Anti-Leishmania major Properties of Nuphar lutea (Yellow Water Lily) Leaf Extracts and Purified 6,6' Dihydroxythiobinupharidine (DTBN).

Cutaneous leishmaniasis (CL) is a zoonotic disease, manifested as chronic ulcers, potentially leaving unattractive scars. There is no preventive vaccination or optimal medication against leishmaniasis. Chemotherapy generally depends upon a small group of compounds, each with its own efficacy, toxicity, and rate of drug resistance. To date, no standardized, simple, safe, and highly effective regimen for treating CL exists. Therefore, there is an urgent need to develop new optimal medication for this disease. Sesquiterpen thio-alkaloids constitute a group of plant secondary metabolites that bear great potential for medicinal uses. The nupharidines found in Nuphar lutea belong to this group of compounds. We have previously published that Nuphar lutea semi-purified extract containing major components of nupharidines has strong anti-leishmanial activity in vitro. Here, we present in vivo data on the therapeutic benefit of the extract against Leishmania major (L. major) in infected mice. We also expanded these observations by establishing the therapeutic effect of the extract-purified nupharidine 6,6'-dihydroxythiobinupharidine (DTBN) in vitro against promastigotes and intracellular amastigotes as well as in vivo in L. major-infected mice. The results suggest that this novel anti-parasitic small molecule has the potential to be further developed against Leishmania.

A Nuphar lutea plant active ingredient, 6,6'-dihydroxythiobinupharidine, ameliorates kidney damage and inflammation in a mouse model of chronic kidney disease.

Chronic Kidney Disease (CKD) associated complications are associated with increased inflammation through the innate immune response, which can be modulated with anti-inflammatory agents. An active ingredient derived from the Nuphar lutea aquatic plant, 6,6'-dihydroxythiobinupharidine (DTBN) has anti-inflammatory properties, mainly through the inhibition of NF-κB. We tested the effects of DTBN on mice with CKD. After preliminary safety and dosing experiments, we exposed 8 weeks old male C57BL/6J mice to adenine diet to induce CKD. Control and CKD animals were treated with IP injections of DTBN (25 µg QOD) or saline and sacrificed after 8 weeks. Serum urea and creatinine were significantly decreased in CKD-DTBN Vs CKD mice. Kidney histology showed a decrease in F4/80 positive macrophage infiltration, damaged renal area, as well as decreased kidney TGF-ß in CKD-DTBN Vs CKD mice. Kidney inflammation indices (IL-1ß, IL-6 and P-STAT3) were significantly decreased in CKD-DTBN as compared to CKD mice. DTBN treatment showed no apparent damage to tissues in control mice, besides a decrease in weight gain and mild hypoalbuminemia without proteinuria. Thus, DTBN significantly improved renal failure and inflammation indices in CKD mice. Therefore, this and similar substances may be considered as an additional treatment in CKD patients.

In Vitro and In Vivo Therapeutic Potential of 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea on Cells and K18-hACE2 Mice Infected with SARS-CoV-2.

We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6'-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-κB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and/or its molecular derivatives against this and other related viruses.

Cytotoxicity of Thioalkaloid-Enriched Nuphar lutea Extract and Purified 6,6'-Dihydroxythiobinupharidine in Acute Myeloid Leukemia Cells: The Role of Oxidative Stress and Intracellular Calcium.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by uncontrolled proliferation of immature myeloid progenitors. Here, we report the in vitro antileukemic effects of the sesquiterpene thioalkaloid-enriched fraction of the Nuphar lutea leaf extract (NUP) and a purified thioalkaloid 6,6'-dihydroxythiobinupharidine (DTBN). Treatment with 0.3-10 µg/mL NUP caused a dose- and time-dependent reduction in proliferation and viability of human AML cells (KG-1a, HL60 and U937). This was associated with apoptosis induction manifested by annexin-V/propidium iodide binding as well as cleavage of caspases 8, 9, and 3 as well as poly (ADP-ribose) polymerase. Caspase-dependence of the apoptotic effect was confirmed using the pan-caspase inhibitor Q-VD-OPH. NUP induced significant biphasic changes in the cytosolic levels of reactive oxygen species (ROS) compared to untreated cells-a decrease at early time points (2-4 h) and an increase after a longer incubation (24 h). ROS accumulation was accompanied by lowering the cellular glutathione (GSH) levels. In addition, NUP treatment resulted in elevation of the cytosolic Ca2+ (Ca2+cyt) levels. The thiol antioxidant and glutathione precursor N-acetyl cysteine prevented NUP-induced ROS accumulation and markedly inhibited apoptosis. A similar antiapoptotic effect was obtained by Ca2+cyt chelating using BAPTA. These data indicate that NUP-induced cell death is mediated, at least in part, by the induction of oxidative stress and Ca2+cyt accumulation. However, a substantial apoptotic activity of pure DTBN (0.05-0.25 µg/mL), was found to be independent of cytosolic ROS or Ca2+, suggesting that alternative mechanisms are involved in DTBN-induced cytotoxicity. Notably, neither NUP nor DTBN treatment significantly induced cell death of normal human peripheral blood mononuclear cells. Our results provide the basis for further investigation of the antileukemic potential of NUP and its active constituents.

Inhibition of Cysteine Proteases by 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea.

The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.

6,6'-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity.

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6'-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.

Nuphar lutea Extracts Exhibit Anti-Viral Activity against the Measles Virus.

Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammatory and pathogen-related diseases. It has shown that Nuphar lutea extracts (NUP) are active against various pathogens including bacteria, fungi, and leishmanial parasites. In an effort to detect novel therapeutic agents against negative-stranded RNA (- RNA) viruses, we have tested the effect of a partially-purified alkaloid mixture of Nuphar lutea leaves on the measles virus (MV). The MV vaccine's Edmonston strain was used to acutely or persistently infect cells. The levels of several MV proteins were detected by a Western blot and immunocytochemistry. Viral RNAs were quantitated by qRT-PCR. Virus infectivity was monitored by infecting African green monkey kidney VERO cells' monolayers. We showed that NUP protected cells from acute infection. Decreases in the MV P-, N-, and V-proteins were observed in persistently infected cells and the amount of infective virus released was reduced as compared to untreated cells. By examining viral RNAs, we suggest that NUP acts at the post-transcriptional level. We conclude, as a proof of concept, that NUP has anti-viral therapeutic activity against the MV. Future studies will determine the mechanism of action and the effect of NUP on other related viruses.

Epigenetic aspects of floral homeotic genes in relation to sexual dimorphism in the dioecious plant Mercurialis annua.

In plants, dioecy characterizes species that carry male and female flowers on separate plants and it occurs in about 6% of angiosperms; however, the molecular mechanisms that underlie dioecy are essentially unknown. The ability for sex-reversal by hormone application raises the hypothesis that the genes required for the expression of both sexes are potentially functional but are regulated by epigenetic means. In this study, proteomic analysis of nuclear proteins isolated from flower buds of females, males, and feminized males of the dioecious plant Mercurialis annua revealed differential expression of nuclear proteins that are implicated in chromatin structure and function, including floral homeotic proteins. Focusing on floral genes, we found that class B genes were mainly expressed in male flowers, while class D genes, as well as SUPERMAN-like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation was found between the expression of class B and D genes and the changes in DNA methylation or chromatin conformation of these genes. Thus, we could not confirm DNA methylation or chromatin conformation of floral genes to be the major determinant regulating sexual dimorphisms. Instead, determination of sex in M. annua might be controlled upstream of floral genes by one or more sex-specific factors that affect hormonal homeostasis. A comprehensive model is proposed for sex-determination in M. annua.

6,6'-Dihydroxythiobinupharidine as a poison of human type II topoisomerases.

A number of natural products with medicinal properties increase DNA cleavage mediated by type II topoisomerases. In an effort to identify additional natural compounds that affect the activity of human type II topoisomerases, a blind screen of a library of 341 Mediterranean plant extracts was conducted. Extracts from Nuphar lutea, the yellow water lily, were identified in this screen. N. lutea has been used in traditional medicine by a variety of indigenous populations. The active compound in N. lutea, 6,6'-dihydroxythiobinupharidine, was found to enhance DNA cleavage mediated by human topoisomerase IIα and IIß âˆ¼8-fold and ∼3-fold, respectively. Mechanistic studies with topoisomerase IIα indicate that 6,6'-dihydroxythiobinupharidine is a "covalent poison" that acts by adducting the enzyme outside of the DNA cleavage-ligation active site and requires the N-terminal domain of the protein for its activity. Results suggest that some of the medicinal properties of N. lutea may result from the interactions between 6,6'-dihydroxythiobinupharidine and the human type II enzymes.

Nupharidine enhances Aggregatibacter actinomycetemcomitans clearance by priming neutrophils and augmenting their effector functions.

OBJECTIVES: Nupharidine (6,6'-Dihydroxythiobinupharidine), purified from the aquatic plant Nuphar lutea leaves (Water lily) prompts antimicrobial activity of immune cells. The aim of the study was to test the effect of Nupharidine on neutrophil function against Aggregatibacter actinomycetemcomitans, JP2 clone (Aa-JP2). METHODS: Neutrophils derived from the human cell line HL60 and human peripheral blood derived from aggressive periodontitis and periodontally healthy subjects were incubated with Nupharidine or vehicle and inoculated with JP2. Bacterial survival was tested using viable counts on blood agar (CFU's). Neutrophils' necrosis/apoptosis, reactive oxygen species (ROS) production, phagocytosis and neutrophil extracellular traps (NET) production following infection were tested, as well as markers of neutrophil priming. RESULTS: Nupharidine had no direct bactericidal effect on JP2, but it enhanced Aa-JP2 clearance by neutrophils. Nupharidine enhanced neutrophil phagocytosis, ROS production and NET formation during JP2 infection. Furthermore, Nupharidine enhanced the expression of certain markers of neutrophils priming, specifically iCAM1, DECTIN-2 and intracellular IL-1ß. CONCLUSION: Nupharidine was shown to promote neutrophil effector bactericidal functions, boosting Aa-JP2 clearance. The results point to the potential of Nupharidine as an adjunctive agent in the treatment of Aa-JP2 periodontitis, but this should be tested initially using pre-clinical and clinical studies.

Anti-Metastatic Effect of Semi-Purified Nuphar Lutea Leaf Extracts.

Nuphar lutea L. SM., leaf and rhizome extracts (NUP), contain nupharidines as active components. Nupharidines belong to the sesquiterpene lactones class of a naturally occurring plant terpenoids. This family of compounds has gained considerable interest for treating infection, inflammation and cancer. NF-κB is a central, downstream regulator of inflammation, cell proliferation and apoptosis. In our previous work we demonstrated strong inhibition of NF-κB activity and induction of apoptosis by NUP. In addition, NUP exhibited anti-inflammatory properties and partial protection from LPS-induced septic shock by modulating ERK pathway and cytokine secretion in macrophages. In the present study, we examined the effect of NUP in a B16 melanoma experimental murine lung metastasis model and its ability to affect the ERK and NF-κB pathways in variety of cell lines. We showed that NUP and cisplatin combined treatment was synergistic and reduced the lung metastatic load. In addition NUP treatment inhibited TNFα-induced IκBα degradation and NF- κB nuclear translocation. We also observed that NUP induced ERK activation. Furthermore, ERK inhibition prevented NF-κB inactivation by NUP. Overall, our work implies that co-administration of NF-κB inhibitors such as NUP, with standard anti-cancer drugs, may act as "sensitizers" for more effective chemotherapy.

Sexual Dimorphism in the Response of Mercurialis annua to Stress.

The research presented stemmed from the observations that female plants of the annual dioecious Mercurialis annua outlive male plants. This led to the hypothesis that female plants of M. annua would be more tolerant to stress than male plants. This hypothesis was addressed in a comprehensive way, by comparing morphological, biochemical and metabolomics changes in female and male plants during their development and under salinity. There were practically no differences between the genders in vegetative development and physiological parameters. However, under salinity conditions, female plants produced significantly more new reproductive nodes. Gender-linked differences in peroxidase (POD) and glutathione transferases (GSTs) were involved in anti-oxidation, detoxification and developmental processes in M. annua. ¹H NMR metabolite profiling of female and male M. annua plants showed that under salinity the activity of the TCA cycle increased. There was also an increase in betaine in both genders, which may be explainable by its osmo-compatible function under salinity. The concentration of ten metabolites changed in both genders, while 'Female-only-response' to salinity was detected for five metabolites. In conclusion, dimorphic responses of M. annua plant genders to stress may be attributed to female plants' capacity to survive and complete the reproductive life cycle.

Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases.

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10-100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption.

Purification of eutrophic water by ryegrass.

The present study was carried out to investigate the growth characteristics of different ryegrass (Lolium perenne L.) cultivars and their ability to remediate eutrophic water using floating plant-bed technology. Greenhouse and lake experiments were conducted to evaluate the grass genotypes for water remediation. Twelve cultivars of ryegrass including Grazer, Secale Cerale, Energa, Rustmaster, AngusI, Abundant, AngusII, Jivet, Gulf, Surrey, Major and Barwoltra were grown in the floating plant-bed system. The plant biomass, plant NP (nitrogen and phosphorus) accumulations and the water purification capacity of selected grasses were significantly different (P < 0.05). Abundant, AngusII and Major showed most efficient purification capacity of eutrophic water. In a greenhouse, after 26 days of growth, the eutrophic water was purified to various extents by the different ryegrass cultivars. Nitrogen removal efficiency varied from 52.20% to 73.82% and phosphorus removal efficiency ranged from 75.12% to 84.77%. In a lake experiment at Huajiachi pond, after 162 days of growth, the plant shoot biomass increased from 321.5 to 922.8 g/m(2) dry weight basis, shoot NP accumulation ranged from 61.5 to 168.2 mg m(-2) d(-1) and 11.9 to 47.2 mg m(-2) d(-1) respectively. NP accumulation rate by the various cultivars of ryegrass was highly positively correlated to their biomass production and water purification capacity. Thus, plant biomass could be used as an indicator for assessing purification capacity of a ryegrass cultivar.

Evolution and ecology meet molecular genetics: adaptive phenotypic plasticity in two isolated Negev desert populations of Acacia raddiana at either end of a rainfall gradient.

BACKGROUND AND AIMS: The ecological, evolutionary and genetic bases of population differentiation in a variable environment are often related to the selection pressures that plants experience. We compared differences in several growth- and defence-related traits in two isolated populations of Acacia raddiana trees from sites at either end of an extreme environmental gradient in the Negev desert. METHODS: We used random amplified polymorphic DNA (RAPD) to determine the molecular differences between populations. We grew plants under two levels of water, three levels of nutrients and three levels of herbivory to test for phenotypic plasticity and adaptive phenotypic plasticity. KEY RESULTS: The RAPD analyses showed that these populations are highly genetically differentiated. Phenotypic plasticity in various morphological traits in A. raddiana was related to patterns of population genetic differentiation between the two study sites. Although we did not test for maternal effects in these long-lived trees, significant genotype × environment (G × E) interactions in some of these traits indicated that such plasticity may be adaptive. CONCLUSIONS: The main selection pressure in this desert environment, perhaps unsurprisingly, is water. Increased water availability resulted in greater growth in the southern population, which normally receives far less rain than the northern population. Even under the conditions that we defined as low water and/or nutrients, the performance of the seedlings from the southern population was significantly better, perhaps reflecting selection for these traits. Consistent with previous studies of this genus, there was no evidence of trade-offs between physical and chemical defences and plant growth parameters in this study. Rather, there appeared to be positive correlations between plant size and defence parameters. The great variation in several traits in both populations may result in a diverse potential for responding to selection pressures in different environments.

Physiological parameters of plants as indicators of water quality in a constructed wetland.

INTRODUCTION: Increasing demand for water has stimulated efforts to treat wastewater for reuse in agriculture. Decentralized facilities for wastewater treatment became popular as a solution to remote and small communities. These systems mimic natural wetlands, cleaning wastewater as they flow through a complex of filter media, microbial fauna, and vegetation. The function of plants in constructed wetlands (CWs) has not been fully elucidated yet. DISCUSSION: In the research reported here, we provide evidence for a new use of plant physiological parameters in CWs as bioindicators of water quality along the system. We measured improved plant performance downstream of the CW by means of photochemical efficiency, CO(2) assimilation rate, and cell membrane stability. In addition, we found evidence for temporal improvement of plant performance, which was correlated to the establishment phase of plants in a newly operating CW. It is suggested that improved monitoring and management of CWs should take into planning consideration the promising potential of phyto-indicators.

Leishmania major: anti-leishmanial activity of Nuphar lutea extract mediated by the activation of transcription factor NF-κB.

Here we report the effect of a partially purified alkaloid fraction (NUP) of Nuphar lutea on nuclear factor kappa B (NF-κB) expression and studied its mechanism of toxicity against Leishmania major in C3H mice peritoneal macrophages. NUP was found to be a mixture of thermo-stable dimeric sesquiterpene thioalkaloids containing mainly thionupharidines. The anti-leishmanial activity was shown to be mediated through the activation of NF-κB and increased iNOS production. Additionally, the nitric oxide inhibitor, N(G)-monomethyl-L-arginine (0.5mM) totally reverted the anti-leishmanial effect of NUP (0.25 and 0.5µg/ml). NUP was also shown to act as an anti-oxidant, almost completely inhibiting the macrophage respiratory burst activity. However, no elevated lysozyme (EC3.2.1.17) or ß-galactosidase (EC3.2.1.23) activities were demonstrated in macrophages treated with NUP. This study suggests, that the activity of NUP is mediated by NF-κB activation and the production of nitric oxide which is dependent on the L-arginine:NO pathway.

Establishment of a constructed wetland in extreme dryland.

BACKGROUND, AIM, AND SCOPE: The project was set to construct an extensive wetland in the southernmost region of Israel at Kibbutz Neot Smadar (30 degree 02'45" N and 35 degree 01'19" E). The results of the first period of monitoring, summary, and perspectives are presented. The constructed wetland (CW) was built and the subsequent monitoring performed in the framework of the Southern Arava Sustainable Waste Management Plan, funded by the EU LIFE Fund. The specific aims were: (1) To end current sewage disposal and pollution of the ground, the aquifer, and the dry river bed (wadi) paths by biologically treating the sewage as part of the creation of a sustainable wetland ecosystem. (2) Serve as an example of CW in the Negev highlands and the Arava Valley climates for neighboring communities and as a test ground for plants and building methods appropriate to hyper arid climate. (3) Serve as an educational resource and tourist attraction for groups to learn about water reuse, recycling, local wildlife and migrating birds, including serving the heart of a planned Ecological-Educational Bird Park. This report is intended to allow others who are planning similar systems in hyper arid climates to learn from our experience. MATERIALS AND METHODS: The project is located in an extreme arid desert with less than 40 mm of rain annually and temperature ranges of -5 degree C to +42 degree C. The site receives 165-185 m3 of municipal and agricultural wastes daily, including cowshed and goat wastes and winery outflow. RESULTS: The CW establishment at Neot Smadar was completed in October 2006. For 8 months, clean water flowed through the system while the plants were taking root. In June 2007, the wetland was connected to the oxidation pond and full operation began. Because of seepage and evaporation, during the first several months, the water level was not high enough to allow free flow from one bed to the next. To bed A, the water was pumped periodically from the oxidation pond (Fig. 1) and from there flowed by gravitation through the rest of the system. The initial results of the monitoring are promising. In nearly all measurements, the system succeeded as expected to reduce levels of contaminants at least to the level acceptable for irrigating fruit trees and often to the level of unlimited irrigation. The introduction of the plants in the system and their physiological performance were evaluated and were found to correlate well to the quality of water in the various beds. DISCUSSION: It should be said at the outset that evaluation of the performance of a CW system is a long-term process. Thus, the main aim of this report is to present the problems, difficulties, preliminary results, and concepts concerned with the first stage of establishment of CW in an extremely dry region. CONCLUSIONS: The CW system was designed to dispose of municipal and agricultural wastes in a way that not merely reduces pollution, but adds to environmental quality by creating accessible parkland for local residents and tourists. Several factors affected the performance of the system at the initial stages of operation: ecological balance between microbes and plants, big seasonal variations, seepage and evaporation reduced the flow in the initial operation of the system. Despite the initial difficulties, the quality of water coming out the system is acceptable for irrigation. RECOMMENDATIONS AND PERSPECTIVES: The CW can function well under extreme dryland conditions. The oxidation pond was the major source of evaporation and bad odors. Therefore, alternatives to the oxidation pond are needed. Cost effectiveness of the system still has to be evaluated systematically.

Salt-driven interactions between Pistacia lentiscus and Salsola inermis.

BACKGROUND, AIM, AND SCOPE: It can be learned from the Pistacia spp. germplasm collection (http://www.bgu.ac.il/pistacia) that the growth of Salsola inermis is inhibited in the vicinity of the evergreen Pistacia lentiscus, but not in the surroundings of the deciduous Pistacia atlantica and Pistacia chinensis. Irrigation of trees during the summer months increases soil salinity around the trees. It was therefore hypothesized that inhibition of S. inermis around P. lentiscus is related to depletion of salt in the vicinity of the latter species. MATERIALS AND METHODS: A multi-approach experimental scheme was carried out which included soil edaphic characterization and germination tests. To test salt tolerance of P. lentiscus, plants were grown in a hydroponic system for a month in medium containing NaCl, while physiological and growth parameters were measured. RESULTS: Conductivity measurements in summer, during the growth season of S. inermis, indicated that soil salinity beneath deciduous Pistacia trees was significantly higher than that below P. lentiscus. Germination of S. inermis seeds on filter paper moistened with P. lentiscus low-conductivity soil filtrate was twice as high as that of the deciduous trees high-conductivity soil filtrates. Nevertheless, fresh and dry weights of mature S. inermis growing next to P. atlantica and P. chinensis were 2.9 to 4.8 times higher than those of plants growing in the vicinity of P. lentiscus. In a hydroponic system, no significant differences were found in growth parameters and stomatal conductance between P. lentiscus growing in control and salt treatments. It was therefore proposed that salt depletion in the vicinity of P. lentiscus inhibits the growth, but not germination, of S. inermis thus confirming the halophylic characteristics of this plant. DISCUSSION: The nature of Salsola-Pistacia interactions cannot be explained by allelopathic effects; hence, plausible salt-driven interactions were considered. Our data showed that S. inermis accumulated salt and has halophytic characteristics. Interestingly, germination of S. inermis was inhibited in medium containing salt, but the salt was obligatory for further growth, development, and fast biomass production. These results explained the observation of large biomass accumulation in the more saline soil around the deciduous P. atlantica and P. chinensis and the lack of development in the salt-depleted soil around the salt-tolerant accumulator P. lentiscus. CONCLUSIONS: Soil salinity around Pistacia trees critically affects the growth of S. inermis. Inhibition of S. inermis growth, but not germination, around the evergreen P. lentiscus, stems from the latter's ability to deplete salt from its surroundings. The results indicated that P. lentiscus is able to tolerate and accumulate salt, which we assume contributes to its wide distribution along the Mediterranean coast in Israel. Recommendations and perspectives While the phytoremediation potential of Salsola spp. has been explored to some extent, this of P. lentiscus has not been tested and proven before. The results suggest that the evergreen perennial salt-tolerant P. lentiscus can be recommended for horticulture purposes and soil stabilization in relatively saline environments.

Nuphar lutea thioalkaloids inhibit the nuclear factor kappaB pathway, potentiate apoptosis and are synergistic with cisplatin and etoposide.

We screened thirty-four methanolic plant extracts for inhibition of the constitutive nuclear factor kappaB (NFkappaB) activity by a NFkappaB-luciferase reporter gene assay. Strong inhibition of NFkappaB activity was found in extracts of leaf and rhizome from Nuphar lutea L. SM. (Nuphar). The inhibitory action was narrowed down to a mixture of thionupharidines and/or thionuphlutidines that were identified in chromatography fractions by one- and two-dimensional NMR analysis. Dimeric sesquiterpene thioalkaloids were identified as the major components of the mixture. The Nuphar alkaloids mixture (NUP) showed a dose dependent inhibition of NFkappaB activity in a luciferase reporter gene assay as well as reduction of nuclear NFkappaB subunits expression as tested by western blots and immunohistochemistry. Decreased DNA binding was demonstrated in electro mobility shift assays. NUP inhibited both inducible and constitutive NFkappaB activation and affected the canonical and alternative pathways. Suppression of NFkappaB was not cell type specific. Induction of apoptosis by the alkaloid mixture was demonstrated by time-dependent and dose-dependent cleavage of procaspase-9 and PARP. Synergistic cytotoxicity of the active mixture with cisplatin and etoposide was demonstrated. Overall, our results show that NUP inhibits the NFkappaB pathway and acts as a sensitizer to conventional chemotherapy, enabling the search for its specific target and application against cancer and inflammation.