MutationalPatterns: The one stop shop for the analysis of mutational processes

Background: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. Results: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. Conclusions: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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Diffusion-Limited Cell Dehydration: Analytical and Numerical Solutions for a Planar Model

10.1115/imece1999-0600 ◽

1999 ◽

Author(s):

Alexander V. Kasharin ◽

Jens O. M. Karlsson

Keyword(s):

Analytical Solution ◽

Numerical Solutions ◽

Planar System ◽

One Dimensional ◽

Diffusion Limited ◽

Dimensional Diffusion ◽

Constant Diffusivity ◽

A Cell ◽

Cell Dehydration ◽

The One

Abstract The process of diffusion-limited cell dehydration is modeled for a planar system by writing the one-dimensional diffusion-equation for a cell with moving, semipermeable boundaries. For the simplifying case of isothermal dehydration with constant diffusivity, an approximate analytical solution is obtained by linearizing the governing partial differential equations. The general problem must be solved numerically. The Forward Time Center Space (FTCS) and Crank-Nicholson differencing schemes are implemented, and evaluated by comparison with the analytical solution. Putative stability criteria for the two algorithms are proposed based on numerical experiments, and the Crank-Nicholson method is shown to be accurate for a mesh with as few as six nodes.

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Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

eLife ◽

10.7554/elife.17290 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 78

Author(s):

Rowena DeJesus ◽

Francesca Moretti ◽

Gregory McAllister ◽

Zuncai Wang ◽

Phil Bergman ◽

...

Keyword(s):

Adaptor Protein ◽

Er Stress Response ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Selection Step ◽

A Cell ◽

Cell Type Specific ◽

Cellular Pathways ◽

Mtor Signalling

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

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Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing, Butyrate-Forming Acetogen, Eubacterium limosum KIST612

Applied and Environmental Microbiology ◽

10.1128/aem.00675-15 ◽

2015 ◽

Vol 81 (14) ◽

pp. 4782-4790 ◽

Cited By ~ 32

Author(s):

Jiyeong Jeong ◽

Johannes Bertsch ◽

Verena Hess ◽

Sunju Choi ◽

In-Geol Choi ◽

...

Keyword(s):

Carbon Monoxide ◽

Atp Synthesis ◽

Binding Motif ◽

Oxidation Of Co ◽

Co Dehydrogenase ◽

Content Type ◽

A Genome ◽

Eubacterium Limosum ◽

The One ◽

Conservation Model

ABSTRACTEubacterium limosumKIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na+dependent. Consistent with the finding of a Na+-dependent Rnf complex is the presence of a conserved Na+-binding motif in thecsubunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding ofetfAandetfBgenes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H2+ CO2. The energetic benefit may be one reason that butyrate is formed only from CO but not from H2+ CO2.

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Three-Dimensional Microfabrication System for Biodegradable Microdevices With High-Resolution and Bio-Applicability

Advances in Bioengineering ◽

10.1115/imece2005-82154 ◽

2005 ◽

Cited By ~ 1

Author(s):

Akira Yamada ◽

Fuminori Niikura ◽

Koji Ikuta

Keyword(s):

Tensile Strength ◽

Biodegradable Polymers ◽

Conventional Method ◽

Three Dimensional ◽

Single Layer ◽

Batch Process ◽

Potential Applications ◽

A Cell ◽

The One ◽

Almost All

Biodegradable polymers are employed in medicine and its further application is expected with eagerness. But the lack of an appropriate processing method retards the progress. To overcome this problem, we have developped a novel three-dimensional microfabrication system. The system design allows us the processing of the free three-dimensional micro-level forms by stacking up melted polymers from the nozzle. Different from the conventional method, we adopted a batch process to supply materials in order to eliminate the prior process that required toxic solvents. In addition, it is possible to handle almost all biodegradable thermoplastic resins by adopting this system. A single layer from the piled-up layers of extruded lines was observed to evaluate the resolution. The lateral and depth resolutions attained are 40 μm and 45 μm, respectively. Biodegradable polymers enable three-dimensional microstructures such as micro-pipes, micro-bends, and micro-coil springs to be manufactured in less than 15 min. The biocompatibility of the newly fabricated structure was evaluated using a cell line (PC12). For this purpose, a small vessel, with a transparent base, was fabricated using PLA and cells were cultivated in it. The results were then compared with the results obtained using the standard method. The mechanical strength of our microstructures was evaluated using a tensile strength test. The tensile strength of the microstructure was lower than the one obtained from the conventional method, but has enough strength for fabrication of medical devices. Our system renders it possible to produce toxic-free, as well as transparent and leakage-free devices. Our system is expected to have potential applications in optimum design and fabrication of implantable devices, especially in tissue engineering.

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Analysis of fragment ends in plasma DNA from patients with cancer

10.1101/2021.04.23.21255935 ◽

2021 ◽

Author(s):

Karan K. Budhraja ◽

Bradon R. McDonald ◽

Michelle D. Stephens ◽

Tania Contente-Cuomo ◽

Havell Markus ◽

...

Keyword(s):

Nucleotide Sequence ◽

Characteristic Curve ◽

Cost Effective ◽

Cancer Diagnostics ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

Plasma Dna ◽

Patients With Cancer ◽

Fragmentation Patterns ◽

Tumor Dna

AbstractFragmentation patterns observed in plasma DNA reflect chromatin accessibility in contributing cells. Since DNA shed from cancer cells and blood cells may differ in fragmentation patterns, we investigated whether analysis of genomic positioning and nucleotide sequence at fragment ends can reveal the presence of tumor DNA in blood and aid cancer diagnostics. We analyzed whole genome sequencing data from >2700 plasma DNA samples including healthy individuals and patients with 11 different cancer types. We observed higher fractions of fragments with aberrantly positioned ends in patients with cancer, driven by contribution of tumor DNA into plasma. Genomewide analysis of fragment ends using machine learning showed overall area under the receiver operative characteristic curve of 0.96 for detection of cancer. Our findings remained robust with as few as 1 million fragments analyzed per sample, suggesting that analysis of fragment ends can become a cost-effective and accessible approach for cancer detection and monitoring.One-sentence summaryAnalyzing the positioning and nucleotide sequence at fragment ends in plasma DNA may enable cancer diagnostics.

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Population sequencing data reveal a compendium of mutational processes in the human germ line

Science ◽

10.1126/science.aba7408 ◽

2021 ◽

pp. eaba7408

Author(s):

Vladimir B. Seplyarskiy ◽

Ruslan A. Soldatov ◽

Evan Koch ◽

Ryan J. McGinty ◽

Jakob M. Goldmann ◽

...

Keyword(s):

Germ Line ◽

Nonnegative Matrix ◽

Replication Timing ◽

Mutagenic Effect ◽

Dna Lesions ◽

Sequencing Data ◽

Biological Interpretation ◽

Mutagenic Process ◽

Mutational Processes ◽

Transcription And Replication

Biological mechanisms underlying human germline mutations remain largely unknown. We statistically decompose variation in the rate and spectra of mutations along the genome using volume-regularized nonnegative matrix factorization. The analysis of a sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. We provide a biological interpretation for seven of these processes. We associate one process with bulky DNA lesions that resolve asymmetrically with respect to transcription and replication. Two processes track direction of replication fork and replication timing, respectively. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions and a mutagenic effect of LINE repeats. We localize a mutagenic process specific to oocytes from population sequencing data. This process appears transcriptionally asymmetric.

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Physiological function of Flo11p domains and the particular role of amyloid core sequences of this adhesin in Saccharomyces cerevisiae

10.1101/2021.04.01.438097 ◽

2021 ◽

Author(s):

Clara Bouyx ◽

Marion Schiavone ◽

Marie-Ange Teste ◽

Etienne Dague ◽

Nathalie Sieczkowski ◽

...

Keyword(s):

Cell Wall ◽

Amyloid Β ◽

Surface Hydrophobicity ◽

Yeast Cells ◽

Invasive Growth ◽

Specific Domain ◽

A Genome ◽

A Cell ◽

Cellular Aggregates

Flocculins are a family of glycosylated proteins that provide yeast cells with several properties such as biofilm formation, flocculation, invasive growth or formation of velum. These proteins are similarly organised with a N-terminal (adhesion) domain, a stalk-like central B-domain with several repeats and a C-terminal sequence carrying a cell wall anchor site. They also contain amyloid β-aggregation-prone sequences whose functional role is still unclear. In this work, we show that Flo11p differs from other flocculins by the presence of unique amyloid-forming sequences, whose the number is critical in the formation of adhesion nanodomains under a physical shear force. Using a genome editing approach to identify the function of domains in Flo11p phenotypes, we show that the formation of cellular aggregates whose density increases with the number of amyloid sequences cannot be attributed to a specific domain of Flo11p. The same is true for plastic adhesion and surface hydrophobicity the intensity of which depends mainly on the abundance of Flo11p on the cell surface. In contrast, the N and C domains of Flo11p are essential for invasive growth in agar, whereas a reduction in the number of repeats of the B domain weakens this phenotype. However, expression of FLO11 alone is not sufficient to trigger this invasion phenotype. Finally, we show that this flocculin contributes to the integrity of the cell wall.

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Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

Open Biology ◽

10.1098/rsob.150230 ◽

2016 ◽

Vol 6 (1) ◽

pp. 150230 ◽

Cited By ~ 17

Author(s):

Laura Wood ◽

Daniel G. Booth ◽

Giulia Vargiu ◽

Shinya Ohta ◽

Flavia deLima Alves ◽

...

Keyword(s):

Protein Function ◽

Mitotic Chromosomes ◽

Protein Levels ◽

Kinetochore Assembly ◽

Rnai Technology ◽

Gradual Loss ◽

A Cell ◽

Stable Association ◽

Or Gene ◽

Regulated Gene Expression

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).

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Comparison of West Balkan adult trout habitat predictions using a Pseudo-2D and a 2D hydrodynamic model

Hydrology Research ◽

10.2166/nh.2016.352 ◽

2016 ◽

Vol 48 (6) ◽

pp. 1697-1709 ◽

Cited By ~ 5

Author(s):

Christina Papadaki ◽

Vasilis Bellos ◽

Lazaros Ntoanidis ◽

Elias Dimitriou

Keyword(s):

Hydrodynamic Model ◽

River Discharge ◽

Environmental Flow ◽

Two Dimensional ◽

Habitat Models ◽

One Dimensional ◽

2D Flow ◽

A Cell ◽

The One ◽

Water Depths

Abstract Hydraulic-habitat models combine the dynamic behavior of river discharge with geomorphological and ecological responses. In this study, they are used for estimating environmental flow requirements. We applied a Pseudo-two-dimensional (2D) model based on the one-dimensional (1D) HEC-RAS model and an in-house 2D (FLOW-R2D) hydrodynamic model to a section of river for several flows in respect of summer conditions of the study reach, and compared the results derived from the models in terms of water depths and velocities as well as habitat predictions in terms of weighted usable area (WUA). In general, 2D models are more promising in habitat studies since they quantify spatial variations and combinations of flow patterns important to stream flora and fauna in a higher detail than the 1D models. Relationships between WUA and discharge for the two models were examined, to compare the similarity as well as the magnitude of predictions over the modelled discharge range. The models predicted differences in the location of maxima and changes in variation of velocity and water depth. Finally, differences in spatial distribution (in terms of suitability indices and WUA) between the Pseudo-2D and the fully 2D modelling results can be considerable on a cell-by-cell basis.

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