This work appeared Dec. 1 in the journal Nature Methods.

Imaging deep tissues with light is challenging. Visible light is often quickly absorbed and scattered by structures and molecules in the body, preventing researchers from seeing deeper than a millimeter within a tissue. If they do manage to probe further, substances like collagen or melanin often muddy the image, creating the equivalent of background noise through their natural fluorescence.

“Biological molecules naturally absorb and emit light in the visible spectrum, which is about 350 to 700 nanometers,” said Junjie Yao, assistant professor of biomedical engineering at Duke. “So when using it to image deep tissue, it’s like trying to observe the stars in daylight. The signals get flooded out.”

To wade out from these muddied waters, Yao and his collaborator Vladislav Verkhusha, professor of genetics at Albert Einstein College of Medicine, New York developed a protein that absorbs and emits longer wavelengths of light in the near-infrared (NIR) spectrum.

“Tissue is the most transparent in the 700-1300 nanometer window of NIR light,” said Yao. “At those wavelengths, light can penetrate deeper into a tissue, and because there is less natural background fluorescence to filter out, we can take longer exposures and capture clearer images.”

Verkhusha and his lab used a process called directed molecular evolution to engineer their proteins, using photoreceptors normally found in bacteria as the basis for the structure. These photoreceptors are useful for imaging research because they can switch between a silent and active state when hit with a specific wavelength of light. They can bind with biliverdin, a biomolecule that appears in high quantities in mammalian and human tissues.

“We studied the structure of the biliverdin to determine how the photoreceptor would best attach to the biomolecule,” explained Verkhusha. “After we understood the binding process, we carefully introduced substitutions of key parts of the molecule connecting to the biliverdin to increase the electron binding, which is necessary to obtain the red-shifted fluorescence. Last, we performed random mutagenesis followed by high-throughput screenings so the proteins would evolve and increase their brightness.”

The brightest found protein, termed miRFP718nano, is easily produced in cells and tissues and emits light just outside of the visible range. But while the NIR activation itself is helpful, what happens after the initial burst of activity holds even more promise for biomedical imaging.

“We’ve seen that the NIR range can be broken up into two main zones,” explained Yao. “When NIR light first hit these proteins, they emit light in the first zone, which is about 700-900 nanometers. But as they decay, the wavelength gradually gets longer, like the tail behind a comet. That’s when they begin to emit light in the second NIR zone, which is from 900-1300 nanometers.”

In this second zone, all benefits from using the shorter wavelength, zone one NIR light are enhanced: light can penetrate tissue two times deeper, background fluorescence is significantly dimmed, and image resolution can be two to three times clearer, allowing for detailed images of complex structures.

As a proof of concept, Yao and his team at Duke used an imaging technique called short-wavelength infrared (SWIR) to test the efficacy of the new protein. This process sends NIR zone-one light deep into the tissue to activate the fluorescent proteins. As the proteins decay, they emit NIR zone-two light that provides information about the structure and composition of the targeted tissues and cells that can be translated into high-resolution images.

After introducing engineered miRFP718nano proteins into their animal models, the team used them to capture images of microbes in the mouse digestive tract, visualize cells in a mouse mammary gland and even track changes to inflammation in a mouse liver. All the images captured were clearer and more detailed than images made using a standard NIR zone-one imaging protein.

Yao and Verkhusha are optimistic that their continued partnership will be a boon for their work in both biomedical imaging and protein engineering. As Verkhusha continues to refine and improve the fluorescent proteins and biosensors, Yao is excited to use the new tool to visualize the brain more closely and to potentially track the movement of cancer cells.

“This is an exciting new front of our decade-long collaboration because we can use the imaging tools to guide the protein engineering decisions, and we can use the advanced protein engineering to improve the imaging capabilities,” Yao said.

This work was supported by grants from the US National Institutes of Health (GM122567, EB028143, NS111039, NS115581), the Chan Zuckerberg Initiative (226178), the Cancer Foundation Finland and Magnus Ehrnrooth Foundation, and the NIH Intramural Research Program for the Vaccine Research Center of the National Institute of Allergy and Infectious Diseases.

BSIs with various microbial pathogens can rapidly escalate to life-threatening sepsis when the body is overwhelmed by the multiplying invaders and shuts down its organs’ functions. In 2017, there were 48.9 million cases and 11 million sepsis-related deaths worldwide. Importantly, almost half of all global sepsis cases occurred among children, with an estimated 20 million cases and 2.9 million global deaths in those under five years of age.

To prevent BSIs from progressing to full-blown sepsis, the infection-causing bacterial or fungal species must be identified as fast as possible. Only then can optimal pathogen-tailored antibacterial or antifungal treatments be applied in time. The conventional method used in clinical laboratories to identify the causative pathogenic species is long and laborious, requiring two time-consuming culture steps that take at least 1 to 3 days to complete.

“For all patients with sepsis, their chances of surviving dramatically shrink the longer it takes to identify the infection-causing pathogen(s) and thus, receive the most promising antimicrobial treatment,” said Nigel Klein, M.D., Ph.D., pls, a Professor of Infectious Disease and Immunology at GOSH, and a senior author on the study. “At Great Ormond Street Hospital we have been working to demonstrate both the importance rapid diagnosis and the fact that with innovative approaches we can identify the causative organism in between 40 minutes and six hours. Compared to adult patients, sepsis in infants and small children progresses much faster, and therefore there is a real need for diagnostic methods that support early detection. Accurate diagnosis is even more significant due to the availability of only small blood volumes from paediatric patients which can make re-sampling challenging.”

In 2020, senior authors Klein and Elaine Cloutman-Green, Ph.D., a Consultant Clinical Scientist and Infection Control Doctor at GOSH, began collaborating with Lead Staff Scientist Michael Super, Ph.D. and Founding Director Donald Ingber, M.D., Ph.D. at Harvard’s Wyss Institute to solve this problem. “Based on our earlier success with FcMBL in isolating pathogens from joints as well as bovine and human blood with extraordinary efficiencies, we hypothesized that building an FcMBL-mediated pathogen capture into a modified clinical blood culture protocol could shorten the time and reduce the size of the required patient samples to yield the same results that time-consuming blood culture protocols provide,” said Super.

In the pathogen identification process currently performed in clinical settings, first, blood samples are added to bottles containing liquid media in which infectious microbes, if present, are amplified to a certain density. Then, the amplified microbes are grown on solid media as isolated colonies whose constituent cells eventually can be identified with a highly sensitive, yet fast and relatively inexpensive analytical method know as MALDI-TOF mass spectrometry (MS). “Indeed, isolating the infectious microbes directly from grown liquid blood cultures using FcMBL makes them available for MALDI-TOF MS analysis much earlier,” added Super.

FcMBL is the key component of a broad-spectrum pathogen capture technology. It consists of a genetically engineered human immune protein called mannose-binding lectin (MBL) that is fused to the Fc fragment of an antibody molecule to produce the resulting FcMBL protein. In this configuration, the MBL portion of FcMBL can capture more than 100 [CHECK WITH MIKE] different microbial species with high efficiency, including virtually all of the bacterial and fungal pathogens causing sepsis. FcMBL’s Fc portion can be used to couple it to magnetic beads, allowing the captured pathogens to be quickly pulled out of patient samples and liquid blood cultures.

In the earlier stages of the project, the Wyss team provided purified bead-coupled FcMBL to the GOSH team, which had access to blood samples from pediatric patients at the hospital. At later stages, the sepsis and infectious disease company BOA Biomedical, co-founded by Super and Ingber to commercialize the Wyss Institute’s FcMBL technology, provided the FcMBL reagent and critical expertise to the project. BOA Biomedical meanwhile developed the manufacturing capabilities for FcMBL that the Food and Drug Administration (FDA) in the US and other federal health agencies require for producing therapeutic and diagnostic products.

“Sepsis is the leading killer in hospitals, and rapidly initiating the right antibiotic saves lives. Using work originally developed at the Wyss Institute, BOA Biomedical’s revolutionary FcMBL technology helps to quickly and accurately identify the pathogen causing sepsis, ushering in a new era of targeted antimicrobial therapy to help individual patients and curb society’s deadly antimicrobial resistance problem,” stated Mike McCurdy, M.D., Chief Medical Officer of BOA Biomedical.

In addition to using the gold standard two-step blood culture in combination with MALDI-TOF MS pathogen identification, the team also included the Bruker Corporation’s MBT Sepsityper® kit as a comparison. Brought to market in 2021, the MBT Sepsityper® essentially eliminates the time-consuming second microbial culture step by lysing microbial cells from the liquid culture and spinning the fragments down in a centrifuge before analyzing them by MALDI-TOF mass spectrometry analysis. Although it accelerates the overall diagnostic process, the MBT Sepsityper® methodproduces lower microbial detection rates than those obtained with the conventional culture method, which means that it may still fail to identify the infection-causing pathogen in a significant fraction of blood samples.

“Our FcMBL approach has opened up the opportunity to identify pathogenic organisms to guide treatment 24 to 48 hours earlier than would be possible using standard culture techniques. It has also enabled us to use this identification to make any ongoing culture for antibiotic sensitivities more tailored to the needs of the patient. This method isn’t tied into a specific platform or manufacturer, and thus we see clear potential for it to become a new standard processing step for clinical pathogen detection,” said Cloutman-Green.

“The FcMBL method identified 94.1% of microbial species found in clinical blood culture analysis with samples from 68 pediatric patients,” said first author Kerry Kite, who performed her graduate work with Klein and Cloutman-Green. “We were able to identify more infectious species in positive liquid blood cultures using the FcMBL method than with the MBT Sepsityper® method (25 of 25 vs 17 of 25), and this trend was even further pronounced in the case of the common fungal pathogen Candida (24 of 24 vs 9 of 24).” Candida species account for about 5% of all cases of severe sepsis and are the fourth most common pathogen isolated from patients’ bloodstreams in the United States. Not only do infections with Candida and other fungi require specific antifungal treatments, distinguishing among the various types of pathogenic fungi helps direct the appropriate antimicrobial therapy. Specifically in neonatal intensive care units, Candidainfections are a major cause of morbidity and mortality, killing as many as 40% of infants and often causing neurodevelopmental impairments in those that survive.

“By continuously adapting the powerful FcMBL pathogen capture technology to unmet and pressing diagnostic needs, such as the rapid diagnosis of sepsis in pediatric patients, we hope to profoundly alter the frequently dismal prospects of patients of all ages,” said Ingber. “Our ultimate goal is to be able to accurately and even more rapidly identify pathogens directly in small samples of blood without the need for any additional microbial cultures.” Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

The study was also authored by Sahil Loomba and Thomas Elliott at Imperial College London; Francis Yongblah, Lily Gates and Dagmar Alber at GOSH; George Downey and James Hill at BOA Biomedical; and Shanda Lightbown and Thomas Doyle at the Wyss Institute. The authors were supported in their work by the clinical microbiology staff at GOSH, as well as Erika Tranfield with MALDI-TOF MS expertise. At GOSH, critical financial help for the project from the Benecare Foundation, philanthropists Luca Albertini and Professor Pauline Barrieu, as well the Office of Vice-President (Advancement) at University College London was coordinated by Simona Santojanni. At the Wyss Institute, the study was funded by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement Number W911NF-16-C-0050, and the Wyss Institute’s technology translation engine. Additional support was provided by BOA Biomedical.

Tiny biological nanoparticles called “small extracellular vesicles” (sEVs) are released from every type of cell in the body and are believed to play a large role in cell-to-cell communication and disease transmission. The new technology, dubbed Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance, or ANSWER for short, not only pulls these nanoparticles from biofluids in under 10 minutes, it also sorts them into size categories believed to have distinct biological roles.

The results appeared online November 23 in the journal Science Advances.

“These nanoparticles have significant potential in medical diagnosis and treatment, but the current technologies for separating and sorting them take several hours or days, are inconsistent, produce low yield or purity, suffer from contamination and sometimes damage the nanoparticles,” said Tony Jun Huang, the William Bevan Distinguished Professor of Mechanical Engineering and Materials Science at Duke.

“We want to make extracting and sorting high-quality sEVs as simple as pushing a button and getting the desired samples faster than it takes to take a shower,” Huang said.

Recent research indicates that sEVs are composed of several subgroups with distinct sizes (e.g., smaller than 50 nanometers, between 60 and 80 nanometers, and between 90 and 150 nanometers). Each size is believed to have different biological properties.

The recent discovery of sEV subpopulations has excited researchers because of their potential to revolutionize the field of non-invasive diagnostics, such as the early detection of cancer and Alzheimer’s disease. But the particles haven’t found their way into clinical settings yet.

Huang said this is largely due to the difficulties associated with separating and isolating these nano-sized sEV subpopulations. To meet this challenge, Huang, his doctoral student Jinxin Zhang, and collaborators at UCLA, Harvard, and Magee-Womens Research Institute, developed the ANSWER platform.

The device uses a single pair of transducers to generate a standing sound wave that envelops a narrow, enclosed channel filled with fluid. The sound wave “leaks” into the liquid center through the channel walls and interacts with the original standing sound wave. With careful design of the wall thickness, channel size and sound frequency, this interaction creates a resonance that forms “virtual pillars” along the center of the channel.

Each of these virtual pillars is essentially a half-egg-shaped region of high pressure. As particles attempt to cross over the pillars, they get pushed toward the edges of the channel. And the bigger the particles, the bigger the push. By tuning the series of virtual pillars to create nuanced forces on the traveling nanoparticles, the researchers can precisely sort them by size into a variety of groups determined by the needs of the experiments at hand.

“The ANSWER EV fractionation technology is the most advanced capability for precise EV fractionation, and it will significantly impact the horizon of EV diagnostics, prognostics and liquid biopsy,” said David Wong, director for UCLA Center for Oral/Head & Neck Oncology Research.

In the new paper, the researchers demonstrate that their ANSWER platform can successfully sort sEVs into three subgroups with 96% accuracy for nanoparticles on the larger end of the spectrum and 80% accuracy for the smallest. They also show flexibility in their system, adjusting the number of groupings and ranges of sizes with simple updates to the sound wave parameters. Each of the experiments only took 10 minutes to complete, whereas other methods such as ultra-centrifugation can take several hours or days.

“Due to its contact-free nature, ANSWER offers a biocompatible approach for the separation of biological nanoparticles.” Zhang said. “Unlike mechanical filtration methods, which have fixed separation cutoff diameters, ANSWER offers a tunable approach to nanoscale separation, and the cutoff diameter can be precisely modified by varying the input acoustic power.”

Moving forward, the researchers will continue improving the ANSWER technology so that it can be efficient in purifying other biologically relevant nanoparticles such as viruses, antibodies and proteins.

This research was supported by the National Institutes of Health (UH3TR002978, R01HD103727, U18TR003778, R01GM132603, R01GM143439 R01GM135486, R01GM144417, R44AG063643) and the National Science Foundation (CMMI-2104295, CMMI-2104526).

Phase transitions describe various phenomena around us, from water turning into ice to magnetic transitions to the superconducting transition where electrical resistance vanishes. In the cases of superconductivity and magnetism, the phase transition is continuous, characterized by “symmetry breaking” that leads to the formation of an ordered state. The ordered state is perfect (defect-free) when this transition is very slow, a regime called the “adiabatic limit.” However, for transitions not satisfying this limit, there appear topological defects, whose generation is described by the Kibble-Zurek (KZ) mechanism. Experimentally, the KZ mechanism manifests as a power-law dependence of the defect density on the cooling rate.

Interestingly, the KZ mechanism, while widely studied for phase transitions at thermal equilibrium, has not yet been demonstrated experimentally for nonequilibrium phase transitions. However, a recent simulation study has suggested that the KZ mechanism can be applied to dynamical ordering transitions between disordered and ordered flow states, a phenomenon that can be experimentally tested in superconducting vortex systems.

To this end, a research group from the Tokyo Institute of Technology (Tokyo Tech), Japan led by Prof. Satoshi Okuma recently showed that the state of motion of a collection of magnetic fluxes (vortices) penetrating a superconductor is in the process of nonequilibrium phase transition from disordered flow to ordered lattice flow, and the lattice defects spontaneously appear in line with the KZ mechanism. Their breakthrough study was published in Physical Review Letters (selected as an Editors’ suggestion).

In their work, the team prepared a 330-nm thick strip-shaped film of amorphous Mo_xGe_1−x (x ≈ 0.78) on a silicon substrate and then cooled it down to enable a superconducting transition at 6.3 K. By applying a magnetic field perpendicular to the surface, the vortices were generated and experiments were performed at 4.1 K under a field strength of 3.5 T.

The team drove the vortices using a driving current that was linearly ramped up at various quench rates (dI/dt). On reaching the end-point of quench, the vortex configuration was frozen by turning off the current abruptly.

“In our study, we tested the simulation predictions by experimentally examining the configurational order of vortices after undergoing dynamical ordering as a function of the quench rate,” explains Prof. Okuma.

The team found that the vortex configuration became less ordered with increasing quench rates, signifying a phase transition. “We investigated the lattice defects appearing during this transition and their variation with the quench rate,” says Prof. Okuma. “We found that the defect density scaled as a power law with the quench rate, consistent with the KZ scenario,” he highlights. The team additionally estimated the power law exponent (≈ 0.4-0.5), which was close to the value predicted by simulation (0.39).

“We also observed an impulse-adiabatic crossover on the ordered side of the transition, another key prediction of the KZ mechanism,” adds Prof. Okuma.

Overall, this study extends the applicability of the Kibble-Zurek mechanism from equilibrium phase transitions to nonequilibrium phase transitions, opening the door to countless new investigations. Given this landmark result, new developments are expected in the research field of nonequilibrium phase transitions.

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Materials provided by Tokyo Institute of Technology. Note: Content may be edited for style and length.

The findings were published in a Royal Society Open Science study by recent RIT alumnus Madhusudan Rajendran ’22 MS (bioinformatics) and Associate Professor Gregory Babbitt from the Thomas H. Gosnell School of Life Sciences. They studied how the viral spike proteins in several SARS-CoV-2 variants interact with the host cell receptors known as ACE2 in both humans and various bats of genus Rhinolophus. Babbitt said the results were surprising.

“We were hoping to see really cool adaptive evolution happening as the virus got more used to humans and less used to bats, but we actually saw that there wasn’t a whole lot of change,” said Babbitt. “Because this binding site has not evolved very much, there’s really not much stopping it from transmitting from humans to bats. If you look at the phylogenetic relationships of bats to humans, we’re pretty far apart on the mammalian tree. So it suggests that there would be pretty widespread cross-species infectivity, and the literature has shown there’s been a lot of evidence of that.”

The scientists used a computer simulation method called molecular dynamics to put proteins in a solvated simulation and then watch them move. The approach uses high performance computing on graphics processors to show what every atom does over time. Babbitt said this approach allows scientists to study questions that cannot be answered in a traditional laboratory.

“It would be dangerous to do experiments where we reinfected bats with human viral strains, so our computer-based simulations offered a much safer alternative,” said Babbitt.

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Materials provided by Rochester Institute of Technology. Original written by Luke Auburn. Note: Content may be edited for style and length.