Signal amplification and optimization of riboswitch-based hybrid inputs by modular and titratable toehold switches

Abstract Background Synthetic biological circuits are widely utilized to control microbial cell functions. Natural and synthetic riboswitches are attractive sensor modules for use in synthetic biology applications. However, tuning the fold-change of riboswitch circuits is challenging because a deep understanding of the riboswitch mechanism and screening of mutant libraries is generally required. Therefore, novel molecular parts and strategies for straightforward tuning of the fold-change of riboswitch circuits are needed. Results In this study, we devised a toehold switch-based modulator approach that combines a hybrid input construct consisting of a riboswitch and transcriptional repressor and de-novo-designed riboregulators named toehold switches. First, the introduction of a pair of toehold switches and triggers as a downstream signal-processing module to the hybrid input for coenzyme B12 resulted in a functional riboswitch circuit. Next, several optimization strategies that focused on balancing the expression levels of the RNA components greatly improved the fold-change from 260- to 887-fold depending on the promoter and host strain. Further characterizations confirmed low leakiness and high orthogonality of five toehold switch pairs, indicating the broad applicability of this strategy to riboswitch tuning. Conclusions The toehold switch-based modulator substantially improved the fold-change compared to the previous sensors with only the hybrid input construct. The programmable RNA-RNA interactions amenable to in silico design and optimization can facilitate further development of RNA-based genetic modulators for flexible tuning of riboswitch circuitry and synthetic biosensors.

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Toehold Switch-Based Genetic Modulator for Tuning of Riboswitch Sensor

10.21203/rs.3.rs-23721/v1 ◽

2020 ◽

Author(s):

Yunhee Hwang ◽

Seong Gyeong Kim ◽

Sungho Jang ◽

Jongmin Kim ◽

Gyoo Yeol Jung

Keyword(s):

Dose Response ◽

Dynamic Range ◽

De Novo ◽

Stoichiometric Ratio ◽

Design And Optimization ◽

Cell Functions ◽

Processing Module ◽

Biological Circuits ◽

Operational Range ◽

Genetic Modulators

Abstract BackgroundSynthetic biological circuits are widely utilized to control microbial cell functions. Natural and synthetic riboswitches are attractive classes of sensor modules for use in synthetic biological applications. However, tuning the dose-response parameters of riboswitch circuits is challenging because considerable understanding of riboswitch mechanism and screening of mutant libraries are generally required. Therefore, novel molecular parts and strategies for controlling the dose-response parameters of riboswitch circuits are needed.ResultsHere, we developed a toehold switch-based genetic modulator that combines a previously reported hybrid input construct, which consists of riboswitch and transcriptional repressor, and de-novo-designed riboregulators named as toehold switches. First, the introduction of a pair of toehold switch and trigger as a downstream signal-processing module resulted in a functional riboswitch circuit. Next, several optimization strategies that focused on the stoichiometric ratio of RNA components greatly improved the fold-change. Finally, further characterizations confirmed low leakiness and high orthogonality for multiple toehold switches, indicating its applicability in riboswitch circuits in a seamless manner.ConclusionsThe toehold switch-based genetic modulator improved the dynamic range and dramatically shifted the operational range compared to the previous sensors only with hybrid input construct. The programmable RNA-RNA interactions amenable to in silico design and optimization can facilitate further development of RNA-based genetic modulators for flexible tuning of riboswitch circuitry and synthetic biosensors.

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Single-cell Long Non-coding RNA Landscape of T Cells in Human Cancer Immunity

10.1101/2020.07.22.215855 ◽

2020 ◽

Author(s):

Haitao Luo ◽

Dechao Bu ◽

Lijuan Shao ◽

Yang Li ◽

Liang Sun ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

De Novo ◽

Human Cancer ◽

Transcriptome Assembly ◽

Deep Understanding ◽

Cell Functions ◽

Cancer Immunity ◽

Cancer Immunotherapies

AbstractThe development of new therapeutic targets for cancer immunotherapies and the development of new biomarkers require deep understanding of T cells. To date, the complete landscape and systematic characterization of long noncoding RNAs (lncRNAs) in T cells in cancer immunity are lacking. Here, by systematically analyzing full-length single-cell RNA sequencing (scRNA-seq) data of more than 20,000 T cell libraries across three cancer types, we provide the first comprehensive catalog and the functional repertoires of lncRNAs in human T cells. Specifically, we developed a custom pipeline for de novo transcriptome assembly obtaining 9,433 novel lncRNA genes that increased the number of current human lncRNA catalog by 16% and nearly doubled the number of lncRNAs expressed in T cells. We found that a portion of expressed genes in single T cells were lncRNAs which have been overlooked by the majority of previous studies. Based on metacell maps constructed by MetaCell algorithm that partition scRNA-seq datasets into disjointed and homogenous groups of cells (metacells), 154 signature lncRNAs associated with effector, exhausted, and regulatory T cell states are identified, 84 of which are functionally annotated based on co-expression network, indicating that lncRNAs might broadly participate in regulation of T cell functions. Our findings provide a new point of view and resource for investigating the mechanisms of T cell regulation in cancer immunity as well as for novel cancer-immune biomarker development and cancer immunotherapies.

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Defect Engineering in 2D Materials: Precise Manipulation and Improved Functionalities

Research ◽

10.34133/2019/4641739 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 10

Author(s):

Jie Jiang ◽

Tao Xu ◽

Junpeng Lu ◽

Litao Sun ◽

Zhenhua Ni

Keyword(s):

High Performance ◽

2D Materials ◽

Optoelectronic Devices ◽

Deep Understanding ◽

Building Blocks ◽

Research Progress ◽

Defect Engineering ◽

Plasma Chemical ◽

Design And Optimization ◽

Novel Structure

Two-dimensional (2D) materials have attracted increasing interests in the last decade. The ultrathin feature of 2D materials makes them promising building blocks for next-generation electronic and optoelectronic devices. With reducing dimensionality from 3D to 2D, the inevitable defects will play more important roles in determining the properties of materials. In order to maximize the functionality of 2D materials, deep understanding and precise manipulation of the defects are indispensable. In the recent years, increasing research efforts have been made on the observation, understanding, manipulation, and control of defects in 2D materials. Here, we summarize the recent research progress of defect engineering on 2D materials. The defect engineering triggered by electron beam (e-beam), plasma, chemical treatment, and so forth is comprehensively reviewed. Firstly, e-beam irradiation-induced defect evolution, structural transformation, and novel structure fabrication are introduced. With the assistance of a high-resolution electron microscope, the dynamics of defect engineering can be visualized in situ. Subsequently, defect engineering employed to improve the performance of 2D devices by means of other methods of plasma, chemical, and ozone treatments is reviewed. At last, the challenges and opportunities of defect engineering on promoting the development of 2D materials are discussed. Through this review, we aim to build a correlation between defects and properties of 2D materials to support the design and optimization of high-performance electronic and optoelectronic devices.

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Multitarget, Selective Compound Design Yields Picomolar Inhibitors of a Kinetoplastid Pteridine Reductase 1

10.26434/chemrxiv.13026797 ◽

2020 ◽

Author(s):

Ina Poehner ◽

Antonio Quotadamo ◽

Joanna Panecka-Hofman ◽

Rosaria Luciani ◽

Matteo Santucci ◽

...

Keyword(s):

Drug Discovery ◽

Dihydrofolate Reductase ◽

Multidisciplinary Approach ◽

Microwave Assisted Synthesis ◽

Crystallographic Structure ◽

Computational Docking ◽

Design And Optimization ◽

Microwave Assisted ◽

Structure Activity ◽

Further Development

The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

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Multitarget, Selective Compound Design Yields Picomolar Inhibitors of a Kinetoplastid Pteridine Reductase 1

10.26434/chemrxiv.13026797.v1 ◽

2020 ◽

Author(s):

Ina Poehner ◽

Antonio Quotadamo ◽

Joanna Panecka-Hofman ◽

Rosaria Luciani ◽

Matteo Santucci ◽

...

Keyword(s):

Drug Discovery ◽

Dihydrofolate Reductase ◽

Multidisciplinary Approach ◽

Microwave Assisted Synthesis ◽

Crystallographic Structure ◽

Computational Docking ◽

Design And Optimization ◽

Microwave Assisted ◽

Structure Activity ◽

Further Development

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Toeholder: a software for automated design and in silico validation of toehold riboswitches

10.1101/2021.11.09.467922 ◽

2021 ◽

Author(s):

Angel Fernando Cisneros Caballero ◽

Francois D. Rouleau ◽

Carla Bautista ◽

Pascale Lemieux ◽

Nathan Dumont-Leblond

Keyword(s):

In Silico ◽

Dynamic Range ◽

Regulatory Elements ◽

Automated Design ◽

Design And Optimization ◽

Potential Applications ◽

Dynamics Simulations ◽

Effective Use ◽

Biological Circuits ◽

High Throughput Experiments

Synthetic biology aims to engineer biological circuits, which often involve gene expression. A particularly promising group of regulatory elements are riboswitches because of their versatility with respect to their targets, but early synthetic designs were not as attractive because of a reduced dynamic range with respect to protein regulators. Only recently, the creation of toehold switches helped overcome this obstacle by also providing an unprecedented degree of orthogonality. However, a lack of automated design and optimization tools prevents the widespread and effective use of toehold switches in high-throughput experiments. To address this, we developed Toeholder, a comprehensive open-source software for toehold design and in silico benchmarking. Toeholder takes into consideration sequence constraints as well as data derived from molecular dynamics simulations of a toehold switch. We describe the software and its in silico validation results, as well as its potential applications and impacts on the management and design of toehold switches.

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Theoretical Assessment of Light Structural Damage due to Ship Collision With Ice

Journal of Offshore Mechanics and Arctic Engineering ◽

10.1115/1.2919897 ◽

1991 ◽

Vol 113 (1) ◽

pp. 61-66 ◽

Cited By ~ 1

Author(s):

A. Moshaiov ◽

M. R. Steinhilber

Keyword(s):

Structural Design ◽

Structural Damage ◽

Rate Sensitivity ◽

Marine Transportation ◽

Design And Optimization ◽

Ice Loads ◽

Ship Collision ◽

Further Development ◽

Theoretical Assessment ◽

Major Consideration

A major consideration in the recent development of marine transportation for ice-infested waters is the strength required for ships’ hulls. Plasticity methods are currently used in conjunction with given design ice loads. In this paper, a new plasticity model is suggested. It is based on the assumption that the kinetic energy of the ice/ship collision is absorbed both by the ice and the structure. During the collision process, the ice/structure contact area varies due to ice crushing, which dissipates some of the energy. At the same time, the plating may deform plastically, absorbing the remaining energy. Other forms of energies and ice failures are not accounted for, allowing a conservative estimate of the damage. A parametric study is performed, revealing the significance of the energy absorbed by the ice in reducing the predicted permanent deflection of the plating. The new model is shown to be useful for ship structural design and optimization in addition to the evaluation of operating restrictions. Several recommendations for further development of the model are discussed, including the incorporation of strain rate sensitivity of the ice-crushing strength and the plating yield strength.

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BGLF2 Increases Infectivity of Epstein-Barr Virus by Activating AP-1 uponDe NovoInfection

mSphere ◽

10.1128/msphere.00138-18 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 12

Author(s):

Natsuno Konishi ◽

Yohei Narita ◽

Fumiya Hijioka ◽

H. M. Abdullah Al Masud ◽

Yoshitaka Sato ◽

...

Keyword(s):

Signaling Pathway ◽

Epstein Barr Virus ◽

De Novo ◽

Physiological Role ◽

Significant Loss ◽

Viral Gene ◽

Barr Virus ◽

Cell Functions ◽

Epstein Barr ◽

Lytic Genes

ABSTRACTEpstein-Barr virus (EBV) is a human gammaherpesvirus that causes infectious mononucleosis and several malignancies, such as endemic Burkitt lymphoma and nasopharyngeal carcinoma. Herpesviruses carry genes that can modify cell functions, including transcription and ubiquitination, thereby facilitating viral growth and survival in infected cells. Using a reporter screening system, we revealed the involvement of several EBV gene products in such processes. Of these, BGLF2 activated the AP-1 signaling pathway through phosphorylation of p38 and c-Jun N-terminal kinase (JNK). Knockout of the BGLF2 gene did not affect viral gene expression and viral genome DNA replication, but resulted in marked reduction of progeny titer. We also found that the BGLF2 disruption resulted in significant loss of infectivity uponde novoinfection. Interestingly, expression of a binding partner, BKRF4, repressed the activation of AP-1 by BGLF2. These results shed light on the physiological role of the tegument protein BGLF2.IMPORTANCEEpstein-Barr virus (EBV), an oncogenic gammaherpesvirus, carries ~80 genes. While several genes have been investigated extensively, most lytic genes remain largely unexplored. Therefore, we cloned 71 EBV lytic genes into an expression vector and used reporter assays to screen for factors that activate signal transduction pathways, viral and cellular promoters. BGLF2 activated the AP-1 signaling pathway, likely by interacting with p38 and c-Jun N-terminal kinase (JNK), and increased infectivity of the virus. We also revealed that BKRF4 can negatively regulate AP-1 activity. Therefore, it is suggested that EBV exploits and modifies the AP-1 signaling pathway for its replication and survival.

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Benign extracerebral fluid collection in infancy as a risk factor for the development of de novo intracranial arachnoid cysts

Journal of Neurosurgery Pediatrics ◽

10.3171/2013.8.peds1399 ◽

2013 ◽

Vol 12 (6) ◽

pp. 555-564 ◽

Cited By ~ 12

Author(s):

Tobias A. Mattei ◽

Brandon J. Bond ◽

Deepak Sambhara ◽

Carlos R. Goulart ◽

Julian J. Lin

Keyword(s):

Risk Factor ◽

De Novo ◽

Multiple Logistic Regression Analysis ◽

Fluid Collection ◽

Arachnoid Cysts ◽

Ct Scans ◽

Delivery Mode ◽

History Of ◽

Fluid Collections ◽

Further Development

Object Intracranial arachnoid cysts are a relatively common benign intracranial pathology, accounting for as many as 0.75%–1% of nontraumatic CNS lesions. Although it has already been demonstrated that rupture of arachnoid cysts may lead to subdural hematomas/hygromas, no study to date has investigated benign extracerebral collection in infancy as a possible predisposing factor for further development of arachnoid cysts. Methods The authors performed a retrospective imaging and chart review of macrocephalic infants 12 months old or younger who were referred to neurosurgical care at OSF St. Francis Medical Center from 2003 to 2010, and who were diagnosed with benign extracerebral fluid collection in infancy on thin-slice (1-mm) head CT scans. Special attention was given to the investigation of risk factors for further development of de novo arachnoid cysts. Several epidemiological factors in the infants and mothers were analyzed, including gestational age at delivery, mode of delivery, mother's age at delivery, delivery complications, birth weight, age of macrocephaly development, degree of macrocephaly, family history of macrocephaly, prenatal and postnatal history of infection, fontanel status, presence of papilledema, previous history of head trauma, and smoking status. Imaging characteristics of the initial scans, such as location of subdural collection (frontal vs frontoparietal and frontotemporal) and presence of ventriculomegaly, were also evaluated. For those patients in whom arachnoid cysts were identified on subsequent CT scans, the size and location of the cysts were also analyzed. Results The authors identified 44 children with benign extracerebral fluid collection in infancy. From this group, over a mean follow-up of 13 months (range 6–13 months), 18 children developed intracranial arachnoid cysts (a 40.9% incidence of de novo development of arachnoid cysts), with 27.8% presenting with bilateral cysts. In the multiple logistic regression analysis, infants who presented with an extracerebral collection restricted to the bilateral frontal region were more likely to develop intracranial arachnoid cysts (p = 0.035) than those with collections involving the frontotemporal and frontoparietal regions (odds ratio [OR] = 5.73). Additionally, children with benign extracerebral fluid collections and plagiocephaly were more likely to develop intracranial arachnoid cysts (p = 0.043) than those without plagiocephaly (OR = 4.96). Conclusions This is the first report in the neurosurgical literature demonstrating that benign extracerebral fluid collections in infancy may constitute a significant risk factor for development of de novo arachnoid cysts. These findings support a 2-hit hypothesis for the development of arachnoid cysts, in which the combination of an embryological defect in arachnoid development followed by a second event leading to impairment of CSF fluid absorption in early childhood could lead to abnormal CSF dynamics and the consequent expansion of fluid collections in the intraarachnoid spaces.

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In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1

Journal of Bacteriology ◽

10.1128/jb.00230-09 ◽

2009 ◽

Vol 191 (12) ◽

pp. 3842-3851 ◽

Cited By ~ 22

Author(s):

Michael J. Gray ◽

Jorge C. Escalante-Semerena

Keyword(s):

Rhodobacter Sphaeroides ◽

High Performance ◽

Biosynthetic Pathway ◽

De Novo ◽

Nutritional Analysis ◽

In Vivo Analysis ◽

Essential Enzyme ◽

Necessary And Sufficient ◽

Coenzyme B

ABSTRACT The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B12). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ + strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.

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