Princeton researchers report new system to study chronic hepatitis B

Posted on by Catherine Zandonella
A co-culture of human hepatocytes
A co-culture of human hepatocytes and non-parenchymal stromal cells self-assembles into liver-like structures that can be infected for extended periods with the hepatitis B virus. Image courtesy of Benjamin Winer and Alexander Ploss, Princeton University Department of Molecular Biology.

By the Department of Molecular Biology

Scientists from Princeton University‘s Department of Molecular Biology have successfully tested a cell-culture system that will allow researchers to perform laboratory-based studies of long-term hepatitis B virus (HBV) infections. The technique, which is described in a paper published July 25 in the journal Nature Communications, will aid the study of viral persistence and accelerate the development of antiviral drugs to cure chronic hepatitis B, a condition that affects over 250 million people worldwide and can cause severe liver disease, including liver cancer.

HBV specifically infects the liver by binding to a protein called sodium-taurocholate co-transporting polypeptide (NTCP) that is only present on the surface of liver cells. Once inside the cell, HBV hijacks its host’s cellular machinery to convert the virus’s DNA into a stable “mini-chromosome.” This allows the virus to establish persistent, long-term infections that can ultimately cause liver fibrosis, cirrhosis and hepatocellular carcinoma. The World Health Organization estimates that 600,000 people die every year as a result of HBV infection.

Researchers have so far failed to develop drugs that can cure chronic HBV infections, partly because they have not been able to study the long-term infection of liver cells grown in the laboratory. Liver cells—also known as hepatocytes—lose their function within days of being isolated from donor livers, preventing researchers from studying anything other than the acute stage of HBV infection. Hepatocytes can be maintained for longer when they are co-cultured with other, supportive cells.

“In previous studies using hepatocytes and cells known as fibroblasts grown on micro-patterned surfaces, HBV infections worked with only a few donors, and infection lasted for no longer than 14-19 days and required the suppression of antiviral cell signaling pathways, which poses problems for studying host-cell responses to HBV and for antiviral drug testing,” said Alexander Ploss, an assistant professor of molecular biology at Princeton University.

Dr. Ploss and colleagues at Princeton and the Hurel Corporation, led by graduate student Benjamin Winer, tested a different system, in which primary human hepatocytes are co-cultured with non-parenchymal stromal cells, which are cells that support the function of the parenchymal hepatocytes in the liver. When plated in collagen-coated labware, the co-cultures self-assemble into liver-like structures. These self-assembling liver-like cultures could be persistently infected with HBV for over 30 days, without the aid of antiviral signaling inhibitors. Moreover, the system worked with hepatocytes grown from a variety of donors and with viruses isolated from chronically-infected patients, which are harder to work with than lab-grown strains of HBV.

“The establishment of a co-culturing system of human primary hepatocytes and non-parenchymal stromal cells for extended HBV infection is a valuable addition to the armamentarium of cell culture model systems for the study of HBV biology and therapeutic development, which has been hampered by a relative lack of efficient infectious cell culture systems,” said T. Jake Liang, a senior investigator at the National Institute of Diabetes and Digestive and Kidney Diseases, who was not involved in the research.

Ploss and colleagues were able to scale down their co-culture infections to volumes as small as a few hundred microliters. This will be important for future high-throughput screens for anti-HBV drug candidates. As a proof-of-principle for these screens, the researchers found that they could block HBV infections in their co-culture system using drugs that either prevent the virus’ entry into hepatocytes or inhibit a viral enzyme that is essential for the virus’ replication. “The platform presented here may aid the identification and testing of novel therapeutic regimens,” Ploss said.

This study is supported in part by grants from the National Institutes of Health (R21AI117213 to Alexander Ploss and R37GM086868 to Tom W. Muir), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (to Alexander Ploss) and funds from Princeton University (to Alexander Ploss). Benjamin Y. Winer is a recipient of F31 NIH/NRSA Ruth L. Kirschstein Predoctoral awarded from the National Institute of Allergy and Infectious Diseases. Felix Wojcik is supported by a German Research Foundation (DFG) postdoctoral fellowship.

The study, “Long-term hepatitis B infection in a scalable hepatic co-culture system,” by Benjamin Y. Winer, Tiffany S. Huang, Eitan Pludwinski, Brigitte Heller, Felix Wojcik, Gabriel E. Lipkowitz, Amit Parekh, Cheul Cho, Anil Shrirao, Tom W. Muir, Eric Novik, Alexander Ploss, was published in Nature Communications on July 25, 2017. DOI: 10.1038/s41467-017-00200-8.

Read more in this commentary in Nature Microbiology.

Study reveals ways in which cells feel their surroundings

Posted on by Catherine Zandonella
Model of fibrin network
Researchers used computer modeling to show how cells can feel their way through their surroundings, important when, for example, a tumor cell invades a new tissue or organ. This computer simulation depicts collagen fibers that make up the extracellular matrix in which cells live. Local arrangements of these fibers are extremely variable in their flexibility, with some fibers (blue) responding strongly to the cell and others (red) responding hardly at all. The surprising amount of variability in a local area makes it difficult for cells (represented by green arrows) to determine the overall amount of stiffness in a local area, and suggests that cells need to move or change shape to sample more of the surrounding area.

By Catherine Zandonella, Office of the Dean for Research

Cells push out tiny feelers to probe their physical surroundings, but how much can these tiny sensors really discover? A new study led by Princeton University researchers and colleagues finds that the typical cell’s environment is highly varied in the stiffness or flexibility of the surrounding tissue, and that to gain a meaningful amount of information about its surroundings, the cell must move around and change shape. The finding aids the understanding of how cells respond to mechanical cues and may help explain what happens when migrating tumor cells colonize a new organ or when immune cells participate in wound healing.

“Our study looks at how cells literally feel their way through an environment, such as muscle or bone,” said Ned Wingreen, Princeton’s Howard A. Prior Professor in the Life Sciences and professor of molecular biology and the Lewis-Sigler Institute for Integrative Genomics. “These tissues are highly disordered on the cellular scale, and the cell can only make measurements in the immediate area around it,” he said. “We wanted to model this process.” The study was published online on July 18 in the journal Nature Communications.

The organs and tissues of the body are enmeshed in a fiber-rich structure known as the extracellular matrix, which provides a scaffold for the cells to live, move and differentiate to carry out specific functions. Cells interact with this matrix by extending sticky proteins out from the cell surface to pull on nearby fibers. Previous work, mostly employing artificial flat surfaces, has shown that cells can use this tactile feedback to determine the elasticity or stiffness in a process called mechanosensing. But because the fibers of the natural matrix are all interconnected in a jumbled, three-dimensional network, it was not clear how much useful information the cell could glean from feeling its immediate surroundings.

To find out, the researchers built a computer simulation that mimicked a typical cell in a matrix made of collagen protein, which is found in skin, bones, muscles and connective tissue. The team also modeled a cell in a network of fibrin, the strong, stringy protein that makes up blood clots. To accurately capture the composition of these networks, the researchers worked with Chase Broedersz, a former Princeton Lewis-Sigler Fellow who is now professor of physics at Ludwig-Maximilians-University of Munich, and his colleagues Louise Jawerth and Stefan Münster to first create physical models of the matrices, using approaches originally developed in the group of collaborator David Weitz, a systems biologist at Harvard University. Princeton graduate student Farzan Beroz then used those models to recreate virtual versions of the collagen and fibrin networks in computer models.

With these virtual networks, Beroz, Broedersz and Wingreen could then ask the question: can cells glean useful information about the elasticity or stiffness of their environment by feeling their surroundings? If the answer is yes, then the finding would shed light on how cells can change in response to those surroundings. For example, the work might help explain how cancer cells are able to detect that they’ve arrived at an organ that has the right type of scaffold to support tumor growth, or how cells that arrive at a wound know to start secreting proteins to promote healing.

Using mathematics, the researchers calculated how the networks would deform when nearby fibers are pulled on by cells. They found that both the collagen and fibrin networks contained configurations of fibers with remarkably broad ranges of collective stiffness, from rather bendable to very rigid, and that these regions could be immediately next to each other. As a result, the cell could have two nearby probes whereby one detects hardness and the other detects softness, making it difficult for a cell to learn by mechanosensing what type of tissue it inhabits. “We were surprised to find that the cell’s environment can vary quite a lot even across a small distance,” Wingreen said.

The researchers concluded that to obtain an accurate assessment of its environment, a cell must move around and also change shape, for example elongating to cover a different area of the matrix. “What we found in our simulation conforms to what experimentalists have found,” Wingreen said, “and reveals new, ‘intelligent’ strategies that cells could employ to feel their way through tissue environments.”

The study was supported in part by the National Science Foundation (grants DMR-1310266, DMR-1420570, PHY-1305525 and PHY-1066293) the German Excellence Initiative, and the Deutsche Forschungsgemeinschaft.

The study, “Physical limits to biomechanical sensing in disordered fiber networks,” by Farzan Beroz, Louise Jawerth, Stefan Münster, David Weitz, Chase Broedersz, and Ned Wingreen, was published in the journal Nature Communications on July 18, 2017. DOI 10.1038/NCOMMS16096.

New model projects an increase in dust storms in the US

Posted on by Catherine Zandonella
Drifting dust burying farm abandoned farm equipment.
Drifting dust burying farm abandoned farm equipment in 1935. Image courtesy of NOAA.

By Pooja Makhijani for the Office of Communications

Could the storms that once engulfed the Great Plains in clouds of black dust in the 1930’s once again wreak havoc in the U.S.? A new statistical model developed by researchers at Princeton University and the National Oceanic and Atmospheric Administration (NOAA)’s Geophysical Fluid Dynamics Laboratory (GFDL) predicts that climate change will amplify dust activity in parts of the U.S. in the latter half of the 21st century, which may lead to the increased frequency of spectacular dust storms that have far-reaching impacts on public health and infrastructure.

The model, detailed in a study published July 17 in the journal Scientific Reports, eliminates some of the uncertainty found in previous dust activity models by using present-day satellite data such as dust optical depth, which measures to what extent dust particles block sunlight, as well as leafy green coverage over land and other factors.

“Few existing climate models have captured the magnitude and variability of dust across North America,” said Bing Pu, the study’s lead author and an associate research scholar in the Program in Atmospheric and Oceanic Sciences (AOS), a collaboration between Princeton and GFDL.

Dust storms happen when wind blows soil particles into the atmosphere. Dust storms are most frequent and destructive in arid climates with loose soil — especially on lands affected by drought and deforestation. Certain regions of the U.S., such as the southwestern deserts and the central plains, are dust-prone. Most importantly, existing climate models predict “unprecedented” dry conditions in the late-21st century due to an increase in greenhouse gases in these very areas.

It is this “perfect storm” of geography and predicted drought and drought-like conditions that led Pu and her colleague Paul Ginoux, a physical scientist at GFDL, to examine the influence of climate change on dust. They analyzed satellite data about the frequency of dust events and the land’s leafy green coverage over the contiguous U.S., as well as precipitation and surface wind speed, and reported that climate change will increase dust activity in the southern Great Plains from spring to fall in the late half of the 21st century due to reduced rainfall, increased land surface bareness and increased surface wind speed. Conversely, they predicted reduced dust activity in the northern Great Plains in spring during the same time period due to increased precipitation and increased surface vegetation.

Although it is still unclear if rising temperatures themselves trigger the release of yet more dust into the atmosphere, this research offers a glimpse of what the future might hold. “This is an early attempt to project future changes in dust activity in parts of the United States caused by increasing greenhouse gases,” Pu said. Nonetheless, these findings are important given the huge economic and health consequences of severe dust storms, as they can disrupt public transportation systems and trigger respiratory disease epidemics. “Our specific projections may provide an early warning on erosion control, and help improve risk management and resource planning,” she said.

The paper, “Projection of American dustiness in the late 21st century due to climate change,” was published July 17, 2017 in the journal Scientific Reports (doi 10.1038/s41598-017-05431-9 ) and is available online.

This research was supported by NOAA, Princeton University’s Cooperative Institute for Climate Science, and NASA grantNNH14ZDA001N-ACMAP.

Read more about the research in this GFDL Research Highlight.