Researchers at Penn State developed and used the method to create complex multi-sectioned compartments and compartments within compartments, all without any physical dividers or membranes. Their results are published in a paper that appears online in the journal Biomacromolecules.

“A cell has a variety of compartments or containers, like the nucleus and mitochondria, with membranes that separate their contents from the rest of the cell,” said Gregory Mountain, graduate student at Penn State and an author of the study. “These compartments help the cell perform different tasks in different locations. But researchers are continuing to discover compartments within the cell that lack physical outer membranes and that can form and then dissolve as needed. Disruption of these membraneless compartments has been implicated in diseases such as ALS, Alzheimer’s, and Type II Diabetes.”

Membraneless compartments within cells result from the separation of molecules like proteins and RNA into different liquid phases. To create membraneless compartments in the lab, the researchers combined simplified charged polymers of repeating amino acids and/or nucleic acids in water. The polymers were fluorescently labeled, which allowed the researchers to monitor the resulting interactions.

As the charged polymers interacted, with opposite charges attracting, separate compartments formed in the liquid with no physical dividers. Combining four polymers allowed the researchers to produce droplets with two compartments as well as compartments-within-compartments. Combining six polymers produced droplets with three compartments.

“This straightforward method allows us to understand the basic chemistry of how multi-phase compartments could form and dissolve within a cell,” said Mountain. “Then we can build up the complexity. Factors like temperature, salt concentration, acidity, and charge strength of the polymers can affect how these compartments behave, so it is important to first understand the common conditions under which their formation is possible.”

To understand how molecules might accumulate within compartments, the researchers added macromolecule probes. They found that the probes tended to accumulate within compartments that contained polymers having the strongest charge-based interactions with the probes.

“Membraneless compartments might form within a cell to perform a specific task using specific components,” said Mountain. “But it’s unclear how the cell controls what components go where and why some molecules end up in one compartment versus another. By identifying the properties that determine where probe molecules end up within multi-phase compartments, we can start to understand how cells might prepare for chemical reactions.”

Next, the researchers are exploring how to selectively dissolve and reform individual compartments using a variety of control mechanisms. For example, manipulating one of the charged polymers within a compartment to make it neutral could cause that compartment to selectively dissolve within a multi-compartment system.

“Eventually we hope to control when compartments appear and what types of molecules they contain.” said Mountain. “We also hope to create systems with multiple kinds of membraneless compartments that can be individually controlled. Our work provides the groundwork for an experimental model of multi-compartment membraneless organelles within living cells and may one day also allow us to create artificial cells with multi-phase compartments.”

In addition to Mountain the research team includes Christine Keating, distinguished professor of chemistry at Penn State. This work was supported by the National Science Foundation.

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The analysis of public genome sequence data from SARS-CoV-2 and related viruses found no evidence that the virus was made in a laboratory or otherwise engineered.

“By comparing the available genome sequence data for known coronavirus strains, we can firmly determine that SARS-CoV-2 originated through natural processes,” said Kristian Andersen, PhD, an associate professor of immunology and microbiology at Scripps Research and corresponding author on the paper.

In addition to Andersen, authors on the paper, “The proximal origin of SARS-CoV-2,” include Robert F. Garry, of Tulane University; Edward Holmes, of the University of Sydney; Andrew Rambaut, of University of Edinburgh; W. Ian Lipkin, of Columbia University.

Coronaviruses are a large family of viruses that can cause illnesses ranging widely in severity. The first known severe illness caused by a coronavirus emerged with the 2003 Severe Acute Respiratory Syndrome (SARS) epidemic in China. A second outbreak of severe illness began in 2012 in Saudi Arabia with the Middle East Respiratory Syndrome (MERS).

On December 31 of last year, Chinese authorities alerted the World Health Organization of an outbreak of a novel strain of coronavirus causing severe illness, which was subsequently named SARS-CoV-2. As of February 20, 2020, nearly 167,500 COVID-19 cases have been documented, although many more mild cases have likely gone undiagnosed. The virus has killed over 6,600 people.

Shortly after the epidemic began, Chinese scientists sequenced the genome of SARS-CoV-2 and made the data available to researchers worldwide. The resulting genomic sequence data has shown that Chinese authorities rapidly detected the epidemic and that the number of COVID-19 cases have been increasing because of human to human transmission after a single introduction into the human population. Andersen and collaborators at several other research institutions used this sequencing data to explore the origins and evolution of SARS-CoV-2 by focusing in on several tell-tale features of the virus.

The scientists analyzed the genetic template for spike proteins, armatures on the outside of the virus that it uses to grab and penetrate the outer walls of human and animal cells. More specifically, they focused on two important features of the spike protein: the receptor-binding domain (RBD), a kind of grappling hook that grips onto host cells, and the cleavage site, a molecular can opener that allows the virus to crack open and enter host cells.

Evidence for natural evolution

The scientists found that the RBD portion of the SARS-CoV-2 spike proteins had evolved to effectively target a molecular feature on the outside of human cells called ACE2, a receptor involved in regulating blood pressure. The SARS-CoV-2 spike protein was so effective at binding the human cells, in fact, that the scientists concluded it was the result of natural selection and not the product of genetic engineering.

This evidence for natural evolution was supported by data on SARS-CoV-2’s backbone — its overall molecular structure. If someone were seeking to engineer a new coronavirus as a pathogen, they would have constructed it from the backbone of a virus known to cause illness. But the scientists found that the SARS-CoV-2 backbone differed substantially from those of already known coronaviruses and mostly resembled related viruses found in bats and pangolins.

“These two features of the virus, the mutations in the RBD portion of the spike protein and its distinct backbone, rules out laboratory manipulation as a potential origin for SARS-CoV-2” said Andersen.

Josie Golding, PhD, epidemics lead at UK-based Wellcome Trust, said the findings by Andersen and his colleagues are “crucially important to bring an evidence-based view to the rumors that have been circulating about the origins of the virus (SARS-CoV-2) causing COVID-19.”

“They conclude that the virus is the product of natural evolution,” Goulding adds, “ending any speculation about deliberate genetic engineering.”

Possible origins of the virus

Based on their genomic sequencing analysis, Andersen and his collaborators concluded that the most likely origins for SARS-CoV-2 followed one of two possible scenarios.

In one scenario, the virus evolved to its current pathogenic state through natural selection in a non-human host and then jumped to humans. This is how previous coronavirus outbreaks have emerged, with humans contracting the virus after direct exposure to civets (SARS) and camels (MERS). The researchers proposed bats as the most likely reservoir for SARS-CoV-2 as it is very similar to a bat coronavirus. There are no documented cases of direct bat-human transmission, however, suggesting that an intermediate host was likely involved between bats and humans.

In this scenario, both of the distinctive features of SARS-CoV-2’s spike protein — the RBD portion that binds to cells and the cleavage site that opens the virus up — would have evolved to their current state prior to entering humans. In this case, the current epidemic would probably have emerged rapidly as soon as humans were infected, as the virus would have already evolved the features that make it pathogenic and able to spread between people.

In the other proposed scenario, a non-pathogenic version of the virus jumped from an animal host into humans and then evolved to its current pathogenic state within the human population. For instance, some coronaviruses from pangolins, armadillo-like mammals found in Asia and Africa, have an RBD structure very similar to that of SARS-CoV-2. A coronavirus from a pangolin could possibly have been transmitted to a human, either directly or through an intermediary host such as civets or ferrets.

Then the other distinct spike protein characteristic of SARS-CoV-2, the cleavage site, could have evolved within a human host, possibly via limited undetected circulation in the human population prior to the beginning of the epidemic. The researchers found that the SARS-CoV-2 cleavage site, appears similar to the cleavage sites of strains of bird flu that has been shown to transmit easily between people. SARS-CoV-2 could have evolved such a virulent cleavage site in human cells and soon kicked off the current epidemic, as the coronavirus would possibly have become far more capable of spreading between people.

Study co-author Andrew Rambaut cautioned that it is difficult if not impossible to know at this point which of the scenarios is most likely. If the SARS-CoV-2 entered humans in its current pathogenic form from an animal source, it raises the probability of future outbreaks, as the illness-causing strain of the virus could still be circulating in the animal population and might once again jump into humans. The chances are lower of a non-pathogenic coronavirus entering the human population and then evolving properties similar to SARS-CoV-2.

Funding for the research was provided by the US National Institutes of Health, the Pew Charitable Trusts, the Wellcome Trust, the European Research Council, and an ARC Australian Laureate Fellowship.

The minuscule fibers, which are made of polyester, polypropylene and other types of plastics, are shed or washed off of synthetic textiles used in clothing and other consumer and industrial products. Once shed, they enter wastewater and accumulate in oceans, rivers and lakes worldwide, accounting for more than 90% of microplastic pollution in some areas.

“Past field studies have shown that many fish eat large quantities of the fibers every day but have protective mechanisms within the gut that seem to be preventing damage,” said David E. Hinton, Nicholas Distinguished Professor of Environmental Quality at Duke University. “But when you extend your study down to the tissue and cellular levels, as we did, harmful changes are observed.”

“In addition to the fibers that fish eat, hundreds or thousands of microfibers also pass through their gills each day, and we find that this is where much of the damage occurs,” said Melissa Chernick, a researcher in Hinton’s lab at Duke’s Nicholas School of the Environment.

The team published its peer-reviewed findings March 9 in the open-access journal PLoS ONE.

Fish exposed to high levels of microfibers in their tank water for 21 days exhibited aneurysms, fused membranes and increased mucus production in their gills as well as significant changes to the epithelial cells lining their gills and other effects.

“There were severe changes, and a lot of them. And each change can affect respiration,” Chernick said. “If you’re a fish in the wild with gill damage and you’re in a low-oxygen environment or being chased by a predator, you’re in trouble. The same goes if you’re competing with other fish for food. Just having these damages would cause you to be less competitive.”

Though the gut itself seems to be protected from similar damage, the new study finds that when microplastic fibers are in the gut, they may release chemical coatings that are taken up into the fish’s bloodstream.

The researchers are still working to identify these chemicals and determine their impacts, but one troubling effect has already been observed. Female fish exposed to fibers containing polypropylene produced more eggs over time, suggesting that chemicals that may be leaching from the microfibers are acting as endocrine disruptors.

Worldwide, nearly six million tons of synthetic fibers such as polyester or polypropylene were produced in 2016. These textiles shed microfibers during washing or regular use. A single garment can shed nearly 2,000 microfibers per wash, Chernick noted, and because wastewater treatment plants aren’t equipped to remove the fibers, they escape into downstream surface waters and accumulate in the environment. They can also enter the environment through sewage release, stormwater runoff or atmospheric deposition.

“Even if they are released miles from the ocean, they can work their way down there. So they affect both freshwater and marine organisms,” Hinton said.

Hinton and Chernick conducted the new study with Lingling Hu of Zhejiang University of Technology in China, and Lee Ferguson and Anne Lewis of Duke. Ferguson is an associate professor of civil and environmental engineering, ecotoxicology and environmental health. Lewis is a doctoral candidate in civil and environmental engineering.

To conduct the research, they placed 27 breeding pairs of healthy Japanese medaka fish (Oryzias latipes) in water tanks with high levels of suspended microplastic fibers. They monitored fish weights, egg production, and ingestion and egestion of fibers — how much fiber went in, how much was excreted out — weekly. After 21 days, they examined the fish’s tissues to see what changes, if any, had occurred. Tank water was changed weekly and stored for chemical analysis, to determine what dyes or additives had been released.

“Microplastic pollution is an environmental threat that poses increasing risks for species and ecosystems worldwide,” Chernick said. “Until now, most studies have focused primarily on looking for the presence of plastics in animals, without identifying what the effects on various tissues might be. But that’s exactly where our study suggests the science needs to go.”

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Materials provided by Duke University. Original written by Tim Lucas. Note: Content may be edited for style and length.