Inhibition Of Colony Growth Research Articles

Determination of Desirable Attributes of an Indigenous Burkholderia Isolate Towards Biological Control of Plant Pathogenic Fungi and Its Microbial Enzyme Production

Purpose: Microorganisms are proven bio-resources for the environmentally-friendly and sustainable biological control of plant diseases and microbial enzyme production. The present study confirms the identity of an indigenous Burkholderia isolate, determines its desirable features as a biological control agent of plant pathogenic fungi and an enzyme producer and analyzes molecular relationships with selected environmental isolates of Burkholderia. Research Method: In vitro antagonism of Burkholderia isolate on colony growth and spore germination of five fungal pathogens causing field and postharvest diseases were tested. Antifungal ability of cell-free filtrate and effect of temperature on antifungal ability were determined. Extracellular enzyme production of Burkholderia isolate was screened and phylogenetic relationships were elucidated. Findings: Burkholderia isolate inhibited colony growth of the five fungal pathogens by a range of 45 - 59 %, reduced the spore germination ability forming spores of a typical morphology. Antifungal ability was lost beyond 57 oC and cell-free filtrates did not show antagonism against the tested fungi. The bacterial isolate gave the best match with Burkholderia A45 strain and it was; catalase, gelatinase, lipase and casein hydrolysis positive. The indigenous bacterial isolate grouped together with Burkholderia strains, having biodegradation ability of environmental pollutants. Research Limitations: Absence of a comprehensive image on extracellular enzyme producing ability and information on biodegradation ability of the bacterial isolate. Originality/value: Findings will be useful to exploit the potential of the bacterium in integrated management of pre- and postharvest fungal diseases and in microbial enzyme production.

In Vitro Studies on the Growth Inhibiting Potential of Some Botanical Extracts Against Macrophomina phaseolina

Natural plant extracts are valuable source of numerous fungitoxic compounds that can substitute synthetic fungicides. In current studies, six plant extracts viz., Zingiber officinalis, Eucalyptus camaldulensis, Azadirachta indica, Allium cepa, Cassia fistula and Allium sativum in three dose levels standard dose (S.D), S/2 and S/3 were tested against colony growth of Macrophomina phaseolina under in vitro conditions. Among all the treatments ginger extract at its standard dose was found highly effective followed by eucalyptus and neem. Percentage colony growth inhibition of (50 %), (38 %) and (29 %) was recorded to produce by ginger, eucalyptus and ginger respectively as compared to control treatment where no growth inhibition was recorded. Extract of Allium sativum was found least effective phytochemical where pathogenic fungal growth inhibition was (24.5 %). Concentration comparison of three dose levels showed that (S.D) was highly effective where mean colony growth of M. phaseolina was (3.72 cm) followed by (4 cm) @ S/2 and maximum diameter was (4.36 cm) @ S/3 concentration.

Isolation, identification, carbon utilization profile and control of Pythium graminicola, the causal agent of chilli damping‐off

AbstractPostemergence damping‐off of chilli caused by Pythium spp. is a common and serious problem in large chilli growing areas of India under the moist conditions that generally prevails during the sowing period. Therefore, in order to better understand this disease, an isolate belonging to the genus Pythium (Pythiales) was isolated from the infected chilli (Capsicum annuum L.) plant root parts collected from the fields of Chandauli district, Uttar Pradesh, India. Based on the congruence of cultural, morphological, cardinal growth rate and the sequence data analysis, the isolate was identified as Pythium graminicola. The molecular phylogenetic analysis based on ITS‐rDNA sequences clustered the isolate with representative sequences for P. graminicola from GenBank in the Pythium clade. The isolate carbon utilization profiles were characterized using Biolog FF MicroPlate method. The results revealed that the isolate used a wide range of carbon sources, mainly carbohydrates, but also amino acids, suggesting the use of metabolic routes that include glycolysis/gluconeogenesis. Moreover, an in vitro colony growth inhibition assay was performed to determine the influence of chemical (fungicides) and biological (bacteria and fungi) antagonists over the pathogen using the poison plate and dual culture method, respectively. Overall, the results revealed that the presence of aggressive broad range biocontrol agents can be used as an effective environmentally friendly approach for management and control of damping‐off in production systems. The antagonist can serve as a bio‐efficient and eco‐friendly alternative to synthetic fungicides for the development of an effective integrated pest management (IPM) system and obtaining higher yields.

Pre-Clinical Efficacy of Co-Treatment with KDM1A (LSD1) Inhibitor and Ruxolitinib or BET Inhibitor Against Post-MPN-sAML Blast Progenitor Cells

LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates active enhancers and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1, mediating transcriptional repression and differentiation block in LSCs that persist in the minimal residual disease (MRD) following attainment of clinical complete remission, leading to relapse and poor outcome in AML. In AML LSCs, genetic alterations and epigenetic dysregulation of enhancers affect levels of myeloid transcriptional regulators, including c-Myc, PU.1, GATA 2 and CEBPα, and their target genes, which are involved in differentiation block in LSCs. Our present studies demonstrate that CRISPR/Cas9-mediated knockout of LSD1 in the AML OCI-AML5 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at super-enhancers and enhancers (SEs/Es) (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed CoREST, c-Myc, CDK6, and c-KIT, while inducing p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq, and protein expression by Western analyses). This led to significant growth inhibition, differentiation and loss of viability of OCI-AML5 and patient-derived AML blasts (p < 0.01). Similar effects were observed following exposure of OCI-AML5 (96 hours) to tet-inducible shRNA to LSD1. Knock-down of GFI1 by shRNA (by 90%) also inhibited growth and induced differentiation, associated with upregulation of PU.1, p21 and CD11b levels. Treatment with irreversible (INCB059872, 0.25 to 1.0 µM) or reversible (SP2577, 1.0 to 2.0 µM) LSD1 inhibitor (LSD1i) inhibited binding of LSD1 to CoREST, and significantly induced growth inhibition, differentiation and loss of viability (over 96 hours) of the OCI-AML5, post-myeloproliferative neoplasm (post-MPN) sAML SET2 and HEL92.1.7 cells, as well as patient-derived AML and post-MPN sAML blasts (p < 0.01). Co-treatment with INCB059872 and ruxolitinib synergistically induced apoptosis of the post-MPN sAML SET2 and HEL92.1.7 cells and patient-derived CD34+ post-MPN sAML blasts (combination indices < 1.0). Notably, pre-treatment with the LSD1i for 48 hours significantly re-sensitized ruxolitinib-persister/resistant SET2 and HEL92.1.7 cells to ruxolitinib (p < 0.001). We previously reported that treatment with the BET inhibitor (BETi) JQ1 or OTX015 represses SE/E-driven AML-relevant oncogenes including MYC, RUNX1, CDK6, PIM1, and Bcl-xL, while inducing p21 and p27 levels in post-MPN sAML blasts (Leukemia 2017;31:678-687). This was associated with inhibition of colony growth and loss of viability of AML and post-MPN sAML blasts (p < 0.01). Here, we determined that INCB059872 treatment induced similar levels of lethality in BETi-sensitive or BETi-persister/resistant AML and post-MPN sAML cells. Since BETi treatment also depleted LSD1 protein levels, co-treatment with the BETi OTX015 and LSD1i INCB059872 or SP2577 induced synergistic lethality in AML and post-MPN sAML blasts (combination indices < 1.0). Co-treatment with INCB059872 (1.5 mg/kg) and OTX015 (50 mg/kg) both orally for 21 days, compared to each agent alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with HEL92.1.7 or HEL92.1.7/OTX015-resistant-GFP/Luc sAML xenografts (p < 0.01). Collectively, these findings strongly support further in vivo testing and pre-clinical development of LSD1i-based combinations with ruxolitinib against post-MPN sAML and with BETi against AML or post-MPN sAML cells. Disclosures Bose: CTI BioPharma: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Constellation: Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Pfizer: Research Funding. Kadia:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Bioline RX: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding. Verstovsek:Ital Pharma: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; CTI BioPharma Corp: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Celgene: Consultancy, Research Funding; NS Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Sierra Oncology: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Roche: Research Funding.

Role of HDAC8 Upregulation in Resistance of FLT3-ITD AML Stem Cells to FLT3 Inhibitors

The FLT3-ITD mutation occurs in 25-30% of acute myeloid leukemia (AML) incidence, and is associated with higher relapse rate and inferior survival. FLT3 inhibitors have shown potent antileukemic effect, but the responses are not sustained in most FLT3-ITD AML patients. Histone deacetylases (HDACs) are crucial for cancer stem cell maintenance through epigenetically regulating gene expression, modulating the function of non-histone proteins via post-translational modification, and maintaining genome stability. Here we investigated the potential role of HDACs in FLT3 inhibitors resistance and FLT3-ITD AML stem cells maintenance. We firstly evaluated mRNA expression alterations of 11 HDACs in FLT3-ITD+ MV4-11 and MOLM-13 AML cells upon FLT3 inhibitor AC220 (Quizartinib) treatment, and found that HDAC8 was the most significantly upregulated gene. Inhibition of FLT3 signaling via siRNA-mediated knockdown of FLT3 also resulted in increased expression of HDAC8. Treatment of primary FLT3-ITD+ CD34+ AML cells with AC220 confirmed HDAC8 upregulation. Targeting HDAC8 using specific HDAC8 inhibitor 22d or knockdown of HDAC8 readily resulted in significant apoptosis of cell lines and primary AML samples, and inhibited colony growth and replating activity in primary FLT3-ITD+ CD34+ AML cells. Moreover, these antileukemic effects were even more pronounced upon 22d and AC220 combination. To explore the mechanism of HDAC8 mediated FLT3 inhibitor resistance, we investigated transcriptomic change of MV4-11 cells upon 22d treatment by RNA-seq. KEGG pathway analysis revealed that the p53 signaling pathway was significantly upregulated. Upstream regulator prediction by Ingenuity pathway analysis also showed p53 as the key activated upstream transcription factor. Western blot analysis on acetylated p53 (Lys382) demonstrated that targeting FLT3 signaling had little effect on the p53 acetylation, while suppression of HDAC8 markedly increased p53 acetylation, and this effect was even more significant when both FLT3 and HDAC8 were inhibited simultaneously. Knockdown of p53 could partially rescued FLT3-ITD AML cells from 22d and/or AC220 induced apoptosis, and offset the inhibitory effect on growth and colony formation. We next studied the mechanism of HDAC8 upregulation upon FLT3 signally inhibition. We found FOXO1 and FOXO3 (FOXOs) binding motifs in HDAC8 promoter. FLT3 signaling inhibition promoted the binding of FOXOs to HDAC8 promoter. Knockdown both FOXOs abrogated HDAC8 upregulation upon AC220 treatment, and caused growth inhibition and apoptosis of FLT3-ITD AML cells. Finally, to translate our experimental results into clinical usage, we tested the in vivo effect of 22d, AC220, and the combination of both on patient-derived xenograft (PDX) model engrafted with four primary FLT3-ITD AML samples. 22d or AC220 treatment reduced the percentage and total number of human CD45+ leukemic cells in murine bone marrow, and prolonged the survival of leukemic mice. The combination of 22d and AC220 significantly reduced AML cell engraftment compared with either 22d or AC220 alone, and further elongated the survival period. Human CD34+ cells in BM were markedly reduced in mice treated with 22d or AC220 alone, with further reduction seen in combination. Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced engraftment compared with AC220 treatment alone, indicating reduced LSC capacity of residual cells. These results show that combination of 22d and AC220 enhances targeting of primitive AML stem cells in vivo. In conclusion, upon FLT3 inhibitor treatment, FLT3-ITD AML cells may upregulate the expression of HDAC8 through FOXOs activation, which in turn inhibit the function of tumor suppressor gene p53, and promote the maintenance of FLT3-ITD AML stem cells and drug resistance. Targeting FLT3 and HDAC8 simultaneously may be promising approach to eliminating FLT3-ITD AML stem cells, and to cure this subset of leukemia. Disclosures No relevant conflicts of interest to declare.

STAT1 Transcriptional Response Predicts Molecular Responses of PB-Derived Clonogenic Cells from MPN Patients to Interferon Alpha

STAT1 Transcriptional Response Predicts Molecular Responses of PB-Derived Clonogenic Cells from MPN Patients to Interferon Alpha

Trichoderma asperellum biocontrol activity and induction of systemic defenses against Sclerotium cepivorum in onion plants under tropical climate conditions

The biocontrol potential of three native Costa Rican Trichoderma asperellum strains has been evaluated against the necrotrophic ascomycete Sclerotium cepivorum, the causal agent of onion (Allium cepa L.) white rot. In Costa Rica, where climatic conditions enhance the development of this pathogen, white rot reduces onion yields up to 50% of total harvest. In our study, the three T. asperellum strains tested in in vitro assays showed their capacity to antagonize S. cepivorum, with BCC1 displaying percentages of colony growth inhibition of the pathogen of 81 and 90% in dual culture and cellophane membrane assays, respectively. In addition, this Trichoderma strain was able to reduce a 74% the plant mortality compared to untreated plants under greenhouse conditions. In field trials, carried out in two consecutive harvest years and in two different tropical locations, the two tested dosages of T. asperellum BCC1 reduced the incidence of white rot in a 3.41% for the lowest dose and 3.61% for the highest dose when compared to onion plants treated with chemical fungicides. Additionally, a significantly increase of 20.4% in onion bulb yield was recorded for the highest dose of BCC1. The potential of T. asperellum BCC1 to induce systemic defenses in onion plants against S. cepivorum was evaluated for four onion defense marker genes in a 21-day time course study by quantitative real-time PCR and using onion plants grown under greenhouse conditions. The expression profile of AcPR1 and AcPAL1, characterized by being undulating, indicates an initial activation of salicylic acid-dependent defense pathways (1 and 7 days) by T. asperellum BCC1 when either applied alone or in combination with the pathogen, while the up-regulation of AcEIN3 observed in those same treatments at day 21 revealed the activation of ethylene-dependent defense pathways by this Trichoderma strain. T. asperellum BCC1 exerts efficient biocontrol against S. cepivorum and activates onion systemic defenses against this pathogen under greenhouse conditions, while it reduces onion white rot incidence and increases crop yield in field trials performed under Costa Rica’s tropical climate conditions.

The Potency of Anredera cordifolia as Botanical Pesticide for Sustainable Blast Disease (Pyricularia oryzae) Management on Paddy

Blast disease (Pyricularia oryzae) is one of the most important diseases of rice that can significantly reduce paddy production. This disease causes a potential loss of 3.65 tonnes/ha with loss percentages ranging from 60-100% if the disease is not handled properly. In spite of the issue of the negative effects of synthetic pesticide usage, the blast disease is commonly controlled using synthetic fungicides. Therefore, the exploration of potential botanical pesticides may provide safer and environmentally friendly blast disease management. This study aimed to test the potential of binahong plant (Anredera cordifolia) as botanical fungicide to inhibit the growth of P. oryzae and the development of blast disease in laboratory testing. The experiments were arranged in a Completely Randomized Design (CRD) consisting of 7 treatments of 5 extract concentrations and 2 controls. All treatments were repeated 5 times. The effectiveness of binahong leaf extract in controlling the pathogen and the disease was tested using poisoned food technique and detached leaf assay. The binahong leaf extract tested in the concentration range of 0.25-2% showed the ability to inhibit the growth of P. oryzae with the highest colony growth inhibition of 25.11%. Characteristics of P. oryzae mycelium and colony in the binahong leaf extract treatment were different from the control treatment. The treatment of paddy leaves with binahong leaf extract suppressed the blast disease development through the lessening of diseased paddy leaf area percentages. This study indicates the potency of binahong leaf extract for sustainable rice blast disease management.

Nitro aspirin (NCX4040) induces apoptosis in PC3 metastatic prostate cancer cells via hydrogen peroxide (H2O2)-mediated oxidative stress

Non-steroidal anti-inflammatory drugs (NSAID) have shown promise as anticancer agents by inducing cell death apart from their antipyretic, anti-inflammatory and anti-thrombogenic effects. In our current study, we investigated the oxidative stress mediated cell death mechanism of a NSAID derivative NCX4040 (a nitric oxide (NO) releasing form of aspirin) in castration-resistant prostate cancer (CRPC) PC3 cell line. Our data revealed that NCX4040 is more potent than its parent compound aspirin or NO releasing compound DETA NONOate. NCX4040 significantly induced hydrogen peroxide formation with ensuing oxidative stress and mitochondrial depolarization resulting in lipid peroxidation, cell cycle arrest, inhibition of colony growth and induction of apoptosis in PC3 cells. Moreover, NCX4040 inhibited migration potential of PC3 cells by depolymerizing F-actin and promoting anoikis. Interestingly, elevated levels of NADPH oxidase 1 (NOX1), superoxide dismutase (SOD) 1 and 2 were observed upon NCX4040 treatment. However, down regulation of anti-apoptotic markers B-cell lymphoma 2 (Bcl2) and anti-oxidant thioredoxin reductase 1 (TXNRD1) expression were observed. In addition, NCX4040 down regulated cyclin D1 expression in PC3 cells further supporting the anticancer effect of NCX4040. Western blot analysis revealed that significant down regulation of key anti-apoptotic markers such as cellular inhibitor of apoptosis protein-1 (cIAP1), X-linked inhibitor of apoptosis (XIAP), survivin, and Cellular-Myc (c-Myc). On the other hand, NCX4040-treated cells showed upregulation of phosho histone H2AX (pH2AX), cleaved caspase3 and cleaved Poly [ADP-ribose] polymerase 1 (PARP1). Taken together, our data demonstrate that NCX4040 treatment enhances free radical formation which in turn induces oxidative stress leading to mitochondrial mediated cell death in metastatic PC3 cells.

Allelopathic effects and potential active substances of Ceratophyllum demersum L. on Chlorella vulgaris Beij.

Morphological responses of green algae to submerged macrophytes were rarely studied, and the potential active substances played roles in the interaction were little known previously. In the current work, according to acetone extract experiments, it was demonstrated that submerged macrophytes (Ceratophyllum demersum L.) could allelopathically inhibit the growth of Chlorella vulgaris Beij. and induce its colony formation, the effects of which were concentration dependent. Gas chromatography–mass spectrometry analysis revealed eight kinds of active substances from the C. demersum extracts, namely hexanoic acid, phthalic acid, octanedioic acid, butenoic acid, azelaic acid, palmitic acid, alpha linolenic acid and pentanedioic acid. Standard compound addition test indicated that palmitic acid and alpha linolenic acid might play important roles in the induction of colony formation and growth inhibition of C. vulgaris. This study provided some more new insights into the photosynthetic organism interaction between submerged macrophytes and green algae, in terms of not only growth but also morphological responses.

Integrated management of Meloidogyne incognita and Fusarium oxysporum in cucumber by combined application of abamectin and fludioxonil

The southern root-knot nematode (Meloidogyne incognita) and Fusarium wilt (Fusarium oxysporum) are major constraints in cucumber production worldwide. Co-infection of these pathogens results in a disease complex which is difficult to control. In China, growers apply nematicides and fungicides separately to control M. incognita and F. oxysporum, respectively, which requires a great amount of labor thus increasing production costs. We hypothesized that the combined application of nematicides and fungicides would significantly reduce the labor cost, and control M. incognita and F. oxysporum. In this study, toxicity of abamectin and fludioxonil was determined against M. incognita and F. oxysporum, respectively, in the laboratory, and the efficacy of a combination of abamectin and fludioxonil (abamectin + fludioxonil) for control of M. incognita and F. oxysporum simultaneously was evaluated under field conditions. In the laboratory, abamectin was highly toxic to second-stage juveniles (J2) of M. incognita with the median lethal concentration (LC50) of 2.60 mg L−1 and fludioxonil inhibited colony growth of F. oxysporum on potato dextrose agar (PDA) with the median effective concentration (EC50) of 0.34 mg L−1. In the field trials, abamectin + fludioxonil at 300 and 375 g ha−1 was effective in reducing population levels of M. incognita and incidence of Fusarium wilt in two consecutive years. Moreover, abamectin + fludioxonil at 375 g ha−1 significantly (P < 0.05) increased cucumber fruit yield by 45.23% and 40.85% in two years, respectively, compared to the untreated control. The yield increasing effect by the combined treatment was significantly greater than either treatment alone. Results from this study indicated the simultaneous application of abamectin + fludioxonil was effective for the control of the M. incognita and F. oxysporum disease complex in cucumber production, while enhancing cucumber fruit yield.

Eckol inhibits Reg3A-induced proliferation of human SW1990 pancreatic cancer cells.

Pancreatic cancer (PaC) is characterized by a highly inflammatory tumor microenvironment, and inflammatory mediators are implicated in the progression of this cancer. Regenerating gene protein (Reg) 3A is significantly upregulated during pancreatic inflammation, and has been demonstrated to serve an important role during PaC progression, based on its increased expression levels in PaC and potent cell proliferation-promoting activity. The aim of the present study was to investigate the effect of eckol, a phlorotannin compound with a variety of biological activities including anti-inflammatory, anti-tumor and cytoprotective effects, on Reg3A-induced proliferation of human SW1990 PaC cells. SW1990 cells were pre-treated with eckol for 48 h at concentrations of 5, 10 and 20 µg/ml. Subsequently, Reg3A protein was added to the culture media at a final concentration of 50 ng/ml in the presence or absence of eckol for 24 h. The cytotoxicity and proliferative capacity of the SW1990 cells was determined using an MTT and flow cytometry analysis. Cell colony formation was also used to determine the effect of eckol on the anchorage-independent growth and colony-forming capacity of Reg3A-treated PaC cells. The expression levels of cyclin D1, STAT3, JAK2, and NF-κB p65 were measured with reverse transcription-quantitative PCR and western blotting. Eckol reduced Reg3A-promoted cell survival, inhibited Reg3A-induced cell cycle progression and inhibited colony growth of SW1990 cells in soft agar in a concentration-dependent manner. Additionally, the Reg3A-mediated upregulation of expression of JAK2, STAT3, NF-κBp65 and cyclin D1 was reduced when treated with eckol. Reg3A is upregulated during pancreatic inflammation and exhibits a pro-growth function and may thus serve a critical role during inflammation-driven PaC malignancies. Eckol may be a potential protective agent against progression of PaC accompanied by pancreatic inflammation.

Soil fumigation with mustard essential oil to control bacterial wilt in tomato

In vitro experiments showed that six isolates, belonging to different biovars, were equally susceptible to mustard essential oil (MEO) vapor. The proportion of cell mortality and colony growth inhibition were directly related to the dose of MEO. A 24 h exposure to 1 or 2 μL of AITC per liter of box volume resulted in mortality of 89% and 92% of cells, respectively, and exposure to 10 μL L−1 resulted in 100% cell mortality. The growth of developing colonies was reduced by 89% at 2 or 4 μL L−1 and by 96% at 6 or 10 μL L−1. The leakage of intracellular materials increased with increasing doses of MEO vapor. The decline in soil inoculum density by fumigation was directly related to the MEO dose. The initial inoculum density of approximately 3 × 105 colony-forming units per gram of soil declined by 67, 91 and 96% at fumigation doses of 25, 50 and 75 μL of allyl isothiocyanate (AITC) per liter of soil, respectively. The pathogen was not detected at a fumigation dose of 100 μL L−1. Disease control was also related to the fumigation dose. After 48 days, the bacterial wilt symptoms did not develop on the tomatoes when the soil was fumigated at 100 μL L−1 or higher.

RSK2-Mediated ELK3 Activation Enhances Cell Transformation and Breast Cancer Cell Growth by Regulation of c-fos Promoter Activity.

Ribosomal S6 kinase 2 (RSK2), regulated by Ras/Raf/MEKs/ERKs, transmits upstream activation signals to downstream substrates including kinases and transcription and epigenetic factors. We observed that ELK members, including ELK1, 3, and 4, highly interacted with RSK2. We further observed that the RSK2-ELK3 interaction was mediated by N-terminal kinase and linker domains of RSK2, and the D and C domains of ELK3, resulting in the phosphorylation of ELK3. Importantly, RSK2-mediated ELK3 enhanced c-fos promoter activity. Notably, chemical inhibition of RSK2 signaling using kaempferol (a RSK2 inhibitor) or U0126 (a selective MEK inhibitor) suppressed EGF-induced c-fos promoter activity. Moreover, functional deletion of RSK2 by knockdown or knockout showed that RSK2 deficiency suppressed EGF-induced c-fos promoter activity, resulting in inhibition of AP-1 transactivation activity and Ras-mediated foci formation in NIH3T3 cells. Immunocytofluorescence assay demonstrated that RSK2 deficiency reduced ELK3 localization in the nucleus. In MDA-MB-231 breast cancer cells, knockdown of RSK2 or ELK3 suppressed cell proliferation with accumulation at the G1 cell cycle phase, resulting in inhibition of foci formation and anchorage-independent cancer colony growth in soft agar. Taken together, these results indicate that a novel RSK2/ELK3 signaling axis, by enhancing c-Fos-mediated AP-1 transactivation activity, has an essential role in cancer cell proliferation and colony growth.

Effect on Colony Growth Inhibition of Soil-Borne Fungal Pathogens by Available Chlorine Content in Sodium Hypochlorite.

Our study investigated the available chlorine content, contact time and difference among strains of each pathogen for sodium hypochlorite (NaOCl) to control chemically against soil-borne fungal pathogens, such as Phytophthora rot by Phytophthora cactorum, violet root rot by Helicobasidium mompa, and white root rot by Rosellinia necatrix, causing die-back symptom on apple trees. As a result, the colony growth of Phytophthora cactorum was inhibited completely by soaking over 5 s in 31.25 ml/l available chlorine content of NaOCl. Those of H. mompa and R. necatrix were inhibited entirely by soaking over 160 s in 62.5 and 125 ml/l available chlorine content in NaOCl, respectively. Also, inhibition effect on available chlorine in NaOCl among strains of each soil-borne pathogen showed no significant difference and was similar to or better than that of fungicides.

TISSUE CULTURE OF MICE KIDNEYS FOR THE TOXICITY EVALUATION OF SOME CYTOSTATICS

The rationale for the present study is explained by a high risk of the development of side effects, including nephrotoxic, in patients that receive the treatment with cytostatics bleocin, lastet, and cisplatin for oncologic diseases. The present article focuses on the evaluation of the toxicity of the mentioned drugs. The method of plasma-free renal tissue cultures in mice on the perforated millipore filters was used. The identification of the toxicity of different doses of the drugs was performed by the comparison of the areas of the grown colonies. The authors presented the data that the inhibition of the growth of renal tissue cultures (by 50%) required a 4.3-fold increase in the dose of lastet in comparison with bleocin. The inhibition of the tissue culture colony growth with cisplatin by 85% was registered in the dose of the drug that corresponded to a 3% solution. The materials of the article have significant practical value because a rational indication of cytostatics bleocin, lastet, and cisplatin with regard to their nephrotoxic effect will allow the specialists to improve the treatment outcome and will contribute to the improvement of the quality of life of patients.

Synergistic Pre-Clinical Activity of Targeted Inhibition of KDM1A and BET Proteins Against Human AML Blast Progenitor Cells

There is a clear need to develop novel therapies that would overcome differentiation block and eliminate AML stem/progenitor cells. Genetic and epigenetic dysregulation of enhancers regulates expressions of myeloid lineage transcriptional regulators and their target genes in AML stem/progenitor cells. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates enhancer maintenance and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1 that mediate transcriptional repression and differentiation block in AML blast progenitor cells (BPCs). We had previously reported that treatment with the reversible LSD1 inhibitor (LSDi) SP2509 increases the permissive H3K4Me3 mark on the chromatin, associated with induction of p21, p27 and CEBPα levels, as well as of differentiation and loss of viability of AML BPCs (Leukemia. 2014; 28: 2155-64). In the present studies, we further evaluated the anti-AML efficacy of LSD1i-based combination with BET protein inhibitor (BETi). First, we determined that tet-inducible shRNA to KDM1A depleted protein levels of KDM1A, repressed c-Myc, but de-repressed p21, CD11b (ITGAM), CD86 and CEBPα, thereby inhibiting colony growth and modestly inducing lethality in genetically diverse cultured AML cell lines. Following sgRNA-directed, CRISPR/Cas9-mediated gene-editing of LSD1 in AML BPCs, surviving clones exhibited ~50% KDM1A levels and decreased c-Myc and DNMT1 expressions compared to the control AML BPCs. Treatment with either the reversible LSDi, SP2577 (Salarius Pharma), or with the irreversible LSDi ORY-1001, disrupted binding of KDM1A with CoREST. Following LSDi treatment, ATAC-Seq analyses demonstrated significant increase in the accessible chromatin of AML BPCs (represented by gained peaks). Gained ATAC-Seq peaks also involved the chromatin of MED11/13, LY96, CEBPB, RARA, CDKN1C and CD86 genes. ChIP-Seq analysis also showed increased H3K27Ac peaks in the chromatin of CD86, ITGAM, SAMHD1, TET2, MED12 and E2F1, and a reduction of peaks in RUNX1, CDK6, KIT, CTNNB1, HOXB5, FLT3 and MEIS1. RNA-Seq analyses after LSD1i treatment also showed significant perturbations (log2 fold-change >1.25 and p<0.05) in the mRNA expressions, including those of ITGAM, LY96, CD86, SAMHD1, IRF8, APAF1, CDK6, and KIT. Gene set enrichment analysis against Hallmark and Transcription Factor-Target datasets showed positive enrichment of E2F and GFI1 targets, as well as of IL2-STAT5 and TGF-beta signaling, but significant depletion (FDR q-values <0.1) of MYC-targets and genes involved in oxidative phosphorylation. QPCR and Western analyses following LSD1i treatment confirmed significant up regulation of mRNA and protein levels, respectively, of ITGAM, CD86 and LY96 in cultured and primary patient-derived AML BPCs. This was associated with morphologic features of differentiation and inhibition of colony growth in AML cells (OCI-AML5, MOLM13, THP1 and MV4-11) (p < 0.01). We also queried for expression mimickers (EMs) through connectivity mapping of the mRNA signature following LSD1i treatment, utilizing the LINCS1000-CMap analyses. Among the top EM hits were BET protein inhibitors (BETis). Treatment with BETi or BET protein degraders (PROTACs) depleted LSD1 levels in AML BPCs. Utilizing ChIP-Seq data, we also noted that LSD1 promoter is occupied by BET protein BRD4 in the AML cells. Consistent with this, treatment with the BETi OTX015 depleted KDM1A expression in AML cells. Notably, co-treatment with LSDi (SP2577 or ORY-1001) and OTX015 induced synergistic lethality in AML BPCs, including of CD34+, CD38-, Lin- AML stem/progenitor cells (combination indices < 1.0). This was associated with greater depletion of c-Myc, c-Myb and PU.1, but greater induction of p21 and p27. LSD1i or BETi treatment significantly improved survival of the immune-depleted mice (compared to the control mice) engrafted with the AML OCI-AML5 cells or patient-derived xenograft (PDX) models of AML (p < 0.01). Collectively, these findings elucidate the molecular mechanisms and strongly support further in vivo testing and pre-clinical development of LSD1i-based combinations with BETi against AML BPCs. DisclosuresSoldi:Beta Cat Pharma: Employment. Han:Beta Cat Pharma: Employment. DiNardo:Agios: Consultancy, Other: Advisory role; Bayer: Other: Advisory role; Celgene: Other: Advisory role; Medimmune: Other: Advisory role; Karyopharm: Other: Advisory role; AbbVie: Consultancy, Other: Advisory role. Kadia:Celgene: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Abbvie: Consultancy; Abbvie: Consultancy; BMS: Research Funding; Jazz: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding; Novartis: Consultancy. Khoury:Stemline Therapeutics: Research Funding.

BCL-2 Inhibitor Venetoclax (ABT-199) and MEK Inhibitor GDC-0973 Synergise to Target AML Progenitors and Overcome Drug Resistance with the Use of PET Scanning in a Mouse Model of HR-MDS to Monitor Response to Treatment

Introduction: Targeted drugs are needed for HR-MDS/AML, particularly in elderly patients and Venetoclax, approved for some CLL, gives promising results in elderly AML. Assays to predict response to treatment may enable us to deliver personalized treatment. We sought to determine the most informative assay to predict response; viability assays can directly measure the effects of reagents on growth. Progenitor assays can potentially determine if the reagents can target diseased primitive cells. PET scanning can be used to follow response to treatment.Methods: Peripheral blood (PB) or bone marrow (BM) from 7 MDS/AML patients were incubated in a) no treatment, b) ABT-199 (1 µM) (Abbvie), c) GDC-0973 (1 µM) (Genentech) or d) ABT-199+GDC-0973 (1 µM of each) and assessed for viability using the MTT assay (n=2); cell death followed using the Incucyte® Zoom System (Essen Bioscience) (n=2) or methocult progenitor assays (Stem Cell Technologies) (n=4). Having shown that RAS:BCL-2 co-localization correlated with prognosis in MDS/AML patients (Leuk Res 37:312-9, 2013), immunofluorescence was undertaken. A micro PET device dedicated to mice was used to measure BM blast proliferation. After injection of 18F-FLT(a thymidine analogue) in mice untreated (n=7) or ABT-199 (75mg/kg)+GDC-0973(10mg/kg) treated (n=5) normal FVB/N, HR-MDS mice treated with vehicle (n=4), 2-month old HR-MDS before (n=5) and 3-month old before (n=4) and after ABT-199 (75mg/kg)+GDC-0973(10mg/kg) treatment (n=8), PET imaging was performed (Inveon Siemens Medical Systems), analyzed for signal and quantified.Results: Patient details and results are summarized on Table 1. Using the MTT assay 2 PB patient samples were found to be sensitive to ABT-199 treatment (Figure 1A, AS, p=0.00042 and YA, 0.00002) and more sensitive to the combination compared to untreated (AS, p=0.00007 and YA, 0.000003). With the incucyte the BM of one patient (AE) was found to be resistant to both ABT-199 and GDC-0973, but sensitive to the combination (Figure 1B). PB and BM from patient JA were assayed for apoptosis with the incucyte and were found to be sensitive to ABT-199 with increased apoptosis, resistant to GDC-0973 with decreased apoptosis and sensitive to the combination. Four bone marrow samples were tested in the 4 conditions using the progenitor assay (Figure 1C). Three patients were sensitive to GDC-0973, inhibiting any colony formation and the fourth had reduced colony numbers. In this assay patient JA appeared to be sensitive to GDC-0973 treatment whereas the incucyte assay scored this sample to be resistant to apoptosis; thus the cytotoxic effects of GDC-0973 may not be via apoptopsis. As the progenitor assay is likely to score the primitive disease population, this assay may prove more informative than the others without prior selection. One patient (DH) was clearly resistant to ABT-199, whereas the other three (JA, CB and FL) had reduced colony growth. All patients were sensitive to the combination treatment and inhibited colony growth. The RAS:BCL-2 co-localization in the PB revealed no complex in either the Mito or PM upon treatment with ABT-199 alone and some localization in the Mito with GDC-0973. With both ABT-199 and GDC-0973, there were hardly any cells confirming the cytotoxic effects of the combination. As we have previously shown that PM co-localization of the complex is associated with drug resistance (Blood 130:2613, 2017Suppl), we used the combination on our HR-MDS mouse model, where the complex co-localizes in the PM and followed the mice by PET scanning (Figure 1D). Weak signal was visualized in the femurs of untreated and ABT-199+GDC-0973 treated FVB/N mice (FBR 1.17+/-0.34 and 1.02+/-0.08 respectively). Mild PET signal was seen in the femurs of 2 month-old HR-MDS mice, (FBR 1.79+/-0.98). Intense PET signal was seen in the femurs and proximal humerus of HR-MDS mice treated with vehicle (3 month-old, FBR=2.35+/-1.32). Low PET signals were seen in the femurs of 5/8 HR-MDS mice treated with ABT-199+GDC-0973 (FBR=1.93+/-0.84). FBRs of the 3 groups of HR-MDS mice were significantly higher than those of FBV/N groups.Conclusion: Combined Venetoclax (ABT-199) and GDC-0973 targets MDS/AML progenitors and can potentially overcome drug resistance with the disruption of the RAS:BCL-2 complex. Bone marrow disease progression in HR-MDS mice can be monitored with 18F-FLT-PET imaging; PET data shows that the combination slows down disease progression. DisclosuresPadua:Abbvie: Research Funding; Genentech: Research Funding. Giraudier:Novartis: Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Reata: Equity Ownership; Celgene: Consultancy; Jazz Pharma: Consultancy; Oncolyze: Equity Ownership; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; SentiBio: Equity Ownership; Astra Zeneca: Research Funding.

In-vitro efficacy of bio-control agent and essential oils against leaf blight of chickpea caused by Alternaria alternata

Alternaria leaf blight is one of the most destructive foliar diseases of chickpea in different countries including Pakistan and has caused huge losses ranging from 5 to 100% around the world. This study was carried out to check the in-vitro effectiveness and sustainability of a bio-control agent Trichoderma viride and some essential oils like Castor, Jasmine, Clove, Sesame, Neem, Coconut, Henna, Black seed, and Mint oil at different doses of 1%, 2%, 4%, and 6% by food poisoned method against the mycelial colony growth inhibition of A. alternata which causes leaf blight in chickpea. The results indicate that maximum germination (80%) was recorded in control, (63%) under soil infestation and a minimum germination of (60%) was recorded in seed infestation. Maximum shoot weight (0.5630 g) in control, (0.2751 g) under soil infestation and (0.2064 g) in seed infestation. Maximum root weight (0.5937 g) in control, (0.4359 g) under soil infestation method, and (0.4102 g) in seed infestation method. Maximum shoot length (20.00 cm) in control, (10.23 cm) under soil infestation method and (7.053 cm) in seed infestation method. Maximum root length (7.19 cm) in control, (4.80 cm) under soil infestation method and (4.80 cm) in seed infestation. Bio-control agent Trichoderma viride showed (74.44%) growth inhibition compared to control (1.00%) growth inhibition of A. alternata. Maximum colony growth inhibition of A. alternata was (80.00%) Sesame, Coconut (77.04%), Henna (72.59%), Mint (66.07%), Black seed (71.85%), Jasmine (64.07%), Clove (70.74%), Neem (73.33%), Castor (58.89%) and minimum of (1.00%) was recorded in control. The results of this study will be very helpful for researchers and farming community for better management of this destructive disease of chickpea.

A description of Mycobacterium chelonae subsp. gwanakae subsp. nov., a rapidly growing mycobacterium with a smooth colony phenotype due to glycopeptidolipids.

Three rapidly growing mycobacterial strains, MOTTH4W, MOTT36WT and MOTT68W, were isolated from the sputa of three independent Korean patients co-infected with Mycobacterium yongonense Type II strains. The 16S rRNA gene sequences of all three strains were unique, which were closest to that of Mycobacterium chelonae subsp. bovis KCTC 39630T (99.9 % similarity). Multilocus sequence typing analysis targeting 10 housekeeping genes including hsp65 and rpoB revealed the distinct phylogenetic location of these strains, which were clustered with M. chelonae subsp. chelonae ATCC 35752T and M. chelonae subsp. bovis KCTC 39630T. Phylogenetic analysis based on whole genome sequences revealed a 95.89 % average nucleotide identity (ANI) value with M. chelonae subsp. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, phenotypic characteristics such as a smooth colony morphology and growth inhibition at 37 °C, distinct MALDI-TOF MS profiles of extracted total lipids due to surface glycopeptidolipids, and distinct drug susceptibility profiles further supported the taxonomic characterization of these strains as representing a novel subspecies of Mycobacterium chelonae. Mycobacterium chelonae subsp. gwanakae subsp. nov. is proposed and the type strain is MOTT36WT (=KCTC 29127T=JCM 32454T).