those studies and his team ’ s work , he says , bolster the case for the clinical trial launched last spring to test famotidine as a treatment for COVID-19 , results of which are still pending .
The Department of Defense under the Discovery of Medical Countermeasures Against Novel Entities , or DOMANE , program is also studying famotidine as a COVID-19 treatment , according to Vanity Fair , and another famotidine trial in Bangladesh is also now recruiting patients .
“ Most people ’ @ @ @ @ @ @ @ than treatments , ” Abrams says , but “ with President Trump having COVID , that brings to light again the issue of treatments , especially since he was getting famotidine .
”

ABOVE: © ISTOCK.COM, BWFOLSOM
The uncertainty of the COVID-19 pandemic has made our stomachs churn, and now, evidence suggests that intense heartburn may be linked with worse symptoms of the disease. Some drugs that neutralize stomach acid, such as famotidine, which President Donald Trump is taking, are associated with reduced severity, but others, such as Prilosec, correlate with higher infection rates and risk of death, at least in patients hospitalized with SARS-CoV-2 infections.
“Everyone has some level of acid reflux,” says Helder Nakaya, a systems biologist at the University of São Paulo in Brazil who has been studying the link between stomach acid and SARS-CoV-2 infection. “But I want to be clear . . . we cannot claim that this increased risk of death would apply to everyone with reflux.” The link does suggest that stomach acid might be a factor that’s often overlooked when it comes to COVID-19, Nakaya says.
In a retrospective analysis posted on medRxiv of roughly 1,300 hospitalized COVID-19 patients, Nakaya and colleagues found that individuals taking proton-pump inhibitors, including Prilosec, had a two- to three-fold higher risk of death compared with hospitalized patients not taking those drugs. This observation falls in line with a study published in late August in the American Journal of Gastroenterology by doctors in the US that also found people taking a proton-pump inhibitor twice a day for acid reflux had higher odds of testing positive for SARS-CoV-2 compared with individuals taking that type of drug once a day or individuals who took a histamine-2 receptor blocker such as Pepcid AC.
Proton-pump inhibitors “may undermine the gastric barrier to SARS-CoV-2 entry and reduce microbial diversity in the gut,” increasing patients’ risk getting COVID-19, the US-based team writes in its report.
Another retrospective analysis published online in Gastroenterology in May found that in a cohort of 1,620 hospitalized patients, proton-pump inhibitors had no relationship to the patients’ outcomes. Meanwhile, famotidine, which blocks the histamine-2 receptor on cells, correlated with a reduced risk of patients being intubated or dying from COVID-19.
Prior to analyzing that larger dataset, the authors had heard anecdotal evidence that famotidine might make COVID-19 symptoms less severe, and they’d heard that a few in vitro experiments also backed up the idea, study coauthor Joseph Conigliaro, the division chief of general internal medicine in the Department of Medicine at Northwell Health in Manhasset, New York, tells The Scientist in an email. In April, Northwell partnered with Alchem Laboratories and launched a randomized clinical trial to test whether giving up to 360 milligrams of famotidine intravenously to COVID-19 patients would improve their health outcomes compared with patients who received standard of care treatment. The trial later came under fire with a government whistleblower complaint for being hastily approved with little evidence and for the high doses of the drug being used, according to the Associated Press. Still, Northwell’s research on famotidine continued.
“Neither the whistleblower complaint or the AP story had much of an effect on us. However, they are part of an overall trend to politicize Covid-19 clinical trials and prospective therapies that is deeply unfortunate,” Matthew Libassi, a spokesperson for the Feinstein Institutes for Medical Research, the research arm of Northwell, writes in an email to The Scientist. “That trend concerns us as it makes it harder to conduct medical science research.”
Despite the pushback, additional evidence for famotidine’s role in fighting viruses has surfaced. A past study published in 1996 had shown that famotidine could reduce viral replication of HIV, and a more recent computational analysis published in May also suggested that the histamine blocker could have some antiviral properties—specifically, that it might inhibit a protease that processes proteins vital for SARS-CoV-2 replication.
The computational results offered “some biological plausibility” to the hypothesis that famotidine could reduce the severity of COVID-19, but “in the end I think the computational models do not tell the whole picture,” Conigliaro says. That’s why he and his colleagues conducted their retrospective study on patients admitted to the hospital between February 25 and April 13 (these patients were not part of Northwell’s clinical trial testing famotidine). Analysis of the data revealed a distinct difference in the overall health outcomes of the patients taking famotidine compared with patients not given the drug. (Why the patients were given famotidine is not clear, though some had a history of gastric reflux and were taking the medication prior to being admitted to the hospital.)
“I expected a difference, but not so pronounced,” Conigliaro says. “I was also surprised that doses as low as what was used is the retrospective study would work that well. We had anticipated that bigger doses would be needed.” In the study, some patients received up to 20 milligrams of famotidine per day for five days intravenously, while others received it orally; when used for acid reflux, patients can take as much as 160 milligrams four times a day. Those who got the drug in the hospital fared better than those who did not, the study found.
Julian Abrams, a gastroenterologist at Columbia University Irving Medical Center-New York Presbyterian Hospital and a collaborator of Conigliaro, notes that the link between famotidine and reduced severity of COVID-19 is still only correlational at this point, but nevertheless warrants more attention.
Stomach acid and ACE2
Nakaya’s work offers a bit more insight into potential mechanisms. He and his team weren’t initially planning to look at the relationship between antacids and COVID-19 at all. Their project began as an offshoot of another one that Nakaya’s graduate student, Leandro Jimenez, had started. Jimenez had been analyzing the transcriptome data of patients with Barrett’s esophagus, a condition in which acid reflux causes damage to the lining of the esophagus.
The data from biopsies and a gene expression repository revealed increased expression of the gene ACE2, which encodes a cell surface protein that SARS-CoV-2 uses to enter human cells, in individuals with Barrett’s esophagus compared with individuals who did not have the condition. That increased gene activity was tied to regulation of intracellular pH pathways, specifically, their enrichment. That connection suggests increased ACE2 expression is linked with lower pH inside cells, possibly a result of exposure to stomach acid.
“And that raised a flag,” Nakaya says, which led his team to hypothesize that Barrett’s esophagus, as well as gastroesophageal reflux disease (GERD), may be a comorbidities of COVID-19 that hadn’t been identified before.
To test the link between stomach acid and SARS-CoV-2 infectivity, Nakaya’s team exposed human monocytes in cell culture to different pH conditions and then to SARS-CoV-2. The researchers used these immune cells because they are known to be susceptible to infection by the coronavirus, Nakaya says. Under normal oxygen levels, the cells in culture with a pH lower than 7.4 had a higher expression of ACE2 and also a higher viral load. The result indicates that intracellular pH may influence the ability of SARS-CoV-2 to infect cells and replicate within them.
But it wasn’t clear, says Nakaya, that the finding would have any clinical relevance, so the team dug into the medical records of patients in Manaus and São Paulo, Brazil, who had been hospitalized for COVID-19 and found that proton-pump inhibitors correlated with an increased risk of death. That association, Nakaya says, suggests that it might not be the drugs themselves that lead to worsening COVID-19 symptoms, but instead that the proton-pump inhibitors are, the team writes, “important markers of hidden comorbidities that involve the damage caused by the excess stomach acid in GI tissues.” In other words, the low pH that cells are dealing with—and that patients are trying to treat with a proton-pump inhibitor—might make the cells more vulnerable to SARS-CoV-2 infection.
The results, however, don’t explain why famotidine correlated with better outcomes in Conigliaro and Abrams’s study, supposing the drug is also used to suppress stomach acid. “We don’t think that stomach acid is the explanation for the findings,” says Abrams. “We really don’t know why we found what we did.”
The past work on HIV and the computational analysis suggest that famotidine works as an antiviral, and a study published today (October 7) also supports that idea. The research shows that ranitidine bismuth citrate, another histamine antagonist and antibiotic combo used to treat stomach acid, suppresses SARS-CoV-2 replication in infected golden Syrian hamsters and improves their virus-related pneumonia symptoms. But famotidine, Conigliaro says, may have benefits other than being antiviral; it may actually prevent patients’ immune systems from overreacting to a SARS-CoV-2 infection and causing a life-threatening cytokine storm. There’s some evidence for this from an observational study in which patients severely ill with COVID-19 were given a cocktail of histamine blockers, one of which was famotidine, and had better outcomes than did patients receiving the standard of care whose cases were reported elsewhere. Those data and other research “seem to suggest that the anti-histamine effect is what prevents patients from getting the cytokine storm,” Conigliaro explains, noting that cells with the histamine-2 receptor are in the lungs as well as the stomach.
The results from those studies and his team’s work, he says, bolster the case for the clinical trial launched last spring to test famotidine as a treatment for COVID-19, results of which are still pending. The Department of Defense under the Discovery of Medical Countermeasures Against Novel Entities, or DOMANE, program is also studying famotidine as a COVID-19 treatment, according to Vanity Fair, and another famotidine trial in Bangladesh is also now recruiting patients.
“Most people’s attention is shifting towards vaccines rather than treatments,” Abrams says, but “with President Trump having COVID, that brings to light again the issue of treatments, especially since he was getting famotidine.”


thescientist--2020-10-07--High Content Phenotypic Screening and Analysis Enhances Drug Discovery

Phenotypic screening identifies potential drug candidates based on their ability to modulate cells or organisms in a desired manner .
@ @ @ @ @ on models that can both induce pathological behavior and facilitate high throughput/high content screening .
@ @ @ @ @ Scientist , Molly Shoichet and Bridget Wagner will discuss strategies for creating such models and @ @ @ @ @ to identify agents to counter chronic diseases , such as cancer and diabetes .
• @ @ @ @ @ discovery in the beta cell Bridget Wagner , PhD Director of Pancreatic Cell Biology and @ @ @ @ @ Institute of MIT and Harvard

FREE Webinar
Wednesday, November 18, 2020
2:30 - 4:00 PM, Eastern Standard Time
Register Now
Phenotypic screening identifies potential drug candidates based on their ability to modulate cells or organisms in a desired manner. However, phenotypic screening depends on models that can both induce pathological behavior and facilitate high throughput/high content screening. In this webinar from The Scientist, Molly Shoichet and Bridget Wagner will discuss strategies for creating such models and outline how they use them to identify agents to counter chronic diseases, such as cancer and diabetes.
Topics to be covered
How 3D cell culture enables predictive drug screening
Phenotypic screening for small molecule discovery in the beta cell
Register Now
Meet the Speakers:
Molly Shoichet, PhD, FRS, OC
Professor, Chemical Engineering & Applied Chemistry
Biomaterials & Biomedical Engineering
University of Toronto
Tier 1 Canada Research Chair in Tissue Engineering
Bridget Wagner, PhD
Director of Pancreatic Cell Biology and Metabolic Disease
Institute Scientist
Broad Institute of MIT and Harvard
Sponsored by


thescientist--2020-10-06--Whole-Slide Imaging and Beyond: A New Open Platform for Scanning and Bioimaging

Whole-slide imaging is an essential task for labs engaged in pathology , histopathology , and molecular biology research , including @ @ @ @ @ assays .
However , commercial systems are often restrictive and researchers must purchase additional expensive @ @ @ @ @ of microscope-based research .
The new CellScan from Molecular Machines and Industries ( MMI ) @ @ @ @ @ for researchers seeking maximum utility from their microscopes .
In this webinar brought to you @ @ @ @ @ scientists use CellScan for a variety of lab applications , such as routine photo-documentation , @ @ @ @ @ , time-resolved live-cell imaging , and combinations of these techniques .
• Introduction to CellScan @ @ @ @ @ An overview of how CellScan contributes to current and future experimental needs

FREE Webinar
Thursday, November 5, 2020
12:00 - 1:00 PM, Eastern Time
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Whole-slide imaging is an essential task for laboratories engaged in pathology, histopathology, and molecular biology research, including sample preparation for Next-Generation Sequencing assays. However, commercial systems are often restrictive and researchers must purchase additional expensive equipment to perform common types of microscope-based research.
The new CellScan from Molecular Machines and Industries (MMI) presents an exciting new option for researchers seeking maximum utility from their microscopes. In this webinar brought to you by MMI, learn how scientists use CellScan for a variety of laboratory applications, such as routine photo-documentation, whole-slide scanning, fluorescence imaging, time-resolved live-cell imaging, and combinations of these techniques.
Topics to be covered
Introduction to CellScan technology and its applications
An overview of how CellScan contributes to current and future experimental needs
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Meet the Speakers:
David Hitrys
Commercial Director, Americas
Molecular Machines and Industries


thescientist--2020-10-08--A Challenge Trial for COVID-19 Would Not Be the First of Its Kind

ABOVE : Volunteer Molly Perry receives an injection of malaria sporozoites from Jim Kublin at the Fred Hutch–based Seattle Malaria Clinical Trials Center as part of a challenge study .
ROBERT HOOD/FRED HUTCH As the 2009 pandemic flu virus infected millions around the world , Matthew Memoli began planning to expose healthy volunteers to the pathogen—a proposal that met with considerable opposition .
The scientific community was divided on whether the approach was ethical or even warranted .
As the director of the Clinical Studies Unit at the Laboratory of Infectious Diseases at the National Institute of Allergy and Infectious @ @ @ @ @ @ @ the value of a so-called challenge trial for flu , and there was precedent for conducting one .
“ Our current vaccine and most of the antivirals that we use for flu were all developed in some part because of challenge studies , ” says Memoli .
But at the time he was applying for regulatory approval in 2010 , it had been a decade since anyone had done a flu challenge study .
He made his case for reviving the approach to address the ongoing pandemic virus .
He argued to numerous @ @ @ @ @ @ @ what people could encounter in the real world would allow researchers to track subjects ’ immune responses and test drugs and vaccines efficiently and with far fewer people than would be needed for traditional clinic trials .
In the end , the US Food and Drug Administration ( FDA ) and the National Institutes of Health ( NIH ) ethics review board agreed with him , and he developed a challenge model using a wildtype strain of H1N1 that has since been sprayed up the noses of more than 400 volunteers .
“ @ @ @ @ @ @ @ I ’ m making people sick on purpose .
It ’ s not a joke ; it ’ s a very serious thing that I ’ m doing .
And I only want to be doing it if it really is truly something that is beneficial , ” Memoli says .
Ten years later , Memoli is once again working on developing a human challenge model for a pandemic virus—this time , SARS-CoV-2 .
It ’ s not yet clear whether it will be used to test out vaccines or even therapeutics for @ @ @ @ @ @ @ I have always believed that we should be preparing to do it.
” Since the early days of the COVID-19 pandemic , a debate has focused on whether a challenge study for COVID-19 vaccines should proceed .
In June , an advisory group to the World Health Organization ( WHO ) issued a report providing guidelines for how such trials could be designed , but the committee was undecided over whether they could ethically proceed .
Late last month , the Financial Times reported that the UK government announced it was considering a @ @ @ @ @ @ @ start early next year .
( Imperial College London and the contract research organization hVivo , reportedly partnering on the trial , did not respond to requests for comment before deadline . )
For many pathogens other than SARS-CoV-2 , challenge studies have already become a key part of research on vaccines and therapeutics .
Besides influenza , scientists have developed challenge models for an array of other viruses as well as bacteria and parasites .
“ I think challenge studies have become more accepted , honestly , ” says Memoli .
“ @ @ @ @ @ @ @ , I think there was a big resurgence.
” The first step in conducting a challenge study is to design the challenge—that is , what the researchers will expose study participants to .
The strain , dose , and administration are considered , and the approach varies widely depending on the pathogen .
The goal is to elicit a consistent response in a majority of volunteers while ensuring everyone ’ s safety .
When clinical researcher Anna Durbin of Johns Hopkins Bloomberg School of Public Health and her collaborators at NIAID began developing @ @ @ @ @ @ @ ago , they started with two naturally attenuated strains they hoped could serve as proxies for the more dangerous forms of the virus to test vaccines ; the goal wasn ’ t to make people sick but simply to infect them .
The researchers wanted to be able to see the virus in cell cultures grown from blood samples from at least 80 percent of volunteers before they began challenge trials to test an experimental vaccine .
The researchers started with 10 participants who received 1,000 virus particles by subcutaneous injection , and @ @ @ @ @ @ @ had an average of only about 300 virus particles per milliliter of blood , however—a far lower concentration than the 10,000 to 100,000 viruses per milliliter of blood typical of natural dengue infections .
Several volunteers got a typical dengue rash over their whole body , but none got a fever .
In 2016 , Durbin and her colleagues published the results of a challenge trial of 41 volunteers showing a vaccine candidate to be 100 percent effective—none of the 21 participants who received the vaccine developed viremia when challenged with the virus @ @ @ @ @ @ @ get vaccinated did—prompting Brazil ’ s Butantan Institute to license the vaccine technology and launch an ongoing Phase 3 trial .
In other cases , researchers use the wildtype pathogen in an attempt to replicate natural disease .
For challenge models of influenza , Memoli says , it ’ s important to actually induce illness .
When it comes to respiratory viruses such as flu and SARS-CoV-2 , he explains , a vaccine might not prevent infection , but rather reduce symptom severity , shorten the course of the disease , or limit @ @ @ @ @ @ @ miss if the challenge simply causes infection but not sickness .
So far , Memoli says , developing a challenge model for SARS-CoV-2 has been “ a bit simpler ” than it was for flu , partly because the novel coronavirus has not yet evolved significantly different strains .
“ Really all the SARS-CoV-2 viruses that have been identified have pretty much the same properties , ” he notes , and moreover , the coronavirus has proven relatively easy to grow in the lab .
But there ’ s still a long way @ @ @ @ @ @ @ that his protocol must still be reviewed by the FDA—which must approve the proposed pathogen challenge as an investigational new drug before it is administered to people for an initial validation study—and an NIH institutional review board , as well as other committees .
“ We have lots to do over the next few months to even determine if everyone felt comfortable doing this before we could even make the decision to move forward , ” he says .
If a challenge trial were to proceed , the first study would simply serve @ @ @ @ @ @ @ 5 to 10 people to receive a relatively low dose of pathogen .
If the exposure proved safe enough , the researchers would expand the study and likely test higher doses until they are able to trigger illness in enough participants—typically at least 60–80 percent—to power future vaccine and drug trials .
With many labs now having characterized viral stocks of SARS-CoV-2 , “ that standardization and the qualification of the challenge material is one of the challenges that the coronavirus field is overcoming right now , ” says Fred Hutchinson Cancer Research @ @ @ @ @ @ @ conducts challenge studies of malaria .
But , he adds , “ given the pace at which we ’ re moving into these [ COVID-19 ] vaccine studies , I ’ m skeptical ” that a challenge trial would accelerate vaccine development .
A consistent and safe exposure is just the first requirement of a challenge study .
After the challenge is delivered to volunteers , researchers must be prepared to care for the participants if they get sick , and make sure they don ’ t transmit the infection to others .
@ @ @ @ @ @ @ easy .
In Kublin ’ s challenge studies of malaria , patients can simply go home after their exposure .
Because Anopheles mosquitos that carry malaria parasites do not live in Washington State , there is no risk of them biting an infected person from the trial and spreading the disease , and the researchers closely monitor the participants ’ blood for evidence of the parasite .
Once it makes it into the person ’ s circulation , it must mature for another two weeks .
By the time a volunteer might start @ @ @ @ @ @ @ been diagnosed and treated , says Kublin , who has used the model to test experimental vaccines and treatments .
The situation is similarly benign in challenge studies of hookworm .
While chronic infections with the parasite can cause gastrointestinal distress , anemia , and other complications in areas where it ’ s endemic , people who have volunteered to allow larvae of the pathogen to burrow through the skin on their arm don ’ t get sick , likely because of the relatively low dose and participants ’ good nutrition , say @ @ @ @ @ @ @ of George Washington University ’ s Vaccine Research Unit who are now conducting a hookworm vaccine challenge trial .
( The Vaccine Research Unit is also a site of the Phase 3 trial of Moderna ’ s COVID-19 vaccine candidate . )
The researchers confirm infection by looking for eggs in participants ’ stool samples , and then treat them with a medication called albendazole that kills the worms .
A few volunteers have agreed to remain infected and provide stool samples from which the team can harvest eggs for a new batch @ @ @ @ @ @ @ .
Diemert notes that it ’ s “ almost impossible ” for participants to spread the disease to others if they are using a toilet .
In other cases , the pathogen presents enough of a risk to the exposed individual or to others that trial participants must stay for a period of time at an in-patient facility under quarantine .
Such is the case when Johns Hopkins University infectious disease researcher David Sack has conducted vaccine challenge studies of cholera and other enteric bacteria .
The team members use strains they know @ @ @ @ @ @ @ which volunteers are given , along with fluids , as soon as they meet the clinical criteria for having developed the illness .
Similarly , participants who receive Memoli ’ s flu challenge stay in quarantine for one to two weeks , until they test negative for the flu on two consecutive days , although they don ’ t receive antiviral treatment so as to observe the infection take its course .
Memoli notes that the need for an extended quarantine of perhaps three weeks for a SARS-CoV-2 challenge would be a hinderance @ @ @ @ @ @ @ The more contentious part of the debate over whether a challenge study for COVID-19 should proceed is the risk to the individuals being deliberately exposed to the novel coronavirus , which is still very much an unknown—and one that can cause fatal disease .
So far , SARS-CoV-2 has claimed more than 1 million lives around the globe , including more than 200,000 in the US .
( In contrast , during the 2019–2020 flu season , the US Centers for Disease Control and Prevention estimates , there were between 24,000 and 62,000 @ @ @ @ @ @ @ reports that the UK trial will treat participants who fall ill with remdesivir , a drug that appears to expedite recovery .
“ Personally , I do not think that it is ethical at this time to conduct COVID-19 challenge vaccine trials , ” says Diemert .
“ There is no effective rescue treatment and we have no way of predicting who may have serious complications due to infection.
” Sack agrees : “ We say in general that young people don ’ t get as sick , but we also know that @ @ @ @ @ @ @ s also the risk that surviving participants could suffer long-term consequences , says Monica McArthur , a physician-scientist in the Center for Vaccine Development and Global Health at the University of Maryland School of Medicine .
“ While I ’ m a proponent of challenge models , I think there are some ethical requirements , and those include having a significant knowledge of the long-term effects of the infection.
” In making the evaluation of whether a COVID-19 challenge trial is warranted , the risks must be balanced with the need .
As @ @ @ @ @ @ @ there are many COVID-19 vaccine candidates in late-stage trials , with results expected over the next several months .
If one or more of these studies yields results that support the vaccine ’ s approval , a challenge trial may not be necessary .
Challenge trials may be warranted when there isn ’ t enough disease transmission in the community to determine if a vaccine is protective , says Durbin , who adds that there is currently no shortage of natural COVID-19 spread in the population .
“ When you ’ ve got @ @ @ @ @ @ @ you can ’ t necessarily predict when or where those outbreaks will occur to plan for a traditional Phase 3 efficacy study , challenge studies can be very useful , ” says Durbin , who is developing a human challenge model for Zika for that reason .
But back in 2016 , when she and her colleagues first proposed a Zika human challenge model because they realized that the Zika outbreak was waning , the FDA told them that “ it was unethical at the time to do a Zika challenge model , @ @ @ @ @ @ @ because there were still so many Zika cases .
“ But now we ’ re talking about a COVID-19 challenge trial .
That kind of blows my mind.
” Nevertheless , Durbin and others say it ’ s too soon to take the possibility of future challenge studies off the table .
With so many moving parts , you never know what the future holds .
“ I count myself among the many who are still waiting for more information on the COVID virus that would be used as the challenge strain , @ @ @ @ @ @ @ future ( i.e.
, How urgent is this by next year ? )
, and any advances in COVID therapeutics , ” Bethony tells The Scientist in an email .
Memoli is also a “ maybe ” on whether the SARS-CoV-2 challenge model he ’ s currently developing should ever be used to give people COVID-19 .
“ As we look to see what happens here over the next few months , in terms of the vaccine , in terms of the disease itself and its spread in the world , we ’ @ @ @ @ @ @ @ ” he says .
For now , he reiterates , “ we should definitely be preparing .
”

ABOVE: Volunteer Molly Perry receives an injection of malaria sporozoites from Jim Kublin at the Fred Hutch–based Seattle Malaria Clinical Trials Center as part of a challenge study.
ROBERT HOOD/FRED HUTCH
As the 2009 pandemic flu virus infected millions around the world, Matthew Memoli began planning to expose healthy volunteers to the pathogen—a proposal that met with considerable opposition. The scientific community was divided on whether the approach was ethical or even warranted.
As the director of the Clinical Studies Unit at the Laboratory of Infectious Diseases at the National Institute of Allergy and Infectious Diseases (NIAID), Memoli recognized the value of a so-called challenge trial for flu, and there was precedent for conducting one. “Our current vaccine and most of the antivirals that we use for flu were all developed in some part because of challenge studies,” says Memoli. But at the time he was applying for regulatory approval in 2010, it had been a decade since anyone had done a flu challenge study.
He made his case for reviving the approach to address the ongoing pandemic virus. He argued to numerous groups that a controlled exposure that mimicked what people could encounter in the real world would allow researchers to track subjects’ immune responses and test drugs and vaccines efficiently and with far fewer people than would be needed for traditional clinic trials. In the end, the US Food and Drug Administration (FDA) and the National Institutes of Health (NIH) ethics review board agreed with him, and he developed a challenge model using a wildtype strain of H1N1 that has since been sprayed up the noses of more than 400 volunteers.
While I’m a proponent of challenge models, I think there are some ethical requirements and those include having a significant knowledge of the long-term effects of the infection. —Monica McArthur, University of Maryland School of Medicine
“I personally take this very seriously. I’m making people sick on purpose. It’s not a joke; it’s a very serious thing that I’m doing. And I only want to be doing it if it really is truly something that is beneficial,” Memoli says.
Ten years later, Memoli is once again working on developing a human challenge model for a pandemic virus—this time, SARS-CoV-2. It’s not yet clear whether it will be used to test out vaccines or even therapeutics for COVID-19, he says, but “I have always believed that we should be preparing to do it.”
Since the early days of the COVID-19 pandemic, a debate has focused on whether a challenge study for COVID-19 vaccines should proceed. In June, an advisory group to the World Health Organization (WHO) issued a report providing guidelines for how such trials could be designed, but the committee was undecided over whether they could ethically proceed. Late last month, the Financial Times reported that the UK government announced it was considering a vaccine challenge trial for COVID-19 that could start early next year. (Imperial College London and the contract research organization hVivo, reportedly partnering on the trial, did not respond to requests for comment before deadline.)
For many pathogens other than SARS-CoV-2, challenge studies have already become a key part of research on vaccines and therapeutics. Besides influenza, scientists have developed challenge models for an array of other viruses as well as bacteria and parasites. “I think challenge studies have become more accepted, honestly,” says Memoli. “Since I started doing the flu challenge, I think there was a big resurgence.”
Determining the challenge
The first step in conducting a challenge study is to design the challenge—that is, what the researchers will expose study participants to. The strain, dose, and administration are considered, and the approach varies widely depending on the pathogen. The goal is to elicit a consistent response in a majority of volunteers while ensuring everyone’s safety.
When clinical researcher Anna Durbin of Johns Hopkins Bloomberg School of Public Health and her collaborators at NIAID began developing a challenge model of dengue several years ago, they started with two naturally attenuated strains they hoped could serve as proxies for the more dangerous forms of the virus to test vaccines; the goal wasn’t to make people sick but simply to infect them. The researchers wanted to be able to see the virus in cell cultures grown from blood samples from at least 80 percent of volunteers before they began challenge trials to test an experimental vaccine.
There is no effective rescue treatment and we have no way of predicting who may have serious complications due to infection. —David Diemert, George Washington University
The researchers started with 10 participants who received 1,000 virus particles by subcutaneous injection, and all 10 developed an infection. They had an average of only about 300 virus particles per milliliter of blood, however—a far lower concentration than the 10,000 to 100,000 viruses per milliliter of blood typical of natural dengue infections. Several volunteers got a typical dengue rash over their whole body, but none got a fever. In 2016, Durbin and her colleagues published the results of a challenge trial of 41 volunteers showing a vaccine candidate to be 100 percent effective—none of the 21 participants who received the vaccine developed viremia when challenged with the virus whereas all 20 who didn’t get vaccinated did—prompting Brazil’s Butantan Institute to license the vaccine technology and launch an ongoing Phase 3 trial.
In other cases, researchers use the wildtype pathogen in an attempt to replicate natural disease. For challenge models of influenza, Memoli says, it’s important to actually induce illness. When it comes to respiratory viruses such as flu and SARS-CoV-2, he explains, a vaccine might not prevent infection, but rather reduce symptom severity, shorten the course of the disease, or limit the pathogen’s spread—effects researchers may miss if the challenge simply causes infection but not sickness.
So far, Memoli says, developing a challenge model for SARS-CoV-2 has been “a bit simpler” than it was for flu, partly because the novel coronavirus has not yet evolved significantly different strains. “Really all the SARS-CoV-2 viruses that have been identified have pretty much the same properties,” he notes, and moreover, the coronavirus has proven relatively easy to grow in the lab. But there’s still a long way to go, Memoli adds, noting that his protocol must still be reviewed by the FDA—which must approve the proposed pathogen challenge as an investigational new drug before it is administered to people for an initial validation study—and an NIH institutional review board, as well as other committees. “We have lots to do over the next few months to even determine if everyone felt comfortable doing this before we could even make the decision to move forward,” he says.
If a challenge trial were to proceed, the first study would simply serve to validate the model, enrolling only 5 to 10 people to receive a relatively low dose of pathogen. If the exposure proved safe enough, the researchers would expand the study and likely test higher doses until they are able to trigger illness in enough participants—typically at least 60–80 percent—to power future vaccine and drug trials.
With many labs now having characterized viral stocks of SARS-CoV-2, “that standardization and the qualification of the challenge material is one of the challenges that the coronavirus field is overcoming right now,” says Fred Hutchinson Cancer Research Center’s Jim Kublin, who conducts challenge studies of malaria. But, he adds, “given the pace at which we’re moving into these [COVID-19] vaccine studies, I’m skeptical” that a challenge trial would accelerate vaccine development.
Ensuring participants’ safety
A consistent and safe exposure is just the first requirement of a challenge study. After the challenge is delivered to volunteers, researchers must be prepared to care for the participants if they get sick, and make sure they don’t transmit the infection to others.
Jim Kublin (left) watches Sanaria Inc.’s Jonathan Jackson and Meghan Marquette unpack malaria samples that will be injected into human test subjects during a clinical trial of a new malaria vaccine at the Fred Hutchinson Cancer Research Center in Seattle on April 21, 2016. ROBERT HOOD/FRED HUTCH
In some cases, that’s easy. In Kublin’s challenge studies of malaria, patients can simply go home after their exposure. Because Anopheles mosquitos that carry malaria parasites do not live in Washington State, there is no risk of them biting an infected person from the trial and spreading the disease, and the researchers closely monitor the participants’ blood for evidence of the parasite. Once it makes it into the person’s circulation, it must mature for another two weeks. By the time a volunteer might start to feel sick, they’ve been diagnosed and treated, says Kublin, who has used the model to test experimental vaccines and treatments.
The situation is similarly benign in challenge studies of hookworm. While chronic infections with the parasite can cause gastrointestinal distress, anemia, and other complications in areas where it’s endemic, people who have volunteered to allow larvae of the pathogen to burrow through the skin on their arm don’t get sick, likely because of the relatively low dose and participants’ good nutrition, say Jeff Bethony and David Diemert, codirectors of George Washington University’s Vaccine Research Unit who are now conducting a hookworm vaccine challenge trial. (The Vaccine Research Unit is also a site of the Phase 3 trial of Moderna’s COVID-19 vaccine candidate.) The researchers confirm infection by looking for eggs in participants’ stool samples, and then treat them with a medication called albendazole that kills the worms. A few volunteers have agreed to remain infected and provide stool samples from which the team can harvest eggs for a new batch of larvae for the next challenge study. Diemert notes that it’s “almost impossible” for participants to spread the disease to others if they are using a toilet.
In other cases, the pathogen presents enough of a risk to the exposed individual or to others that trial participants must stay for a period of time at an in-patient facility under quarantine. Such is the case when Johns Hopkins University infectious disease researcher David Sack has conducted vaccine challenge studies of cholera and other enteric bacteria. The team members use strains they know can be killed by an antibiotic, which volunteers are given, along with fluids, as soon as they meet the clinical criteria for having developed the illness.
Similarly, participants who receive Memoli’s flu challenge stay in quarantine for one to two weeks, until they test negative for the flu on two consecutive days, although they don’t receive antiviral treatment so as to observe the infection take its course. Memoli notes that the need for an extended quarantine of perhaps three weeks for a SARS-CoV-2 challenge would be a hinderance, though not an insurmountable obstacle.
Considerations for a COVID-19 challenge trial
The more contentious part of the debate over whether a challenge study for COVID-19 should proceed is the risk to the individuals being deliberately exposed to the novel coronavirus, which is still very much an unknown—and one that can cause fatal disease. So far, SARS-CoV-2 has claimed more than 1 million lives around the globe, including more than 200,000 in the US. (In contrast, during the 2019–2020 flu season, the US Centers for Disease Control and Prevention estimates, there were between 24,000 and 62,000 flu-related deaths.) The Financial Times reports that the UK trial will treat participants who fall ill with remdesivir, a drug that appears to expedite recovery.
Researchers must be prepared to care for the participants if they get sick, and make sure they don’t transmit the infection to others.
“Personally, I do not think that it is ethical at this time to conduct COVID-19 challenge vaccine trials,” says Diemert. “There is no effective rescue treatment and we have no way of predicting who may have serious complications due to infection.” Sack agrees: “We say in general that young people don’t get as sick, but we also know that some young people die.”
There’s also the risk that surviving participants could suffer long-term consequences, says Monica McArthur, a physician-scientist in the Center for Vaccine Development and Global Health at the University of Maryland School of Medicine. “While I’m a proponent of challenge models, I think there are some ethical requirements, and those include having a significant knowledge of the long-term effects of the infection.”
In making the evaluation of whether a COVID-19 challenge trial is warranted, the risks must be balanced with the need. As several researchers noted to The Scientist, there are many COVID-19 vaccine candidates in late-stage trials, with results expected over the next several months. If one or more of these studies yields results that support the vaccine’s approval, a challenge trial may not be necessary.
Challenge trials may be warranted when there isn’t enough disease transmission in the community to determine if a vaccine is protective, says Durbin, who adds that there is currently no shortage of natural COVID-19 spread in the population. “When you’ve got diseases that occur in sporadic outbreaks and you can’t necessarily predict when or where those outbreaks will occur to plan for a traditional Phase 3 efficacy study, challenge studies can be very useful,” says Durbin, who is developing a human challenge model for Zika for that reason. But back in 2016, when she and her colleagues first proposed a Zika human challenge model because they realized that the Zika outbreak was waning, the FDA told them that “it was unethical at the time to do a Zika challenge model,” Durbin says, in large part because there were still so many Zika cases. “But now we’re talking about a COVID-19 challenge trial. That kind of blows my mind.”
Nevertheless, Durbin and others say it’s too soon to take the possibility of future challenge studies off the table. With so many moving parts, you never know what the future holds. “I count myself among the many who are still waiting for more information on the COVID virus that would be used as the challenge strain, the state of vaccines in the near future (i.e., How urgent is this by next year?), and any advances in COVID therapeutics,” Bethony tells The Scientist in an email.
Memoli is also a “maybe” on whether the SARS-CoV-2 challenge model he’s currently developing should ever be used to give people COVID-19. “As we look to see what happens here over the next few months, in terms of the vaccine, in terms of the disease itself and its spread in the world, we’ll have to make some decisions,” he says. For now, he reiterates, “we should definitely be preparing.”