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1.  Population Diversification in a Yeast Metabolic Program Promotes Anticipation of Environmental Shifts 
PLoS Biology  2015;13(1):e1002042.
Detailed study of the dynamic response of yeast to combinations of sugars reveals an anticipatory population diversification strategy that allows rapid adaptation to shifts in environmental carbon source availability.
Author Summary
To survive in resource-limited and dynamic environments, microbial populations implement a diverse repertoire of regulatory strategies. These strategies often rely on anticipating impending environmental shifts, enabling the population to be prepared for a future change in conditions. It has long been known that cells optimize nutritional value from mixtures of carbon sources, for example glucose and galactose, by sequential activation of regulatory programs that allow for metabolizing the preferred carbon source first before metabolizing the secondary carbon source. Using automated flow-cytometry, we mapped the dynamical behavior of populations simultaneously presented with a large panel of different glucose and galactose concentrations. We show that, counter to expectations, in populations presented with glucose and galactose simultaneously, the galactose regulatory pathway is activated in a fraction of the cell population hours before glucose is fully consumed. We demonstrate that the size of this fraction of cells is tuned by the concentration of the two sugars. This population diversification may constitute a tradeoff between the benefit of rapid galactose consumption once glucose is depleted and the cost of expressing the galactose pathway.
Delineating the strategies by which cells contend with combinatorial changing environments is crucial for understanding cellular regulatory organization. When presented with two carbon sources, microorganisms first consume the carbon substrate that supports the highest growth rate (e.g., glucose) and then switch to the secondary carbon source (e.g., galactose), a paradigm known as the Monod model. Sequential sugar utilization has been attributed to transcriptional repression of the secondary metabolic pathway, followed by activation of this pathway upon depletion of the preferred carbon source. In this work, we demonstrate that although Saccharomyces cerevisiae cells consume glucose before galactose, the galactose regulatory pathway is activated in a fraction of the cell population hours before glucose is fully consumed. This early activation reduces the time required for the population to transition between the two metabolic programs and provides a fitness advantage that might be crucial in competitive environments.
PMCID: PMC4307983  PMID: 25626086
2.  Human vision is attuned to the diffuseness of natural light 
Journal of Vision  2014;14(9):15.
All images are highly ambiguous, and to perceive 3-D scenes, the human visual system relies on assumptions about what lighting conditions are most probable. Here we show that human observers' assumptions about lighting diffuseness are well matched to the diffuseness of lighting in real-world scenes. We use a novel multidirectional photometer to measure lighting in hundreds of environments, and we find that the diffuseness of natural lighting falls in the same range as previous psychophysical estimates of the visual system's assumptions about diffuseness. We also find that natural lighting is typically directional enough to override human observers' assumption that light comes from above. Furthermore, we find that, although human performance on some tasks is worse in diffuse light, this can be largely accounted for by intrinsic task difficulty. These findings suggest that human vision is attuned to the diffuseness levels of natural lighting conditions.
PMCID: PMC4141658  PMID: 25139864
lightness; prior; lighting statistics; diffuseness; ideal observer
3.  Synthetic circuit for exact adaptation and fold-change detection 
Nucleic Acids Research  2014;42(9):6078-6089.
Biological organisms use their sensory systems to detect changes in their environment. The ability of sensory systems to adapt to static inputs allows wide dynamic range as well as sensitivity to input changes including fold-change detection, a response that depends only on fold changes in input, and not on absolute changes. This input scale invariance underlies an important strategy for search that depends solely on the spatial profile of the input. Synthetic efforts to reproduce the architecture and response of cellular circuits provide an important step to foster understanding at the molecular level. We report the bottom-up assembly of biochemical systems that show exact adaptation and fold-change detection. Using a malachite green aptamer as the output, a synthetic transcriptional circuit with the connectivity of an incoherent feed-forward loop motif exhibits pulse generation and exact adaptation. A simple mathematical model was used to assess the amplitude and duration of pulse response as well as the parameter regimes required for fold-change detection. Upon parameter tuning, this synthetic circuit exhibits fold-change detection for four successive rounds of two-fold input changes. The experimental realization of fold-change detection circuit highlights the programmability of transcriptional switches and the ability to obtain predictive dynamical systems in a cell-free environment for technological applications.
PMCID: PMC4027175  PMID: 24728988
4.  Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology 
Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."
PMCID: PMC3960857  PMID: 24084388
Cellular Biology; Issue 79; Bioengineering; Synthetic Biology; Chemistry Techniques; Synthetic; Molecular Biology; control theory; TX-TL; cell-free expression; in vitro; transcription-translation; cell-free protein synthesis; synthetic biology; systems biology; Escherichia coli cell extract; biological circuits; biomolecular breadboard
5.  Discriminating External and Internal Causes for Heading Changes in Freely Flying Drosophila 
PLoS Computational Biology  2013;9(2):e1002891.
As animals move through the world in search of resources, they change course in reaction to both external sensory cues and internally-generated programs. Elucidating the functional logic of complex search algorithms is challenging because the observable actions of the animal cannot be unambiguously assigned to externally- or internally-triggered events. We present a technique that addresses this challenge by assessing quantitatively the contribution of external stimuli and internal processes. We apply this technique to the analysis of rapid turns (“saccades”) of freely flying Drosophila melanogaster. We show that a single scalar feature computed from the visual stimulus experienced by the animal is sufficient to explain a majority (93%) of the turning decisions. We automatically estimate this scalar value from the observable trajectory, without any assumption regarding the sensory processing. A posteriori, we show that the estimated feature field is consistent with previous results measured in other experimental conditions. The remaining turning decisions, not explained by this feature of the visual input, may be attributed to a combination of deterministic processes based on unobservable internal states and purely stochastic behavior. We cannot distinguish these contributions using external observations alone, but we are able to provide a quantitative bound of their relative importance with respect to stimulus-triggered decisions. Our results suggest that comparatively few saccades in free-flying conditions are a result of an intrinsic spontaneous process, contrary to previous suggestions. We discuss how this technique could be generalized for use in other systems and employed as a tool for classifying effects into sensory, decision, and motor categories when used to analyze data from genetic behavioral screens.
Author Summary
Researchers have spent considerable effort studying how specific sensory stimuli elicit behavioral responses and how other behaviors may arise independent of external inputs in conditions of sensory deprivation. Yet an animal in its natural context, such as searching for food or mates, turns both in response to external stimuli and intrinsic, possibly stochastic, decisions. We show how to estimate the contribution of vision and internal causes on the observable behavior of freely flying Drosophila. We developed a dimensionality reduction scheme that finds a one-dimensional feature of the visual stimulus that best predicts turning decisions. This visual feature extraction is consistent with previous literature on visually elicited fly turning and predicts a large majority of turns in the tested environment. The rarity of stimulus-independent events suggests that fly behavior is more deterministic than previously suggested and that, more generally, animal search strategies may be dominated by responses to stimuli with only modest contributions from internal causes.
PMCID: PMC3585425  PMID: 23468601
6.  The protocol for the Be Our Ally Beat Smoking (BOABS) study, a randomised controlled trial of an intensive smoking cessation intervention in a remote Aboriginal Australian health care setting 
BMC Public Health  2012;12:232.
Australian Aboriginal peoples and Torres Strait Islanders (Indigenous Australians) smoke at much higher rates than non-Indigenous people and smoking is an important contributor to increased disease, hospital admissions and deaths in Indigenous Australian populations. Smoking cessation programs in Australia have not had the same impact on Indigenous smokers as on non-Indigenous smokers. This paper describes the protocol for a study that aims to test the efficacy of a locally-tailored, intensive, multidimensional smoking cessation program.
This study is a parallel, randomised, controlled trial. Participants are Aboriginal and Torres Strait Islander smokers aged 16 years and over, who are randomly allocated to a 'control' or 'intervention' group in a 2:1 ratio. Those assigned to the 'intervention' group receive smoking cessation counselling at face-to-face visits, weekly for the first four weeks, monthly to six months and two monthly to 12 months. They are also encouraged to attend a monthly smoking cessation support group. The 'control' group receive 'usual care' (i.e. they do not receive the smoking cessation program). Aboriginal researchers deliver the intervention, the goal of which is to help Aboriginal peoples and Torres Strait Islanders quit smoking. Data collection occurs at baseline (when they enrol) and at six and 12 months after enrolling. The primary outcome is self-reported smoking cessation with urinary cotinine confirmation at 12 months.
Stopping smoking has been described as the single most important individual change Aboriginal and Torres Strait Islander smokers could make to improve their health. Smoking cessation programs are a major priority in Aboriginal and Torres Strait Islander health and evidence for effective approaches is essential for policy development and resourcing. A range of strategies have been used to encourage Aboriginal peoples and Torres Strait Islanders to quit smoking however there have been few good quality studies that show what approaches work best. More evidence of strategies that could work more widely in Indigenous primary health care settings is needed if effective policy is to be developed and implemented. Our project will make an important contribution in this area.
Trial Registration
Australian New Zealand Clinical Trials Registry (ACTRN12608000604303)
PMCID: PMC3349500  PMID: 22439653
Indigenous; Aboriginal; Torres Strait Islander; Randomised controlled trial; Smoking cessation; Study protocol; Be Our Ally Beat Smoking (BOABS) Study
7.  Characterization of Novel Bovine Gastrointestinal Tract Treponema Isolates and Comparison with Bovine Digital Dermatitis Treponemes ▿  
This study aimed to isolate and characterize treponemes present in the bovine gastrointestinal (GI) tract and compare them with bovine digital dermatitis (BDD) treponemes. Seven spirochete isolates were obtained from the bovine GI tract, which, on the basis of 16S rRNA gene comparisons, clustered within the genus Treponema as four novel phylotypes. One phylotype was isolated from several different GI tract regions, including the omasum, colon, rumen, and rectum. These four phylotypes could be divided into two phylotype pairs that clustered closest with each other and then with different, previously reported rumen treponemes. The treponemes displayed great genotypic and phenotypic diversity between phylotypes and differed considerably from named treponeme species and those recently reported by metagenomic studies of the bovine GI tract. Phylogenetic inference, based on comparisons of 16S rRNA sequences from only bovine treponemes, suggested a marked divergence between two important groups. The dendrogram formed two major clusters, with one cluster containing GI tract treponemes and the other containing BDD treponemes. This division among the bovine treponemes is likely the result of adaptation to different niches. To further differentiate the bovine GI and BDD strains, we designed a degenerate PCR for a gene encoding a putative virulence factor, tlyC, which gave a positive reaction only for treponemes from the BDD cluster.
PMCID: PMC3019712  PMID: 21057019
8.  Design and Validation of a Data Simulation Model for Longitudinal Healthcare Data 
AMIA Annual Symposium Proceedings  2011;2011:1176-1185.
Evaluating performance characteristics of analytic methods developed to identify treatment effects in longitudinal healthcare data has been hindered by lack of an objective benchmark to measure performance. Relationships between drugs and subsequent treatment effects are not precisely quantified in real-world data, and simulated data offer potential to augment method development by providing data with known, measurable characteristics. However, the use of simulated data has been limited due to its inability to adequately reflect the complexities inherent in real-world databases that are necessary for effective method development. The goal of this study was to develop and evaluate a model for simulating longitudinal healthcare data that adequately captures these complexities. An empiric design was chosen that utilizes the characteristics of a real healthcare database as simulation input. This model demonstrates the potential for simulated data with known characteristics to adequately reflect complex relationships among diseases and treatments as recorded in healthcare databases.
PMCID: PMC3243118  PMID: 22195178
9.  The role of Foxg1 in the development of neural stem cells of the olfactory epithelium 
The olfactory epithelium (OE) of the mouse is an excellent model system for studying principles of neural stem cell biology because of its well-defined neuronal lineage and its ability to regenerate throughout life. To approach the molecular mechanisms of stem cell regulation in the OE, we have focused on Foxg1, also known as brain factor-1, which is a member of the Forkhead transcription factor family. Foxg1−/− mice show major defects in the OE at birth, suggesting that Foxg1 plays an important role in OE development. We find that Foxg1 is expressed in cells within the basal compartment of the OE, the location where OE stem and progenitor are known to reside. Since FoxG1 is known to regulate proliferation of neuronal progenitor cells during telencephalon development, we performed BrdU pulse-chase of Sox2-expressing neural stem cells during primary OE neurogenesis. We found the percentage of Sox2-expressing cells that retained BrdU was twice as high in Foxg1−/− OE as in wildtypes, suggesting that these cells are delayed and/or halted in their development in the absence of Foxg1. Our findings suggest that the proliferation and/or subsequent differentiation of Sox2-expressing neural stem cells in the OE are regulated by Foxg1.
PMCID: PMC2878634  PMID: 19686101
Mouse; neurogenesis; olfactory epithelium; neuronal progenitor; neural stem cell; proliferation; Forkhead; transcription factor; BrdU; Mash1; Ngn1; Sox2; TGF-β; FGF; olfactory receptor neuron
10.  Regulatory Activity Revealed by Dynamic Correlations in Gene Expression Noise 
Nature genetics  2008;40(12):1493-1498.
Gene regulatory interactions are context–dependent, active in some cellular states but not others. Stochastic fluctuations, or ‘noise,’ in gene expression propagate through active, but not inactive, regulatory links [1, 2]. Thus, correlations in gene expression noise could provide a non-invasive means to probe the activity states of regulatory links. However, global, ‘extrinsic’, noise sources generate correlations even without direct regulatory links. Here we show that single-cell time-lapse microscopy, by revealing time lags due to regulation, can discriminate between active regulatory connections and extrinsic noise. We demonstrate this principle mathematically, using stochastic modeling, and experimentally, using simple synthetic gene circuits. We then use this approach to analyze dynamic noise correlations in the galactose metabolism genes of E. coli. We find that the CRP-GalS-GalE feed-forward loop is inactive in standard conditions, but can become active in a GalR mutant. These results show how noise can help analyze the context-dependence of regulatory interactions in endogenous gene circuits.
PMCID: PMC2829635  PMID: 19029898
11.  Association of Unique, Isolated Treponemes with Bovine Digital Dermatitis Lesions▿  
Journal of Clinical Microbiology  2009;47(3):689-696.
This study used a PCR-based approach targeting 16S rRNA gene fragments to determine the occurrence and association of the three bovine digital dermatitis (BDD) treponeme phylogroups within lesions found in cattle from the United Kingdom. Examination of 51 BDD lesions collected from infected cattle across the United Kingdom revealed that BDD treponeme group 1 (Treponema medium/Treponema vincentii-like), group 2 (Treponema phagedenis-like), and group 3 (Treponema putidum/Treponema denticola-like) were present in 96.1%, 98%, and 76.5% of BDD lesions, respectively. The three phylogroups were present together in 74.5% of lesions. The PCR assays enabled the isolation of further treponeme strains from previously mixed primary BDD lesion cultures. Here a representative from each of the three distinct treponeme phylogroups was isolated from a single BDD lesion for the first time. These data highlight the extent to which this disease is polytreponemal. Immunohistochemistry and electron microscopy were used to investigate lesional hoof tissues, resulting in treponemes being identified copiously in hair follicles and sebaceous glands, suggesting a potential route of exit and/or entry for these pathogens. This study gives further evidence for the importance of the three treponeme groups in BDD pathogenesis and reiterates the value of molecular genetic approaches for isolating and identifying fastidious anaerobes.
PMCID: PMC2650952  PMID: 19144804
12.  Matrilysin-dependent Elastolysis by Human Macrophages 
Human macrophages found in juxtaposition to fragmented elastin in vivo express the elastolytic matrix metalloproteinases (MMPs) progelatinase B, prometalloelastase, and promatrilysin. Though MMPs can degrade a range of extracellular matrix components, increasing evidence suggests that preferred targets in vivo include nonmatrix substrates such as chemokines and growth factors. Hence, the means by which MMPs participate in elastin turnover remain undefined as does the identity of the elastolysins. Herein, human macrophage cultures have been established that express a complement of elastolytic proteinases similar, if not identical, to that found in vivo. Under plasminogen-free conditions, macrophages preferentially use metalloelastase to mediate elastolysis via a process that deposits active enzyme on elastin surfaces. By contrast, in the presence of plasminogen, human macrophages up-regulate proteolysis 10-fold by processing promatrilysin to an active elastolysin via a urokinase-type plasminogen activator-dependent pathway. Matrilysin-deficient human macrophages fail to mediate an elastolytic response despite the continued expression of gelatinase B and metalloelastase. Thus, acting in concert with cosecreted cysteine proteinases whose activities are constrained to sites of macrophage-elastin contact (Punturieri, A., S. Filippov, E. Allen, I. Caras, R. Murray, V. Reddy, and S.J. Weiss. 2000. J. Exp. Med. 192:789–799), matrilysin confers macrophages with their most potent MMP-dependent elastolytic system.
PMCID: PMC2194197  PMID: 12963695
cysteine proteinase; elastin; macrophage; matrix metalloproteinase; plasminogen
13.  Regulation of Elastinolytic Cysteine Proteinase Activity in Normal and Cathepsin K–Deficient Human Macrophages 
Human macrophages mediate the dissolution of elastic lamina by mobilizing tissue-destructive cysteine proteinases. While macrophage-mediated elastin degradation has been linked to the expression of cathepsins L and S, these cells also express cathepsin K, a new member of the cysteine proteinase family whose elastinolytic potential exceeds that of all known elastases. To determine the relative role of cathepsin K in elastinolysis, monocytes were differentiated under conditions in which they recapitulated a gene expression profile similar to that observed at sites of tissue damage in vivo. After a 12-d culture period, monocyte-derived macrophages (MDMs) expressed cathepsin K in tandem with cathepsins L and S. Though cysteine proteinases are acidophilic and normally confined to the lysosomal network, MDMs secreted cathepsin K extracellularly in concert with cathepsins L and S. Simultaneously, MDMs increased the expression of vacuolar-type H+-ATPase components, acidified the pericellular milieu, and maintained extracellular cathepsin K in an active form. MDMs from a cathepsin K–deficient individual, however, retained the ability to express, process, and secrete cathepsins L and S, and displayed normal elastin-degrading activity. Thus, matrix-destructive MDMs exteriorize a complex mix of proteolytic cysteine proteinases, but maintain full elastinolytic potential in the absence of cathepsin K by mobilizing cathepsins L and S.
PMCID: PMC2193285  PMID: 10993910
cysteine proteinases; macrophages; cathepsin K; elastinolysis; pycnodysostosis
14.  Protective Immunity to Rotavirus Shedding in the Absence of Interleukin-6: Th1 Cells and Immunoglobulin A Develop Normally 
Journal of Virology  2000;74(11):5250-5256.
We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 × O1a)F2 backgrounds]. Naive IL-6− mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6− and IL-6+ mice exhibited efficient Th1-type gamma interferon responses in Peyer's patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4+ T cells from IL-6− mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyer's patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6− mice in comparison with IL-6+ mice. Following oral inoculation with murine rotavirus, IL-6− and IL-6+ mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6− and IL-6+ mice [(C57BL/6 × O1a)F2 background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.
PMCID: PMC110879  PMID: 10799601
15.  Infection of Mice Lacking Interleukin-7 (IL-7) Reveals an Unexpected Role for IL-7 in the Development of the Parasite Schistosoma mansoni 
Infection and Immunity  1999;67(8):4183-4190.
A single intradermal administration of recombinant interleukin-7 (IL-7) has been shown to aggravate the course of murine schistosomiasis, to favor the development of Th2-associated antibodies specific for the parasite, and to alter migration kinetics and/or migratory route of the parasite within its vertebrate host. Here we show that after infection of IL-7-deficient mice with Schistosoma mansoni, the predominant parasite-specific humoral response follows a Th1 pattern, and the development of the parasite is greatly impaired. In IL-7-deficient mice, increased numbers of larvae reach the lungs and fewer larvae reach the liver, compared to control mice. In the absence of IL-7, female worms show an altered fecundity, leading to decreased numbers of eggs trapped in the tissues and to an amelioration of the pathology of the infected host. The most striking observation is the blockade of parasite growth in an IL-7-defective environment, leading to dwarf male and female worms. The results of this study have important implications for the role of IL-7 in the host-parasite relationship and show how parasites can disable or evade the host immune response.
PMCID: PMC96723  PMID: 10417190

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