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A reliable clock for your microbiome

first_img Harvard researchers discover a biomarker that can determine both chronological and biological age Related For all the attention the human microbiome has been getting over the last few years, one aspect of the research rarely makes headlines: the difficulty of observing how it changes over time in response to various stimuli. The most common analysis method is extracting bacteria from fecal samples and then sequencing their genomes, but this approach, while minimally invasive, loses crucial information about where and when bacterial changes occur in the gut, giving scientists an incomplete picture of the dynamics of the microbiome.Now, a new tool created by researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School (HMS) provides a solution to this problem in the form of a set of bacterial genes that have been engineered to detect and record changes in the growth of different populations of bacteria over time in the guts of living mice with single-cell precision, and can serve as a platform for complex, synthetic-biology-based diagnostics and therapeutics for a variety of applications in the gut. The tool is described in a study published in Nature Communications.Keeping timeThe system uses an oscillating gene circuit, called a repressilator, as a kind of genetic clock to measure bacterial growth. The repressilator consists of three bacterial genes that code for three proteins (tetR, cl, and lacI), each of which blocks the expression of one of the other proteins. The genes are linked into a negative feedback loop, so that when the concentration of one of the repressor proteins falls below a certain level, the protein it had been repressing is expressed. This blocks the expression of the third protein, and the process repeats in a cyclical fashion.This gif shows how the position of the fluorescent rings changes when the repressilator is in a different part of its cycle at the beginning of colony growth. Credit: Silver Lab and Wyss Institute at Harvard UniversityWhen all three genes are inserted into a plasmid and introduced into bacteria, the number of negative feedback loop cycles completed can serve as a record of how many cell divisions the bacteria have undergone. Every time the bacteria divide, any repressor proteins present in their cytoplasm are diluted, so their concentration gradually falls and triggers the expression of the next protein in the repressilator cycle. Crucially, the repressilator cycle repeats after 15.5 bacterial generations regardless of the rate at which the bacteria are growing. This allows it to act as an objective measurement of time, much like a clock or a watch.“Imagine if you had two people wearing two different watches, and the second hand on one person’s watch was moving twice as fast as the other person’s,” explained first author David Riglar, a former postdoc at the Wyss Institute and HMS who now leads a research group as a Sir Henry Dale Fellow at Imperial College London. “If you stopped both watches after one hour, they wouldn’t agree on what time it was, because their measurement of time varies based on the rate of the second hand’s movement. In contrast, our repressilator is like a watch that always moves at the same speed, so no matter how many different people are wearing one, they will all give a consistent measurement of time. This quality allows us to more precisely study the behavior of bacteria in the gut.”This visual schematic tracks the repressilator cycle through individual bacterial samples over time: the expression of each of the repressor proteins is represented by a different fluorescent color. Credit: Paulsson Lab and Wyss Institute at Harvard UniversityThe researchers coupled each of the three repressor proteins to a differently colored fluorescent molecule, and developed an imaging workflow called RINGS (Repressilator-based Inference of Growth at Single-cell level) to track which protein was expressed at different time points during the bacteria’s growth. “As a bacterial colony grows outward, the repressilator circuit creates these different fluorescent, tree-ring-like signatures based on which repressor protein was active in the single bacterium that started the colony,” said Riglar. “The pattern of the fluorescent rings records how many repressilator cycles have occurred since growth began, and we can analyze that pattern to study how growth rates vary between different bacteria and in different environments.”Using RINGS, the team was able to successfully track cell divisions in several different bacterial species grown in vitro, and observed that the length of the bacteria’s repressilator cycle remained consistent when they were grown on extracted samples of mouse intestine (to simulate a complex microenvironment) or exposed to an antibiotic (to simulate stress conditions and inconsistent growth patterns).Tracking changeTo evaluate the repressilator’s performance in vivo, the team administered E. coli containing the repressilator circuit to mice orally, then analyzed bacteria extracted from fecal samples. The repressilator remained active for up to 16 days after introduction, showing that long-term oscillatory gene expression could be maintained in gut bacteria in living mammals. The RINGS analysis successfully detected changes in bacterial growth patterns, and bacteria whose repressilator circuits were in different stages could be “synchronized” by giving the mice a compound in their drinking water that arrested the repressilator cycle at a given stage.Finally, the researchers tested the repressilator’s ability to detect differences in bacterial growth rates that have been observed as a result of gut inflammation. Mice were given an inflammation-inducing compound, followed by repressilator-loaded bacteria. After 15 hours, RINGS analysis showed that the bacteria from mice with inflammation had repressilators in a wider range of phases than bacteria from control mice, suggesting that inflammation produces an environment that drives inconsistencies in bacterial growth, potentially leading to imbalances in the gut microbiome.“This repressilator allows us to really probe the intricacies of bacterial behavior in the living gut, not only in both healthy and diseased states, but also spatially and temporally,” said corresponding author Pamela Silver, a core faculty member at the Wyss Institute and the Elliot T. and Onie H. Adams Professor of Biochemistry and Systems Biology at HMS. “The fact that we can resynchronize the repressilator when it’s already in the gut, as well as maintain it without the need to administer selective antibiotics, also means that we can study the microbiome in a more natural state with minimal disruption.”In addition to understanding the dynamics of the microbiome, the repressilator unlocks the potential for complex, synthetic-biology-based diagnostics and therapeutics for the human gut. Potential applications include the creation of a system that is programmed to initiate a gene transcription cascade at a certain point in the circadian rhythm, or a diagnostic that records how much time has elapsed since the detection of a given biomarker.“Not only does this research solve a specific problem related to monitoring dynamic changes in microbiome physiology within the living gut, it provides a platform that could lead to entirely new types of diagnostics and even time-dependent therapeutics,” said Wyss Director Donald Ingber, who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as professor of bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.Additional authors of the paper include David Richmond, Laurent Potvin-Trottier, Andrew Verdegaal, Somenath Bakshi, Emanuele Leoncini, Lorena Lyon, and Johan Paulsson from HMS, and Alexander Naydich from the Wyss Institute, HMS, and the Harvard Paulson School.This research was supported by a Human Frontier Science Program Long-Term Fellowship, the Menzies Foundation, the Wellcome Trust, the National Science Foundation, the Defense Advanced Research Projects Agency, Harvard Medical School, and the Wyss Institute for Biologically Inspired Engineering. The Daily Gazette Sign up for daily emails to get the latest Harvard news. Discovery may open door to probiotics that enhance results from exercise and athletics You are what you eat — and how you cook it Finding our genomic clockwork Study finds performance-enhancing bacteria in human microbiome Research suggests gut microbes adapt quickly to changes in diet and preparation, particularly in starchy vegetables last_img read more

2016 Rio Olympics: Brazil Assuage Fears over Zika Virus

first_imgAlex Enumah in AbujaWith barely days to the commencement of the 2016 Olympics Games in Rio de Janeiro, the Brazilian government has assured all participants and visitors alike of its determination to host a historic and disease-free sports fiesta despite fears of the dreaded Zika virus disease in that region. He gave the assurance that the protection of the health of Brazilians and tourists coming to the global event is a priority and as such all necessary measures have been put in place to protect the health of everyone.In a statement made available to THISDAY by the Deputy Head of Mission, Embassy of Brazil in Abuja, Roberto Affaia, Brazil’s Minister of Health, Ricardo Barros said that protecting the health of Brazilians and tourists coming to this world event is a priority to the federal government, which has pledged to put into effect appropriate measures to protect peoples’ health.“I was recently in Geneva, Switzerland, and reaffirmed to the International Olympic Committee (IOC) that we would never risk the health of athletes and tourists. Brazil is keeping all necessary care and measures so that the games are a historical milestone in sports”.Barros claimed that the circulation of the Zika virus, spread by the Aedesaegypti mosquito, will not hinder the country from having a safe and unforgettable event for athletes, participants and spectators as according to him, the risks are minimal.Adding: The country’s healthcare system is duly prepared for this big moment, with preventive actions in place against the Aedesaegypti, 24/7 monitoring in the six cities hosting the games and trained professionals who are qualified to attend to emergencies.According to the statement a study published by the University of Cambridge makes a forecast of less than one case of infection among the 500,000 tourists.The statement further revealed that specialists from the World Health Organisation (WHO) on June 14 corroborated that the risk of propagation of the disease is very low. It said during the games, trips to Rio will represent 0.25 per cent of all travel to Zika-affected areas, according to United States’ CDC while noting that on the overall, the virus is already circulating in 60 countries, and Brazil represents 15 per cent of the exposed population.The minister also observed that during the games, Brazil will be in winter time, which is when diseases spread by the Aedesaegypti hit their lower rate.“In addition to that, the mobilisation actions to fight the mosquito, like home visits and public investment in monitoring and prevention, have caused an early fall of Zika rates – infection cases dropped 87 per cent between February and May this year.“The monitoring and follow-up of these data are still in progress by means of a partnership with the WHO, in an absolutely transparent manner. The proper measures to fight the Aedesaegypti are still in effect, with the backup of three thousand health agents in Rio.“During the event preparation phase, 51 test events were performed, monitored by the Ministry of Health, and no case of infection resulted from them. Since May 3, the Olympic torch has passed through more than 100 cities, and not a single case has been reported either” he said.While stating that Brazil has experience in organising big events, he said a similar fear was entertained over the possible epidemic of the dengue fever when Brazil hosted the last World Cup but, only three cases were reported in tourists.He therefore called on all tourists to feel free to visit the country during the games.The Olympics is perceived to be the greatest sporting event in the planet and over 200 countries and 500,000 international tourists are expected to be in Brazil for the games.Share this:FacebookRedditTwitterPrintPinterestEmailWhatsAppSkypeLinkedInTumblrPocketTelegramlast_img read more

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