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COVID-19 studies should also focus on mucosal immunity, researchers argue




BUFFALO, N.Y. — Anyone who has undergone a nasal swab or saliva test for COVID-19 knows that the virus is most easily detected in the nose and mouth. That’s why, University at Buffalo researchers argue in a new paper, more COVID-19 studies should be devoted to how immunity emerges to SARS-CoV-2 in the mucous membranes of the nose and mouth.

The paper was published Nov. 30 in Frontiers in Immunology.

Noting that the mucosal immune system is the immune system’s largest component, the researchers expressed concern that it hasn’t been a focus of much of the research on COVID-19 to date.

“We think it is a serious omission to ignore the mucosal immune response to SARS-CoV-2, given its initial sites of infection,” said Michael W. Russell, PhD, emeritus professor, Department of Microbiology and Immunology in the Jacobs School of Medicine and Biomedical Sciences at UB, and senior author on the paper. “Clearly the response of the systemic immunoglobulin G antibody [the most abundant circulating antibody] is important — we do not deny that — but on its own it is insufficient.”

Russell noted that naturally, the initial focus of research on the disease was on cases of severe disease when the virus descends into the lower respiratory tract, especially the lungs, where the cellular immune responses exacerbate the inflammation rather than fight the infection.

But since the upper respiratory tract, including the nose, tonsils and adenoids are the initial point of infection for the SARS-CoV-2 virus, the immune responses that are triggered there are of special interest.

In addition, the high rate of asymptomatic transmission of COVID-19, which the Centers for Disease Control and Prevention recently estimated at more than 50%, is another reason why mucosal immunity is so important, according to the authors.

‘Something, somewhere, does a fairly good job of controlling the virus’

“Given that many infected people remain asymptomatic, and that a large number of those who develop symptoms suffer only mild to moderate disease, this suggests that something, somewhere, does a fairly good job of controlling the virus,” said Russell.

“Could it be that this is due to early mucosal immune responses that succeed in containing and eliminating the infection before it becomes serious?” he asked. “We will not know unless these questions are addressed.”

The paper recommends that studies are needed to determine the nature of mucosal secretory immunoglobulin A (SIgA) antibody responses over the course of infection, including asymptomatic or pre-symptomatic infection, and mild and moderate cases of COVID-19 disease. In addition, the authors point out that the mucosal immune responses may vary depending on different age groups and populations.

A focus on mucosal immunity might also make it possible to develop a type of vaccine, such as a nasal vaccine, that could be easier to store, transport and administer. Several such vaccines are now under development for COVID-19 but how far along they are is unknown.

Russell added that these vaccines might not have special temperature requirements and might be more palatable for large swaths of the population, especially children, because they would not require an injection.

Potential advantages of a mucosal vaccine

“The potential advantage of a mucosal vaccine – especially one that is intranasal – is that it should induce immune responses, including SIgA antibodies, in the mucosal tracts, in this case especially the upper respiratory tract, where the coronavirus makes first contact,” explained Russell, adding that injected vaccines usually do not do this.

Among the areas of study that the authors suggest would be constructive are molecular studies on IgA antibodies and their relationship to the disease stage of COVID-19, and determining the characteristics of cells that secrete IgA antibodies and other mucosal immune cells induced by the infection or by vaccination.

“As mucosal immunologists with several decades of experience behind us, we have been perturbed at the lack of attention to this, and we hope to draw attention to this glaring omission,” said Russell. “After all, the mucosal immune system is by far the largest component of the entire immune system, and it has evolved to protect the mucosal surfaces where the great majority of infections arise.”


Co-authors with Russell are Pearay L. Ogra of UB and Zina Moldoveanu and Jiri Mestecky of the University of Alabama at Birmingham.



Regulating the ribosomal RNA production line




Cryo-electron microscopy study allows researchers to visualize structural changes in an E. coli enzyme synthesizing ribosomal RNA that shift it between turbo- and slow-modes depending on the bacteria’s growth rate

The enzyme that makes RNA from a DNA template is altered to slow the production of ribosomal RNA (rRNA), the most abundant type of RNA within cells, when resources are scarce and the bacteria Escherichia coli needs to slow its growth. Researchers used cryo-electron microscopy (cryo-EM) to capture the structures of the RNA polymerase while in complex with DNA and showed how its activity is changed in response to poor-growth conditions. A paper describing the research led by Penn State scientists appears January 22, 2020 in the journal Nature Communications.

“RNA polymerase is an enzyme that produces a variety of RNAs using information encoded in DNA,” said Katsuhiko Murakami, professor of biochemistry and molecular biology at Penn State and the leader of the research team. “This is one of the key steps in the central dogma of molecular biology: transferring genetic information from DNA to RNA, which in turn often codes for protein. It’s required for life and the process is basically shared from bacteria to humans. We are interested in understanding how the structure of RNA polymerase is changed for modulating its activity and function, but it’s been difficult to capture using traditional methods like X-ray crystallography, which requires crystallizing a sample to determine its structure.”

RNA polymerase functions by binding to specific DNA sequences called “promoters” found near the beginning of genes that are going to be made into RNA. To understand the structure and function of the polymerase during this interaction, researchers need to capture the polymerase while it is bound to the promoter DNA, but the interaction can be very unstable at some promoters. Crystallography can only capture RNA polymerase bound to a promoter if the complex is very stable, but for ribosomal RNA promoters this interaction tends to be unstable so that the polymerase can quickly escape to begin making the RNA. To see these interactions the researchers turned to cryo-EM, a method that allows them to visualize the structure of macromolecules in solution.

“When you talk about RNA, most people think about messenger RNA (mRNA), which is the template for making proteins,” said Murakami. “But the most abundant type of RNA in cells doesn’t actually code for protein. Ribosomal RNA is the major structural component of the ribosome, which is the cellular machinery that builds proteins using messenger RNAs as templates. Ribosomal RNA synthesis accounts for up to 70 percent of total RNA synthesis in E. coli cells.”

When a cell divides, which E. coli can do every twenty minutes in nutrient-rich growth conditions, it needs to provide the two resulting daughter cells with enough ribosomes to function, so it is continually making ribosomal RNAs.

“If you do some back-of-the-envelope calculations, an E. coli cell needs to make around 70,000 ribosomes every 20 minutes,” said Murakami. “This means RNA polymerase starts ribosomal RNA synthesis every 1.7 seconds from each ribosomal RNA promoter. So, the polymerase has to bind the ribosomal RNA promoter transiently in order to quickly move onto the ribosomal RNA synthesis step. This is not an ideal for a crystallographic approach, but in a cryo-EM study, we could capture this interaction and, in fact, see different several stages of the interaction in a single sample.”

The researchers were able to determine the three-dimensional structures of the RNA polymerase-promoter complex at two different stages. One when the DNA was still “closed,” before the two strands of the DNA molecule are separated allowing access to the template strand (they refer to this as a closed complex), and one when the DNA was “open” (called an open complex) and primed for RNA synthesis to begin.

“We found a large conformational change in part of the polymerase called the ? (sigma) factor when it binds to promoter DNA, which has never been observed before” said Murakami. “This change opens a gate that allows the DNA to enter a cleft in the polymerase and form the open complex quickly.”

When E. coli needs to slow its growth due to limited resources, two molecules–a global transcription regulator called DksA and a bacterial signaling molecule called ppGpp, bind directly with the polymerase to reduce production of ribosomal RNA. The research team investigated how the binding of these two factors alters the conformation of the polymerase and affects its activity in a promoter-specific manner.

“DksA and ppGpp binding to the polymerase alters its conformation, which prevents the opening of a gate and therefor the polymerase has to follow an alternative pathway to form the open complex,” said Murakami. “This is not an ideal pathway for the ribosomal RNA promoter and thus slow its activity. It’s exciting to see these conformational changes to the polymerase that have direct functional consequences. We couldn’t do this without the cryo-EM, so I’m very thankful to have access to this technology here at Penn State for optimizing experimental conditions for preparing cryo-EM specimens before sending them to the National Cryo-EM Facility at NCI/NIH for high-resolution data collections. We are going to be able to continue to analyze cellular components and complexes that were previously inaccessible.”


In addition to Murakami, the research team includes Yeonoh Shin and M. Zuhaib Qayyum at Penn State and Danil Pupov, Daria Esyunina, and Andrey Kulbachinskiy at the Russian Academy of Sciences. The research was funded by the U.S. National Institutes of Health, the Russian Science Foundation, and the Russian Foundation for Basic Research. Additional support was provided by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research.


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A professor from RUDN University developed new liquid crystals




A professor from RUDN University together with his Indian colleagues synthesized and studied new dibenzophenazine-based liquid crystals that could potentially be used in optoelectronics and solar panels. The results of the study were published in the Journal of Molecular Liquids.

Liquid crystals are an intermediate phase between a liquid and a solid body. They are ordered like regular chrystals but at the same time have a flow like liquids. It is this duality that allows them to be used in organic LEDs and LCDs. Unlike other liquid crystals, discotic ones (DLC) are capable of self-assembly into ordered structures. This makes them a promising material for industrial electronics, namely, for the production of displays. A professor from RUDN University together with his Indian colleagues synthesized and described new dibenzophenazine-based DLCs.

“Discotic liquid crystals are interesting because of their ability to form self-assembled ordered columnar structures. In such structures, an electric charge can move along the column, which makes them useful for optoelectronic devices such as organic LEDs, organic field-effect transistors (OFET), photoelectric solar elements, and sensors,” said Prof. Viktor Belyaev, a Ph.D. in Technical Sciences from the Department of Mechanics and Mechatronics at RUDN University.

DLCs consist of disc-shaped molecules aligned in columns. In the center of each disc, there is an aromatic ring (a cyclical organic fragment) surrounded by chains of other organic fragments. Due to this aromatic center, a DLC can transfer a charge along the axis of a column. Prof. Belyaev developed discotic liquid crystals with an aromatic compound called dibenzophenazine in the center. As for the chains that surrounded it, the team tried three different types of fragments. The molecular structure of the new DLCs was studied using spectral, X-ray diffraction, and elementary analysis. Then, the team tested the three groups of DLCs in a set of experiments.

The experiments showed that alkoxy thiol chains increased the polarity of the molecules in liquid crystals thus improving the internal structure of the columns and making them more even. All new DLCs were able to withstand temperatures up to 330?. However, the crystals that consisted of smaller molecules (i.e. the ones with their aromatic center surrounded by alkyl thiols) lost their intermediary status and transitioned from the liquid crystal to the liquid form at lower temperatures (55.1 ?) that the crystals from the other two groups. This is due to the size of the molecules in the columns: the bigger they are, the more stable is the liquid crystal state.

“The new discotic liquid crystals could play an important role in organic optoelectronic devices and solar panels,” added Prof. Viktor Belyaev from RUDN University.



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New technique builds super-hard metals from nanoparticles




PROVIDENCE, R.I. [Brown University] — Metallurgists have all kinds of ways to make a chunk of metal harder. They can bend it, twist it, run it between two rollers or pound it with a hammer. These methods work by breaking up the metal’s grain structure — the microscopic crystalline domains that form a bulk piece of metal. Smaller grains make for harder metals.

Now, a group of Brown University researchers has found a way to customize metallic grain structures from the bottom up. In a paper published in the journal Chem, the researchers show a method for smashing individual metal nanoclusters together to form solid macro-scale hunks of solid metal. Mechanical testing of the metals manufactured using the technique showed that they were up to four times harder than naturally occurring metal structures.

“Hammering and other hardening methods are all top-down ways of altering grain structure, and it’s very hard to control the grain size you end up with,” said Ou Chen, an assistant professor of chemistry at Brown and corresponding author of the new research. “What we’ve done is create nanoparticle building blocks that fuse together when you squeeze them. This way we can have uniform grain sizes that can be precisely tuned for enhanced properties.”

For this study, the researchers made centimeter-scale “coins” using nanoparticles of gold, silver, palladium and other metals. Items of this size could be useful for making high-performance coating materials, electrodes or thermoelectric generators (devices that convert heat fluxes into electricity). But the researchers think the process could easily be scaled up to make super-hard metal coatings or larger industrial components.

The key to the process, Chen says, is the chemical treatment given to the nanoparticle building blocks. Metal nanoparticles are typically covered with organic molecules called ligands, which generally prevent the formation of metal-metal bonds between particles. Chen and his team found a way to strip those ligands away chemically, allowing the clusters to fuse together with just a bit of pressure.

The metal coins made with the technique were substantially harder than standard metal, the research showed. The gold coins, for example, were two to four times harder than normal. Other properties like electrical conduction and light reflectance were virtually identical to standard metals, the researchers found.

The optical properties of the gold coins were fascinating, Chen says, as there was a dramatic color change when the nanoparticles were compressed into bulk metal.

“Because of what’s known as the plasmonic effect, gold nanoparticles are actually purplish-black in color,” Chen said. “But when we applied pressure, we see these purplish clusters suddenly turn to a bright gold color. That’s one of the ways we knew we had actually formed bulk gold.”

In theory, Chen says, the technique could be used to make any kind of metal. In fact, Chen and his team showed that they could make an exotic form of metal known as a metallic glass. Metallic glasses are amorphous, meaning they lack the regularly repeating crystalline structure of normal metals. That gives rise to remarkable properties. Metallic glasses are more easily molded than traditional metals, can be much stronger and more crack-resistant, and exhibit superconductivity at low temperatures.

“Making metallic glass from a single component is notoriously hard to do, so most metallic glasses are alloys,” Chen said. “But we were able to start with amorphous palladium nanoparticles and use our technique to make a palladium metallic glass.”

Chen says he’s hopeful that the technique could one day be widely used for commercial products. The chemical treatment used on the nanoclusters is fairly simple, and the pressures used to squeeze them together are well within the range of standard industrial equipment. Chen has patented the technique and hopes to continue studying it.

“We think there’s a lot of potential here, both for industry and for the scientific research community,” Chen said.


Chen’s coauthors on the paper were Yasutaka Nagaoka, Masayuki Suda, Insun Yoon, Na Chen, Hanjun Yang, Yuzi Liu, Brendan A. Anzures, Stephen W. Parman, Zhongwu Wang, Michael Grünwald and Hiroshi M. Yamamoto. The research was supported by the National Science Foundation (CMMI-1934314, DMR-1332208, DMR-1848499) and the U.S. Department of Energy (DE-AC02-06CH11357).


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No more needles for diagnostic tests?




Nearly pain-free microneedle patch can test for antibodies and more in the fluid between cells

Blood draws are no fun.

They hurt. Veins can burst, or even roll — like they’re trying to avoid the needle, too.

Oftentimes, doctors use blood samples to check for biomarkers of disease: antibodies that signal a viral or bacterial infection, such as SARS-CoV-2, the virus responsible for COVID-19; or cytokines indicative of inflammation seen in conditions such as rheumatoid arthritis and sepsis.

These biomarkers aren’t just in blood, though. They can also be found in the dense liquid medium that surrounds our cells, but in a low abundance that makes it difficult to be detected.

Until now.

Engineers at the McKelvey School of Engineering at Washington University in St. Louis have developed a microneedle patch that can be applied to the skin, capture a biomarker of interest and, thanks to its unprecedented sensitivity, allow clinicians to detect its presence.

The technology is low cost, easy for a clinician or patients themselves to use, and could eliminate the need for a trip to the hospital just for a blood draw.

The research, from the lab of Srikanth Singamaneni, the Lilyan & E. Lisle Hughes Professor in the Department of Mechanical Engineering & Material Sciences, was published online Jan. 22 in the journal Nature Biomedical Engineering.

In addition to the low cost and ease of use, these microneedle patches have another advantage over blood draws, perhaps the most important feature for some: “They are entirely pain-free,” Singamaneni said.

Finding a biomarker using these microneedle patches is similar to blood testing. But instead of using a solution to find and quantify the biomarker in blood, the microneedles directly capture it from the liquid that surrounds our cells in skin, which is called dermal interstitial fluid (ISF). Once the biomarkers have been captured, they’re detected in the same way — using fluorescence to indicate their presence and quantity.

ISF is a rich source of biomolecules, densely packed with everything from neurotransmitters to cellular waste. However, to analyze biomarkers in ISF, conventional method generally requires extraction of ISF from skin. This method is difficult and usually the amount of ISF that can be obtained is not sufficient for analysis. That has been a major hurdle for developing microneedle-based biosensing technology.

Another method involves direct capture of the biomarker in ISF without having to extract ISF. Like showing up to a packed concert and trying to make your way up front, the biomarker has to maneuver through a crowded, dynamic soup of ISF before reaching the microneedle in the skin tissue. Under such conditions, being able to capture enough of the biomarker to see using the traditional assay isn’t easy.

But the team has a secret weapon of sorts: “plasmonic-fluors,” an ultrabright fluorescence nanolabel. Compared with traditional fluorescent labels, when an assay was done on microneedle patch using plasmonic-fluor, the signal of target protein biomarkers shined about 1,400 times as bright and become detectable even when they are present at low concentrations.

“Previously, concentrations of a biomarker had to be on the order of a few micrograms per milliliter of fluid,” Zheyu (Ryan) Wang, a graduate student in the Singamaneni lab and one of the lead authors of the paper, said. That’s far beyond the real-world physiological range. But using plasmonic-fluor, the research team was able to detect biomarkers on the order of picograms per milliliter.

“That’s orders of magnitude more sensitive,” Ryan said.

These patches have a host of qualities that can make a real impact on medicine, patient care and research.

They would allow providers to monitor biomarkers over time, particularly important when it comes to understanding how immunity plays out in new diseases.

For example, researchers working on COVID-19 vaccines need to know if people are producing the right antibodies and for how long. “Let’s put a patch on,” Singamaneni said, “and let’s see whether the person has antibodies against COVID-19 and at what level.”

Or, in an emergency, “When someone complains of chest pain and they are being taken to the hospital in an ambulance, we’re hoping right then and there, the patch can be applied,” Jingyi Luan, a student who recently graduated from the Singamaneni lab and one of the lead authors of the paper, said. Instead of having to get to the hospital and have blood drawn, EMTs could use a microneedle patch to test for troponin, the biomarker that indicates myocardial infarction.

For people with chronic conditions that require regular monitoring, microneedle patches could eliminate unnecessary trips to the hospital, saving money, time and discomfort — a lot of discomfort.

The patches are almost pain-free. “They go about 400 microns deep into the dermal tissue,” Singamaneni said. “They don’t even touch sensory nerves.”

In the lab, using this technology could limit the number of animals needed for research. Sometimes research necessitates a lot of measurements in succession to capture the ebb and flow of biomarkers — for example, to monitor the progression of sepsis. Sometimes, that means lot of small animals.

“We could significantly lower the number of animals required for such studies,” Singamaneni said.

The implications are vast — and Singamaneni’s lab wants to make sure they are all explored.

There is a lot of work to do, he said: “We’ll have to determine clinical cutoffs,” that is, the range of biomarker in ISF that corresponds to a normal vs. abnormal level. “We’ll have to determine what levels of biomarker are normal, what levels are pathological.” And his research group is working on delivery methods for long distances and harsh conditions, providing options for improving rural healthcare.

“But we don’t have to do all of this ourselves,” Singamaneni said. Instead, the technology will be available to experts in different areas of medicine.

“We have created a platform technology that anyone can use,” he said. “And they can use it to find their own biomarker of interest.”

We don’t have to do all of this ourselves

Singamaneni and Erica L. Scheller, assistant professor of Medicine in the Division of Bone and Mineral Disease at the School of Medicine, worked together to investigate the concentration of biomarkers in local tissues.

Current approaches for such evaluation require the isolation of local tissues and do not allow successive and continuous inspection. Singamaneni and Scheller are developing a better platform to achieve long term monitoring of local biomarker concentration.

Working together

Srikanth Singamaneni, the Lilyan E. Lisle Hughes Professor in the Department of Mechanical Engineering & Materials Science, and Jai S. Rudra, assistant professor in the Department of Biomedical Engineering, worked together to look at cocaine vaccines, which work by blocking cocaine’s ability to enter the brain.

Current candidates for such a vaccine don’t confer long-lasting results; they require frequent boosting. Singamaneni and Rudra wanted a better way to determine when the effects of the vaccine had waned. “We’ve shown that we can use the patches to understand whether a person is still producing the necessary antibodies,” Singamaneni said. “No blood draw necessary.”



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