Culture

During the COVID-19 pandemic, food systems faced disruptions from staff shortages and supply chain issues. Now, a Virginia Tech researcher is assisting with efforts to help plants themselves from facing their own pandemic.

Just like human diseases, plant diseases don't have arbitrary boundaries. These diseases don't stop at a border crossing or a port of entry. That's why plant disease surveillance, improved plant disease detection systems, and predictive plant disease modeling - integrated at the global scale - are necessary to mitigate future plant disease outbreaks and protect the global food supply, according to a team of researchers in a new commentary published in "Proceedings of the National Academy of Sciences."

"The manuscript offers a unique and timely perspective on plant diseases, particularly in the context of the COVID-19 pandemic," said David Schmale, a co-author on the paper and a professor in the Virginia Tech School of Plant and Environmental Sciences in the College of Agriculture and Life Sciences. "What would happen if the world lost a staple crop, such as wheat, to a plant disease pandemic? The manuscript considers current tools and capabilities in the context of climate change and growing human populations. There is a clear opportunity to bring researchers together that work on the epidemiology of human diseases and plant diseases."

Schmale was a part of a team of experts, led by North Carolina State University, that met in Raleigh, North Carolina, a few years ago to discuss plant diseases and their impacts on food security. This manuscript is the result of that meeting, and many of the experts that were there in Raleigh are co-authors on the paper.

The idea is to "detect these plant disease outbreak sources early and stop the spread before it becomes a pandemic," said Jean Ristaino, William Neal Reynolds Distinguished Professor of Plant Pathology at North Carolina State University and the paper's lead author. Once an epidemic occurs, it is difficult to control, Ristaino said, likening the effort to the one undertaken to stop the spread of COVID-19.

Ristaino said that the efforts from a wide range of scholars - so-called convergence science - are needed to prevent plant disease pandemics. That means economists, engineers, crop scientists, crop disease specialists, geneticists, geographers, data analysts, statisticians, and others working together to protect crops, the farmers growing crops, and the people fed by those crops.

While some diseases are already under some sort of global surveillance - such as wheat rust and late blight, an important pathogen that affects potatoes and caused the Irish Potato Famine - other crop diseases are not routinely monitored.

A new strain of the fungal pathogen that causes wheat rust turned up in 1999 (Ug99) and has moved quickly throughout Africa, recently flying over the Red Sea into Yemen.

Globalization of food trade is another factor for the jump of plant pathogens and further risks a food supply already strained by a growing world population and human diseases.

Research is underway to model the risk of plant-pathogen spread and help predict and then prevent outbreaks, the researchers report in the paper. Modeling and forecasting disease spread can help mobilize mitigation strategies more precisely to stop these plant pandemics.

"Our work extends the concept of One Health to plant disease pandemics and the threat they pose to global food security," Schmale said. "People, domestic animals, and plants are all tightly connected through each other and the environments that they share. These connections are changing, and we must address the threat of high-risk plant pathogens to staple food crops to safeguard global food security."

That internal nagging feeling that drives you to seek sleep at night and wake in the morning to eat, work, and play, is, it turns out, genetic, and it's not just in people. Nearly every living organism - from animals to plants as well as several microorganisms and fungi - has an internal body clock, or a circadian rhythm.

Yet, scientists have been perplexed out how these genes operate. Now, Virginia Tech scientists have taken a step closer to an answer thanks to the DNA of a mouse, a petri dish, and much patience. In a new study published in the journal Genes & Development, Shihoko Kojima, an assistant professor in the Department of Biological Sciences, part of the
Virginia Tech College of Science, and a researcher with the Fralin Life Sciences Institute, and her team has identified a novel gene, Per2AS, that controls the sleep/wake cycle in mice. Per2AS appears to be a new type of gene, known as a non-coding gene. Unlike most other genes, Per2AS is not translated from RNA into a subsequent protein, thus making its function unclear until now.
(Circadian rhythms derives from the Latin circa diem, or "around a day.")

The study has been in the works for several years. Nine, exactly. Why the long tenure? Well, it's complicated. Literally. "It was difficult to find out what its job is because Per2AS was a noncoding gene," Kojima said. "Scientists have accumulated a lot of knowledge and tools to figure out the function of traditional genes. However, these tools cannot be readily applicable to nontraditional genes, such as Per2AS, because most tools are made based on the unique characteristics common to traditional genes."

In addition to Kojima, the study includes authorship of 13 members of the Virginia Tech, including faculty, former staff, and alumni. They are University Distinguished Professor John Tyson of the Department of Biological Sciences and former director of the systems biology in the Academy of Integrated Science, research specialist Rebecca Mosig; undergraduate alums Allison Castaneda, Jacob Deslauriers, Landon Frazier, Kevin He, Naseem Maghzian, and Camille Schrier, most of who are seeking advanced degrees in health care or research; and Blacksburg High School alums Aarati Pokharel, now at the University of Virginia, and Lily Zhu, now at Johns Hopkins University.

According to Kojima, when Human Genome Project started some 30 years ago, scientists then thought most of our genome is made out of traditional genes, because these genes were believed to control unique traits that we all have - eye and hair color, height and weight, personality. That didn't turn out to be true.

"It turned out that only 2 percent of our genome is used for traditional genes and the rest appears to be nontraditional genes. There has been a hot debate whether these nontraditional genes are also important for our traits - some say it is DNA junk, while others say they have important functions," she said.

Growing evidence suggests that at least some nontraditional genes are important for various biological processes, such as neuronal activities, immune functions, and cell differentiation, as well as disease development including cancer, neurodegeneration, and congenital genetic diseases."

The big takeaway: A nontraditional gene can have functions to control our body clock and therefore is important for our genome to have. In other words, nontraditional genes are as vital as their more basic counterparts.

"People also have an equivalent gene," Kojima said. "However, it is unclear at this point whether the human version has the same function(s) as the mouse version. Most organisms living on the Earth have a circadian clock because this is an internal timing system important to adapt to the daily environmental changes caused by the Earth's rotation. The circadian clock of human is not much different from that of rodents or insects."

What's next? Kojima wants to study the gene in a live mouse model. Not just from a petri dish. "We also want to know if this gene is in many other organisms. If so, that would mean this gene is very important."

If your idea of conspiracy theories entails aliens, UFOs, governmental cover-ups at Roswell Air Force base, and the melody of The X-Files--you're not alone. That was, indeed, the classic notion, says Scott Tyson, an assistant professor of political science at the University of Rochester.

But over the course of the last five years, he noticed a watershed. For starters, the term "theory" no longer applied to the convoluted ideas spouted by today's conspiracist groups such as QAnon, the Proud Boys, and the Oath Keepers, all of whom Tyson calls largely "theoryless."

For example, Tyson, a game theorist whose research focuses on authoritarian politics, conspiracies, and radicalization, points out that those who believe erroneously that former President Donald Trump's "victory was stolen," usually do not believe that votes cast on that same ballot for successful Republican congressional candidates have been tampered with.

Yet, these conspiracies have entered the mainstream discourse and are driving the growing radicalization of average Americans that manifested itself most visibly in the storming of the US Capitol on January 6, he says.

In a recent study, "Sowing the Seeds: Radicalization as a Political Tool" published in the American Journal of Political Science, Tyson--together with University of Michigan coauthor Todd Lehmann--looks at two common policy interventions--economic and psychological--designed to counter the growing radicalization among the US population.

The duo finds that improving economic conditions reduces both radicalization efforts and dissent. However, trying to render people psychologically less susceptible to radicalization attempts can backfire and instead increase the efforts by radical leaders to influence and radicalize more followers.

While radical assertions of a "deep state" and "stolen elections" have long bubbled quietly beneath the public discourse, Tyson says those ideas have now moved into the mainstream. That shift--from fringe to center stage--Tyson argues, happened during the Trump presidency.

Q&A

What's the nutshell definition of "radicalization"?

"Radicalization" is used interchangeably with "indoctrination." Essentially, it's creating self-motivation among people to do certain things. You would call someone radicalized when those things that you would normally have to motivate someone to do--you don't have to do anymore because they've become self-motivated. That's where conspiracism comes in--it restructures the way that people perceive the social world around them. Radicalization involves an element of extremism and is fundamentally a political thought with an ecosystem to it: there needs to be a political group, or a set of political leaders who are trying to restructure people's beliefs or their values in such a way that it helps their own political goals or causes.

How can radicalization be countered?

The way to combat it is not to hope for the easy solution. It's a false idea that we can just take out the leaders and it'll all go away, akin to simply cutting off the head off the snake. That doesn't actually work. You have to go from the bottom up to start trying to siphon off radicalized people, and treat the organization more as a terrorist group, in terms of any hearts and minds policies.

Does leadership "decapitation" work against a radical group such as QAnon?

We looked in our research at what happens when you threaten leadership decapitation and found that you actually provide an incentive for leaders to increase their efforts to radicalize others. The reason is very simple: if we think of radicalized people as having the self-motivation to do things against the government--whether it's protests, attacks, or to bomb things--if more people become radicalized the actual leaders are less important in these kinds of antigovernment actions. Our theory suggests that leaders are less important in the actual production of antigovernment actions, so that the government is essentially forced to divert attention from the leaders and toward these other threats. The leaders intentionally take away from their own control.

Why were conspiracies able to enter the American mainstream so pervasively?

Trump was incredibly important in giving a megaphone to conspiracists who had been on the fringe beforehand until he became a political force and essentially weaponized a lot of those ideas. When Trump unleashed all these conspiracies on the public--many people didn't know that they were really fringe ideas. One other reason they were able to spread so quickly is our so-called "media ecosystem." We have media outlets like Fox News, OAN, and Newsmax who are perfectly willing to spout conspiracies. When it all started back in 2015, the mainstream media wasn't ready to deal with this kind of weaponization. That's why conspiracists were able to misuse the mainstream media to essentially launder their claims: the conspiracists would make a bunch of unfounded assertions and accusations, which the mainstream media would pick up in turn to report on. Part of the debunking, however, was retelling the untrue story. That way a lot of these conspiracy narratives ended up reaching a much larger audience.

What role did the pandemic play in the spread of conspiracies and the radicalization of US citizens?

QAnon was around before the pandemic, and the radicalization campaigns of far-right groups were already under way beforehand. But it certainly accelerated these efforts and made them more effective. Because of the pandemic people were more isolated, which means they were talking to fewer people, and the echo chamber became narrower. That in turn, made people more susceptible to becoming radicalized. It's very similar to how cults recruit people: they isolate them from their family and friends who are not involved in the cult. They keep new recruits in that echo chamber long enough until they've been able to radicalize them. The number of QAnon members and radicalized people through other far-right groups today would be much, much lower if the pandemic hadn't forced us all to isolate in the way that it did.

DARIEN, IL - A recent study of teens and young adults found that several factors related to sleep timing and sleep duration are associated with an increased risk of cannabis use and binge drinking of alcohol during the following year.

Results show that a greater late-night preference predicted a greater likelihood of any cannabis use the following year. Greater late-night preference, greater daytime sleepiness, later sleep timing on the weekend, and shorter sleep duration during weekdays and on the weekend, all predicted an increased risk for more severe binge drinking the following year.

For further analysis, the sample was stratified into two groups: middle school/high school students (age 12-18) and high school graduates (age 18-27). Results show that sleep variables predicted marijuana use only in the middle school and high school students, while different patterns of sleep characteristics predicted binge drinking in the two stratified samples.

"Overall, the results suggest that teens in middle and high school may be more vulnerable to sleep-related risk for substance use," said lead author Brant P. Hasler, who has a doctorate in clinical psychology and is an associate professor of psychiatry, psychology, and clinical and translational science in the Center for Sleep and Circadian Science at the University of Pittsburgh. "The particular pattern of sleep predictors in the middle school and high school sample is consistent with the 'circadian misalignment' caused by early school start times."

Multiple years of data were analyzed from the National Consortium on Alcohol and Neurodevelopment in Adolescence. The sample comprised 831 participants, including 423 females. Participants were between 12 and 21 years of age at baseline. Results were controlled for factors such as age, sex, race, parental education, and previous year's substance use.

"Sleep is modifiable behavior, and perhaps easier to modify than going after substance use directly," said Hasler. "Furthermore, other studies show college-age teens are more willing to hear about changing their sleep than changing their substance use. Thus, focusing on improving teen sleep -- including through delaying school start times -- may be an underutilized but effective approach to reducing risk for problematic substance use."

CDC data show that only 25% of students in grades 9 through 12 get sufficient sleep on an average school night, and early school start times are one factor associated with insufficient sleep in teens. The American Academy of Sleep Medicine recommends that middle school and high school start times should be 8:30 a.m. or later to support an adequate opportunity for adolescents to obtain sufficient sleep on school nights. Sleep problems such as insufficient sleep duration, irregular sleep timing, and insomnia also are common among college students, and these problems are associated with anxiety and depression symptoms.

The research abstract was published recently in an online supplement of the journal Sleep and will be presented as an oral presentation on Friday, June 11, during Virtual SLEEP 2021. SLEEP is the annual meeting of the Associated Professional Sleep Societies, a joint venture of the American Academy of Sleep Medicine and the Sleep Research Society.

This press release includes data that has been updated since the publication of the abstract.

DARIEN, IL - Preliminary results from a study of more than 700,000 patients with traumatic brain injury (TBI) show that those with a sleep disorder had an increased risk of developing dementia.

Results show that over a median follow-up period of more than four years, TBI patients with a diagnosed sleep disorder were 25% more likely to develop dementia. The results were similar when stratified by sex: Having a sleep disorder was associated with a 25.5% increase in the risk of incident dementia in male persons with TBI and a 23.4% increase in the risk of developing dementia in female persons with TBI.

"Our study's novelty is its confirmation of sleep disorders' association with incident dementia in both male and female patients, independently of other known dementia risks," said lead author and primary investigator Dr. Tatyana Mollayeva, an affiliate scientist at the Kite Research Institute, the research arm of the Toronto Rehabilitation Institute and one of the principal research institutes at the University Health Network. Mollayeva is part of the Acquired Brain Injury & Society team at KITE. She is also an assistant professor at the Dalla Lana School of Public Health. "We are also the first to report on the risks that sleep disorders and other factors pose separately for male and female patients with TBI."

The retrospective study involved a province-wide cohort of all adult patients who were free of dementia when admitted to the emergency department or acute care hospital with a diagnosis of TBI between May 2003 and April 2013. The total sample comprised 712,708 patients with TBI of all severities. Their median age was 44 years, and 59% were male.

Over a median follow-up period of 52 months, 32,834 patients -- or 4.6% -- developed dementia. Analyses controlled for age, sex, income level, injury severity, and known comorbidity risks.

"The strong links to incidence of dementia in both sexes suggest a need for more targeted sleep disorders risk awareness in patients with TBI," said Mollayeva.

The research abstract was published recently in an online supplement of the journal Sleep and will be presented as an oral presentation on June 13 during Virtual SLEEP 2021. SLEEP is the annual meeting of the Associated Professional Sleep Societies, a joint venture of the American Academy of Sleep Medicine and the Sleep Research Society.

According to the National Alliance on Mental Illness and the World Health Organization, depression affects 16 million Americans and 322 million people worldwide. Emerging evidence suggests that the COVID-19 pandemic is further exacerbating the prevalence of depression in the general population. With this trajectory, it is evident that more effective strategies are needed for therapeutics that address this critical public health issue.

In a recent study, publishing in the June 9, 2021 online edition of Nature Translational Psychiatry, researchers at University of California San Diego School of Medicine used a combination of modalities, such as measuring brain function, cognition and lifestyle factors, to generate individualized predictions of depression.

The machine learning and personalized approach took into account several factors related to an individual's subjective symptoms, such as sleep, exercise, diet, stress, cognitive performance and brain activity.

"There are different underlying reasons and causes for depression," said Jyoti Mishra, PhD, senior author of the study, director of NEATLabs and assistant professor in the Department of Psychiatry at UC San Diego School of Medicine. "Simply put, current health care standards are mostly just asking people how they feel and then writing a prescription for medication. Those first-line treatments have been shown to be only mild to moderately effective in large trials.

"Depression is a multifaceted illness, and we need to approach it with personalized treatment whether that be therapy with a mental health professional, more exercise or a combination of approaches."

The one-month study collected data from 14 participants with depression using smartphone applications and wearables (like smart watches) to measure mood and lifestyle variables of sleep, exercise, diet and stress, and paired these with cognitive evaluations and electroencephalography, using electrodes on the scalp to record brain activity.

The goal was not to make any comparisons across individuals, but to model the predictors of each person's daily fluctuations in depressed mood.

The researchers developed a new machine-learning pipeline to systematically identify distinct predictors of low mood in each individual.

As an example, exercise and daily caffeine intake emerged as strong predictors of mood for one participant, but for another, it was sleep and stress that were more predictive, while in a third subject, the top predictors were brain function and cognitive responses to rewards.

"We should not be approaching mental health as one size fits all. Patients will benefit by having more direct and quantified insight onto how specific behaviors may be feeding their depression. Clinicians can leverage this data to understand how their patients might be feeling and better integrate medical and behavioral approaches for improving and sustaining mental health," said Mishra.

"Our study shows that we can use the technology and tools that are readily available, like cell phone apps, to collect information from individuals with or at risk for depression, without significant burden to them, and then harness that information to design personalized treatment plans."

Mishra said next steps include examining if the personalized treatment plans guided by the data and machine learning are effective.

"Our findings could have broader implications than depression. Anyone seeking greater well-being could benefit from insights quantified from their own data. If I don't know what is wrong, how do I know how to feel better?"

Researchers from the University of Liège (Belgium) have recently developed a new artificial neuron inspired by the different modes of operation of human neurons. Called a Bistable Recurrent Cell (BRC), this process has enabled recurrent networks to learn temporal relationships of more than a thousand discrete time units where classical methods failed after only a hundred time units. These important results are published in the journal PLOS One.

The enormous interest in artificial intelligence (AI) in recent years has led to the development of extremely powerful machine learning techniques. For example, time series - any series of data where a time component is present, such as stock prices, weather patterns or electroencephalograms - are by their nature extremely common and of great interest due to their wide range of applications. Time-series analysis is a type of task for which machine learning techniques are of particular interest, enabling the prediction of future events based on past events. Given the diversity of potential applications, it is logical that the processing of such data via AI algorithms has become very popular in recent years.

A particular type of artificial neural network, called a recurrent neural network (RNN), has been specially developed in recent years to have a memory that enables the network to retain information over time in order to correctly process a time series. Each time new data is received, the network updates its memory to retain this new information. Despite these developments, it is still difficult to train such networks and their memory capability is limited in time. &laquo We can imagine the example of a network that receives new information every day, » explains Nicolas Vecoven, a doctoral student in the Systems and Modeling lab at the University of Liège and first author of the study. &laquo but after the fiftieth day, we notice that the information from the first day had already been forgotten. »

&laquo However, human neurons capable of retaining information over an almost infinite period of time thanks to the bi-stability mechanism. This allows neurons to stabilise in two different states, depending on the history of the electrical currents they have been subjected to, and this for an infinite period of time. In other words, thanks to this mechanism, human neurons can retain a bit (a binary value) of information for an infinite time. », Nicolas further explains. Based on this bi-stability mechanism, Nicolas Vecoven and his colleagues Damien Ernst (an AI specialist) and Guillaume Drion (a neuroscience specialist) from ULiège, have constructed a new artificial neuron with this same mechanism and have integrated it into recurrent artificial networks. Called a Bistable Recurrent Cell (BRC), this new artificial neuron has enabled recurrent networks to learn temporal relationships of more than 1000 time steps, where classical methods have failed after only about 100 time steps. These are important and promising results that have been published in the journal PLOS One. The three researchers are continuing their research in this particular field and are continuing to develop technologies to improve the memories of RNNs, by promoting the emergence of equilibrium points within them.

DURHAM, N.H.--Why did the deer cross the road? According to research from the University of New Hampshire to keep going and going and going. Researchers have discovered the longest distance ever recorded by an adult male white-tailed deer--300 kilometers, or close to 200 miles, in just over three weeks. The finding has important implications for population management and the transmission of disease, especially chronic wasting disease, a fatal neurological disease.

"Deer are one of the most abundant, well-known and intensely managed species of wildlife in the United States," said Remington Moll, assistant professor of wildlife ecology and lead author. "So, to make this discovery despite the fact that they are so well studied is pretty surprising."

In their study, published in the journal Ecology and Evolution, researchers analyzed data from GPS radio collars on more than 600 deer in Missouri. One dispersal, or long-distance journey, of an adult white-tailed deer stood out for its length, duration and age of the deer. The buck travelled close to 300 kilometers over 22 days by moving an average of 13.6 kilometers per day (almost eight and a half miles), crossing a major river seven times, an interstate highway, a railroad and eight state highways. To confirm the findings, the researchers surveyed the scientific literature for other dispersals of white-tailed deer. The deer, known as N17003, stood head and antlers above others; his walkabout was 174 kilometers longer than any other recorded for an adult male deer.

"This extraordinary movement just jumped out from the others we tracked," said Moll. "At first, we thought it was an error. It looks like someone took the GPS collar and drove across the state of Missouri."

The findings were remarkable not only for the deer's range--he roamed a distance equal to that between New York City and Baltimore--but also because unlike juvenile males, who move to seek breeding opportunities, adult males tend to stay put. Movements were faster and more directional than those in their home territory and were faster and more directional at night than during the day when the deer frequently sheltered in forest cover. The journey, which happened in November 2017, occurred during hunting season.

"We call this a rare event, but we haven't been putting collars out for that long, and not in these large numbers," said Moll. "It's entirely possible that it could be happening with greater frequency than we've known."

Nearly eight million Americans hunt deer which contributes more than $20 billion to the U.S. economy. The researchers say that understanding the distance deer travel and how they do it is important for managing the species and controlling chronic wasting disease, a fatal neurological disease spread by direct contact and the environment. Knowing that deer are crossing county or even state lines highlights a need for regional management coordination.

Funding for this study was provided by the Missouri Department of Conservation, the U.S. Fish and Wildlife Service and the University of Montana.

Co-authors are Jon Roberts and Joshua Millspaugh, University of Montana; Kevyn Wiskirchen, Jason Sumners, Jason Isabelle and Barbara Keller, Missouri Department of Conservation; and Robert Montgomery, Michigan State University.

The >University of New Hampshire inspires innovation and transforms lives in our state, nation, and world. More than 16,000 students from all 50 states and 71 countries engage with an award-winning faculty in top-ranked programs in business, engineering, law, health and human services, liberal arts and the sciences across more than 200 programs of study. As one of the nation's highest-performing research universities, UNH partners with NASA, NOAA, NSF and NIH, and receives more than $110 million in competitive external funding every year to further explore and define the frontiers of land, sea and space.

The bad odors produced by the Waste Water Treatment Plants, known as WWTPs, have become a growing concern in the cities and towns that host these facilities and are considered by citizens to be the main cause of the perception of pollution, along with the dust and noise.

Now, and thanks to a collaboration between the Institute for Bioengineering of Catalonia (IBEC) and the company DAM, a new way is being opened to detect and treat these odors.

According to the researchers, "the results obtained in the SNIFFDRONE project (Odor monitoring by drones for environmental purposes) are very positive and represent a significant advance in the field of odor management in the WWTP. The new system will help to take appropriate control actions and therefore improve the management of the plant compared to current practices "

This is how the technicians from the DAM R & D & i Department express themselves, when assessing the work carried out in this European research that ended on October 31st. Specifically, a drone has been developed capable of predicting "the odor concentration from the readings of chemical sensors and providing measurements that help to locate the sources of origin", indicate from DAM.

The study, carried out together with the IBEC (Institute for Bioengineering of Catalonia) and which is part of the ATTRACT initiative (GA 777222, Horizon 2020 program of the European Union), starts from the reality that current odor evaluation methodologies use measurements infrequent olfactometric measurements that do not allow a precise characterization, so an effective monitoring of the plant cannot be carried out.

"The system provides odor concentration maps that help to take appropriate control actions and therefore improve plant management compared to current practices," explain the researchers from DAM's R + D + i department.

Investigation details

The drone is configured with an electronic nose made up of 21 chemical sensors, plus temperature, humidity and pressure sensors, in a miniature sensor chamber. It also contains a sampling system, GPS positioning, and connects to a base station for signal processing and data analysis in real time.

"The system has been calibrated and validated in real operating conditions through several measurement campaigns at the Molina de Segura WWTP (Murcia). The results obtained allow us to obtain a prototype capable of predicting the intensity of the odor of ambient air samples in real time and simultaneously collecting samples for analysis in the laboratory after the flight, which allows us to calibrate and validate the operation of the system " , stand out from the Valencian water purification company.

"The developed drone allows, thanks to a combination of chemical sensors and artificial intelligence, to quickly assess the intensity of the odor emitted by a waste management plant in large areas that are sometimes difficult to access. This information is relevant for the plant operators with the ultimate goal of minimizing the impact on neighboring communities ", declares Santi Marco, Head of the Group of Signal and Information Processing for Sensing Systems at IBEC and Professor at the University of Barcelona .

For all these reasons, the DAM Innovation department technicians consider that the results of SNIFFDRONE represent a significant advance in the field of odor management in WWTP, since until now "the odor detection robots had been tested with Single-odor chemical sources in relatively simple controlled scenarios and in most cases using ground-based robots ", they conclude.

DARIEN, IL - Dreams result from a process that often combines fragments of multiple life experiences and anticipates future events, according to novel evidence from a new study.

Results show that 53.5% of dreams were traced to a memory, and nearly 50% of reports with a memory source were connected to multiple past experiences. The study also found that 25.7% of dreams were related to specific impending events, and 37.4% of dreams with a future event source were additionally related to one or more specific memories of past experiences. Future-oriented dreams became proportionally more common later in the night.

"Humans have struggled to understand the meaning of dreams for millennia," said principal investigator Erin Wamsley, who has a doctorate in cognitive neuroscience and is an associate professor in the department of psychology and program in neuroscience at Furman University in Greenville, South Carolina. "We present new evidence that dreams reflect a memory-processing function. Although it has long been known that dreams incorporate fragments of past experience, our data suggest that dreams also anticipate probable future events."

The study involved 48 students who spent the night in the laboratory for overnight sleep evaluation using polysomnography. During the night, participants were awakened up to 13 times to report on their experiences during sleep onset, REM sleep, and non-REM sleep. The following morning, participants identified and described waking life sources for each dream reported the previous evening. A total of 481 reports were analyzed.

"This is a new description of how dreams draw simultaneously from multiple waking-life sources, utilizing fragments of past experience to construct novel scenarios anticipating future events," said Wamsley.

According to Wamsley, the proportional increase of future-oriented dreams later in the night may be driven by temporal proximity to the upcoming events. While these dreams rarely depict future events realistically, the activation and recombination of future-relevant memory fragments may nonetheless serve an adaptive function.

The research abstract was published recently in an online supplement of the journal Sleep and will be presented as a poster beginning June 9 during Virtual SLEEP 2021. SLEEP is the annual meeting of the Associated Professional Sleep Societies, a joint venture of the American Academy of Sleep Medicine and the Sleep Research Society.

ROCHESTER, Minn. -- Mayo Clinic researchers and colleagues at the University of Minnesota showed that COVID-19 exacerbates the damaging impact of senescent cells in the body. In preclinical studies, the senolytic drugs discovered at Mayo significantly reduced inflammation, illness, and mortality from COVID infection in older mice. The findings appear in the journal Science.

Senescent cells (damaged or non-functioning cells that persist in the body) contribute to many aspects of aging and illness, including inflammation and multiple chronic diseases. Based on the "Amplifier/Rheostat Hypothesis" of senescent cells developed at Mayo, the researchers sought to discover how COVID-19 causes much higher mortality in the elderly and chronically-ill. They showed that human senescent cells have an amplified response to the SARS spike protein, provoking increased production of factors causing inflammation and tissue damage by senescent cells.

The researchers also found that older mice infected with viruses, including a coronavirus related to SARS-CoV-2 using a model developed at University of Minnesota, showed an amplified reaction, with increased senescent cells, inflammation, and nearly 100 % mortality. When the researchers treated similar mice - before or after the infection - with senolytics, drugs that selectively remove senescent cells from the body, the result was the opposite. Anti-viral antibodies increased, while signs of inflammation and senescent cells significantly decreased along with mortality, so survival of the old, infected mice became more like that of younger mice.

The researchers suggest that reducing the existing burden of senescent cells in older or chronically-diseased patients may increase their resilience and decrease their risk of dying from viral infections, including SARS-CoV-2. Three such clinical trials are now underway.

"Even though vaccine use is growing, senolytics could still be helpful to those who cannot receive the vaccine, and especially to older individuals in nursing homes with comorbidities or immunity issues," says James Kirkland, M.D., Ph.D., director of the Kogod Center on Aging and, together with Tamar Tchkonia, Ph.D., senior author for Mayo Clinic on the study. The study suggests senolytics could also improve response of the elderly to vaccines and help them fight bacterial and other viral infections.

The research was supported by the National Institutes of Health; the Connor Fund; Robert J. and Theresa W. Ryan; Robert P. and Arlene R. Kogod; the Noaber Foundation; the University of Minnesota Clinical and Translational Science Institute; the Medical Discovery Team on the Biology of Aging; and The Irene Diamond Fund/American Federation on Aging Research Postdoctoral Transition 10 Award. Also, by the Fesler-Lampert Chair in Aging Studies and the AFAR Junior Faculty Award.

UPTON, NY--Scientists at the U.S. Department of Energy's (DOE) Brookhaven National Laboratory have published the first detailed atomic-level model of the SARS-CoV-2 "envelope" protein bound to a human protein essential for maintaining the lining of the lungs. The model showing how the two proteins interact, just published in the journal Nature Communications, helps explain how the virus could cause extensive lung damage and escape the lungs to infect other organs in especially vulnerable COVID-19 patients. The findings may speed the search for drugs to block the most severe effects of the disease.

"By obtaining atomic-level details of the protein interactions we can explain why the damage occurs, and search for inhibitors that can specifically block these interactions," said study lead author Qun Liu, a structural biologist at Brookhaven Lab. "If we can find inhibitors, then the virus won't cause nearly as much damage. That may give people with compromised health a much better chance for their immune systems to fight the virus successfully."

Scientists discovered the details and developed the molecular model using one of the new cryo-electron microscopes at Brookhaven Lab's Laboratory for BioMolecular Structure (LBMS), a new research facility built with funding from New York State adjacent to Brookhaven's National Synchrotron Light Source II (NSLS-II).

"LBMS opened last summer ahead of schedule because of its importance in the battle against COVID-19," said Sean McSweeney, director of LBMS and a coauthor on the paper. "LBMS and NSLS-II offer complementary protein-imaging techniques and both are playing important roles in deciphering the details of proteins involved in COVID-19. This is the first paper published based on results from the new facility."

Liguo Wang, scientific operations director of LBMS and another coauthor on the paper, explained that "cryo-electron microscopy (cryo-EM) is particularly useful for studying membrane proteins and dynamic protein complexes, which can be difficult to crystallize for protein crystallography, another common technique for studying protein structures. With this technique we created a 3-D map from which we could see how the individual protein components fit together."

"Without cryo-EM, we couldn't have gotten a structure to capture the dynamic interactions between these proteins," Liu said.

Triggering lung disruption

The SARS-CoV-2 envelope protein (E), which is found on the virus's outer membrane alongside the now-infamous coronavirus spike protein, helps to assemble new virus particles inside infected cells. Studies published early in the COVID-19 pandemic showed that it also plays a crucial role in hijacking human proteins to facilitate virus release and transmission. Scientists hypothesize that it does this by binding to human cell-junction proteins, pulling them away from their usual job of keeping the junctions between lung cells tightly sealed.

"That interaction can be good for the virus, and very bad for humans--especially elderly COVID-19 patients and those with pre-existing medical conditions," Liu said.

When lung cell junctions are disrupted, immune cells come in to try to fix the damage, releasing small proteins called cytokines. This immune response can make matters worse by triggering massive inflammation, causing a so-called "cytokine storm" and subsequent acute respiratory distress syndrome.

Also, because the damage weakens the cell-cell connections, it might make it easier for the viruses to escape from the lungs and travel through the bloodstream to infect other organs, including the liver, kidneys, and blood vessels.

"In this scenario, most damage would occur in patients with more viruses and more E proteins being produced," Liu said. And this could become a vicious cycle: More viruses making more E proteins and more cell-junction proteins being pulled out, causing more damage, more transmission, and more viruses again. Plus, any existing damage, such as lung-cell scarring, would likely make it harder for COVID patients to recover from the damage.

"That's why we wanted to study this interaction--to understand the atomic-level details of how E interacts with one of these human proteins to learn how to interrupt the interactions and reduce or block these severe effects," Liu said.

From specks to blobs to map to model

The scientists obtained atomic-level details of the interaction between E and a human lung-cell-junction protein called PALS1 by mixing the two proteins together, freezing the sample rapidly, and then studying the frozen sample with the cryo-EM. The electron microscopes use high-energy electrons to interact with the sample in much the same way that regular light microscopes use beams of light. But electrons allow scientists to see things at a much smaller scale due to their extremely short wavelength (100,000 times shorter than that of visible light).

The first images didn't look like much more than specks. But image-processing techniques allowed the team to select specks that were actual complexes of the two proteins.

"We used two-dimensional averaging and started to see some structural features that are shared among these particles. Our images showed the complex from different orientations but at fairly low resolution," Liu said. "Then we use computational tools and computation infrastructure at Brookhaven's Computational Science Initiative to perform three-dimensional reconstructions. These give us a 3-D model--an experimental map of the structure."

With an overall resolution of 3.65 Angstroms (the size of just a few atoms), the map had enough information about the unique characteristics of the individual amino acids that make up the two proteins for the scientists to fit the known structures of those amino acids into the map.

"We can see how the chain of amino acids that makes up the PALS1 protein folds to form three structural components, or domains, and how the much smaller chain of amino acids that makes up the E protein fits in a hydrophobic pocket between two of those domains," Liu said.

The model provides both the structural details and an understanding of the intermolecular forces that allow E proteins deep within an infected cell to wrench PALS1 from its place at the cell's outer boundary.

"Now we can explain how the interactions pull PALS1 from the human lung-cell junction and contribute to the damage," Liu said.

Implications for drugs and evolution

"This structure provides the foundation for our computational science colleagues to run docking studies and molecular dynamics simulations to search for drugs or drug-like molecules that might block the interaction," said John Shanklin, chair of Brookhaven Lab's Biology Department and a coauthor on the paper. "And if they identify promising leads, we have the analytical capabilities to rapidly screen through such candidate drugs to identify ones that might be key to preventing severe consequences of COVID-19."

Understanding the dynamics of this protein interaction will also help scientists track how viruses like SARS-CoV-2 evolve.

"When the virus protein pulls PALS1 out of the cell junction, it could help the virus spread more easily. That would provide a selective advantage for the virus. Any traits that increase the survival, spread, or release of the virus are likely to be retained," Liu said.

The longer the virus continues to circulate, the more chances there are for new evolutionary advantages to arise.

"This is one more reason it is so essential for us to identify and implement promising therapeutics," Liu said. "In addition to preventing the most severe infections, drugs that effectively treat COVID-19 will keep us ahead of these mutations."

An unprecedented analysis of almost 10,000 Harmful Algal Bloom (HAB) events worldwide over the past 33 years was launched today by UNESCO's Intergovernmental Oceanographic Commission.

The first-ever global statistical analysis examined ~9,500 HABs events over 33 years and found that the harm caused by HABs rises in step with growth of the aquaculture industry and marine exploitation and calls for more research on linkages.

Conducted over seven years by 109 scientists in 35 countries, the study found that reported HAB events have increased in some regions and decreased or held steady in others. A widely-stated view that HABs are on the rise throughout the world, perhaps due to climate change, isn't confirmed.

However, the study, "Perceived global increase in algal blooms is attributable to intensified monitoring and emerging bloom impacts," published in the Nature journal Communications Earth & Environment, creates the world's first baseline against which to track future shifts in the location, frequency and impacts of HABs, which differ depending on which of the 250 harmful marine algae species is involved and where, requiring assessment on a species-by-species and site-by-site basis.

A public webinar on Global HAB Status Report will take place Tuesday Jun 15, 2021 at 1 PM, Paris time. To register: https://bit.ly/3z3kjCB

Databases mined

The scientists mined both the global Harmful Algae Event Database (HAEDAT), consisting of 9,503 events with one or more impacts on human society, and the Ocean Biodiversity Information System (OBIS) database, containing 7 million microalgal observation records, including 289,668 toxic algal species occurrences.

The study found that regionally-recorded HAB events, after being corrected for higher levels of monitoring effort, have

Increased:

Central America/Caribbean

South America

Mediterranean

North Asia

Decreased:

West Coast America

Australia/New Zealand

No significant change:

East Coast America

South East Asia

Europe

The 9,503 events' impacts on humans break down as follows:

48% involved seafood toxins

43% high phytoplankton counts and/or water discolorations with a socio-economic impact

7% mass animal or plant mortalities

2% caused other impacts (including foam and mucilage production)

(As well, in 11% of events, a single incident had multiple impacts, e.g. both water discoloration and mass mortality)

Of the event records linked to seafood toxins:

35% were Paralytic Shellfish Toxins (PST)

30% Diarrhetic Shellfish Toxins (DST)

9% Ciguatera Poisoning (CP)

9% marine and brackish water cyanobacterial toxins

7% Amnesic Shellfish Toxins (AST)

10% others, including Neurotoxic Shellfish Toxins (NST), Azaspiracid Shellfish Toxins (AZA), and toxic aerosols

By region, the largest number of records came from, in order:

Europe

North Asia

Mediterranean

The east and west coasts of North America

Caribbean

Pacific/Oceania

Southeast Asia

With more limited data sets for South America, and Australia/New Zealand

All geographic regions were impacted by multiple HAB types, but in varying proportions.

50% of regional HAEDAT records in the Caribbean, Benguela, Mediterranean Sea, North and South East Asia related to high phytoplankton density problems.

Seafood toxins and fish kill impacts dominated in all other regions

Among toxin-related impacts:

Paralytic Shellfish Toxins (PST) prevailed in North America, the Caribbean, South America, South East Asia, and North Asia

Diarrhetic Shellfish Toxins (DST) were the most frequently recorded in Europe and the Mediterranean (and are an emerging threat in the USA)
Neurotoxic Shellfish Toxins (NST) were confined to the US State of Florida, with a single outbreak also reported from New Zealand

Human poisonings from Ciguatera were prominent in the tropical Pacific, the Indian Ocean, Australia and the Caribbean.

For the most part, however, the impacts were confined to shellfish harvesting area closures; rarely to human poisonings. The exception: Ciguatera event records are almost exclusively based on medical reports of human poisonings.

HAB events over time

Eight of nine regions used in the study showed increases in reports logged via HAEDAT of harmful events per year, of which six were statistically significant.

The OBIS dataset, meanwhile, generally showed an increase in sampling effort in five of the nine regions.

When all the information was combined, the researchers could find no statistically significant global trend overall.

They also found, however, that aquaculture production increased 16-fold from a global total 11.35 million tonnes of seafood in 1985 to 178.5 million tonnes in 2018, with the largest increases occurring in Southeast Asia and South America/Caribbean and Central America, with North America and Europe stabilising.

The number of recorded harmful algal bloom events over time was strongly correlated with intensified aquaculture production in all regions with data suitable for the study.

However, says lead author Gustaaf M. Hallegraeff of the University of Tasmania: Intensified aquaculture clearly drives an increase in HAB monitoring efforts essential to sustaining the industry and protecting human health.

"And, just as clearly, a secondary effect of aquaculture is nutrient pollution. But a major data gap exists here. Conducting a meta-analysis of HABs vs aquaculture we had data on HAB monitoring efforts using OBIS records as a proxy but data on nutrient pollution is inadequate. The relationship between aquaculture-related nutrients and HABs therefore represents an important direction for further research."

Greater monitoring efforts

The study revealed

A 4-fold increase from 1985 to 2018 in observations of organisms mainly responsible for Diarrhetic Shellfish Poisoning (84,392 OBIS records)

A 7-fold increase in observations of organisms mainly responsible for Amnesic Shellfish Poisoning (128,282 OBIS records)

A 6-fold increase in observations of organisms mainly responsible for Paralytic Shellfish Poisoning (9,887 OBIS records)

(Note: Some observations may include non-toxic species or strains.)

In each case, the clear increase in the number of observations of problematic organisms paralleled an increase in records of associated toxic syndrome impacts.

They also found that the presence of toxic HAB species doesn't always accurately predict cases of human shellfish poisonings, which the study credits to the food safety risk management strategies in many affected countries. Some 11,000 non-fatal events related to Diarrhetic Shellfish Poisoning were reported worldwide, mostly from Europe, South America and Japan, with impacts consisting mostly of shellfish harvesting area closures.

Also, the study says, despite widespread distribution of the responsible algal species, there have been no human fatalities from Amnesic Shellfish Poisoning since the original 1987 incident in Prince Edward Island, Canada (150 illnesses, three fatalities). But ASP-associated mortalities of important marine mammals are of growing concern in Alaska and other parts of western North America, and ASP toxins have been linked to marine mammal calf mortalities in Argentina.

Of the world's 3,800 human Paralytic Shellfish Poisonings from 1985 to 2018, the largest number (2,555 from 1983 to 2013, including 165 fatalities) occurred in the Philippines, which depends strongly on aquaculture for human food protein.

DNA and other advanced detection methods have improved knowledge of the global distribution of ciguatera- causing organisms. Ciguatera poisonings, rarely fatal but annually affecting 10,000 to 50,000 people, have been decreasing in Hawaii and remained stable in French Polynesia and the Caribbean but constitute a new phenomenon in the Canary Islands.

Farmed fish killed by algal blooms: Largely a human-generated problem.

Aquacultured finfish mortalities account for much greater economic damage than HAB-contaminated seafood. Notes the study: wild marine finfish can simply swim away from blooms but those held captive in intensive aquaculture operations are vulnerable. Recorded losses include US $71 million in Japan in 1972, $70 million in Korea in 1995, $290 million in China in 2012, and $100 million in Norway in 2019.

A 2016 Chilean salmon mortality event caused a record $800 million loss, causing major social unrest.

Again, the presence of fish-killing HAB species doesn't accurately predict economic losses, the study shows. For example, Heterosigma blooms occur on the west and east coasts of Canada and the US, but fish mortalities are mostly confined to the west coast. In large part, the difference reflects the differences between sites where blooms occur and the relative location and size of aquaculture operations.

A harmful algae species that caused no problems in Australian lagoons killed 50,000 caged fish in Malaysia in 2014. It is now also known in Japan and the Philippines.

The authors note that some troublesome algal species may thrive, others decline, as ocean waters warm and acidify.

Commentary

"There has been a widely-stated contention that HABs worldwide are increasing in distribution, frequency or intensity, so a quantitative global assessment is long overdue," says lead author Prof. Hallegraeff of the Institute for Marine and Antarctic Studies, University of Tasmania.

"While some of the HAB literature over the past 30 years has handpicked selected examples to claim a global increase and expansion in HABs, this new big data approach shows a much more nuanced trend," he adds.

"Our study concludes that the health and economic damages caused by harmful microalgae -- seafood poisoning, water discolouration that blights tourism, and the death of finfish in aquaculture operations, for example -- differ between regions."

Adds co-author Adriana Zingone: "We also found that overexploitation acts as a natural multiplier of the effects of HABs, leading to an increase in impacts independent of an actual trend in HABs."

"It should be noted that over the last 40 years capacity and monitoring efforts to detect harmful species and harmful events have also increased, thus increasing the reporting of harmful events across the world's seas," she says.

"The absence of events and decreasing trends, like all negative results, are rarely published. Whether or not HABs are increasing globally, however, their impacts are a growing concern all around the globe."

Says co-author Henrik Oksfeldt Enevoldsen: "As the human population continues to increase in tandem with resource demands, HABs will predictably constitute a serious threat in terms of seafood safety and security, a hindrance to recreational uses of the sea, and a problem for the tourism industry."

"Occurrences of harmful species over time and their human impacts can be expected to change locally, regionally and globally alongside the effects that climate, hydrography and human pressure impose on the coastal environment."

"Understanding the trends and distribution patterns of harmful species and events at multiple spatial and temporal scales will help predict whether, where and when to expect HABs, their frequency and intensity. This knowledge is fundamental for effective management of HABs and to optimise the uses and values of the maritime space in coastal areas."

Johan Hanssens, Secretary-General Flanders Department of Economy, Science and Innovation, a sponsor of this report, concluded: "This status report is a very timely reminder, at the start of the UN Decade of Ocean Science for Sustainable Development, that a thorough understanding of natural and ecological processes in the ocean is crucial for the development of the blue economy, now that many coastal countries are turning to the sea for additional resources, including food provisioning. International scientific collaboration is essential and most efficient to address the associated challenges."

Durham, NC - Critically ill COVID-19 patients treated with non-altered stem cells from umbilical cord connective tissue were more than twice as likely to survive as those who did not have the treatment, according to a study published today in STEM CELLS Translational Medicine.

The clinical trial, carried out at four hospitals in Jakarta, Indonesia, also showed that administering the treatment to COVID-19 patients with an added chronic health condition such as diabetes, hypertension or kidney disease increased their survival more than fourfold.

All 40 patients who took part in the double-blind, controlled, randomized study were adults in intensive care who had been intubated due to COVID-19-induced pneumonia. Half were given intravenous infusions containing umbilical mesenchymal stromal cells, or stem cells derived from the connective tissue of a human birth cord, and half were given infusions without them.

The survival rate of those receiving the stem cells was 2.5 times higher and climbed even more - 4.5 times - in the COVID-19 patients who had other chronic health conditions, said Ismail Hadisoebroto Dilogo, professor of medicine at Cipto Mangunkusumo Central Hospital-Universitas Indonesia and research team member.

The stem cell infusion also was found to be safe and well-tolerated with no life-threatening complications or acute allergic reactions in seven days of post-infusion monitoring, he said.

Previous clinical trials have shown that treating COVID-19 pneumonia patients with stem cells from umbilical cord connective tissue may help them survive and recover more quickly, but the Indonesian study is the first to treat intubated, critically ill COVID-19 pneumonia patients with a naive, or non-genetically manipulated, form of the stem cells.

"Unlike other studies, our trial used stem cells obtained through explants from actual umbilical cord tissue and we did not manipulate them to exclude ACE2, a cellular protein thought to be an entry point for COVID-19," Dilogo said.

Some research suggests that one of the main causes of acute respiratory distress in COVID-19 patients is "cytokine storm," a condition in which infection prompts the body's immune system to flood the bloodstream with inflammatory proteins.

"The exact cause of cytokine storm is still unknown, but our study indicates that the presence of non-manipulated umbilical cord stromal stem cells improves patient survival by modulating the immune system toward an anti-inflammatory immune state," Dilogo said.

Since there is no cure for COVID-19, supportive care has been the only help available for patients who are critically ill with the virus.

"Although our study focused on a small number of patients, we think this experimental treatment could potentially lead to an effective adjuvant therapy for COVID-19 patients in intensive care who do not respond to conventional supportive treatment," he said.

Dilogo's research team launched the clinical trial last year after the COVID-19 occupancy rate in Jakarta's intensive care units climbed to 80 percent and the mortality rate of critically ill COVID-19 pneumonia patients in the ICUs reached 87 percent.

"This study, which assessed the potential therapeutic effect of human umbilical-cord mesenchymal stem cells on critically-ill COVID-19 patients, provides promising results that could inform a potential treatment to increase survival rates," said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and Director of the Wake Forest Institute for Regenerative Medicine. "Having additional potential therapies, such as MSCs, could be highly beneficial for these patients."

The incidence rate of lung adenocarcinoma (LUAD) is increasing gradually and the mortality is still high. Recent advances in the genomic profile of LUAD have identified a number of driver alterations in specific genes, enabling molecular classification and targeted therapy accordingly. However, only a fraction of LUAD patients with those driver mutations could benefit from targeted therapy, and the remaining large numbers of patients were unclassified. RNA editing events are those nucleotide changes in the RNA. Currently, the role of RNA editing events in tumorigenesis and their potential clinical utility have been reported in a series of studies. However, the profiles of the RNA editing events and their clinical relevance in LUAD remained largely unknown.

"We describe a comprehensive landscape of RNA editing events in LUAD by integrating transcriptomic and genomic data from our NJLCC project and TCGA project. We find that the global RNA editing level is significantly increased in tumor tissues and is highly heterogeneous across LUAD patients. The high RNA editing level in tumors can be attributed to both RNA and DNA alterations." said Dr. Cheng Wang, the first author for this work. The results indicated that the pattern of RNA editing events could represent the global characteristics of lung adenocarcinoma. "We then define a new molecular subtype, EC3, based on most variable RNA editing sites. The patients of this subtype show the poorest prognosis. Importantly, the subtype is independent of classic molecular subtypes based on gene expression or DNA methylation. We further propose a simplified prediction model including eight RNA editing sites to accurately distinguish EC3 subtype. " said Dr. Wang. Molecular typing based on a few RNA editing sites may have enormous potential in the clinics. "By applying the simplified model, we find that the EC3 subtype is associated with the sensitivity of specific chemotherapy drugs." said Dr. Wang.

"Our study comprehensively describes the general pattern of RNA editing in LUAD. More importantly, we propose a novel molecular subtyping strategy of LUAD based on RNA editing that could predict the prognosis of patients. A simplified model with a few editing sites makes the strategy potentially available in the clinics." said Professor Hongbing Shen, the corresponding author.