Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei.

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

The Effect of Dads Matter-HV on Father Engagement in Home Visiting Services.

Engaging fathers early in child and family services has the potential to promote positive father contributions towards positive child development, improve family well-being, and enhance service outcomes over time. However, low father engagement in child and family services remains a persistent problem, and few interventions designed to improve father engagement in these services have been rigorously tested. The current study assesses the effect of a service enhancement intervention called Dads Matter-Home Visiting (Dads Matter-HV) on biological father engagement in home visiting services when compared to home visiting services delivered as usual. To assess the efficacy of the Dads Matter-HV intervention, the research team used a stratified cluster randomized clinical trial design with five agencies delivering early home visiting service programs. Seventeen teams across the five agencies were randomly assigned to either the control group condition (i.e., standard home visiting services as usual) or the intervention condition (i.e., Dads Matter-HV). Data were collected from a total of 204 families at baseline, 4 months postbaseline (92% retention rate), and 12 months postbaseline (84% retention rate). The results suggest that Dads Matter-HV increases biological father engagement for fathers who begin services in the postnatal period, but reduces engagement when services are initiated prenatally. Findings suggest some pathways through which the intervention effects engagement.

Evaluating the effect of curcumin on the metacestode of Taenia crassiceps.

Curcumin, a curcuminoid present in the rhizome of the plant Curcuma longa has multiple pharmacological effects including anticarcinogenic and anti-inflammatory properties. This work evaluates the anthelmintic effect of the curcumin molecule (98% pure) on Taenia crassiceps cysticerci viability in vitro. Cysticerci incubated in the presence of increasing concentrations of curcumin showed a dose-dependent mortality correlated with a significant increase in the production of reactive oxygen species and a partial inhibition of thioredoxin-glutathione reductase, the only disulfide reductase present in these parasites. At 500 µM curcumin, a 100% of cysticerci lethality was obtained after 2 h of treatment. These results suggest the curcumin-induced oxidative stress could be in the origin of the anthelminthic effect of curcumin. Mice with cysticerci were injected intraperitoneally with 20, 40, or 60 mM curcumin daily for 30 days. A decrease in the burden of cysticerci (46%) was observed with a 60 mM dose of curcumin, supporting this compound as a potential anthelmintic drug.

Overcoming bacteriophage insensitivity in Staphylococcus aureus using clindamycin and azithromycinat subinhibitory concentrations.

BACKGROUND: Staphylococcus aureus is a pathogen of major concern in both acute infections and chronic conditions such as chronic rhinosinusitis (CRS). Bacteriophage (phage) therapy has recently regained interest for its potential to treat infections caused by antibiotic resistant strains including Methicillin Resistant Staphylococcus aureus (MRSA). However, bacteria can adapt and become resistant to phages. The aim of this study is to determine the potential for antibiotics to overcome phage resistance. METHODS: The susceptibility of S. aureus clinical isolates (CIs) to phages J-Sa36, Sa83 and Sa87 alone or in combination with protein synthesis inhibitor (PSI) antibiotics clindamycin, azithromycin and erythromycin was assessed using plaque spot assays, minimum inhibitory concentration (MIC) assays, double layer spot assays and resazurin assays. The safety and efficacy of subinhibitory PSI antibiotics in combination with phage was tested in a Sprague Dawley rat model of sinusitis infected with a phage resistant S. aureus CI. RESULTS: All three antibiotics at subinhibitory concentrations showed synergy when combined with all 3 phages against S. aureus CIs in planktonic and biofilm form and could sensitize phage-resistant S. aureus to promote phage infection. The combination of topical subinhibitory clindamycin or azithromycin and phage was safe and could eradicate S. aureus sinonasal biofilms in vivo. CONCLUSION: Subinhibitory concentrations of PSI antibiotics could sensitize phage-resistant S. aureus and MRSA strains to phages in vitro and in vivo. This data supports the potential use of phage-PSI antibiotic combination therapies, in particular for difficult-to-treat infections with phage-resistant S. aureus and MRSA strains.

Fine capsule variation affects bacteriophage susceptibility in Klebsiella pneumoniae ST258.

Multidrug resistant (MDR) carbapenemase-producing (CP) Klebsiella pneumoniae, belonging to clonal group CG258, is capable of causing severe disease in humans and is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as therapeutic alternatives to antibiotics. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the genomes of 18 Kumoniae target strains and tested them against novel lytic bacteriophages (n = 65). The genomes of K pneumoniae carrying blaNDM and blaKPC were sequenced and CG258 isolates selected for bacteriophage susceptibility testing. The local K pneumoniae CG258 population was dominated by sequence type ST258 clade 1 (86%) with variations in capsular locus (cps) and prophage content. CG258-specific bacteriophages primarily targeted the capsule, but successful infection is also likely blocked in some by immunity conferred by existing prophages. Five tailed bacteriophages against K pneumoniae ST258 clade 1 were selected for further characterization. Our findings show that effective control of K pneumoniae CG258 with bacteriophage will require mixes of diverse lytic viruses targeting relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic bacteriophage candidates against this serious pathogen.

Acute peritoneal dialysis, complications and outcomes in 389 children with STEC-HUS: a multicenter experience.

BACKGROUND: Management of acute kidney injury (AKI) in children with hemolytic uremic syndrome induced by a Shiga toxin-producing Escherichia coli infection (STEC-HUS) is supportive; however, 40 to 60% of cases need kidney replacement therapy (KRT). The aim of this study was to analyze procedure complications, especially peritonitis, and clinical outcomes in children with AKI secondary to STEC-HUS treated with acute PD. METHODS: This is a multicenter retrospective study conducted among thirty-seven Argentinian centers. We reviewed medical records of 389 children with STEC-HUS hospitalized between January 2015 and February 2019 that required PD. RESULTS: Complications associated with PD were catheter malfunction (n = 93, 24%), peritonitis (n = 75, 19%), fluid leaks (n = 45, 11.5%), bleeding events (n = 23, 6%), and hyperglycemia (n = 8, 2%). In the multivariate analysis, the use of antibiotic prophylaxis was independently associated with a decreased risk of peritonitis (hazard ratio 0.49, IC 95% 0.29-0.81; p = 0.001), and open-surgery catheter insertion was independently associated with a higher risk (hazard ratio 2.8, IC 95% 1.21-6.82; p = 0.001). Discontinuation of PD due to peritonitis, severe leak, or mechanical complications occurred in 3.8% of patients. No patient needed to be transitioned to other modality of KRT due to inefficacy of the technique. Mortality during the acute phase occurred in 2.8% patients due to extrarenal complications (neurological and cardiac involvement), not related to PD. CONCLUSIONS: Acute PD was a safe and effective method to manage AKI in children with STEC-HUS. Prophylactic antibiotics prior to insertion of the PD catheter should be considered to decrease the incidence of peritonitis.

Bacteriophage AB-SA01 Cocktail in Combination with Antibiotics against MRSA-VISA Strain in an In Vitro Pharmacokinetic/Pharmacodynamic Model.

This study aimed to test the efficacy of bacteriophage-antibiotic combinations (BACs) in vitro in 24-h time-kill settings and in ex vivo simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic models for 96 h. BACs prevented the development of bacteriophage resistance, while some bacteriophage resistance emerged in bacteriophage-alone treatments. In addition, BACs resulted in an enhancement of bacterial eradication in SEV models. Our findings support the potential activity of BAC therapy for combating serious methicillin-resistant Staphylococcus aureus (MRSA) infections.

Bacteriophage-Antibiotic Combination Strategy: an Alternative against Methicillin-Resistant Phenotypes of Staphylococcus aureus.

Comparative time-kill experiments with Staphylococcus aureus bacteriophage (phage) Sb-1 alone and phage-antibiotic combinations (PACs) against two methicillin-resistant S. aureus (MRSA) strains have shown synergy with both daptomycin-phage and vancomycin-phage combinations. PACs prevented development of phage resistance and demonstrated bactericidal activity for all triple combinations. In addition, the extracellular membrane vesicle (MV) formation and the potential impact of phage on MV suppression were examined. Our results demonstrate the potential of PAC for combating MRSA infections.

Pharmacokinetics and Time-Kill Study of Inhaled Antipseudomonal Bacteriophage Therapy in Mice.

Inhaled bacteriophage (phage) therapy is a potential alternative to conventional antibiotic therapy to combat multidrug-resistant (MDR) Pseudomonas aeruginosa infections. However, pharmacokinetics (PK) and pharmacodynamics (PD) of phages are fundamentally different from antibiotics and the lack of understanding potentially limits optimal dosing. The aim of this study was to investigate the in vivo PK and PD profiles of antipseudomonal phage PEV31 delivered by pulmonary route in immune-suppressed mice. BALB/c mice were administered phage PEV31 at doses of 107 and 109 PFU by the intratracheal route. Mice (n = 4) were sacrificed at 0, 1, 2, 4, 8, and 24 h posttreatment and various tissues (lungs, kidney, spleen, and liver), bronchoalveolar lavage fluid, and blood were collected for phage quantification. In a separate study combining phage with bacteria, mice (n = 4) were treated with PEV31 (109 PFU) or phosphate-buffered saline (PBS) at 2 h postinoculation with MDR P. aeruginosa Infective PEV31 and bacteria were enumerated from the lungs. In the phage-only study, the PEV31 titer gradually decreased in the lungs over 24 h, with a half-life of approximately 8 h for both doses. In the presence of bacteria, in contrast, the PEV31 titer increased by almost 2-log10 in the lungs at 16 h. Furthermore, bacterial growth was suppressed in the PEV31-treated group, while the PBS-treated group showed exponential growth. Of the 10 colonies tested, four phage-resistant isolates were observed from the lung homogenates sampled at 24 h after phage treatment. These colonies had a different antibiogram to the parent bacteria. This study provides evidence that pulmonary delivery of phage PEV31 in mice can reduce the MDR bacterial burden.

Bacteriophage-Antibiotic Combinations for Enterococcus faecium with Varying Bacteriophage and Daptomycin Susceptibilities.

Concerns regarding increased prevalence of daptomycin (DAP)-resistant strains necessitate novel therapies for Enterococcus faecium infections. Obligately lytic bacteriophages are viruses that target, infect, and kill bacterial cells. Limited studies have evaluated phage-antibiotic combinations against E. faecium After an initial screen of eight E. faecium strains, three strains with varying DAP/phage susceptibilities were selected for further experiments. Phage-to-strain specificity contributed to synergy with antibiotics by time-kill analyses and was associated with lower development of phage resistance.

Comparative transcriptome analysis suggests convergent evolution of desiccation tolerance in Selaginella species.

BACKGROUND: Desiccation tolerant Selaginella species evolved to survive extreme environmental conditions. Studies to determine the mechanisms involved in the acquisition of desiccation tolerance (DT) have focused on only a few Selaginella species. Due to the large diversity in morphology and the wide range of responses to desiccation within the genus, the understanding of the molecular basis of DT in Selaginella species is still limited. RESULTS: Here we present a reference transcriptome for the desiccation tolerant species S. sellowii and the desiccation sensitive species S. denticulata. The analysis also included transcriptome data for the well-studied S. lepidophylla (desiccation tolerant), in order to identify DT mechanisms that are independent of morphological adaptations. We used a comparative approach to discriminate between DT responses and the common water loss response in Selaginella species. Predicted proteomes show strong homology, but most of the desiccation responsive genes differ between species. Despite such differences, functional analysis revealed that tolerant species with different morphologies employ similar mechanisms to survive desiccation. Significant functions involved in DT and shared by both tolerant species included induction of antioxidant systems, amino acid and secondary metabolism, whereas species-specific responses included cell wall modification and carbohydrate metabolism. CONCLUSIONS: Reference transcriptomes generated in this work represent a valuable resource to study Selaginella biology and plant evolution in relation to DT. Our results provide evidence of convergent evolution of S. sellowii and S. lepidophylla due to the different gene sets that underwent selection to acquire DT.

Efficacy of phage cocktail AB-SA01 therapy in diabetic mouse wound infections caused by multidrug-resistant Staphylococcus aureus.

BACKGROUND: Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus. Antibiotic-resistant Staphylococcus aureus is frequently isolated from DFU infections. Bacteriophages (phages) represent an alternative or adjunct treatment to antibiotic therapy. Here we describe the efficacy of AB-SA01, a cocktail of three S. aureus Myoviridae phages, made to current good manufacturing practice (cGMP) standards, and which has undergone two phase I clinical trials, in treatment of multidrug-resistant (MDR) S. aureus infections. RESULTS: Wounds of saline-treated mice showed no healing, but expanded and became inflamed, ulcerated, and suppurating. In contrast, AB-SA01 treatment decreased the bacterial load with efficacy similar or superior to vancomycin treatment. At the end of the treatment period, there was a significant decrease (p < 0.001) in bacterial load and wound size in infected phage- and vancomycin-treated groups compared with infected saline-treated mice. In phage-treated mice, wound healing was seen similar to vancomycin treatment. No mortality was recorded associated with infections, and post-mortem examinations did not show any evident pathological lesions other than the skin wounds. No adverse effects related to the application of phages were observed. CONCLUSION: Topical application of phage cocktail AB-SA01 is effective, as shown by bacterial load reduction and wound closure, in the treatment of diabetic wound infections caused by MDR S. aureus. Our results suggest that topical phage cocktail treatment may be effective in treating antibiotic-resistant S. aureus DFU infections.

In Silico Selection and In Vitro Evaluation of New Molecules That Inhibit the Adhesion of Streptococcus mutants through Antigen I/II.

Streptococcus mutans is the main early colonizing cariogenic bacteria because it recognizes salivary pellicle receptors. The Antigen I/II (Ag I/II) of S. mutans is among the most important adhesins in this process, and is involved in the adhesion to the tooth surface and the bacterial co-aggregation in the early stage of biofilm formation. However, this protein has not been used as a target in a virtual strategy search for inhibitors. Based on the predicted binding affinities, drug-like properties and toxicity, molecules were selected and evaluated for their ability to reduce S. mutans adhesion. A virtual screening of 883,551 molecules was conducted; cytotoxicity analysis on fibroblast cells, S. mutans adhesion studies, scanning electron microscopy analysis for bacterial integrity and molecular dynamics simulation were also performed. We found three molecules ZINC19835187 (ZI-187), ZINC19924939 (ZI-939) and ZINC19924906 (ZI-906) without cytotoxic activity, which inhibited about 90% the adhesion of S. mutans to polystyrene microplates. Molecular dynamic simulation by 300 nanoseconds showed stability of the interaction between ZI-187 and Ag I/II (PDB: 3IPK). This work provides new molecules that targets Ag I/II and have the capacity to inhibit in vitro the S. mutans adhesion on polystyrene microplates.

Early clinical experience of bacteriophage therapy in 3 lung transplant recipients.

Bacteriophage therapy (BT) uses bacteriophages to treat pathogenic bacteria and is an emerging strategy against multidrug-resistant (MDR) infections. Experience in solid organ transplant is limited. We describe BT in 3 lung transplant recipients (LTR) with life-threatening MDR infections caused by Pseudomonas aeruginosa (n = 2) and Burkholderia dolosa (n = 1). For each patient, lytic bacteriophages were selected against their bacterial isolates. BT was administered for variable durations under emergency Investigational New Drug applications and with patient informed consent. Safety was assessed using clinical/laboratory parameters and observed clinical improvements described, as appropriate. All patients received concurrent antibiotics. Two ventilator-dependent LTR with large airway complications and refractory MDR P. aeruginosa pneumonia received BT. Both responded clinically and were discharged from the hospital off ventilator support. A third patient had recurrent B. dolosa infection following transplant. Following BT initiation, consolidative opacities improved and ventilator weaning was begun. However, infection relapsed on BT and the patient died. No BT-related adverse events were identified in the 3 cases. BT was well tolerated and associated with clinical improvement in LTRs with MDR bacterial infection not responsive to antibiotics alone. BT may be a viable adjunct to antibiotics for patients with MDR infections.

High Resolution Nanoscale Probing of Bacteriophages in an Inhalable Dry Powder Formulation for Pulmonary Infections.

Use of powder phage formulations for the treatment of multiple-drug-resistant pulmonary infections is gaining attention. To achieve therapeutic benefits, it is critical for phages to remain stable in the formulation. Assessment of phage stability relies on plaque assay (bioactivity), which requires powder samples to be reconstituted in liquid. The purpose of this study was to develop an innovative approach using photothermal-induced resonance-enhanced atomic force microscopy infrared spectroscopy (AFM-IR) to assess the presence of phages and investigate their protein conformation in the solid state. Staphylococcal phage S83 was spray-dried with lactose and sodium stearate using spray-drying. The phage powder recrystallized at 60% relative humidity (RH), so it was stored and handled below this RH. For the AFM-IR measurements, spray-dried Staphylococcal phage Sa83 powder was embedded in resin, followed by microtome sectioning. AFM-IR spectra collected from different regions within the microtomed sections revealed the presence of phage proteins with amide I and amide II bands at 1640 and 1550 cm-1, respectively. The phages were confirmed to be stable, as the plaque assay showed negligible titer reduction after spray-drying. Our results thus demonstrated the utility of AFM-IR for characterization of nanosized phages present in extremely low quantity in spray-dried particles. These biologically active phages were shown to retain their physical and chemical integrity in the spray-dried particles.

Successful adjunctive use of bacteriophage therapy for treatment of multidrug-resistant Pseudomonas aeruginosa infection in a cystic fibrosis patient.

INTRODUCTION: We describe the use of bacteriophage therapy in a 26-year-old cystic fibrosis (CF) patient awaiting lung transplantation. HOSPITAL COURSE: The patient developed multidrug resistant (MDR) Pseudomonas aeruginosa pneumonia, persistent respiratory failure, and colistin-induced renal failure. We describe the use of intravenous bacteriophage therapy (BT) along with systemic antibiotics in this patient, lack of adverse events, and clinical resolution of infection with this approach. She did not have recurrence of pseudomonal pneumonia and CF exacerbation within 100 days following the end of BT and underwent successful bilateral lung transplantation 9 months later. CONCLUSION: Given the concern for MDR P. aeruginosa infections in CF patients, BT may offer a viable anti-infective adjunct to traditional antibiotic therapy.

CNNs for automatic glaucoma assessment using fundus images: an extensive validation.

BACKGROUND: Most current algorithms for automatic glaucoma assessment using fundus images rely on handcrafted features based on segmentation, which are affected by the performance of the chosen segmentation method and the extracted features. Among other characteristics, convolutional neural networks (CNNs) are known because of their ability to learn highly discriminative features from raw pixel intensities. METHODS: In this paper, we employed five different ImageNet-trained models (VGG16, VGG19, InceptionV3, ResNet50 and Xception) for automatic glaucoma assessment using fundus images. Results from an extensive validation using cross-validation and cross-testing strategies were compared with previous works in the literature. RESULTS: Using five public databases (1707 images), an average AUC of 0.9605 with a 95% confidence interval of 95.92-97.07%, an average specificity of 0.8580 and an average sensitivity of 0.9346 were obtained after using the Xception architecture, significantly improving the performance of other state-of-the-art works. Moreover, a new clinical database, ACRIMA, has been made publicly available, containing 705 labelled images. It is composed of 396 glaucomatous images and 309 normal images, which means, the largest public database for glaucoma diagnosis. The high specificity and sensitivity obtained from the proposed approach are supported by an extensive validation using not only the cross-validation strategy but also the cross-testing validation on, to the best of the authors' knowledge, all publicly available glaucoma-labelled databases. CONCLUSIONS: These results suggest that using ImageNet-trained models is a robust alternative for automatic glaucoma screening system. All images, CNN weights and software used to fine-tune and test the five CNNs are publicly available, which could be used as a testbed for further comparisons.

Regulatory network analysis reveals novel regulators of seed desiccation tolerance in Arabidopsis thaliana.

Desiccation tolerance (DT) is a remarkable process that allows seeds in the dry state to remain viable for long periods of time that in some instances exceed 1,000 y. It has been postulated that seed DT evolved by rewiring the regulatory and signaling networks that controlled vegetative DT, which itself emerged as a crucial adaptive trait of early land plants. Understanding the networks that regulate seed desiccation tolerance in model plant systems would provide the tools to understand an evolutionary process that played a crucial role in the diversification of flowering plants. In this work, we used an integrated approach that included genomics, bioinformatics, metabolomics, and molecular genetics to identify and validate molecular networks that control the acquisition of DT in Arabidopsis seeds. Two DT-specific transcriptional subnetworks were identified related to storage of reserve compounds and cellular protection mechanisms that act downstream of the embryo development master regulators LEAFY COTYLEDON 1 and 2, FUSCA 3, and ABSCICIC ACID INSENSITIVE 3. Among the transcription factors identified as major nodes in the DT regulatory subnetworks, PLATZ1, PLATZ2, and AGL67 were confirmed by knockout mutants and overexpression in a desiccation-intolerant mutant background to play an important role in seed DT. Additionally, we found that constitutive expression of PLATZ1 in WT plants confers partial DT in vegetative tissues.

Proof-of-Principle Study in a Murine Lung Infection Model of Antipseudomonal Activity of Phage PEV20 in a Dry-Powder Formulation.

Bacteriophage therapy is a promising alternative treatment to antibiotics, as it has been documented to be efficacious against multidrug-resistant bacteria with minimal side effects. Several groups have demonstrated the efficacy of phage suspension in vivo to treat lung infections using intranasal delivery; however, phage dry-powder administration to the lungs has not yet been explored. Powder formulations provide potential advantages over a liquid formulation, including easy storage, transport, and administration. The purpose of this study was to assess the bactericidal activities of phage dry-powder formulations against multidrug-resistant (MDR) strain Pseudomonas aeruginosa FADDI-PA001 in a mouse lung infection model. Phage PEV20 spray dried with lactose and leucine produced an inhalable powder at a concentration of 2 × 107 PFU/mg. P. aeruginosa lung infection was established by intratracheal administration of the bacterial suspension to neutropenic mice. At 2 h after the bacterial challenge, the infected mice were treated with 2 mg of the phage powder using a dry-powder insufflator. At 24 h after the phage treatment, the bacterial load in the lungs was decreased by 5.3 log10 (P < 0.0005) in the phage-treated group compared with that in the nontreated group. Additionally, the phage concentration in the lungs was increased by 1 log10 at 24 h in the treated group. These results demonstrate the feasibility of a pulmonary delivery of phage PEV20 dry-powder formulation for the treatment of lung infection caused by antibiotic-resistant P. aeruginosa.

Methylome analysis reveals an important role for epigenetic changes in the regulation of the Arabidopsis response to phosphate starvation.

Phosphate (Pi) availability is a significant limiting factor for plant growth and productivity in both natural and agricultural systems. To cope with such limiting conditions, plants have evolved a myriad of developmental and biochemical strategies to enhance the efficiency of Pi acquisition and assimilation to avoid nutrient starvation. In the past decade, these responses have been studied in detail at the level of gene expression; however, the possible epigenetic components modulating plant Pi starvation responses have not been thoroughly investigated. Here, we report that an extensive remodeling of global DNA methylation occurs in Arabidopsis plants exposed to low Pi availability, and in many instances, this effect is related to changes in gene expression. Modifications in methylation patterns within genic regions were often associated with transcriptional activation or repression, revealing the important role of dynamic methylation changes in modulating the expression of genes in response to Pi starvation. Moreover, Arabidopsis mutants affected in DNA methylation showed that changes in DNA methylation patterns are required for the accurate regulation of a number of Pi-starvation-responsive genes and that DNA methylation is necessary to establish proper morphological and physiological phosphate starvation responses.