The mycelium of the Trametes versicolor synn. Coriolus versicolor (Turkey tail mushroom) exhibit anti-melanoma activity in vitro.

Melanoma is one of the most aggressive forms of skin cancer and is characterized by high metastatic potential. Despite improvements in early diagnosis and treatment, the mortality rate among metastatic melanoma patients continues to represent a significant clinical challenge. Therefore, it is imperative that we search for new forms of treatment. Trametes versicolor is a mushroom commonly used in Chinese traditional medicine due to its numerous beneficial properties. In the present work, we demonstrate T. versicolor fruiting body and mycelium ethanol extracts exhibit potent cytotoxic activity towards A375 (IC50 = 663.3 and 114.5 µg/mL respectively) and SK-MEL-5 (IC50 = 358.4 and 88.6 µg/mL respectively) human melanoma cell lines. Further studies revealed that T. versicolor mycelium extract induced apoptotic cell death and poly (ADP-ribose) polymerase cleavage, upregulated the expression of autophagy-associated marker LC3-II, increased the presentation of major histocompatibility complex II and expression of programmed death-ligand receptor, and inhibited cell migration in SK-MEL-5 cells. Therefore, our present findings highlight the therapeutic potential of T. versicolor mycelium extract for the treatment of melanoma and merit further study.

Development of Biodegradable/Biocompatible Nanoliposome-Encapsulated Antimicrobial Essential Oils for Topical Creams and Gels.

Nanoencapsulation with safe materials improves delivery, stability, and activity of bioactive components. We report a novel safe, and effective method for the development of encapsulated antimicrobial essential oils (EO) for topical creams and gels. The method developed features three aspects that, to our knowledge, had not been previously demonstrated: (1) use of novel liposomes (LPs) to encapsulate EOs, (2) use of the EOs to replace synthetic organic solvents that are potentially toxic and/or leave harmful residues, and (3) an encapsulation process at temperatures below the boiling point of water. The LPs were made from soy lecithin, phytosterol, and α-tocopherol (vitamin E) that were synthesized using the EOs as the solvent. The liposomes were converted to nanoliposomes (NLPs) through a series of sonication, homogenization, and extrusion steps. Transmission electron microscopy indicated that the NLPs alone and nanoliposome encapsulated EOs (NLP-EOs) were spherical in shape with sizes ranging between 50 and 115 nm diameter and with negative zeta potentials ranging from -34 to -43 mV. There was no significant heavy metal contamination [As, Pb, Cd, Hg] based on inductively coupled plasma (ICP) mass spectrometry MS analyses. Nearly complete EO encapsulation (95% encapsulation efficiency) was achieved and confirmed by GC/MS. Three of the NLP-EOs made of various essential oils were used to make topical formulations (cream and gel) which exhibited antimicrobial activities against Escherichia coli (Gram negative) and Bacillus subtilis (Gram positive) bacteria. The creams with NLP-EOs were as active against the two bacteria in the antimicrobial assays as the conventional antibiotic Kanamycin that was used as positive control.

THE DEVELOPMENT OF A HIGH-RESOLUTION MASS SPECTROMETRY METHOD FOR ULTRA-TRACE ANALYSIS OF CHLORINATED DIOXINS IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES INCLUDING VIET NAM ERA VETERANS.

Chlorinated dioxins are labeled and recognized by both the World Health Organization and the United Nations Environmental Programme (UNEP) as "persistent organic pollutants". Their potential for high toxicity is one of the primary factors behind intense public and regulatory scrutiny and the need to measure the compounds at very low limits, specifically the isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). This article highlights the early mass spectrometry methods to investigate, detect, confirm, and quantify chlorinated dioxins and the initial applications involving human biomonitoring, as attempts were made to attribute health effects to TCDD exposure. This effort represented a complex and difficult scientific response to the pressing need to investigate expected exposures and alleged subsequent medical effects, which in the case of the Viet Nam veterans was being attempted a decade or more after their exposure. It is noteworthy that this method and its development touched on delicate issues involving human subjects, war veterans, environmental contamination, and was difficult not only scientifically, but for ethical and political reasons as well. Stable-isotope dilution with analysis by gas chromatography/high-resolution mass spectrometry (GC/HRMS) became the method of choice because of its ability to monitor characteristic ions and isotope ratios to quantify and qualify/confirm the analyte in the presence of coextracting and coeluting interferences at these low levels with the highest possible confidence. This method was rigorously tested and validated before it was used to discover and monitor levels in the environment and in various populations at then unprecedented low levels. These early studies demonstrated the feasibility of monitoring dioxins in humans even decades after exposure, and led to the detection of 2,3,7,8-TCDD in the general population as well as specific overexposed populations. These studies also provided strong evidence regarding the origins of the 2,3,7,8-isomer in the environment. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Matrix-assisted ionization Fourier transform mass spectrometry for the analysis of lipids.

RATIONALE: Assessing the utility of vacuum matrix-assisted ionization (MAI) for the direct and rapid analysis of lipids in complex samples with emphasis on bacterial taxonomy. METHODS: Matrix-assisted ionization Fourier transform mass spectrometry (MAI-FTMS) was used to characterize polar and non-polar lipids in mixtures. RESULTS: For non-polar lipid triacylglycerols (TAGs), MAI-FTMS produced lipid-specific ions for eight different edible oils and allowed these oils to be identified based on their MAI-FTMS profiles. For polar lipids from bacteria, MAI-FTMS of crude lipid extracts allowed taxonomic identification of eight blind-coded samples based on taxonomy-specific phospholipid profiles. MAI produced results comparable and complementary to benchmark MALDI and ESI methods currently used for characterization of polar and non-polar lipids in the same mixtures. CONCLUSIONS: The newly developed MAI technique is a rapid, simple and complementary method for the characterization of polar and non-polar lipids in complex mixtures.

Thymosin ß4 dynamics during chicken enteroid development.

The sheared avian intestinal villus-crypts exhibit high tendency to self-repair and develop enteroids in culture. Presuming that this transition process involves differential biomolecular changes, we employed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) to find whether there were differences in the spectral profiles of sheared villi versus the enteroids, assessed in the mass range of 2-18 kDa. The results showed substantial differences in the intensities of the spectral peaks, one particularly corresponding to the mass of 4963 Da, which was significantly low in the sheared villus-crypts compared with the enteroids. Based on our previous results with other avian tissues and further molecular characterization by LC-ESI-IT-TOF-MS, and multiple reaction monitoring (MRM), the peak was identified to be thymosin ß4 (Tß4), a ubiquitously occurring regulatory peptide implicated in wound healing process. The identity of the peptide was further confirmed by immunohistochemistry which showed it to be present in a very low levels in the sheared villi but replete in the enteroids. Since Tß4 sequesters G-actin preventing its polymerization to F-actin, we compared the changes in F-actin by its immunohistochemical localization that showed no significant differences between the sheared villi and enteroids. We propose that depletion of Tß4 likely precedes villous reparation process. The possible mechanism for the differences in Tß4 profile in relation to the healing of the villus-crypts to developing enteroids is discussed.

The Arabidopsis Proteins AtNHR2A and AtNHR2B Are Multi-Functional Proteins Integrating Plant Immunity With Other Biological Processes.

AtNHR2A (Arabidopsis thaliana nonhost resistance 2A) and AtNHR2B (Arabidopsis thaliana nonhost resistance 2B) are two proteins that participate in nonhost resistance, a broad-spectrum mechanism of plant immunity that protects plants against the majority of potential pathogens. AtNHR2A and AtNHR2B are localized to the cytoplasm, chloroplasts, and other subcellular compartments of unknown identity. The multiple localizations of AtNHR2A and AtNHR2B suggest that these two proteins are highly dynamic and versatile, likely participating in multiple biological processes. In spite of their importance, the specific functions of AtNHR2A and AtNHR2B have not been elucidated. Thus, to aid in the functional characterization of these two proteins and identify the biological processes in which these proteins operate, we used immunoprecipitation coupled with mass spectrometry (IP-MS) to identify proteins interacting with AtNHR2A and AtNHR2B and to generate their interactome network. Further validation of three of the identified proteins provided new insights into specific pathways and processes related to plant immunity where AtNHR2A and AtNHR2B participate. Moreover, the comprehensive analysis of the AtNHR2A- and AtNHR2B-interacting proteins using published empirical information revealed that the functions of AtNHR2A and AtNHR2B are not limited to plant immunity but encompass other biological processes.

Cold tolerance response mechanisms revealed through comparative analysis of gene and protein expression in multiple rice genotypes.

Due to its tropical origin and adaptation, rice is significantly impacted by cold stress, and consequently sustains large losses in growth and productivity. Currently, rice is the second most consumed cereal in the world and production losses caused by extreme temperature events in the context of "major climatic changes" can have major impacts on the world economy. We report here an analysis of rice genotypes in response to low-temperature stress, studied through physiological gas-exchange parameters, biochemical changes in photosynthetic pigments and antioxidants, and at the level of gene and protein expression, towards an understanding and identification of multiple low-temperature tolerance mechanisms. The first effects of cold stress were observed on photosynthesis among all genotypes. However, the tropical japonica genotypes Secano do Brazil and Cypress had a greater reduction in gas exchange parameters like photosynthesis and water use efficiency in comparison to the temperate japonica Nipponbare and M202 genotypes. The analysis of biochemical profiles showed that despite the impacts of low temperature on tolerant plants, they quickly adjusted to maintain their cellular homeostasis by an accumulation of antioxidants and osmolytes like phenolic compounds and proline. The cold tolerant and sensitive genotypes showed a clear difference in gene expression at the transcript level for OsGH3-2, OsSRO1a, OsZFP245, and OsTPP1, as well as for expression at the protein level for LRR-RLKs, bHLH, GLYI, and LTP1 proteins. This study exemplifies the cold tolerant features of the temperate japonica Nipponbare and M202 genotypes, as observed through the analysis of physiological and biochemical responses and the associated changes in gene and protein expression patterns. The genes and proteins showing differential expression response are notable candidates towards understanding the biological pathways affected in rice and for engineering cold tolerance, to generate cultivars capable of maintaining growth, development, and reproduction under cold stress. We also propose that the mechanisms of action of the genes analyzed are associated with the tolerance response.

Phorbol 12-Myristate 13-Acetate-Induced Changes in Chicken Enterocytes.

Increased intestinal epithelial permeability has been linked to many enteric diseases because it allows easy access of microbial pathogens and toxins into the system. In poultry production, the restrictions in the use of antibiotic growth promoters have increased the chances of birds being susceptible to different enteric diseases. Thus, understanding the mechanisms which compromise intestinal function is pertinent. Based on our previous observation which showed the primary chicken enterocytes in culture undergoing dystrophic changes on treatment with phorbol myristate acetate (PMA), we surmised that this model, which appeared to mimic increased intestinal permeability, may help to understand the mechanisms of this problem. As genomic and proteomic changes are associated with many physiological and pathological problems, we were interested to find whether certain proteomic changes underlie the morphological alterations in the enterocytes induced by PMA. We exposed primary enterocyte cultures to a sub-lethal concentration of PMA, extracted the proteins, and analyzed by mass spectrometry for differentially regulated proteins. Our results showed that PMA affected several biological processes which negatively affected their energy metabolism, nuclear activities, and differentially regulated the levels of several stress proteins, chaperon, cytoskeletal, and signal transduction proteins that appear to be relevant in the cause of enterocyte dystrophy. Phorbol myristate acetate-affected signal transduction activities also raise the possibilities of their increased susceptibility to pathogens. The changes in enterocyte integrity can make intestine vulnerable to invasion by microbial pathogens and disrupt gut homeostasis.

Microdialysis Sampling of Quorum Sensing Homoserine Lactones during Biofilm Formation.

Bacteria communicate chemically through a system called quorum sensing. In this work, microdialysis sampling procedures were optimized to collect quorum sensing molecules produced during in situ biofilm formation directly on the polymeric semipermeable membrane of the microdialysis probe. V. harveyi, a Gram-negative bacterium, was used as the model organism and releases variable chain length acylhomoserine lactones (AHLs) and acyl-oxohomoserine lactones (AOHLs) as signaling molecules during quorum sensing. Eliciting biofilm formation required coating fetal bovine serum onto the poly(ether sulfone) microdialysis membrane. Dialysates were collected in different experiments either during or after biofilm formation directly on a microdialysis probe. Continuous sampling of C4-AHL, C6-AHL, C8-AHL, C6-OXO-AHL, and C12-OXO-AHL was achieved over a period of up to 4 days. The AHLs and AOHLs in dialysates were concentrated with solid-phase extraction and quantified using LC-MS. Dialysate concentrations obtained for the AOHLs and AHLs ranged between 1 and 100 ppb (ng/mL) and varied between sampling days. This work demonstrates the initial use of microdialysis sampling to collect quorum sensing signaling chemicals during biofilm formation by a Gram-negative bacterial species.

A method to culture chicken enterocytes and their characterization.

Enterocytes function as both absorptive and protective components of intestine that come in close contact with a variety of enteric factors, such as dietary, microbial, and parasites, that have potential to affect the organismal health. Understanding how enterocytes interact with this complex array of factors may help improve gut health particularly in the context of poultry production where it is also linked to food safety issues. The enterocyte in vitro culture can help screen different factors and their interactions with microbiome, and potentially be utilized in the development of interventions strategies for pathogens such as antibiotic alternatives. We developed a method to culture primary chicken enterocytes and conducted their characterization using cytochemical and proteomic methods, and investigated their potential to respond to different chemical stimuli. Using selected micronutrients, microbial toxins, and metabolic modulators, we assessed their effects on the viability and morphological changes in enterocytes. We found that whereas some nutritional factors (calcitriol, retinoic acid) produced different morphological changes, toxins such as aflatoxin B1 and deoxynivalenol produced enterocyte degeneration and death, and the bacterial lipopolysaccharide had very little effect compared on the basis of their mass. Both cyclic AMP and phorbol myristate acetate exhibited some cachectic effects on enterocytes with the later showing more severe changes. Thyroxin induced distinct morphological changes making the cells more cuboidal and Na-butyrate produced no significant change in morphology. The cytochemical and proteomic characterization suggest that these enterocytes largely belong to epithelial cell categories which may be amenable to analysis of biochemical paths and mechanisms of action of different factors that affect these cells. Based on these results we conclude that chicken enterocyte culture can be a useful in vitro model to study intestinal physiology.

A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide Using Proteogenomics.

Salmonella is an intracellular pathogen infecting a wide range of hosts and can survive in macrophages. An essential mechanism used by macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2O2. For Tn-seq, a saturated Tn5 insertion library was grown in vitro under either 2.5 (H2O2L) or 3.5 mM H2O2 (H2O2H). We identified two sets of overlapping genes required for resistance of S. Typhimurium to H2O2L and H2O2H, and the results were validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for H2O2 resistance included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), and H2O2 scavenging enzymes. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H2O2. On the contrary, a subset of conditionally essential proteins identified by Tn-seq were analyzed by targeted proteomics, and 70% of them were upregulated by H2O2. The identified genes will deepen our understanding on S. Typhimurium survival mechanisms in macrophages, and can be exploited to develop new antimicrobial drugs.

Changes in polyphenolics during maturation of Java plum (Syzygium cumini Lam.).

Java plum (Syzygium cumini Lam.) is a rich source of polyphenolics with many purported health benefits, but the effect of maturation on polyphenolic content is unknown. Freeze-dried samples of Java plum from seven different maturity stages were analyzed for anthocyanin, flavonol, flavanonol and hydrolysable tannin composition by HPLC. Anthocyanins were first detected at the green-pink stage of maturity and increased throughout maturation with the largest increase occurring from the dark purple to black stages of maturation. Levels of gallotannins, ellagitannins, flavonols, gallic acid and ellagic acid were highest at early stages of maturation and decreased as the fruit ripened. For production of antioxidant-rich nutraceutical ingredients, fruit should be harvested immature to obtain extracts rich in hydrolysable tannins and flavonols. The exceptional anthocyanin content of black fruit may prove useful as a source of a natural colorant.

Proteomic Changes in the Plasma of Broiler Chickens with Femoral Head Necrosis.

Femoral head necrosis (FHN) is a skeletal problem in broiler chickens, where the proximal femoral head cartilage shows susceptibility to separation from its growth plate. The selected birds with FHN showed higher body weights and reduced plasma cholesterol. The proteomic differences in the plasma of healthy and FHN-affected chickens were explored using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography/electrospray ionization-tandem mass spectrometry (LC-MS/MS) to prospect for protein biomarkers. We isolated two differentially expressed low molecular weight proteins and identified them by MALDI peptide mass fingerprinting as fibrinogen- and fetuin-derived peptides, respectively. These peptides were reduced in birds susceptible to femoral head problems. Quantitation of LC-MS/MS spectra showed elevated levels of gallinacin-9, apolipoprotein A1, and hemoglobin and reduced levels of alpha-1-acid glycoprotein, albumin, and SPINK7 proteins in FHN. These results suggest that the bodyweight and the lipid profiles along with the above proteins can be useful as noninvasive biomarkers of FHN.

Proteomic Changes in Chicken Plasma Induced by Salmonella typhimurium Lipopolysaccharides.

Lipopolysaccharides (LPS) are cell wall components of Gram-negative bacteria that produce inflammation and sickness in higher animals. The objective was to identify plasma proteomic changes in an avian model of inflammation. Chickens were treated with either saline or LPS, and blood was collected at 24 hours postinjection. The pooled plasma samples were depleted of high-abundant proteins and analyzed by matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). MALDI analyses showed an increase in fibrinogen beta-derived peptide and a decrease in apolipoprotein-AII-derived peptide in LPS samples. Label-free quantitation of LC-MS/MS spectra revealed an increase in the levels of α1-acid glycoprotein, a chemokine CCLI10, and cathelicidin-2, but a decrease in an interferon-stimulated gene-12-2 protein in the LPS group. These differentially expressed proteins are associated with immunomodulation, cytokine changes, and defense mechanisms, which may be useful as candidate biomarkers of infection and inflammation.

Isolation and Characterization of Chicken Yolk Vitelline Membrane Lipids Using Eggs Enriched With Conjugated Linoleic Acid.

The vitelline membrane (VM) encloses the chicken egg yolk, separating it from albumen. The VM weakens during storage, and dietary lipid modification significantly affects its strength. However, no studies have characterize the fatty acyl residue (FA) composition of the VM, and reports of VM isolation and quantified lipid content are inconsistent. Therefore, the objectives of this study were: (1) to develop a washing and isolation method that removes residual yolk from VM without damage; (2) to determine the FA and lipid composition of CLA-rich egg yolk VM, relative to controls; (3) to determine the effect of 20 days of refrigeration on VM FA and lipid composition. To determine VM FA and lipid composition, 36 hens received either a corn-soybean meal-based control diet ("Control"), or the Control supplemented with either 10 % soy oil ("Soy control"), or 10 % CLA-rich soy oil ("CLA") for 30 days. VM were analyzed the day of collection ("fresh"), or after 20 days of refrigeration ("refrigerated"). There were no differences in FA compositions of fresh and refrigerated membranes within a treatment. CLA-rich yolk VM contains CLA, greater SFA, and significantly greater DHA relative to controls. Direct MALDI-TOF-MS identified 15 phosphatidylcholines, three phosphatidylethanolamines, one sphingomyelin, and 15 triacylglycerols in VM. Lipid species that showed significant differences among egg types included nine phosphatidylcholines and six triacylglycerols. MALDI analysis indicated significant differences in nine lipid classes on the VM inner layer. After refrigeration, five lipid classes on the inner layer and seven lipid classes on the outer layer had statistically significant differences among VM types.

Simple radiometric method for accurately quantitating epitope densities of hapten-protein conjugates with sulfhydryl linkages.

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses (14)C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten-BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (r(2) = 0.98, y = 0.98x + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten-protein conjugations, and choosing the most effective epitope densities for conjugate vaccines.

Biomass and RRR-α-tocopherol production in Stichococcus bacillaris strain siva2011 in a balloon bioreactor.

BACKGROUND: Green microalgae represent a renewable natural source of vitamin E. Its most bioactive form is the naturally occurring RRR-α-tocopherol which is biosynthesized in photosynthetic organisms as a single stereoisomer. It is noteworthy that the natural and synthetic α-tocopherols are different biomolecular entities. This article focuses on RRR-α-tocopherol production in Stichococcus bacillaris strain siva2011 biomass in a bioreactor culture with methyl jasmonate (MeJa) elicitor. Additionally, a nonlinear mathematical model was used to quantitatively scale-up and predict the biomass production in a 20 L balloon bioreactor with dual variables such as time and volume. RESULTS: Approximately 0.6 mg/g dry weight (DW) of RRR-α-tocopherol was enhanced in S. bacillaris strain siva2011 biomass with the MeJa 50 µL/L for 24 hrs elicitations when compared to the control. The R2 value from the nonlinear model was enhanced up to 95% when compared to the linear model which significantly improved the accuracy for estimating S. bacillaris strain siva2011 biomass production in a balloon bioreactor. CONCLUSIONS: S. bacillaris strain siva2011 is a new green microalga which biosynthesizes significant amounts of RRR-α-tocopherol. Systematically validated dual variable empirical data should provide key insights to multivariable or fourth order modeling for algal biomass scale-up. This bioprocess engineering should provide valuable information for industrial production of RRR-α-tocopherol from green cells.

Isolation and characterization of chicken bile matrix metalloproteinase.

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.

Bioprocessing of Stichococcus bacillaris strain siva2011.

BACKGROUND: Globally, the development of a cost-effective long-term renewable energy infrastructure is one of the most challenging problems faced by society today. Microalgae are rich in potential biofuel substrates such as lipids, including triacylglycerols (TAGs). Some of these algae also biosynthesize small molecule hydrocarbons. These hydrocarbons can often be used as liquid fuels, often with more versatility and by a more direct approach than some TAGs. However, the appropriate TAGs, accumulated from microalgae biomass, can be used as substrates for different kinds of renewable liquid fuels such as biodiesel and jet fuel. RESULTS: This article describes the isolation and identification of a lipid-rich, hydrocarbon-producing alga, Stichococcus bacillaris strain siva2011, together with its bioprocessing, hydrocarbon and fatty acid methyl ester (FAME) profiles. The S. bacillaris strain siva2011 was scaled-up in an 8 L bioreactor with 0.2% CO2. The C16:0, C16:3, C18:1, C18:2 and C18:3 were 112.2, 9.4, 51.3, 74.1 and 69.2 mg/g dry weight (DW), respectively. This new strain produced a significant amount of biomass of 3.79 g/L DW on day 6 in the 8 L bioreactor and also produced three hydrocarbons. CONCLUSIONS: A new oil-rich microalga S. bacillaris strain siva2011 was discovered and its biomass has been scaled-up in a newly designed balloon-type bioreactor. The TAGs and hydrocarbons produced by this organism could be used as substrates for jet fuel or biodiesel.

Gas chromatography-mass spectrometry of JWH-018 metabolites in urine samples with direct comparison to analytical standards.

JWH-018 (1-pentyl-3-(1-naphthoyl)indole) is one of numerous potential aminoalkylindoles contained in products marketed as 'K2' or 'Spice'. Investigation of the urinary metabolites from consumption of these compounds is important because they are banned in the United States and many European countries. An efficient extraction procedure and gas chromatography-mass spectrometry (GC-MS) method were developed for detection of 'K2' metabolites in urine from individuals suspected of using these products. Analytical standards were used to elucidate the structure-specific mass spectral fragmentations and retention properties to confirm proposed identifications and support quantitative studies. A procedure for the synthesis of one of these metabolites (5-hydroxypentyl JWH-018) was also developed. Results are comparable to existing LC-MS/MS methods, with the same primary metabolites detected. The specific metabolite hydrolysis products include 4-hydroxpentyl, 5-hydroxypentyl, and N-pentanoic acid derivatives.