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The study, by a team of Brown University researchers, used computer models to simulate the airflow inside a compact car with various combinations of windows open or closed. The simulations showed that opening windows — the more windows the better — created airflow patterns that dramatically reduced the concentration of airborne particles exchanged between a driver and a single passenger. Blasting the car’s ventilation system didn’t circulate air nearly as well as a few open windows, the researchers found.

“Driving around with the windows up and the air conditioning or heat on is definitely the worst scenario, according to our computer simulations,” said Asimanshu Das, a graduate student in Brown’s School of Engineering and co-lead author of the research. “The best scenario we found was having all four windows open, but even having one or two open was far better than having them all closed.”

Das co-led the research with Varghese Mathai, a former postdoctoral researcher at Brown who is now an assistant professor of physics at the University of Massachusetts, Amherst. The study is published in the journal Science Advances.

The researchers stress that there’s no way to eliminate risk completely — and, of course, current guidance from the U.S. Centers for Disease Control (CDC) notes that postponing travel and staying home is the best way to protect personal and community health. The goal of the study was simply to study how changes in airflow inside a car may worsen or reduce risk of pathogen transmission.

The computer models used in the study simulated a car, loosely based on a Toyota Prius, with two people inside — a driver and a passenger sitting in the back seat on the opposite side from the driver. The researchers chose that seating arrangement because it maximizes the physical distance between the two people (though still less than the 6 feet recommended by the CDC). The models simulated airflow around and inside a car moving at 50 miles per hour, as well as the movement and concentration of aerosols coming from both driver and passenger. Aerosols are tiny particles that can linger in the air for extended periods of time. They are thought to be one way in which the SARS-CoV-2 virus is transmitted, particularly in enclosed spaces.

Part of the reason that opening windows is better in terms of aerosol transmission is because it increases the number of air changes per hour (ACH) inside the car, which helps to reduce the overall concentration of aerosols. But ACH was only part of the story, the researchers say. The study showed that different combinations of open windows created different air currents inside the car that could either increase or decrease exposure to remaining aerosols.

Because of the way air flows across the outside of the car, air pressure near the rear windows tends to be higher than pressure at the front windows. As a result, air tends to enter the car through the back windows and exit through the front windows. With all the windows open, this tendency creates two more-or-less independent flows on either side of the cabin. Since the occupants in the simulations were sitting on opposite sides of the cabin, very few particles end up being transferred between the two. The driver in this scenario is at slightly higher risk than the passenger because the average airflow in the car goes from back to front, but both occupants experience a dramatically lower transfer of particles compared to any other scenario.

The simulations for scenarios in which some but not all windows are down yielded some possibly counterintuitive results. For example, one might expect that opening windows directly beside each occupant might be the simplest way to reduce exposure. The simulations found that while this configuration is better than no windows down at all, it carries a higher exposure risk compared to putting down the window opposite each occupant.

“When the windows opposite the occupants are open, you get a flow that enters the car behind the driver, sweeps across the cabin behind the passenger and then goes out the passenger-side front window,” said Kenny Breuer, a professor of engineering at Brown and a senior author of the research. “That pattern helps to reduce cross-contamination between the driver and passenger.”

It’s important to note, the researchers say, that airflow adjustments are no substitute for mask-wearing by both occupants when inside a car. And the findings are limited to potential exposure to lingering aerosols that may contain pathogens. The study did not model larger respiratory droplets or the risk of actually becoming infected by the virus.

Still, the researchers say the study provides valuable new insights into air circulation patterns inside a car’s passenger compartment — something that had received little attention before now.

“This is the first study we’re aware of that really looked at the microclimate inside a car,” Breuer said. “There had been some studies that looked at how much external pollution gets into a car, or how long cigarette smoke lingers in a car. But this is the first time anyone has looked at airflow patterns in detail.”

The research grew out of a COVID-19 research task force established at Brown to gather expertise from across the University to address widely varying aspects of the pandemic. Jeffrey Bailey, an associate professor of pathology and laboratory medicine and a coauthor of the airflow study, leads the group. Bailey was impressed with how quickly the research came together, with Mathai suggesting the use of computer simulations that could be done while laboratory research at Brown was paused for the pandemic.

“This is really a great example of how different disciplines can come together quickly and produce valuable findings,” Bailey said. “I talked to Kenny briefly about this idea, and within three or four days his team was already doing some preliminary testing. That’s one of the great things about being at a place like Brown, where people are eager to collaborate and work across disciplines.”

One of the major hurdles to combating the COVID-19 pandemic and fully reopening communities across the country is the availability of mass rapid testing. Knowing who is infected would provide valuable insights about the potential spread and threat of the virus for policymakers and citizens alike.

Yet, people must often wait several days for their results, or even longer when there is a backlog in processing lab tests. And, the situation is worsened by the fact that most infected people have mild or no symptoms, yet still carry and spread the virus.

In a new study published in the scientific journal Cell, the team from Gladstone, UC Berkeley, and UCSF has outlined the technology for a CRISPR-based test for COVID-19 that uses a smartphone camera to provide accurate results in under 30 minutes.

“It has been an urgent task for the scientific community to not only increase testing, but also to provide new testing options,” says Melanie Ott, MD, PhD, director of the Gladstone Institute of Virology and one of the leaders of the study. “The assay we developed could provide rapid, low-cost testing to help control the spread of COVID-19.”

The technique was designed in collaboration with UC Berkeley bioengineer Daniel Fletcher, PhD, as well as Jennifer Doudna, PhD, who is a senior investigator at Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and an investigator of the Howard Hughes Medical Institute. Doudna recently won the 2020 Nobel Prize in Chemistry for co-discovering CRISPR-Cas genome editing, the technology that underlies this work.

Not only can their new diagnostic test generate a positive or negative result, it also measures the viral load (or the concentration of SARS-CoV-2, the virus that causes COVID-19) in a given sample.

“When coupled with repeated testing, measuring viral load could help determine whether an infection is increasing or decreasing,” says Fletcher, who is also a Chan Zuckerberg Biohub Investigator. “Monitoring the course of a patient’s infection could help health care professionals estimate the stage of infection and predict, in real time, how long is likely needed for recovery.”

A Simpler Test through Direct Detection

Current COVID-19 tests use a method called quantitative PCR — the gold standard of testing. However, one of the issues with using this technique to test for SARS-CoV-2 is that it requires DNA. Coronavirus is an RNA virus, which means that to use the PCR approach, the viral RNA must first be converted to DNA. In addition, this technique relies on a two-step chemical reaction, including an amplification step to provide enough of the DNA to make it detectable. So, current tests typically need trained users, specialized reagents, and cumbersome lab equipment, which severely limits where testing can occur and causes delays in receiving results.

As an alternative to PCR, scientists are developing testing strategies based on the gene-editing technology CRISPR, which excels at specifically identifying genetic material.

All CRISPR diagnostics to date have required that the viral RNA be converted to DNA and amplified before it can be detected, adding time and complexity. In contrast, the novel approach described in this recent study skips all the conversion and amplification steps, using CRISPR to directly detect the viral RNA.

“One reason we’re excited about CRISPR-based diagnostics is the potential for quick, accurate results at the point of need,” says Doudna. “This is especially helpful in places with limited access to testing, or when frequent, rapid testing is needed. It could eliminate a lot of the bottlenecks we’ve seen with COVID-19.”

Parinaz Fozouni, a UCSF graduate student working in Ott’s lab at Gladstone, had been working on an RNA detection system for HIV for the past few years. But in January 2020, when it became clear that the coronavirus was becoming a bigger issue globally and that testing was a potential pitfall, she and her colleagues decided to shift their focus to COVID-19.

“We knew the assay we were developing would be a logical fit to help the crisis by allowing rapid testing with minimal resources,” says Fozouni, who is co-first author of the paper, along with Sungmin Son and María Díaz de León Derby from Fletcher’s team at UC Berkeley. “Instead of the well-known CRISPR protein called Cas9, which recognizes and cleaves DNA, we used Cas13, which cleaves RNA.”

In the new test, the Cas13 protein is combined with a reporter molecule that becomes fluorescent when cut, and then mixed with a patient sample from a nasal swab. The sample is placed in a device that attaches to a smartphone. If the sample contains RNA from SARS-CoV-2, Cas13 will be activated and will cut the reporter molecule, causing the emission of a fluorescent signal. Then, the smartphone camera, essentially converted into a microscope, can detect the fluorescence and report that a swab tested positive for the virus.

“What really makes this test unique is that it uses a one-step reaction to directly test the viral RNA, as opposed to the two-step process in traditional PCR tests,” says Ott, who is also a professor in the Department of Medicine at UCSF. “The simpler chemistry, paired with the smartphone camera, cuts down detection time and doesn’t require complex lab equipment. It also allows the test to yield quantitative measurements rather than simply a positive or negative result.”

The researchers also say that their assay could be adapted to a variety of mobile phones, making the technology easily accessible.

“We chose to use mobile phones as the basis for our detection device since they have intuitive user interfaces and highly sensitive cameras that we can use to detect fluorescence,” explains Fletcher. “Mobile phones are also mass-produced and cost-effective, demonstrating that specialized lab instruments aren’t necessary for this assay.”

Accurate and Quick Results to Limit the Pandemic

When the scientists tested their device using patient samples, they confirmed that it could provide a very fast turnaround time of results for samples with clinically relevant viral loads. In fact, the device accurately detected a set of positive samples in under 5 minutes. For samples with a low viral load, the device required up to 30 minutes to distinguish it from a negative test.

“Recent models of SARS-CoV-2 suggest that frequent testing with a fast turnaround time is what we need to overcome the current pandemic,” says Ott. “We hope that with increased testing, we can avoid lockdowns and protect the most vulnerable populations.”

Not only does the new CRISPR-based test offer a promising option for rapid testing, but by using a smartphone and avoiding the need for bulky lab equipment, it has the potential to become portable and eventually be made available for point-of-care or even at-home use. And, it could also be expanded to diagnose other respiratory viruses beyond SARS-CoV-2.

In addition, the high sensitivity of smartphone cameras, together with their connectivity, GPS, and data-processing capabilities, have made them attractive tools for diagnosing disease in low-resource regions.

“We hope to develop our test into a device that could instantly upload results into cloud-based systems while maintaining patient privacy, which would be important for contact tracing and epidemiologic studies,” Ott says. “This type of smartphone-based diagnostic test could play a crucial role in controlling the current and future pandemics.”

About the Research Project

The study entitled “Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy,” was published online by Cell on December 4, 2020.

Other authors of the study include Gavin J. Knott, Michael V. D’Ambrosio, Abdul Bhuiya, Max Armstrong, and Andrew Harris from UC Berkeley; Carley N. Gray, G. Renuka Kumar, Stephanie I. Stephens, Daniela Boehm, Chia-Lin Tsou, Jeffrey Shu, Jeannette M. Osterloh, Anke Meyer-Franke, and Katherine S. Pollard from Gladstone Institutes; Chunyu Zhao, Emily D. Crawford, Andreas S. Puschnick, Maira Phelps, and Amy Kistler from the Chan Zuckerberg Biohub; Neil A. Switz from San Jose State University; and Charles Langelier and Joseph L. DeRisi from UCSF.

The research was supported by the National Institutes of Health (NIAID grant 5R61AI140465-03 and NIDA grant 1R61DA048444-01); the NIH Rapid Acceleration of Diagnostics (RADx) program; the National Heart, Lung, and Blood Institute; the National Institute of Biomedical Imaging and Bioengineering; the Department of Health and Human Services (Grant No. 3U54HL143541-02S1); as well as through philanthropic support from Fast Grants, the James B. Pendleton Charitable Trust, The Roddenberry Foundation, and multiple individual donors. This work was also made possible by a generous gift from an anonymous private donor in support of the ANCeR diagnostics consortium.

The study — recently published online in JAMA Network Open — is, according to its authors, one of the first to examine the impact of comorbid conditions and neighborhood socioeconomic status (SES) on outcomes for Black, Hispanic and Asian patients hospitalized for COVID-19. Findings indicate that Black and Hispanic populations are not inherently more susceptible to poor COVID-19 outcomes compared to other groups, and that once hospitalized, their outcomes are equal to or better than their White counterparts.

“We know that Black and Hispanic populations account for a disproportionate share of COVID-19-related deaths relative to their population size in New York and major cities across the country,” says Gbenga Ogedegbe, MD, MPH, Dr. Adolph and Margaret Berger Professor of Medicine and Population Health at NYU Langone Health, and the study’s lead author. “We were, however, surprised to find that Black and Hispanic patients were no more likely to be hospitalized across NYU Langone than White patients, which means we need to look at other structural factors at play that are negatively affecting outcomes in these communities. These factors include poor housing conditions, unequal access to health care, differential employment opportunities, and poverty — and they must be addressed,” says Ogedegbe, who is also director of NYU Langone’s Institute for Excellence in Health Equity.

How the Study Was Conducted

The team of investigators obtained all data from NYU Langone Health’s electronic health record (EHR) of 9,722 patients tested for COVID-19 at the health system’s 260 outpatient office sites and four acute care hospitals in Manhattan, Brooklyn, and Long Island between March 1, 2020 and April 8, 2020, and followed them through May 13, 2020. The patients’ race and ethnicity data was self-reported.

For every patient who tested positive for COVID-19 , the researchers compiled race/ethnicity data, patient characteristics such as body mass index (BMI), age and sex, and neighborhood socioeconomic (SES) data contained in a weighted index of seven indicators (including median household income, level of education and housing value, among others).

Study Findings

• Among the 4,843 patients who tested positive for COVID-19, 39 percent were White, 15.7 percent were Black, 25.9 percent were Hispanic, 7 percent were Asian, and 7.4 percent were multiracial/other; 2,623 patients were hospitalized.
• Of 2,623 patients hospitalized, 39.9 percent were White, 14.3 percent were Black, 27.3 percent were Hispanic, 6.9 percent were Asian, and 7.9 percent were multiracial/other. Hospitalized patients were older and had higher comorbidity than patients who tested positive but were not hospitalized. 70.8 percent were discharged, 36.3 percent experienced critical illness, 24.7 percent died or were discharged to hospice, and 4.5 percent remained hospitalized as of May 13, 2020.
• Black and Hispanic patients had a lower risk of critical illness and were less likely to die or be discharged to hospice compared to White patients. After adjusting for age, sex, insurance status and comorbidity, Black patients continued to have lower risk of death compared to White patients, while Hispanics and Asian patients had similar rates to White patients.
• After adjusting for all the above factors, Asian patients had higher odds of being hospitalized than White patients even though they were less likely to test positive for COVID-19.

“Our findings provide more evidence that the social determinants of health play a critical role in determining patient outcomes, particularly for Black patients, before they ever get to the hospital,” said Joseph Ravenell, MD, associate professor in the Department of Population Health and associate dean for Diversity Affairs and Inclusion at NYU Langone.

“However, we do see a bit of a paradox,” said Ravenell. “In keeping with other research, we’ve found that once Black patients with COVID-19 make it to the hospital — despite coming from lower-income neighborhoods — their odds of dying are similar to or lower than White patients. Meanwhile, we also know that Black and Hispanic people are disproportionately contracting and dying of COVID-19 across the country.”

According to Ogedegbe and Ravenell, Black populations are more likely to be uninsured and underinsured than White populations and thus more likely to die at home as opposed to in hospital due to poorer access to care. Another predictor of poor outcomes for patients hospitalized with COVID-19 is male sex. In this particular study cohort, 62 percent of Black hospitalized patients were female, which could explain their relatively better outcomes. The study population may also not be representative of the overall New York City population, they said.

Study senior author Leora Horwitz, MD, associate professor in the Departments of Population Health and Medicine and director of the Center for Healthcare Innovation and Delivery Science at NYU Langone, says that future studies need to better examine the direct impact of structural inequities on racial and ethnic disparities in COVID-19 related hospitalization, morbidity, and mortality.

In addition to Ogedegbe, Horwitz, and Ravenell, additional co-authors from NYU Langone Health are Samrachana Adhikari, PhD, Mark Butler, PhD, Tiffany Cook, MA, Fritz Francois, MD, Eduardo Iturrate, MD, Girardin Jean-Louis, PhD, Simon Jones, PhD, Deborah Onakomaiya, MPH, Christopher Petrilli, MD, Claudia Pulgarin, MS, Seann Reagan, MA, Harmony Reynolds, MD, Azizi Seixas, PhD, and Frank Michael Volpicelli, MD.

The idea of stimulating the brain via an implant to generate artificial visual percepts is not new and dates back to the 1970s. However, existing systems are only able to generate a small number of artificial ‘pixels’ at a time. At the NIN, researchers from a team led by Pieter Roelfsema are now using new implant production and implantation technologies, cutting-edge materials engineering, microchip fabrication, and microelectronics, to develop devices that are more stable and durable than previous implants. The first results are very promising.

Electrical stimulation

When electrical stimulation is delivered to the brain via an implanted electrode, it generates the percept of a dot of light at a particular location in visual space, known as a ‘phosphene.’ The team developed high-resolution implants consisting of 1024 electrodes and implanted them in the visual cortex of two sighted monkeys. Their goal was to create interpretable images by delivering electrical stimulation simultaneously via multiple electrodes, to generate a percept that was composed of multiple phosphenes. “The number of electrodes that we have implanted in the visual cortex, and the number of artificial pixels that we can generate to produce high-resolution artificial images, is unprecedented,” says Roelfsema.

Recognizing dots, lines and letters

The monkeys first had to perform a simple behavioral task in which they made eye movements to report the location of a phosphene that was elicited during electrical stimulation via an individual electrode. They were also tested on more complex tasks such as a direction-of-motion task, in which micro-stimulation was delivered on a sequence of electrodes, and a letter discrimination task, in which micro-stimulation was delivered on 8-15 electrodes simultaneously, creating a percept in the form of a letter. The monkeys successfully recognized shapes and percepts, including lines, moving dots, and letters, using their artificial vision.

“Our implant interfaces directly with the brain, bypassing prior stages of visual processing via the eye or the optic nerve. Hence, in the future, such technology could be used for the restoration of low vision in blind people who have suffered injury or degeneration of the retina, eye, or optic nerve, but whose visual cortex remains intact,” explains Xing Chen, postdoctoral researcher in Roelfsema’s team.

This research lays the foundations for a neuroprosthetic device that could allow profoundly blind people to regain functional vision and to recognize objects, navigate in unfamiliar surroundings, and interact more easily in social settings, significantly improving their independence and quality of life.

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Materials provided by Netherlands Institute for Neuroscience – KNAW. Note: Content may be edited for style and length.

Scientists have long pondered just what the brain’s tree of cell types looks like. Now, an international collaboration led by Dr. Andreas Tolias from Baylor College of Medicine, Dr. Philipp Berens from the University of Tübingen in Germany and Dr. Rickard Sandberg from the Karolinska Institute in Stockholm, Sweden, has published an article in Nature that provides one of the most detailed and complete characterizations of the diversity of neural types in the brain so far.

Uncovering the shape of the tree of cortical cell types with Patch-seq

Neuroscientists mostly use three fundamental features to describe neurons: their anatomy, or how they look under a microscope; their physiology, or how they respond when stimulated; and, more recently, the genes they express, which are known as their transcriptome.

For this study, the research team used an experimentally challenging technique that they developed several years ago, called Patch-seq. This technique allowed them to collect a large multimodal database including genetic, anatomical and physiological information from single cells in the mouse motor cortex.

“Gathering all these three fundamental features from the same set of neurons was the key that enabled us to get a much deeper understanding of how neurons in the motor cortex are related to each other and a clearer view of how the tree of cell types looks like,” said co-first author Dr. Federico Scala, postdoctoral associate in Tolias lab at Baylor.

Dr. Dmitry Kobak, also co-first author and a research scientist in Berens lab, described that while the broad genetic families of neurons had distinct anatomical and physiological properties, within each family the neurons exhibited extensive anatomical and physiological diversity. Importantly, all the three basic neuronal characteristics (anatomy, physiology and transcriptome) were correlated, which enabled the team to find interesting links between them.

“Our data supports the view that the tree of cortical cell types may look more like a banana tree with few big leaves rather than an olive tree with many small ones. This view provides a simpler model to describe the diversity of neurons we find in the brain. We believe that this simpler view will lead to a more principled understanding of why we have so many cell types in the brain to begin with and what they are used for,” said Tolias, Brown Foundation Endowed Chair of Neuroscience and director of the Center for Neuroscience and Artificial Intelligence at Baylor.

In this metaphor, neurons follow a hierarchy consisting of distinct, non-overlapping branches at the level of families, the large leaves of the banana tree. Within each family, neurons show continuous changes in their genetic, anatomical and physiological features, and all three features within a family are correlated. In parallel, work published simultaneously in Cell, scientists from the Allen Institute of Brain Science in Seattle obtained very similar results from mouse visual cortex underscoring that this view of cell types may be a general building principle of brain circuits.

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Materials provided by Baylor College of Medicine. Original written by Graciela Gutierrez. Note: Content may be edited for style and length.

At a time when the incidence and social costs of dementia and Alzheimer’s disease in particular continue to rise, this breakthrough is very timely as it could enable the detection of the disease much earlier than current approaches. The findings are also important for the testing of therapies against this devastating disease.

Alzheimer’s disease is characterized by two pathological changes in brain tissue. One is a protein called tau while the other involves the amyloid beta peptide. Both can form clumps of aggregates that progressively accumulate in specific areas of the brain. For tau, individual units of the protein can aggregate into finely-ordered fibrillar structures facilitated by a biochemical process called phosphorylation. Throughout the disease process, amyloid beta and phosphorylated tau (p-tau) are released from the brain into cerebrospinal fluid; the amount of the released proteins are used as reliable surrogate markers for clinical diagnoses of Alzheimer’s disease.

Normally, amyloid beta levels in cerebrospinal fluid become abnormal several years before p-tau. The current clinical tests for p-tau become abnormal when memory failings develop. This makes it difficult to identify people with the disease at the very early stages before it is too late. How can we, therefore, reliably detect these sub-threshold disease changes?

To address these challenges, the scientists discovered that there are specific forms of p-tau that undergo very minute increases in cerebrospinal fluid and blood in people with emerging Alzheimer pathology. Consequently, the researchers developed highly sensitive techniques to measure these biological markers that precede clinical signs by several years.

In the first study, conducted in the Alfa parent cohort study at the Barcelona Beta Research Centre (BBRC), with the support of “la Caixa” Foundation, about a third of the 381 people evaluated had brain evidence of Alzheimer pathology but without any cognitive problems, meaning that these changes could not be detected in the clinic by memory assessments. Remarkably, the new p-tau markers correctly identified these emerging abnormalities measured in cerebrospinal fluid and regular blood samples.

Subsequent studies performed in Gothenburg, Paris and Ljubljana revealed that these new markers continue to increase from the preclinical stage through the onset of cognitive problems to the late dementia stages. For this reason, progressive increases in p-tau could provide insights into the biological and clinical development of Alzheimer’s disease. The studies are now published in the leading journals EMBO Molecular Medicine and Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association.

Kaj Blennow, professor of neurochemistry at the University of Gothenburg, who directed the work said “A possible way to improve the chances of future therapies is to test them on people in the very early stages of the disease with elusive biological changes but lacking clinical symptoms including memory failings. Candidate drug trials have not been too successful.” He added that: “The practical challenge, however, is that these very tiny initial changes are incredibly difficult to measure reliably. This compromises our chances to identify and recruit preclinical AD patients for clinical trials.”

Dr. Thomas Karikari, an assistant professor at the University of Gothenburg, who co-led the discovery commented: “The remarkable findings reported in these publications show that the new highly sensitive tools capture the earliest Alzheimer disease changes in the brain in clinically normal people. These tools therefore have the potential to advance population screening and clinical trials.”

According to Dr. Marc Suárez-Calvet, neurologist and ERC researcher at BBRC, “the biomarker detected in blood may change clinical practice in the coming years, since it will improve the diagnosis of patients with Alzheimer’s disease, both in its asymptomatic and symptomatic phases.”

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Materials provided by University of Gothenburg. Note: Content may be edited for style and length.

Researchers at Texas A&M University have invented a high-throughput cell separation method that can be used in conjunction with droplet microfluidics, a technique whereby tiny drops of fluid containing biological or other cargo can be moved precisely and at high speeds. Specifically, the researchers successfully isolated pathogens attached to host cells from those that were unattached within a single fluid droplet using an electric field.

“Other than cell separation, most biochemical assays have been successfully converted into droplet microfluidic systems that allow high-throughput testing,” said Arum Han, professor in the Department of Electrical and Computer Engineering and principal investigator of the project. “We have addressed that gap, and now cell separation can be done in a high-throughput manner within the droplet microfluidic platform. This new system certainly simplifies studying host-pathogen interactions, but it is also very useful for environmental microbiology or drug screening applications.”

The researchers reported their findings in the August issue of the journal Lab on a Chip.

Microfluidic devices consist of networks of micron-sized channels or tubes that allow for controlled movements of fluids. Recently, microfluidics using water-in-oil droplets have gained popularity for a wide range of biotechnological applications. These droplets, which are picoliters (or a million times less than a microliter) in volume, can be used as platforms for carrying out biological reactions or transporting biological materials. Millions of droplets within a single chip facilitate high-throughput experiments, saving not just laboratory space but the cost of chemical reagents and manual labor.

Biological assays can involve different cell types within a single droplet, which eventually need to be separated for subsequent analyses. This task is extremely challenging in a droplet microfluidic system, Han said.

“Getting cell separation within a tiny droplet is extremely difficult because, if you think about it, first, it’s a tiny 100-micron diameter droplet, and second, within this extremely tiny droplet, multiple cell types are all mixed together,” he said.

To develop the technology needed for cell separation, Han and his team chose a host-pathogen model system consisting of the salmonella bacteria and the human macrophage, a type of immune cell. When both these cell types are introduced within a droplet, some of the bacteria adhere to the macrophage cells. The goal of their experiments was to separate the salmonella that attached to the macrophage from the ones that did not.

For cell separation, Han and his team constructed two pairs of electrodes that generated an oscillating electric field in close proximity to the droplet containing the two cell types. Since the bacteria and the host cells have different shapes, sizes and electrical properties, they found that the electric field produced a different force on each cell type. This force resulted in the movement of one cell type at a time, separating the cells into two different locations within the droplet. To separate the mother droplet into two daughter droplets containing one type of cells, the researchers also made a downstream Y-shaped splitting junction.

Han said although these experiments were carried with a host and pathogen whose interaction is well-established, their new microfluidic system equipped with in-drop separation is most useful when the pathogenicity of bacterial species is unknown. He added that their technology enables quick, high-throughput screening in these situations and for other applications where cell separation is required.

“Liquid handling robotic hands can conduct millions of assays but are extremely costly. Droplet microfluidics can do the same in millions of droplets, much faster and much cheaper,” Han said. “We have now integrated cell separation technology into droplet microfluidic systems, allowing the precise manipulation of cells in droplets in a high-throughput manner, which was not possible before.”

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Materials provided by Texas A&M University. Original written by Vandana Suresh. Note: Content may be edited for style and length.

Now, in a study on mice conducted by the Neural Computation Unit at the Okinawa Institute of Science and Technology Graduate University (OIST), the authors, Dr. Katsuhiko Miyazaki and Dr. Kayoko Miyazaki, pinpoint specific areas of the brain that individually promote patience through the action of serotonin. Their findings were published 27th November in Science Advances.

“Serotonin is one of the most famous neuromodulators of behavior, helping to regulate mood, sleep-wake cycles and appetite,” said Dr. Katsuhiko Miyazaki. “Our research shows that release of this chemical messenger also plays a crucial role in promoting patience, increasing the time that mice are willing to wait for a food reward.”

Their most recent work draws heavily on previous research, where the unit used a powerful technique called optogenetics — using light to stimulate specific neurons in the brain — to establish a causal link between serotonin and patience.

The scientists bred genetically engineered mice which had serotonin-releasing neurons that expressed a light-sensitive protein. This meant that the researchers could stimulate these neurons to release serotonin at precise times by shining light, using an optical fiber implanted in the brain.

The researchers found that stimulating these neurons while the mice were waiting for food increased their waiting time, with the maximum effect seen when the probability of receiving a reward was high but when the timing of the reward was uncertain.

“In other words, for the serotonin to promote patience, the mice had to be confident that a reward would come but uncertain about when it would arrive,” said Dr. Miyazaki.

In the previous study, the scientists focused on an area of the brain called the dorsal raphe nucleus — the central hub of serotonin-releasing neurons. Neurons from the dorsal raphe nucleus reach out into other areas of the forebrain and in their most recent study, the scientists explored specifically which of these other brain areas contributed to regulating patience.

The team focused on three brain areas that had been shown to increase impulsive behaviors when they were damaged — a deep brain structure called the nucleus accumbens, and two parts of the frontal lobe called the orbitofrontal cortex and the medial prefrontal cortex.

“Impulse behaviors are intrinsically linked to patience — the more impulsive an individual is, the less patient — so these brain areas were prime candidates,” explained Dr. Miyazaki.

Good things come to those who wait (or not…)

In the study, the scientists implanted optical fibers into the dorsal raphe nucleus and also one of either the nucleus accumbens, the orbitofrontal cortex, or the medial prefrontal cortex.

The researchers trained mice to perform a waiting task where the mice held with their nose inside a hole, called a “nose poke,” until a food pellet was delivered. The scientists rewarded the mice in 75% of trials. In some test conditions, the timing of the reward was fixed at six or ten seconds after the mice started the nose poke and in other test conditions, the timing of the reward varied.

In the remaining 25% of trials, called the omission trials, the scientists did not provide a food reward to the mice. They measured how long the mice continued performing the nose poke during omission trials — in other words, how patient they were — when serotonin-releasing neurons were and were not stimulated.

When the researchers stimulated serotonin-releasing neural fibers that reached into the nucleus accumbens, they found no increase in waiting time, suggesting that serotonin in this area of the brain has no role in regulating patience.

But when the scientists stimulated serotonin release in the orbitofrontal cortex and the medial prefrontal cortex while the mice were holding the nose poke, they found the mice waited longer, with a few crucial differences.

In the orbitofrontal cortex, release of serotonin promoted patience as effectively as serotonin activation in the dorsal raphe nucleus; both when reward timing was fixed and when reward timing was uncertain, with stronger effects in the latter.

But in the medial prefrontal cortex, the scientists only saw an increase in patience when the timing of the reward was varied, with no effect observed when the timing was fixed.

“The differences seen in how each area of the brain responded to serotonin suggests that each brain area contributes to the overall waiting behavior of the mice in separate ways,” said Dr. Miyazaki.

Modelling patience

To investigate this further, the scientists constructed a computational model to explain the waiting behavior of the mice.

The model assumes that the mice have an internal model of the timing of reward delivery and keep estimating the probability that a reward will be delivered. They can therefore judge over time whether they are in a reward or non-reward trial and decide whether or not to keep waiting. The model also assumes that the orbitofrontal cortex and the medial prefrontal cortex use different internal models of reward timing, with the latter being more sensitive to variations in timing, to calculate reward probabilities individually.

The researchers found that the model best fitted the experimental data of waiting time by increasing the expected reward probability from 75% to 94% under serotonin stimulation. Put more simply, serotonin increased the mice’s belief that they were in a reward trial, and so they waited longer.

Importantly, the model showed that stimulation of the dorsal raphe nucleus increased the probability from 75% to 94% in both the orbital frontal cortex and the medial prefrontal cortex, whereas stimulation of the brain areas separately only increased the probability in that particular area.

“This confirmed the idea that these two brain areas are calculating the probability of a reward independently from each other, and that these independent calculations are then combined to ultimately determine how long the mice will wait,” explained Dr. Miyazaki. “This sort of complementary system allows animals to behave more flexibly to changing environments.”

Ultimately, increasing our knowledge of how different areas of the brain are more or less affected by serotonin could have vital implications in future development of drugs. For example, selective serotonin reuptake inhibitors (SSRIs) are drugs that boost levels of serotonin in the brain and are used to treat depression.

“This is an area we are keen to explore in the future, by using depression models of mice,” said Dr. Miyazaki. “We may find under certain genetic or environmental conditions that some of these identified brain areas have altered functions. By pinning down these regions, this could open avenues to provide more targeted treatments that act on specific areas of the brain, rather than the whole brain.”

The finding concerns a key process essential to life: the transcription phase of gene expression, which enables cells to live and do their jobs.

During transcription, an enzyme called RNA polymerase wraps itself around the double helix of DNA, using one strand to match nucleotides to make a copy of genetic material — resulting in a newly synthesized strand of RNA that breaks off when transcription is complete. That RNA enables production of proteins, which are essential to all life and perform most of the work inside cells.

Just as with any coherent message, RNA needs to start and stop in the right place to make sense. A bacterial protein called Rho was discovered more than 50 years ago because of its ability to stop, or terminate, transcription. In every textbook, Rho is used as a model terminator that, using its very strong motor force, binds to the RNA and pulls it out of RNA polymerase. But a closer look by these scientists showed that Rho wouldn’t be able to find the RNAs it needs to release using the textbook mechanism.

“We started studying Rho, and realized it cannot possibly work in ways people tell us it works,” said Irina Artsimovitch, co-lead author of the study and professor of microbiology at The Ohio State University.

The research, published online by the journal Science today, Nov. 26, 2020, determined that instead of attaching to a specific piece of RNA near the end of transcription and helping it unwind from DNA, Rho actually “hitchhikes” on RNA polymerase for the duration of transcription. Rho cooperates with other proteins to eventually coax the enzyme through a series of structural changes that end with an inactive state enabling release of the RNA.

The team used sophisticated microscopes to reveal how Rho acts on a complete transcription complex composed of RNA polymerase and two accessory proteins that travel with it throughout transcription.

“This is the first structure of a termination complex in any system, and was supposed to be impossible to obtain because it falls apart too quickly,” Artsimovitch said.

“It answers a fundamental question — transcription is fundamental to life, but if it were not controlled, nothing would work. RNA polymerase by itself has to be completely neutral. It has to be able to make any RNA, including those that are damaged or could harm the cell. While traveling with RNA polymerase, Rho can tell if the synthesized RNA is worth making — and if not, Rho releases it.”

Artsimovitch has made many important discoveries about how RNA polymerase so successfully completes transcription. She didn’t set out to counter years of understanding about Rho’s role in termination until an undergraduate student in her lab identified surprising mutations in Rho while working on a genetics project.

Rho is known to silence the expression of virulence genes in bacteria, essentially keeping them dormant until they’re needed to cause infection. But these genes do not have any RNA sequences that Rho is known to preferentially bind. Because of that, Artsimovitch said, it has never made sense that Rho looks only for specific RNA sequences, without even knowing if they are still attached to RNA polymerase.

In fact, the scientific understanding of the Rho mechanism was established using simplified biochemical experiments that frequently left out RNA polymerase — in essence, defining how a process ends without factoring in the process itself.

In this work, the researchers used cryo-electron microscopy to capture images of RNA polymerase operating on a DNA template in Escherichia coli, their model system. This high-resolution visualization, combined with high-end computation, made accurate modeling of transcription termination possible.

“RNA polymerase moves along, matching hundreds of thousands of nucleotides in bacteria. The complex is extremely stable because it has to be — if the RNA is released, it is lost,” Artsimovitch said. “Yet Rho is able to make the complex fall apart in a matter of minutes, if not seconds. You can look at it, but you can’t get a stable complex to analyze.”

Using a clever method to trap complexes just before they fall apart enabled the scientists to visualize seven complexes that represent sequential steps in the termination pathway, starting from Rho’s engagement with RNA polymerase and ending with a completely inactive RNA polymerase. The team created models based on what they saw, and then made sure that these models were correct using genetic and biochemical methods.

Though the study was conducted in bacteria, Artsimovitch said this termination process is likely to occur in other forms of life.

“It appears to be common,” she said. “In general, cells use similar working mechanisms from a common ancestor. They all learned the same tricks as long as these tricks were useful.”

Artsimovitch, working with an international research team of collaborators, co-led the study with Markus Wahl, a former Ohio State graduate student now at Freie Universität Berlin.

This work was supported by grants from the German Research Foundation; the German Federal Ministry of Education and Research; the Indian Council of Medical Research; the Department of Biotechnology, Government of India; the National Institutes of Health; and the Sigrid Jusélius Foundation.

UQ PhD candidate Hubert Cheung said efforts to shift entrenched values and beliefs about Chinese medicine are not achieving conservation gains in the short term.

He said a better understanding of traditional practices was critical for conservationists to form more effective strategies.

“The use of endangered species in traditional Chinese medicine threatens species’ survival and is a challenge for conservationists,” Mr Cheung said.

“Pushing messages of inefficacy, providing various forms of scientific evidence or promoting biomedical alternatives doesn’t seem to be drastically influencing decisions and behaviours.

“And, although many practices and treatments continue to be criticised for lacking scientific support, the World Health Organization approved the inclusion of traditional Chinese medicine in its global compendium of medical practices last year.

“The challenge now is for conservationists to work proactively with practitioners and others in the industry to find sustainable solutions.

“However, most conservation scientists and organisations are unfamiliar with traditional Chinese medicine, which makes it difficult to devise effective and culturally-nuanced interventions.”

The researchers have examined the core theories and practices of traditional Chinese medicine, in a bid to make it more accessible.

They hope their study — and the nuances within — will influence policy and campaigning.

“Today, traditional Chinese medicine is formally integrated into China’s healthcare system, and has been central to China’s response to the ongoing pandemic,” Mr Cheung said.

“In fact, the Chinese government’s COVID-19 clinical guidance has included recommendations for the use of a product containing bear bile, which has raised concerns among conservation groups.”

UQ’s Professor Hugh Possingham said traditional Chinese medicine was now not only entrenched in the social and cultural fabric of Chinese society, but also gaining users elsewhere.

“A better understanding of traditional Chinese medicine will empower conservationists to engage more constructively with stakeholders in this space,” Professor Possingham said.

“We’re hoping that this work can help all parties develop more effective and lasting solutions for species threatened by medicinal use.”

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Materials provided by University of Queensland. Note: Content may be edited for style and length.