Caenorhabditis Elegans Exhibits Morphine Addiction-like Behavior via the Opioid-like Receptor NPR-17

Addiction has become a profound societal problem worldwide, and few effective treatments are available. The nematode Caenorhabditis elegans (C. elegans) is an excellent invertebrate model to study neurobiological disease states. C. elegans reportedly developed a preference for cues that had previously been paired with addictive drugs, similar to place conditioning findings in rodents. Moreover, several recent studies discovered and reported the existence of an opioid-like system in C. elegans. Still unclear, however, is whether C. elegans exhibits addictive-like behaviors for opioids, such as morphine. In the present study, we found that C. elegans exhibited dose-dependent preference for morphine using the conditioned chemosensory-cue preference (CCP) test. This preference was blocked by co-treatment with the opioid receptor antagonist naloxone. C. elegans also exhibited aversion to naloxone-precipitated withdrawal from chronic morphine exposure. The expression of morphine-induced CCP and morphine withdrawal were abolished in worms that lacked the opioid-like receptor NPR-17. Dopamine-deficient mutant (cat-2 (e1112)) worms also did not exhibit morphine-induced CCP. These results indicate that the addictive function of the opioid system exists in C. elegans, which may serve as a useful model of opioid addiction.

Effects of the noncoding subgenomic RNA of red clover necrotic mosaic virus in virus infection

In recent years, a new class of viral noncoding subgenomic (ncsg)RNA has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant (xr) RNA structure, which blocks the progression of 5’→3’ exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate: (i) absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wildtype RCNMV or an SR1f-deficient mutant RCNMV revealed that wt RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10 to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3’ region of RCNMV RNA1 support the premature mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses. IMPORTANCE Knowledge of how RNA viruses manipulate host and viral gene expression is crucial to our understanding of infection and disease. Unlike viral protein-host interactions, little is known about the control of gene expression by viral RNA. Here we begin to address this question by investigating the noncoding subgenomic (ncsg)RNA of red clover necrotic mosaic virus (RCNMV), called SR1f. Similar exoribonuclease-resistant RNAs of flaviviruses are well-studied, but the roles of plant viral ncsgRNAs, and how they arise, are poorly understood. Surprisingly, we find the likely exonuclease candidate, XRN4, is not required to generate SR1f, and we assess the effects of SR1f on virus accumulation and symptom development. Finally, we compare the effects of infection by wildtype RCNMV vs an SR1f-deficient mutant on host gene expression in Nicotiana benthamiana , which reveals that ncsgRNAs such as SR1f are key players in virus-host interactions to facilitate productive infection.

Combined Inhibition of Bcl-2 and Mcl-1 Circumvents Resistance of TP53 Deficient/Mutant AML to BH3 Mimetics

AML patients with TP53 mutations have extremely poor clinical outcomes. This is due primarily to limited responses to available therapies including the highly promising FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents (DiNardo CD et al., Blood 2020), which resulted in CR/CRi rates of 70-95% and good tolerability in elderly patients (DiNardo CD et al., Lancet Oncol 2018 and Blood 2019). Apoptosis is regulated by anti- and pro-apoptotic proteins. While p53 does not directly regulate anti-apoptotic Bcl-2 proteins that are resistance factors for VEN, p53 transcriptionally up-regulates pro-apoptotic Bcl-2 proteins. Reverse phase protein array analysis of samples from newly-diagnosed AML patients found that pro-apoptotic Bax was significantly decreased in patients with TP53 mutations (Carter BZ, ASH 2019), which, as expected, diminished the effectiveness of Bcl-2 inhibition. Thus, strategies to target additional anti-apoptotic proteins, or increase pro-apoptotic proteins, are needed to enhance the efficacy of Bcl-2 inhibition in these patients. We determined protein levels of Bcl-2 family members in isogeneic Molm13 cells with TP53-knockout (KO), or with various hotspot TP53 mutations including R175H, Y220C, M237I, R248Q, R273H, and R282W. We observed markedly decreased Bax expression, to a less degree Bak decrease, and variable alterations in other Bcl-2 proteins in these cells compared to TP53-wild-type (WT) controls. We treated the aforementioned cells with VEN or the Mcl-1 inhibitor AMG 176 and found that TP53-KO or mutant cells were more resistant to both VEN and AMG 176 compared to WT controls. However, the combination of two inhibitors was highly synergistic in both settings, controls (CI = 0.2) and TP53-KO and mutant cells (CI < 0.1). To demonstrate that the decreased sensitivity to BH3 mimetics was, at least in part, mediated through Bax reduction in the TP53-mutant cells, we treated Bax knockdown (KD) Molm13 cells with VEN, AMG 176, or both. The Bax KD cells were resistant to VEN and AMG 176, while the combination of the two agents synergistically induced cell death. To establish potential clinical relevance of co-targeting Bcl-2 and Mcl-1 in TP53-mutant AML, we co-cultured cells from various TP53-mutant AML patients (n = 8) with mesenchymal stromal cells and treated them with VEN, AMG 176, or both. The combination synergistically induced cell death in both CD45 + leukemia blasts (CI values between 0.04 ± 0.04 to 0.34 ± 0.10) and CD34 + AML stem/progenitor cells (CI values between 0.07 ± 0.08 to 0.28 ± 0.14). RNA-sequencing of mononuclear and MRD cells of clinical samples (Issa G, ASH 2019) collected after induction therapy revealed that Mcl-1 expression was significantly higher in the TP53-mutated mononuclear and MRD cells compared to their WT counterparts (Fig. 1), which suggests that Mcl-1 contributes to treatment resistance and disease relapse. This further suggests that Mcl-1 inhibition should be incorporated in AML treatment, including VEN-based therapies, for patients with TP53 mutations. Finally, we treated NSG mice inoculated with isogeneic TP53-WT luciferase/GFP-labeled Molm13 and BFP-labeled TP53 R248W/R213* Molm13 cells (10:1) with VEN, AMG 176, or their combination. Only the combination treatment markedly decreased the number of GFP- and BFP-labeled cells in circulation and significantly prolonged mouse survival (median 23 d, 25 d, 24.5 d for control, VEN, AMG 176, respectively; and 45 d for VEN + AMG 176: P = 0.0007, 0.0009, and 0.0011 of combination vs. control, VEN, and AMG 176, respectively) (Fig. 2). Collectively, we demonstrate that decreased Bax contributes to resistance of TP53-mutant AML to BH3 mimetics. Mcl-1 expression positively impacts therapy resistance and disease reoccurrence in TP53-mutant AML. Thus, targeting Bcl-2 or Mcl-1 individually is insufficient and inhibition of both proteins is needed to shift cell fate from survival to death and circumvents resistance of TP53 deficient/mutant AML and AML stem/progenitor cells to BH3 mimetics. The concept warrants further clinical evaluation. Figure 1 Figure 1. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Andreeff: Medicxi: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Karyopharm: Research Funding; Syndax: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; Glycomimetics: Consultancy; Oxford Biomedica UK: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii

Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin) acting as an electron donor for nitrogenase. However, the relative contribution of Rnf into nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We have constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated a markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defect under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between FMN and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE.

Characterization of Virulence Function of Pseudomonas cichorii Avirulence Protein E1 (AvrE1) during Host Plant Infection

<i>Pseudomonas cichorii</i> secretes effectors that suppress defense mechanisms in host plants. However, the function of these effectors, including avirulence protein E1 (AvrE1), in the pathogenicity of <i>P. cichorii</i>, remains unexplored. In this study, to investigate the function of <i>avrE1</i> in <i>P. cichorii</i> JBC1 (PcJBC1), we created an <i>avrE1</i>-deficient mutant (JBC1^ΔavrE1) using CRISPR/Cas9. The disease severity caused by JBC1^ΔavrE1 in tomato plants significantly decreased by reducing water soaking during early infection stage, as evidenced by the electrolyte leakage in infected leaves. The disease symptoms caused by JBC1^ΔavrE1 in the cabbage midrib were light-brown spots compared to the dark-colored ones caused by PcJBC1, which indicates the role of AvrE1 in cell lysis. The <i>avrE1</i>-deficient mutant failed to elicit cell death in non-host tobacco plants. Disease severity and cell death caused by JBC1^ΔavrE1 in host and non-host plants were restored through heterologous complementation with <i>avrE1</i> from <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 (PstDC3000). Overall, our results indicate that <i>avrE1</i> contributes to cell death during early infection, which consequently increases disease development in host plants. The roles of PcJBC1 AvrE1 in host cells remain to be elucidated.

Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii

Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.

Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album

Sandalwood (Santalum album L.) is highly valued for its fragrant heartwood and extracted oil. Santalols, which are the main components of that oil, are terpenoids, and these are biosynthesized via the mevalonic acid (MVA) pathway. Mevalonate kinase (MK) and phosphomevalonate kinase (PMK) are key enzymes in the MVA pathway. Little is known about the genes that encode MK and PMK in S. album or the mechanism that regulates their expression. To isolate and identify the functional genes involved in santalol biosynthesis in S. album, an MK gene designated as SaMK, and a PMK gene designated as SaPMK, were cloned from S. album. The sequences of these genes were analyzed. A bioinformatics analysis was conducted to assess the homology of SaMK and SaPMK with MK and PMK genes from other plants. The subcellular localization of SaMK and SaPMK proteins was also investigated, as was the functional complementation of SaMK and SaPMK in yeast. Our results show that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp long, respectively. SaMK contained a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and SaPMK contained a 1527 bp ORF encoding a polypeptide of 508 amino acids. SaMK and SaPMK showed high homology with MK and PMK genes of other plant species. Functional complementation of SaMK in a MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strain YMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively, mediating MVA biosynthesis in yeast. An analysis of tissue expression patterns revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but weakly expressed in sapwood. SaPMK was highly expressed in roots and mature leaves, but weakly expressed in young leaves. Induction experiments with several elicitors showed that SaMK and SaPMK expression was upregulated by methyl jasmonate. These results will help to further study the role of MK and PMK genes during santalol biosynthesis in S. album.

Influence of Bacillus subtilis strain on abscisic acid content in ABA-deficient barley mutant and its wild type

The ability to produce phytohormones and influence their metabolism in plants is an important property of rhizosphere bacteria that determines their plant growth promoting effect. Since abscisic acid (ABA) reduces stomatal conductance and increases the ability of tissues to conduct water, maintenance of water balance in lettuce plants on the background of activation of their growth was associated with the accumulation of ABA under the influence of bacteria. The aim of the study is to test the hypothesis that the growth-stimulating effect of bacteria on plants depends on their ability to synthesize the hormone ABA. The plants were grown on a light platform; seedlings were treated with a bacterial suspension simultaneously with planting. The ABA content, the relative water content, the chlorophyll content, the level of non-photochemical quenching, the leaf area and the weight of the shoots were measured. The level of transcripts of the HvNCED1, HvNCED2, and HvCYP707A1 genes responsible for ABA metabolism in barley was assessed using real-time PCR. Comparison of the ABA-deficient mutant of barley and plants of its wild type revealed the stimulation of the growth of plants of both genotypes upon bacterial treatment. The shoot mass and leaf area of the untreated mutant with bacteria were about 30 % less compared to Steptoe. The stimulating effect of bacteria was manifested in an increase in leaf area by 15 % in Steptoe and by 35 % in Az 34; shoot mass – by 18 % and 41 %, respectively. As a result, the phenotype difference between plants of two genotypes decreased. In the deficient mutant, the ABA level increased under the influence of Bacillus subtilis IB-22 more than twice. It was due to the ability of bacteria to produce ABA and reduce the activity of ABA degradation in barley plants. The results obtained in this study indicate that certain bacterial strains are able to increase the level of ABA in plants, compensating for the genetically determined deficiency of this hormone.

Structure-function characterization of Streptococcus intermedius surface antigen Pas

Streptococcus intermedius , an oral commensal bacterium, is found at various sites including subgingival dental plaque, purulent infections, and in cystic fibrosis lungs. Oral streptococci utilize proteins on their surface to adhere to tissues and/or surfaces localizing the bacteria, which subsequently leads to the development of biofilms, colonization and infection. Among the 19 genomically annotated cell-wall attached surface proteins on S. intermedius , Pas is an adhesin that belongs to the Antigen I/II (AgI/II) family. Here we have structurally and functionally characterized Pas, particularly focusing on its microbial-host as well as microbial-microbial interactions. The crystal structures of V Pas and C 123 Pas show high similarity with AgI/II of S. mutans . V Pas hosts a conserved metal binding site, and likewise the C 123 Pas structure retains its conserved metal binding sites and isopeptide bonds within its three DEv-IgG domains. Pas interacts with nanomolar affinity to lung alveolar glycoprotein 340 (Gp340), its scavenger receptor cysteine rich domains (SRCRs) and with fibrinogen. Both Candida albicans and Pseudomonas aeruginosa , the opportunistic pathogens that cohabitate with S. intermedius in the lungs of CFTR patients were studied in dual-species biofilm studies. The Pas deficient mutant (Δ pas ) displayed significant reduction in dual biofilm formation with C. albicans . In similar studies with P. aeruginosa , Pas did not mediate the biofilm formation with either the acute isolate (PAO1), or the chronic isolate (FRD1). However, the Sortase A deficient mutant (Δ srtA ) displayed reduced biofilm formation with both C. albicans and P. aeruginosa FRD1. Taken together, our findings highlight the role of Pas in both microbial-host and interkingdom interactions and expose its potential role in disease outcomes. Importance Streptococcus intermedius , an oral commensal bacterium, has been clinically observed in subgingival dental plaque, purulent infections, and in cystic fibrosis lungs. In this study, we have (a) determined the crystal structure of the V- and C-regions of Pas; (b) shown that its surface protein Pas adheres to fibrinogen, which could potentially ferry the microbe through the blood stream from the oral cavity; (c) characterized Pas’s high affinity adherence to lung alveolar protein Gp340 that could fixate the microbe on lung epithelial cells; and (d) most importantly shown that these surface proteins on the oral commensal S. intermedius enhances biofilms of known pathogens Candida albicans and Pseudomonas aeruginosa .

ExoS/ChvI Two-Component Signal-Transduction System Activated in the Absence of Bacterial Phosphatidylcholine

Sinorhizobium meliloti contains the negatively charged phosphatidylglycerol and cardiolipin as well as the zwitterionic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) as major membrane phospholipids. In previous studies we had isolated S. meliloti mutants that lack PE or PC. Although mutants deficient in PE are able to form nitrogen-fixing nodules on alfalfa host plants, mutants lacking PC cannot sustain development of any nodules on host roots. Transcript profiles of mutants unable to form PE or PC are distinct; they differ from each other and they are different from the wild type profile. For example, a PC-deficient mutant of S. meliloti shows an increase of transcripts that encode enzymes required for succinoglycan biosynthesis and a decrease of transcripts required for flagellum formation. Indeed, a PC-deficient mutant is unable to swim and overproduces succinoglycan. Some suppressor mutants, that regain swimming and form normal levels of succinoglycan, are altered in the ExoS sensor. Our findings suggest that the lack of PC in the sinorhizobial membrane activates the ExoS/ChvI two-component regulatory system. ExoS/ChvI constitute a molecular switch in S. meliloti for changing from a free-living to a symbiotic life style. The periplasmic repressor protein ExoR controls ExoS/ChvI function and it is thought that proteolytic ExoR degradation would relieve repression of ExoS/ChvI thereby switching on this system. However, as ExoR levels are similar in wild type, PC-deficient mutant and suppressor mutants, we propose that lack of PC in the bacterial membrane provokes directly a conformational change of the ExoS sensor and thereby activation of the ExoS/ChvI two-component system.