The study led by Professor Dr. Ana J. Garcia-Saez (CECAD — Cluster of Excellence for Aging Research at the University of Cologne) and Junior Professor Dr. Katia Cosentino (CellNanOs — Center for Cellular Nanoanalytics at the Osnabrück University) has been published in the journal Molecular Cell under the title ‘The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial DNA-mediated inflammation’.

Normally, the body initiates apoptosis — a form of programmed cell death — to get rid of damaged cells. Knowledge of how exactly this clean-up mechanism functions at the level of the molecules involved can make a significant contribution to the therapy of diseases. In cancer, for example, the cells are not systematically removed from the body, but continue to live and spread. Radiotherapy treatment is routinely used to kill cancer cells, but it induces inflammation in the body. Research is being carried out worldwide to find out how cancer therapies cause inflammation and how treatments can be less harmful to the body.

In this study, the research team from CECAD and CellNanOs focused on two proteins that are already known to be involved in cell death. BAX and BAK are the two proteins that regulate the cell death mechanism in mitochondria, the power-houses of the cell. Using high-resolution imaging techniques, the researchers were able to observe for the first time how dynamically BAX and BAK formed structures in mitochondria that influenced cell death and inflammation.

Due to their great similarity, BAX and BAK have so far been referred to as twin proteins. That they differ systematically in their mechanism of action is a new finding of this study. The research team was able to show that BAK proteins organize into smaller structures more quickly than BAX, and that the two proteins influence each other.

‘We could clearly observe under the microscope how both proteins interacted with each other to form a pore in the mitochondrion, so that the mitochondrial DNA exited through the pore to trigger inflammation,’ explained Andreas Jenner of the University of Cologne. This dynamic interplay of BAK and BAX proteins was previously unknown, and it regulates the formation of the pore and thus the amount of mitochondrial DNA released. This, in turn, determines whether an inflammatory response is triggered in the body.

‘The relative availability of BAX and BAK proteins in cells determines the growth of the pore and the rate at which mitochondrial DNA is released. Possibly, our findings open up new perspectives to control inflammation during cancer treatments,’ explained Katia Cosentino of the Osnabrück University.

‘Our results highlight how BAX and BAK contribute to cell death in different ways and suggest that these two proteins should be well balanced in therapeutic cancer treatments,’ said Ana J. Garcia-Saez of the University of Cologne.

However, such therapy does not yet exist. For now, further investigation is needed to determine whether additional molecules contribute to the dynamics of BAX and BAK pore formation and regulate pore growth and size.

Work for the study began at the IFIB (Interfaculty Institute of Biochemistry) in Tübingen and was completed at the CECAD Research Center, Institute of Genetics, in the Garcia-Saez laboratory in Cologne and at the Center for Cellular Nanoanalytics (CellNanOs) at the Osnabrück University.

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The new, modified charge pumping technique can detect single defects as small as the diameter of a hydrogen atom (one-tenth of a billionth of a meter) and can indicate where they’re located in the transistor. Researchers could also use the new capability to detect and manipulate a property in each electron known as quantum spin. The ability to manipulate individual spins has applications in both basic research and quantum engineering and computing.

Transistors act as electrical switches. In the on position, which represents the “1” of binary digital information, a designated amount of current flows from one side of a semiconductor to the other. In the off position, representing the “0” of binary logic, current ceases to flow.

Defects in a transistor can interfere with the reliable flow of current and significantly degrade the performance of transistors. These defects could be broken chemical bonds in the transistor material. Or they could be atomic impurities that trap electrons in the material. Scientists have devised several ways to categorize defects and minimize their impact, tailored to the structure of the transistor under study.

In the traditional design known as the metal oxide semiconductor field effect transistor (MOSFET), a metal electrode called the gate sits atop a thin insulating layer of silicon dioxide. Below the insulating layer lies the interface region that separates the insulating layer and the main body of the semiconductor. In a typical transistor, current travels through a narrow channel, only one billionth of a meter thick, that extends from the source, which lies on one side of the gate, to a “drain” on the other side. The gate controls the amount of current in the channel.

Charge pumping is a two-step process in which the examiner alternately pulses the gate with a positive test voltage, then a negative one. (The transistor does not act as an on/off switch during this testing mode.) In traditional charge pumping, the alternating voltage pulses are applied at a single, set frequency.

In the first step of the test, the positive voltage attracts or pumps electrons, which are negatively charged, to the boundary or interface between the gate’s insulating layer and the body of the transistor. Some of the pumped electrons become trapped in defects at the interface, but there are many electrons left over. In the second step, a negative voltage is applied, to rid the interface of the excess electrons, leaving only the trapped ones behind. The negative voltage also attracts positive charge carriers, known as “holes,” to the region, where they combine with electrons trapped in the defects. This activity generates a current proportional to the number of defects. The greater the output current, the larger the number of defects.

In the recent past, the current was indeed a reliable measure of defects. However, the insulating oxide layer in modern transistors is now so thin — just 10 to 20 hydrogen atoms wide — that an effect from the realm of quantum mechanics comes into play, confounding measurements using the traditional charge-pumping method.

According to quantum theory, electrons and other subatomic particles can never be truly trapped; there’s always some probability they will escape or “tunnel” out of an enclosure or boundary layer. The thinner the material, the higher the probability that electrons will escape, creating a tunneling current. As transistor dimensions shrank, the tunneling current leaking through the insulating oxide layer made it nearly impossible to detect defects with ordinary charge pumping. Scientists all but abandoned the technique.

NIST researchers James Ashton, Mark Anders and Jason Ryan have now found a way to salvage the technique so that it not only works for ultrathin transistor components but is also more sensitive, enabling scientists to record signals from a single defect. The solution arose when the scientists came to a key realization: The current that results from quantum tunneling remains virtually the same, regardless of the frequency at which charge pumping pulses the positive and negative voltages.

Armed with that knowledge, the team revised the charge pumping technique by alternately applying the method’s positive and negative voltages at two different frequencies rather than the single frequency used in the traditional method. Applying the voltages at two different frequencies gave the researchers two different output currents. By subtracting one output current from the other, the constant signal from the quantum tunneling current dropped out. With the confounding tunneling current eliminated, the researchers were able to detect defects in transistors with ultrasmall features. The researchers reported their development of the frequency-modulated charge pumping technique online in the Feb. 2 Applied Physics Letters.

“We’ve given charge pumping a new lease on life,” said Ashton.

“The modulated-frequency technique is now useful for looking at single interface defects, which gives engineers control of single electron charges in a very sensitive measurement scheme,” he added.

Since only one electron is involved, the output current is equal to multiples of the electron’s charge, a fundamental physics constant calculated by NIST and other institutions.

Because the method can detect single electrons, it may serve as a sensitive probe of an electron’s quantum spin. Modulated-frequency charge pumping may provide a valuable guide to scientists who are now exploring how electron spin might store and transfer information in a computer of the future. It may also prove useful in quantum metrology, as a potential new way of determining a quantum standard of electrical current.

‘In our study, we investigated which immune cells are activated in severe cases and in what way the endothelium, in other words the blood vessels, and their activation play a role in the disease progress’, explains Prof. Christine Falk, Scientist at the Hanover Medical School (MHH) and the German Center for Infection Research (DZIF). Many clinical symptoms, such as the destruction of blood vessels in the lungs and acute respiratory distress syndrome, pointed to an impact on the endothelium.

The endothelium is a thin layer of cells that line blood vessels, forming a barrier between blood flow and the surrounding tissues. Infection with SARS-CoV-2 appears to cause strong activation of immune and endothelial cells in the lungs, resulting in the release of various soluble plasma proteins. Severe COVID-19 cases are associated with a dysfunction of the endothelium, wherein the barrier between the alveoli and the surrounding vessels is no longer intact.

The scientists studied 25 patients with severe COVID-19 and 17 recovered patients in the intensive care unit (ICU). They were able to prove that the severity of the disease is linked to disruption of the endothelial barrier and can be measured by looking at inflammatory and endothelial plasma proteins. A pattern of seven plasma proteins appears to be associated with a severe form of the disease, which is characterised by strong inflammatory processes and in which the endothelium is permanently damaged. Furthermore, recovery from severe COVID-19 cases seems to be related to the regeneration of this endothelial barrier.

Which immune cells were detected in the COVID-19 ICU patients? The study showed excessive activation of T-lymphocytes and natural killer cells as well as development of memory T-cells and strong proliferation of plasmablasts, cells that can produce large amounts of antibodies. Furthermore, ICU patients infected with SARS-CoV-2 had high titres of spike- and nucleocapsid-specific antibodies. The researchers found particularly interesting that the immune cell phenotype of these patients mainly changed over time and was less related to progressive severity of the disease. The progression of COVID-19, on the other hand, was closely linked to increased levels of various soluble plasma proteins, namely certain inflammatory mediators and especially endothelial factors.

‘We were able to demonstrate that ICU patients with COVID-19 can be divided into different groups based on their plasma protein profile, which are associated with disease severity’, explains lead author Louisa Ruhl, a PhD doctoral student at MHH. This finding is of great importance for identification of potential biomarkers for severe COVID-19 courses as well as for the development and use of new therapeutic concepts.

Christine Falk’s team now wants to investigate which elements of the immune system lead to activation and damage of the endothelium and whether the strong activation of the immune system also leads to the development of virus-specific T-lymphocytes that can recognise and destroy infected cells and thus contribute to overreaction. Moreover, the study has shown that there are also shifts in the immune cell repertoire in recovered ICU patients with COVID-19. This could be related to the development of Long-COVID cases. These aspects are currently being promoted as part of the COFONI initiative of the state of Lower Saxony with a Fasttrack and a Flexfund project. In collaboration with partners from pneumology (Prof. Tobias Welte, MHH and DZL) and neurology (Prof. Günter Höglinger, MHH), they are not only investigating as to whether the endothelial inflammation with the overreaction of the T-lymphocytes and natural killer cells causes lasting damage, but also to what extent the regeneration of the lung is impaired and the nervous system is affected.

The study was conducted jointly with scientists and clinicians at the MHH, the German Center for Lung Research (DZL) and scientists at the Erlangen University Hospital.

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A multi-institutional team of researchers, including virologist Marc Johnson, a professor of molecular microbiology and immunology at the University of Missouri, has detected at least four “cryptic” variants of SARS-CoV-2, the virus that causes COVID-19, in samples of wastewater from New York City’s public sewer system. Their findings were recently published in Nature Communications, a journal of Nature.

According to the Centers for Disease Control and Prevention (CDC), viruses such as SARS-CoV-2 can continually evolve by acquiring mutations. Variants, such as delta or omicron, may contain one or more mutations within their viral sequence that help to distinguish themselves from other variants of SARS-CoV-2.

Johnson, a co-corresponding author on the study, believes the results suggest the “cryptic” mutations they identified in New York City could be linked to possible animal origins. While these origins have not been verified yet, he believes one possible source could be the rats that frequent New York City’s sewer system.

“For instance, we still don’t know where the omicron variant came from, but it had to come from somewhere,” Johnson said. “These variants are bubbling up everywhere, including omicron, which eventually spilled into the general population and wreaked havoc. We think these weird lineages could be where the next variant of concern for COVID-19 comes from.”

Hunting for virus mutations

The idea for this project started in March 2020 after John Dennehy, a virologist and professor of biology at Queens College, City University of New York, began looking for different ways to analyze the impact of the COVID-19 pandemic. Monica Trujillo, an associate professor at Queensborough Community College, City University of New York, shared with Dennehy a study from Australia that described using wastewater to track the spread of a coronavirus, and it inspired Trujillo to ask officials from the New York City Department of Environmental Protection to send her wastewater samples in order to conduct similar work.

In the summer of 2020, Dennehy and Trujillo teamed up with Davida Smyth, the lead author on the study. Smyth, now an associate professor at Texas A&M University-San Antonio, was at The New School, New York at the time. They put together a team of researchers to begin tracking the spread of coronavirus via New York City’s wastewater. Since then, Dennehy, a co-corresponding author on the study, found some interesting results. When he shared some of the more unusual results during a March 2021 episode of Vincent Racaniello’s podcast “This Week in Virology,” a popular podcast with virologists nationwide, Johnson happened to be listening.

“The mutations that we were observing in our sample weren’t typical among any of the known sequences circulating at that time,” Dennehy said.

Ironically, Johnson was simultaneously reaching out to various researchers across the United States who were doing similar work with wastewater after he observed some unusual results from his analysis of wastewater in some of his samples from the greater St. Louis area. On the podcast, he heard Dennehy describe their targeted approach, which happened to be the same method MU was using to test samples of wastewater for SARS-CoV-2 in Missouri, but with one key difference — MU was analyzing a larger region of the virus genome for possible mutations. Within a week, Johnson had samples of NYC wastewater delivered to MU for further analysis.

“When we first started with the samples from New York City, I was looking to see if they had the same virus sequences that I saw in some of my samples from St. Louis,” Johnson said. “They were different, but all of them had similar mutations in common at one particular location on the virus — Q498. What’s amazing is that in most of the samples from New York City, the Q in Q498 had turned into a Y, or glutamine into tyrosine. If you look at the database, there was not, and continues to not be, a human patient who has had that mutation.”

Dennehy believes a possible explanation could be a biological process called convergent evolution.

“An animal in Missouri is not going to mix with the same type of animal in New York City,” Dennehy said. “Therefore, the evolution of the virus in each geographic area is independent of each other, but because it’s the same animal, the virus looks the same in both places. For instance, we think conditions in South Africa that gave rise to the omicron variant are the same conditions in New York City that gave rise to our cryptic variants. As a biologist, I thought the spread of delta was menacing, but the speed in which omicron took over New York City is on another level.”

Understanding the broader impact

Smyth joined the research team in part due to her passion for taking science from inside a laboratory to showing how it can be applied through a broader, real-world impact. A proud Irish immigrant, she transitioned from studying the basics of biology early in her career to now pursuing her passion by engaging her students in real-world health issues, such as antibiotic resistance, and tracking coronavirus in wastewater.

“I’m interested in how we can take what we know and apply it to the real world where it is needed the most,” said Smyth, who is also the deputy director for the National Center for Science and Civic Engagement. “Wastewater surveillance is really important in the context of community-based, public health measures where we have the ability to communicate health information at a level where decisions, practices and interventions can be implemented. Wastewater surveillance is fast, inexpensive and unbiased, and for that reason it has the ability to be implemented depending on resource availability in a variety of contexts, especially in areas with limited resources such as low testing and vaccine availability.”

“Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater,” was published in Nature Communications. Funding was provided in part by the New York City Department of Environmental Protection, a donation from the Linda Markeloff Charitable Fund, and a grant from the National Institutes of Health (U01DA053893-01).

Co-authors include Devon Gregory, Maddie Graham, Yue Guan, Caitlyn Guldenpfennig, Terry Lyddon, Clayton Rushford, Reinier Suarez, Emma Teixeiro and Mark Daniels at MU; Davida Smyth and Geena Sompanya at Texas A&M University-San Antonio; Monica Trujillo at Queensborough Community College, City University of New York; Kristen Cheung, Anna Gao, Irene Hoxie, Sherin Kannoly, Nanami Kubota, Michelle Markman, and Kaung Myat San at the City University of New York; and Fabrizio Spagnolo at Long Island University.

Broadly neutralizing antibodies (bNAbs) targeting virus envelope (Env) protein have significant potential for treating HIV-1. They were initially identified in rare cases of patients whose serum was capable of inhibiting numerous HIV strains. These antibodies exhibit multiple antiviral activities. As well as neutralizing the virus, i.e. preventing it from infecting new cells, they also kill infected cells. Consequently, they are referred to as polyfunctional molecules. It is necessary to fully understand the scope of these antiviral activities in order to use existing antibodies more effectively or refine the selection criteria for new antibodies. It is moreover useful to further investigate the polyfunctionality of anti-HIV-1 antibodies in order to improve our understanding of the role played by antibodies and thus tackle other viral infections.

Initially, teams at the Institut Pasteur, CNRS, VRI and Université de Paris sought to determine whether antibodies were capable of preventing infected cells from producing viral particles. To that end, they cultured CD4 T cells (HIV’s natural target) in vitro with various antibodies for 24 hours. They subsequently measured the quantity of viral particles produced by the cells in the culture medium and the quantity of viral particles remaining in the cells. As a result of these experiments, the scientists were able to demonstrate that certain antibodies increased virus quantity in cells but reduced it in the culture medium. This intriguing finding led them to believe that certain antibodies impeded the release of viral particles without preventing their production.

To test this theory, the scientists used various microscopy techniques to observe cells’ production of viral particles. They initially examined the cells by fluorescence microscopy, a technique used to differentiate virus proteins. This enabled them to demonstrate that infected cells accumulate large quantities of mature viral protein. This finding suggests that full viral particles accumulate in cells. To determine the precise location of these viral particles, the scientists subsequently used scanning electron microscopy to observe the surface of infected cells. “Using this method, we observed that these antibodies (bNAbs) prompt an accumulation of viral particles at the surface of cells, forming clusters and highly atypical structures (see illustration),” comments Timothée Bruel, co-last author of the study and scientist in the Virus and Immunity Unit[2] at the Institut Pasteur.

Next, the scientists combined a transmission electron microscopy technique with immunogold labeling. This enabled them to demonstrate that antibodies interpose themselves between viral particles and the infected cell, forming a chain cluster. Experiments with mutant antibodies subsequently demonstrated that the antibodies’ Y shape creates this clustered structure. Their arms are capable of linking two viruses, or one virus to the infected cell membrane, and their attachment points are sufficiently strong to prompt this phenomenon.

“We have demonstrated that only the most powerful antibodies tether viral particles at the surface of infected cells. Trapped viral particles can no longer infect new cells,” concludes Olivier Schwartz, co-last author of the study and Head of the Virus and Immunity Unit at the Institut Pasteur.

This work has revealed a new antiviral activity for broadly neutralizing anti-HIV-1 antibodies. It deepens our understanding of these antibodies’ mechanism of action and explains their efficacy in clinical trials. The scientists are now examining antibodies targeting other viruses, including SARS-CoV-2, to determine whether they also inhibit viral spread through this mechanism.

[1] HIV-1 is the pandemic form of the human immunodeficiency virus. The other less common type is HIV-2.

[2] This is named the “Virology Unit” at the CNRS (CNRS/Institut Pasteur).

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