Efficient Construction and Utilization of k-Ordered FM-indexes with kISS for Ultra-Fast Read Mapping in Large Genomes.

MOTIVATION: The Full-text index in Minute space (FM-index) is a memory-efficient data structure widely used in bioinformatics for solving the fundamental pattern-matching task of searching for short patterns within a long reference. With the demand for short query patterns, the k-ordered concept has been proposed for FM-indexes. However, few construction algorithms in the state of the art fully exploit this idea to achieve significant speedups in the pan-genome era. RESULTS: We introduce the k-ordered Induced Suffix Sorting (kISS) for efficient construction and utilization of k-ordered FM-indexes. We present an algorithmic workflow for building k-ordered suffix arrays, incorporating two novel strategies to improve time and memory efficiency. We also demonstrate the compatibility of integrating k-ordered FM-indexes with locate operations in FMtree. Experiments show that kISS can improve the construction time, and the generated k-ordered suffix array can also be applied to FMtree without any additional in computation or memory usage. AVAILABILITY: https://github.com/jhhung/kISS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A gene silencing screen uncovers diverse tools for targeted gene repression in Arabidopsis.

DNA methylation has been utilized for target gene silencing in plants. However, it is not well understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain-of-function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression through DNA methylation, histone H3K27me3 deposition, H3K4me3 demethylation, histone deacetylation, inhibition of RNA polymerase II transcription elongation or Ser-5 dephosphorylation. These proteins also silenced many other genes with different efficacies, and a machine learning model could accurately predict the efficacy of each silencer on the basis of various chromatin features of the target loci. Furthermore, some proteins were also able to target gene silencing when used in a dCas9-SunTag system. These results provide a more comprehensive understanding of epigenetic regulatory pathways in plants and provide an armament of tools for targeted gene manipulation.

AC measurements and simulations of hepatic radiofrequency ablation.

INTRODUCTION: The radiofrequency ablation (RFA) of liver cancer is a desirable treatment option, as it is minimally invasive. An accurate numerical simulation can greatly help physicians better plan their surgical protocols. Previously, the displacement current in the RFA process was considered negligible, and therefore RFA simulation was modeled as a direct current (DC) system instead of an alternating current (AC) system. Our study investigated the hypothesis that the displacement current in the RFA process should not always be considered negligible. METHODS: AC measurements of ex vivo bovine liver ablation were performed, and numerical simulations were also conducted to test the hypothesis that the relative permittivity would significantly decrease after the liver tissue reached a high temperature. RESULTS: The displacement current was observed to be a sizable fraction of the conduction current, especially before the onset of the first pause. The simulation results indicated that the relative permittivity is likely to decrease to several hundred or lower at elevated temperatures. CONCLUSIONS: Our study results suggest that the DC model may be inadequate, especially before the first roll-off and that additional information could be available during RFA treatment by considering the AC nature of RFA, which could lead to improved numerical simulation. Additional measurements of tissue parameters are needed to reach the full potential of the AC model for further development of ablation control.

Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain.

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF-TET1cd fusions, the SunTag-TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.

RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

Characteristics and processing of Pol IV-dependent transcripts in Arabidopsis.

RNAi is a highly conserved machinery in eukaryotes for transcriptional or post-transcriptional silencing. In plants, RNA-directed DNA Methylation (RdDM) pathway deploys small interfering RNAs (siRNAs) to target DNA methylation. This process is initiated by the plant-specific RNA polymerase IV (Pol IV) to produce RNA precursors that are later processed into siRNAs. Until recently, characterization of Pol IV transcript had been difficult due to its presumed rapid turnover. This review summarizes five recent reports on Pol IV-dependent RNAs that explore the biogenesis and features of Pol IV transcripts, as well as alternative dicer-independent processing of Pol IV products.

DNA methylome of the 20-gigabase Norway spruce genome.

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.

Differential regulation of nuclear factor-kappa B subunits on epidermal keratinocytes by ultraviolet B and tacrolimus.

Modulation of nuclear factor-kappa B (NF-κB) expression has important clinical implications including anti-inflammation. Recently, we have shown that direct regulation of NF-κB/p65 subunit may account for tacrolimus ointment's remarkable clinical efficacy on treating inflammatory dermatoses. However, NF-κB is a dimeric transcription factor formed by hetero- or homodimeration of the five Rel family proteins. The complete operational scheme of different NF-κB subunits remains obscure. It has been shown that homodimers consist of NF-κB/p50 may serve an inhibitory role in suppressing inflammation while dimers consisting of NF-κB/p65 activate inflammatory pathway. Our current study aimed to explore the effects of ultraviolet B (UVB) on epidermal keratinocytes in terms of specific NF-κB subunits NF-κB/p50 and NF-κB/p65. Additionally, the effects of tacrolimus on differential regulation of NF-κB subunits of UVB irradiated keratinocytes were also investigated. Our result showed that UVB sequentially regulated the activities of different subunits of NF-κB: the activity of NF-κB/p50 was downregulated in the early stage (6 hours), followed by upregulation of NF-κB/p65 in the later stage (12 hours). The results from immunofluorescence, immunocytochemical, and immunohistochemical analyses indicated that the nuclear expression of NF-κB/p50 could be seen constitutively while the nuclear expression of NF-κB/p65 could only be seen after UVB irradiation. Furthermore, treatment with tacrolimus didn't affect the nuclear activation and translocation of NF-κB/p50, while the UVB induced NF-κB/p65 nuclear expression was suppressed by tacrolimus. In summary, we have shown that UVB irradiation sequentially regulated different NF-κB subunits. The clinical efficacy of tacrolimus may be attributed to its specific regulatory effect on NF-κB/p65 but not NF-κB/p50 of the NF-κB pathway.

High-Glucose Environment Inhibits p38MAPK Signaling and Reduces Human ß-Defensin-3 Expression [corrected] in Keratinocytes.

Diabetes mellitus is characterized by elevated plasma glucose and increased rates of skin infections. Altered immune responses have been suggested to contribute to this prevalent complication, which involves microbial invasion. In this study we explored the effects of a high-glucose environment on the innate immunity of keratinocytes by focusing on ß defensin-3 (BD3) using in vivo and in vitro models. Our results demonstrated that the perilesional skins of diabetic rats failed to show enhanced BD3 expression after wounding. In addition, high-glucose treatment reduced human BD3 (hBD3) expression of cultured human keratinocytes. This pathogenic process involved inhibition of p38MAPK signaling, an event that resulted from increased formation of advanced glycation end products. On the other hand, toll-like receptor-2 expression and function of cultured keratinocytes were not significantly affected by high-glucose treatment. In summary, high-glucose conditions inhibited the BD3 expression of epidermal keratinocytes, which in turn contributed to the frequent occurrences of infection associated with diabetic wounding.