Parkinson's disease is a neurodegenerative disorder associated with misfolding and aggregation of α-synuclein as a hallmark protein. Two yeast strain collections comprising conditional alleles of essential genes were screened for the ability of each allele to reduce or improve yeast growth upon α-synuclein expression. The resulting 98 novel modulators of α-synuclein toxicity clustered in several major categories including transcription, rRNA processing and ribosome biogenesis, RNA metabolism and protein degradation. Furthermore, expression of α-synuclein caused alterations in pre-rRNA transcript levels in yeast and in human cells. We identified the nucleolar DEAD-box helicase Dbp4 as a prominent modulator of α-synuclein toxicity. Downregulation of DBP4 rescued cells from α-synuclein toxicity, whereas overexpression led to a synthetic lethal phenotype. We discovered that α-synuclein interacts with Dbp4 or its human ortholog DDX10, sequesters the protein outside the nucleolus in yeast and in human cells, and stabilizes a fraction of α-synuclein oligomeric species. These findings provide a novel link between nucleolar processes and α-synuclein mediated toxicity with DDX10 emerging as a promising drug target.

DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.

The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.

Vasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation.

The RNA helicases, which help to unwind stable RNA duplexes, and have important roles in RNA metabolism, belong to a class of motor proteins that play important roles in plant development and responses to stress. Although this family of genes has been the subject of systematic investigation in Arabidopsis, rice, and tomato, it has not yet been characterized in cotton. In this study, we identified 161 putative RNA helicase genes in the genome of the diploid cotton species Gossypium raimondii. We classified these genes into three subfamilies, based on the presence of either a DEAD-box (51 genes), DEAH-box (52 genes), or DExD/H-box (58 genes) in their coding regions. Chromosome location analysis showed that the genes that encode RNA helicases are distributed across all 13 chromosomes of G. raimondii. Syntenic analysis revealed that 62 of the 161 G. raimondii helicase genes (38.5%) are within the identified syntenic blocks. Sixty-six (40.99%) helicase genes from G. raimondii have one or several putative orthologs in tomato. Additionally, GrDEADs have more conserved gene structures and more simple domains than GrDEAHs and GrDExD/Hs. Transcriptome sequencing data demonstrated that many of these helicases, especially GrDEADs, are highly expressed at the fiber initiation stage and in mature leaves. To our knowledge, this is the first report of a genome-wide analysis of the RNA helicase gene family in cotton.

Invasive species can have devastating effects on native ecosystems and therefore impose a significant threat to human welfare. The introduction rate of invasive species has accelerated dramatically in recent times due to human activity (anthropogenic effects), with a steadily growing pool of widespread tramp species. We present an in-depth analysis of four pantropical species of Xyleborus ambrosia beetles (Xyleborus volvulus, Xyleborus perforans, Xyleborus ferrugineus, and Xyleborus affinis) with similar ecology (fungus cultivation in dead wood), reproductive biology (permanent inbreeding) and genetic system (haplodiploidy). The unique combination of reproductive traits and broad host plant usage pre-adapts these beetles for colonizing of new areas.

Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.

Acidithiobacillus ferrivorans is an acidophilic bacterium that represents a substantial proportion of the microbial community in a low temperature mining waste stream. Due to its ability to grow at temperatures below 15 °C, it has previously been classified as 'psychrotolerant'. Low temperature-adapted microorganisms have strategies to grow at cold temperatures such as the production of cold acclimation proteins, DEAD/DEAH box helicases, and compatible solutes plus increasing their cellular membrane fluidity. However, little is known about At. ferrivorans adaptation strategies employed during culture at its temperature extremes. In this study, we report the transcriptomic response of At. ferrivorans SS3 to culture at 8 °C compared to 20 °C. Analysis revealed 373 differentially expressed genes of which, the majority were of unknown function. Only few changes in transcript counts of genes previously described to be cold adaptation genes were detected. Instead, cells cultured at cold (8 °C) altered the expression of a wide range of genes ascribed to functions in transcription, translation, and energy production. It is, therefore, suggested that a temperature of 8 °C imposed little cold stress on At. ferrivorans, underlining its adaptation to growth in the cold as well as suggesting it should be classified as a 'eurypsychrophile'.

Cotton yields are greatly reduced under high salinity stress conditions, although cotton is considered a moderately salt-tolerant crop. Understanding at the molecular level how cotton responds to salt stress will help in developing salt tolerant varieties. Here, we combined physiological analysis with isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics of seedling leaves of 2 genotypes differing in salinity tolerance to 200 mM (18.3 dS/m) NaCl stress. Salt stress produced significant stress symptoms in the sensitive genotype Nan Dan Ba Di Da Hua (N), including lower relative water and chlorophyll contents and higher relative electrolyte leakage and Na+/K+ ratio in leaf samples, compared with those in the tolerant genotype Earlistaple 7 (Z). A total of 58 differentially abundant salt-responsive proteins were identified. Asp-Glu-Ala-Asp (DEAD)-box ATP-dependent RNA helicase 3 and protochlorophyllide reductase were markedly suppressed after salt treatment, whereas the phosphate-related differentially abundant proteins (DAPs) phosphoethanolamine N-methyltransferase 1 and 14-3-3-like protein E were induced, and all these proteins may play significant roles in salt stress. Twenty-nine salt-responsive proteins were also genotype specific, and 62.1 and 27.6% of these were related to chloroplast and defense responses, respectively. Based on the Arabidopsis thaliana protein interaction database, orthologs of 25 proteins showed interactions in Arabidopsis, and among these, a calmodulin protein was predicted to have 212 functional partners. In addition, the Golgi apparatus and calcium may be important for salt secretion in cotton. Through integrative proteome and transcriptome analysis, 16 DAPs were matched to differentially expressed genes and verified using qRT-PCR. On the basis of these findings, we proposed that some proteins related to chloroplast, ATP, ribosomal, and phosphate metabolism as well as to the Golgi apparatus and calcium may play key roles in the short-term salt stress response of cotton seedling leaves.

Atopic dermatitis (AD) is a severe inflammatory skin disorder, characterized by elevated levels of proinflammatory cytokines that fuel a vicious cycle of inflammation. While inflammatory recombinant human epidermal (RHE) models relevant to AD have been established, comprehensive understanding remains limited. To illuminate changes and identify potential hub genes involved in AD-related inflammation, RHE models, stimulated by an inflammatory cocktail including polyinosinic-polycytidylic acid, tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4) and interleukin 13 (IL-13), were constructed and examined using tandem mass tags-proteomic coupled with RNA-seq transcriptomic analyses. Principal component analysis (PCA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment were employed for the analysis of related genes and proteins. Protein-protein interaction networks helped identify hub genes, which were further confirmed by qPCR and western blot. We observed high expression of thymic stromal lymphopoietin in the inflammatory RHE. Our study identified 2369 differentially expressed genes and 880 differentially expressed proteins in the cocktail-induced group versus the normal control group. A total of 248 overlapping symbols were enriched in various biological processes and signaling pathways, including cornification envelope, cell-cell junction, calcium ion binding, extracellular matrix receptor, terpenoid backbone biosynthesis, and peroxisome proliferator-activated receptors signaling pathway, among others. Among the 248 overlapping symbols, CytoHubba identified 10 hub molecules, namely signal transducer and activator of transcription 3 (STAT3), integrin subunit beta 1 (ITGB1), filaggrin (FLG), involucrin (IVL), DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58), small proline rich protein 1B (SPRR1B), interferon induced with helicase C domain 1 (IFIH1), desmoglein 1 (DSG1), collagen type XVII alpha 1 chain (COL17A1), and integrin subunit alpha 6 (ITGA6), based on the degree. These integrated results offer valuable insights into the molecular mechanisms of AD and present potential tools for screening cosmetic formulations intended for the treatment of AD.

Primary high-grade gliomas possess invasive growth and lead to unfavorable survival outcome. The investigation of biomarkers for prediction of survival outcome in patients with gliomas is important for clinical assessment. The DEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linked (DDX3X) controls tumor migration, proliferation, and progression. However, the role of DDX3X in defining the pathological grading and survival outcome in patients with human gliomas is not yet clarified. We analyzed the DDX3X gene expression, WHO pathological grading, and overall survival from de-linked data. Further validation was done using quantitative RT-PCR of cDNA from normal brain and glioma, and immunohistochemical (IHC) staining of tissue microarray. Statistical analysis of GEO datasets showed that DDX3X mRNA expression demonstrated statistically higher in WHO grade IV (n = 81) than in non-tumor controls (n = 23, p = 1.13 × 10(-10)). Moreover, DDX3X level was also higher in WHO grade III (n = 19) than in non-tumor controls (p = 2.43 × 10(-5)). Kaplan-Meier survival analysis showed poor survival in patients with high DDX3X mRNA levels (n = 24) than in those with low DDX3X expression (n = 53) (median survival, 115 vs. 58 weeks, p = 0.0009, by log-rank test, hazard ratio: 0.3507, 95% CI: 0.1893-0.6496). Furthermore, DDX3X mRNA expression and protein production significantly increased in glioma cells compared with normal brain tissue examined by quantitative RT-PCR, and Western blot. IHC staining showed highly staining of high-grade glioma in comparison with normal brain tissue. Taken together, DDX3X expression level positively correlates with WHO pathologic grading and poor survival outcome, indicating that DDX3X is a valuable biomarker in human gliomas.

P21-activated kinase 5 (PAK5) plays an important role in tumors. However, the functional role of PAK5 in mammary tumorigenesis in vivo remains unclear. Here, we show that PAK5 deficiency represses MMTV-PyVT-driven breast tumorigenesis. DEAD-box RNA helicase 5 (DDX5) is a substrate of PAK5, which is phosphorylated on threonine 69. PAK5-mediated DDX5 phosphorylation promotes breast cancer cell proliferation and metastasis. The increased expression levels of PAK5 and phospho-DDX5 threonine 69 are associated with metastasis and poor clinical outcomes of patients. PAK5 facilitates the phosphorylation-dependent sumoylation of DDX5 to stabilize DDX5. Both the phosphorylation and sumoylation of DDX5 enhance the formation of a DDX5/Drosha/DGCR8 complex, thus promoting microRNA-10b processing. Finally, we verify decreased expression of DDX5 phosphorylation and sumoylation and mature miR-10b in PAK5-/-/MMTV-PyVT transgenic mice. Our findings provide insights into the function of PAK5 in microRNA (miRNA) biogenesis, which might be a potential therapeutic target for breast cancer.

Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved.

DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

Triple-negative breast cancer (TNBC) is a great detriment to women's health due to the lack of effective therapeutic targets. In this study, we employed an integrated genetic screen to identify a pivotal oncogenic factor, heterogeneous nuclear ribonucleoprotein U (HNRNPU), which is required for the progression of TNBC. We elucidated the pro-oncogenic role of HNRNPU, which can induce the proliferation and migration of TNBC cells via its association with DEAD box helicase 5 (DDX5) protein. Elevated levels of the HNRNPU-DDX5 complex prohibited the intron retention of minichromosome maintenance protein 10 (MCM10) pre-mRNA, decreased nonsense-mediated mRNA decay, and activated Wnt/β-catenin signalling; on the other hand, HNRNPU-DDX5 is located in the transcriptional start sites (TSS) of LIM domain only protein 4 (LMO4) and its upregulation promoted the transcription of LMO4, consequently activating PI3K-Akt-mTOR signalling. Our data highlight the synergetic effects of HNRNPU in RNA transcription and splicing in regulating cancer progression and suggest that HNRNPU may act as a potential molecular target in the treatment of TNBC.

DDX3 is a DEAD box RNA helicase with oncogenic properties. RK-33 is developed as a small-molecule inhibitor of DDX3 and showed potent radiosensitizing activity in preclinical tumor models. This study aimed to assess DDX3 as a target in breast cancer and to elucidate how RK-33 exerts its anti-neoplastic effects. High DDX3 expression was present in 35% of breast cancer patient samples and correlated with markers of aggressiveness and shorter survival. With a quantitative proteomics approach, we identified proteins involved in the mitochondrial translation and respiratory electron transport pathways to be significantly downregulated after RK-33 or DDX3 knockdown. DDX3 localized to the mitochondria and DDX3 inhibition with RK-33 reduced mitochondrial translation. As a consequence, oxygen consumption rates and intracellular ATP concentrations decreased and reactive oxygen species (ROS) increased. RK-33 antagonized the increase in oxygen consumption and ATP production observed after exposure to ionizing radiation and reduced DNA repair. Overall, we conclude that DDX3 inhibition with RK-33 causes radiosensitization in breast cancer through inhibition of mitochondrial translation, which results in reduced oxidative phosphorylation capacity and increased ROS levels, culminating in a bioenergetic catastrophe.

Aberrant expression of P68 RNA helicase (p68), a prototypical member of the DEAD box family of RNA helicases, contributes to tumor development and progression. P68 tyrosine phosphorylation induced by PDGF signaling facilitates cancer metastasis by promoting EMT. In this report, we show that p68 promotes breast cancer cell EMT and cell migration by upregulation of PDGF receptor β (PDGFR-β). Knockdown of p68 in MDA-MB-231 and BT549 cells significantly decreases PDGFR-β both in mRNA and protein levels. P68 promotes EMT and cell migration in response to PDGF-BB stimulation via upregulation of PDGFR-β, suggesting that p68 enhances PDGF signaling by a positive feedback loop in cancer cells. Furthermore, our study reveals that p68 mediates the effects of PDGFR-β in regulation of androgen receptor (AR) in breast cancer cells. We demonstrate that p68 and PDGFR-β co-regulate AR expression and promote androgen-mediated proliferation in breast cancer cells. Our studies uncover an important pathway of p68-PDGFR-β axis in promoting breast cancer progression.

Breast cancer is the most common cancer for women in Taiwan and post-lumpectomy radiotherapy is one of the therapeutic strategies for this malignancy. Although the 10-year overall survival of breast cancer patients is greatly improved by radiotherapy, the locoregional recurrence is around 10% and triple negative breast cancers (TNBCs) are at a high risk for relapse. The aim of this paper is to understand the mechanisms of radioresistance in breast cancers which may facilitate the development of new treatments in sensitizing breast cancer toward radiation therapy. Tribbles homolog 3 (TRIB3) is a pseudokinase protein and known to function as a protein scaffold within cells. It has been reported that higher TRIB3 expression is a poor prognostic factor in breast cancer patients with radiotherapy. In this study, we investigate the involvement of TRIB3 in the radiation response of TNBC cells. We first found that the expression of TRIB3 and the activation of Notch1, as well as Notch1 target genes, increased in two radioresistant TNBC cells. Knockdown of TRIB3 in radioresistant MDA-MB-231 TNBC cells decreased Notch1 activation, as well as the CD24-CD44⁺ cancer stem cell population, and sensitized cells toward radiation treatment. The inhibitory effects of TRIB3 knockdown in self-renewal or radioresistance could be reversed by forced expression of the Notch intracellular domain. We also observed an inhibition in cell growth and accumulated cells in the G₀/G¹ phase in radioresistant MDA-MB-231 cells after knockdown of TRIB3. With immunoprecipitation and mass spectrometry analysis, we found that, BCL2-associated transcription factor 1 (BCLAF1), BCL2 interacting protein 1 (BNIP1), or DEAD-box helicase 5 (DDX5) were the possible TRIB3 interacting proteins and immunoprecipitation data also confirmed that these proteins interacted with TRIB3 in radioresistant MDA-MB-231 cells. In conclusion, the expression of TRIB3 in radioresistant TNBC cells participated in Notch1 activation and targeted TRIB3 expression may be a strategy to sensitize TNBC cells toward radiation therapy.

Emerging studies demonstrated the roles of long non-coding RNAs (LncRNAs) are being implicated in the progression of many cancers. Here we report the discovery of a critical role for the linc00630 in the development of Non-Small-Cell Lung Cancers (NSCLCs). Screening from the microarray of six paired NSCLCs and adjacent non-tumor tissues, linc00630 showed a significantly higher RNA levels in NSCLCs. With the higher level confirmed in a separate cohort 90 NSCLCs patients, overexpressed of linc00630 also positive associated with tumor size, TNM tumor stage, lymph node status positive and overall patient outcomes. Linc00630 overexpression increased cell proliferation and metastasis in vitro and in vivo whereas linc00630 silencing had opposite effects. By RNA pull-down and mass spectrometry we identified Histone deacetylases 1 (HDAC1) and DEAD-box helicase 23 (DDX23) as the linc00630-binding protein that associated with mechanism of linc00630. DDX23 can specific bind with the promoter of Linc00630 to up-regulate the RNA level and high level of linc00630 strength the protein stability of HDAC1 to regulate the downstream pathway.Our study demonstrates the effectiveness of Linc00630 oligonucleotide-based promotion of NSCLCs metastasis and proliferation, illuminating a new basis of DDX23-Linc00630-HDAC1 signal axis for understanding its pathogenicity, which could be further developed as a valuable therapeutic strategy.

Cannabis (Cannabis sativa L.) offers many industrial, agricultural, and medicinal applications, but is commonly threatened by the gray mold disease caused by the fungus Botrytis cinerea. With few effective control measures currently available, the use of beneficial rhizobacteria represents a promising biocontrol avenue for cannabis. To counter disease development, plants rely on a complex network of inducible defense pathways, allowing them to respond locally and systemically to pathogens attacks. In this study, we present the first attempt to control gray mold in cannabis using beneficial rhizobacteria, and the first investigation of cannabis defense responses at the molecular level. Four promising Pseudomonas (LBUM223 and WCS417r) and Bacillus strains (LBUM279 and LBUM979) were applied as single or combined root treatments to cannabis seedlings, which were subsequently infected by B. cinerea. Symptoms were recorded and the expression of eight putative defense genes was monitored in leaves by reverse transcription quantitative polymerase chain reaction. The rhizobacteria did not significantly control gray mold and all infected leaves were necrotic after a week, regardless of the treatment. Similarly, no systemic activation of putative cannabis defense genes was reported, neither triggered by the pathogen nor by the rhizobacteria. However, this work identified five putative defense genes (ERF1, HEL, PAL, PR1, and PR2) that were strongly and sustainably induced locally at B. cinerea's infection sites, as well as two stably expressed reference genes (TIP41 and APT1) in cannabis. These markers will be useful in future researches exploring cannabis defense pathways.