Now, researchers at MIT and the Woods Hole Oceanographic Institution (WHOI), in collaboration with oceanographers and marine biologists in Cuba, have identified microbes living within the slimy biofilms of some coral species that may help protect the coral against certain nutrient imbalances.

The team found these microbes can take up and “scrub out” nitrogen from a coral’s surroundings. At low concentrations, nitrogen can be an essential nutrient for corals, providing energy for them to grow. But an overabundance of nitrogen, for instance from the leaching of nitrogen-rich fertilizers into the ocean, can trigger mats of algae to bloom. The algae can outcompete coral for resources, leaving the reefs stressed and bleached of color.

By taking up excess nitrogen, the newly identified microbes may prevent algal competition, thereby serving as tiny protectors of the coral they inhabit. While corals around the world are experiencing widespread stress and bleaching from global warming, it seems that some species have found ways to protect themselves from other, nitrogen-related sources of stress.

“One of the aspects of finding these organisms in association with corals is, there’s a natural way that corals are able to combat anthropogenic influence, at least in terms of nitrogen availability, and that’s a very good thing,” says Andrew Babbin, the Doherty Assistant Professor in Ocean Utilization in MIT’s Department of Earth, Atmospheric and Planetary Sciences. “This could be a very natural way that reefs can protect themselves, at least to some extent.”

Babbin and his colleagues have reported their findings in the the ISME Journal.

Dead zone analogues

Babbin’s group studies how marine communities in the ocean cycle nitrogen, a key element for life. Nitrogen in the ocean can take various forms, such as ammonia, nitrite, and nitrate. Babbin has been especially interested in studying how nitrogen cycles, or is taken up, in anoxic environments — low-oxygen regions of the ocean, also known as “dead zones,” where fish are rarely found and microbial life can thrive.

“Locations without enough oxygen for fish are where bacteria start doing something different, which is exciting to us,” Babbin says. “For instance, they can start to consume nitrate, which has then an impact on how productive a specific part of the water can be.”

Dead zones are not the only anoxic regions of the ocean where bacteria exhibit nitrogen-feasting behavior. Low-oxygen environments can be found at smaller scales, such as within biofilms, the microbe-rich slime that covers marine surfaces from shipwrecked hulls to coral reefs.

“We have biofilms inside us that allow different anaerobic processes to happen,” Babbin notes. “The same is true of corals, which can generate a ton of mucus, which acts as this retardation barrier for oxygen.”

Despite the fact that corals are close to the surface and within reach of oxygen, Babbin wondered whether coral slime would serve to promote “anoxic pockets,” or concentrated regions of low oxygen, where nitrate-consuming bacteria might thrive.

He broached the idea to WHOI marine microbiologist Amy Apprill, and in 2017, the researchers set off with a science team on a cruise to Cuba, where Apprill had planned a study of corals in the protected national park, Jardines de la Reina, or Gardens of the Queen.

“This protected area is one of the last refuges for healthy Caribbean corals,” Babbin says. “Our hope was to study one of these less impacted areas to get a baseline for what kind of nitrogen cycle dynamics are associated with the corals themselves, which would allow us to understand what an anthropogenic perturbation would do to that system.”

Swabbing for scrubbers

In exploring the reefs, the scientists took small samples from coral species that were abundant in the area. Onboard the ship, they incubated each coral specimen in its own seawater, along with a tracer of nitrogen — a slightly heavier version of the molecules found naturally in seawater.

They brought the samples back to Cambridge and analyzed them with a mass spectrometer to measure how the balance of nitrogen molecules changed over time. Depending on the type of molecule that was consumed or produced in the sample, the researchers could estimate the rate at which nitrogen was reduced and essentially denitrified, or increased through other metabolic processes.

In almost every coral sample, they observed rates of denitrification were higher than most other processes; something on the coral itself was likely taking up the molecule.

The researchers swabbed the surface of each coral and grew the slimy specimens on Petri dishes, which they examined for specific bacteria that are known to metabolize nitrogen. This analysis revealed multiple nitrogen-scrubbing bacteria, which lived in most coral samples.

“Our results would imply that these organisms, living in association with the corals, have a way to clean up the very local environment,” Babbin says. “There are some coral species, like this brain coral Diploria, that exhibit extremely rapid nitrogen cycling and happen to be quite hardy, even through an anthropogenic change, whereas Acropora, which is in rough shape throughout the Caribbean, exhibits very little nitrogen cycling. “

Whether nitrogen-scrubbing microbes directly contribute to a coral’s health is still unclear. The team’s results are the first evidence of such a connection. Going forward, Babbin plans to explore other parts of the ocean, such as the tropical Pacific, to see whether similar microbes exist on other corals, and to what extent the bacteria help to preserve their hosts. His guess is that their role is similar to the microbes in our own systems.

“The more we look at the human microbiome, the more we realize the organisms that are living in association with us do drive our health,” Babbin says. “The exact same thing is true of coral reefs. It’s the coral microbiome that defines the health of the coral system. And what we’re trying to do is reveal just what metabolisms are part of this microbial network within the coral system.”

This research was supported, in part, by MIT Sea Grant, the Simons Foundation, the MIT Montrym, Ferry, and mTerra funds, and by Bruce Heflinger ’69, SM ’71, PhD ’80.

Although the principles of DNA origami are straightforward, the technique’s tools and methods for designing new structures are not always easy to grasp and have not been well documented. In addition, scientists new to the method have had no single reference they could turn to for the most efficient way of building DNA structures and how to avoid pitfalls that could waste months or even years of research.

That’s why Jacob Majikes and Alex Liddle, researchers at the National Institute of Standards and Technology (NIST) who have studied DNA origami for years, have compiled the first detailed tutorial on the technique. Their comprehensive report provides a step-by-step guide to designing DNA origami nanostructures, using state-of-the-art tools. Majikes and Liddle described their work in the Jan .8 issue of the Journal of Research of the National Institute of Standards and Technology.

“We wanted to take all the tools that people have developed and put them all in one place, and to explain things that you can’t say in a traditional journal article,” said Majikes. “Review papers might tell you everything that everyone’s done, but they don’t tell you how the people did it. “

DNA origami relies on the ability of complementary base pairs of the DNA molecule to bind to each other. Among DNA’s four bases — adenine (A), cytosine (C), guanine (G) and thymine (T) — A binds with T and G with C. This means that a specific sequence of As, Ts, Cs and Gs will find and bind to its complement.

The binding enables short strands of DNA to act as “staples,” keeping sections of long strands folded or joining separate strands. A typical origami design may require 250 staples. In this way, the DNA can self-assemble into a variety of shapes, forming a nanoscale framework to which an assortment of nanoparticles — many useful in medical treatment, biological research and environmental monitoring — can attach.

The challenges in using DNA origami are twofold, said Majikes. First, researchers are fabricating 3D structures using a foreign language — the base pairs A, G, T and C. In addition, they’re using those base-pair staples to twist and untwist the familiar double helix of DNA molecules so that the strands bend into specific shapes. That can be difficult to design and visualize. Majikes and Liddle urge researchers to strengthen their design intuition by building 3D mock-ups, such as sculptures made with bar magnets, before they start fabrication. These models, which can reveal which aspects of the folding process are critical and which ones are less important, should then be “flattened” into 2D to be compatible with computer-aided design tools for DNA origami, which typically use two-dimensional representations.

DNA folding can be accomplished in a variety of ways, some less efficient than others, noted Majikes. Some strategies, in fact, may be doomed to failure.

“Pointing out things like ‘You could do this, but it’s not a good idea’ — that type of perspective isn’t in a traditional journal article, but because NIST is focused on driving the state of technology in the nation, we’re able to publish this work in the NIST journal,” Majikes said. “I don’t think there’s anywhere else that would have given us the leeway and the time and the person hours to put all this together.”

Liddle and Majikes plan to follow up their work with several additional manuscripts detailing how to successfully fabricate nanoscale devices with DNA.

The gene, called TSC2, codes for tuberin, a protein that acts to inhibit cell growth and proliferation. When mutations occur in TSC2, resulting in a lack of tuberin in cells, the cells enlarge and multiply, leading to the formation of tumors.

To restore the function of TSC2 and tuberin in a mouse model of tuberous sclerosis complex, researchers developed a form of gene therapy using an adeno-associated virus vector carrying the DNA that codes for a condensed form of tuberin (which fits within the vector’s carrying capacity) and functions like the normal full-length tuberin protein. Mice with tuberous sclerosis complex had a shortened life span of about 58 days on average, and they showed signs of brain abnormalities consistent with those that are often seen in patients with the disease. When the mice were injected intravenously with the gene therapy treatment, however, their average survival was extended to 462 days, and their brains showed reduced signs of damage.

“Current treatments for tuberous sclerosis complex include surgery and/or lifelong treatment with drugs that cause immune suppression and potentially compromise early brain development. Therefore, there is a clear need to identify other therapeutic approaches for this disease,” says co-lead author Shilpa Prabhakar, an investigator in the MGH departments of Neurology and Radiology. “Adeno-associated virus vectors have been used widely in clinical trials for many hereditary diseases with little to no toxicity, long-term action in nondividing cells, and improvement in symptoms,” adds Prabhakar. She notes that benefits can be seen after a single injection, and some forms of the viral vector can efficiently enter the brain and peripheral organs after intravenous injection.

The U.S. Food and Drug Administration has approved a limited number of gene therapy products for use in humans, and the results from this study suggest that clinical trials are warranted to test the strategy’s potential in patients with tuberous sclerosis complex.

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That’s according to an international study published Jan. 8 in The Lancet Respiratory Medicine, led by researchers at Vanderbilt University Medical Center in coordination with researchers in Spain.

The study, which is far the largest of its kind to date, tracks the incidence of delirium and coma in 2,088 COVID-19 patients admitted before April 28, 2020, to 69 adult intensive care units across 14 countries.

ICU delirium is associated with higher medical costs and greater risk of death and long-term ICU-related dementia. Seminal studies at VUMC over the past two decades have spurred widespread interest in ICU delirium research, and the resulting body of evidence has come to inform critical care guidelines endorsed by medical societies in several countries. These guidelines include well calibrated pain management with prompt discontinuation of analgesics and sedatives, daily spontaneous awakening trials, daily spontaneous breathing trials, delirium assessments throughout the day, early mobility and exercise, and family engagement.

Some 82% of patients in this observational study were comatose for a median of 10 days, and 55% were delirious for a median of three days. Acute brain dysfunction (coma or delirium) lasted for a median of 12 days.

“This is double what is seen in non-COVID ICU patients,” said VUMC’s Brenda Pun, DNP, RN, co-first author on the study with Rafael Badenes MD, PhD, of the University of Valencia in Spain. The authors cite a previous large, multi-site ICU study, also led by VUMC, where acute brain dysfunction lasted a median of five days, including four days of coma and one day of delirium.

The authors note that COVID-19 disease processes could predispose patient to a higher burden of acute brain dysfunction. But they also note that a number of patient care factors, some of which are related to pressures posed on health care by the pandemic, also appear to have played a significant role.

The study appears to show a reversion to outmoded critical care practices, including deep sedation, widespread use of benzodiazepine infusions (benzodiazepine is a nervous system depressant), immobilization, and isolation from families. The authors find that, where COVID-19 is concerned, there has been an apparent widespread abandonment of newer clinical protocols that are proven to help ward off the acute brain dysfunction that stalks many critically ill patients.

“It is clear in our findings that many ICUs reverted to sedation practices that are not in line with best practice guidelines,” Pun said, “and we’re left to speculate on the causes. Many of the hospitals in our sample reported shortages of ICU providers informed about best practices. There were concerns about sedative shortages, and early reports of COVID-19 suggested that the lung dysfunction seen required unique management techniques including deep sedation. In the process, key preventive measures against acute brain dysfunction went somewhat by the boards.”

Using electronic health records, investigators were able to closely examine patient characteristics, care practices and findings from clinical assessments. Some 88% of patients tracked in the study were invasively mechanical ventilated at some point during hospitalization, 67% on the day of ICU admission. Patients receiving benzodiazepine sedative infusions were at 59% higher risk of developing delirium. Patients who received family visitation (in-person or virtual) were at 30% lower risk of delirium.

“There’s no reason to think that, since the close of our study, the situation for these patients has changed,” said one of the study’s senior authors, Pratik Pandharipande, MD, MSCI, professor of Anesthesiology.

“These prolonged periods of acute brain dysfunction are largely avoidable. Our study sounds an alarm: as we enter the second and third waves of COVID-19, ICU teams need above all to return to lighter levels of sedation for these patients, frequent awakening and breathing trials, mobilization and safe in-person or virtual visitation.”

Pandharipande is co-director, with the study’s other senior author, Wesley Ely, MD, MPH, of the Critical Illness, Brain Dysfunction, and Survivorship Center. Pun is director of data quality with the center. Other VUMC investigators on the study include Onur Orun, MS, Wencong Chen, PhD, Rameela Raman, PhD, Beata-Gabriela Simpson, MPH, Stephanie Wilson-Linville, BSN, Nathan Brummel, MD, and Timothy Girard, MD.