Novel melatonin-producing Bacillus safensis EH143 mitigates salt and cadmium stress in soybean.

Recently, microorganism and exogenous melatonin application has been recognized as an efficient biological tool for enhancing salt tolerance and heavy metal detoxification in agriculture crops. Thus, the goal of this study was to isolate and evaluate a novel melatonin-producing plant growth promoting bacterium. With high-throughput whole genome sequencing, phytohormone measurements, expression profiling, and biochemical analysis, we can identify a novel PGPB that produces melatonin and unravel how it promotes soybean growth and development and protects against salt and Cd stress. We identify the melatonin synthesis pathway (tryptophan→tryptamine→serotonin melatonin) of the halotolerant (NaCl > 800 mM) and heavy metal-resistant (Cd >3 mM) rhizobacterium Bacillus safensis EH143 and use it to treat soybean plants subjected to Cd and NaCl stresses. Results show that EH143 will highly bioaccumulate heavy metals and significantly improve P and Ca2+ uptake and the K+/Na+ (93%↑under salt stress) ratio while reducing Cd uptake (49% under Cd stress) in shoots. This activity was supported by the expression of the ion regulator HKT1, MYPB67, and the calcium sensors CDPK5 and CaMK1 which ultimately led to increased plant growth. EH143 significantly decreased ABA content in shoots by 13%, 20%, and 34% and increased SA biosynthesis in shoots by 14.8%, 31%, and 48.2% in control, salt, and Cd-treated plants, upregulating CYP707A1 and CYP707A2 and PAL1 and ICS, respectively. The melatonin content significantly decreased along with a reduced expression of ASMT3 following treatment with EH143; moreover, reduced expression of peroxidase (POD) and superoxide dismutase (SOD) by 134.5% and 39% under salt+Cd stress, respectively and increased level of total amino acids were observed. Whole-genome sequencing and annotation of EH143 revealed the presence of the melatonin precursor tryptophan synthase (trpA, trpB, trpS), metal and other ion regulators (Cd: cadA, potassium: KtrA and KtrB, phosphate: glpT, calcium: yloB, the sodium/glucose cotransporter: sgIT, and the magnesium transporter: mgtE), and enzyme activators (including the siderophore transport proteins yfiZ and yfhA, the SOD sodA, the catalase katA1, and the glutathione regulator KefG) that may be involved in programming the plant metabolic system. As a consequence, EH143 treatment significantly reduced the contents of lipid peroxidation (O2-, MDA, and H2O2) up to 69%, 46%, and 29% in plants under salt+Cd stress, respectively. These findings suggest that EH143 could be a potent biofertilizer to alleviate NaCl and Cd toxicity in crops and serve as an alternative substitute for exogenous melatonin application.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Chitosan-GSNO nanoparticles: a positive modulator of drought stress tolerance in soybean.

BACKGROUND: Chitosan biopolymer is an emerging non-toxic and biodegradable plant elicitor or bio-stimulant. Chitosan nanoparticles (CSNPs) have been used for the enhancement of plant growth and development. On the other hand, NO is an important signaling molecule that regulates several aspects of plant physiology under normal and stress conditions. Here we report the synthesis, characterization, and use of chitosan-GSNO nanoparticles for improving drought stress tolerance in soybean. RESULTS: The CSGSNONPs released NO gas for a significantly longer period and at a much lower rate as compared to free GSNO indicating that incorporation of GSNO in CSNPs can protect the NO-donor from rapid decomposition and ensure optimal NO release. CS-GSNONPs improved drought tolerance in soybean plants reflected by a significant increase in plant height, biomass, root length, root volume, root surface area, number of root tips, forks, and nodules. Further analyses indicated significantly lower electrolyte leakage, higher proline content, higher catalase, and ascorbate peroxidase activity, and reduction in MDA and H2O2 contents after treatment with 50 µM CS-GSNONPs under drought stress conditions. Quantitative real-time PCR analysis indicated that CS-GSNONPs protected against drought-induced stress by regulating the expression of drought stress-related marker genes such as GmDREB1a, GmP5CS, GmDEFENSIN, and NO-related genes GmGSNOR1 and GmNOX1. CONCLUSIONS: This study highlights the potential of nano-technology-based delivery systems for nitric oxide donors to improve plant growth, and development and protect against stresses.

PARP Inhibitor Sensitizes BRCA-mutant Pancreatic Cancer to Oxaliplatin by Suppressing the CDK1/BRCA1 Axis.

BACKGROUND/AIM: Currently, olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, has been approved as maintenance therapy for patients with germline BRCA mutations and metastatic pancreatic cancer. However, platinum-based chemotherapy, which induces synthetic lethality with PARP inhibitor treatment, is still controversial. Hence, we aimed to examine a platinum-based drug in combination with a PARP inhibitor and generate data regarding the use of a PARP inhibitor in the overall treatment of pancreatic cancer. MATERIALS AND METHODS: Using the Capan-1 cell line (BRCA2-mutant pancreatic cancer cell line), we evaluated the combinatorial effects of olaparib, a PARP inhibitor, and oxaliplatin by cell viability, combination index, western blotting, immunocytochemistry, flow cytometry, apoptosis assays and in vivo experiments. RESULTS: Capan-1 cells showed high sensitivity to olaparib due to the alteration in PARP activity, which led to cell death through the accumulation of oxaliplatin-induced DNA damage. Beyond DNA damage, oxaliplatin also suppressed the CDK1/BRCA1 signaling axis, which induced defects in homologous recombination repair. Additionally, inhibition of CDK1, a biomarker for oxaliplatin efficacy, induced cell death regardless of the BRCA mutation profile. CONCLUSION: Oxaliplatin may be used in combination with olaparib in PDAC patients with DNA damage repair mutations. Our findings highlight CDK1 as a potential therapeutic target for pancreatic cancer.

Discovery and Feasibility Study of Medical Fluorophore 33 as a Novel Theranostic Agent.

Fluorescent dyes have garnered significant attention as theranostic platforms owing to their inherent characteristics. In this study, we present the discovery of Medical Fluorophore 33 (MF33), a novel and potent theranostic agent with a phenaleno-isoquinolinium salt structure that can serve as a cancer therapeutic strategy. The synthesis of MF33 is readily achievable through a simple Rh(III)-catalyzed reaction. Moreover, MF33 displayed strong fluorescence signals, excellent microsomal stability, and high biocompatibility in vivo. It induces significant apoptosis in cancer cells via the p53/p21/caspase-3 signaling pathway, leading to selective cytotoxicity in various cancer cells. In vivo fluorescence imaging with MF33 enabled the visualization of sentinel lymph nodes in living mice. Notably, repeated intraperitoneal administration of MF33 resulted in antitumor activity in mice with colorectal cancer. Collectively, our findings suggest that phenaleno-isoquinolinium salt-based MF33 is a viable theranostic agent for biomedical imaging and cancer treatment.

S-Nitrosoglutathione (GSNO)-Mediated Lead Detoxification in Soybean through the Regulation of ROS and Metal-Related Transcripts.

Heavy metal toxicity, including lead (Pb) toxicity, is increasing in soils, and heavy metals are considered to be toxic in small amounts. Pb contamination is mainly caused by industrialization (e.g., smelting and mining), agricultural practices (e.g., sewage sludge and pests), and urban practices (e.g., lead paint). An excessive concentration of Pb can seriously damage and threaten crop growth. Furthermore, Pb adversely affects plant growth and development by affecting the photosystem, cell membrane integrity, and excessive production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2-). Nitric oxide (NO) is produced via enzymatic and non-enzymatic antioxidants to scavenge ROS and lipid peroxidation substrates to protect cells from oxidative damage. Thus, NO improves ion homeostasis and confers resistance to metal stress. In the present study, we investigated the effect of exogenously applied NO and S-nitrosoglutathione in soybean plants Our results demonstrated that exogenously applied NO aids in better growth under lead stress due to its ability in sensing, signaling, and stress tolerance in plants under heavy metal stress along with lead stress. In addition, our results showed that S-nitrosoglutathione (GSNO) has a positive effect on soybean seedling growth under lead-induced toxicity and that NO supplementation helps to reduce chlorophyll maturation and relative water content in leaves and roots following strong bursts under lead stress. GSNO supplementation (200 µM and 100 µM) reduced compaction and approximated the oxidative damage of MDA, proline, and H2O2. Moreover, under plant stress, GSNO application was found to relieve the oxidative damage by reactive oxygen species (ROS) scavenging. Additionally, modulation of NO and phytochelatins (PCS) after prolonged metal reversing GSNO application confirmed detoxification of ROS induced by the toxic metal lead in soybean. In summary, the detoxification of ROS caused by toxic metal concentrations in soybean is confirmed by using NO, PCS, and traditionally sustained concentrations of metal reversing GSNO application.

Enhanced Resistance of atnigr1 against Pseudomonas syringae pv. tomato Suggests Negative Regulation of Plant Basal Defense and Systemic Acquired Resistance by AtNIGR1 Encoding NAD(P)-Binding Rossmann-Fold in Arabidopsis thaliana.

Nitric oxide (NO) regulates several biological and physiological processes in plants. This study investigated the role of Arabidopsis thaliana Negative Immune and Growth Regulator 1 (AtNIGR1), encoding an NAD(P)-binding Rossmann-fold superfamily, in the growth and immunity of Arabidopsis thaliana. AtNIGR1 was pooled from the CySNO transcriptome as a NO-responsive gene. Seeds of the knockout (atnigr1) and overexpression plants were evaluated for their response to oxidative [(hydrogen peroxide (H2O2) and methyl viologen (MV)] or nitro-oxidative [(S-nitroso-L-cysteine (CySNO) and S-nitroso glutathione (GSNO)] stress. Results showed that the root and shoot growth of atnigr1 (KO) and AtNIGR1 (OE) exhibited differential phenotypic responses under oxidative and nitro-oxidative stress and normal growth conditions. To investigate the role of the target gene in plant immunity, the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000 virulent (Pst DC3000 vir) was used to assess the basal defense, while the Pst DC3000 avirulent (avrB) strain was used to investigate R-gene-mediated resistance and systemic acquired resistance (SAR). Data revealed that AtNIGR1 negatively regulated basal defense, R-gene-mediated resistance, and SAR. Furthermore, the Arabidopsis eFP browser indicated that the expression of AtNIGR1 is detected in several plant organs, with the highest expression observed in germinating seeds. All results put together suggest that AtNIGR1 could be involved in plant growth, as well as basal defense and SAR, in response to bacterial pathogens in Arabidopsis.

N-methyl-D-aspartate receptors induce M1 polarization of macrophages: Feasibility of targeted imaging in inflammatory response in vivo.

BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are considered to be involved in several physiological and pathophysiological processes in addition to the progression of neurological disorders. However, how NMDARs are involved in the glycolytic phenotype of M1 macrophage polarization and the possibility of using them as a bio-imaging probe for macrophage-mediated inflammation remain unclear. METHODS: We analyzed cellular responses to NMDAR antagonism and small interfering RNAs using mouse bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS). An NMDAR targeting imaging probe, N-TIP, was produced via the introduction of NMDAR antibody and the infrared fluorescent dye FSD Fluor™ 647. N-TIP binding efficiency was tested in intact and LPS-stimulated BMDMs. N-TIP was intravenously administered to mice with carrageenan (CG)- and LPS-induced paw edema, and in vivo fluorescence imaging was conducted. The anti-inflammatory effects of dexamethasone were evaluated using the N-TIP-mediated macrophage imaging technique. RESULTS: NMDARs were overexpressed in LPS-treated macrophages, subsequently inducing M1 macrophage polarization. Mechanistically, NMDAR-mediated Ca2+ accumulation resulted in LPS-stimulated glycolysis via upregulation of PI3K/AKT/mTORC1 signaling. In vivo fluorescence imaging with N-TIP showed LPS- and CG-induced inflamed lesions at 5 h post-inflammation, and the inflamed lesions could be detected until 24 h. Furthermore, our N-TIP-mediated macrophage imaging technique helped successfully visualize the anti-inflammatory effects of dexamethasone in mice with inflammation. CONCLUSION: This study demonstrates that NMDAR-mediated glycolysis plays a critical role in M1 macrophage-related inflammation. Moreover, our results suggest that NMDAR targeting imaging probe may be useful in research on inflammatory response in vivo.

Exogenous SA Applications Alleviate Salinity Stress via Physiological and Biochemical changes in St John's Wort Plants.

The plant St. John's wort contains high levels of melatonin, an important biochemical that has both beneficial and adverse effects on stress. Therefore, a method for increasing melatonin levels in plants without adversely affecting their growth is economically important. In this study, we investigated the regulation of melatonin levels in St. John's wort by exposing samples to salinity stress (150 mM) and salicylic acid (0.25 mM) to augment stress tolerance. The results indicated that salinity stress significantly reduced the plant chlorophyll content and damaged the photosystem, plant growth and development. Additionally, these were reconfirmed with biochemical indicators; the levels of abscisic acid (ABA) and proline were increased and the activities of antioxidants were reduced. However, a significant increase was found in melatonin content under salinity stress through upregulation in the relative expression of tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT), and N-acetylserotonin methyltransferase (ASMT). The salicylic acid (SA) treatment considerably improved their photosynthetic activity, the maximum photochemical quantum yield (133%), the potential activity of PSⅡ (294%), and the performance index of electron flux to the final PS I electron acceptors (2.4%). On the other hand, SA application reduced ABA levels (32%); enhanced the activity of antioxidant enzymes, such as superoxide dismutase (SOD) (15.4%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (120%); and increased polyphenol (6.4%) and flavonoid (75.4%) levels in salinity-stressed St. John's wort plants. Similarly, SA application under NaCl stress significantly modulated the melatonin content in terms of ion balance; the level of melatonin was reduced after SA application on salt-treated seedlings but noticeably higher than on only SA-treated and non-treated seedlings. Moreover, the proline content was reduced considerably and growth parameters, such as plant biomass, shoot length, and chlorophyll content, were enhanced following treatment of salinity-stressed St. John's wort plants with salicylic acid. These findings demonstrate the beneficial impact of salt stress in terms of a cost-effective approach to extract melatonin in larger quantities from St. John's wort. They also suggest the efficiency of salicylic acid in alleviating stress tolerance and promoting growth of St. John's wort plants.

Epithelial-Mesenchymal Transition Phenotype and Peritumoral Immune Cell Infiltration in Advanced Biliary Tract Cancer.

BACKGROUND/AIM: This study evaluated the clinical implications of epithelial-mesenchymal transition (EMT) markers and peritumoral immune cell infiltration in patients with biliary tract cancer (BTC) treated with gemcitabine plus cisplatin (GemCis). MATERIALS AND METHODS: Forty-five patients with advanced BTC who received GemCis were included as the study population. We conducted multiplex immunohistochemistry and examined EMT markers and their correlations with immune cell infiltrate at the invasive tumor margin. Study population was subdivided into two groups: twenty-four patients with overall survival (OS) less than 10 months (short-term survivor group, SS) and 21 with OS of 20 months or longer (long-term survivor group, LS). RESULTS: The density of tumor cells expressing epithelial marker E-cadherin (E-cadherin+ CK+) at the invasive tumor margin tended to be higher in the LS group than that in the SS group (p=0.065). The density of tumor cells expressing mesenchymal marker vimentin (vimentin+ CK+) was significantly higher in the SS group than that in the LS group (p=0.021). The density of E-cadherin- vimentin+ tumor cells (E-cadherin- vimentin+ CK+) was also significantly higher in the SS group (p=0.020). The density of OX40 expressing cells was significantly higher in the SS group compared to that in the LS group (p=0.006). The density of vimentin-expressing tumor cells was positively correlated with FoxP3+ CD4+ regulatory T-cells (r=0.29, p=0.047) and OX40+ cells (r=0.48, p<0.001). CONCLUSION: EMT-related features were enriched in BTC patients with poor survival outcomes and associated with regulatory T-cell infiltration.

Preceptor Perceptions of Pharmacy Student Performance Before and After a Curriculum Transformation.

Objective. To explore preceptors' perceptions about the performance of undergraduate pharmacy students during experiential placements in Australia, before and after curricular transformation.Methods. Using a semi-structured approach, we interviewed 26 preceptors who had recently supervised students who took part in the transformed curriculum and students from the previous curriculum. A directed content analysis approach was used to analyze the transcripts.Results. Preceptors described students from the transformed curriculum as having improved professional skills, behaviors, and attitudes and as having an increased ability to perform clinical activities compared to students of the previous curriculum. Preceptors also perceived that students in the transformed curriculum had improved clinical knowledge and knowledge application. They less frequently expressed that students in the transformed curriculum had lower-than-expected knowledge levels.Conclusion. The results of this study suggest that curricular transformation with a focus on skill-based and active learning can improve the performance of pharmacy students in terms of their professional behaviors and attitudes, skills, knowledge, and clinical abilities, as perceived by preceptors.

Heavy metal toxicity in plants and the potential NO-releasing novel techniques as the impending mitigation alternatives.

Environmental pollutants like heavy metals are toxic, persistent, and bioaccumulative in nature. Contamination of agricultural fields with heavy metals not only hampers the quality and yield of crops but also poses a serious threat to human health by entering the food chain. Plants generally cope with heavy metal stress by regulating their redox machinery. In this context, nitric oxide (NO) plays a potent role in combating heavy metal toxicity in plants. Studies have shown that the exogenous application of NO donors protects plants against the deleterious effects of heavy metals by enhancing their antioxidative defense system. Most of the studies have used sodium nitroprusside (SNP) as a NO donor for combating heavy metal stress despite the associated concerns related to cyanide release. Recently, NO-releasing nanoparticles have been tested for their efficacy in a few plants and other biomedical research applications suggesting their use as an alternative to chemical NO donors with the advantage of safe, slow and prolonged release of NO. This suggests that they may also serve as potential candidates in mitigating heavy metal stress in plants. Therefore, this review presents the role of NO, the application of chemical NO donors, potential advantages of NO-releasing nanoparticles, and other NO-release strategies in biomedical research that may be useful in mitigating heavy metal stress in plants.

Exogenously Applied Sodium Nitroprusside Mitigates Lead Toxicity in Rice by Regulating Antioxidants and Metal Stress-Related Transcripts.

Sustainable agriculture is increasingly being put in danger by environmental contamination with dangerous heavy metals (HMs), especially lead (Pb). Plants have developed a sophisticated mechanism for nitric oxide (NO) production and signaling to regulate hazardous effects of abiotic factors, including HMs. In the current study, we investigated the role of exogenously applied sodium nitroprusside (SNP, a nitric oxide (NO) donor) in ameliorating the toxic effects of lead (Pb) on rice. For this purpose, plants were subjected to 1.2 mM Pb alone and in combination with 100 µM SNP. We found that under 1.2 mM Pb stress conditions, the accumulation of oxidative stress markers, including hydrogen peroxide (H2O2) (37%), superoxide anion (O2-) (28%), malondialdehyde (MDA) (33%), and electrolyte leakage (EL) (34%), was significantly reduced via the application of 100 µM SNP. On the other hand, under the said stress of Pb, the activity of the reactive oxygen species (ROS) scavengers such as polyphenol oxidase (PPO) (60%), peroxidase (POD) (28%), catalase (CAT) (26%), superoxide dismutase (SOD) (42%), and ascorbate peroxidase (APX) (58%) was significantly increased via the application of 100 µM SNP. In addition, the application of 100 µM SNP rescued agronomic traits such as plant height (24%), number of tillers per plant (40%), and visible green pigments (44%) when the plants were exposed to 1.2 mM Pb stress. Furthermore, after exposure to 1.2 mM Pb stress, the expression of the heavy-metal stress-related genes OsPCS1 (44%), OsPCS2 (74%), OsMTP1 (83%), OsMTP5 (53%), OsMT-I-1a (31%), and OsMT-I-1b (24%) was significantly enhanced via the application of 100 µM SNP. Overall, our research evaluates that exogenously applied 100 mM SNP protects rice plants from the oxidative damage brought on by 1.2 mM Pb stress by lowering oxidative stress markers, enhancing the antioxidant system and the transcript accumulation of HMs stress-related genes.

The Casein Kinase 2 Inhibitor CX-4945 Promotes Cholangiocarcinoma Cell Death Through PLK1.

BACKGROUND/AIM: Casein Kinase 2 (CK2) is a prosurvival protein kinase involved in cell growth/proliferation through the regulation of the cell cycle and apoptosis. CK2 is over-expressed in various cancers, which correlates with a poor prognosis. This study examined the anti-cancer effects of silmitasertib (CX-4945), a CK2 inhibitor, on cholangiocarcinoma (CCA) cells. MATERIALS AND METHODS: The effects of CX-4945 on cell viability, cell cycle arrest, and apoptosis in the human cholangiocarcinoma cell lines TFK-1 and SSP-25 were evaluated. Alterations in posttranslational modifications and the levels of cell cycle regulators including p21, Polo-like kinase 1 (PLK1), andp53 were assessed by western blotting. Apoptotic responses were examined using Propidium iodine/Annexin V staining. RESULTS: TFK-1 and SSP-25 cells exposed to CX-4945 showed morphologic changes and a more than 50% decrease in cell viability (p<0.05). Cell cycle arrest at the G2 phase was detected following an increase in phosphorylated PLK1 and p21. Furthermore, phospho-PLK1 induced the degradation of p53, which led to the dissociation of Bax from Bcl-xL. The cleavage of Caspase3 and PARP were also induced by CX-4945 treatment. CONCLUSION: CX-4945 induces cell cycle arrest and cell death in cholangiocarcinoma cells via the regulation of PLK1 and p53. This may provide a novel therapeutic strategy for advanced cholangiocarcinoma.

Correction: Pande et al. Nitric Oxide Signaling and Its Association with Ubiquitin-Mediated Proteasomal Degradation in Plants. Int. J. Mol. Sci. 2022, 23, 1657.

The authors wish to make the following corrections to the original publication [...].

Phytohormonal Regulation Through Protein S-Nitrosylation Under Stress.

The liaison between Nitric oxide (NO) and phytohormones regulates a myriad of physiological processes at the cellular level. The interaction between NO and phytohormones is mainly influenced by NO-mediated post-translational modifications (PTMs) under basal as well as induced conditions. Protein S-nitrosylation is the most prominent and widely studied PTM among others. It is the selective but reversible redox-based covalent addition of a NO moiety to the sulfhydryl group of cysteine (Cys) molecule(s) on a target protein to form S-nitrosothiols. This process may involve either direct S-nitrosylation or indirect S-nitrosylation followed by transfer of NO group from one thiol to another (transnitrosylation). During S-nitrosylation, NO can directly target Cys residue (s) of key genes involved in hormone signaling thereby regulating their function. The phytohormones regulated by NO in this manner includes abscisic acid, auxin, gibberellic acid, cytokinin, ethylene, salicylic acid, jasmonic acid, brassinosteroid, and strigolactone during various metabolic and physiological conditions and environmental stress responses. S-nitrosylation of key proteins involved in the phytohormonal network occurs during their synthesis, degradation, or signaling roles depending upon the response required to maintain cellular homeostasis. This review presents the interaction between NO and phytohormones and the role of the canonical NO-mediated post-translational modification particularly, S-nitrosylation of key proteins involved in the phytohormonal networks under biotic and abiotic stresses.

Nitric Oxide Signaling and Its Association with Ubiquitin-Mediated Proteasomal Degradation in Plants.

Nitric oxide (NO) is a versatile signaling molecule with diverse roles in plant biology. The NO-mediated signaling mechanism includes post-translational modifications (PTMs) of target proteins. There exists a close link between NO-mediated PTMs and the proteasomal degradation of proteins via ubiquitylation. In some cases, ubiquitin-mediated proteasomal degradation of target proteins is followed by an NO-mediated post-translational modification on them, while in other cases NO-mediated PTMs can regulate the ubiquitylation of the components of ubiquitin-mediated proteasomal machinery for promoting their activity. Another pathway that links NO signaling with the ubiquitin-mediated degradation of proteins is the N-degron pathway. Overall, these mechanisms reflect an important mechanism of NO signal perception and transduction that reflect a close association of NO signaling with proteasomal degradation via ubiquitylation. Therefore, this review provides insight into those pathways that link NO-PTMs with ubiquitylation.

Blockade of GRP78 Translocation to the Cell Surface by HDAC6 Inhibition Suppresses Proliferation of Cholangiocarcinoma Cells.

BACKGROUND/AIM: HDAC6, a cytoplasmic localized deacetylase, is a positive regulator of cancer progression via modification of various substrates. We evaluated how the interaction between HDAC6 and glucose regulatory protein 78 (GRP78) affects the growth of cholangiocarcinoma (CCA). MATERIALS AND METHODS: The anti-tumor effects of ACY-1215, an HDAC6 specific inhibitor, in CCA cell lines were analyzed by cell viability assay, western blotting, flow cytometry, co-immunoprecipitation, and biotinylation assays. In vivo effects of ACY-1215 were evaluated in a xenograft model using CCA cell line TFK-1. RESULTS: ACY-1215 increased the acetyl-form of GRP78 by approximately 50% compared to control, which impaired the translocation of GRP78 to the plasma membrane by 50% through alteration of cellular proliferative signaling via PI3K/AKT. Furthermore, ACY-1215 suppressed tumor growth by 50% compared to vehicle control in a CCA xenograft model. CONCLUSION: Increase in GRP78 acetylation by HDAC6 inhibition suppressed GRP78 translocation to the cell surface, which inhibited proliferation and promoted apoptosis in CCA.

Discovery of novel phenaleno isoquinolinium-based fluorescence imaging agents for sentinel lymph node mapping.

Fluorescence imaging agents have recently received huge attention due to their important role in disease diagnostics. However, the intrinsic problems of these probes, such as complex synthetic routes and high molecular weight, remain challenging. Here, we developed novel phenaleno isoquinolinium-based fluorescent agents, Medical Fluorophores 37-41 (MF37-41), applicable to the quantitative and sensitive detection of sentinel lymph nodes (SLNs). These imaging agents showed no adverse effects on the proliferation of immune and normal cells and did not induce in vivo toxicity. In vivo fluorescence lifetime imaging demonstrated the accumulation of phenaleno isoquinolinium salts in the SLNs of nude mice within 15 min postinjection, consistent with our biodistribution findings. These results suggest that phenaleno isoquinolinium salts are feasible fluorescence imaging agents that can be used as potential lymphatic tracers.