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Pimenta A.C.,University of Porto | Dourado D.F.A.R.,Computational and Systems Biology | Martins J.M.,University of Porto | Melo A.,University of Porto | And 5 more authors.
Journal of Chemical Information and Modeling | Year: 2014

Crystallographic structures of NGF/p75NTR and proNGF/p75NTR were previously obtained in 2:1 and 2:2 stoichiometries, respectively. However, evidence shows that both stoichiometries can occur for mature neurotrophins and pro-neurotrophins. We used Molecular Dynamics (MD) simulations to examine the energetic and structural characteristics of these two complete systems as well as the uncomplexed forms of NGF and understand how these could translate in a new view of different biological outcomes. Here, we show that one chain at the 2:2 proNGF complex seems to be preferentially lost creating a 2:1 structure able to interact with sortilin. We also demonstrated that the structure of the neurotrophin dimers is not pre-established and suffers large structural modifications upon p75NTR binding. Moreover, our data suggests an elegant explanation for the dual role of NGF in neuronal cell death and survival, where different stoichiometries induce conformational changes that might be the basis for the different biological outcomes observed with the mature and proforms of neurotrophins. © 2014 American Chemical Society. Source


Ting Tan R.S.,National University of Singapore | Lin B.,National University of Singapore | Lin B.,U.S. National Institutes of Health | Liu Q.,National University of Singapore | And 5 more authors.
Immunology and Cell Biology | Year: 2013

Although specific single Toll-like receptor (TLR) ligands are known to drive the development of Th1 or Th2 immunity, the outcome of different combinations of TLR ligands on innate immunity is not well defined. Spatiotemporal dynamics are critical in determining the specificity of the immune response, but the mechanisms underlying combinatorial TLR stimulation remain unclear. Here, we tested pairwise combinations of TLR ligands separated by different time intervals for their effect on cytokine production in macrophages. We observed that stimulation via a combination of MyD88- and TRIF-utilizing adaptors leads to a highly synergistic cytokine response. On a timescale of 4-24 h, macrophages pretreated with poly(I:C) (TLR3 ligand) are cross-primed to a second stimulation with R848 (TLR7 ligand) and vice versa, and each condition exhibits different optimal time windows of synergistic response for each cytokine. We show that the synergy resulting from combinatorial stimuli (poly(I:C) and R848 is also regulated by the order and dosage of the TLR agonists. Secondary response genes, which depend on new protein synthesis for transcription, show greater synergy than primary response genes, and such enhancement is abolished when new protein synthesis is inhibited. Synergistic cytokine production appears concordant with sustained ERK phosphorylation, suggesting that the de novo factors act via inhibition of ERK dephosphorylation, for example, by the downregulation of dual specificity phosphatase 6. Taken together, our findings illustrate a checkpoint in the innate immune system, where the synchronization of timing of both MyD88 and TRIF pathways is required for a maximal cytokine response and potential memory effect in macrophages. © 2013 Australasian Society for Immunology Inc. All rights reserved. Source


News Article
Site: http://news.mit.edu/topic/mitmechanical-engineering-rss.xml

A new technique invented at MIT can precisely measure the growth of many individual cells simultaneously. The advance holds promise for fast drug tests, offers new insights into growth variation across single cells within larger populations, and helps track the dynamic growth of cells to changing environmental conditions. The technique, described in a paper published in Nature Biotechnology, uses an array of suspended microchannel resonators (SMR), a type of microfluidic device that measures the mass of individual cells as they flow through tiny channels. A novel design has increased throughput of the device by nearly two orders of magnitude, while retaining precision. The paper’s senior author, MIT professor Scott Manalis, and other researchers have been developing SMRs for nearly a decade. In the new study, the researchers used the device to observe the effects of antibiotics and antimicrobial peptides on bacteria, and to pinpoint growth variations of single cells among populations, which has important clinical applications. Slower-growing bacteria, for instance, can sometimes be more resistant to antibiotics and may lead to recurrent infections. “The device provides new insights into how cells grow and respond to drugs,” says Manalis, the Andrew (1956) and Erna Viterbi Professor in the MIT departments of Biological Engineering and Mechanical Engineering and a member of the Koch Institute for Integrative Cancer Research. The paper’s lead authors are Nathan Cermak, a recent PhD graduate from MIT’s Computational and Systems Biology Program, and Selim Olcum, a research scientist at the Koch Institute. There are 13 other co-authors on the paper, from the Koch Institute, MIT’s Microsystems Technology Laboratory, the Dana-Farber Cancer Institute, Innovative Micro Technology, and CEA LETI in France. Manalis and his colleagues first developed the SMR in 2007 and have since introduced multiple innovations for different purposes, including to track single cell growth over time, measure cell density, weigh cell-secreted nanovesicles, and, most recently, measure the short-term growth response of cells in changing nutrient conditions. All of these techniques have relied on a crucial scheme: One fluid-filled microchannel is etched in a tiny silicon cantilever sensor that vibrates inside a vacuum cavity. When a cell enters the cantilever, it slightly alters the sensor’s vibration frequency, and this signal can be used to determine the cell’s weight. To measure a cell’s growth rate, Manalis and colleagues could pass an individual cell through the channel repeatedly, back and forth, over a period of about 20 minutes. During that time, a cell can accumulate mass that is measurable by the SMR. But while the SMR weighs cells 10 to 100 times more accurately than any other method, it has been limited to one cell at a time, meaning it could take many hours, or even days, to measure enough cells. The key to the new technology was designing and controlling an array of 10 to 12 cantilever sensors that act like weigh stations, recording the mass of a cell as it flows through the postage-stamp-sized device. Between each sensor are winding “delay channels,” each about five centimeters in length, through which the cells flow for about two minutes, giving them time to grow before reaching the next sensor. Whenever one cell exits a sensor, another cell can enter, increasing the device’s throughput. Results show the mass of each cell at each sensor, graphing the extent to which they’ve grown or shrunk. In the study, the researchers were able to measure about 60 mammalian cells and 150 bacteria per hour, compared to single SMRs, which measured only a few cells in that time. “Being able to rapidly measure the full distribution of growth rates shows us both how typical cells are behaving, an­­d also lets us detect outliers — which was previously very difficult with limited throughput or precision,” Cermak says. One comparable method for measuring masses of many individual cells simultaneously is called quantitative phase microscopy (QPM), which calculates the dry mass of cells by measuring their optical thickness. Unlike the SMR-based approach, QPM can be used on cells that grow adhered to surfaces. However, the SMR-based approach is significantly more precise. “We can reliably resolve changes of less than one-tenth of a percent of a cancer cell’s mass in about 20 minutes. This precision is proving to be essential for many of the clinical applications that we’re pursuing,” Olcum says. In one experiment using the device, the researchers observed the effects of an antibiotic, called kanamycin, on E. coli. Kanamycin inhibits protein synthesis in bacteria, eventually stopping their growth and killing the cells. Traditional antibiotic tests require growing a culture of bacteria, which could take a day or more. Using the new device, within an hour the researchers recorded a change in rate in which the cells accumulate mass. The reduced recording time is critical in testing drugs against bacterial infections in clinical settings, Manalis says: “In some cases, having a rapid test for selecting an antibiotic can make an important difference in the survival of a patient.” Similarly, the researchers used the device to observe the effects of an antimicrobial peptide called CM15, a relatively new protein-based candidate for fighting bacteria. Such candidates are increasingly important as bacteria strains become resistant to common antibiotics. CM15 makes microscopic holes in bacteria cell walls, such that the cell’s contents gradually leak out, eventually killing the cell. However, because only the mass of the cell changes and not its size, the effects may be missed by traditional microscopy techniques. Indeed, the researchers observed the E. coli cells rapidly losing mass immediately following exposure to CM15. Such results could lend validation to the peptide and other novel drugs by providing some insight into the mechanism, Manalis says. The researchers are currently working with members of the Dana Farber Cancer Institute, through the the Koch Institute and Dana Farber/Harvard Cancer Center Bridge Project, to determine if the device could be used to predict patient response to therapy by weighing tumor cells in the presence of anticancer drugs. Marc Kirschner, a professor and chair of the Department of Systems Biology at Harvard Medical School, who was not involved in the study, said the new microfluidics device will open up new avenues for studying the “physiology and pharmacology of cell growth. … Since growth is related to proliferation and to the stress a cell is under, it is a natural feature to study, but it has been difficult before this method.” “The technical problems to get this working were significant and it is still incredible for me to think that they pulled this off,” Kirschner adds. “I expect that when it is … into biology labs it will be useful for many problems in cancer, metabolism, cell death, and cell stress.” The research was sponsored, in part, by the U.S. Army Research Office, the Koch Institute and Dana Farber/Harvard Cancer Center Bridge Project, the National Science Foundation, and the National Cancer Institute.


Angel A.,Computational and Systems Biology | Angel A.,John Innes Center | Angel A.,University of Oxford | Song J.,John Innes Center | And 6 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

A fundamental property of many organisms is an ability to sense, evaluate, and respond to environmental signals. In some situations, generation of an appropriate response requires long-term information storage. A classic example is vernalization, where plants quantitatively sense long-term cold and epigenetically store this cold-exposure information to regulate flowering time. In Arabidopsis thaliana, stable epigenetic memory of cold is digital: following long-term cold exposure, cells respond autonomously in an all-or-nothing fashion, with the fraction of cells that stably silence the floral repressor FLOWERING LOCUS C (FLC) increasing with the cold exposure duration. However, during cold exposure itself it is unknown whether vernalizing cold is registered at FLC in individual cells in an all-or-nothing (digital) manner or is continuously varying (analog). Using mathematical modeling, we found that analog registration of cold temperature is problematic due to impaired analog-to-digital conversion into stable memory. This disadvantage is particularly acute when responding to short cold periods, but is absent when cold temperatures are registered digitally at FLC. We tested this prediction experimentally, exposing plants to short periods of cold interrupted with even shorter warm breaks. For FLC expression, we found that the system responds similarly to both interrupted and uninterrupted cold, arguing for a digital mechanism integrating long-term temperature exposure. Source


News Article | September 6, 2016
Site: http://www.biosciencetechnology.com/rss-feeds/all/rss.xml/all

A new technique invented at MIT can precisely measure the growth of many individual cells simultaneously. The advance holds promise for fast drug tests, offers new insights into growth variation across single cells within larger populations, and helps track the dynamic growth of cells to changing environmental conditions. The technique, described in a paper published in Nature Biotechnology, uses an array of suspended microchannel resonators (SMR), a type of microfluidic device that measures the mass of individual cells as they flow through tiny channels. A novel design has increased throughput of the device by nearly two orders of magnitude, while retaining precision. The paper’s senior author, MIT professor Scott Manalis, and other researchers have been developing SMRs for nearly a decade. In the new study, the researchers used the device to observe the effects of antibiotics and antimicrobial peptides on bacteria, and to pinpoint growth variations of single cells among populations, which has important clinical applications. Slower-growing bacteria, for instance, can sometimes be more resistant to antibiotics and may lead to recurrent infections. “The device provides new insights into how cells grow and respond to drugs,” says Manalis, the Andrew (1956) and Erna Viterbi Professor in the MIT departments of Biological Engineering and Mechanical Engineering and a member of the Koch Institute for Integrative Cancer Research. The paper’s lead authors are Nathan Cermak, a recent Ph.D. graduate from MIT’s Computational and Systems Biology Program, and Selim Olcum, a research scientist at the Koch Institute. There are 13 other co-authors on the paper, from the Koch Institute, MIT’s Microsystems Technology Laboratory, the Dana-Farber Cancer Institute, Innovative Micro Technology, and CEA LETI in France. Manalis and his colleagues first developed the SMR in 2007 and have since introduced multiple innovations for different purposes, including to track single cell growth over time, measure cell density, weigh cell-secreted nanovesicles, and, most recently, measure the short-term growth response of cells in changing nutrient conditions. All of these techniques have relied on a crucial scheme: One fluid-filled microchannel is etched in a tiny silicon cantilever sensor that vibrates inside a vacuum cavity. When a cell enters the cantilever, it slightly alters the sensor’s vibration frequency, and this signal can be used to determine the cell’s weight. To measure a cell’s growth rate, Manalis and colleagues could pass an individual cell through the channel repeatedly, back and forth, over a period of about 20 minutes. During that time, a cell can accumulate mass that is measurable by the SMR. But while the SMR weighs cells 10 to 100 times more accurately than any other method, it has been limited to one cell at a time, meaning it could take many hours, or even days, to measure enough cells. The key to the new technology was designing and controlling an array of 10 to 12 cantilever sensors that act like weigh stations, recording the mass of a cell as it flows through the postage-stamp-sized device. Between each sensor are winding “delay channels,” each about five centimeters in length, through which the cells flow for about two minutes, giving them time to grow before reaching the next sensor. Whenever one cell exits a sensor, another cell can enter, increasing the device’s throughput. Results show the mass of each cell at each sensor, graphing the extent to which they’ve grown or shrunk. In the study, the researchers were able to measure about 60 mammalian cells and 150 bacteria per hour, compared to single SMRs, which measured only a few cells in that time. “Being able to rapidly measure the full distribution of growth rates shows us both how typical cells are behaving, an­­d also lets us detect outliers — which was previously very difficult with limited throughput or precision,” Cermak says. One comparable method for measuring masses of many individual cells simultaneously is called quantitative phase microscopy (QPM), which calculates the dry mass of cells by measuring their optical thickness. Unlike the SMR-based approach, QPM can be used on cells that grow adhered to surfaces. However, the SMR-based approach is significantly more precise. “We can reliably resolve changes of less than one-tenth of a percent of a cancer cell’s mass in about 20 minutes. This precision is proving to be essential for many of the clinical applications that we’re pursuing,” Olcum says. In one experiment using the device, the researchers observed the effects of an antibiotic, called kanamycin, on E. coli. Kanamycin inhibits protein synthesis in bacteria, eventually stopping their growth and killing the cells. Traditional antibiotic tests require growing a culture of bacteria, which could take a day or more. Using the new device, within an hour the researchers recorded a change in rate in which the cells accumulate mass. The reduced recording time is critical in testing drugs against bacterial infections in clinical settings, Manalis says: “In some cases, having a rapid test for selecting an antibiotic can make an important difference in the survival of a patient.” Similarly, the researchers used the device to observe the effects of an antimicrobial peptide called CM15, a relatively new protein-based candidate for fighting bacteria. Such candidates are increasingly important as bacteria strains become resistant to common antibiotics. CM15 makes microscopic holes in bacteria cell walls, such that the cell’s contents gradually leak out, eventually killing the cell. However, because only the mass of the cell changes and not its size, the effects may be missed by traditional microscopy techniques. Indeed, the researchers observed the E. coli cells rapidly losing mass immediately following exposure to CM15. Such results could lend validation to the peptide and other novel drugs by providing some insight into the mechanism, Manalis says. The researchers are currently working with members of the Dana Farber Cancer Institute, through the MIT/DFCI Bridge program, to determine if the device could be used to predict patient response to therapy by weighing tumor cells in the presence of anticancer drugs. Marc Kirschner, a professor and chair of the Department of Systems Biology at Harvard Medical School, who was not involved in the study, said the new microfluidics device will open up new avenues for studying the “physiology and pharmacology of cell growth. … Since growth is related to proliferation and to the stress a cell is under, it is a natural feature to study, but it has been difficult before this method.” “The technical problems to get this working were significant and it is still incredible for me to think that they pulled this off,” Kirschner adds. “I expect that when it is … into biology labs it will be useful for many problems in cancer, metabolism, cell death, and cell stress.” The research was sponsored, in part, by the U.S. Army Research Office, the Koch Institute and Dana Farber/Harvard Cancer Center Bridge Project, the National Science Foundation, and the National Cancer Institute.

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