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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2016

A Re-examination of the Role of AUXIN RESPONSE FACTOR 8 in Developmental Abnormalities Caused by the P1/HC-Pro Viral Suppressor of RNA Silencing

Plant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA (miRNA) pathway. These abnormalities were originally thought to reflect a pleiotropic impact of silencing suppressors on miRNA control of plant development. However, subsequent work with the P1/HC-Pro potyviral suppressor of silencing showed that global impairment of the miRNA pathway was not responsible for the phenotypical anomalies. More recently, developmental defects caused by a P1/HC-Pro transgene under control of the 35S promoter were attributed to moderate upregulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence in that work was that the developmental defects in the 35S-pro:P1/HC-Pro transgenic Arabidopsis were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. Here we report a re-investigation of the role of ARF8 in P1/HC-Pro-mediated developmental defects. We characterized the progeny of a cross between our 35S-pro:P1/HC-Pro transgenic Arabidopsis line and the same arf8-6 T-DNA insertion mutant used in the earlier study. The T-DNA mutation had little effect in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the P1/HC-Pro phenotype in the F2 generation. However, this loss of phenotype was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the 35S-pro:P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA2accompanied by the appearance of 35S promoter siRNAs. Furthermore, arf8-8, an independent loss-of-function point mutation, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together these data argue against the reported role of increased ARF8 expression in mediating developmental defects in P1/HC-Pro transgenic plants.\n\nAuthor SummaryRNA silencing is an important antiviral defense in plants that uses small RNA molecules to target the invading RNA. Plant viruses, however, have countered with proteins that suppress RNA silencing, and one of the best-studied plant viral suppressors of silencing is P1/HC-Pro. When the genetic model plant Arabidopsis thaliana is bioengineered to express P1/HC-Pro, the resulting plants display distinct developmental abnormalities. These abnormalities are thought to arise because P1/HC-Pro also interferes with the arm of RNA silencing that uses small RNAs called microRNAs (miRNAs) to regulate expression of the plant's own genes. Earlier work, however, showed that interference with all miRNAs in general could not be responsible for these developmental defects. More recently, it was reported that enhanced expression of a single miRNA-controlled gene, AUXIN RESPONSE FACTOR 8 (ARF8), underlies the developmental defects caused by P1/HC-Pro. However, using the same ARF8 mutation as that report, as well as a second, independent ARF8 loss-of function mutation, we now show that mis-regulation of ARF8 is not responsible for those defects. One or a few key miRNA-controlled factors might, in fact, underlie the developmental defects caused by P1/HC-Pro; however, our results show that ARF8 is not one of the key factors.

Plant Biology↗
bioRxiv2016

How do Research Faculty in the Biosciences Evaluate Paper Authorship Criteria?

Authorship of peer-reviewed journal articles and abstracts has become the primary currency and reward unit in academia. Such a reward is crucial for students and postdocs who are often under-compensated and thus highly value authorship as an incentive. While numerous scientific and publishing organizations have written guidelines for determining author qualifications and author order, there remains much ambiguity when it comes to how these criteria are weighed by research faculty. Here, we sought to provide some initial insight on how faculty view the relative importance of 11 criteria for scientific authorship. We distributed an online survey to 564 biomedical engineering, biology, and bioengineering faculty members at 10 research institutions across the United States. The response rate was approximately 18%, resulting in a final sample of 102 respondents. Results revealed a consensus on some criteria, such as time spent conducting experiments, but there was a lack of agreement regarding the role of funding procurement. This study provides quantitative assessments of how faculty members in the biosciences evaluate authorship criteria. We discuss the implications of these findings for researchers, especially new graduate students, to help navigate the discrepancy between official policies for authorship and the contributions that faculty truly value.

Preprint↗
bioRxiv2016

PhysiCell: an Open Source Physics-Based Cell Simulator for 3-D Multicellular Systems

Many multicellular systems problems can only be understood by studying how cells move, grow, divide, interact, and die. Tissue-scale dynamics emerge from systems of many interacting cells as they respond to and influence their microenvironment. The ideal \"virtual laboratory\" for such multicellular systems simulates both the biochemical microenvironment (the \"stage\") and many mechanically and biochemically interacting cells (the \"players\" upon the stage).\n\nPhysiCell--physics-based multicellular simulator--is an open source agent-based simulator that provides both the stage and the players for studying many interacting cells in dynamic tissue microenvironments. It builds upon a multi-substrate biotransport solver to link cell phenotype to multiple diffusing substrates and signaling factors. It includes biologically-driven sub-models for cell cycling, apoptosis, necrosis, solid and fluid volume changes, mechanics, and motility \"out of the box.\" The C++ code has minimal dependencies, making it simple to maintain and deploy across platforms. PhysiCell has been parallelized with OpenMP, and its performance scales linearly with the number of cells. Simulations up to 105-106 cells are feasible on quad-core desktop workstations; larger simulations are attainable on single HPC compute nodes.\n\nWe demonstrate PhysiCell by simulating the impact of necrotic core biomechanics, 3-D geometry, and stochasticity on the dynamics of hanging drop tumor spheroids and ductal carcinoma in situ (DCIS) of the breast. We demonstrate stochastic motility, chemical and contact-based interaction of multiple cell types, and the extensibility of PhysiCell with examples in synthetic multicellular systems (a \"cellular cargo delivery\" system, with application to anti-cancer treatments), cancer heterogeneity, and cancer immunology. PhysiCell is a powerful multicellular systems simulator that will be continually improved with new capabilities and performance improvements. It also represents a significant independent code base for replicating results from other simulation platforms. The PhysiCell source code, examples, documentation, and support are available under the BSD license at http://PhysiCell.MathCancer.org and http://PhysiCell.sf.net.\n\nAuthor SummaryThis paper introduces PhysiCell: an open source, agent-based modeling framework for 3-D multicellular simulations. It includes a standard library of sub-models for cell fluid and solid volume changes, cycle progression, apoptosis, necrosis, mechanics, and motility. PhysiCell is directly coupled to a biotransport solver to simulate many diffusing substrates and cell-secreted signals. Each cell can dynamically update its phenotype based on its microenvironmental conditions. Users can customize or replace the included sub-models.\n\nPhysiCell runs on a variety of platforms (Linux, OSX, and Windows) with few software dependencies. Its computational cost scales linearly in the number of cells. It is feasible to simulate 500,000 cells on quad-core desktop workstations, and millions of cells on single HPC compute nodes. We demonstrate PhysiCell by simulating the impact of necrotic core biomechanics, 3-D geometry, and stochasticity on hanging drop tumor spheroids (HDS) and ductal carcinoma in situ (DCIS) of the breast. We demonstrate contact- and chemokine-based interactions among multiple cell types with examples in synthetic multicellular bioengineering, cancer heterogeneity, and cancer immunology.\n\nWe developed PhysiCell to help the scientific community tackle multicellular systems biology problems involving many interacting cells in multi-substrate microenvironments. PhysiCell is also an independent, cross-platform codebase for replicating results from other simulators.

systems biology↗
bioRxiv2017

Synthetic OCP Heterodimers are Photoactive and Recapitulate the Fusion of Two Primitive Carotenoproteins in the Evolution of Cyanobacterial Photoprotection

The Orange Carotenoid Protein (OCP) governs photoprotection in the majority of cyanobacteria. It is structurally and functionally modular, comprised of a C-terminal regulatory domain (CTD), an N-terminal effector domain (NTD) and a ketocarotenoid; the chromophore spans the two domains in the ground state and translocates fully into the NTD upon illumination. Using both the canonical OCP1 and the presumably more primitive OCP2 from Fremyella diplosiphon, we show that an NTD-CTD heterodimer forms when the domains are expressed as separate polypeptides. The carotenoid is required for the heterodimeric association, assembling an orange complex which is stable in the dark. Both OCP1 and OCP2 heterodimers are photoactive, undergoing light-driven heterodimer dissociation, but differ in their ability to reassociate in darkness, setting the stage for bioengineering photoprotection in cyanobacteria as well as for developing new photoswitches for biotechnology. Additionally, we reveal that homodimeric CTD can bind carotenoid in the absence of NTD, and name this truncated variant the C-terminal domain-like Carotenoid Protein (CCP). This finding supports the hypothesis that the OCP evolved from an ancient fusion event between genes for two different carotenoid-binding proteins ancestral to the NTD and CTD. We suggest that the CCP and its homologs constitute a new family of carotenoproteins within the NTF2-like superfamily found across all kingdoms of life.

plant biology↗
bioRxiv2017

Finding analytic stationary solutions to the chemical master equation by gluing state spaces at one or two states recursively

Noise is often indispensable to key cellular activities, such as gene expression, necessitating the use of stochastic models to capture its dynamics. The chemical master equation (CME) is a commonly used stochastic model that describes how the probability distribution of a chemically reacting system varies with time. Knowing analytic solutions to the CME can have benefits, such as expediting simulations of multiscale biochemical reaction networks and aiding the design of distributional responses. However, analytic solutions are rarely known. A recent method of computing analytic stationary solutions relies on gluing simple state spaces together recursively at one or two states. We explore the capabilities of this method and introduce algorithms to derive analytic stationary solutions to the CME. We first formally characterise state spaces that can be constructed by performing single-state gluing of paths, cycles or both sequentially. We then study stochastic biochemical reaction networks that consist of reversible, elementary reactions with two-dimensional state spaces. We also discuss extending the method to infinite state spaces and designing stationary distributions that satisfy user-specified constraints. Finally, we illustrate the aforementioned ideas using examples that include two interconnected transcriptional components and chemical reactions with two-dimensional state spaces.\n\nSubject AreasSystems biology, synthetic biology, biomathematics, bioengineering

systems biology↗
bioRxiv2017

High-Throughput Laboratory Evolution Of Escherichia coli Under Multiple Stress Environments

Bacterial cells have a remarkable capacity to adapt and to evolve to environmental changes. Although many mutations contributing to adaptive evolution have been identified, the relationship between the mutations and the phenotypic changes responsible for fitness gain has yet to be fully elucidated. For a better understanding of phenotype-genotype relationship in evolutionary dynamics, we performed high-throughput laboratory evolution of Escherichia coli under various stress conditions using an automated culture system. One measure of phenotype, transcriptome analysis, revealed that the expression changes which occurred during the evolution were generally similar among the strains evolved in the same stress environment. We also found several genes and gene functions for which mutations were commonly fixed in the strains resistant to the same stress, and whose effects on resistance were verified experimentally. We demonstrated that the integration of transcriptome and genome data enables us to extract the mechanisms for stress resistance.\n\nAuthor summaryUnderstanding the relationship between phenotypic and genetic changes is a fundamental goal in evolutionary biology, which can provide insights into the past and future evolutionary trajectories. Evolution of microorganisms in a laboratory has been the primary approach to clarify the mappings of phenotypic and genotypic changes. Here, we performed high-throughput laboratory evolution with bacteria using an automated culture system, to quantify phenotypic and genotypic changes occurred under various stress conditions. We identified various stress-specific gene expression changes and mutations, and contributions of them to fitness gain were validated. These results demonstrated that the integration of phenotypic and genotypic changes makes it possible to extract the mechanisms for stress resistance evolution, which will contribute to bioengineering applications.

evolutionary biology↗
bioRxiv2017

Silver fluoride as a treatment for the disease dental caries

The current paradigm of treatment for dental caries (tooth decay) in primary teeth is dangerous, fails to reach many children, and suffers high recurrence. Acceptance of the paradigm arises from a misperception that untreated caries in primary teeth is a threat to life. We show a linear relationship between age and deaths in the United States from 1999 through 2015 caused by dental caries, pulpal / periapical abscess, or facial cellulitis. The intercept of 6 years coincides with emergence of the first permanent tooth: it appears that caries in primary teeth is not a threat to life. Thus, treatment goals should be to avoid pain, which is not possible with operative dentistry, as it causes pain. Medical management of caries is a distinct treatment philosophy which employs topical minimally invasive therapies that treat the disease, and is not merely prevention. Silver diamine fluoride (SDF) is a central agent to enable effective non-invasive treatment. The announcement of FDA Breakthrough Therapy designation suggests that SDF will become the first FDA approved drug for treating the disease dental caries. Since our last review, 4 clinical trials have been completed, which inform an update to the application protocol and frequency regimen. Suggestions from these studies are to skip the rinsing step due to demonstration of safety and concern of diminished effectiveness by dilution, and to start patients with an intensive regimen of multiple applications over the first few weeks. Breakthroughs in elucidating the impact of SDF on tooth structure and the plaque microbiome inform potential opportunities for bioengineering and understanding caries arrest, respectively. Dentists have been surprised by preference of this treatment over traditional invasive approaches. Renewed interest in this old material has delivered progress to optimize the judicious use of SDF, and enable a revolution in caries management - particularly for primary teeth.\n\nONE SENTENCE SUMMARYAnesthesia is inappropriate for first-line treatment of early childhood caries now that safe topical treatments such as silver diamine fluoride are available.

pharmacology and toxicology↗
bioRxiv2017

A Synthetic Microbial Operational Amplifier

Synthetic biology has created oscillators, latches, logic gates, logarithmically linear circuits, and load drivers that have electronic analogs in living cells. The ubiquitous operational amplifier, which allows circuits to operate robustly and precisely has not been built with bio-molecular parts. As in electronics, a biological operational-amplifier could greatly improve the predictability of circuits despite noise and variability, a problem that all cellular circuits face. Here, we show how to create a synthetic 3-stage inducer-input operational amplifier with a differential transcription-factor stage, a CRISPR-based push-pull stage, and an enzymatic output stage with just 5 proteins including dCas9. Our Bio-OpAmp expands the toolkit of fundamental circuits available to bioengineers or biologists, and may shed insight into biological systems that require robust and precise molecular homeostasis and regulation.\n\nOne Sentence SummaryA synthetic bio-molecular operational amplifier that can enable robust, precise, and programmable homeostasis and regulation in living cells with just 5 protein parts is described.

synthetic biology↗
bioRxiv2017

Mechanism of genome interrogation: How CRISPR RNA-guided Cas9 proteins locate specific targets on DNA

The ability to precisely edit and modify genome opens endless opportunities to investigate fundamental properties of living systems as well as to advance various medical techniques and bioengineering applications. This possibility is now close to reality due to a recent discovery of the adaptive bacterial immune system, which is based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that utilize RNA to find and cut the double-stranded DNA molecules at specific locations. Here we develop a quantitative theoretical approach to analyze the mechanism of target search on DNA by CRISPR RNA-guided Cas9 proteins, which is followed by a selective cleavage of nucleic acids. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the target search is presented. It is found that the location of specific sites on DNA by CRISPR Cas9 proteins is governed by binding first to protospacer adjacent motif (PAM) sequences on DNA, which is followed by reversible transitions into DNA interrogation states. In addition, the search dynamics is strongly influenced by the off-target cutting. Our theoretical calculations allows us to explain the experimental observations and to give experimentally testable predictions. Thus, the presented theoretical model clarifies some molecular aspects of the genome interrogation by CRISPR RNA-guided Cas9 proteins.\n\nInsert Received for publication Date and in final form Date.

biophysics↗
bioRxiv2017

Native and engineered clifednamide biosynthesis in multiple Streptomyces spp.

Polycyclic tetramate macrolactam (PTM) natural products are produced by actinomycetes and other bacteria. PTMs are often bioactive, and the simplicity of their biosynthetic clusters make them attractive for bioengineering. Clifednamide-type PTMs from Streptomyces sp. JV178 contain a distinctive ketone group, suggesting the existence of a novel PTM oxidizing enzyme. Here, we report the new cytochrome P450 enzyme (CftA) is required for clifednamide production. Genome mining was used to identify several new clifednamide producers, some having improved clifednamide yields. Using a parallel synthetic biology approach, CftA isozymes were used to engineer the ikarugamycin pathway of Streptomyces sp. NRRL F-2890 to yield clifednamides. Further, we observed that strong CftA expression leads to the production of a new PTM, clifednamide C. We demonstrate the utility of both genome mining and synthetic biology to rapidly increase clifednamide production and identify a PTM tailoring enzyme for rational molecule design.

synthetic biology↗
bioRxiv2017

The response to selection in Glycoside Hydrolase Family 13 structures: A comparative quantitative genetics approach

The Glycoside Hydrolase Family 13 (GH13) is both evolutionary diverse and relevant to many industrial applications. Its members perform the hydrolysis of starch into smaller carbohydrates. Members of the family have been bioengineered to improve catalytic function under industrial environments. We introduce a framework to analyze the response to selection of GH13 protein structures given some phylogenetic and simulated dynamic information. We found that the TIM-barrel is not selectable since it is under purifying selection. We also show a method to rank important residues with higher inferred response to selection. These residues can be altered to effect change in properties. In this work, we define fitness as inferred thermodynamic stability. We show that under the developed framework, residues 112Y, 122K, 124D, 125W, and 126P are good candidates to increase the stability of the truncated protein 4E2O. Overall, this paper demonstrate the feasibility of a framework for the analysis of protein structures for any other fitness landscape.

bioinformatics↗
bioRxiv2017

Spin coated chitin films for biosensors and its analysis are depended on chitin-surface interactions

Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans, and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.

biophysics↗
bioRxiv2017

Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a \"peripheral infrastructure\" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.

plant biology↗
bioRxiv2018

Investigation of Electrical Stimulation Effect on Intracellular Calcium Dynamics Using Locally Uniform Mesh Refinement

Modeling-based methods are conventionally exploited to simulate and predict the dynamics of a system in various fields of engineering. However, due to the intricacy of biological systems, these methods were rarely utilized in bioengineering until recently. By developing advanced computers with the ability to cope with enormous calculations and extending our knowledge about biological systems, modeling-based approaches have been adopted to manipulate biological systems. While utilizing the models to investigate the behavior of a system has some advantages including lower cost and time consumption, such methods were used for tissue engineering scarcely. Exploiting modeling-based methods to tissue engineering field requires developing and utilizing efficient computational methods to model gene regulation and signaling networks, which determine cell response to environmental changes and cellular fate.\n\nIn the present work, a novel spatio-temporal method was proposed, predicated on locally uniform mesh refinement. A benchmark comparison was performed with the previously used method and the results indicated the better performance of the incipiently adopted method. Besides, the model was utilized to investigate the effect of a popular differentiating stimulator, namely electrical stimulation, on mesenchymal stem cells as a type of stem cells widely used in tissue engineering. The results of the simulations demonstrated the puissance of such computational models in analyzing and predicting the effect of biochemical and biophysical perturbations on the cells and hence, their potential utility in tissue engineering.

systems biology↗
bioRxiv2018

Characterization of Diverse Homoserine Lactone Synthases in Escherichia coli

Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux Receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach is still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum sensing synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.

synthetic biology↗
bioRxiv2018

Genome-wide fitness assessment during diurnal growth reveals an expanded role of the cyanobacterial circadian clock protein KaiA

The recurrent pattern of light and darkness generated by Earths axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium Synechococcus elongatus PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses, but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a night-time metabolic program that is essential for S. elongatus to survive in the dark.\n\nSignificanceUnderstanding how photosynthetic bacteria respond to and anticipate natural light-dark cycles is necessary for predictive modeling, bioengineering, and elucidating metabolic strategies for diurnal growth. Here, we identify the genetic components that are important specifically under light-dark cycling conditions and determine how a properly functioning circadian clock prepares metabolism for darkness, a starvation period for photoautotrophs. This study establishes that the core circadian clock protein KaiA is necessary to enable rhythmic de-repression of a night-time circadian program.

microbiology↗
bioRxiv2018

An unexpectedly effective Monte Carlo technique for the RNA inverse folding problem

Solving the RNA inverse folding problem, also known as the RNA design problem, is critical to advance several scientific fields like bioengineering, yet existing approaches have had limited success. The problem has several features that resist traditional computational techniques, such as its exponential complexity and the chaotic behavior of its cost function. Although some state-of-the-art AI approaches have reported promising results, all existing computational methods substantially underperform expert human designers. I combine a different technique, Nested Monte Carlo Search (NMCS), with domain-specific knowledge to create an algorithm that outperforms all prior published methods by wide margins and solves 95 of the 100 puzzles listed in a recently proposed RNA solving difficulty benchmark.

bioinformatics↗
bioRxiv2018

eQTL network analysis reveals that regulatory genes are evolutionarily older and bearing more types of PTM sites in Coprinopsis cinerea

Understanding the DNA variation in regulation of carbohydrate-active enzymes (CAZymes) is fundamental to the use of wood-decaying basidiomycetes in lignocellulose conversion into renewable energy. Our goal is to identify the regulators of lignocellulolytic enzymes in Coprinopsis cinerea, of which the genome harbors high number of Auxiliary Activities enzymes.\n\nThe DNA sequence of C. cinerea family including 46 single spore isolates (SSIs) from crosses of two homozygous strains are used to develop a panel of SNP markers. Then the RNA sequence were used to characterize the gene expression profiles. The RNA were extracted from cultures grown on softwood-enriched sawdust to induce lignocellulolytic enzymes and CCR de-repression genes. To assess the genetic contribution to enzyme expression variations among the 46 SSIs, associations between SNPs and gene expressions were examined genome-widely. 5148 local eQTLs and 7738 distant eQTLs were obtained. By analyzing these eQTLs, the potential regulatory factors of the CAZymes expression and the de-repression of Carbon Catabolism Repression (CCR) were identified.\n\nThe eQTL network is characterized in terms of hotspots, evolutionary age and post-translational modifications (PTMs). In the eQTL network of C. cinerea, the non-regulatory genes are younger than the regulatory genes. The proteins regulated by combinational multiple types of PTMs are more likely to function as super regulatory hotspots in protein-protein interactions. The evolutionary age analysis and the PTMome analysis could serve as alternative methods to identify master regulators from genomic data.\n\nThis work demonstrates a comprehensive bioinformatics approach to identify regulatory factors with next-generation sequencing data. The results provide candidate genes for bioengineering to increase the enzyme production, which will practically benefit the bioethanol production from lignocellulose.\n\nSignificanceThis eQTL analysis is designed to study the fungal CAZymes and carbon catabolism repression, especially during the mycelium stage.\n\nO_LIIn Coprinopsis cinerea, only the regions near two ends of the chromosomes have high recombination rate, and suitable for family based eQTL analysis.\nC_LIO_LIA sugar transporter is a hotspot controlling many CCR genes.\nC_LIO_LICAZymes are not regulated by a master regulator, but by individual regulators. This indicates that CAZymes are under specific regulatory pathways, so can response to specific conditions.\nC_LIO_LIIn the eQTL network, the rGenes are evolutionarily older, with more types of PTM sites than eGenes.\nC_LIO_LIIn the eQTL network, the proteins with more types of PTM sites are more likely associated with Information Storage and Processing, and act as super-hub in the network.\nC_LI

bioinformatics↗