A Month After Emergency Declaration, Trump's Promises Largely Unfulfilled
PROMISE MADE PROMISE NOT KEPT 

NPR April 13, 2020

President Trmp speaks during a news conference about the coronavirus

 pandemic in the Rose Garden of the White House on March 13, 2020.

Chip Somodevilla/Getty Images

One month ago today, President Trump declared a national emergency.

In a Rose Garden address, flanked by leaders from giant retailers and medical testing companies, he promised a mobilization of public and private resources to attack the coronavirus.

"We've been working very hard on this. We've made tremendous progress," Trump said. "When you compare what we've done to other areas of the world, it's pretty incredible."

But few of those promises have come to pass.

NPR's Investigations Team dug into each of the claims made from the podium that day. And rather than a sweeping national campaign of screening, drive-through sample collection and lab testing, it found a smattering of small pilot projects and aborted efforts.

In some cases, no action was taken at all.


SHOTS - HEALTH NEWS
Map: Tracking The Spread Of The Coronavirus In The U.S.


SHOTS - HEALTH NEWS
Coronavirus State-By-State Projections: When Will Each State Peak?

Target did not partner with the federal government, for example.

And a lauded Google project turned out to not to be led by Google at all, and then once launched was limited to a smattering of counties in California.

The remarks in the Rose Garden highlighted the Trump administration's strategic approach: a preference for public-private partnerships. But as the White House defined what those private companies were going to do, in many cases it promised more than they could pull off.

"What became clear in the days and weeks or even in some cases the hours following that event was that they had significantly over-promised what the private sector was ready to do," said Jeremy Konyndyk, senior policy fellow at the Center for Global Development.

The White House declined to comment on this story.

Drive-Through Testing Largely Nonexistent At Retail Partners

During the Rose Garden address, the president introduced a series of leaders from major retailers to suggest there would be cooperation between the federal government and private sector companies for drive-through testing.

"We've been in discussions with pharmacies and retailers to make drive-through tests available in the critical locations identified by public health professionals," President Trump said.

NPR contacted the retailers who were there and found that discussions have not led to any wide-scale implementation of drive-through tests.


In the month since the announcement, Walmart has opened two testing sites — one in the Chicago area and another in Bentonville, Ark. Walgreens has opened two in Chicago; CVS has opened four sites.

Brian Cornell, board chairman and CEO of Target Corp., speaks during the March 13 news conference with President Trump at the White House. Target has so far not opened any COVID-19 testing sites.Andrew Harrer/Bloomberg via Getty Images

Target has not opened any. In fact, the company said it had no formal partnership with the federal government, and suggested they were waiting for the government to take the lead.

"At this time, federal, state and local officials continue to lead the planning for additional testing sites," a Target spokesperson said. "We stand committed to offering our parking lot locations and supporting their efforts when they are ready to activate."

Home Testing Promised, But Not Implemented

The president also welcomed Bruce Greenstein, an executive vice president of the LHC Group, to the microphone.

Greenstein's organization primarily provides in-home health care, and he pledged that it would be helping with testing "for Americans that can't get to a test site or live in rural areas far away from a retail establishment."

NPR called more than 20 LHC sites in 12 states, and none of them are doing in-home testing one month following the Rose Garden address. Employees at the LHC sites said they lacked both testing kits and the training to administer kits.

In response to NPR's reporting, Greenstein said that their primary focus so far has been getting proper personal protective equipment, or PPE, for their nurses, and working with hospitals on transitioning recovered COVID patients home. He says they'll start working with one New Orleans hospital "as soon as next week" to provide in-home testing, and to expand the service later.

LHC Group Executive Vice President Bruce Greenstein bumps elbows with President Trump during the March 13 news conference.Chip Somodevilla/Getty Images

No Screening Website To Facilitate Drive-Through Testing

During the March 13 Rose Garden address, the president also promised that Google was working to develop a website to determine whether a COVID-19 test would be warranted, and if so, to direct individuals to nearby testing.

The president said there were 1,700 Google engineers working on it, and the vice president said that guidance on the website would be available in two days.

"Google is helping to develop a website," the president said. "It's going to be very quickly done, unlike websites of the past, to determine whether a test is warranted and to facilitate testing at a nearby convenient location."

Dr. Deborah Birx, the coronavirus response coordinator at the White House, said that the website would screen patients, tell them where to receive drive-through testing, and provide testing results.

No such screening and testing website was ever developed by Google.

A pilot program was developed by Verily, a sister company to Google owned by the same parent company: Alphabet. Verily's program, called Project Baseline, was created to support California community-based COVID-19 testing from screening to testing to delivery of test results.

Verily has rolled out six testing sites primarily in coordination with the California state government — not the federal government — and is currently only available to residents of five counties in California.

During the March 13 news conference, Deborah Birx, the White House coronavirus response coordinator, outlined a website that would screen patients, tell them where to receive testing, and provide results. No such screening service came to exist.Andrew Harrer/Bloomberg via Getty Images
"We work in partnership with local public health agencies, the California Governor's office, and the California Department of Public Health," a spokesperson for Verily said, adding that their COVID-19 testing program was "federally supported."

There were not 1,700 engineers ever engaged in the project, as the president had claimed, according to Verily.

"As we initially ramped this program, we had nearly 1,000 volunteers from across Alphabet supporting a variety of functions," a Verily spokesperson told NPR.

Verily is in discussions with other health care organizations to support this kind of testing project outside of California, but there has been no announcement of future plans to do so.

A Department of Health and Human Services spokesperson pointed out that Apple had released a screening tool in collaboration with the CDC and the White House. That screening tool does not have the functions outlined in the March 13 Rose Garden address.

The President's Federal Agency Promises

In declaring the national emergency last month, the president also proposed several policy changes that were solely within the realm of the federal government to execute. On these, the administration largely followed through.

President Trump promised to waive interest on student loans held by government agencies, for instance. That policy was implemented by the secretary of education on March 20.

And the president made good on pledges to waive regulations and laws to give medical providers flexibility to respond to the healthcare crisis.

But there were exceptions. The president said he would waive license requirements so that doctors could practice in states with the greatest needs, for example. But medical licensing is a state issue, and the president does not have the authority to waive it.


"There's no statutory authority for the federal government to take over the delivery of health care services" says Dale Van Demark, a partner advising health industries at the law firm McDermott Will & Emery. Added Iris Hentze, policy specialist at The National Conference of State Legislatures: "These occupational licenses are really more or less completely controlled and regulated by states." What the federal government was able to do is to waive in-state requirements for healthcare providers that serve people enrolled in Medicare, Medicaid and CHIP, so they can get reimbursed for the out-of-state care they provided.

The promises weren't limited to matters of health care. The president announced that his administration would "purchase, at a very good price, large quantities of crude oil for storage in the U.S. Strategic Reserve."

"We're going to fill it right up to the top," he said, "saving the American taxpayer billions and billions of dollars."

The Trump administration has not done so. The president made the promise without first securing the funds from Congress, and t=he Department of Energy puts the responsibility on Congress' shoulders.

"Despite strong efforts from the Administration, Congress would not provide funding for the purchase of oil for SPR in the Stimulus bill," a Department of Energy spokesperson said. "The Department continues to work with Congress to deliver on the President's directive to provide relief to the American energy industry during this tumultuous time."

A Failure In Public-Private Partnerships

Later in that March 13 press conference, when asked whether he took responsibility for the apparent lag in coronavirus testing in the United States, the president responded, "I don't take responsibility at all."

He also suggested that laboratory capacity for testing would soon greatly expand. And he singled out two companies:

"I want to thank Roche, a great company, for their incredible work. I'd also like to thank Thermo Fisher," he said.

Roche Diagnostics Corporation President and CEO Matthew Sause speaks at the March 13 news conference. Roche and Thermo Fisher Scientific said they distributed millions of tests to labs, but that didn't increase testing because the U.S. lags behind in sample collection kits.Chip Somodevilla/Getty Images

Trump noted that the FDA was approving their processes, and then made a prediction: "It'll go very quickly," he said. "It's going very quickly — which will bring, additionally, 1.4 million tests on board next week and 5 million within a month. I doubt we'll need anywhere near that."

Roche and Thermo Fisher Scientific said they were able to get millions of tests distributed on schedule to labs in the United States, one of the rare bright spots in the coronavirus crisis. These tests are what are used at labs to check whether samples contain the coronavirus.

But those tests were not the primary reason for inadequate testing. The United States lags behind in sample collection kits — the swabs and tubes that frontline medical workers send to labs.

And those labs themselves struggled with processing capacity.

In the days before the March 13 Rose Garden address, leaders of diagnostic testing labs like LabCorp and Quest went to the White House with three core requests. And during the Rose Garden address, the CEOs of those two organizations stood with the president as the coronavirus task force pledged to wield government resources for their partnership.

More than a month later, the diagnostic testing labs — and the group that represents them in Washington, the American Clinical Laboratory Association — still have those three requests: government funds to build new testing facilities, national standards to prioritize who gets tested, and government support for the supply chain.

President Trump leaves the Rose Garden after the March 13 news conference about the ongoing coronavirus pandemic. Few of the promises made at the conference have been fulfilled.Chip Somodevilla/Getty Images

Konyndyk said it was an indication that the public-private partnerships the president touted on March 13 were a one-way street.

"What you want to have is a constructive partnership between the federal government and the private sector. Instead, what we see, I think, is a game of 'not it,'" said Konyndyk, who served in the Obama administration at USAID, leading the government response to international disasters.

Although the federal government needs the help of the private sector, the federal government has only limited power over those companies. So to make things work, there needs to be close cooperation and advanced negotiation before announcing what companies will do, and that didn't happen, Konyndyk said.

Private companies did part of what was promised in the Rose Garden address — there is more testing today than a month ago.

But by over-promising what private sector companies would do — and in some cases, without adequate consultation about what they could do — the White House left other pledges that day unfulfilled.

White House Seeks To Lower Farmworker Pay To Help Agriculture Industry

April 10, 2020

FRANCO ORDOÑEZ

Work continues at a winery in Clarksburg, Calif., last month. Farms are operating as essential businesses amid the coronavirus pandemic.Rich Pedroncelli/AP

New White House Chief of Staff Mark Meadows is working with Agriculture Secretary Sonny Perdue to see how to reduce wage rates for foreign guest workers on American farms, in order to help U.S. farmers struggling during the coronavirus, according to U.S. officials and sources familiar with the plans.

Opponents of the plan argue it will hurt vulnerable workers and depress domestic wages.

The measure is the latest effort being pushed by the U.S. Department of Agriculture to help U.S farmers who say they are struggling amid disruptions in the agricultural supply chain compounded by the outbreak; the industry was already hurting because of President Trump's tariff war with China.

"The administration is considering all policy options during this unprecedented crisis to ensure our great farmers are protected, and President Trump has done and will do everything he can to support their vital mission," a White House official told NPR.

The nation's roughly 2.5 million agricultural laborers have been officially declared "essential workers" as the administration seeks to ensure that Americans have food to eat and that U.S. grocery stores remain stocked. Workers on the H-2A seasonal guest-worker program are about 10% of all farmworkers.


The effort to provide "wage relief" to U.S. farmers follows an announcement Friday by the USDA to develop a program that will include direct payments to farmers and ranchers hurt by the coronavirus. Trump said Friday that he has directed Perdue to provide at least $16 billion in relief.

Last month, the U.S. State Department said it will start processing more applicants seeking H-2A temporary guest worker visas to ensure U.S. farmers have foreign workers in time for spring planting.

The most recent push to lower wage rates for workers on H-2A visas has drawn pushback from some strange bedfellows: immigrant-rights advocates and immigration hard-liners usually aligned with Trump.

Erik Nicholson, national vice president for the United Farm Workers, says people who have worked in agriculture for decades are concerned they are going to lose their jobs. And he said vulnerable guest workers are not being provided proper hand-washing facilities and still being forced to live in cramped housing.

"So in the middle of a pandemic, rather than trying to figure out the cheap way to do things, we need to make sure we live up to the expectations society has of us as an industry to keep the food flowing," Nicholson said.

Groups on the right fear Trump is succumbing to the will of the agriculture lobby that is demanding lower wages for foreign and domestic farmworkers at a time of record high unemployment in the United States.
The Department of Labor reports that 16.6 million Americans have filed for unemployment aid in the past few weeks.

"President Trump should see right through what the agriculture lobby is demanding in the name of 'food security' at the height of a health crisis – lower wages for American workers and more cheap foreign labor," said Dan Stein of the Federation for American Immigration Reform, which supports immigration restrictions. "These appalling demands underscore that the whole way this nation produces food should be reexamined."

It's unclear how the reforms would be made, including whether they would be taken through executive action or through the federal regulatory process. But Perdue has pushed for adjusting what is known as the adverse effect wage rate, which prevents farmers using the H-2A program from paying all workers — U.S. and guest workers — wages below the prevailing rates in the surrounding area.
Earlier this year, Perdue said the adverse wage rate has set almost a $15 minimum wage for agriculture, noting "no other business in the country has that," according to the agriculture trade journal DTN.

The "adverse effect wage rates" are based on a USDA survey of what agricultural workers are paid in each state. It's $11.71 in Florida, $12.67 in North Carolina and $14.77 in California.

A USDA official told NPR that Perdue is working with Trump to "resolve long-standing challenges facing the agriculture industry, including reforms to the H-2A program.

"These challenges have been exacerbated by these uncertain times," the official said in a statement.

U.S. farmers say they have had to cut back on production because of the high number of restaurant and hotel closures. Cory Lunde, a spokesman for the Western Growers Association, said U.S. farmers are fighting to keep "our farms afloat in the face of the near-total collapse of the food-service sector" and more recent slowdown in the retail market.

Lee Wicker, deputy director of the North Carolina Growers Association, said Trump administration officials are trying to look at ways to help because "they understand that we're in trouble and they want to secure the food supply for the American people."

"When a farmer goes out of business, you know, he doesn't come back," Wicker said. "Food supply is a national security issue and, as bad as this COVID-19 crisis is, perhaps it can be a catalyst to start a conversation about our agriculture policy and having sustainable agriculture and diversity."

Scientists Program CRISPR to Fight Viruses in Human Cells

A common gene-editing enzyme could be used to disable RNA viruses such as flu or Ebola

By Tanya Lewis on October 23, 2019

Researchers modified the enzyme Cas13 to target and inactivate viruses such as influenza (shown here). Credit: Kateryna Kon Getty Images

CRISPR is usually thought of as a laboratory tool to edit DNA in order to fix genetic defects or enhance certain traits—but the mechanism originally evolved in bacteria as a way to fend off viruses called bacteriophages. Now scientists have found a way to adapt this ability to fight viruses in human cells.

In a recent study, Catherine Freije, Cameron Myhrvold and Pardis Sabeti at the Broad Institute of the Massachusetts Institute of Technology and Harvard University, and their colleagues programmed a CRISPR-related enzyme to target three different single-stranded RNA viruses in human embryonic kidney cells (as well as human lung cancer cells and dog kidney cells) grown in vitro and chop them up, rendering them largely unable to infect additional cells. If further experiments show this process works in living animals, it could eventually lead to new antiviral therapies for diseases such as Ebola or Zika in humans.

Viruses come in many forms, including DNA and RNA, double-stranded and single-stranded. About two thirds of the ones that infect humans are RNA viruses, and many have no approved treatment. Existing therapies often use a small molecule that interferes with viral replication—but this approach does not work for newly emerging viruses or ones that are evolving rapidly.

“CRISPR” refers to a series of DNA sequences in bacterial genomes that were left behind from previous bacteriophage infections. When the bacteria encounter these pathogens again, enzymes called CRISPR-associated (Cas) proteins recognize and bind to these sequences in the virus and destroy them. In recent years, researchers (including study co-author Feng Zhang) have reengineered one such enzyme, called Cas9, to cut and paste DNA in human cells. The enzyme binds to a short genetic tag called a guide RNA, which directs the enzyme to a particular part of the genome to make cuts. Previous studies have used Cas9 to prevent replication of double-stranded DNA viruses or of single-stranded RNA viruses that produce DNA in an intermediate step during replication. Only a small fraction of RNA viruses that infect humans produce such DNA intermediates—but another CRISPR enzyme, called Cas13, can be programmed to cleave single-stranded RNA viruses.

“The nice thing about CRISPR systems and systems like Cas13 is that their initial purpose in bacteria was to defend against viral infection of bacteria, and so we sort of wanted to bring Cas13 back to its original function—and apply this to mammalian viruses in mammalian cells,” says Freije, who is a doctoral student in virology at Harvard. “Because CRISPR systems rely on guide RNAs to specifically guide the CRISPR protein to a target, we saw this as a great opportunity to use it as a programmable antiviral.”

Freije and her colleagues programmed Cas13 to target three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV) and vesicular stomatitis virus (VSV). LCMV is an RNA virus that mostly infects mice—but it is in the same family as the virus that causes Lassa fever, which is found in West Africa and is much more dangerous to study in the lab. IAV is a flu virus; although some antiviral medications for flu already exist, such viruses evolve rapidly, so there is a need for better options. Finally, VSV is a model for many other single-stranded RNA viruses.

To determine how effective Cas13 was at destroying the viruses, the researchers also used it as a diagnostic tool to see how much viral RNA was being released from infected cells. They saw a twofold to 44-fold reduction in RNA, depending on which virus they were looking at and the time point. They also looked at how well the released RNA was able to go on and infect new cells. In most cases, they saw a 100-fold reduction in infectivity—and in some cases, more than 300-fold—according to Freije. The findings were published online on October 10 in Molecular Cell.

“The results are very impressive,” says Chen Liang, a professor at the Lady Davis Institute at Jewish General Hospital and the department of microbiology and immunology at McGill University in Montreal, who was not involved in the study. His own laboratory has used the Cas9 enzyme to deactivate DNA viruses. The concept is very similar, but Cas13 has a few advantages, he says. For one, Cas13 can be used to target one virus using several guide RNAs, making it difficult for the virus to “escape.” Secondly, the new study also used Cas13 to detect how much viral RNA was left over to infect cells. The amount of viral knockdown the group achieved is “very significant,” Liang says. “If you can target and inactivate all three [of these] viruses, in principle, you can inactivate any virus.

As with any approach, there are limitations. One is the question of how to deliver the Cas13 to target a virus in a living person, Liang notes, and the researchers have not yet done any animal studies. Another is the fact that viruses will eventually develop resistance. But Cas13 has an advantage here: when Cas9 cuts viral DNA, mammalian cells repair it and can cause mutations that make the virus more resistant. Yet with Cas13, these cells do not have the mechanism to repair the RNA and introduce errors that would help the virus escape being destroyed. Even if a virus does evolve resistance, or if a new virus is encountered, the method could be quickly adapted.

“One of the things that’s most exciting about this approach is the programmability,” says Myhrvold, a postdoctoral fellow at Harvard. “Once you figure out how to do this well for one virus it’s not that hard to design sequences against another virus—or another one. Furthermore, if the virus changes its own sequence—as viruses are known to do, just during an outbreak or in response to therapy—you can very easily update the CRISPR RNA sequence and keep up with the virus.”

Freije agrees. “We are definitely excited about future prospects of optimizing the system and trying it out in mouse models,” she says. Beyond therapeutics, the team hopes to understand more about how viruses operate—how they replicate and what parts of their genomes are most important. Using approaches like this, “you can really start to get a better picture of what parts of these viruses are and, most importantly, what really makes them tick.”

How do bacteria defend themselves against viruses?

The CRISPR-Cas system in some bacteria helps to form an effective barrier to invading viruses.

DIGEST Apr 3, 2019



A transmission electron microscopy image of bacteriophages taken at The University of Alabama’s Optical Analysis Facility. Image credit: Chou-Zheng and Hatoum-Aslan, 2019 (CC BY 4.0)

Just as humans are susceptible to viruses, bacteria have their own viruses to contend with. These viruses – known as phages – attach to the surface of bacterial cells, inject their genetic material, and use the cells’ enzymes to multiply while destroying their hosts.

To defend against a phage attack, bacteria have evolved a variety of immune systems. For example, when a bacterium with an immune system known as CRISPR-Cas encounters a phage, the system creates a ‘memory’ of the invader by capturing a small snippet of the phage’s genetic material. The pieces of phage DNA are copied into small molecules known as CRISPR RNAs, which then combine with one or more Cas proteins to form a group called a Cas complex. This complex patrols the inside of the cell, carrying the CRISPR RNA for comparison, similar to the way a detective uses a fingerprint to identify a criminal. Once a match is found, the Cas proteins chop up the invading genetic material and destroy the phage.

There are several different types of CRISPR-Cas systems. Type III systems are among the most widespread in nature and are unique in that they provide a nearly impenetrable barrier to phages attempting to infect bacterial cells. Medical researchers are exploring the use of phages as alternatives to conventional antibiotics and so it is important to find ways to overcome these immune responses in bacteria. However, it remains unclear precisely how Type III CRISPR-Cas systems are able to mount such an effective defense.

Chou-Zheng and Hatoum-Aslan used genetic and biochemical approaches to study the Type III CRISPR-Cas system in a bacterium called Staphylococcus epidermidis. The experiments showed that two enzymes called PNPase and RNase J2 played crucial roles in the defense response triggered by the system. PNPase helped to generate CRISPR RNAs and both enzymes were required to help to destroy genetic material from invading phages.

Previous studies have shown that PNPase and RNase J2 are part of a machine in bacterial cells that usually degrades damaged genetic material. Therefore, these findings show that the Type III CRISPR-Cas system in S. epidermidis has evolved to coordinate with another pathway to help the bacteria survive attack from phages. CRISPR-Cas immune systems have formed the basis for a variety of technologies that continue to revolutionize genetics and biomedical research. Therefore, along with aiding the search for alternatives to antibiotics, this work may potentially inspire the development of new genetic technologies in the future.

Phage-Encoded Anti-CRISPR Defenses
Annual Review of Genetics

Vol. 52:445-464 (Volume publication date November 2018)
First published as a Review in Advance on September 12, 2018
https://doi.org/10.1146/annurev-genet-120417-031321

Sabrina Y. Stanley1 and Karen L. Maxwell2
1Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
2Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1M1, Canada; email: karen.maxwell@utoronto.ca

Abstract

The battle for survival between bacteria and bacteriophages (phages) is an arms race where bacteria develop defenses to protect themselves from phages and phages evolve counterstrategies to bypass these defenses. CRISPR-Cas adaptive immune systems represent a widespread mechanism by which bacteria protect themselves from phage infection. In response to CRISPR-Cas, phages have evolved protein inhibitors known as anti-CRISPRs. Here, we describe the discovery and mechanisms of action of anti-CRISPR proteins. We discuss the potential impact of anti-CRISPRs on bacterial evolution, speculate on their evolutionary origins, and contemplate the possible next steps in the CRISPR-Cas evolutionary arms race. We also touch on the impact of anti-CRISPRs on the development of CRISPR-Cas-based biotechnological tools.

FULL ARTICLE HERE



Biological Sciences
RESEARCH ARTICLE
Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria
Ido Yosef, Miriam Manor, Ruth Kiro, and View ORCID Profile Udi Qimron
PNAS June 9, 2015 112 (23) 7267-7272; first published May 18, 2015 https://doi.org/10.1073/pnas.1500107112

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved April 28, 2015 (received for review January 25, 2015)

Significance

Antibiotic resistance of pathogens is a growing concern to human health, reviving interest in phage therapy. This therapy uses phages (natural bacterial enemies) to kill pathogens. However, it encounters many obstacles such as delivery barriers into the tissues and bacterial resistance to phages. Here, we use phages for delivering a programmable DNA nuclease, clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas), to reverse antibiotic resistance and eliminate the transfer of resistance between strains. This approach combines CRISPR-Cas delivery with lytic phage selection of antibiotic-sensitized bacteria. The strategy may reduce the prevalence of antibiotic-resistant bacteria in treated surfaces and on skin of medical personnel, as it uses phages in a unique way that overcomes many of the hurdles encountered by phage therapy.

Abstract

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.

DOWNLOAD WHOLE PAPER AS PDF FROM HERE 

   Genetically Engineered Phages: a Review of Advances over the Last Decade

Diana P. Pires, Sara Cleto, Sanna Sillankorva, Joana Azeredo, Timothy K. Lu

DOI: 10.1128/MMBR.00069-15

 

SUMMARY

Soon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.

INTRODUCTION

Bacteriophages (phages) are among the most abundant biological particles on earth. They are also highly versatile and adaptable to a great number of applications. Phages are viruses that infect bacteria; their self-replication depends on access to a bacterial host. Phages were discovered independently by Frederick Twort in 1915 (1) and by Félix d'Hérelle in 1917 (2), and they were used early on as antimicrobial agents. Although the initial results of phage therapy were promising (3, 4), poorly controlled trials and inconsistent results generated controversy within the scientific community about the efficacy and reproducibility of using phages to treat bacterial infections (5–7). The discovery of penicillin in 1928 and the subsequent arrival of the antibiotic era further cast a shadow on phage therapy (5, 6). As a result, phage therapy was discontinued in Western countries, even as its use continued in Eastern Europe and the former Soviet Union (8–10).

Despite the important success of antibiotics in improving human health, antibiotic resistance has emerged with steadily increasing frequency, rendering many antibiotics ineffective (11–14). Multidrug-resistant bacteria currently constitute one of the most widespread global public health concerns (15–17). More than 2 million people are sickened every year in the United States alone as a result of antibiotic-resistant infections, resulting in at least 23,000 deaths per year (16). The rising tide of antibiotic resistance coupled with the low rate of antibiotic discovery (17, 18) has revived interest in phages as antibacterial agents (19–21).

Unlike most antibiotics, phages are typically highly specific for a particular set of bacterial species or strains and are thus expected to have fewer off-target effects on commensal microflora than antibiotics do (22). The self-replicating nature of phages and the availability of simple, rapid, and low-cost phage production processes are additional advantages for their use as antimicrobials (22). Phages have been used not only to treat and prevent human bacterial infections (9, 23–25) but also to control plant diseases (26–29), detect pathogens (30–33), and assess food safety (34–37).

READ/DOWNLOAD THE ARTICLE 

REVIEW ARTICLE
Microbiol., 03 May 2019 | https://doi.org/10.3389/fmicb.2019.00954

Genetic Engineering of Bacteriophages Against Infectious Diseases

Yibao Chen1,2, Himanshu Batra3, Junhua Dong1,2, Cen Chen1,2, Venigalla B. Rao3 and Pan Tao1,2,3*

Bacteriophages (phages) are the most abundant and widely distributed organisms on Earth, constituting a virtually unlimited resource to explore the development of biomedical therapies. The therapeutic use of phages to treat bacterial infections (“phage therapy”) was conceived by Felix d’Herelle nearly a century ago. However, its power has been realized only recently, largely due to the emergence of multi-antibiotic resistant bacterial pathogens. Progress in technologies, such as high-throughput sequencing, genome editing, and synthetic biology, further opened doors to explore this vast treasure trove. Here, we review some of the emerging themes on the use of phages against infectious diseases. In addition to phage therapy, phages have also been developed as vaccine platforms to deliver antigens as part of virus-like nanoparticles that can stimulate immune responses and prevent pathogen infections. Phage engineering promises to generate phage variants with unique properties for prophylactic and therapeutic applications. These approaches have created momentum to accelerate basic as well as translational phage research and potential development of therapeutics in the near future.

Introduction

Bacteriophages (phages), discovered in the early 20th century independently by Frederick Twort and Felix d’Herelle, are the most abundant organisms on earth with up to 2.5 × 108 phages per ml in natural waters (Bergh et al., 1989). It is well accepted that phages specifically infect bacteria and, therefore, were considered for the development of natural approaches to treat bacterial infections since their discovery (Wittebole et al., 2014; Salmond and Fineran, 2015). However, due to the discovery of antibiotics that provided greater breadth and potency, phage therapy lagged behind although research continued in some Eastern European countries (Chanishvili, 2012, 2016; Wittebole et al., 2014). Therefore, in the following several decades, phages were mainly used as model organisms to explore the basic mechanisms of life and led to the birth of modern molecular biology. One classical example is the demonstration of a central biological question in the early 20th century, the nature of a gene, by “Hershey-Chase experiment” (also called “Waring blender experiment”) (Salmond and Fineran, 2015). This elegant experiment demonstrated that DNA, not protein, is the genetic material of T2 phage.

Recently, the emergence of multi-antibiotic resistant bacterial pathogens and the low rate of new antibiotic discovery brought new urgency to develop phage-based therapies (Lu and Koeris, 2011; Viertel et al., 2014; Domingo-Calap and Delgado-Martinez, 2018). A striking example is the recent San Diego patient who was infected by multi-drug resistant Acinetobacter baumannii stain during travelling to Egypt. The patient went into a coma for nearly 2 months but awoke 2 days after intravenous injection of a phage cocktail that lyses this bacterium and finally completely recovered (Schooley et al., 2017). With recent advances, particularly the genome engineering (Martel and Moineau, 2014; Ando et al., 2015; Lemay et al., 2017; Tao et al., 2017b; Kilcher et al., 2018), the applications of phages have greatly expanded. In addition to its use in antibacterial therapy, phages were used in synthetic biology (Lemire et al., 2018), material science (Cao et al., 2016), and biomedical fields (Cao et al., 2018; Tao et al., 2018c). Considering the abundance and diversity, there is vast potential to engineer phages for different applications. In this review, we will focus on the applications of phages in infectious disease, in particular, vaccine development and phage therapy. We will discuss the phage engineering strategies and how these can equip the phages with the ability to advance the vaccine and phage therapy fields.

1College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
2The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, China
3Department of Biology, The Catholic University of America, Washington, DC, United States

READ / DOWNLOAD THE REST OF THE ARTICLE HERE 

Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides
Yan-ru Cui,
Shao-jie Wang,
Jun Chen,
Jie Li,
Wenzhang Chen,
Shuyue Wang,
Bing Meng,
Wei Zhu,
Zhuhong Zhang,
Bei Yang,
Biao Jiang,
Guang Yang,
Peixiang Ma &
Jia Liu 

Genome Biology volume 21, Article number: 51 (2020) Cite this article
Research
Open Access
Published: 26 February 2020

Abstract

Background

CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.

ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.

Conclusion

G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

READ/DOWNLOAD HERE

Heterogeneous Diversity of Spacers within CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
Article (PDF Available) in Physical Review Letters 105(12):128102 · September 2010 with 231
Download full-text PDF

DOI: 10.1103/PhysRevLett.105.128102 · Source: PubMedCite this publication

Jiankui he
Southern University of Science and Technology


Michael Deem
Rice University

Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) in bacterial and archaeal DNA have recently been shown to be a new type of antiviral immune system in these organisms. We here study the diversity of spacers in CRISPR under selective pressure. We propose a population dynamics model that explains the biological observation that the leader-proximal end of CRISPR is more diversified and the leader-distal end of CRISPR is more conserved. This result is shown to be in agreement with recent experiments. Our results show that the CRISPR spacer structure is influenced by and provides a record of the viral challenges that bacteria face.
https://www.researchgate.net/publication/46424214_Heterogeneous_Diversity_of_Spacers_within_CRISPR_Clustered_Regularly_Interspaced_Short_Palindromic_Repeats

Volume 366
Issue 9
May 2019

Article Contents
ABSTRACT
INTRODUCTION
BIOLOGICAL RELEVANCE OF ANTI-CRISPR PROTEINS
MECHANISMS AND STRUCTURES OF ANTI-CRISPR PROTEINS
APPLICATIONS OF ANTI-CRISPR PROTEINS
OUTLOOK
FUNDING
REFERENCES


MINI REVIEW

Keeping CRISPR in check: diverse mechanisms of phage-encoded anti-CRISPRS 
Despoina Trasanidou, Ana Sousa Gerós, Prarthana Mohanraju, Anna Cornelia Nieuwenweg, Franklin L Nobrega, Raymond H J Staals

FEMS Microbiology Letters, Volume 366, Issue 9, May 2019, fnz098, https://doi.org/10.1093/femsle/fnz098

Published: 11 May 2019

ABSTRACT

CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.

READ/DOWNLOAD

The physicist's guide to one of biotechnology's hottest new topics: CRISPR-Cas

Melia E Bonomo1,3 and Michael W Deem1,2,3,4

Published 30 April 2018 • © 2018 IOP Publishing Ltd
Physical Biology, Volume 15, Number 4

DownloadArticle PDF

Article information

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute a multi-functional, constantly evolving immune system in bacteria and archaea cells. A heritable, molecular memory is generated of phage, plasmids, or other mobile genetic elements that attempt to attack the cell. This memory is used to recognize and interfere with subsequent invasions from the same genetic elements. This versatile prokaryotic tool has also been used to advance applications in biotechnology. Here we review a large body of CRISPR-Cas research to explore themes of evolution and selection, population dynamics, horizontal gene transfer, specific and cross-reactive interactions, cost and regulation, non-immunological CRISPR functions that boost host cell robustness, as well as applicable mechanisms for efficient and specific genetic engineering. We offer future directions that can be addressed by the physics community. Physical understanding of the CRISPR-Cas system will advance uses in biotechnology, such as developing cell lines and animal models, cell labeling and information storage, combatting antibiotic resistance, and human therapeutics.

Export citation and abstract BibTeX RIS



Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.


1. Introduction


In 1987, Ishino and colleagues had set out to identify the encoded protein and primary structure of a particular gene in Escherichia coli by analyzing its chromosomal DNA segment and flanking regions [1]. They found an interesting sequence structure at the gene's 3'-end flanking region, in which five homologous sequences of 29 nucleotides were arranged as direct repeats with 32-nucleotide sequences spaced between them. Little did they know that their discovery would prove to have critical immunological significance. It was not until 2000 that these mysterious repeated genomic elements were revisited when Mojica and colleagues searched the available microbial genome database and found many organisms that contained partially palindromic sequences of 24–40 basepairs with 20–58 basepair sequences spaced between them [2]. These were found in almost all archaea, about half of bacteria, no viruses, and no eukaryotes. Related and unrelated species had nearly identical structure in these repeat sequence units. The sequences in between, called 'spacers', were unique to an individual locus and were not found in other genomes [3]. After many suggested abbreviations, including SRSRs, short regularly spaced repeats, and SPIDR, spacers interspersed direct repeats, the scientific community settled on calling these elements clustered regularly interspaced short palindromic repeats, or CRISPR.

SEE 
https://plawiuk.blogspot.com/search?q=BACTERIOPHAGES

https://plawiuk.blogspot.com/search?q=PHAGES

From Pleistocene to trophic rewilding: A wolf in sheep’s clothing


Dustin R. Rubenstein and Daniel I. Rubenstein

PNAS January 5, 2016 https://doi.org/10.1073/pnas.1521757113

This Letter has a Reply and related content. Please see:
Reply to Rubenstein and Rubenstein: Time to move on from ideological debates on rewilding - December 16, 2015
Science for a wilder Anthropocene: Synthesis and future directions for trophic rewilding research - October 26, 2015

Nearly 10 y ago, we (1) critiqued the idea of Pleistocene rewilding (2), a misguided attempt to resurrect bygone ecosystems. Much has happened to the Earth’s biodiversity over the decade since the term “Pleistocene rewilding” was coined, most of it bad. More than half a billion people have been added to the world’s population, and ecosystems continue to be degraded at an alarming rate. A sixth mass extinction is underway, and poaching of megafauna has increased across sub-Saharan Africa. Unfortunately, one thing that has not happened is any serious attempt to scientifically study Pleistocene rewilding. Despite a number of publicized Pleistocene rewilding projects (Oostvaardersplassen in The Netherlands and Pleistocene Park in Siberia), we have yet to see any quantitative data concerning the impacts of megafauna reintroductions.

Svenning et al. (3) again revive the Pleistocene rewilding debate. No longer calling it Pleistocene rewilding, they repackage the sensational into something more palatable: “trophic rewilding.” “Trophic” refers to a “trophic cascade”—when the removal of a top predator or herbivore has indirect and cascading effects on lower tropic levels. Over the past decade, there has been much scientific study of trophic cascades, including those created by the removal of megafauna. However, even these studies recognize that ecosystems are no longer pristine, especially those harboring large mammals. Today’s reality is that wildlife and people must coexist. Setting aside large tracts to bring back communities of disrupted cascades is a luxury.

As the metaanalysis of Svenning et al. (3) shows, rewilding—especially when trophic cascades are reinstated—can alter ecosystem function, often for the better, even if the mechanism is incompletely understood. However, using proxy species when mechanisms are uncertain to recreate ancient ecosystems could have many unintended consequences (1). Simply repackaging Pleistocene rewilding as trophic rewilding does nothing to change this fact. Without good science, such large-scale reintroductions could be as untested as dumping iron into the sea, or placing particles in the sky to attenuate the effects of climate change. We cannot afford to co-opt good science (research on trophic cascades) to justify bad science (Pleistocene rewilding) at a time when species are in peril.

There is no doubt that today’s ecosystems are different from those of 10,000 y ago. However, they are also quite different from the ecosystems of just 10 y ago, when rhinoceros and elephant poaching in Africa seemed under control. In another 10 y, there may be no rhinoceros left. Rather than continuing to promote the sensational by repackaging a failed conservation strategy in shiny new clothing, we should direct our efforts toward preserving the ecosystems and wildlife that remain. We should focus on ways to feed the millions of new mouths appearing each year without destroying more biodiversity (4). We should stop talking about trophic or Pleistocene rewilding, or its next rebadging. We were criticized (5) for drawing the analogy to Jurasssic Park (1). However, Svenning et al. (3) argue for “a framework for integrating synthetic biology and trophic rewilding science.” It is time to be practical, not sensational. It is time to move on.

Footnotes
↵1To whom correspondence should be addressed. Email: dr2497@columbia.edu.


Author contributions: D.R.R. and D.I.R. wrote the paper.


The authors declare no conflict of interest, though they are related.

References
↵

Rubenstein DR,
Rubenstein DI,
Sherman PW,
Gavin TA

(2006) Pleistocene park: Does re-wilding North America represent sound conservation for the 21st century? Biol Conserv 132:232–238

CrossRefGoogle Scholar
↵

Donlan J, et al.

(2005) Re-wilding North America. Nature 436(7053):913–914

.


CrossRefPubMedGoogle Scholar
↵

Svenning J-C, et al.

(2015) Science for a wilder Anthropocene: Synthesis and future directions for trophic rewilding research. Proc Natl Acad Sci USA doi:10.1073/pnas.1502556112

.


Abstract/FREE Full TextGoogle Scholar
↵

Searchinger TD, et al.

(2015) High carbon and biodiversity costs from converting Africa’s wet savannahs to cropland. Nat Clim Change 5:481–486

.


CrossRefGoogle Scholar
↵

Donlan CJ

(2007) Restoring America’s big, wild animals. Sci Am 296(6):70–77

.


PubMedGoogle Scholar

Elaborately decorated eggs predate Easter by thousands of years

JONONMAC46 (CC BY-SA 3.0)

By Michael Price Apr. 8, 2020

If you wanted to make an impression on a high-ranking Bronze or Iron Age chieftain, mere jewelry or gems wouldn’t cut it. Instead, you’d present them with an egg—an elaborately carved and embellished ostrich eggshell, to be exact. Such oologic offerings have been found inside the tombs of Mediterranean and Middle Eastern elites who lived from about 2500 to 500 B.C.E., equally thrilling and perplexing archaeologists. Who made them, and how did they wind up in the hands of ancient nobility?

To crack the case, a team of archaeologists and museum curators took a closer look at decorated eggshells in the collection of the British Museum, which includes five prized eggs in outstanding condition. The intact eggs were all discovered in a burial site known as the Isis Tomb in Vulci, Italy, that was uncovered in 1839 by Napoleon Bonaparte’s brother, Prince Lucien. The tomb dates to about 600 B.C.E. and was filled with other luxury items, including gold jewelry and bronze dinnerware. All five of the ostrich eggs were painted, and four were engraved with repeating geometric patterns (as seen above), animal motifs, and chariots and soldiers.

On other, fragmented pieces found in about a dozen other burial sites around the Mediterranean and Middle East, the researchers used stable isotope analysis—a technique that matches chemical markers in bones and teeth to specific regions—to trace the eggs’ origins. Researchers already suspected they were made by Assyrian and Phoenician craftworkers, and the isotope analysis bore that out. But they found that even within the same tomb, eggshells came from several different regions, indicating a more complex supply chain than previously thought, the researchers report today in Antiquity. A scanning electron microscope also revealed the engravers used a multitude of tools and techniques, underlining the intense effort and skill that went into making these ovular ornaments.

---30---