A calmodulin-gated calcium channel links pathogen patterns to plant immunity.

Pathogen-associated molecular patterns (PAMPs) activate innate immunity in both animals and plants. Although calcium has long been recognized as an essential signal for PAMP-triggered immunity in plants, the mechanism of PAMP-induced calcium signalling remains unknown1,2. Here we report that calcium nutrient status is critical for calcium-dependent PAMP-triggered immunity in plants. When calcium supply is sufficient, two genes that encode cyclic nucleotide-gated channel (CNGC) proteins, CNGC2 and CNGC4, are essential for PAMP-induced calcium signalling in Arabidopsis3-7. In a reconstitution system, we find that the CNGC2 and CNGC4 proteins together-but neither alone-assemble into a functional calcium channel that is blocked by calmodulin in the resting state. Upon pathogen attack, the channel is phosphorylated and activated by the effector kinase BOTRYTIS-INDUCED KINASE1 (BIK1) of the pattern-recognition receptor complex, and this triggers an increase in the concentration of cytosolic calcium8-10. The CNGC-mediated calcium entry thus provides a critical link between the pattern-recognition receptor complex and calcium-dependent immunity programs in the PAMP-triggered immunity signalling pathway in plants.

A New Methodology for High Spatiotemporal Resolution Measurements of Air Volatile Organic Compounds: From Sampling to Data Deconvolution.

Monitoring of volatile organic compounds (VOCs) in air is crucial for understanding their atmospheric impacts and advancing their emission reduction plans. This study presents an innovative integrated methodology suitable for achieving semireal-time high spatiotemporal resolution three-dimensional measurements of VOCs from ground to hundreds of meters above ground. The methodology integrates an active AirCore sampler, custom-designed for deployment from unmanned aerial vehicles (UAV), a proton-transfer-reaction mass spectrometry (PTR-MS) for sample analysis, and a data deconvolution algorithm for improved time resolution for measurements of multiple VOCs in air. The application of the deconvolution technique significantly improves the signal strength of data from PTR-MS analysis of AirCore samples and enhances their temporal resolution by 4 to 8 times to 4-11 s. A case study demonstrates that the methodology can achieve sample collection and analysis of VOCs within 45 min, resulting in >120-360 spatially resolved data points for each VOC measured and achieving a horizontal resolution of 20-55 m at a UAV flight speed of 5 m/s and a vertical resolution of 5 m. This methodology presents new possibilities for acquiring 3-dimensional spatial distributions of VOC concentrations, effectively tackling the longstanding challenge of characterizing three-dimensional VOC distributions in the lowest portion of the atmospheric boundary layer.

Unveiling the absorption, translocation, and metabolism of penthiopyrad in pakchoi under hydroponic and soil-cultivated conditions.

The efficient use of pesticides has long been a topic of public concern, necessitating a thorough understanding of their movement in plants. This study investigates the translocation and distribution of penthiopyrad in pakchoi plants cultivated both in hydroponic and soil-cultivated conditions. Results indicate that penthiopyrad predominantly accumulates in the roots, with concentrations of 11.3-53.9 mg/kg following root application, and in the leaves, with concentrations of 2.0-17.1 mg/kg following foliar application. The bioconcentration factor exceeded 1, with values ranging from 1.2 to 23.9 for root application and 6.4 to 164.0 for foliar application, indicating a significant role in the absorption and accumulation processes. The translocation factor data, which were <1, suggest limited the translocations within pakchoi plants. The limitation may be attributed to the hydrophobic properties of penthiopyrad (log Kow = 3.86), as evidenced by its predominant distribution in the subcellular solid fractions of pakchoi tissues, accounting for 93.1% to 99.5% of the total proportion. Six metabolites (753-A-OH, M12, 754-T-DO, M11, PCA, and PAM) were identified in this study as being formed during this process. These findings provide valuable insights into the absorption, translocation, and metabolism of penthiopyrad in pakchoi.

PE (0:0/14:0), an endogenous metabolite of the gut microbiota, exerts protective effects against sepsis-induced intestinal injury by modulating the AHR/CYP1A1 pathway.

Sepsis is known to cause damage to the intestinal mucosa, leading to bacterial translocation, and exacerbation of both local and remote organ impairments. In the present study, fecal samples were collected from both septic and healthy individuals. Analysis through 16s rRNA sequencing of the fecal microbiota revealed that sepsis disrupts the balance of the gut microbial community. Recent research has highlighted the association of lipid metabolism with disease. By analyzing the fecal metabolome, four lipid metabolites that showed significant differences between the two groups were identified: PE (O-16:0/0:0), PE (17:0/0:0), PE (0:0/14:0), and PE (12:0/20:5 (5Z, 8Z, 11Z, 14Z, 17Z)). Notably, the serum levels of PE (0:0/14:0) were higher in the healthy group. Subsequent in vitro and in vivo experiments demonstrated the protective effects of this compound against sepsis-induced intestinal barrier damage. Label-free proteomic analysis showed significant differences in the expression levels of the aryl hydrocarbon receptor (AHR), a protein implicated in sepsis pathogenesis, between the LPS-Caco-2 and LPS-Caco-2 + PE (0:0/14:0) groups. Further analysis, with the help of Discovery Studio 3.5 software and co-immunoprecipitation assays, confirmed the direct interaction between AHR and PE (0:0/14:0). In the cecal ligation and puncture (CLP) model, treatment with PE (0:0 /14:0) was found to up-regulate the expression of tight junction proteins through the AHR/Cytochrome P450, family 1, subfamily A, and polypeptide 1 (CYP1A1) pathway. This highlights the potential therapeutic use of PE (0:0/14:0) in addressing sepsis-induced intestinal barrier damage.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2 harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.

Clinical outcomes of an olive wire and external fixator versus a metallic screw in the treatment of inferior tibiofibular syndesmosis injuries in ankle fractures.

BACKGROUND: Metal screws are the most widely used in treating syndesmotic injuries, but failure and the rigidity of the screws can threaten the success of the treatment and increase the cost of care. We have provided an alternative with an olive wire and external fixator(OWEF) used for syndesmotic fixation. METHODS: A retrospective longitudinal follow-up study was conducted. From February 2011 to January 2018, 58 of 72 patients with ankle fractures and associated syndesmotic disruption were treated with either screw or OWEF fixation. The costs, complications, and clinical outcomes using Olerud-Molander score and Visual Analog score in screw and OWEF fixation group were compared. RESULTS: We found the severity of the injury, BMI of the patients and the different fixation methods were determinants of the complications and clinical outcomes. But if no malreduction of the syndesmosis was present, no difference in clinical result was detected. CONCLUSION: The OWEF method appeared to be at least equally functional and effective to screw fixation while maintaining possible lower complication rate. LEVELS OF CLINICAL EVIDENCE: Level 3.

Antinociceptive effects of carbidopa levodopa on normal rats and Parkinson's disease mice.

Carbidopa levodopa is widely used to ameliorate motor symptoms of Parkinson's disease (PD) patients. Pain is one of common symptoms of PD. The aim of this experiment is to study antinociceptive effects of carbidopa levodopa on normal rats and PD mice. Rats were intragastrically treated with carbidopa levodopa and the hind paw withdrawal latency (HWL) was investigated. PD mouse model was prepared with MPTP and then the antinociceptive effects of carbidopa levodopa on PD mice were evaluated. In normal rats, the HWL to thermal stimulus was augmented after carbidopa levodopa administration (p<0.05 or p<0.01) and carbidopa levodopa increased the HWL (p<0.05 or p<0.01) to mechanical stimulus. In PD mice, carbidopa levodopa elevated the HWL of the thermal stimulus in PD mice (p<0.05). Furthermore, the HWL in the inflammatory pain of PD mice was also increased by carbidopa levodopa treatmet (p<0.01). The current findings indicate that carbidopa levodopa has an antinociceptive effects in normal rats and PD mice. The analgesic effect of carbidopa levodopa on patients with or without PD is worth studying in further research.

Relationship between Moderate-to-Vigorous Physical Activity with Health-Related Physical Fitness Indicators among Pakistani School Adolescents: Yaali-Pak Study.

Background: The current study is the foremost study exploring the relationships between moderate-to-vigorous physical activity and health-related physical fitness indicators among 12-16-year-old adolescents of the South Punjab region of Pakistan. Methods: The researcher adopted the cross-sectional research design for the study. A total of 2970 participants (1477 boys and 1493 girls) aged adolescents from South Punjab, Pakistan, completed health-related physical fitness indicators measuring strength, endurance, and aerobic capacity through a hand-grip strength test, modified pull-up test, plank test, and 20-m shuttle run test, and physical activity were subjectively assessed by International Physical Activity Questionnaire Short Form (IPAQ-SF). Linear regression models were used to explore the between moderate-to-vigorous physical activity (MVPA) with health-related physical fitness indicators. Results: Positive associations were observed between hand-grip strength (p < 0.001), modified pull-up (p < 0.001), plank exercise (p < 0.001), and 20-m shuttle run test (p < 0.001), with MVPA. The gender-specific comparison also indicated a significant (p < 0.001) and positive relationship. The results revealed that as MVPA increases, body composition, muscular strength, core muscular endurance, and aerobic capacity could improve in both genders. Conclusions: MVPA appears to be an effective and reliable predictor of health-related physical fitness among school adolescents.

FAM129B-dependent activation of NRF2 promotes an invasive phenotype in BRAF mutant melanoma cells.

Incidence of melanoma continues to rise in the United States with ~100,000 new cases diagnosed in 2019. While the 5-year survival rate of melanoma is 99% when localized, the rate of survival drops to 22.5% when distant disease is detected. As such, an area of great interest is understanding the mechanisms that promote melanoma metastasis so that better potential therapeutic targets can be discovered. Herein, we demonstrate that activation of NRF2 by FAM129B contributes to increased metastatic potential of BRAF V600E mutant melanoma cells. Specifically, FAM129B induces NRF2 by competing for Kelch-like ECH-associated protein 1 (KEAP1) binding (the negative regulator of NRF2) via an ETGE motif. Furthermore, we show that phosphorylation of FAM129B plays a role in mediating the interaction between FAM129B and KEAP1, as the phosphorylation status of FAM129B dictates its subcellular localization. When phosphorylated, FAM129B is found primarily in the cytosol where it can bind to KEAP1, but upon inhibition of mitogen-activated protein kinase kinase activity, FAM129B is localized to the cell membrane and no longer interacts with KEAP1. In BRAF V600E mutant melanoma, the mitogen-activated protein kinase pathway leads to hyperphosphorylation of FAM129B, and therefore FAM129B localizes to the cytosol, binds KEAP1, and upregulates NRF2. Importantly, genetic modulation or pharmacological inhibition that results in a decrease in FAM129B protein level or its phosphorylation decreases migration and invasion of mutant melanoma in an NRF2-dependent manner. Overall, these data indicate that phosphorylation of FAM129B plays a significant role in driving the metastatic potential of BRAF V600E melanoma via upregulation of the NRF2 signaling pathway.

USP15 negatively regulates Nrf2 through deubiquitination of Keap1.

Nrf2 is a master regulator of the antioxidant response. Under basal conditions, Nrf2 is polyubiquitinated by the Keap1-Cul3 E3 ligase and degraded by the 26S proteasome. In response to Nrf2 inducers there is a switch in polyubiquitination from Nrf2 to Keap1. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. Here we report that the deubiquitinating enzyme, USP15, specifically deubiquitinates Keap1, which suppresses the Nrf2 pathway. We demonstrated that deubiquitinated Keap1 incorporates into the Keap1-Cul3-E3 ligase complex more efficiently, enhancing the complex stability and enzymatic activity. Consequently, there is an increase in Nrf2 protein degradation and a reduction in Nrf2 target gene expression. Furthermore, USP15-siRNA enhances chemoresistance of cells through upregulation of Nrf2. These findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.

A chloride efflux transporter, BIG RICE GRAIN 1, is involved in mediating grain size and salt tolerance in rice.

Grain size is determined by the size and number of cells in the grain. The regulation of grain size is crucial for improving crop yield; however, the genes and molecular mechanisms that control grain size remain elusive. Here, we report that a member of the detoxification efflux carrier /Multidrug and Toxic Compound Extrusion (DTX/MATE) family transporters, BIG RICE GRAIN 1 (BIRG1), negatively influences grain size in rice (Oryza sativa L.). BIRG1 is highly expressed in reproductive organs and roots. In birg1 grain, the outer parenchyma layer cells of spikelet hulls are larger than in wild-type (WT) grains, but the cell number is unaltered. When expressed in Xenopus laevis oocytes, BIRG1 exhibits chloride efflux activity. Consistent with this role of BIRG1, the birg1 mutant shows reduced tolerance to salt stress at a toxic chloride level. Moreover, grains from birg1 plants contain a higher level of chloride than those of WT plants when grown under normal paddy field conditions, and the roots of birg1 accumulate more chloride than those of WT under saline conditions. Collectively, the data suggest that BIRG1 in rice functions as a chloride efflux transporter that is involved in mediating grain size and salt tolerance by controlling chloride homeostasis.

Differential and overlapping targets of the transcriptional regulators NRF1, NRF2, and NRF3 in human cells.

The nuclear factor (erythroid 2)-like (NRF) transcription factors are a subset of cap'n'collar transcriptional regulators. They consist of three members, NRF1, NRF2, and NRF3, that regulate the expression of genes containing antioxidant-response elements (AREs) in their promoter regions. Although all NRF members regulate ARE-containing genes, each is associated with distinct roles. A comprehensive study of differential and overlapping DNA-binding and transcriptional activities of the NRFs has not yet been conducted. Here, we performed chromatin immunoprecipitation (ChIP)-exo sequencing, an approach that combines ChIP with exonuclease treatment to pinpoint regulatory elements in DNA with high precision, in conjunction with RNA-sequencing to define the transcriptional targets of each NRF member. Our approach, done in three U2OS cell lines, identified 31 genes that were regulated by all three NRF members, 27 that were regulated similarly by all three, and four genes that were differentially regulated by at least one NRF member. We also found genes that were up- or down-regulated by only one NRF member, with 84, 84, and 22 genes that were regulated by NRF1, NRF2, and NRF3, respectively. Analysis of the ARE motifs identified in ChIP peaks revealed that NRF2 prefers binding to AREs flanked by GC-rich regions and that NRF1 prefers AT-rich flanking regions. Thus, sequence preference, likely in combination with upstream signaling events, determines NRF member activation under specific cellular contexts. Our analysis provides a comprehensive description of differential and overlapping gene regulation by the transcriptional regulators NRF1, NRF2, and NRF3.

The Maize MID-COMPLEMENTING ACTIVITY Homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF Coordinates Organ Growth and Tissue Patterning.

Organogenesis occurs through cell division, expansion, and differentiation. How these cellular processes are coordinated remains elusive. The maize (Zea mays) leaf provides a robust system to study cellular differentiation due to its distinct tissues and cell types. The narrow odd dwarf (nod) mutant displays defects at both the cellular and tissue level that increase in severity throughout growth. nod mutant leaves have reduced size due to fewer and smaller cells compared with the wild type. The juvenile-to-adult transition is delayed, and proximal distal-patterning is abnormal in this mutant. Differentiation of specialized cells such as those forming stomata and trichomes is incomplete. Analysis of nod-1 sectors suggests that NOD plays a cell-autonomous function in the leaf. We cloned nod positionally and found that it encodes CELL NUMBER REGULATOR13 (CNR13), the maize MID-COMPLEMENTING ACTIVITY homolog. CNR13/NOD is localized to the membrane and is enriched in dividing tissues. Transcriptome analysis of nod mutants revealed overrepresentation of cell wall, hormone metabolism, and defense gene categories. We propose that NOD coordinates cell activity in response to intrinsic and extrinsic cues.

The interaction of CaM7 and CNGC14 regulates root hair growth in Arabidopsis.

Oscillations in cytosolic free calcium determine the polarity of tip-growing root hairs. The Ca2+ channel cyclic nucleotide gated channel 14 (CNGC14) contributes to the dynamic changes in Ca2+ concentration gradient at the root hair tip. However, the mechanisms that regulate CNGC14 are unknown. In this study, we detected a direct interaction between calmodulin 7 (CaM7) and CNGC14 through yeast two-hybrid and bimolecular fluorescence complementation assays. We demonstrated that the third EF-hand domain of CaM7 specifically interacts with the cytosolic C-terminal domain of CNGC14. A two-electrode voltage clamp assay showed that CaM7 completely inhibits CNGC14-mediated Ca2+ influx, suggesting that CaM7 negatively regulates CNGC14-mediated calcium signaling. Furthermore, CaM7 overexpressing lines phenocopy the short root hair phenotype of a cngc14 mutant and this phenotype is insensitive to changes in external Ca2+ concentrations. We, thus, identified CaM7-CNGC14 as a novel interacting module that regulates polar growth in root hairs by controlling the tip-focused Ca2+ signal.

Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2-related factors 1 and 2 (NRF1 and NRF2).

Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1+/+ than in KEAP1-/- isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1-/- cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.

Brusatol overcomes chemoresistance through inhibition of protein translation.

The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.

Transport and homeostasis of potassium and phosphate: limiting factors for sustainable crop production.

Potassium (K) and phosphate (Pi) are both macronutrients essential for plant growth and crop production, but the unrenewable resources of phosphorus rock and potash have become limiting factors for food security. One critical measure to help solve this problem is to improve nutrient use efficiency (NUE) in plants by understanding and engineering genetic networks for ion uptake, translocation, and storage. Plants have evolved multiple systems to adapt to various nutrient conditions for growth and production. Within the NUE networks, transport proteins and their regulators are the primary players for maintaining nutrient homeostasis and could be utilized to engineer high NUE traits in crop plants. A large number of publications have detailed K+ and Pi transport proteins in plants over the past three decades. Meanwhile, the discovery and validation of their regulatory mechanisms are fast-track topics for research. Here, we provide an overview of K+ and Pi transport proteins and their regulatory mechanisms, which participate in the uptake, translocation, storage, and recycling of these nutrients in plants.

Nitric oxide negatively regulates AKT1-mediated potassium uptake through modulating vitamin B6 homeostasis in Arabidopsis.

Nitric oxide (NO), an active signaling molecule in plants, is involved in numerous physiological processes and adaptive responses to environmental stresses. Under high-salt conditions, plants accumulate NO quickly, and reorganize Na(+) and K(+) contents. However, the molecular connection between NO and ion homeostasis is largely unknown. Here, we report that NO lowers K(+) channel AKT1-mediated plant K(+) uptake by modulating vitamin B6 biosynthesis. In a screen for Arabidopsis NO-hypersensitive mutants, we isolated sno1 (sensitive to nitric oxide 1), which is allelic to the previously noted mutant sos4 (salt overly sensitive 4) that has impaired Na(+) and K(+) contents and overproduces pyridoxal 5'-phosphate (PLP), an active form of vitamin B6. We showed that NO increased PLP and decreased K(+) levels in plant. NO induced SNO1 gene expression and enzyme activity, indicating that NO-triggered PLP accumulation mainly occurs through SNO1-mediated vitamin B6 salvage biosynthetic pathway. Furthermore, we demonstrated that PLP significantly repressed the activity of K(+) channel AKT1 in the Xenopus oocyte system and Arabidopsis root protoplasts. Together, our results suggest that NO decreases K(+) absorption by promoting the synthesis of vitamin B6 PLP, which further represses the activity of K(+) channel AKT1 in Arabidopsis. These findings reveal a previously unidentified pivotal role of NO in modulating the homeostasis of vitamin B6 and potassium nutrition in plants, and shed light on the mechanism of NO in plant acclimation to environmental changes.

Weighting and indirect effects identify keystone species in food webs.

Species extinctions are accelerating globally, yet the mechanisms that maintain local biodiversity remain poorly understood. The extinction of species that feed on or are fed on by many others (i.e. 'hubs') has traditionally been thought to cause the greatest threat of further biodiversity loss. Very little attention has been paid to the strength of those feeding links (i.e. link weight) and the prevalence of indirect interactions. Here, we used a dynamical model based on empirical energy budget data to assess changes in ecosystem stability after simulating the loss of species according to various extinction scenarios. Link weight and/or indirect effects had stronger effects on food-web stability than the simple removal of 'hubs', demonstrating that both quantitative fluxes and species dissipating their effects across many links should be of great concern in biodiversity conservation, and the potential for 'hubs' to act as keystone species may have been exaggerated to date.

Molecular mechanisms of Nrf2 regulation and how these influence chemical modulation for disease intervention.

Nrf2 (nuclear factor erytheroid-derived-2-like 2) transcriptional programmes are activated by a variety of cellular stress conditions to maintain cellular homoeostasis. Under non-stress conditions, Nrf2 is under tight regulation by the ubiquitin proteasome system (UPS). Detailed mechanistic investigations have shown the Kelch-like ECH-associated protein 1 (Keap1)-cullin3 (Cul3)-ring-box1 (Rbx1) E3-ligase to be the primary Nrf2 regulatory system. Recently, both beta-transducin repeat-containing E3 ubiquitin protein ligase (ß-TrCP) and E3 ubiquitin-protein ligase synoviolin (Hrd1) have been identified as novel E3 ubiquitin ligases that negatively regulate Nrf2 through Keap1-independent mechanisms. In addition to UPS-mediated regulation of Nrf2, investigations have revealed a cross-talk between Nrf2 and the autophagic pathway resulting in activation of Nrf2 in a non-canonical manner. In addition to regulation at the protein level, Nrf2 was recently shown to be regulated at the transcriptional level by oncogenic K-rat sarcoma (Ras). A consequence of these differential regulatory mechanisms is the dual role of Nrf2 in cancer: the canonical, protective role and the non-canonical 'dark-side' of Nrf2. Based on the protective role of Nrf2, a vast effort has been dedicated towards identifying novel chemical inducers of Nrf2 for the purpose of chemoprevention. On the other hand, upon malignant transformation, some cancer cells have a constitutively high level of Nrf2 offering a growth advantage, as well as rendering cancer cells resistant to chemotherapeutics. This discovery has led to a new paradigm in cancer treatment; the initially counterintuitive use of Nrf2 inhibitors as adjuvants in chemotherapy. Herein, we will discuss the mechanisms of Nrf2 regulation and how this detailed molecular understanding can be leveraged to develop Nrf2 modulators to prevent diseases, mitigate disease progression or overcome chemoresistance.