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.

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.

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.

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.

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.

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.

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.

Triolein emulsion enhances temozolomide brain delivery: an experimental study in rats.

PURPOSE: To evaluate the enhancement of temozolomide (TMZ) delivery in the rat brain using a triolein emulsion. MATERIALS AND METHODS: Rats were divided into the five groups as following: group 1 (negative control), group 2 (treated with triolein emulsion and TMZ 20 mg/kg), and group 3 (TMZ 20 mg/kg treatment without triolein), group 4 (treated with triolein emulsion and TMZ 10 mg/kg), and group 5 (TMZ 10 mg/kg treatment without triolein). Triolein emulsion was infused into the right common carotid artery. One hour later, the TMZ concentration was evaluated quantitatively and qualitatively using high-performance liquid chromatography (HPLC-MS) and desorption electrospray ionization mass spectrometry (DESI-MS) imaging, respectively. The concentration ratios of the ipsilateral to contralateral hemisphere in each group were determined and the statistical analysis was conducted using an unpaired t-test. RESULTS: Quantitatively, the TMZ concentration ratio of the ipsilateral to the control hemisphere was 2.41 and 1.13 in groups 2 and 3, and were 2.49 and 1.14 in groups 4 and 5, respectively. Thus, the TMZ signal intensities of TMZ in group 2 and 4 were statistically high in the ipsilateral hemispheres. Qualitatively, the signal intensity of TMZ was remarkably high in the ipsilateral hemisphere in group 2 and 4. CONCLUSIONS: The triolein emulsion efficiently opened the blood-brain barrier and could provide a potential new strategy to enhance the therapeutic effect of TMZ. HPLC-MS and DESI-MS imaging were shown to be suitable for analyses of enhancement of brain TMZ concentrations.

Novel quinoline-based fluorescent bioimaging probe, KSNP117, for sentinel lymph node mapping.

Fluorescent imaging agents with biocompatibility and high sensitivity are urgently required for the accurate detection of sentinel lymph nodes (SLNs). Herein, we report the design of a novel quinoline-based fluorescent probe, designated KSNP117, which can be applied as a biomedical imaging agent in the sensitive and quantitative detection of SLNs. KSNP117 exerted no adverse effects on the proliferation of ovary and immune cells and also showed excellent serum stability with photo-brightening effects. In vivo fluorescent imaging revealed the accumulation of KSNP117 in the SLNs of nude mice within 10 min post injection, without in vivo toxicity, which was consistent with the findings of ex vivo imaging. These results support the potential of KSNP117 as a promising lymphatic tracer for biomedical imaging applications.

Development of a Noninvasive KIM-1-Based Live-Imaging Technique in the Context of a Drug-Induced Kidney-Injury Mouse Model.

The development of reliable methods to diagnose acute kidney injury is essential to allow the adoption of early therapeutic interventions and evaluate their effectiveness. Based on the fact that kidney injury molecule-1 (KIM-1) expression levels in kidneys are markedly upregulated early after a damage event, here we developed a noninvasive KIM-1-based molecular imaging technique to detect kidney injury. First, we took advantage of a phage-display platform to select small peptides demonstrating a specific high binding affinity to KIM-1. The promising candidate was conjugated with fluorescent probes, and its imaging potential was validated in vitro and in vivo. This peptide, with the sequence CNRRRA, not only showed a high imaging potential in vitro, allowing a strong detection of KIM-1 expressing cells by microscopy and flow cytometry but also generated a strong kidney-specific signal in live-imaging in vivo experiments in the context of a drug-induced kidney-injury mouse model. Our data overall suggest that the CNRRRA peptide is a promising probe to use in the context of in vivo imaging for the detection of KIM-1 overexpression in damaged kidneys.