The heterogeneity and complexity of Cannabis extracts as antitumor agents.

The Cannabis plant contains over 100 phytocannabinoids and hundreds of other components. The biological effects and interplay of these Cannabis compounds are not fully understood and yet influence the plant's therapeutic effects. Here we assessed the antitumor effects of whole Cannabis extracts, which contained significant amounts of differing phytocannabinoids, on different cancer lines from various tumor origins. We first utilized our novel electrospray ionization liquid chromatography mass spectrometry method to analyze the phytocannabinoid contents of 124 Cannabis extracts. We then monitored the effects of 12 chosen different Cannabis extracts on 12 cancer cell lines. Our results show that specific Cannabis extracts impaired the survival and proliferation of cancer cell lines as well as induced apoptosis. Our findings showed that pure (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) did not produce the same effects on these cell lines as the whole Cannabis extracts. Furthermore, Cannabis extracts with similar amounts of Δ9-THC produced significantly different effects on the survival of specific cancer cells. In addition, we demonstrated that specific Cannabis extracts may selectively and differentially affect cancer cells and differing cancer cell lines from the same organ origin. We also found that cannabimimetic receptors were differentially expressed among various cancer cell lines and suggest that this receptor diversity may contribute to the heterogeneous effects produced by the differing Cannabis extracts on each cell line. Our overall findings indicate that the effect of a Cannabis extract on a specific cancer cell line relies on the extract's composition as well as on certain characteristics of the targeted cells.

Antifouling Properties of a Self-Assembling Glutamic Acid-Lysine Zwitterionic Polymer Surface Coating.

There is a need for the development of antifouling materials to resist adsorption of biomacromolecules. Here we describe the preparation of a novel zwitterionic block copolymer with the potential to prevent or delay the formation of microbial biofilms. The block copolymer comprised a zwitterionic (hydrophilic) section of alternating glutamic acid (negatively charged) and lysine (positively charged) units and a hydrophobic polystyrene section. Cryo-TEM and dynamic-light-scattering (DLS) results showed that, on average, the block copolymer self-assembled into 7-nm-diameter micelles in aqueous solutions (0 to 100 mM NaCl, pH 6). Quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and contact angle measurements demonstrated that the block copolymer self-assembled into a brush-like monolayer on polystyrene surfaces. The brush-like monolayer produced from a 100 mg/L block copolymer solution exhibited an average distance, d, of approximately 4-8 nm between each block copolymer molecule (center to center). Once the brush-like monolayer self-assembled, it reduced EPS adsorption onto the polystyrene surface by ∼70% (mass), reduced the rate of bacterial attachment by >80%, and inhibited the development of thick biofilms. QCM-D results revealed that the EPS molecules penetrate between the chains of the brush and adsorb onto the polystyrene surface. Additionally, AFM analyses showed that the brush-like monolayer prevents the adhesion of large (> d) hydrophilic colloids onto the surface via hydration repulsion; however, molecules or colloids small enough to fit between the brush polymers (< d) were able to be adsorbed onto the surface via van der Waals interactions. Overall, we found that the penetration of extracellular organelles, as well as biopolymers through the brush, is critical for the failure of the antifouling coating, and likely could be prevented through tuning of the brush density. Stability and biofilm development testing on multiple surfaces (polypropylene, glass, and stainless steel) support practical applications of this novel block copolymer.

A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis.

Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic effects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufficient to comprehend the medicinal and pharmacological effects of the whole plant. In this study we suggest a new ESI-LC/MS/MS approach to identify phytocannabinoids from 10 different subclasses, and comprehensively profile the identified compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identified and used for profiling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant effects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same effects. This study's approach for phytocannabinoid profiling can enable researchers and physicians to analyze the effects of specific Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis.

Potential microbial hazards from graywater reuse and associated matrices: A review.

Millions of decentralized graywater-reuse systems are operating worldwide. This water is directly accessible to household inhabitants, raising environmental and public health concerns. Graywater may contain a variety of harmful organisms, the types and numbers of which vary with source-type, storage time, and background levels of infection in the community source. In this review, we find that most studies indicate high amounts of microbial pathogens in raw graywater and therefore treatment and disinfection are recommended to lower possible health risks. Where these recommendations have been followed, epidemiological and quantitative microbial risk-assessment studies have found negligible health risks of bacterial pathogens in treated graywater. Chlorine is currently suggested as the most cost-effective disinfection agent for inactivating graywater bacterial pathogens and preventing regrowth. Various studies demonstrate that the introduction and diversity of pathogenic bacteria in the soil via irrigation can be affected by several factors, but treated graywater may not be a major contributor of bacterial contamination or antibiotic resistance. However, an accurate assessment of the infectious capabilities, exposure pathways, and resistance of specific pathogens, particularly viruses and antibiotic-resistant bacteria found in treated graywater after disinfection, as well as in the graywater piping, irrigated soils, plants, and associated aerosols is largely lacking in the literature. In addition, research shows that fecal bacterial indicators might not reliably indicate the presence or quantities of pathogens in graywater and thus, the indicator standard for graywater contamination should be revised.

Quantification and risks associated with bacterial aerosols near domestic greywater-treatment systems.

Greywater (GW) reuse can alleviate water stress by lowering freshwater consumption. However, GW contains pathogens that may compromise public health. During the GW-treatment process, bioaerosols can be produced and may be hazardous to human health if inhaled, ingested, or come in contact with skin. Using air-particle monitoring, BioSampler®, and settle plates we sampled bioaerosols emitted from recirculating vertical flow constructed wetlands (RVFCW) - a domestic GW-treatment system. An array of pathogens and indicators were monitored using settle plates and by culturing the BioSampler® liquid. Further enumeration of viable pathogens in the BioSampler® liquid utilized a newer method combining the benefits of enrichment with molecular detection (MPN-qPCR). Additionally, quantitative microbial risk assessment (QMRA) was applied to assess risks of infection from a representative skin pathogen, Staphylococcus aureus. According to the settle-plate technique, low amounts (0-9.7×10(4)CFUm(-2)h(-1)) of heterotrophic bacteria, Staphylococcus spp., Pseudomonas spp., Klebsiella pneumoniae, Enterococcus spp., and Escherichia coli were found to aerosolize up to 1m away from the GW systems. At the 5m distance amounts of these bacteria were not statistically different (p>0.05) from background concentrations tested over 50m away from the systems. Using the BioSampler®, no bacteria were detected before enrichment of the GW-aerosols. However, after enrichment, using an MPN-qPCR technique, viable indicators and pathogens were occasionally detected. Consequently, the QMRA results were below the critical disability-adjusted life year (DALY) safety limits, a measure of overall disease burden, for S. aureus under the tested exposure scenarios. Our study suggests that health risks from aerosolizing pathogens near RVFCW GW-treatment systems are likely low. This study also emphasizes the growing need for standardization of bioaerosol-evaluation techniques to provide more accurate quantification of small amounts of viable, aerosolized bacterial pathogens.

The question of pathogen quantification in disinfected graywater.

Graywater (GW) reuse for irrigation is recognized as a sustainable solution for water conservation. One of the major impediments to GW reuse is the presence of pathogenic microorganisms. This study monitored three similar on-site GW treatment systems bi-monthly over the course of a year to compare the presence of pathogens and indicators in raw, biologically treated, and biologically treated and disinfected [by chlorine and ultraviolet light (UV)] GW. The systems were designed to allow the testing of the same batch (collection) of water as it passed through the treatment chain. The samples were analyzed using standard culture-dependent methods and the data were compared to culture-independent DNA-based methods. Results suggested that the presence and abundance of fecal coliforms, Escherichia coli, Salmonella enterica, Enterococcus spp., Staphylococcus aureus and Pseudomonas aeruginosa differ among the various GW streams (e.g. raw, biologically treated, and disinfected). The culture-dependent analyses suggested that both chlorine and UV inactivate most of the bacteria tested in the biologically treated GW, albeit at different efficiencies. Conversely, the DNA-based analyses indicated no significant differences in pathogenic bacterial abundance between the biologically treated GW with or without disinfection. To better understand the discrepancies between the results, we repeated the analysis in the laboratory under controlled conditions using Enterococcus faecalis as a model bacterium and obtained similar results. We suggest that disinfection of biologically treated GW with chlorine or UV is effective for treating pathogens, but that the inactivation efficiency cannot be estimated by DNA-based qPCR.

Adapting enzyme-based microbial water quality analysis to remote areas in low-income countries.

Enzyme-substrate microbial water tests, originally developed for efficiency gains in laboratory settings, are potentially useful for on-site analysis in remote settings. This is especially relevant in developing countries where water quality is a pressing concern and qualified laboratories are rare. We investigated one such method, Colisure, first for sensitivity to incubation temperatures in order to explore alternative incubation techniques appropriate for remote areas, and then in a remote community of Zambia for detection of total coliforms and Escherichia coli in drinking-water samples. We sampled and analyzed 352 water samples from source, transport containers and point-of-use from 164 random households. Both internal validity (96-100%) and laboratory trials (zero false negatives or positives at incubation between 30 and 40 °C) established reliability under field conditions. We therefore recommend the use of this and other enzyme-based methods for remote applications. We also found that most water samples from wells accessing groundwater were free of E. coli whereas most samples from surface sources were fecally contaminated. We further found very low awareness among the population of the high levels of recontamination in household storage containers, suggesting the need for monitoring and treatment beyond the water source itself.

Assessment of pathogenic bacteria in treated graywater and irrigated soils.

Reuse of graywater (GW) for irrigation is recognized as a sustainable solution for water conservation. One major impediment for reuse of GW is the possible presence of pathogenic microorganisms. The presence and abundance of six pathogens and indicators were investigated in three GW recirculating vertical flow constructed wetland treatment systems and their respective irrigated yard soils. The treated GW and soils were monitored once every two months for six months using real-time quantitative PCR. As a control, samples from four soils irrigated with fresh water (FW) were similarly analyzed for pathogens and indicators. Comparable types of pathogens and fecal indicator bacteria, including Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa, Enterococcus faecalis, and Shigella spp., were found in the treated GW, their corresponding irrigated soils and the FW-irrigated soils. Moreover, the abundance of these bacteria in the GW- and FW-irrigated soils was of the same order of magnitude, suggesting that the source of the pathogens cannot be established. Our results suggest that GW irrigation has no effect on the diversity and abundance of the tested pathogens and indicators in yard soils.