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bioRxiv2018

A method for genome editing in the anaerobic magnetotactic bacterium Desulfovibrio magneticus RS-1

ABSTRACTMagnetosomes are complex bacterial organelles that serve as model systems for studying cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacterial Magnetospirillum spp. that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have also been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing Deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified ten genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed many interesting mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration has not been feasible due to low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.\n\nIMPORTANCEMagnetotactic bacteria (MTB) are a group of organisms that form small, intracellular magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membrane, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization as well as potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply-branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from the cubooctahedral-shaped magnetite crystals of the genetically tractable Alphaproteobacteria MTB. Here, we present a method for genome editing in the anaerobic Deltaproteobacterium Desulfovibrio magneticus RS-1, the first cultured representative of the deeply-branching MTB. This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.

microbiology↗

bioRxiv2018

The finite state projection based Fisher information matrix approach to estimate and maximize the information in single-cell experiments

Modern optical imaging experiments not only measure single-cell and single-molecule dynamics with high precision, but they can also perturb the cellular environment in myriad controlled and novel settings. Techniques, such as single-molecule fluorescence in-situ hybridization, microfluidics, and optogenetics, have opened the door to a large number of potential experiments, which begs the question of how best to choose the best possible experiment. The Fisher information matrix (FIM) estimates how well potential experiments will constrain model parameters and can be used to design optimal experiments. Here, we introduce the finite state projection (FSP) based FIM, which uses the formalism of the chemical master equation to derive and compute the FIM. The FSP-FIM makes no assumptions about the distribution shapes of single-cell data, and it does not require precise measurements of higher order moments of such distributions. We validate the FSP-FIM against well-known Fisher information results for the simple case of constitutive gene expression. We then use numerical simulations to demonstrate the use of the FSP-FIM to optimize the timing of single-cell experiments with more complex, non-Gaussian fluctuations. We validate optimal simulated experiments determined using the FSP-FIM with Monte-Carlo approaches and contrast these to experiment designs chosen by traditional analyses that assume Gaussian fluctuations or use the central limit theorem. By systematically designing experiments to use all of the measurable fluctuations, our method enables a key step to improve co-design of experiments and quantitative models.\n\nAuthor summaryA main objective of quantitative modeling is to predict the behaviors of complex systems under varying conditions. In a biological context, stochastic fluctuations in expression levels among isogenic cell populations have required modeling efforts to incorporate and even rely upon stochasticity. At the same time, new experimental variables such as chemical induction and optogenetic control have created vast opportunities to probe and understand gene expression, even at single-molecule and single-cell precision. With many possible measurements or perturbations to choose from, researchers require sophisticated approaches to choose which experiment to perform next. In this work, we provide a new tool, the finite state projection based Fisher information matrix (FSP-FIM), which considers all cell-to-cell fluctuations measured in modern data sets, and can design optimal experiments under these conditions. Unlike previous approaches, the FSP-FIM does not make any assumptions about the shape of the distribution being measured. This new tool will allow experimentalists to optimally perturb systems to learn as much as possible about single-cell processes with a minimum of experimental cost or effort.

systems biology↗

bioRxiv2015

Quorum-sensing synchronization of synthetic toggle switches: A design based on monotone dynamical systems theory

AbstractSynthetic constructs in biotechnology, biocomputing, and modern gene therapy interventions are often based on plasmids or transfected circuits which implement some form of \"on-off\" switch. For example, the expression of a protein used for therapeutic purposes might be triggered by the recognition of a specific combination of inducers (e.g., antigens), and memory of this event should be maintained across a cell population until a specific stimulus commands a coordinated shut-off. The robustness of such a design is hampered by molecular (\"intrinsic\") or environmental (\"extrinsic\") noise, which may lead to spontaneous changes of state in a subset of the population and is reflected in the bimodality of protein expression, as measured for example using flow cytometry. In this context, a \"majority-vote\" correction circuit, which brings deviant cells back into the desired state, is highly desirable, and quorum-sensing has been suggested as a way for cells to broadcast their states to the population as a whole so as to facilitate consensus.\n\nIn this paper, we propose what we believe is the first such design that has mathematically guaranteed properties of stability and auto-correction. Our approach is guided by concepts and theory from the field of \"monotone\" dynamical systems developed by M. Hirsch, H. Smith, and others. We benchmark our design by comparing it to an existing design which has been the subject of experimental and theoretical studies, illustrating its superiority in stability and self-correction of synchronization errors. Our stability analysis, based on dynamical systems theory, guarantees global convergence to steady states, ruling out unpredictable (\"chaotic\") behaviors and even sustained oscillations. These results are valid no matter what are the values of parameters, and are based only on the wiring diagram. The theory is complemented by extensive computational bifurcation analysis, performed for a biochemically-detailed and biologically-relevant model that we developed. Another novel feature of our approach is that our theorems on stability of steady states for homogeneous or mixed populations are valid independently of the number of cells in the population, and depend only on the relative proportions of each type of state.\n\nWhile monotone systems theory has been used previously for systems biology analysis, the current work illustrates its power for synthetic biology design, and thus has wider significance well beyond the application to the important problem of coordination of toggle switches.\n\nAuthor SummaryFor the last decade, outstanding progress has been made, and considerable practical experience has accumulated, in the construction of elementary genetic circuits that perform various tasks, such as memory storage and logical operations, in response to both exogenous and endogenous stimuli. Using modern molecular \"plug-and-play\" technologies, various (re-)programmable cellular populations can be engineered in a routine manner, and they can be combined into more complex cellular systems.\n\nAmong all engineered synthetic circuits, a toggle, a robust bistable switch leading to a binary response dynamics, is the simplest basic synthetic biology device, analogous to the \"flip-flop\" or latch in electronic design, and it plays a key role in biotechnology, biocomputing, and proposed gene therapies. However, despite many remarkable properties of the existing toggle designs, they must be tightly controlled in order to avoid spontaneous switching between different expression states (loss of long-term memory) or even the breakdown of stability through the generation of stable oscillations.\n\nTo address this concrete challenge, we have developed a new design for quorum-sensing synthetic toggles, based on monotone dynamical systems theory. Our design is endowed with strong theoretical guarantees that completely exclude unpredictable chaotic behaviors, as well as undesired stable oscillations, and leads to robust consensus states.

Synthetic Biology↗

bioRxiv2015

GreA and GreB enhance Escherichia coli RNA polymerase transcription rate in a reconstituted transcription-translation system

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC=\"FIGDIR/small/024604_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@53902org.highwire.dtl.DTLVardef@e1ba85org.highwire.dtl.DTLVardef@19c961eorg.highwire.dtl.DTLVardef@143928d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract\n\nC_FIG

Synthetic Biology↗

bioRxiv2014

Metabolic free energy and deterministic-but-for-error biological codes: a ‘Data Rate Theorem’ aging model

The living state is cognitive at every scale and level of organization. Since it is possible to associate a broad class of cognitive processes with dual information sources, many pathologies can be addressed using statistical models based on the Shannon Coding, the Shannon-McMillan Source Coding, the Rate Distortion, and the Data Rate Theorems, as these impose powerful necessary condition constraints on information generation and exchange, and on system control. Deterministic-but-for-error biological codes do not directly invoke cognition, although they may be essential subcomponents within larger cognitive processes. A formal argument, however, places such codes within a similar framework, with metabolic free energy serving as a control signal stabilizing biochemical code-and-translator dynamics in the presence of noise. Demand beyond available energy supply then expresses itself in punctuated destabilization of the coding channel, affecting a spectrum of essential biological functions. Aging, normal or prematurely driven by psychosocial or environmental stressors, must eventually interfere with the routine operation of such mechanisms, triggering chronic diseases associated with senescence. Amyloid fibril formation, intrinsically disordered protein logic gates, and cell surface glycan/lectin kelp bed logic gates are reviewed from this perspective. The results, however, generalize beyond coding systems having easily recognizable symmetry modes.

Systems Biology↗

bioRxiv2016

Inferring hidden causal relations between pathway members using reduced Google matrix of directed biological networks

Signaling pathways represent parts of the global biological network which connects them into a seamless whole through complex direct and indirect (hidden) crosstalk whose structure can change during development or in pathological conditions. We suggest a novel methodology, called Googlomics, for the structural analysis of directed biological networks using spectral analysis of their Google matrices, using parallels with quantum scattering theory, developed for nuclear and mesoscopic physics and quantum chaos. We introduce the reduced Google matrix method for the regulatory biological networks and demonstrate how its computation allows inferring hidden causal relations between the members of a signaling pathway or a functionally related group of genes. We investigate how the structure of hidden causal relations can be reprogrammed as the result of changes in the transcriptional network layer during cancerogenesis. The suggested Googlomics approach rigorously characterizes complex systemic changes in the wiring of large causal biological networks.

systems biology↗

bioRxiv2018

Depletion of microbiome-derived molecules in the host using Clostridium genetics

The gut microbiota produce hundreds of molecules that are present at high concentrations in circulation and whose levels vary widely among humans. In most cases, molecule production has not been linked to specific bacterial strains or metabolic pathways, and unraveling the contribution of each molecule to host biology remains difficult. A general system to toggle molecules in this pool on/off in the host would enable interrogation of the mechanisms by which they modulate host biology and disease processes. Such a system has been elusive due to limitations in the genetic manipulability of Clostridium and its relatives, the source of many molecules in this pool. Here, we describe a method for reliably constructing clean deletions in a model commensal Clostridium, C. sporogenes (Cs), including multiply mutated strains. We demonstrate the utility of this method by using it to toggle off the production of ten Cs-derived molecules that accumulate in host tissues. By comparing mice colonized by wild-type Cs versus a mutant deficient in the production of branched short-chain fatty acids, we discover a previously unknown IgA-modulatory activity of these abundant microbiome-derived molecules. Our method opens the door to interrogating and sculpting a highly concentrated pool of chemicals from the microbiome.

microbiology↗

bioRxiv2018

Strength in numbers: collaborative science for new experimental model systems

Our current understanding of biology is heavily based on the contributions from a small number of genetically tractable model organisms. Most eukaryotic phyla lack such experimental models, and this limits our ability to explore the molecular mechanisms that ultimately define their biology, ecology, and diversity. In particular, marine protists suffer from a paucity of model organisms despite playing critical roles in global nutrient cycles, food webs, and climate. To address this deficit, an initiative was launched in 2015 to foster development of ecologically and taxonomically diverse marine protist genetic models. This multifaceted, complex but important challenge required a highly collaborative community-based approach. Herein we describe this approach, the advances achieved, and the lessons learned by participants in this novel community-based model for research.

microbiology↗

bioRxiv2026

Predictability failure in glucose-insulin system for ICU patients

Modern medicine implicitly assumes that physiological responses to intervention are predictably determined by administered treatments. However, physiological systems containing intrinsic delays between the detection of a stimulus and the biological response may violate this assumption. We investigate the human glucose-insulin system as described by the Ultradian model and mathematically demonstrate that clinically relevant forcing protocols-such as pulsatile insulin delivery and step-wise glucose infusion, both commonly used in intensive care units (ICUs)-can induce sustained temporal chaos that may hamper accurate prediction of the physiological response. If not accounted for, these chaotic dynamics could create difficulties in achieving optimal dosing and timing when administering glucose and insulin in clinical or home care settings. This phenomenon, termed delay-induced uncertainty (DIU), arises from the interaction between physiological delay, intrinsic shear near a limit cycle, and external forcing. Using the Ultradian glucose-insulin model, we compute top Lyapunov exponents to quantify predictability. Across a range of pulsatile and step-wise forcing regimes, including stochastic amplitudes drawn from Markov processes, we observe positive Lyapunov exponents, indicating sustained chaos. Our results suggest that delayed endocrine regulation may fundamentally limit the predictive value of the models used to develop glycemic management strategies, with implications for clinical protocols in the ICU.

systems biology↗

bioRxiv2017

Long-Term Memory In The Migration Movements Of Enucleated Amoeba proteus

How motile, free unicellular organisms maximize the rate at which they encounter resources and develop optimal search strategies remains largely unknown. In fact, cell foraging is a very complex activity in which unicellular organisms integrate a diversity of external cues and develop efficient systemic movements to localize nourishment. These foraging strategies are critical when cells face conditions of scarce resources or they dont possess information on where food is located. Here, in order to determine whether nuclear activity is directly involved in cell migration, we placed single, well-isolated, enucleated and non-enucleated starved Amoeba proteus on nutrient-free petri dishes, and we then analyzed their trajectories of movement using non-linear dynamic tools. We found that despite being enucleated, the systemic responses of the protoplasm exhibited typical biological behaviors, moving with apparent normality, creeping along the substrate, developing pseudopodia and gobbling up prey. Our quantitative studies show that both the non-enucleated and enucleated amoebas display a similar migration structure, characterized by super-diffusivity, non-trivial long-term correlations and move-step fluctuations with scale invariant properties. In conclusion, the nuclear activity does not seem to directly control the systemic cellular movements involved in locating sparse resources.

systems biology↗

bioRxiv2014

Mucosal effects of tenofovir 1% gel

BACKGROUNDTenofovir gel is being evaluated for vaginal and rectal pre-exposure prophylaxis against sexual HIV transmission. Because this is a new prevention strategy targeting large numbers of healthy people, we broadly assessed its effects on the mucosa.\n\nMETHODS AND FINDINGSIn MTN-007, a phase 1, randomized, double-blinded rectal microbicide trial, we used systems genomics/proteomics to determine the effect of tenofovir 1% gel, nonoxynol-9 2% gel, placebo gel or no treatment on rectal biopsies taken at baseline, after one application or after seven daily applications (15 subjects/arm). Experiments were repeated using primary vaginal epithelial cells from four healthy women. After seven days of administration, tenofovir 1% gel had broad-ranging biological effects on the rectal mucosa, which were much more pronounced than--but different from--those caused by the detergent nonoxynol-9. Tenofovir profoundly suppressed anti-inflammatory mediators such as interleukin 10; increased T cell densities; caused mitochondrial dysfunction, possibly by blocking PNPT1 expression; and altered regulatory pathways of cell differentiation and survival. Except for leukocyte-derived factors, all these effects were replicated in primary vaginal epithelial cells, which also proliferated significantly faster in tenofovir's presence.\n\nCONCLUSIONSTenofovir's suppression of anti-inflammatory activity could diminish its prophylactic efficacy over time. The breadth of mucosal changes, including mitochondrial dysfunction and epithelial proliferation, raises questions about its safety for long-term topical use. These findings suggest that a systems biology evaluation of mucosal effects may be beneficial before advancing to large-scale efficacy trials with topical HIV prevention agents that achieve high, long-lasting local drug concentrations.

Microbiology↗

bioRxiv2017

The hominoid-specific gene DSCR4 is involved in regulation of human leukocyte migration

DSCR4 (Down syndrome critical region 4) is an orphan retrotransposon-derived de-novo originated protein coding gene present only in hominoids (humans and great apes). Despite being located on the medically critical genomic region and abundance of evidences indicating its functionality, the role of this gene in human cells was utterly unknown. Due to absence of any prior knowledge regarding the function of DSCR4, for the first time here we used a gene-overexpression approach to discover biological importance and cellular roles of this gene. Our analysis strongly indicates DSCR4 to be mainly involved in regulation of the interconnected biological pathways related to cell migration, coagulation and immune system. We also showed that the predicted biological functions are consistent with tissue-specific expression of DSCR4 in migratory immune system leukocyte cells and neural crest cells that shape facial morphology of human embryo. Immune system and neural crest cells are also shown to be affected in Down syndrome patients who suffer from the same type of DSCR4 misregulation as in our study which further support our findings. Providing evidence for the critical roles of DSCR4 in human cells, our findings establish the basis for further investigations on the roles of DSCR4 in etiology of Down syndrome and unique characteristics of hominoids.

genetics↗

bioRxiv2017

Mapping the phenotypic repertoire of the cytoplasmic 2-Cys peroxiredoxin - thioredoxin system. 1. Understanding commonalities and differences among cell types.

The system (PTTRS) formed by typical 2-Cys peroxiredoxins (Prx), thioredoxin (Trx), Trx reductase (TrxR), and sulfiredoxin (Srx) is central in antioxidant protection and redox signaling in the cytoplasm of eukaryotic cells. Understanding how the PTTRS integrates these functions requires tracing phenotypes to molecular properties, which is non-trivial. Here we analyze this problem based on a model that captures the PTTRS conserved features. We have mapped the conditions that generate each distinct response to H2O2 supply rates ({nu}sup), and estimated the parameters for thirteen human cell types and for Saccharomyces cerevisiae. The resulting composition-to-phenotype map yielded the following experimentally testable predictions. The PTTRS permits many distinct responses including ultra-sensitivity and hysteresis. However, nearly all tumor cell lines showed a similar response characterized by limited Trx-S- depletion and a substantial but self-limited gradual accumulation of hyperoxidized Prx at high{nu} sup. This similarity ensues from strong correlations between the TrxR, Srx and Prx activities over cell lines, which contribute to maintain the Prx-SS reduction capacity in slight excess over the maximal steady state Prx-SS production. In turn, in erythrocytes, hepatocytes and HepG2 cells high{nu} sup depletes Trx-S- and oxidizes Prx mainly to Prx-SS. In all nucleated human cells the Prx-SS reduction capacity defined a threshold separating two different regimes. At sub-threshold{nu} sup cytoplasmic H2O2 is determined by Prx, nM-range and spatially localized, whereas at supra-threshold{nu} sup it is determined by much less active alternative sinks and M-range throughout the cytoplasm. The yeast shows a distinct response where the Prx Tsa1 accumulates in sulfenate form at high{nu} sup. This is mainly due to an exceptional stability of Tsa1s sulfenate.\n\nThe implications of these findings for thiol redox regulation and cell physiology are discussed. All estimates were thoroughly documented and provided, together with analytical approximations for system properties, as a resource for quantitative redox biology.\n\nAbbreviationsASK1, apoptosis signal-regulating kinase 1; Cat, catalase; GSH, glutathione; GPx1, glutathione peroxidase 1; Grx, glutaredoxin; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; Prx, typical 2-Cys peroxiredoxin; PTTRS, peroxiredoxin / thioredoxin / thioredoxin reductase system; Srx, sulfiredoxin; Trx, thioredoxin; TrxR, thioredoxin reductase.

systems biology↗

bioRxiv2017

Synthetic negative feedback circuits using engineered small RNAs

Negative feedback is known to endow biological and man-made systems with robust performance in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules which can inhibit translation of target messenger RNAs (mRNAs). In this paper, we designed, modelled and built two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA which negatively regulates the translation of the mRNA encoding this output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

synthetic biology↗

bioRxiv2018

Evidence for a unique DNA-dependent RNA polymerase in cereal crops

Gene duplication is an important driver for the evolution of new genes and protein functions. Duplication of DNA-dependent RNA polymerase (Pol) II subunits within plants led to the emergence of RNA Pol IV and V complexes, each of which possess unique functions necessary for RNA-directed DNA Methylation. Comprehensive identification of Pol V subunit orthologs across the monocot radiation revealed a duplication of the largest two subunits within the grasses (Poaceae), including critical cereal crops. These paralogous Pol subunits display sequence conservation within catalytic domains, but their carboxy terminal domains differ in length and character of the Ago-binding platform, suggesting unique functional interactions. Phylogenetic analysis of the catalytic region indicates positive selection on one paralog following duplication, consistent with retention via neofunctionalization. Positive selection on residue pairs that are predicted to interact between subunits suggests that paralogous subunits have evolved specific assembly partners. Additional Pol subunits as well as Pol-interacting proteins also possess grass-specific paralogs, supporting the hypothesis that a novel Pol complex with distinct function has evolved in the grass family, Poaceae.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC=\"FIGDIR/small/272708_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (32K):\norg.highwire.dtl.DTLVardef@13d3515org.highwire.dtl.DTLVardef@16831d5org.highwire.dtl.DTLVardef@19f99b8org.highwire.dtl.DTLVardef@1253e70_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG Significance statementThe grass family is critically important for humans, as this group contains cereal grains such as rice, wheat, and corn that form the bulk of the human diet. Here we provide evidence that grasses have evolved a unique polymerase complex of unknown function, suggesting a novel mechanism of gene regulation in the grass lineage. In addition to implications for the biology of grasses, this system offers an opportunity to understand how evolution shapes multi-subunit complexes through duplication of individual components.

plant biology↗

bioRxiv2018

The UBR-1 Ubiquitin Ligase Regulates Glutamate Metabolism to Generate Coordinated Motor Pattern in C. elegans

UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.\n\nAuthor SummaryUbiquitin-mediated protein degradation is central to diverse biological processes. The selection of substrates for degradation is carried out by the E3 ubiquitin ligases, which target specific groups of proteins for ubiquitination. The human genome encodes hundreds of E3 ligases; many exhibit sequence conservation across animal species, including one such ligase called UBR1. Patients carrying mutations in UBR1 exhibit severe systemic defects, but the biology behinds UBR1s physiological function remains elusive. Here we found that the C. elegans UBR-1 regulates glutamate level. When UBR-1 is defective, C. elegans exhibits increased glutamate; this leads to synchronization of motor neuron activity, hence defective locomotion when animals reach adulthood. UBR1-mediated glutamate metabolism may contribute to the physiological defects of UBR1 mutations.

neuroscience↗

bioRxiv2018

A Phantom System Designed to Assess the Effects of Membrane Lipids on Water Proton Relaxation

PurposeQuantitative magnetic resonance imaging (qMRI) provides a method for the non-invasive study of brain structure and associated changes, expressed in physical units. qMRI parameters have been shown to reflect brain tissue composition such as myelin. Nevertheless, it remains a major challenge to identify and quantify the contributions of specific molecular components to the MRI signal. Here, we describe a phantom system that can be used to evaluate the contribution of human brain lipids to qMRI parameters.\n\nMethodsA thin layer evaporation-hydration technique was used to formulate liposomes that mimic the physiological bi-layered membrane lipid environment. We then applied quantitative clinical MRI techniques with adjusted bias corrections in order to test the ability of the phantom system to estimate multiple qMRI parameters such as proton density (PD), T1, T2, T2* and magnetization transfer (MT).\n\nResultsThe results indicated that phantoms composed of various lipids could provide a stable and reliable estimation of qMRI parameters. In addition, the calculated water fraction (WF) maps for the phantoms were found to accurately represent the true WF volumes.\n\nConclusionWe have successfully created a biologically relevant liposome phantom system whose lipid composition can be fully controlled. This system can be used to measure the contributions of lipids to qMRI parameters under conditions that are relevant to in-vivo human scans.

neuroscience↗

bioRxiv2018

Engineering Translational Resource Allocation Controllers: Mechanistic Models, Design Guidelines, and Potential Biological Implementations

The use of orthogonal ribosomes in combination with dynamic resource allocation controllers is a promising approach for relieving the negative effects of cellular resource limitations on the modularity of synthetic gene circuits. Here, we develop a detailed mechanistic model of gene expression and resource allocation, which when simplified to a tractable level of complexity, allows the rational design of translational resource allocation controllers. Analysis of this model reveals a fundamental design trade-off; that reducing coupling acts to decrease gene expression. Through a sensitivity analysis of the experimentally tuneable controller parameters, we identify how each controller design parameter affects the overall closed-loop behaviour of the system, leading to a detailed set of design guidelines for optimally managing this trade-off. Based on our designs, we evaluated a number of alternative potential experimental implementations of the proposed system using commonly available biological components. Finally, we show that the controller is capable of dynamically allocating ribosomes as needed to restore modularity in a number of more complex synthetic circuits, such as the repressilator, and activation cascades composed of multiple interacting modules.

synthetic biology↗