Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.

CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat.

PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.

Whole-genome assembly of Klebsiella pneumoniae coproducing NDM-1 and OXA-232 carbapenemases using single-molecule, real-time sequencing.

The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the blaNDM-1, armA, and qnrB1 genes, along with tellurium and mercury resistance operons. blaNDM-1 is carried on a unique structure in which Tn125 is further bracketed by IS26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including blaCTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying blaOXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies.

Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs.

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio's previous "no-amp targeted sequencing utilizing the CRISPR-Cas9 system." • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription.

BACKGROUND: Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis. RESULTS: Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template. CONCLUSIONS: Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.

Direct growth of nano-worm-like Cu2S on copper mesh as a hierarchical 3D catalyst for Fenton-like degradation of an imidazolium room-temperature ionic liquid in water.

The increasing consumption of room-temperature ionic liquids (RTILs) inevitably releases RTILs into the water environment, posing serious threats to aquatic ecology due to the toxicities of RTILs. Thus, urgent needs are necessitated for developing useful processes for removing RTILs from water, and 1-butyl-3-methylimidazolium chloride (C4mimCl), the most common RTIL, would be the most representative RTIL for studying the removal of RTILs from water. As advanced oxidation processes with hydrogen peroxide (HP) are validated as useful approaches for eliminating emerging contaminants, developing advantageous heterogeneous catalysts for activating HP is the key to the successful degradation of C4mim. Herein, a hierarchical structure is fabricated by growing Cu2S on copper mesh (CSCM) utilizing CM as a Cu source. Compared to its precursor, CuO@CM, this CSCM exhibited tremendously higher catalytic activity for catalyzing HP to degrade C4mim efficiently because CSCM exhibits much more superior electrochemical properties and reactive sites, allowing CSCM to degrade C4mim rapidly. CSCM also exhibits a smaller Ea of C4mim elimination than all values in the literature. CSCM also shows a high capacity and stability for activating HP to degrade C4mim in the presence of NaCl and seawater. Besides, the mechanistic investigation of C4mim elimination by CSCM-activated HP has also been clarified and ascribed to OH and 1O2. The elimination route could also be examined and disclosed in detail through the quantum computational chemistry, confirming that CSCM is a useful catalyst for catalyzing HP to degrade RTILs.

Identification of a CCG-Enriched Expanded Allele in Patients with Myotonic Dystrophy Type 1 Using Amplification-Free Long-Read Sequencing.

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1.

Modulating Direct Growth of Copper Cobaltite Nanostructure on Copper Mesh as a Hierarchical Catalyst of Oxone Activation for Efficient Elimination of Azo Toxicant.

As cobalt (Co) has been the most useful element for activating Oxone to generate SO4•-, this study aims to develop a hierarchical catalyst with nanoscale functionality and macroscale convenience by decorating nanoscale Co-based oxides on macroscale supports. Specifically, a facile protocol is proposed by utilizing Cu mesh itself as a Cu source for fabricating CuCo2O4 on Cu mesh. By changing the dosages of the Co precursor and carbamide, various nanostructures of CuCo2O4 grown on a Cu mesh can be afforded, including nanoscale needles, flowers, and sheets. Even though the Cu mesh itself can be also transformed to a Cu-Oxide mesh, the growth of CuCo2O4 on the Cu mesh significantly improves its physical, chemical, and electrochemical properties, making these CuCo2O4@Cu meshes much more superior catalysts for activating Oxone to degrade the Azo toxicant, Acid Red 27. More interestingly, the flower-like CuCo2O4@Cu mesh exhibits a higher specific surface area and more superior electrochemical performance, enabling the flower-like CuCo2O4@Cu mesh to show the highest catalytic activity for Oxone activation to degrade Acid Red 27. The flower-like CuCo2O4@Cu mesh also exhibits a much lower Ea of Acid Red 27 degradation than the reported catalysts. These results demonstrate that CuCo2O4@Cu meshes are advantageous heterogeneous catalysts for Oxone activation, and especially, the flower-like CuCo2O4@Cu mesh appears as the most effective CuCo2O4@Cu mesh to eliminate the toxic Acid Red 27.

ATTCT and ATTCC repeat expansions in the ATXN10 gene affect disease penetrance of spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal-dominant disorder caused by an expanded pentanucleotide repeat in the ATXN10 gene. This repeat expansion, when fully penetrant, has a size of 850-4,500 repeats. It has been shown that the repeat composition can be a modifier of disease, e.g., seizures. Here, we describe a Mexican kindred in which we identified both pure (ATTCT)n and mixed (ATTCT)n-(ATTCC)n expansions in the same family. We used amplification-free targeted sequencing and optical genome mapping to decipher the composition of these repeat expansions. We found a considerable degree of mosaicism of the repeat expansion. This mosaicism was confirmed in skin fibroblasts from individuals with ATXN10 expansions with RNAScope in situ hybridization. All affected family members with the mixed ATXN10 repeat expansion showed typical clinical signs of spinocerebellar ataxia and epilepsy. In contrast, individuals with the pure ATXN10 expansion present with Parkinson's disease or are unaffected, even in individuals more than 20 years older than the average age at onset for SCA10. Our findings suggest that the pure (ATTCT)n expansion is non-pathogenic, while repeat interruptions, e.g., (ATTCC)n, are necessary to cause SCA10. This mechanism has been recently described for several other repeat expansions including SCA31 (BEAN1), SCA37 (DAB1), and three loci for benign adult familial myoclonic epilepsy BAFME (SAMD12, TNRC6A, RAPGEF2). Therefore, long-read sequencing and optical genome mapping of the entire genomic structure of repeat expansions are critical for clinical practice and genetic counseling, as variations in the repeat can affect disease penetrance, symptoms, and disease trajectory.

Hierarchical ZIF-decorated nanoflower-covered 3-dimensional foam for enhanced catalytic reduction of nitrogen-containing contaminants.

Metal Organic Frameworks (MOFs) represent a promising class of metallic catalysts for reduction of nitrogen-containing contaminants (NCCs), such as 4-nitrophenol (4-NP). Nevertheless, most researches involving MOFs for 4-NP reduction employ noble metals in the form of fine powders, making these powdered noble metal-based MOFs impractical and inconvenient for realistic applications. Thus, it would be critical to develop non-noble-metal MOFs which can be incorporated into macroscale and porous supports for convenient applications. Herein, the present study proposes to develop a composite material which combines advantageous features of macroscale/porous supports, and nanoscale functionality of MOFs. In particular, copper foam (CF) is selected as a macroscale porous medium, which is covered by nanoflower-structured CoO to increase surfaces for growing a cobaltic MOF, ZIF-67. The resultant composite comprises of CF covered by CoO nanoflowers decorated with ZIF-67 to form a hierarchical 3D-structured catalyst, enabling this ZIF-67@Cu foam (ZIF@CF) a promising catalyst for reducing 4-NP, and other NCCs. Thus, ZIF@CF can readily reduce 4-NP to 4-AP with a significantly lower Ea of 20 kJ/mol than reported values. ZIF@CF could be reused over 10 cycles and remain highly effective for 4-NP reduction. ZIF@CF also efficiently reduces other NCCs, such as 2-nitrophenol, 3-nitrophenol, methylene blue, and methyl orange. ZIF@CF can be adopted as catalytic filters to enable filtration-type reduction of NCCs by passing NCC solutions through ZIF@CF to promptly and conveniently reduce NCCs. The versatile and advantageous catalytic activity of ZIF@CF validates that ZIF@CF is a promising and practical heterogeneous catalyst for reductive treatments of NCCs.

Metal organic framework-derived 3D nanostructured cobalt oxide as an effective catalyst for soot oxidation.

While Co3O4 represents one of the most promising catalysts for soot oxidation, conventional Co3O4 nanoparticles (NPs) tend to aggregate, losing their activities. Herein, an alternative approach is proposed for preparing three-dimensional nanostructured Co3O4 (NSCo) using the hierarchically-structured Co-based Metal Organic Frameworks as a precursor. Specifically, ZIF-67 is chosen as the precursor as ZIF-67 can be conveniently synthesized with high yields and it can be easily converted to NSCo via calcination. The resulting NSCo exhibits a unique morphology which enables NSCo to possess more porosities and surface areas than the typical Co3O4 NPs. Consequently, NSCo shows a much higher catalytic activity than the typical Co3O4 NPs for soot oxidation because of superior textural properties of NSCo. Besides, when the soot oxidation by the typical Co3O4 NPs produced a significant amount of unwanted CO, soot can be completely combusted into CO2 using NSCo. In comparison with other reported Co-related catalysts, NSCo also achieves a higher soot oxidation efficiency (100% conversion) at lower temperatures with Tig of 331 °C. NSCo can be reused over many continuous cycles and still retains its catalytic activities. These features validate that NSCo is an easy-to-prepare 3D nanostructured Co3O4 catalyst, which possesses advantageous capabilities for soot oxidation at lower temperatures.

Correction: Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10.

[This corrects the article DOI: 10.1371/journal.pone.0228789.].

Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10.

Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.

Highly accurate long-read HiFi sequencing data for five complex genomes.

The PacBio® HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10-25 kb and accuracies greater than 99.5%. These accurate long reads can be used to improve results for complex applications such as single nucleotide and structural variant detection, genome assembly, assembly of difficult polyploid or highly repetitive genomes, and assembly of metagenomes. Currently, there is a need for sample data sets to both evaluate the benefits of these long accurate reads as well as for development of bioinformatic tools including genome assemblers, variant callers, and haplotyping algorithms. We present deep coverage HiFi datasets for five complex samples including the two inbred model genomes Mus musculus and Zea mays, as well as two complex genomes, octoploid Fragaria × ananassa and the diploid anuran Rana muscosa. Additionally, we release sequence data from a mock metagenome community. The datasets reported here can be used without restriction to develop new algorithms and explore complex genome structure and evolution. Data were generated on the PacBio Sequel II System.

Sex chromosome evolution in parasitic nematodes of humans.

Sex determination mechanisms often differ even between related species yet the evolution of sex chromosomes remains poorly understood in all but a few model organisms. Some nematodes such as Caenorhabditis elegans have an XO sex determination system while others, such as the filarial parasite Brugia malayi, have an XY mechanism. We present a complete B. malayi genome assembly and define Nigon elements shared with C. elegans, which we then map to the genomes of other filarial species and more distantly related nematodes. We find a remarkable plasticity in sex chromosome evolution with several distinct cases of neo-X and neo-Y formation, X-added regions, and conversion of autosomes to sex chromosomes from which we propose a model of chromosome evolution across different nematode clades. The phylum Nematoda offers a new and innovative system for gaining a deeper understanding of sex chromosome evolution.

Nearly Complete Genome Sequence of Brugia malayi Strain FR3.

Lymphatic filariasis affects ∼120 million people and can result in elephantiasis and hydrocele. Here, we report the nearly complete genome sequence of the best-studied causative agent of lymphatic filariasis, Brugia malayi The assembly contains four autosomes, an X chromosome, and only eight gaps but lacks a contiguous sequence for the known Y chromosome.

Site-specific labeling of T7 DNA polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms.

Like most enzymes, DNA polymerases undergo a large conformational change on the binding of a correct nucleotide. To determine how the conformational change contributes to substrate specificity, we labeled the T7 DNA polymerase with a conformationally sensitive fluorophore at a position that provides a signal coincident with structural changes following nucleotide binding and distinguishes correct base pairs from incorrect ones by the sign of the fluorescence change. Here we describe methods to document that only one site on the polymerase was labeled with the fluorophore based on mass spectral analysis of tryptic peptides. In addition, we show by equilibrium titrations of opposing signals that mismatches and correct bases compete for the same site. This analysis forms an essential basis for characterization of a fluorescently labeled enzyme intended for mechanistic studies. Finally, we show that the labeled enzyme can be used to identify single-nucleotide mutations in a procedure that could be automated.

Regulation of shear-induced nuclear translocation of the Nrf2 transcription factor in endothelial cells.

BACKGROUND: Vascular endothelial cells (ECs) constantly experience fluid shear stresses generated by blood flow. Laminar flow is known to produce atheroprotective effects on ECs. Nrf2 is a transcription factor that is essential for the antioxidant response element (ARE)-mediated induction of genes such as heme-oxygenase 1 (HO-1). We previously showed that fluid shear stress increases intracellular reactive oxygen species (ROS) in ECs. Moreover, oxidants are known to stimulate Nrf2. We thus examined the regulation of Nrf2 in cultured human ECs by shear stress. RESULTS: Exposure of human umbilical vein endothelial cells (HUVECs) to laminar shear stress (12 dyne/cm2) induced Nrf2 nuclear translocation, which was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, a protein kinase C (PKC) inhibitor, and an antioxidant agent N-acetyl cysteine (NAC), but not by other protein kinase inhibitors. Therefore, PI3K, PKC, and ROS are involved in the signaling pathway that leads to the shear-induced nuclear translocation of Nrf2. We also found that shear stress increased the ARE-binding activity of Nrf2 and the downstream expression of HO-1. CONCLUSION: Our data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system.