Colder-than-average temperatures in the Antarctic stratosphere created ideal conditions for destroying ozone this year, but declining levels of ozone-depleting chemicals prevented the hole from as being as large as it would have been 20 years ago.

“Chlorine levels in the Antarctic stratosphere have fallen about 11 percent from the peak year in 2000,” said Paul A. Newman, chief scientist for Earth Sciences at NASA’s Goddard Space Flight Center in Greenbelt, Maryland. “This year’s colder temperatures would have given us a much larger ozone hole if chlorine was still at levels we saw back in the year 2000.”

According to NASA, the annual ozone hole reached an average area coverage of 8.83 million square miles (22.9 square kilometers) in 2018, almost three times the size of the contiguous United States. It ranks 13th largest out of 40 years of NASA satellite observations. Nations of the world began phasing out the use of ozone-depleting substances in 1987 under an international treaty known as the Montreal Protocol.

The 2018 ozone hole was strongly influenced by a stable and cold Antarctic vortex — the stratospheric low pressure system that flows clockwise in the atmosphere above Antarctica. These colder conditions — among the coldest since 1979 — helped support formation of more polar stratospheric clouds, whose cloud particles activate ozone-destroying forms of chlorine and bromine compounds.

In 2016 and 2017, warmer temperatures in September limited the formation of polar stratospheric clouds and slowed the ozone hole’s growth. In 2017, the ozone hole reached a size of 7.6 million square miles (19.7 square kilometers) before starting to recover. In 2016, the hole grew to 8 million square miles (20.7 square kilometers).

However, the current ozone hole area is still large compared to the 1980s, when the depletion of the ozone layer above Antarctica was first detected. Atmospheric levels of human-made ozone-depleting substances increased up to the year 2000. Since then, they have slowly declined but remain high enough to produce significant ozone loss.

NOAA scientists said colder temperatures in 2018 allowed for near-complete elimination of ozone in a deep, 3.1-mile (5-kilometer) layer over the South Pole. This layer is where the active chemical depletion of ozone occurs on polar stratospheric clouds. The amount of ozone over the South Pole reached a minimum of 104 Dobson units on Oct. 12 — making it the 12th lowest year out of 33 years of NOAA ozonesonde measurements at the South Pole, according to NOAA scientist Bryan Johnson.

“Even with this year’s optimum conditions, ozone loss was less severe in the upper altitude layers, which is what we would expect given the declining chlorine concentrations we’re seeing in the stratosphere,” Johnson said.

A Dobson unit is the standard measurement for the total amount of ozone in the atmosphere above a point on Earth’s surface, and it represents the number of ozone molecules required to create a layer of pure ozone 0.01 millimeters thick at a temperature of 32 degrees Fahrenheit (0 degrees Celsius) at an atmospheric pressure equivalent to Earth’s surface. A value of 104 Dobson units would be a layer that is 1.04 millimeters thick at the surface, less than the thickness of a dime.

Prior to the emergence of the Antarctic ozone hole in the 1970s, the average amount of ozone above the South Pole in September and October ranged from 250 to 350 Dobson units.

What is ozone and why does it matter?

Ozone comprises three oxygen atoms and is highly reactive with other chemicals. In the stratosphere, roughly 7 to 25 miles (about 11 to 40 kilometers) above Earth’s surface, a layer of ozone acts like sunscreen, shielding the planet from ultraviolet radiation that can cause skin cancer and cataracts, suppress immune systems and damage plants. Ozone can also be created by photochemical reactions between the Sun and pollution from vehicle emissions and other sources, forming harmful smog in the lower atmosphere.

NASA and NOAA use three complementary instrumental methods to monitor the growth and breakup of the ozone hole each year. Satellite instruments like the Ozone Monitoring Instrument on NASA’s Aura satellite and the Ozone Mapping Profiler Suite on the NASA-NOAA Suomi National Polar-orbiting Partnership satellite measure ozone across large areas from space. The Aura satellite’s Microwave Limb Sounder also measures certain chlorine-containing gases, providing estimates of total chlorine levels.

The total amount of ozone in the atmosphere is exceedingly small. All of the ozone in a column of the atmosphere extending from the ground to space would be 300 Dobson units, approximately the thickness of two pennies stacked one on top of the other.

NOAA scientists monitor the thickness of the ozone layer and its vertical distribution above the South Pole by regularly releasing weather balloons carrying ozone-measuring “sondes” up to 21 miles (~34 kilometers) in altitude, and with a ground-based instrument called a Dobson spectrophotometer.

In a paper in the journal Science, the researchers show that varying the spacing between twin boundaries, as opposed to maintaining consistent spacing throughout, produces dramatic improvements in a metal’s strength and rate of work hardening — the extent to which a metal strengthens when deformed.

Huajian Gao, a professor in Brown’s School of Engineering who co-led the work, says the research could point toward new manufacturing techniques for high-performance materials.

“This work deals with what’s known as a gradient material, meaning a material in which there’s some gradual variation in its internal makeup,” Gao said. “Gradient materials are a hot research area because they often have desirable properties compared to homogeneous materials. In this case, we wanted to see if a gradient in nanotwin spacing produced new properties.”

Gao and his colleagues have already shown that nanotwins themselves can improve material performance. Nanotwinned copper, for example, has shown to be significantly stronger than standard copper, with an unusually high resistance to fatigue. But this is the first study to test the effects of variable nanotwin spacing.

Gao and his colleagues created copper samples using four distinct components, each with different nanotwin boundary spacing. Spacings ranging from 29 nanometers between boundaries to 72 nanometers. The copper samples were comprised of different combinations of the four components arranged in different orders across the thickness of the sample. The researchers then tested the strength of each composite sample, as well as the strength of each of the four components.

The tests showed that all of the composites were stronger than the average strength of the four components from which they were made. Remarkably, one of the composites was actually stronger than the strongest of its constituent components.

“To give an analogy, we think of a chain as being only as strong as its weakest link,” Gao said. “But here, we have a situation in which our chain is actually stronger than its strongest link, which is really quite amazing.”

Other tests showed that the composites also had higher rates of work hardening than the average of their constituent components.

To understand the mechanism behind these increases in performance, the researchers used computer simulations of their samples’ atomic structure under strain. At the atomic level, metals respond to strain through the motion of dislocations — line defects in the crystalline structure where atoms are pushed out of place. The way in which those dislocations grow and interact with each other is what determines a metal’s strength.

The simulations revealed that the density of dislocations is much higher in the gradient copper than in a normal metal.

“We found a unique type of dislocation we call bundles of concentrated dislocations, which lead to dislocations an order of magnitude denser than normal,” Gao said. “This type of dislocation doesn’t occur in other materials and it’s why this gradient copper is so strong.”

Gao said that while the research team used copper for this study, nanotwins can be produced in other metals as well. So it’s possible that nanotwin gradients could improve the properties of other metals.

“We’re hoping that these findings will motivate people to experiment with twin gradients in other types of materials,” Gao said.

Other authors on the study were Zhao Cheng, Haofei Zhou, Qiuhong Lu and Lei Lu. The research was funded by the U.S. National Science Foundation and National Natural Science Foundation of China.

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Hao Yan, a researcher at ASU, is a practiced hand in this delicate and exotic field, operating at the crossroads of nanotechnology and fine art.

In new research appearing in the journal Nature Communications, Yan and his colleagues Fei Zhang, Xiaodong Qi and others describe a method for coaxing segments of single-stranded DNA into complex 2- and 3D knotted structures.

The results represent an important advance in the fast-paced field of DNA nanotechnology, in which the molecule of life is used as a structural building material for a vast array of tiny configurations. Among these are miniscule robotic devices, photonic applications, drug delivery systems, logic gates, as well as diagnostic and therapeutic applications.

“The knotted DNA structures demonstrated in this work exhibit unprecedented topological complexity, far beyond what has been achieved before using single stranded folding,” Yan says. “Indeed, it is not only amazing but also surprising that the single-stranded DNA and RNA can thread through its own chains and find a way to form such highly knotted structures, given the fact that the single strand has to weave through so many tangles.”

Yan directs the Biodesign Center for Molecular Design and Biomimetics and is the Martin D. Glick Distinguished Professor in the School of Molecular Sciences at ASU.

Bringing DNA into the fold

The new study involves innovations in the field of DNA origami, which, as the name implies, uses nucleic acids like DNA and RNA to fold and self-assemble into complex forms. This occurs when complementary nucleotide bases in DNA’s 4-letter alphabet come in contact and bind, according to a strict regimen: C bases always pair with G and A bases always pair with T.

In nature, strings of nucleic acids provide the code needed to make complex proteins. This basic biology provides the underpinning for all earthly life. Taking advantage of the simple base-pairing properties of DNA, it is possible to design structures that will self-assemble in the lab. The method has been applied to both single-stranded and double-stranded DNA forms, resulting in nanostructures of increasing complexity and sophistication.

While DNA origami has made startling advances since its inception, one technical innovation has been vexingly difficult to achieve. Until now, creating complex knotted structures in DNA in a predictable and programmable way has eluded researchers.

The new work overcomes this hurdle, establishing precise design rules that permit single-stranded segments of DNA (or RNA) ranging from 1800-7500 nucleotides to form knot-like nanostructures with crossing numbers (where the DNA strand weaves in and out of its own length) ranging from 9 to 57.

The group further demonstrated that these nucleic acid nanostructures can be replicated and amplified, both under laboratory conditions and within living systems.

Nature’s knots

Knotted structures, like those Yan has fabricated, (but much simpler than the synthetic ones), have correlates in the natural world. They have been observed in DNA and proteins and generally form during replication and transcription, (when a DNA sequence is copied into messenger RNA). They can also occur in the genomes of phages — viruses that infect bacterial cells.

Nevertheless, the construction of molecular knots at the nanometer scale, displaying well-defined and consistent geometries requires enormous control and precision. As it happens, nucleic acids like DNA are ideal for the design and synthesis of such molecular knots.

Previously, lengths of double-stranded DNA have been used for nanoscale constructions, with the addition of short pieces or “staple strands” to fasten resulting structures together. The new study instead uses a single length of DNA designed to wrap around itself in a precise, pre-programmed sequence of steps.

Once the knotted DNA nanostructures successfully assemble themselves, they are imaged using atomic force microscopy. Careful calculation allows the researchers to optimize the folding pathways to produce the highest yield for each synthetic structure. The use of single- rather than double-stranded DNA allows the structures to be produced in abundance at much lower cost.

A single-stranded approach opens the door for the design of nanoarchitectures with specific, well-defined functions, which can be produced through successive rounds of in vitro evolution, where desired attributes are selected for in a repetitive process of refinement. Further, the approach outlined in the new study provides a general platform for the design of molecular structures of increased size and unprecedented complexity, paving the way for advances in nanophotonics, drug delivery, cryo-EM analysis and DNA-based memory storage.

Designer DNA (and RNA)

For one of the initial knot designs the strategy Yan and his colleagues developed involved threading a single strand of DNA or RNA through itself 9 times according to a pre-programmed sequence, demonstrating that the new method is capable of producing intricate geometric shapes that are programmable, replicable and scalable.

The design strategy was subsequently expanded to include single-stranded RNA structures and 3D DNA knots, whose forms were reconstructed using a technique known as cryogenic transmission electron microscopy, confirming their proper folding into the desired shapes.

“One of the challenges in this work is how to increase the assembly yield of highly knotted structures.” Said Fei. Unlike classic DNA nanostructures, the single-stranded knots are less forgiving in terms of precise folding order due to the topological complexity. If a single crossing is misfolded during the process, the error will hardly be self-corrected and most of the misfoldings will remain in the completed structure. “We developed a hierarchical folding strategy to guide the correct formation of knots. We compared the folding efficiency of a knot with 23 crossings by using different folding pathways. The AFM images showed a dramatic increase in the folding yield of well-form structures from 0.9% to 57.9% by applying optimized hierarchical folding pathway.” Fei added.

The design rules used to optimize the folding pathways are based on the number of crossing points, the length of DNA and the number of base pairs in the designed structure. Three primary rules were established. First, linear folding paths were found to be preferable to branched paths. Second, the unfolded section of a DNA strand should not thread through itself in the early stages when the strand is still long. Finally, edges of the desired form that have three crossings should fold before those with two crossings.

Following the design strategy, the team was able to create more complex DNA knots with increasing crossing numbers.

Longer chains of single-stranded DNA pose unique challenges for designing programmed nanostructures due to the increased likelihood of unintended self-complementarity of the bases making up the chain. A DNA knot structure boasting 57 crossed nodes successfully assembled, though with lower yield and less precision. When the crossing number was increased to 67, the yield significantly dropped and the resulting structures, imaged by AFM, showed more errors of assembly.

The study reports the largest DNA knots yet assembled, formed from up to 7.5k bases, featuring the most complicated topologies, with up to 57 crossing regions. The single-stranded DNA sequences can be mass produced in living cells for greater efficiency at lower cost. Ultimately, DNA nanostructures of diverse function may be formed within cells, innovations to be pursued in future work.

This includes a deadly poultry influenza virus in China called the H7N9 avian flu virus. Since 2013, H7N9 has infected 1,625 people, killing 623. While not highly contagious for humans, just three mutations could change that, turning H7N9 into the feared Disease X, the term health experts use for the next unknown cause of a worldwide epidemic.

In research to improve influenza therapies against H7N9 and other influenza strains, Petit and his University of Alabama at Birmingham colleagues have detailed the binding site and mechanism of inhibition for two small-molecule experimental inhibitors of influenza viruses. Their report is published in the Journal of Biochemistry,

The two experimental inhibitors studied by Petit, a UAB assistant professor of biochemistry and molecular genetics, are small molecules whose precise mechanism of action was unknown. The inhibitors target the function of a key influenza protein called NS1, which has multiple roles to block the body’s immune response during influenza infection. Thus, NS1 is essential to the survival and adaptability of the influenza virus.

Petit and colleagues used nuclear magnetic resonance, or NMR, spectroscopy to probe interactions of the inhibitors with NS1. They first showed that the inhibitors — called A9 and A22 — interacted with just one of the two independently folded domains of NS1, the NS1 effector domain.

The researchers noted that the structures of both small-molecule inhibitors were very similar to a fragment of a host protein called CPSF30 that the NS1 effector domain binds in order to short-circuit the body’s immune response. Therefore, the researchers hypothesized that A9 and A22 block influenza viral replication and block NS1 function by interfering with the interaction between the NS1 effector domain and CPSF30.

NMR data revealed the particular amino acids of the NS1 effector domain that are involved in inhibitor binding. The researchers — using two significantly different NS1 proteins from distinct influenza strains, including the H7N9 strain — showed that similar sequences of amino acids in the two NS1 proteins were involved in inhibitor binding.

The 1918 “Spanish” flu NS1 protein

Besides the Chinese H7N9 NS1, the other NS1 protein tested was the NS1 effector domain from the 1918 “Spanish” flu, which infected one-third of the world’s population a century ago and killed 50 million to 100 million people.

The UAB researchers then used X-ray crystallography, led by UAB Microbiology assistant professor Todd Green, Ph.D., to determine the three-dimensional structure of the NS1 effector domain from the 1918 “Spanish” flu. This allowed them to map the A9/A22-binding site onto that structure, which confirmed their hypothesis — A9 and A22 interact with the NS1 effector domain hydrophobic pocket that is known to bind the host protein CPSF30.

The crystallography data also showed that the NS1 effector domain is able to dimerize, using an interface different from two other known dimers of the NS1 effector domain. Biological significance of this new dimer form is unknown.

“Altogether, our findings provide strong evidence for the mechanism of action of two anti-influenza compounds that target NS1, and the findings contribute significant structural insights into NS1 that we hope will promote and inform the development and optimization of influenza therapies based on A9 and A22,” Petit said.

The need for novel antiviral compounds is great. Each year, influenza strains kill 250,000 to 500,000 people worldwide, and the virus is noted for quick changes to produce pandemic strains that few people have immunity against. Viral resistance has limited the effectiveness of several earlier antiviral compounds that were developed to treat influenza.