ABSTRACT
The medicinal use of Cannabis sativa L. can be traced back thousands of years to ancient China and Egypt. While marijuana has recently shown promise in managing chronic pain and nausea, scientific investigation of cannabis has been restricted due its classification as a schedule 1 controlled substance. A major breakthrough in understanding the pharmacology of cannabis came with the isolation and characterization of the phytocannabinoids trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). This was followed by the cloning of the cannabinoid CB1 and CB2 receptors in the 1990s and the subsequent discovery of the endocannabinoid system. In addition to the major phytocannabinoids, Δ9-THC and CBD, cannabis produces over 120 other cannabinoids that are referred to as minor and/or rare cannabinoids. These cannabinoids are produced in smaller amounts in the plant and are derived along with Δ9-THC and CBD from the parent cannabinoid cannabigerolic acid (CBGA). While our current knowledge of minor cannabinoid pharmacology is incomplete, studies demonstrate that they act as agonists and antagonists at multiple targets including CB1 and CB2 receptors, transient receptor potential (TRP) channels, peroxisome proliferator-activated receptors (PPARs), serotonin 5-HT1a receptors and others. The resulting activation of multiple cell signaling pathways, combined with their putative synergistic activity, provides a mechanistic basis for their therapeutic actions. Initial clinical reports suggest that these cannabinoids may have potential benefits in the treatment of neuropathic pain, neurodegenerative diseases, epilepsy, cancer and skin disorders. This review focuses on the molecular pharmacology of the minor cannabinoids and highlights some important therapeutic uses of the compounds.
ABSTRACT
Currently, in the state of Colorado and all other states within the United States of America with legalized marijuana programs, testing is required for bacteria, yeast, and mold on marijuana products. The Code of Colorado Regulations, 1 CCR 212-1, considers a passing result when a 1â¯g sample contains <104 colony forming units (CFU) for the total yeast and mold count (TYMC). These measurements are usually obtained by manually counting colonies on petri-dishes or 3â¯M™ Petrifilms™, which is a time consuming and user subjective process. Therefore, an automated counting method utilizing ImageJ has been developed for CFU analysis of TYMC on Petrifilms. The performance of this colony counting method was demonstrated by comparing manual and automated counts from marijuana flower samples containing spikes of Candida albicans as well as samples that tested positive for the presence of yeast and mold. Fifteen images of Petrifilms showing various concentrations of colonies were studied by fifteen users at two institutions using both the automated and manual counting methods. All counts from the automated ImageJ procedure were within 12% of those obtained manually. In twelve out of fifteen Petrifilms, the average count of the automated method was statistically similar to the manual counts. The statistical differences of the other three samples were observed to be random and caused by user errors. The automated counting method could be used to quickly count numbers that are as high as 400 CFUs, reducing time of analysis with improved documentation because the images and the electronic colony counts can be saved on a computer or cloud for long term storage and data access.
Subject(s)
Cannabis/microbiology , Colony Count, Microbial/methods , Flowers/microbiology , Image Processing, Computer-Assisted/methods , Yeasts/growth & development , Candida albicans/growth & development , Colony Count, Microbial/instrumentation , Image Processing, Computer-Assisted/instrumentation , Microbiological Techniques/methods , Stem CellsABSTRACT
A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.
Subject(s)
Green Fluorescent Proteins/metabolism , Microfluidics/methods , Pseudomonas aeruginosa/physiology , Stress, Mechanical , Automation , Biofilms/growth & development , Bioreactors , Entropy , Image Processing, Computer-Assisted , Microscopy, Fluorescence , Pseudomonas aeruginosa/growth & development , SoftwareABSTRACT
The "International Research Experience for Students (IRES)" at Doane University (DU) located in Crete, Nebraska, exposed undergraduate science, technology, engineering, and mathematics (STEM) students to international research at the Karlsruhe Institute of Technology (KIT) in Germany. The international collaboration team included three undergraduate researchers per year from DU, one faculty member and one postdoctoral fellow from DU, two faculty mentors at KIT, and several graduate, post-doctoral, and technical staff at KIT. Prior to departure to Germany, the students received extensive research training, as well as culture and language preparation from the mentors at DU. While in Germany, the students received an in-depth orientation to Karlsruhe, Germany, Europe, the research setting at KIT, and the international collaborators. The eight week summer projects over three years involved nanolithography, nano- to microsized array fabrication, organic synthesis using click chemistry, and surface modifications for sensing and other biomedical research applications. When the students returned from Germany, they continued to conduct research at DU and train other undergraduate students using the expertise acquired from KIT. The DU research students, including the IRES scholars, learned oral and written communication skills. They presented their KIT and DU research results at weekly seminars and at local and national meetings. An external assessment firm evaluated the program, the students, and mentors on a yearly basis before and after the summer research. This enabled all participants to continuously improve the learning objectives and the program execution including three program adjustments implemented in year 2 or 3. The survey data shows that the IRES program provided an enriching experience for the students in research and international culture and established a successful base of collaboration for mentors.
ABSTRACT
Chemical warfare agents pose significant threats in the 21st century, especially for armed forces. A colorimetric detection array was developed to identify warfare mimics, including mustard gas and nerve agents. In total, 188 sensors were screened to determine the best sensor performance, in order to identify warfare mimics 2-chloro ethyl ethylsulfide, 2-2'-thiodiethanol, trifluoroacetic acid, methylphosphonic acid, dimethylphosphite, diethylcyanophosphonate, and diethyl (methylthiomethyl)phosphonate. The highest loadings in the principle component analysis (PCA) plots were used to identify the sensors that were most effective in analyzing the RGB data to classify the warfare mimics. The dataset was reduced to only twelve sensors, and PCA results gave comparable results as the large data did, demonstrating that only twelve sensors are needed to classify the warfare mimics.
Subject(s)
Chemical Warfare Agents/analysis , Colorimetry/methods , Chemical Warfare Agents/chemistry , Color , Principal Component AnalysisABSTRACT
Solid supported colorimetric sensing arrays have the advantage of portability and ease of use when deployed in the field, such as crime scenes, disaster zones, or in war zones, but many sensor arrays require complex fabrication methods. Here, we report a practical method for the fabrication of 4 × 4 colorimetric sensor arrays, which are printed on nylon membranes, using a commercially available inkjet printer. In order to test the efficacy of the printed arrays, they were exposed to 43 analytes at concentrations ranging from 0.001 to 3.0 M for a total of 559 samples of inorganic and organic acids or bases including hydrochloric, acetic, phthalic, malonic, picric, and trifluoroacetic acid, ammonium hydroxide, sodium hydroxide, lysine, and water as the control. Colorimetric data from the imaged arrays was analyzed with linear discriminant analysis and k-nearest neighbors to determine the analyte and concentration with â¼88-90% accuracy. Overall, the arrays have impressive analytical power to identify a variety of analytes at different concentrations while being simple to fabricate.
Subject(s)
Carboxylic Acids/analysis , Colorimetry/methods , Hydrochloric Acid/analysis , Hydroxides/analysis , Lysine/analysis , Colorimetry/instrumentation , Discriminant Analysis , PrintingABSTRACT
With the increasing availability of digital imaging devices, colorimetric sensor arrays are rapidly becoming a simple, yet effective tool for the identification and quantification of various analytes. Colorimetric arrays utilize colorimetric data from many colorimetric sensors, with the multidimensional nature of the resulting data necessitating the use of chemometric analysis. Herein, an 8 sensor colorimetric array was used to analyze select acid and basic samples (0.5 - 10 M) to determine which chemometric methods are best suited for classification quantification of analytes within clusters. PCA, HCA, and LDA were used to visualize the data set. All three methods showed well-separated clusters for each of the acid or base analytes and moderate separation between analyte concentrations, indicating that the sensor array can be used to identify and quantify samples. Furthermore, PCA could be used to determine which sensors showed the most effective analyte identification. LDA, KNN, and HQI were used for identification of analyte and concentration. HQI and KNN could be used to correctly identify the analytes in all cases, while LDA correctly identified 95 of 96 analytes correctly. Additional studies demonstrated that controlling for solvent and image effects was unnecessary for all chemometric methods utilized in this study.
ABSTRACT
A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP). The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD600) and plate counting (colony-forming units (CFUs)). While both OD600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.
ABSTRACT
Colorimetric sensor arrays incorporating red, green, and blue (RGB) image analysis use value changes from multiple sensors for the identification and quantification of various analytes. RGB data can be easily obtained using image analysis software such as ImageJ. Subsequent chemometric analysis is becoming a key component of colorimetric array RGB data analysis, though literature contains mainly principal component analysis (PCA) and hierarchical cluster analysis (HCA). Seeking to expand the chemometric methods toolkit for array analysis, we explored the performance of nine chemometric methods were compared for the task of classifying 631 solutions (0.1 to 3 M) of acetic acid, malonic acid, lysine, and ammonia using an eight sensor colorimetric array. PCA and LDA (linear discriminant analysis) were effective for visualizing the dataset. For classification, linear discriminant analysis (LDA), (k nearest neighbors) KNN, (soft independent modelling by class analogy) SIMCA, recursive partitioning and regression trees (RPART), and hit quality index (HQI) were very effective with each method classifying compounds with over 90% correct assignments. Support vector machines (SVM) and partial least squares - discriminant analysis (PLS-DA) struggled with ~85 and 39% correct assignments, respectively. Additional mathematical treatments of the data set, such as incrementally increasing the exponents, did not improve the performance of LDA and KNN. The literature precedence indicates that the most common methods for analyzing colorimetric arrays are PCA, LDA, HCA, and KNN. To our knowledge, this is the first report of comparing and contrasting several more diverse chemometric methods to analyze the same colorimetric array data.
ABSTRACT
Slippery, porous polymeric antimicrobial surfaces for biofilm attachment inhibition of the clinical strain Pseudomonas aeruginosa (PA14) have been prepared. Porous BMA-EDMA, characterized for its hydrophobic properties, was infused with a slippery liquid creating a hydrophobic liquid interface and characterized by water contact angle and SEM. A low shear force bioreactor was used to prepare biofilms on these antimicrobial surfaces. Biofilm attachment was studied using fluorescence microscopy coupled with image analysis in ImageJ. While the literature presents that these slippery polymers work well as antimicrobial surfaces for several strains of Pseudomonas aeruginosa, it has been found to be strain dependent. This report demonstrates that slippery surfaces do not work well for the strain PA14, and biofilm covered >3.5 times more area as compared to the control glass surfaces.
ABSTRACT
Plant root exudates are important mediators in the interactions that occur between plants and microorganisms in the soil, yet much remains to be learned about spatial and temporal variation in their production. This work outlines a method utilizing a novel colorimetric paper to detect spatial and temporal changes in the production of nitrogen-containing compounds on the root surface. While existing methods have made it possible to conduct detailed analysis of root exudate composition, relatively less is known about where in the root system exudates are produced and how this localization changes as the root grows. Furthermore, there is much to learn about how exudate localization and composition varies in response to stress. Root exudates are chemically diverse secretions composed of organic acids, amino acids, proteins, sugars, and other metabolites. The sensor utilized for the method, ninhydrin, is a colorless substance in solution that reacts with free amino groups to form a purple dye. A detection paper was developed by formulating ninhydrin into a print solution that was uniformly deposited onto paper with a commercial ink jet printer. This "ninhydrin paper" was used to analyze the chemical makeup of root surfaces from maize seedlings grown vertically on germination paper. Through contact between the ninhydrin paper and seedling root surfaces, combined with images of both the seedlings and dried ninhydrin papers captured using a standard flatbed scanner, nitrogen-containing substances on the root surface can be localized and concentration of signal estimated for over 2 weeks of development. The method was found to be non-inhibiting to plant growth over the analysis period although damage to root hairs was observed. The method is sensitive in the detection of free amines at concentrations as little as 140 µM. Furthermore, ninhydrin paper is stable, showing consistent color changes up to 2 weeks after printing. This relatively simple, low-cost method could contribute to a better understanding of root exudates and mechanisms used by plants to interact with the complex soil environment during growth and development.
ABSTRACT
Protein-repellent reactive surfaces that promote localized specific binding are highly desirable for applications in the biomedical field. Nonspecific adhesion will compromise the function of bioactive surfaces, leading to ambiguous results of binding assays and negating the binding specificity of patterned cell-adhesive motives. Localized specific binding is often achieved by attaching a linker to the surface, and the other side of the linker is used to bind specifically to a desired functional agent, as e.g. proteins, antibodies, and fluorophores, depending on the function required by the application. We present a protein-repellent polymer brush enabling highly specific covalent surface immobilization of biorecognition elements by strain-promoted alkyne-azide cycloaddition click chemistry for selective protein adhesion. The protein-repellent polymer brush is functionalized by highly localized molecular binding sites in the low micrometer range using polymer pen lithography (PPL). Because of the massive parallelization of writing pens, the tunable PPL printed patterns can span over square centimeter areas. The selective binding of the protein streptavidin to these surface sites is demonstrated while the remaining polymer brush surface is resisting nonspecific adsorption without any prior blocking by bovine serum albumin (BSA). In contrast to the widely used BSA blocking, the reactive polymer brushes are able to significantly reduce nonspecific protein adsorption, which is the cause of biofouling. This was achieved for solutions of single proteins as well as complex biological fluids. The remarkable fouling resistance of the polymer brushes has the potential to improve the multiplexing capabilities of protein probes and therefore impact biomedical research and applications.
ABSTRACT
There is a significant demand for devices that can rapidly detect chemical-biological-explosive (CBE) threats on-site and allow for immediate responders to mitigate spread, risk, and loss. The key to an effective reconnaissance mission is a unified detection technology that analyzes potential threats in real time. In addition to reviewing the current state of the art in the field, this review illustrates the practicality of colorimetric arrays composed of sensors that change colors in the presence of analytes. This review also describes an outlook toward future technologies, and describes how they could possibly be used in areas such as war zones to detect and identify hazardous substances.
Subject(s)
Chemical Warfare , Colorimetry/methods , Explosive Agents/analysis , Colorimetry/instrumentation , Hazardous Substances/analysisABSTRACT
Bacterial biofilms pose a significant health risk when they grow on devices placed or implanted in the human body. There is a need to develop new materials that can be used as surface coatings on such devices to inhibit biofilm growth. We report on measurements of the biofilm growth rate on a new polymeric material, slippery BMA-EDMA, which can be used as a surface coating for medical devices. Growth rate measurements are also reported for polycarbonate and glass surfaces, for comparison. Measurements are made in a medium shear stress fluid environment. The physical properties of the surfaces are characterized using contact angle, surface roughness, surface skewness and surface kurtosis. Growth rate on the slippery BMA-EDMA is found to be the smallest of the three surfaces. Growth rate is weakly correlated with surface hydrophobicity and surface roughness, while it is strongly correlated with surface skewness and kurtosis.
ABSTRACT
Biofilms are microbial communities attached to a surface and embedded in an extracellular polymeric substance which provides for the protection, stability and nutrients of the various bacterial species indwelling. These communities can build up in a variety of different environments from industrial equipment to medical devices resulting in damage, loss of productivity and disease. They also have great potential for economic and societal benefits as bioremediation agents and renewable energy sources. The great potential benefits and threats of biofilms has encouraged researchers across disciplines to study biofilm characteristics and antibiofilm strategies resulting in chemists, physicists, material scientists, and engineers, to develop beneficial biofilm applications and prevention methods. The ultimate outcome is a wealth of knowledge and innovative technology. However, without extensive formal training in microbes and biofilm research, these scientists find a daunting array of established techniques for growing, quantifying and characterizing biofilms while trying to design experiments and develop innovative laboratory protocols. This mini-review focuses on enriching interdisciplinary efforts and understanding by overviewing a variety of quantitative and qualitative biofilm characterization methods to assist the novice researcher in assay selection. This review consists of four parts. Part 1 is a brief overview of biofilms and the unique properties that demand a highly interdisciplinary approach. Part 2 describes the classical quantification techniques including colony forming unit (CFU) counting and crystal violet staining, but also introduces some modern methods including ATP bioluminescence and quartz crystal microbalance. Part 3 focuses on the characterization of biofilm morphology and chemistry including scanning electron microscopy and spectroscopic methods. Finally, Part 4 illustrates the use of software, including ImageJ and predictive modeling platforms, for biofilm analysis. Each section highlights the most common methods, including literature references, to help novice biofilm researchers make choices which commensurate with their study goals, budget and available equipment.
ABSTRACT
In order to determine if electronic circular dichroism (ECD) is a good tool for the qualitative evaluation of absolute configuration and enantiopurity in the absence of chiral high performance liquid chromatography (HPLC), ECD studies were performed on several prescriptions and over-the-counter drugs. Cotton effects (CE) were observed for both S and R isomers between 200 and 300 nm. For the drugs examined in this study, the S isomers showed a negative CE, while the R isomers displayed a positive CE. The ECD spectra of both enantiomers were nearly mirror images, with the amplitude proportional to the enantiopurity. Plotting the differential extinction coefficient (Δε) versus enantiopurity at the wavelength of maximum amplitude yielded linear standard curves with coefficients of determination (R (2)) greater than 97% for both isomers in all cases. As expected, Equate, Advil, and Motrin, each containing a racemic mixture of ibuprofen, yielded no chiroptical signal. ECD spectra of Suphedrine and Sudafed revealed that each of them is rich in 1S,2S-pseudoephedrine, while the analysis of Equate vapor inhaler is rich in R-methamphetamine.
ABSTRACT
The work described herein examines a rapid mix-and-measure method called DETECHIP suitable for screening of steroids and metabolites. The addition of steroids and metabolites to reactive arrays of colorimetric sensors generated characteristic color "fingerprints" that were used to identify the analyte. A color analysis tool was used to identify the analyte pool that now includes biologically relevant analytes. The mix-and-measure arrays allowed the detection of disease metabolites, orotic acid and argininosuccinic acid; and the steroids androsterone, 1,4-androstadiene, testosterone, stanozolol, and estrone. The steroid 1,4-androstadiene was also detected by this method while dissolved in synthetic urine. Some of the steroids, such as androstadiene, stanozolol, and androsterone were co-dissolved with (2-hydroxypropyl)-ß-cyclodextrin in order to increase solubility in aqueous buffered solutions. The colorimetric arrays do not intend to eliminate ELISA or mass spectroscopy based screening, but to possibly provide an alternative analytical detection method for steroids and metabolites.
ABSTRACT
We explored the potentials of microarray printing using quill-like microcantilevers onto solid supports that are typically used in microspot printing, including paper, polymeric nitrocellulose and nylon membranes. We compared these membranes with a novel porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) support (HEMA) with narrow pore size distribution in the 150 nm range, which demonstrated advantages in pattern definition, spot homogeneity, and consistent spot delivery of different dyes (phloxine B and bromophenol blue) with diameters of several micrometres. The bromophenol blue arrays on HEMA support were used to detect the presence of bovine serum albumin (BSA). In the presence of BSA, the fluorescence spectrum observed from the bromophenol blue microarray exhibited a significant red shift of the maximum emission wavelength. Our results show that the porous HEMA substrates can improve the fidelity and quality of microarrays prepared by using the quill-like microcantilevers. The presented method sets the stage for further studies using chemical and biochemical recognition elements, along with colorimetric and fluorometric sensors that can be spotted by this method onto flat porous polymer substrates.
ABSTRACT
BACKGROUND: Novel, blue fluorescent solids referred to as Wild Plum compounds can camouflage skin imperfections when incorporated into cosmetic products. We evaluated the relationship between sun exposure and skin fluorescence and determined if the application of Wild Plum formulations could restore lost fluorescence without harming the skin. METHODS: The forehead skin of two groups of volunteers of mixed gender and age was examined for fluorescence and redness. In addition, subjects answered questions describing any adverse sensations they experienced after their skin was exposed to Wild Plum formulations for extended periods of time. RESULTS: Fluorescence measurements of both solar and non-solar skin indicated that repeated sun exposure causes a loss of skin fluorescence. Application of Wild Plum formulations caused an increase in skin fluorescence at all concentrations, restoring solar skin fluorescence to values well beyond that of non-solar skin. Photo analysis and interview questions indicated that these formulations did not cause any symptoms of irritancy. CONCLUSION: Wild Plum compounds have the ability to restore fluorescence of solar skin to a level significantly higher than that associated with non-solar skin. Skin appears more luminous and therefore more youthful. This fluorescence restoration is achieved at relatively low concentrations, without any harmful side effects.
Subject(s)
Cosmetics/administration & dosage , Fluorescent Dyes/administration & dosage , Skin Aging/drug effects , Sunlight/adverse effects , Ultraviolet Rays/adverse effects , Adult , Aged , Chemistry, Pharmaceutical , Color , Cosmetics/adverse effects , Emollients/administration & dosage , Emollients/adverse effects , Female , Fluorescent Dyes/adverse effects , Forehead , Humans , Male , Middle Aged , Prunus , Skin Care/methods , Spectrometry, Fluorescence , Young AdultABSTRACT
DETECHIP® is a detection system made of various sensors that has been shown to detect and discriminate between small molecules of interest, including various illicit and over-the-counter drugs. Previously, detection was normalized to a single concentration of analyte. Now this detection assay can detect concentration differences in analytes via red, green, and blue color value changes and shifts in the UV-Vis spectra of the assay. To determine the concentrations differences, the exposed assays were scanned on a flatbed scanner and the images were analyzed for individual RGB values with a custom macro in ImageJ, an image analysis program. Increasing concentrations of the analyte resulted in greater differences in color values between control and analyte wells. These differences showed a linear relationship to concentration change, some with correlation coefficients greater than 98%. This work expands the capability of DETECHIP to give information about the concentration of analyte when the analyte identity is known.