Flex Meta-Storms elucidates the microbiome local beta-diversity under specific phenotypes.

MOTIVATION: Beta-diversity quantitatively measures the difference among microbial communities thus enlightening the association between microbiome composition and environment properties or host phenotypes. The beta-diversity analysis mainly relies on distances among microbiomes that are calculated by all microbial features. However, in some cases, only a small fraction of members in a community plays crucial roles. Such a tiny proportion is insufficient to alter the overall distance, which is always missed by end-to-end comparison. On the other hand, beta-diversity pattern can also be interfered due to the data sparsity when only focusing on nonabundant microbes. RESULTS: Here, we develop Flex Meta-Storms (FMS) distance algorithm that implements the "local alignment" of microbiomes for the first time. Using a flexible extraction that considers the weighted phylogenetic and functional relations of microbes, FMS produces a normalized phylogenetic distance among members of interest for microbiome pairs. We demonstrated the advantage of FMS in detecting the subtle variations of microbiomes among different states using artificial and real datasets, which were neglected by regular distance metrics. Therefore, FMS effectively discriminates microbiomes with higher sensitivity and flexibility, thus contributing to in-depth comprehension of microbe-host interactions, as well as promoting the utilization of microbiome data such as disease screening and prediction. AVAILABILITY AND IMPLEMENTATION: FMS is implemented in C++, and the source code is released at https://github.com/qdu-bioinfo/flex-meta-storms.

Rare variations within the serine/arginine-rich splicing factor PtoRSZ21 modulate stomatal size to determine drought tolerance in Populus.

Rare variants contribute significantly to the 'missing heritability' of quantitative traits. The genome-wide characteristics of rare variants and their roles in environmental adaptation of woody plants remain unexplored. Utilizing genome-wide rare variant association study (RVAS), expression quantitative trait loci (eQTL) mapping, genetic transformation, and molecular experiments, we explored the impact of rare variants on stomatal morphology and drought adaptation in Populus. Through comparative analysis of five world-wide Populus species, we observed the influence of mutational bias and adaptive selection on the distribution of rare variants. RVAS identified 75 candidate genes correlated with stomatal size (SS)/stomatal density (SD), and a rare haplotype in the promoter of serine/arginine-rich splicing factor PtoRSZ21 emerged as the foremost association signal governing SS. As a positive regulator of drought tolerance, PtoRSZ21 can recruit the core splicing factor PtoU1-70K to regulate alternative splicing (AS) of PtoATG2b (autophagy-related 2). The rare haplotype PtoRSZ21hap2 weakens binding affinity to PtoMYB61, consequently affecting PtoRSZ21 expression and SS, ultimately resulting in differential distribution of Populus accessions in arid and humid climates. This study enhances the understanding of regulatory mechanisms that underlie AS induced by rare variants and might provide targets for drought-tolerant varieties breeding in Populus.

Suppression of circular RNA serum and glucocorticoid-induced kinase 1 elevates antioxidant molecules and angiogenesis in trophoblast cells to attenuate preeclampsia via microRNA-508-3p to target and restrain PUM homolog 1.

AIM: Preeclampsia (PE) is a pregnancy-specific syndrome characterized by hypertension and proteinuria. Recently, multiple circular RNAs (circRNAs) were considered latent clinical diagnostic markers or therapeutic targets. This study was to explore the impact of circRNA serum and glucocorticoid-induced kinase 1 (SGK1) on PE via influencing the microRNA (miR)-508-3p/PUM homolog 1 (PUM1) axis. METHODS: Placental tissues of 34 pregnant women with PE and 34 normal pregnant women were collected to measure circRNA SGK1 (circSGK1), miR-508-3p, and PUM1. Human placental trophoblasts HTR-8/SVneo were transfected with plasmids, thereafter to observe proliferation, migration, invasion, and apoptosis, analyze antioxidant molecules Troxerutin (TXN), Glutamate-cysteine ligase catalytic subunit (GCLC), NAD (P) H-quinone oxidoreductase 1 (NQO1), and determine angiogenesis. After the construction of the PE rat model, antioxidant molecules TXN, GCLC, and NQO1, vascular-associated factor vascular endothelial growth factor A (VEGF-A), and histopathological conditions were tested. Examination of the binding of circSGK1 and miR-508-3p with PUM1 was performed. RESULTS: Our data showed that circSGK1 expression was elevated in the placenta of patients with PE. Silenced circSGK1 or elevated miR-508-3p promoted the growth and antioxidant molecules and angiogenesis in trophoblast cells; CircSGK1 combined with miR-508-3p, and miR-508-3p targeted PUM1. CONCLUSIONS: In summary, suppression of circSGK1 augments antioxidant molecules and angiogenesis in trophoblast cells to attenuate PE via miR-508-3p to target PUM1.

Supramolecular Exosome Array for Efficient Capture and In Situ Detection of Protein Biomarkers.

Exosomes are an emerging source for disease biomarker discovery due to the high stability of proteins protected by phospholipid bilayers. However, liquid biopsy with exosomes remains challenging due to the extreme complexity of biological samples. Herein, we introduced an amphiphile-dendrimer supramolecular probe (ADSP) for the efficient capture and high-throughput analysis of exosomes, enabling the array-based assay for marker proteins. Amphiphilic amphotericin B was functionalized onto highly branched globular dendrimers, which can then insert into the exosome membrane efficiently, forming a supramolecular complex through multivalent interactions between the probe and the bilayer of exosomes. The ADSP can be easily coated onto magnetic beads or the nitrocellulose membrane, facilitating the capture of exosomes from a minimum amount of clinical samples. The captured exosomes can be detected with target protein antibodies via Western blotting or in a high-throughput array-based dot blotting format. This new strategy exhibited excellent extraction capability from trace body fluids with superior sensitivity (less than 1 µL plasma), good quantitation ability (R2 > 0.99), and high throughput (96 samples in one batch) using clinical plasma samples. The combination of proteomics and ADSP will provide a platform for the discovery and validation of protein biomarkers for cancer diagnosis and prognosis.

The influence of verapamil on the pharmacokinetics of the pan-HER tyrosine kinase inhibitor neratinib in rats: the role of P-glycoprotein-mediated efflux.

Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been approved for the treatment of HER2-positive (HER2+) early-stage and brain metastatic breast cancer. Thus far, the pharmacology effects and pharmacodynamics of neratinib have been well studied. However, the disposition of neratinib and its influencing factors in vivo remain unclear. P-glycoprotein (P-gp), one of the most extensively studied transporters, substantially restricts penetration of drugs into the body or deeper compartments (i.e., blood-brain barrier, BBB), regarding drug resistance and drug-drug interactions. Thereby, the aim of this study was to investigate the influence of verapamil (a P-gp inhibitor) on the pharmacokinetics of neratinib in rats. Here, we have established a high specific, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify plasma concentrations of neratinib in rats. Pharmacokinetic results showed that verapamil significantly increased the system exposure of neratinib, as Cmax increased by 2.09-fold and AUC0-t increased by 1.64-fold, respectively. Additionally, the in vitro transport of neratinib was evaluated using Madin-Darby canine kidney II (MDCK II) and human MDR1 gene overexpressed MDCK (MDCK-MDR1) cell line models. As a result, the net flux ratio was over than 2 and decreased over 50% by verapamil, suggesting that neratinib was a substrate of P-gp. Hence, our findings have highlighted the important role of P-gp in the system exposure of neratinib in vivo, and drug-drug interaction should be considered when coadministration of P-gp inhibitors with neratinib. These findings may support the further clinical development and application of neratinib.

Chemical Fuel Mediated Self-Regulatory Polymer Brushes for Autonomous Fluorescence Modulator and Wettability Switcher.

Synthetic systems of nonequilibrium self-assembly have made considerable progress; however, the achievement of innovative materials with self-regulated functions analogous to living systems remains a grand challenge. Herein, a versatile nonequilibrium system of polymer brushes with spatiotemporally programmable properties and functions driven by chemical fuels is reported. By combining a responsive polymer with an autonomous pH regulator, the polymer brushes self-regulate their swelling and deswelling process with tunable lifetimes. By using patterned copolymer brushes with pH-responsive fluorescence moiety, an autonomous fluorescence modulator is created that self-regulates its fluorescence in spatiotemporally programmable fashion driven by a chemical or an enzymatic reaction. Furthermore, a self-regulated wettability switcher of polymer brushes both in air and in an aqueous solution is implemented. The methodology and results in the work provide a useful avenue into the exploration of nonequilibrium synthetic materials with programmable functions and would accelerate the transformative developments of nonequilibrium materials and systems in practical applications.

Dynamic topology of double-stranded telomeric DNA studied by single-molecule manipulation in vitro.

The dynamic topological structure of telomeric DNA is closely related to its biological function; however, no such structural information on full-length telomeric DNA has been reported due to difficulties synthesizing long double-stranded telomeric DNA. Herein, we developed an EM-PCR and TA cloning-based approach to synthesize long-chain double-stranded tandem repeats of telomeric DNA. Using mechanical manipulation assays based on single-molecule atomic force microscopy, we found that mechanical force can trigger the melting of double-stranded telomeric DNA and the formation of higher-order structures (G-quadruplexes or i-motifs). Our results show that only when both the G-strand and C-strand of double-stranded telomeric DNA form higher-order structures (G-quadruplexes or i-motifs) at the same time (e.g. in the presence of 100 mM KCl under pH 4.7), that the higher-order structure(s) can remain after the external force is removed. The presence of monovalent K+, single-wall carbon nanotubes (SWCNTs), acidic conditions, or short G-rich fragments (∼30 nt) can shift the transition from dsDNA to higher-order structures. Our results provide a new way to regulate the topology of telomeric DNA.

HGF/c-MET pathway contributes to cisplatin-mediated PD-L1 expression in hepatocellular carcinoma.

Cisplatin has been reported to promote the expression of programmed cell death ligand-1 (PD-L1) in some cancer cells. However, the underlying mechanism through which PD-L1 is transcriptionally regulated by cisplatin in hepatocellular carcinoma (HCC) cells remains largely unknown. In the present study, we found that the expression of hepatocyte growth factor (HGF), p-Akt, p-ERK, and PD-L1 was increased in cisplatin-treated SNU-368 and SNU-739 cells. HGF stimulation also increased PD-L1 expression in these cells. Moreover, Inhibition of HGF/c-MET, PI3K/Akt, and MEK/ERK signaling pathways can dramatically block cisplatin or HGF-induced PD-L1 expression in SNU-368 and SNU-739 cells. In vivo, combination PHA665752 with cisplatin significantly reduced tumor weight with increased infiltration of CD8+ T cells in the tumor. Taken together, our study suggested that HGF/c-Met axis-induced the activation of PI3K/Akt and MEK/ERK pathways contributes to cisplatin-mediated PD-L1 expression. These findings may provide an alternative avenue for the treatment of HCC.

A Polymer with Mechanochemically Active Hidden Length.

Incorporating hidden length into polymer chains can improve their mechanical properties, because release of the hidden length under mechanical loads enables localized strain relief without chain fracture. To date, the design of hidden length has focused primarily on the choice of the sacrificial bonds holding the hidden length together. Here we demonstrate the advantages of adding mechanochemical reactivity to hidden length itself, using a new mechanophore that integrates (Z)-2,3-diphenylcyclobutene-1,4-dicarboxylate, with hitherto unknown mechanochemistry, into macrocyclic cinnamate dimers. Stretching a polymer of this mechanophore more than doubles the chain contour length without fracture. DFT calculations indicate that the sequential dissociation of the dimer, followed by cyclobutene isomerization at higher forces yields a chain fracture energy 11 times that of a simple polyester of the same initial contour length and preserves high energy-dissipating capacity up to ∼3 nN. In sonicated solutions cyclobutene isomerizes to two distinct products by competing reaction paths, validating the computed mechanochemical mechanism and suggesting an experimental approach to quantifying the distribution of single-chain forces under diverse loading scenarios.

Effects of Psoralen on Histone-DNA Interactions Studied by Using Atomic Force Microscopy.

The investigation of the DNA-histone interactions and factors that affect such interactions in the nucleosome is essential for understanding the role of chromatin organization in all cellular processes involved in the repair, transcription, and replication of the eukaryotic genome. As a kind of photosensitive molecule, psoralen (PSO) is used in the treatment of skin disease with ultraviolet light (PSO and ultra violet light, type A). The effect of treatment is remarkable, but the side effect is also obvious. PSO can be embedded in a 5' TA sequence in double-stranded DNA (dsDNA), and dsDNA is mainly wrapped around a histone octamer to form a nucleosome structure in human cells. Therefore, it is very necessary to explore the influence of PSO on DNA-histone interactions. To this end, the binding specificity and mode of DNA and histone in the presence or absence of PSO are investigated systematically. The results show that the presence of PSO (no matter if there is ultra violet light treatment) can increase the overall probability of histone binding to dsDNA while lowering the selectivity of histone binding to the specific DNA sequence in vitro. In addition, the increase of solution ionic strength can lower the ratio of histone binding to nonspecific DNA.

Nanomechanical Properties of a Supramolecular Helix Stabilized by Non-Covalent Interactions.

Supramolecular helices have unique properties and many potential applications, such as chiral separation and asymmetric catalysis. Mechanical property (stability) of the supramolecular helix plays important roles in their functions. Due to the limitation of detection method, it is quite challenging to investigate nanomechanical properties of individual supramolecular helices stabilized by pure supramolecular interactions. Here atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) is used to study the nanomechanical properties of a thermal-responsive supramolecular helix. The unwinding force plateau is observed in the force-extension curve, and the rupture force of the helix is dependent on the loading rate. In addition, the force-induced unwinding process is reversible and there is almost no energy dissipation in the process. Furthermore, the result of thermal shape-fluctuation analysis shows that the persistence length of the supramolecular helix is about 222 nm, which is much larger than helical structure formed by double-stranded DNA (dsDNA). However, because of its unique backbone structure, the supramolecular helix exhibits higher dynamic flexibility during force-induced deformation, since the persistence length determined from the stretching experiment is much smaller (1.1 nm).

Repression of Kisspeptin1 weakens hydrogen peroxide-caused injury in HTR8 cells via adjusting PI3K/AKT/mTOR pathway.

Kisspeptin1 (KISS1) is a tumor metastatic suppressor, and its increased expression is validated in human placenta trophoblast cells. Nonetheless, the actions of KISS1 in hydrogen peroxide (H2 O2 )-impaired human trophoblast HTR8 cells still remain imprecise. This research aims to uncover whether KISS1 can mitigate H2 O2 -triggered cell injury. HTR8 cells were pretreated with 250 µM H2 O2 for 4 hours; the autophagic markers (Beclin-1 and LC3B), cell viability, invasion and apoptosis were appraised. Real-time quantitative polymerase chain reaction and Western blot trials were enforced for the valuation of KISS1 mRNA and protein levels. After si-KISS1 transfection and 3-MA manipulation, the aforesaid biological processes were reassessed for ascertaining the influences of repressed KISS1 in H2 O2 -impaired HTR8 cells. Phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway was eventually estimated. H2 O2 enhanced Beclin-1 and LC3B expression, restricted cell viability, and invasion, and meanwhile caused apoptosis. The elevation of KISS1 evoked by H2 O2 was observed in HTR8 cells. In addition, silencing KISS1 was distinctly annulled the function of H2 O2 in HTR8 cells. Eventually, we observed that the repression of KISS1 triggered the activation of PI3K/AKT/mTOR in HTR8 cells under H2 O2 management. The diverting research unveiled that KISS1 repression eased H2 O2 -caused HTR8 cells injury via mediating PI3K/AKT/mTOR pathway.

Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit.

Single-molecule force spectroscopy is a powerful tool to directly measure protein-protein interactions (PPI). The high specificity and precision of PPI measurements made it possible to reveal detailed mechanisms of intermolecular interactions. However, protein aggregation due to specific or nonspecific interactions is among the most challenging problems in PPI examination. Here, we propose a strategy of a parallel DNA circuit to probe PPI using single-molecule magnetic tweezers. In contrast to PPI examination using atomic force microscopy, microspheres as probes used in magnetic tweezers avoided the single-probe issue of a cantilever. Negatively charged DNA as a linker circumvented the severe aggregation in the PPI construct with a protein linker. The unnatural amino acid encoded in proteins of interest expanded the choices of biorthogonal conjugation. We demonstrated how to apply our strategy to probe the PPI between the PHD3-Bromo and the histone H3 methylated at K4, a critical epigenetic event in leukemia development. We found a rupture force of 12 pN for breaking the PPI, which is much higher than that required to peel DNA off from a nucleosome, 3 pN. We expect that our methods will make PPI measurements of mechanics and kinetics with great precision, facilitating PPI-related research, e.g., PPI-targeted drug discovery.

Evidence that growth hormone can improve mitochondrial function in oocytes from aged mice.

Decline in successful conception decreases more rapidly after 38 years of age owing to follicular depletion and decreased oocyte quality. However, limited information is available regarding the underlying mechanism and the useful treatment. This study aimed to evaluate the effects of growth hormone supplementation on oocyte maturation in vivo in aged and young mice and to determine its effect on mitochondrial function. The influence of three different doses of recombinant human growth hormone (rhGH) (0.4, 0.8 and 1.6 mg/kg/day) for 8 weeks before ovarian stimulation was analyzed. Superovulated oocytes were released from the oviduct of 12-week-old and 40-week-old female C57BL/6J mice 14-16 h after administration of human chorionic gonadotropin. Ovarian follicle and morphological analysis and oocyte maturation parameters were then evaluated. This study is the first, to our knowledge, to report that medium- and high-dose rhGH significantly increases antral follicles in aged mice but anti-Müllerian hormone (AMH) levels. Furthermore, derived oocytes, MII-stage oocyte rate, ATP levels, mitochondrial membrane potential and frequencies of homogeneous mitochondrial distribution increased. In contrast, in both aged and young mice, the mtDNA copy numbers per oocyte were similar before rhGH administration, and upon saline administration, they did not differ significantly. We conclude that medium-dose rhGH supplementation before standard ovarian stimulation regimens improves oocyte quality in aged mice, probably by enhancing mitochondrial functionality.

Atomic Force Microscopy Imaging Study of Aligning DNA by Dumbbell-like Au-Fe3O4 Magnetic Nanoparticles.

Studies on nucleic acid structure and interactions between nucleic acid and its binding molecules are of great importance for understanding and controlling many important biological processes. Atomic force microscopy (AFM) imaging is one of the most efficient methods to disclose the DNA structure and binding modes between DNA and DNA-binding molecules. Long-chain DNA tends to form a random coiled structure, which prevents direct AFM imaging observation of the subtle structure formed by DNA itself or protein binding. Aligning DNA from the random coiled state into the extended state is not only important for applications in DNA nanotechnology but also for elucidating the interaction mechanism between DNA and other molecules. Here, we developed an efficient method based on the magnetic field to align long-chain DNA on a silicon surface. We used AFM imaging to study the alignment of DNA at the single-molecule level, showing that DNA can be stretched and highly aligned by the manipulation of magnetic nanoparticles tethered to one end of DNA and that the aligned DNA can be imaged clearly by AFM. In the absence of the magnetic field, the aligned DNA can relax back to a random coiled state upon rinsing. Such alignment and relaxation can be repeated many times, which provides an efficient method for the manipulation of individual DNA molecules and the investigation of DNA and DNA-binding molecule interactions.

Effect of Chain Conformation on the Single-Molecule Melting Force in Polymer Single Crystals: Steered Molecular Dynamics Simulations Study.

Understanding the relationship between polymer chain conformation as well as the chain composition within the single crystal and the mechanical properties of the corresponding single polymer chain will facilitate the rational design of high performance polymer materials. Here three model systems of polymer single crystals, namely poly(ethylene oxide) (PEO), polyethylene (PE), and nylon-66 (PA66) have been chosen to study the effects of chain conformation, helical (PEO) versus planar zigzag conformation (PE, PA66), and chain composition (PE versus PA66) on the mechanical properties of a single polymer chain. To do that, steered molecular dynamics simulations were performed on those polymer single crystals by pulling individual polymer chains out of the crystals. Our results show that the patterns of force-extension curve as well as the chain moving mode are closely related to the conformation of the polymer chain in the single crystal. In addition, hydrogen bonds can enhance greatly the force required to stretch the polymer chain out of the single crystal. The dynamic breaking and reformation of multivalent hydrogen bonds have been observed for the first time in PA66 at the single molecule level.

Single Molecule Study on Polymer-Nanoparticle Interactions: The Particle Shape Matters.

The study on the nanoparticle-polymer interactions is very important for the design/preparation of high performance polymer nanocomposite. Here we present a method to quantify the polymer-particle interaction at single molecule level by using AFM-based single molecule force spectroscopy (SMFS). As a proof-of-concept study, we choose poly-l-lysine (PLL) as the polymer and several different types of polyoxometalates (POM) as the model particles to construct several different polymer nanocomposites and to reveal the binding mode and quantify the binding strength in these systems. Our results reveal that the shape of the nanoparticle and the binding geometry in the composite have significantly influenced the binding strength of the PLL/POM complexes. Our dynamic force spectroscopy studies indicate that the disk-like geometry facilitate the unbinding of PLL/AlMo6 complexes in shearing mode, while the unzipping mode becomes dominate in spherical PLL-P8W48 system. We have also systematically investigated the effects of charge numbers, particle size, and ionic strength on the binding strength and binding mode of PLL/POM, respectively. Our results show that electrostatic interactions dominate the stability of PLL/POM complexes. These findings provide a way for tuning the mechanical properties of polyelectrolyte-nanoparticle composites.

Clinical application of next-generation sequencing in preimplantation genetic diagnosis cycles for Robertsonian and reciprocal translocations.

PURPOSE: The purpose of this study was to apply next-generation sequencing (NGS) technology to identify chromosomally normal embryos for transfer in preimplantation genetic diagnosis (PGD) cycles for translocations. METHODS: A total of 21 translocation couples with a history of infertility and repeated miscarriage presented at our PGD clinic for 24-chromosome embryo testing using copy number variation sequencing (CNV-Seq). RESULTS: Testing of 98 embryo samples identified 68 aneuploid (69.4 %) and 30 (30.6 %) euploid embryos. Among the aneuploid embryos, the most common abnormalities were segmental translocation imbalances, followed by whole autosomal trisomies and monosomies, segmental imbalances of non-translocation chromosomes, and mosaicism. In all unbalanced embryos resulting from reciprocal translocations, CNV-Seq precisely identified both segmental imbalances, extending from the predicted breakpoints to the chromosome termini. From the 21 PGD cycles, eight patients had all abnormal embryos and 13 patients had at least one normal/balanced and euploid embryo available for transfer. In nine intrauterine transfer cycles, seven healthy babies have been born. In four of the seven children tested at 18 weeks gestation, the karyotypes matched with the original PGD results. CONCLUSION: In clinical PGD translocation cycles, CNV-Seq displayed the hallmarks of a comprehensive diagnostic technology for high-resolution 24-chromosome testing of embryos, capable of identifying true euploid embryos for transfer.

Molecular analysis of DNA in blastocoele fluid using next-generation sequencing.

BACKGROUND: Preimplantation genetic testing (PGT) requires an invasive biopsy to obtain embryonic material for genetic analysis. The availability of a less invasive procedure would increase the overall efficacy of PGT. The aim of the study was to explore the potential of blastocoele fluid (BF) as an alternative source of embryonic DNA for PGT. METHODS: Collection of BF was performed by aspiration with a fine needle prior to vitrification. BF DNA was subjected to whole-genome amplification (WGA) and analyzed by high-resolution next-generation sequencing (NGS). RESULTS: A high-quality WGA product was obtained from 8 of 11 (72.7 %) samples. Comparison of matching BF and blastomere samples showed that the genomic representation of sequencing reads was consistently similar with respect to density and regional coverage across the 24 chromosomes. A genome-wide survey of the sample sequencing data also indicated that BF was highly representative of known single gene sequences, and this observation was validated by PCR analyses of ten randomly selected genes, with an overall efficiency of 84 %. CONCLUSION: This study provides further evidence that BF is a promising alternative source of DNA for PGT.

Stress signaling in response to polycyclic aromatic hydrocarbon exposure in Arabidopsis thaliana involves a nucleoside diphosphate kinase, NDPK-3.

MAIN CONCLUSION: The study is the first to reveal the proteomic response in plants to a single PAH stress, and indicates that NDPK3 is a positive regulator in the Arabidopsis response to phenanthrene stress. Polycyclic aromatic hydrocarbons (PAHs) are highly carcinogenic pollutants that are byproducts of carbon-based fuel combustion, and tend to persist in the environment for long periods of time. PAHs elicit complex, damaging responses in plants, and prior research at the physiological, biochemical, and transcriptional levels has indicated that reactive oxygen species (ROS) and oxidative stress play major roles in the PAH response. However, the proteomic response has remained largely unexplored. This study hypothesized that the proteomic response in Arabidopsis thaliana to phenanthrene, a model PAH, would include a strong oxidative stress signature, and would provide leads to potential signaling molecules involved. To explore that proteomic signature, we performed 2D-PAGE experiments and identified 30 proteins levels that were significantly altered including catalases (CAT), ascorbate peroxidase (APX), peroxiredoxins (POD), glutathione-S-transferase, and glutathione reductase. Also upregulated was nucleoside diphosphate kinase 3 (NDPK-3), a protein known to have metabolic and stress signaling functions. To address whether NDPK-3 functions upstream of the oxidative stress response, we measured levels of stress-responsive enzymes in NDPK-3 overexpressor, loss-of-function knockout, and wild-type plant lines. In the NDPK-3 overexpressor, the enzyme activities of APX, CAT, POD, as well as superoxide dismutase were all increased compared to wild type; in the NDPK-3 knockout line, these enzymes had reduced activity. This pattern occurred in untreated as well as phenanthrene-treated plants. These data support a model in which NDPK-3 is a positive regulator of the Arabidopsis stress response to PAHs.