Integrated transcriptome and cell phenotype analysis suggest involvement of PARP1 cleavage, Hippo/Wnt, TGF-ß and MAPK signaling pathways in ovarian cancer cells response to cannabis and PARP1 inhibitor treatment.

Introduction: Cannabis sativa is utilized mainly for palliative care worldwide. Ovarian cancer (OC) is a lethal gynecologic cancer. A particular cannabis extract fraction ('F7') and the Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor niraparib act synergistically to promote OC cell apoptosis. Here we identified genetic pathways that are altered by the synergistic treatment in OC cell lines Caov3 and OVCAR3. Materials and methods: Gene expression profiles were determined by RNA sequencing and quantitative PCR. Microscopy was used to determine actin arrangement, a scratch assay to determine cell migration and flow cytometry to determine apoptosis, cell cycle and aldehyde dehydrogenase (ALDH) activity. Western blotting was used to determine protein levels. Results: Gene expression results suggested variations in gene expression between the two cell lines examined. Multiple genetic pathways, including Hippo/Wnt, TGF-ß/Activin and MAPK were enriched with genes differentially expressed by niraparib and/or F7 treatments in both cell lines. Niraparib + F7 treatment led to cell cycle arrest and endoplasmic reticulum (ER) stress, inhibited cell migration, reduced the % of ALDH positive cells in the population and enhanced PARP1 cleavage. Conclusion: The synergistic effect of the niraparib + F7 may result from the treatment affecting multiple genetic pathways involving cell death and reducing mesenchymal characteristics.

Hexavalent chromium-reducing plant growth-promoting rhizobacteria are utilized to bio-fortify trivalent chromium in fenugreek by promoting plant development and decreasing the toxicity of hexavalent chromium in the soil.

BACKGROUND: Fenugreek is known to have good anti-diabetes properties. Moreover, several studies accounted that the trivalent form of chromium [Cr(III)] also have anti-diabetic properties. However, its hexavalent form i.e., Cr(VI) is known to be highly toxic and carcinogenic to living beings and retarded plant growth even if it is present in low concentration in soil. Many plant growth-promoting rhizobacteria (PGPR) are reported to have the potential to reduce the Cr(VI) into Cr(III) in soil. In view of the above, the present objective was designed to effectively utilize Cr(VI) reducing PGPRs for the growth and development of fenugreek plant in Cr(VI) amended soil, apart from reducing Cr(VI) in soil and fortification of Cr(III) in the aerial part of plants. METHODS: The experiment was carried out to evaluate the effect of Cr(VI)-reducing PGPRs viz. Bacillus cereus (SUCR44); Microbacterium sp. (SUCR140); Bacillus thuringiensis (SUCR186) and B. subtilis (SUCR188) on growth, uptake and translocation of Cr as well as other physiological parameters in fenugreek grown under artificially Cr(VI) amended soil (100 mg kg-1 of Cr(VI) in soil). RESULTS: The aforementioned concentration of Cr(VI) in soil cause severe reduction in root length (41 %), plant height (43 %), dry root (38 %) and herb biomass (48 %), when compared with control negative (CN; uninoculated plant not grown in Cr(VI) contaminated soil). However, the presence of Microbacterium sp.-SURC140 (MB) mitigates the Cr toxicity resulting in improved root length (92 %), plant height (86 %), dry root (74 %) and herb biomass (99 %) as compared with control positive (CP; uninoculated plants grown in Cr(VI) contaminated soil). The maximum reduction in bioavailability (82 %) of Cr(VI) in soil and its uptake (50 %) by the plant were also observed in MB-treated plants. However, All Cr(VI)-reducing PGPRs failed to decrease the translocation of Cr to the aerial parts. Moreover, the plant treated with MB observed diminution in relative water content (13 %), electrolyte leakage (16%) and lipid peroxidation (38 %) as well as higher chlorophyll (37 %) carotenoids (17 %) contents and antioxidants (18%) potential. CONCLUSION: This study demonstrates that MB can lower the Cr(VI) toxicity to the plant by reducing the bioavailable Cr(VI), consequently reducing the Cr(VI) toxicity level in soil and helping in improving the growth and yield of fenugreek. Additionally, Cr(III) uptakes and translocation may improve the effectiveness of fenugreek in treating diabetes.

Phytocannabinoids Act Synergistically with Non-Steroidal Anti-Inflammatory Drugs Reducing Inflammation in 2D and 3D In Vitro Models.

Lung inflammation is associated with elevated pro-inflammatory cytokines and chemokines. Treatment with FCBD:std (standard mix of cannabidiol [CBD], cannabigerol [CBG] and tetrahydrocannabivarin [THCV]) leads to a marked reduction in the inflammation of alveolar epithelial cells, but not in macrophages. In the present study, the combined anti-inflammatory effect of FCBD:std with two corticosteroids (dexamethasone and budesonide) and two non-steroidal anti-inflammatory drugs (NSAID; ibuprofen and diclofenac), was examined. Enzyme-linked immunosorbent assay (ELISA) was used to determine protein levels. Gene expression was determined by quantitative real-time PCR. Inhibition of cyclo-oxygenase (COX) activity was determined in vitro. FCBD:std and diclofenac act synergistically, reducing IL-8 levels in macrophages and lung epithelial cells. FCBD:std plus diclofenac also reduced IL-6, IL-8 and CCL2 expression levels in co-cultures of macrophages and lung epithelial cells, in 2D and 3D models. Treatment by FCBD:std and/or NSAID reduced COX-1 and COX-2 gene expression but not their enzymatic activity. FCBD:std and diclofenac exhibit synergistic anti-inflammatory effects on macrophages and lung epithelial cells, yet this combined activity needs to be examined in pre-clinical studies and clinical trials.

Phytocannabinoid Compositions from Cannabis Act Synergistically with PARP1 Inhibitor against Ovarian Cancer Cells In Vitro and Affect the Wnt Signaling Pathway.

Ovarian cancer (OC) is the single most lethal gynecologic malignancy. Cannabis sativa is used to treat various medical conditions, and is cytotoxic to a variety of cancer types. We sought to examine the effectiveness of different combinations of cannabis compounds against OC. Cytotoxic activity was determined by XTT assay on HTB75 and HTB161 cell lines. Apoptosis was determined by flow cytometry. Gene expression was determined by quantitative PCR and protein localization by confocal microscopy. The two most active fractions, F5 and F7, from a high Δ9-tetrahydrocannabinol (THC) cannabis strain extract, and their standard mix (SM), showed cytotoxic activity against OC cells and induced cell apoptosis. The most effective phytocannabinoid combination was THC+cannabichromene (CBC)+cannabigerol (CBG). These fractions acted in synergy with niraparib, a PARP inhibitor, and were ~50-fold more cytotoxic to OC cells than to normal keratinocytes. The F7 and/or niraparib treatments altered Wnt pathway-related gene expression, epithelial-mesenchymal transition (EMT) phenotype and ß-catenin cellular localization. The niraparib+F7 treatment was also effective on an OC patient's cells. Given the fact that combinations of cannabis compounds and niraparib act in synergy and alter the Wnt signaling pathway, these phytocannabinoids should be examined as effective OC treatments in further pre-clinical studies and clinical trials.

Management of chromium(VI)-contaminated soils through synergistic application of vermicompost, chromate reducing rhizobacteria and Arbuscular mycorrhizal fungi (AMF) reduced plant toxicity and improved yield attributes in Ocimum basilicum L.

An integrated approach involving vermicompost, chromate reducing bacteria and AMF was tested to manage the toxic impacts of Cr(VI) on Ocimum basilicum as a model plant. Pot experiments were conducted on O. basilicum plants in an artificially Cr(VI)-contaminated soil in two phases of experiment as bioinoculants experiment and vermicompost experiment. In the first phase of the bioinoculants experiment the series of gradient concentrations of Cr(VI) (0, 25, 50 and 100 mg kg-1 in soil) were evaluated with previously isolated four efficient Cr(VI)-reducing rhizo-bacterial strains (Bacillus Cereus strain SUCR 44, BC; Microbacterium sp. strain SUCR 140, MB; Bacillus thuringiensis strain SUCR186, BT; and Bacillus subtilis strain SUCR188; BS) along with Arbuscular Mycorrhizal Fungus-Glomus fasciculatum (GF) in alone and in co-inoculation form. In the second experiment (vermicompost) the best performing strain (MB) was tested alone or in combination with GF along with different doses of vermicompost. It was observed that vermicompost by itself could be useful in decreasing the bioavailable Cr(VI), uptake of Cr besides improving the nutritional status of plants. The vermicompost also played an important and indirect role and improved herb yield by supporting the multiplication of MB (Microbacterium sp.), an efficient chromate reducing rhizobacteria, that further decreased the bioavailable and toxic form of Cr and improved population and colonization of GF too. The translocation of Cr(VI) was averted through improved colonization of GF, also prevented higher accumulation of Cr in aerial parts (leafy herb) of O. basilicum.

Papaya Leaf Curl Virus (PaLCuV) Infection on Papaya (Carica papaya L.) Plants Alters Anatomical and Physiological Properties and Reduces Bioactive Components.

Papaya leaves are used frequently for curing scores of ailments. The medicinal properties of papaya leaves are due to presence of certain bioactive/pharmacological compounds. However, the papaya leaf curl virus (PaLCuV), a geminivirus, is a major threat to papaya cultivation globally. During the present investigation, we observed that PaLCuV infection significantly altered the anatomy, physiology, and bioactive properties of papaya leaves. As compared to healthy leaves, the PaLCuV-infected leaves were found to have reduced stomatal density (76.83%), stomatal conductance (78.34%), photosynthesis rate (74.87%), water use efficiency (82.51%), chlorophyll (72.88%), carotenoid (46.63%), osmolality (48.55%), and soluble sugars (70.37%). We also found lower enzymatic activity (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)-56.88%, 85.27%, and 74.49%, respectively). It was found that the size of guard cells (50%), transpiration rate (45.05%), intercellular CO2 concentration (47.81%), anthocyanin (27.47%), proline content (74.17%), malondialdehyde (MDA) (106.65%), and electrolyte leakage (75.38%) was elevated in PaLCuV-infected leaves. The chlorophyll fluorescence analysis showed that the infected plant leaves had a significantly lower value of maximal quantum yield of photosystem II (PSII (Fv/Fm), photochemical quantum yield of photosystem I (PSI (Y(I)), and effective quantum yield of PSII (Y(II)). However, in non-photochemical quenching mechanisms, the proportion of energy dissipated in heat form (Y(NPQ)) was found to be significantly higher. We also tested the bioactivity of infected and healthy papaya leaf extracts on a Caenorhabditis elegans (C. elegans) model system. It was found that the crude extract of papaya leaves significantly enhanced the life span of C. elegans (29.7%) in comparison to virus-infected leaves (18.4%) on application of 100 µg/mL dose of the crude extract. Our research indicates that the PaLCuV-infected leaves not only had anatomical and physiological losses, but that pharmacological potential was also significantly decreased.

Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.

Direct foliar uptake of phosphorus from desert dust.

Phosphorus (P) scarcity constrains plant growth in many ecosystems worldwide. In P-poor ecosystems, the biogeochemical paradigm links plant productivity with the deposition of P-rich dust originating from desert storms. However, dust P usually has low bioavailability and is thought to be utilized solely via roots. We applied desert dust on the leaf surface of P-deficient and P-sufficient wheat, chickpea and maize to test the feasibility of direct foliar uptake of dust-P and investigate its related acquisition mechanisms. Foliar dust doubled the growth of P-deficient chickpea and wheat, crops originating near the Syrian Desert. P deficiency stimulated several leaf modifications that enabled acquisition of up to 30% of the sparingly soluble dust-P that is conventionally perceived as unavailable. These modifications increased foliar dust capture, acidified the leaf surface and, in chickpea, enhanced exudation of P-solubilizing organic acids. Maize (originating far from deserts) displayed only a marginal response to dust. The dramatic response of chickpea and wheat in comparison to maize suggests that plants that evolved in dust-rich ecosystems adopted specialized utilization strategies. Interestingly, the abovementioned foliar responses are comparable to known P uptake root responses. Given that P limitation is almost universal, a foliar P uptake pathway will have significant ecological and agricultural implications.

C. elegans protein interaction network analysis probes RNAi validated pro-longevity effect of nhr-6, a human homolog of tumor suppressor Nr4a1.

Protein-protein interaction (PPI) studies are gaining momentum these days due to the plethora of various high-throughput experimental methods available for detecting PPIs. Proteins create complexes and networks by functioning in harmony with other proteins and here in silico network biology hold the promise to reveal new functionality of genes as it is very difficult and laborious to carry out experimental high-throughput genetic screens in living organisms. We demonstrate this approach by computationally screening C. elegans conserved homologs of already reported human tumor suppressor and aging associated genes. We select by this nhr-6, vab-3 and gst-23 as predicted longevity genes for RNAi screen. The RNAi results demonstrated the pro-longevity effect of these genes. Nuclear hormone receptor nhr-6 RNAi inhibition resulted in a C. elegans phenotype of 23.46% lifespan reduction. Moreover, we show that nhr-6 regulates oxidative stress resistance in worms and does not affect the feeding behavior of worms. These findings imply the potential of nhr-6 as a common therapeutic target for aging and cancer ailments, stressing the power of in silico PPI network analysis coupled with RNAi screens to describe gene function.

Withanolide A offers neuroprotection, ameliorates stress resistance and prolongs the life expectancy of Caenorhabditis elegans.

Withanolide A (steroidal lactone) forms the major constituent of the most popular herbal drug in Ayurvedic medicine, Ashwagandha. It has been used since ancient times as an alternative medicine for the treatment of a variety of age related disorders. Here we provide multiple lines of evidence indicating that Withanolide A improves healthspan, delays age-associated physiological changes and also extends the lifespan of Caenorhabditis elegans. We also report several neuroprotective benefits of this natural product, including its anti-amyloidogenic effects, alleviation of α-synuclein aggregation and neuroprotection through modulation of neural mediators like acetylcholine. We observed that Withanolide A mediates lifespan extension and promotes stress resistance via insulin/insulin-like growth factor signaling pathway. Such findings could be helpful to develop a therapeutic medicine from this natural product for the prevention or reversal of age-related ailments and to improve the survival of patients suffering from Alzheimer's or Parkinson's disease.

Bacopa monnieri promotes longevity in Caenorhabditis elegans under stress conditions.

BACKGROUND: Bacopa monnieri (L.) Pennell, commonly known as Brahmi is an important medicinal plant traditionally used as memory enhancer and antiepileptic agent. OBJECTIVE: The present study investigated antioxidant and stress resistance potentials of B. monnieri aqueous extract (BMW) using Caenorhabditis elegans animal model system. MATERIALS AND METHODS: The antioxidant activity of the BMW was measured using in vitro (DPPH, reducing power and total polyphenol content) and in vivo (DCF-DA assay) assays. The antistress potential of BMW (0.1, 0.01, and 0.001 mg/ml) was evaluated through thermal stress (37°C) and oxidative stress (10 mM paraquat) using C. elegans. Quantification of the HSP-16.2 level was done using CL2070 transgenic worms. RESULTS: Present study reveals that BMW possess in vitro and in vivo antioxidant activities. BMW significantly enhanced stress tolerance and increased the mean lifespan of worms during thermal and oxidative stress, although it did not extend lifespan at 20°C and attenuated age dependent decline in physiological behaviors. Moreover, it was shown that BMW was able to up-regulate expression of stress associated gene hsp-16.2, which significantly (P < 0.001) extends the mean lifespan of worms under stress conditions. CONCLUSION: The study strongly suggests that BMW acts as an antistressor and potent reactive oxygen species scavenger which enhances the survival of the worms in different stress conditions.

Exploitation of microbes for enhancing bacoside content and reduction of Meloidogyne incognita infestation in Bacopa monnieri L.

Despite the vast exploration of rhizospheric microbial wealth for crop yield enhancement, knowledge about the efficacy of microbial agents as biocontrol weapons against root-knot disease is scarce, especially in medicinal plants, viz., Bacopa monnieri. In the present investigation, rhizospheric microbes, viz., Bacillus megaterium, Glomus intraradices, Trichoderma harzianum ThU, and their combinations were evaluated for the management of Meloidogyne incognita (Kofoid and White) Chitwood and bacoside content enhancement in B. monnieri var CIM-Jagriti. A novel validated method Fourier transform near infrared was used for rapid estimation of total bacoside content. A significant reduction (2.75-fold) in root-knot indices was observed in the combined treatment of B. megaterium and T. harzianum ThU in comparison to untreated control plants. The same treatment also showed significant enhancement (1.40-fold) in total bacoside contents (plant active molecule) content using Fourier transform near-infrared (FT-NIR) method that analyses samples rapidly in an hour without solvent usage and provides ample scope for natural product studies.

Isolation, structure determination, and antiaging effects of 2,3-pentanediol from endophytic fungus of Curcuma amada and docking studies.

An endophytic fungus was isolated from the rhizomes of Curcuma amada (Zingiberaceae), which was identified as Fusarium oxysporum on the basis of its morphological and molecular characters. Chromatographic separation and spectroscopic analysis of the fungal metabolite (chloroform extract) led to the identification of one pure compound having molecular formula C5H12O2, i.e., 2,3-pentanediol (1). Activity analysis of compound 1 demonstrated improved antiaging (antioxidant, thermotolerance) properties against Caenorhabditis elegans, in comparison to a similar, commercially available molecule i.e., 1,5-pentanediol (2). The effective (lower) concentration of 1 significantly showed (28.6%) higher survival percentage of the worms under thermal stress (37 ºC) compared to its higher concentration (25.3%), while similar trends were followed in oxidative stress where (22.2%) higher survival percentage was recorded in comparison to untreated control. The compound 1, however, lacked potential antimicrobial activity, indicating the plausible ramification of the position of OH group in such bioactive molecules. In silico evaluation of these molecules against common as well as unique targets corroborated better antiaging potential of 1 in comparison to that of 2. The results for the first time indicated that the utilization of the endophytic fungi of C. amada could, thus, be a possible source for obtaining non-plant-based bioactive compounds having broader therapeutic applications pertaining to age-related progressions.

Iridoid compound 10-O-trans-p-coumaroylcatalpol extends longevity and reduces α synuclein aggregation in Caenorhabditis elegans.

Aging, the major cause of several ailments has led to intense exploration of potential drugs that delay aging and its associated effects. We mined the information on traditional Indian medicines and identified an iridoid, 10-O-trans-p-Coumaroylcatalpol (OCC), a major ingredient of Premna integrifolia Linn. (syn: Premna serratifolia). OCC forms an important constituent of famous herbal formulation 'Dashmula', a ten herb formulation, commonly used for its various medicinal properties. Employing model system C. elegans, the effect of OCC on life span, stress resistance, chemotaxis, the content of reactive oxygen species (ROS) and on the aggregation of alpha synuclein was studied. OCC extended the mean life span of nematodes, increased their tolerance against chemical induced stress, improved the chemotaxis index and reduced the ROS content. Further, the aggregation of Parkinson's disease (PD) associated protein, alpha synuclein (asyn), was decreased when transgenic a-syn expressing worms were raised on OCC mixed diet. We extended the studies further to explore the possible genetic mechanism that mediates the observed effects of OCC. Employing the genetic knockout mutants TK22 [mev-1(kn1)III]; GR1307 [daf-16(mgDf50)I]; VC199 [sir-2.1(ok434)IV] and transgenic GFP expressing strain TJ356 [zls356; DAF-16::GFP], our studies revealed that the effects were mediated by daf-16 and not by sir-2.1 or mev-1. Our results indicate that OCC has the ability to ameliorate a-syn aggregation, reduce oxidative stress and promote longevity in C. elegans via activation of longevity promoting transcription factor DAF-16. Thus, OCC may serve as a lead compound of plant origin for important nutraceutical intervention against aging and age associated PD.