TY - JOUR TI - An automated programmable platform enabling multiplex dynamic stimuli delivery and cellular response monitoring for high-throughput suspension single-cell signaling studies AU - He, Luye AU - Kniss, Ariel AU - San-Miguel, Adriana AU - Rouse, Tel AU - Kemp, Melissa L. AU - Lu, Hang T2 - Lab on a Chip AB - Cell signaling events are orchestrated by dynamic external biochemical cues. By rapidly perturbing cells with dynamic inputs and examining the output from these systems, one could study the structure and dynamic properties of a cellular signaling network. Conventional experimental techniques limit the implementation of these systematic approaches due to the lack of sophistication in manipulating individual cells and the fluid microenvironment around them; existing microfluidic technologies thus far are mainly targeting adherent cells. In this paper we present an automated platform to interrogate suspension cells with dynamic stimuli while simultaneously monitoring cellular responses in a high-throughput manner at single-cell resolution. We demonstrate the use of this platform in an experiment to measure Jurkat T cells in response to distinct dynamic patterns of stimuli; we find cells exhibit highly heterogeneous responses under each stimulation condition. More interestingly, these cells act as low-pass filters, only entrained to the low frequency stimulus signals. We also demonstrate that this platform can be easily programmed to actively generate arbitrary dynamic signals. We envision our platform to be useful in other contexts to study cellular signaling dynamics, which may be difficult using conventional experimental methods. DA - 2015/// PY - 2015/// DO - 10.1039/C4LC01070A VL - 15 IS - 6 SP - 1497-1507 J2 - Lab Chip LA - en OP - SN - 1473-0197 1473-0189 UR - http://dx.doi.org/10.1039/C4LC01070A DB - Crossref ER - TY - JOUR TI - Widespread Macromolecular Interaction Perturbations in Human Genetic Disorders AU - Sahni, Nidhi AU - Yi, Song AU - Taipale, Mikko AU - Fuxman Bass, Juan I. AU - Coulombe-Huntington, Jasmin AU - Yang, Fan AU - Peng, Jian AU - Weile, Jochen AU - Karras, Georgios I. AU - Wang, Yang AU - Kovács, István A. AU - Kamburov, Atanas AU - Krykbaeva, Irina AU - Lam, Mandy H. AU - Tucker, George AU - Khurana, Vikram AU - Sharma, Amitabh AU - Liu, Yang-Yu AU - Yachie, Nozomu AU - Zhong, Quan AU - Shen, Yun AU - Palagi, Alexandre AU - San-Miguel, Adriana AU - Fan, Changyu AU - Balcha, Dawit AU - Dricot, Amelie AU - Jordan, Daniel M. AU - Walsh, Jennifer M. AU - Shah, Akash A. AU - Yang, Xinping AU - Stoyanova, Ani K. AU - Leighton, Alex AU - Calderwood, Michael A. AU - Jacob, Yves AU - Cusick, Michael E. AU - Salehi-Ashtiani, Kourosh AU - Whitesell, Luke J. AU - Sunyaev, Shamil AU - Berger, Bonnie AU - Barabási, Albert-László AU - Charloteaux, Benoit AU - Hill, David E. AU - Hao, Tong AU - Roth, Frederick P. AU - Xia, Yu AU - Walhout, Albertha J.M. AU - Lindquist, Susan AU - Vidal, Marc T2 - Cell AB - How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread. DA - 2015/4// PY - 2015/4// DO - 10.1016/J.CELL.2015.04.013 VL - 161 IS - 3 SP - 647-660 J2 - Cell LA - en OP - SN - 0092-8674 UR - http://dx.doi.org/10.1016/J.CELL.2015.04.013 DB - Crossref ER - TY - JOUR TI - Multi-modal contributions to detoxification of acute pharmacotoxicity by a triglyceride micro-emulsion AU - Fettiplace, M.R. AU - Lis, K. AU - Ripper, R. AU - Kowal, K. AU - Pichurko, A. AU - Vitello, D. AU - Rubinstein, I. AU - Schwartz, D. AU - Akpa, B.S. AU - Weinberg, G. T2 - Journal of Controlled Release AB - Triglyceride micro-emulsions such as Intralipid® have been used to reverse cardiac toxicity induced by a number of drugs but reservations about their broad-spectrum applicability remain because of the poorly understood mechanism of action. Herein we report an integrated mechanism of reversal of bupivacaine toxicity that includes both transient drug scavenging and a cardiotonic effect that couple to accelerate movement of the toxin away from sites of toxicity. We thus propose a multi-modal therapeutic paradigm for colloidal bio-detoxification whereby a micro-emulsion both improves cardiac output and rapidly ferries the drug away from organs subject to toxicity. In vivo and in silico models of toxicity were combined to test the contribution of individual mechanisms and reveal the multi-modal role played by the cardiotonic and scavenging actions of the triglyceride suspension. These results suggest a method to predict which drug toxicities are most amenable to treatment and inform the design of next-generation therapeutics for drug overdose. DA - 2015/// PY - 2015/// DO - 10.1016/j.jconrel.2014.11.018 VL - 198 SP - 62-70 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-84949117634&partnerID=MN8TOARS KW - Lipid emulsion KW - Local anesthetic KW - Bupivacaine KW - Triglyceride KW - Antidote KW - Biodetoxification ER - TY - JOUR TI - Chromatin regulation at the frontier of synthetic biology AU - Keung, Albert J. AU - Joung, J. Keith AU - Khalil, Ahmad S. AU - Collins, James J. T2 - Nature Reviews Genetics AB - Synthetic biology approaches to characterize gene regulation have largely used transcription factor circuits in bacteria. However, the multilayered regulation of genes by chromatin in eukaryotes provides opportunities for more sophisticated control of gene expression. This Review describes diverse approaches for engineering eukaryotic chromatin states, the insights gained into physiological gene regulation principles, and the broad potential applications throughout biomedical research and industry. As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication. DA - 2015/2/10/ PY - 2015/2/10/ DO - 10.1038/NRG3900 VL - 16 IS - 3 SP - 159-171 J2 - Nat Rev Genet LA - en OP - SN - 1471-0056 1471-0064 UR - http://dx.doi.org/10.1038/NRG3900 DB - Crossref ER - TY - JOUR TI - Confusion About Infusion: Rational Volume Limits for Intravenous Lipid Emulsion During Treatment of Oral Overdoses. AU - Fettiplace, M.R. AU - Akpa, B.S. AU - Rubinstein, I. AU - Weinberg, G. T2 - Annals of emergency medicine AB - After the initial report of treatment of bupropion and lamotrigine overdose by intravenous lipid emulsion, 1 Sirianni A.J. Osterhoudt K.C. Calello D.P. et al. Use of lipid emulsion in the resuscitation of a patient with prolonged cardiovascular collapse after overdose of bupropion and lamotrigine. Ann Emerg Med. 2008; 51: 412-415 Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar additional case reports have asserted the usefulness of lipid emulsion for enteral poisonings. 2 Cave G. Harvey M. Intravenous lipid emulsion as antidote: beyond local anesthetic toxicity: a systematic review. Acad Emerg Med. 2009; 16: 815-824 Crossref PubMed Scopus (159) Google Scholar , 3 Weinberg G. Lipid emulsion infusion resuscitation for local anesthetic and other drug overdose. Anesthesiology. 2012; 117: 180-187 Crossref PubMed Scopus (169) Google Scholar In accordance with these and other reports, the American College of Medical Toxicology offered interim guidelines on lipid resuscitation therapy 4 American College of Medical ToxicologyACMT position statement: interim guidance for the use of lipid resuscitation therapy. J Med Toxicol. 2011; 7: 81-82 Crossref PubMed Scopus (72) Google Scholar ; they recommended a bolus of 1.5 mL/kg, followed by an infusion of 0.25 mL/kg/min of intravenous lipid emulsion if toxicity persists. This recommendation was based on guidelines for treatment of local anesthetic toxicity, 5 Harrop-Griffiths W, Harbey M, Meek T, et al. AAGBI safety guideline: management of severse local anaesthetic toxicity. Association of Anaesthetists of Great Britain and Ireland. 2010. Google Scholar , 6 Neal J.M. Bernards C.M. Butterworth J.F. et al. ASRA practice advisory on local anesthetic systemic toxicity. Reg Anesth Pain Med. 2010; 35: 152-161 Crossref PubMed Scopus (413) Google Scholar in which absorption is quick and toxicity short lived. In contrast to local anesthetic toxicity, prolonged absorption during enteral overdose can result in extended toxicity, with a need for continuing medical support, including a protracted infusion of lipid emulsion. Guidelines for lipid resuscitation therapy in local anesthetic toxicity set an upper limit of 10 to 12 mL/kg during the first half hour, but because of the aforementioned nature of oral overdoses, the American College of Medical Toxicology did not provide a limit on total lipid infusion volume or duration of infusion. In the absence of limits, an increasing number of cases have reported the use of large volumes of lipid to treat oral overdose. However, until definitive studies can be conducted, there is a need for rational volume limits to prevent undisciplined use of intravenous lipid emulsion. Is a 1% Plasma Lipid Concentration Helpful to Treat the Intoxicated Patient?Annals of Emergency MedicineVol. 67Issue 3PreviewWe read with interest the article by Fettiplace et al.1 The authors propose a lipid emulsion infusion regimen to induce a 1% plasma triglyceride concentration to treat a prolonged absorption after oral overdose resulting in a lengthened increased plasma concentration. Full-Text PDF DA - 2015/2/27/ PY - 2015/2/27/ DO - 10.1016/j.annemergmed.2015.01.020 VL - 66 IS - 2 SP - 185-188 UR - https://doi.org/10.1016/j.annemergmed.2015.01.020 ER - TY - JOUR TI - Bird and Scarecrow proteins organize tissue formation in Arabidopsis roots AU - Moreno-Risueno, MA. AU - Sozzani, R. AU - Yardimici, GG. AU - Petricka, JJ. AU - Vernoux, T. AU - Blilou, I. AU - Alonso, J. AU - Winter, CM. AU - Ohler, U. AU - Scheres, B. AU - Benfey, P.N. T2 - Science AB - Tissue patterns are dynamically maintained. Continuous formation of plant tissues during postembryonic growth requires asymmetric divisions and the specification of cell lineages. We show that the BIRDs and SCARECROW regulate lineage identity, positional signals, patterning, and formative divisions throughout Arabidopsis root growth. These transcription factors are postembryonic determinants of the ground tissue stem cells and their lineage. Upon further activation by the positional signal SHORT-ROOT (a mobile transcription factor), they direct asymmetric cell divisions and patterning of cell types. The BIRDs and SCARECROW with SHORT-ROOT organize tissue patterns at all formative steps during growth, ensuring developmental plasticity. DA - 2015/// PY - 2015/// DO - 10.1126/science.aad1171 VL - 350 SP - 426–430 ER - TY - JOUR TI - Methods for RNA Profiling of Gravi-Responding Plant Tissues AU - Dalal, Jyoti AU - Land, Eric AU - Vasani, Naresh AU - He, Luyan AU - Smith, Caroline AU - Rodriguez-Welsh, Maria AU - Perera, Imara Y. AU - Sederoff, Heike T2 - PLANT GRAVITROPISM: METHODS AND PROTOCOLS AB - Plant transcriptional responses to gravity stimulation by reorientation are among the fastest measured in any tissue or species. Upon reorientation, changes in abundance of specific mRNAs can be measured within seconds or minutes, for plastid or nuclear encoded genes, respectively. Identifying fast gravity-induced transcripts has been made possible by the development of high-throughput technology for qualitative and quantitative RNA analysis. RNA profiling has undergone further rapid development due to its enormous potential in basic sciences and medical applications. We describe here the current and most widely used methods to profile the changes in an entire transcriptome by high-throughput sequencing of RNA fractions (RNAseq) and single gene transcript analysis using real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). DA - 2015/// PY - 2015/// DO - 10.1007/978-1-4939-2697-8_9 VL - 1309 SP - 91-117 SN - 1940-6029 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-84929650489&partnerID=MN8TOARS KW - RNA KW - Transcript KW - Transcriptome KW - Sequencing KW - PCR KW - RNAseq KW - sRNA KW - miRNA ER - TY - JOUR TI - Algal ancestor of land plants was preadapted for symbiosis AU - Delaux, Pierre-Marc AU - Radhakrishnan, Guru V. AU - Jayaraman, Dhileepkumar AU - Cheem, Jitender AU - Malbreil, Mathilde AU - Volkening, Jeremy D. AU - Sekimoto, Hiroyuki AU - Nishiyama, Tomoaki AU - Melkonian, Michael AU - Pokorny, Lisa AU - Rothfels, Carl J. AU - Sederoff, Heike Winter AU - Stevenson, Dennis W. AU - Surek, Barbara AU - Zhang, Yong AU - Sussman, Michael R. AU - Dunand, Christophe AU - Morris, Richard J. AU - Roux, Christophe AU - Wong, Gane Ka-Shu AU - Oldroyd, Giles E. D. AU - Ane, Jean-Michel T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - Significance Colonization of land by plants was a critical event for the emergence of extant ecosystems. The innovations that allowed the algal ancestor of land plants to succeed in such a transition remain unknown. Beneficial interaction with symbiotic fungi has been proposed as one of these innovations. Here we show that the genes required for this interaction appeared in a stepwise manner: Some evolved before the colonization of land by plants and others first appeared in land plants. We thus propose that the algal ancestor of land plants was preadapted for interaction with beneficial fungi and employed these gene networks to colonize land successfully. DA - 2015/10/27/ PY - 2015/10/27/ DO - 10.1073/pnas.1515426112 VL - 112 IS - 43 SP - 13390-13395 SN - 0027-8424 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-84945402783&partnerID=MN8TOARS KW - symbiosis KW - plant evolution KW - algae KW - plant-microbe interactions KW - phylogeny ER - TY - JOUR TI - A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants AU - Dalal, J. AU - Yalamanchili, R. AU - Hovary, C. La AU - Ji, M. AU - Rodriguez-Welsh, M. AU - Aslett, D. AU - Ganapathy, S. AU - Grunden, A. AU - Sederoff, Heike AU - Qu, R. D. AU - al. T2 - PLASMID AB - The rapidly advancing field of plant synthetic biology requires transforming plants with multiple genes. This has sparked a growing interest in flexible plant transformation vectors, which can be used for multi-gene transformations. We have developed a novel binary vector series, named the PC-GW series (GenBank: KP826769-KP826773), for Agrobacterium-mediated plant transformation. The PC-GW vectors use the pCAMBIA vector backbone, and contain NPTII, hpt, bar, mCherry or egfp genes as selectable markers for plant transformation. In a modified multiple cloning site (MCS) of the T-DNA region, we have placed the attR1, attR2 and ccdB sequences for rapid cloning of one to four genes by Gateway™-assisted recombination. In addition, we have introduced four meganuclease sites, and other restriction sites for multi-gene vector construction. Finally, we have placed a CaMV 35S promoter and a 35S terminator on the 5' and 3' ends of the MCS. The CaMV 35S promoter is flanked by PstI restriction sites that can be used to replace it with another promoter sequence if needed. The PC-GW vectors provide choices for selectable markers, cloning methods, and can accommodate up to eight gene constructs in a single T-DNA, thereby significantly reducing the number of transformations or crosses needed to generate multi-transgene expressing plants. DA - 2015/9// PY - 2015/9// DO - 10.1016/j.plasmid.2015.06.003 VL - 81 SP - 55-62 SN - 1095-9890 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-84938634755&partnerID=MN8TOARS KW - Gateway KW - Plant transformation KW - Agrobacterium KW - Gene stacking ER - TY - JOUR TI - A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa AU - Dalal, Jyoti AU - Lopez, Harry AU - Vasani, Naresh B. AU - Hu, Zhaohui AU - Swift, Jennifer E. AU - Yalamanchili, Roopa AU - Dvora, Mia AU - Lin, Xiuli AU - Xie, Deyu AU - Qu, Rongda AU - Sederoff, Heike W. T2 - BIOTECHNOLOGY FOR BIOFUELS AB - Camelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits.Expressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50-57 %, while expression of full bypass increased seed yield by 57-73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions.The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops. DA - 2015/10/29/ PY - 2015/10/29/ DO - 10.1186/s13068-015-0357-1 VL - 8 IS - 1 SP - SN - 1754-6834 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-84945972179&partnerID=MN8TOARS KW - Camelina KW - Seed yield KW - Biofuel KW - Photorespiratory bypass KW - Photosynthesis ER - TY - JOUR TI - Interaction of Temperature and Photoperiod Increases Growth and Oil Content in the Marine Microalgae Dunaliella viridis AU - Srirangan, Soundarya AU - Sauer, Marie-Laure AU - Howard, Brian AU - Dvora, Mia AU - Dums, Jacob AU - Backman, Patrick AU - Sederoff, Heike T2 - PLOS ONE AB - Eukaryotic marine microalgae like Dunaliella spp. have great potential as a feedstock for liquid transportation fuels because they grow fast and can accumulate high levels of triacylgycerides with little need for fresh water or land. Their growth rates vary between species and are dependent on environmental conditions. The cell cycle, starch and triacylglycerol accumulation are controlled by the diurnal light:dark cycle. Storage compounds like starch and triacylglycerol accumulate in the light when CO2 fixation rates exceed the need of assimilated carbon and energy for cell maintenance and division during the dark phase. To delineate environmental effects, we analyzed cell division rates, metabolism and transcriptional regulation in Dunaliella viridis in response to changes in light duration and growth temperatures. Its rate of cell division was increased under continuous light conditions, while a shift in temperature from 25 °C to 35 °C did not significantly affect the cell division rate, but increased the triacylglycerol content per cell several-fold under continuous light. The amount of saturated fatty acids in triacylglycerol fraction was more responsive to an increase in temperature than to a change in the light regime. Detailed fatty acid profiles showed that Dunaliella viridis incorporated lauric acid (C12:0) into triacylglycerol after 24 hours under continuous light. Transcriptome analysis identified potential regulators involved in the light and temperature-induced lipid accumulation in Dunaliella viridis. DA - 2015/5/19/ PY - 2015/5/19/ DO - 10.1371/journal.pone.0127562 VL - 10 IS - 5 SP - SN - 1932-6203 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-84930682335&partnerID=MN8TOARS ER -