Abiotic and Biotic Damage of Microalgae Generate Different Volatile Organic Compounds (VOCs) for Early Diagnosis of Algal Cultures for Biofuel Production.

Open microalgal ponds used in industrial biomass production are susceptible to a number of biotic and abiotic environmental stressors (e.g., grazers, pathogens, pH, temperature, etc.) resulting in pond crashes with high economic costs. Identification of signature chemicals to aid in rapid, non-invasive, and accurate identification of the stressors would facilitate targeted and effective treatment to save the algal crop from a catastrophic crash. Specifically, we were interested in identifying volatile organic compounds (VOCs) that can be used to as an early diagnostic for algal crop damage. Cultures of Microchloropsis gaditana were subjected to two forms of algal crop damage: (1) active grazing by the marine rotifer, Brachionus plicatilis, or (2) repeated freeze-thaw cycles. VOCs emitted above the headspace of these algal cultures were collected using fieldable solid phase microextraction (SPME) fibers. An untargeted analysis and identification of VOCs was conducted using gas chromatography-mass spectrometry (GC-MS). Diagnostic VOCs unique to each algal crop damage mechanism were identified. Active rotifer grazing of M. gaditana was characterized by the appearance of carotenoid degradation products, including ß-cyclocitral and various alkenes. Freeze-thaw algae produced a different set of VOCs, including palmitoleic acid. Both rotifer grazing and freeze-thawed algae produced ß-ionone as a VOC, possibly suggesting a common stress-induced cellular mechanism. Importantly, these identified VOCs were all absent from healthy algal cultures of M. gaditana. Early detection of biotic or abiotic environmental stressors will facilitate early diagnosis and application of targeted treatments to prevent algal pond crashes. Thus, our work further supports the use of VOCs for monitoring the health of algal ponds to ultimately enhance algal crop yields for production of biofuel.

Structural determination of four manufacturing impurities of D&C Red No. 33.

Four manufacturing impurities of D&C Red No. 33 isolated by counter-current chromatography were analyzed by NMR and ESI mass spectrometry. Three of these impurities were reported previously with minimal details of structural determination. All four are structurally related to the main component of the dye. The fourth exhibited an unusual discrepancy between the NMR structure and its chemical formula suggested by ESI-MS results. Structural determination and assignment of the main component and four impurities are discussed as well as resolution of the discrepancy between the NMR and ESI-MS results of the fourth impurity.

Metabolic Profiling of Volatile Organic Compounds (VOCs) Emitted by the Pathogens Francisella tularensis and Bacillus anthracis in Liquid Culture.

We conducted comprehensive (untargeted) metabolic profiling of volatile organic compounds (VOCs) emitted in culture by bacterial taxa Francisella tularensis (F. tularensis) subspecies novicida and Bacillus anthracis (B. anthracis) Sterne, surrogates for potential bacterial bioterrorism agents, as well as selective measurements of VOCs from their fully virulent counterparts, F. tularensis subspecies tularensis strain SCHU S4 and B. anthracis Ames. F. tularensis and B. anthracis were grown in liquid broth for time periods that covered logarithmic growth, stationary, and decline phases. VOCs emitted over the course of the growth phases were collected from the headspace above the cultures using solid phase microextraction (SPME) and were analyzed using gas chromatography-mass spectrometry (GC-MS). We developed criteria for distinguishing VOCs originating from bacteria versus background VOCs (originating from growth media only controls or sampling devices). Analyses of collected VOCs revealed methyl ketones, alcohols, esters, carboxylic acids, and nitrogen- and sulfur-containing compounds that were present in the bacterial cultures and absent (or present at only low abundance) in control samples indicating that these compounds originated from the bacteria. Distinct VOC profiles where observed for F. tularensis when compared with B. anthracis while the observed profiles of each of the two F. tularensis and B. anthracis strains exhibited some similarities. Furthermore, the relative abundance of VOCs was influenced by bacterial growth phase. These data illustrate the potential for VOC profiles to distinguish pathogens at the genus and species-level and to discriminate bacterial growth phases. The determination of VOC profiles lays the groundwork for non-invasive probes of bacterial metabolism and offers prospects for detection of microbe-specific VOC biomarkers from two potential biowarfare agents.

Chemical Profiling of Volatile Organic Compounds in the Headspace of Algal Cultures as Early Biomarkers of Algal Pond Crashes.

Algae ponds used in industrial biomass production are susceptible to pathogen or grazer infestation, resulting in pond crashes with high economic costs. Current methods to monitor and mitigate unhealthy ponds are hindered by a lack of early indicators that precede culture crash. We used solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) to identify volatiles emitted from healthy and rotifer infested cultures of Microchloropsis salina. After 48 hours of algal growth, marine rotifers, Brachionus plicatilis, were added to the algae cultures and volatile organic compounds (VOC) were sampled from the headspace using SPME fibers. A GC-MS approach was used in an untargeted analysis of VOCs, followed by preliminary identification. The addition of B. plicatilis to healthy cultures of M. salina resulted in decreased algal cell numbers, relative to uninfected controls, and generated trans-ß-ionone and ß-cyclocitral, which were attributed to carotenoid degradation. The abundances of the carotenoid-derived VOCs increased with rotifer consumption of algae. Our results indicate that specific VOCs released by infected algae cultures may be early indicators for impending pond crashes, providing a useful tool to monitor algal biomass production and pond crash prevention.

Detecting opioid metabolites in exhaled breath condensate (EBC).

Exhaled breath condensate (EBC) collection provides a promising matrix for bioanalysis of endogenous biomarkers of health and also for exogenous compounds like drugs. There is little information regarding drugs and their metabolites contained in breath, as well as their pharmacokinetics. In this present work, we use a simple and non-invasive technique to collect EBC from chronic pain patients using different analgesic opioid drugs to manage pain. Six patients received continuous infusion of morphine and hydromorphone intravenously (IV), together with other analgesic drugs (IV and orally). Repeated sampling of serum and EBC was done at two time points separated by 90 min. The EBC was collected using a glass tube surrounded by dry ice, and an ethanol solvent wash of the glass was performed after EBC extraction to retrieve the apolar compounds stuck to the glass surface. All samples were analyzed with liquid chromatography coupled to mass spectrometry (LC-MS/MS) to identify possible metabolites present in the sample, and to quantify the drugs being used. Several metabolites, such as normorphine (norM), norhydromorphone (norHM) and dihydromorphone (diHM) were detected in both fractions, while hydromorphone 3-glucuronide (HM 3G) was only detected in the solvent rinse fraction. Results were correlated to explain the pharmacokinetics of the main drugs administered. This pilot study presented promising correlations between drug concentrations in blood and breath at different time points for norM, norHM and HM 3G.

Characterization of smokeless powders using multiplexed collision-induced dissociation mass spectrometry and chemometric procedures.

This work demonstrates a non-targeted mass spectrometry approach for identification of organic compounds in smokeless powders. Unburned powders were removed from various commercial ammunitions of different brand, primer composition, caliber, and age. The unburned powders and corresponding fired residues were analyzed by liquid chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry (LC-APCI-TOFMS). Multiplexed collision-induced dissociation was performed at increasing collision potentials resulting in successive fragmentation that provided structural information for compound identification in a non-targeted manner. Nine compounds were identified in the powders, including akardite II, ethyl centralite, diphenylamine, N-nitrosodiphenylamine, and dibutyl phthalate. Multivariate statistical procedures were performed to first investigate association and discrimination of the unburned powders. Principal components analysis (PCA) of the chemical profiles suggested nine distinct groups of powders, according to the dominant organic compounds present. The clusters formed in hierarchical cluster analysis (HCA) were mostly in agreement with PCA groupings although HCA provided a metric to quantify the similarity. Finally, association of the fired residue to the corresponding unburned powder was possible although the success was highly dependent on the composition of the unburned powder and the extent of compound depletion as a result of firing.

A "Resident-as-Teacher" Curriculum Using a Flipped Classroom Approach: Can a Model Designed for Efficiency Also Be Effective?

PROBLEM: The Accreditation Council for Graduate Medical Education requires training that enhances resident teaching skills. Despite this requirement, many residency training programs struggle to implement effective resident-as-teacher (RAT) curricula, particularly within the context of the 80-hour resident workweek. APPROACH: In 2013, the authors developed and evaluated an intensive one-day RAT curriculum using a flipped classroom approach. Twenty-nine second-year residents participated in daylong RAT sessions. The curriculum included four 1-hour workshops focusing on adult learning principles, giving feedback, teaching a skill, and orienting a learner. Each workshop, preceded by independent reading, featured peer co-teaching, application, and feedback. The authors evaluated the curriculum using pre- and postworkshop objective structured teaching examinations (OSTEs) and attitudinal and self-efficacy teaching questionnaires. OUTCOMES: Residents demonstrated statistically significant improvements in performance between pre- and postworkshop OSTEs on each of three core skills: giving feedback (P = .005), orienting a learner (P < .001), and teaching a skill (P < .001). Residents expressed positive attitudes surrounding teaching on the retrospective pre-post attitudinal instrument (P < .001) and rated themselves as more effective teachers (P < .001) after the training. NEXT STEPS: The authors have demonstrated that the flipped classroom approach is an efficient and effective method for training residents to improve teaching skills, especially in an era of work hour restrictions. They have committed to the continuation of this curriculum and are planning to include assessment of its long-term effects on resident behavior change and educational outcomes.

Effect of reduced retinal VLC-PUFA on rod and cone photoreceptors.

PURPOSE: Autosomal dominant Stargardt-like macular dystrophy (STGD3) is a juvenile-onset disease that is caused by mutations in Elovl4 (elongation of very long fatty acids-4). The Elovl4 catalyzes the first step in the conversion of C24 and longer fatty acids (FAs) to very long-chain FAs (VLC-FAs, ≥C26). Photoreceptors are particularly rich in VLC polyunsaturated FAs (VLC-PUFA). To explore the role of VLC-PUFAs in photoreceptors, we conditionally deleted Elovl4 in the mouse retina. METHODS: Proteins were analyzed by Western blotting and lipids by gas chromatography (GC)-mass spectrometry, GC-flame ionization detection, and tandem mass spectrometry. Retina function was assessed by electroretinography (ERG), and structure was evaluated by bright field, immunofluorescence, and transmission electron microscopy. RESULTS: Conditional deletion (KO) of retinal Elovl4 reduced RNA and protein levels by 91% and 96%, respectively. Total retina VLC-PUFAs were reduced by 88% compared to the wild type (WT) levels. Retinal VLC-PUFAs incorporated in phosphatidylcholine were less abundant at 12 months compared to 8-week-old levels. Amplitudes of the ERG a-wave were reduced by 22%, consistent with photoreceptor degeneration (11% loss of photoreceptors). Analysis of the rod a-wave responses gave no evidence of a role for VLC-PUFA in visual transduction. However, there were significant reductions in rod b-wave amplitudes (>30%) that could not be explained by loss of rod photoreceptors. There was no effect of VLC-PUFA reduction on cone ERG responses, and cone density was not different between the WT and KO mice at 12 months of age. CONCLUSIONS: The VLC-PUFAs are important for rod, but not cone, function and for rod photoreceptor longevity.

Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: evidence for a role for hyaluronidase 3 in mouse and human sperm.

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or "reproductive" hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic "somatic" Hyaluronidase 3 (HYAL3), a homolog of SPAM1 with 74.6% structural similarity, exists in two isoforms in human ( approximately 47 and approximately 55 kDa) and mouse ( approximately 44 and approximately 47 kDa) sperm, where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at pH 7. At pH 4, a second acid-active hyase band at approximately 57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4, where Spam1 nulls had significantly (P < 0.01) diminished activity implicating an acidic optima for murine SPAM1. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3 is expressed in epididymal tissue/fluid, from where it is acquired by caudal mouse sperm in vitro. Our results reveal concerted activity of both neutral- and acid-active hyaluronidases in sperm.

Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model.

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.