scholarly journals Investigating viral attenuation by promoter knockout: a systems approach

2017 ◽  
Author(s):  
Matthew L Paff ◽  
Benjamin R. Jack ◽  
Daniel R Boutz ◽  
Bartram L Smith ◽  
Claus O Wilke ◽  
...  
Keyword(s):  
Genome Editing ◽  
Systems Approach ◽  
A Priori ◽  
Rna Seq ◽  
Genome Composition ◽  
T7 Promoter ◽  
Highly Expressed Genes ◽  
The One ◽  
Viral Attenuation ◽  
Gene Expression Levels

Live attenuated viral vaccines provide the most robust and longest lasting immune response. Yet designing them a priori to have reduced growth capacity and also to be robust to evolutionary reversion can be challenging. On the one hand, genome editing methods now enable us to create almost any conceivable viral genome composition. Yet understanding and predicting how engineered genomes will behave and evolve is a challenge. Here we adopt a systems approach in studying a simple attenuation design in bacteriophage T7: promoter knockout. Either or both promoters for the two most highly expressed genes were abolished. Overall fitnesses, major phenotypes and gene expression levels were measured for all initial genomes and for genomes evolved toward for fitness recovery. Initial genomes behaved broadly as expected, but the genomes showed an unexpected ability to evolve back to high fitness. Genome sequences, RNA Seq and proteomics reveal the molecular foundations of the attenuations and recoveries. Overall, the work suggests that a systems approach is ultimately yielding to understanding, if not predicting the consequences of genome editing and evolutionary recoveries of simple genomes.

scholarly journals Investigating viral attenuation by promoter knockout: a systems approach

2017 ◽  
Author(s):  
Matthew L Paff ◽  
Benjamin R. Jack ◽  
Daniel R Boutz ◽  
Bartram L Smith ◽  
Claus O Wilke ◽  
...  
Keyword(s):  
Genome Editing ◽  
Systems Approach ◽  
A Priori ◽  
Rna Seq ◽  
Genome Composition ◽  
T7 Promoter ◽  
Highly Expressed Genes ◽  
The One ◽  
Viral Attenuation ◽  
Gene Expression Levels

Live attenuated viral vaccines provide the most robust and longest lasting immune response. Yet designing them a priori to have reduced growth capacity and also to be robust to evolutionary reversion can be challenging. On the one hand, genome editing methods now enable us to create almost any conceivable viral genome composition. Yet understanding and predicting how engineered genomes will behave and evolve is a challenge. Here we adopt a systems approach in studying a simple attenuation design in bacteriophage T7: promoter knockout. Either or both promoters for the two most highly expressed genes were abolished. Overall fitnesses, major phenotypes and gene expression levels were measured for all initial genomes and for genomes evolved toward for fitness recovery. Initial genomes behaved broadly as expected, but the genomes showed an unexpected ability to evolve back to high fitness. Genome sequences, RNA Seq and proteomics reveal the molecular foundations of the attenuations and recoveries. Overall, the work suggests that a systems approach is ultimately yielding to understanding, if not predicting the consequences of genome editing and evolutionary recoveries of simple genomes.

scholarly journals A broad introduction to RNA-Seq

2021 ◽  
Vol 4 (1) ◽  
pp. 4
Author(s):  
Felix Richter
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Messenger Rna ◽  
Expressed Sequence Tag ◽  
A Priori ◽  
Nucleotide Polymorphisms ◽  
Rna Seq ◽  
Cross Hybridization ◽  
Fixed Tissue ◽  
Highly Expressed Genes

RNA-Seq, named as an abbreviation of "RNA sequencing" and sometimes spelled RNA-seq, RNAseq, or RNASeq, uses next-generation sequencing (NGS) to reveal the presence and quantity of ribonucleic acid (RNA) in a biological sample at a given moment.[1][2] RNA-Seq is used to analyze the continuously changing cellular transcriptome (Figure 1). Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/single nucleotide polymorphisms (SNPs) and changes in gene expression over time, or differences in gene expression in different groups or treatments.[3] In addition to messenger RNA (mRNA) transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as microRNA (miRNA), transfer RNA (tRNA), and ribosomal profiling.[4] RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include single cell sequencing, in situ sequencing of fixed tissue, and native RNA molecule sequencing with single-molecule real-time sequencing.[5] Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know the sequence a priori.[6] Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology, next-gen sequencing of complementary DNA ( cDNA), notably RNA-Seq.

Comparative expression profiling of three early inflorescence stages of oil palm indicates that vegetative to reproductive phase transition of meristem is regulated by sugar balance

2015 ◽  
Vol 42 (6) ◽  
pp. 589 ◽  
Author(s):  
Walter Ajambang ◽  
Sintho W. Ardie ◽  
Hugo Volkaert ◽  
Georges F. Ngando-Ebongue ◽  
Sudarsono Sudarsono
Keyword(s):  
Oil Palm ◽  
Rna Seq ◽  
Illumina Platform ◽  
Reproductive Phase ◽  
Underlying Network ◽  
Highly Expressed Genes ◽  
Three Stages ◽  
Stage 1 ◽  
And Control ◽  
Gene Expression Levels

Breeding and seed production activities in oil palm have been hampered because of the inability of the male parent Pisifera to produce male inflorescence as source of pollen under normal conditions. Researchers are using complete defoliation to induce male inflorescences, but the biological and molecular processes responsible for this morphological change are yet to be revealed. To understand the underlying network of genes that initiate and control this phenotypically documented activity, we initiated a study aimed at identifying differentially expressed genes (DEGs) in three stages of an oil palm inflorescence under complete defoliation stress using RNA-seq. Sequencing on an Illumina platform produced 82 631 476 reads consisting of 8 345 779 076 bases. A total of 60 700 genes were obtained after transcript filtering and normalisation and 54% of them were downregulated. Differences in gene expression levels were significant between tissues under stress. The farther the distance between tissues, the more DEGs recorded. Comparison between stage 2 and stage 1 induced 3893 DEGs whereas 10 136 DEGs were induced between stage 3 and stage 1. Stress response genes and flower development genes were among the highly expressed genes. This study suggests a link between complete defoliation and meristem differentiation from vegetative to reproductive phase in oil palm.

scholarly journals Minkowski box from Yangian bootstrap

2021 ◽  
Vol 2021 (4) ◽  
Author(s):  
Luke Corcoran ◽  
Florian Loebbert ◽  
Julian Miczajka ◽  
Matthias Staudacher
Keyword(s):  
Minkowski Space ◽  
Wigner Function ◽  
Functional Form ◽  
A Priori ◽  
Alternative Methods ◽  
Feynman Integrals ◽  
The One

Abstract We extend the recently developed Yangian bootstrap for Feynman integrals to Minkowski space, focusing on the case of the one-loop box integral. The space of Yangian invariants is spanned by the Bloch-Wigner function and its discontinuities. Using only input from symmetries, we constrain the functional form of the box integral in all 64 kinematic regions up to twelve (out of a priori 256) undetermined constants. These need to be fixed by other means. We do this explicitly, employing two alternative methods. This results in a novel compact formula for the box integral valid in all kinematic regions of Minkowski space.

scholarly journals High heterogeneity undermines generalization of differential expression results in RNA-Seq analysis

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Weitong Cui ◽  
Huaru Xue ◽  
Lei Wei ◽  
Jinghua Jin ◽  
Xuewen Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Small Sample ◽  
Differentially Expressed ◽  
Cancer Type ◽  
Rna Seq ◽  
Sample Sizes ◽  
Large Sample ◽  
Expression Levels ◽  
Gene Expression Levels

Abstract Background RNA sequencing (RNA-Seq) has been widely applied in oncology for monitoring transcriptome changes. However, the emerging problem that high variation of gene expression levels caused by tumor heterogeneity may affect the reproducibility of differential expression (DE) results has rarely been studied. Here, we investigated the reproducibility of DE results for any given number of biological replicates between 3 and 24 and explored why a great many differentially expressed genes (DEGs) were not reproducible. Results Our findings demonstrate that poor reproducibility of DE results exists not only for small sample sizes, but also for relatively large sample sizes. Quite a few of the DEGs detected are specific to the samples in use, rather than genuinely differentially expressed under different conditions. Poor reproducibility of DE results is mainly caused by high variation of gene expression levels for the same gene in different samples. Even though biological variation may account for much of the high variation of gene expression levels, the effect of outlier count data also needs to be treated seriously, as outlier data severely interfere with DE analysis. Conclusions High heterogeneity exists not only in tumor tissue samples of each cancer type studied, but also in normal samples. High heterogeneity leads to poor reproducibility of DEGs, undermining generalization of differential expression results. Therefore, it is necessary to use large sample sizes (at least 10 if possible) in RNA-Seq experimental designs to reduce the impact of biological variability and DE results should be interpreted cautiously unless soundly validated.

scholarly journals Supergraph calculation of one-loop divergences in higher-derivative 6D SYM theory

2020 ◽  
Vol 2020 (8) ◽  
Author(s):  
I. L. Buchbinder ◽  
E. A. Ivanov ◽  
B. S. Merzlikin ◽  
K. V. Stepanyantz
Keyword(s):  
Effective Action ◽  
A Priori ◽  
Coupling Constant ◽  
Harmonic Superspace ◽  
Classical Action ◽  
Higher Derivative ◽  
Component Approach ◽  
Dimensionless Coupling ◽  
The One ◽  
Dimensionless Coupling Constant

Abstract We apply the harmonic superspace approach for calculating the divergent part of the one-loop effective action of renormalizable 6D, $$ \mathcal{N} $$ N = (1, 0) supersymmetric higher-derivative gauge theory with a dimensionless coupling constant. Our consideration uses the background superfield method allowing to carry out the analysis of the effective action in a manifestly gauge covariant and $$ \mathcal{N} $$ N = (1, 0) supersymmetric way. We exploit the regularization by dimensional reduction, in which the divergences are absorbed into a renormalization of the coupling constant. Having the expression for the one-loop divergences, we calculate the relevant β-function. Its sign is specified by the overall sign of the classical action which in higher-derivative theories is not fixed a priori. The result agrees with the earlier calculations in the component approach. The superfield calculation is simpler and provides possibilities for various generalizations.

scholarly journals Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Rasmus Rydbirk ◽  
Jonas Folke ◽  
Kristian Winge ◽  
Susana Aznar ◽  
Bente Pakkenberg ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Reference Genes ◽  
Target Gene ◽  
Structural Changes ◽  
A Priori ◽  
Brain Regions ◽  
Quantitative Real Time Pcr ◽  
Human Brain Tissue ◽  
Web Based ◽  
Gene Expression Levels

Abstract Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer’s disease (AD), Parkinson’s disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies.

Three Kantian Routes to the Synthetic A Priori

2021 ◽  
pp. 1-28
Author(s):  
Robert Audi
Keyword(s):  
A Priori ◽  
Pure Reason ◽  
Critique Of Pure Reason ◽  
Insufficient Attention ◽  
Synthetic A Priori ◽  
The One ◽  
Philosophical Importance

Abstract Kant influentially distinguished analytic from synthetic a priori propositions, and he took certain propositions in the latter category to be of immense philosophical importance. His distinction between the analytic and the synthetic has been accepted by many and attacked by others; but despite its importance, a number of discussions of it since at least W. V. Quine’s have paid insufficient attention to some of the passages in which Kant draws the distinction. This paper seeks to clarify what appear to be three distinct conceptions of the analytic (and implicitly of the synthetic) that are presented in Kant’s Critique of Pure Reason and in some other Kantian texts. The conceptions are important in themselves, and their differences are significant even if they are extensionally equivalent. The paper is also aimed at showing how the proposed understanding of these conceptions—and especially the one that has received insufficient attention from philosophers—may bear on how we should conceive the synthetic a priori, in and beyond Kant’s own writings.

WTLS-Bayes Filter-based Relative Pose Estimation for Space Non-cooperative Unknown Target Without a Priori Knowledge

2021 ◽  
Author(s):  
CHENGGUANG ZHU ◽  
zhongpai Gao ◽  
Jiankang Zhao ◽  
Haihui Long ◽  
Chuanqi Liu
Keyword(s):  
Pose Estimation ◽  
A Priori ◽  
Convergence Time ◽  
Estimation Accuracy ◽  
A Priori Knowledge ◽  
One Step ◽  
Relative Pose ◽  
The One ◽  
Negative Factors ◽  
Priori Knowledge

Abstract The relative pose estimation of a space noncooperative target is an attractive yet challenging task due to the complexity of the target background and illumination, and the lack of a priori knowledge. Unfortunately, these negative factors have a grave impact on the estimation accuracy and the robustness of filter algorithms. In response, this paper proposes a novel filter algorithm to estimate the relative pose to improve the robustness based on a stereovision system. First, to obtain a coarse relative pose, the weighted total least squares (WTLS) algorithm is adopted to estimate the relative pose based on several feature points. The resulting relative pose is fed into the subsequent filter scheme as observation quantities. Second, the classic Bayes filter is exploited to estimate the relative state except for moment-of-inertia ratios. Additionally, the one-step prediction results are used as feedback for WTLS initialization. The proposed algorithm successfully eliminates the dependency on continuous tracking of several fixed points. Finally, comparison experiments demonstrate that the proposed algorithm presents a better performance in terms of robustness and convergence time.

scholarly journals Low-Bias RNA Sequencing of the HIV-2 Genome from Blood Plasma

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Katherine L. James ◽  
Thushan I. de Silva ◽  
Katherine Brown ◽  
Hilton Whittle ◽  
Stephen Taylor ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Blood Plasma ◽  
De Novo ◽  
A Priori ◽  
Pcr Amplification ◽  
Hiv Vaccine ◽  
Whole Genome ◽  
Plasma Samples ◽  
Target Enrichment ◽  
Rna Seq

ABSTRACTAccurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target enrichment through PCR requiresa priorisequence knowledge, which is lacking for HIV-2. Therefore, a target enrichment free method of library preparation would be valuable for the field. We applied an RNA shotgun sequencing (RNA-Seq) method without PCR amplification to cultured viral stocks and patient plasma samples from HIV-2-infected individuals. Libraries generated from total plasma RNA were analyzed with a two-step pipeline: (i)de novogenome assembly, followed by (ii) read remapping. By this approach, whole-genome sequences were generated with a 28× to 67× mean depth of coverage. Assembled reads showed a low level of GC bias, and comparison of the genome diversities at the intrahost level showed low diversity in the accessory genevpxin all patients. Our study demonstrates that RNA-Seq is a feasible full-genomede novosequencing method for blood plasma samples collected from HIV-2-infected individuals.IMPORTANCEAn accurate picture of viral genetic diversity is critical for the development of a globally effective HIV vaccine. However, sequencing strategies are often complicated by target enrichment prior to sequencing, introducing biases that can distort variant frequencies, which are not easily corrected for in downstream analyses. Additionally, detaileda priorisequence knowledge is needed to inform robust primer design when employing PCR amplification, a factor that is often lacking when working with tropical diseases localized in developing countries. Previous work has demonstrated that direct RNA shotgun sequencing (RNA-Seq) can be used to circumvent these issues for hepatitis C virus (HCV) and norovirus. We applied RNA-Seq to total RNA extracted from HIV-2 blood plasma samples, demonstrating the applicability of this technique to HIV-2 and allowing us to generate a dynamic picture of genetic diversity over the whole genome of HIV-2 in the context of low-bias sequencing.

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