Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis.

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography-mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

Effect of Ultralight Dark Matter on g-2 of the Electron.

If dark matter is ultralight, the number density of dark matter is very high, and the techniques of zero-temperature field theory are no longer valid. The dark matter number density modifies the vacuum, giving it a non-negligible particle occupation number. For fermionic dark matter, this occupation number can be no larger than one. However, in the case of bosons, the occupation number is unbounded. If there is a large occupation number, the Bose enhancement needs to be taken into consideration for any process involving particles which interact with the dark matter. Because the occupation number scales inversely with the dark matter mass, this effect is most prominent for ultralight dark matter. In fact, the Bose enhancement effect from the background is so significant for ultralight dark matter that, if dark matter is a dark photon, the correction to the anomalous magnetic moment is larger than experimental uncertainties for a mixing parameter of the order of 10^{-16} and a dark photon mass of the order of 10^{-20} eV. Furthermore, the constraint on the mixing parameter scales linearly with the dark photon mass, and so new significant constraints can be placed on the dark matter mass all the way up to 10^{-14} eV. Future experiments measuring g-2 will probe even smaller gauge mixing parameters.

The impact of the complexing agent on the sensitivity of collision-induced dissociation spectra to fatty acid position for a set of XYZ-type triglycerides.

RATIONALE: The development of an automated platform for the positional analysis of triglycerides (TAGs) based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) continues to be pursued. This work evaluates the positional sensitivities of the collision-induced dissociation (CID) spectra of a representative set of XYZ triglycerides using sodium, lithium, and ammonium salts as complexing agents. METHODS: A set of triglycerides were synthesized and analyzed via ESI-MS/MS using an ion trap mass spectrometer. Using three different complexing agents, the product ion spectra of the corresponding precursor ions for twelve XYZ TAGs were collected, where X, Y, and Z represent C16:0 , C18:1(c-9) , C18:2(cc-9,12) , and C20:4(cccc-5,8,11,14) fatty acid chains. These data were then used to prepare ternary plots for four positional isomer systems to evaluate the positional sensitivity differences among the three different complexing agents. RESULTS: The positional sensitivities for each of the four positional isomer systems were robust for the sodium and lithium adducts. The CID data for the sodium and lithium TAGs demonstrated an unfavorable loss of the fatty acid in the center position and showed a higher sensitivity to fatty acid position, when compared with the CID data for ammonium adducts, especially for the arachidonic acid containing triglycerides. CONCLUSIONS: The data shows that that the relative abundances of the DAG product ions for the XYZ-type TAGs when using sodium and lithium complexing agent adducts are sensitive to fatty acid position and are consistent for the diverse array of TAGs studied in this work. This suggests that using sodium or lithium as the complexing agent may be advantageous for the development of an automated platform for the positional analysis of complex TAG mixtures based on ESI-MS/MS.

Conserving alpha and beta diversity in wood-production landscapes.

International demand for wood and other forest products continues to grow rapidly, and uncertainties remain about how animal communities will respond to intensifying resource extraction associated with woody bioenergy production. We examined changes in alpha and beta diversity of bats, bees, birds, and reptiles across wood production landscapes in the southeastern United States, a biodiversity hotspot that is one of the principal sources of woody biomass globally. We sampled across a spatial gradient of paired forest land-uses (representing pre and postharvest) that allowed us to evaluate biological community changes resulting from several types of biomass harvest. Short-rotation practices and residue removal following clearcuts were associated with reduced alpha diversity (-14.1 and -13.9 species, respectively) and lower beta diversity (i.e., Jaccard dissimilarity) between land-use pairs (0.46 and 0.50, respectively), whereas midrotation thinning increased alpha (+3.5 species) and beta diversity (0.59). Over the course of a stand rotation in a single location, biomass harvesting generally led to less biodiversity. Cross-taxa responses to resource extraction were poorly predicted by alpha diversity: correlations in responses between taxonomic groups were highly variable (-0.2 to 0.4) with large uncertainties. In contrast, beta diversity patterns were highly consistent and predictable across taxa, where correlations in responses between taxonomic groups were all positive (0.05-0.4) with more narrow uncertainties. Beta diversity may, therefore, be a more reliable and information-rich indicator than alpha diversity in understanding animal community response to landscape change. Patterns in beta diversity were primarily driven by turnover instead of species loss or gain, indicating that wood extraction generates habitats that support different biological communities.

Conservación de la Diversidad Alfa y Beta en Paisajes de Producción Maderera Resumen La demanda internacional de madera y otros productos forestales sigue creciendo rápidamente mientras permanecen las incertidumbres sobre cómo responderán las comunidades animales a la intensificación de la extracción de recursos asociada con la producción de bioenergía leñosa. Examinamos los cambios en la diversidad alfa y beta de murciélagos, abejas, aves y reptiles en los paisajes de producción maderera en el sureste de los Estados Unidos, un punto caliente de biodiversidad y una de las fuentes principales de biomasa leñosa a nivel mundial. Muestreamos a lo largo de un gradiente espacial de usos de suelo forestales emparejados (representando la pre- y postcosecha) que nos permitió evaluar los cambios en las comunidades biológicas resultantes de varios tipos de recolección de biomasa. Las prácticas de corta rotación y de eliminación de residuos después de la tala estuvieron asociadas con la reducción de la diversidad alfa (−14.1 y −13.9 especies, respectivamente) y una diversidad beta más baja (es decir, diferencia de Jaccard) entre los pares de uso de suelo (0.46 y 0.50, respectivamente), mientras que el raleo de rotación media incrementó la diversidad alfa (+3.5 especies) y beta (0.59). Durante la duración de una rotación permanente en una sola ubicación, la cosecha de biomasa generalmente derivó en menos biodiversidad. La respuesta de los taxones a la extracción de recursos estuvo muy mal pronosticada por la diversidad alfa: la correlación de las respuestas entre los grupos taxonómicos fue altamente variable (−0.2 a 0.4) con muchas incertidumbres. Como contraste, los patrones de diversidad beta fueron fuertemente coherentes y predecibles en todos los taxones, mientras que la correlación de las respuestas entre los grupos taxonómicos siempre fue positiva (0.05 a 0.4) con incertidumbres más limitadas. Por lo tanto, la diversidad beta puede ser un indicador más confiable y rico en información que la diversidad alfa para entender las respuestas de la comunidad animal a los cambios en el paisaje. Los patrones de la diversidad beta estuvieron impulsados principalmente por la rotación en lugar de la pérdida o ganancia de especies, lo que indica que la extracción de madera genera hábitats que mantienen a diferentes comunidades biológicas.

Shifting landscapes of risk: Quantifying pluvial flood vulnerability beyond the regulated floodplain.

Floods are recognized as the costliest type of natural hazard both worldwide and in the United States, with projected increases in frequency and magnitude in the absence of effective adaptation strategies. In the fall of 2018, Hurricane Florence made landfall in southeastern North Carolina, USA, bringing record rainfall and resulting in widespread inundation that impacted many areas outside of the federally designated Special Flood Hazard Area (SFHA). Much of this flooding was from inland pluvial inundation, which is an understudied component of coastal risk and vulnerability assessments primarily due to the scarcity of infrastructure data and historically lower flooding recurrence rates. This has resulted in severe damages in areas that residents and local officials considered at low risk from flooding. Using nearly-coincident high-spatial, high-temporal resolution CubeSat satellite imagery, we quantified the areal extent of post-Hurricane Florence floodwater within and beyond the 100-year floodplain (SFHA) and the proportion of residential structures exposed to flooding within an eight-county study area. We propose a novel approach to estimate flood risk resulting from this singular event (termed an actualized risk index) when compared to a published empirical model of vulnerability. We show that 24.3% of detected floodwater was outside the 100-year floodplain, 43.4% of exposed residential structures are outside the 100-year floodplain, and communities of highest vulnerability are not only along the coast but also inland along the Cape Fear, Northeast Cape Fear, Trenton, and Neuse Rivers. This suggests that the SFHA may not adequately show the spatial distribution of pluvial flood risk in riverine areas, and that misunderstanding of this risk has led to a pattern of development in which houses have a higher than expected risk of flooding. Moreover, this additional flood risk may disproportionately affect lower-income residents of these largely rural areas. These results have important implications in light of recent policy guidance in southeastern USA states that mandate that predictive coastal vulnerability assessments to sea level rise be conducted relative to 100-year SFHA zones.

Pseudo-Five-Component Reaction for Diastereoselective Synthesis of Butterfly Shaped Bispiro[Oxindole-Pyrrolidine]s.

A pseudo-five-component reaction involving double decarboxylative 1,3-dipolar cycloaddition of azomethine ylides with olefinic oxindoles for the diastereoselective synthesis of bispiro[oxindole-pyrrolidine]s is developed. The major diastereomers are unique butterfly shaped compounds with a plane of symmetry. Recyclable zeolite HY acid catalyst is used to promote the reaction, and only CO2 and H2O are generated as byproducts.

Towards advancing scientific knowledge of climate change impacts on short-duration rainfall extremes.

A large number of recent studies have aimed at understanding short-duration rainfall extremes, due to their impacts on flash floods, landslides and debris flows and potential for these to worsen with global warming. This has been led in a concerted international effort by the INTENSE Crosscutting Project of the GEWEX (Global Energy and Water Exchanges) Hydroclimatology Panel. Here, we summarize the main findings so far and suggest future directions for research, including: the benefits of convection-permitting climate modelling; towards understanding mechanisms of change; the usefulness of temperature-scaling relations; towards detecting and attributing extreme rainfall change; and the need for international coordination and collaboration. Evidence suggests that the intensity of long-duration (1 day+) heavy precipitation increases with climate warming close to the Clausius-Clapeyron (CC) rate (6-7% K-1), although large-scale circulation changes affect this response regionally. However, rare events can scale at higher rates, and localized heavy short-duration (hourly and sub-hourly) intensities can respond more strongly (e.g. 2 × CC instead of CC). Day-to-day scaling of short-duration intensities supports a higher scaling, with mechanisms proposed for this related to local-scale dynamics of convective storms, but its relevance to climate change is not clear. Uncertainty in changes to precipitation extremes remains and is influenced by many factors, including large-scale circulation, convective storm dynamics andstratification. Despite this, recent research has increased confidence in both the detectability and understanding of changes in various aspects of intense short-duration rainfall. To make further progress, the international coordination of datasets, model experiments and evaluations will be required, with consistent and standardized comparison methods and metrics, and recommendations are made for these frameworks. This article is part of a discussion meeting issue 'Intensification of short-duration rainfall extremes and implications for flash flood risks'.

Nonenzymatic Spontaneous Oxidative Transformation of 5,6-Dihydroxyindole.

Melanin is an important phenolic skin pigment found throughout the animal kingdom. Tyrosine and its hydroxylated product dopa provide the starting material for melanin biosynthesis in all animals. Through a set of well-established reactions, they are converted to 5,6-dihydroxyindole (DHI) and DHI-2-carboxylic acid (DHICA). Oxidative polymerization of these two indoles produces the brown to black eumelanin pigment. The steps associated with these transformations are complicated by the extreme instability of the starting materials and the transient and highly reactive nature of the intermediates. We have used mass spectral studies to explore the nonenzymatic mechanism of oxidative transformation of DHI in water. Our results indicate the facile production of not only dimeric and trimeric products but also higher oligomeric forms of DHI upon exposure to air in solution, even under nonenzymatic conditions. Such instantaneous polymerization of DHI avoids toxicity to self-matter and ensures the much-needed deposition of melanin at (a) the wound site and (b) the infection site in arthropods. The rapid deposition of DHI melanin is advantageous for arthropods given their open circulatory system; the process limits blood loss during wounding and prevents the spread of parasites by encapsulating them in melanin, limiting the damage.

Oxidative Oligomerization of DBL Catechol, a potential Cytotoxic Compound for Melanocytes, Reveals the Occurrence of Novel Ionic Diels-Alder Type Additions.

The exposure of human skin to 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone, RK) is known to cause chemical/occupational leukoderma. RK is a carbonyl derivative of 4-(4-hydroxyphenyl)-2-butanol (rhododendrol), a skin whitening agent that was found to cause leukoderma in skin of many consumers. These two phenolic compounds are oxidized by tyrosinase and the resultant products seem to cause cytotoxicity to melanocytes by producing reactive oxygen species and depleting cellular thiols through o-quinone oxidation products. Therefore, it is important to understand the biochemical mechanism of the oxidative transformation of these compounds. Earlier studies indicate that RK is initially oxidized to RK quinone by tyrosinase and subsequently converted to a side chain desaturated catechol called 3,4-dihydroxybenzalacetone (DBL catechol). In the present study, we report the oxidation chemistry of DBL catechol. Using UV-visible spectroscopic studies and liquid chromatography mass spectrometry, we have examined the reaction of DBL catechol with tyrosinase and sodium periodate. Our results indicate that DBL quinone formed in the reaction is extremely reactive and undergoes facile dimerization and trimerization reactions to produce multiple isomeric products by novel ionic Diels-Alder type condensation reactions. The production of a wide variety of complex quinonoid products from such reactions would be potentially more toxic to cells by causing not only oxidative stress, but also melanotoxicity through exhibiting reactions with cellular macromolecules and thiols.

Long-lived particles at the energy frontier: the MATHUSLA physics case.

We examine the theoretical motivations for long-lived particle (LLP) signals at the LHC in a comprehensive survey of standard model (SM) extensions. LLPs are a common prediction of a wide range of theories that address unsolved fundamental mysteries such as naturalness, dark matter, baryogenesis and neutrino masses, and represent a natural and generic possibility for physics beyond the SM (BSM). In most cases the LLP lifetime can be treated as a free parameter from the [Formula: see text]m scale up to the Big Bang Nucleosynthesis limit of [Formula: see text] m. Neutral LLPs with lifetimes above [Formula: see text]100 m are particularly difficult to probe, as the sensitivity of the LHC main detectors is limited by challenging backgrounds, triggers, and small acceptances. MATHUSLA is a proposal for a minimally instrumented, large-volume surface detector near ATLAS or CMS. It would search for neutral LLPs produced in HL-LHC collisions by reconstructing displaced vertices (DVs) in a low-background environment, extending the sensitivity of the main detectors by orders of magnitude in the long-lifetime regime. We study the LLP physics opportunities afforded by a MATHUSLA-like detector at the HL-LHC, assuming backgrounds can be rejected as expected. We develop a model-independent approach to describe the sensitivity of MATHUSLA to BSM LLP signals, and compare it to DV and missing energy searches at ATLAS or CMS. We then explore the BSM motivations for LLPs in considerable detail, presenting a large number of new sensitivity studies. While our discussion is especially oriented towards the long-lifetime regime at MATHUSLA, this survey underlines the importance of a varied LLP search program at the LHC in general. By synthesizing these results into a general discussion of the top-down and bottom-up motivations for LLP searches, it is our aim to demonstrate the exceptional strength and breadth of the physics case for the construction of the MATHUSLA detector.

Erythrofordins D and E, two new cassaine-type diterpenes from Erythrophleum suaveolens.

Two new cassaine-type diterpenoids, namely erythrofordins D (1) and E (2), sourced from a Cameroon collection of Erythrophleum suaveolens were isolated and assessed for anti-tumor activity. In the NCI-60 cancer cell assay, erythrofordins D (1) and E (2) were found to be cytotoxic in the low micro molar ranges with a mean GI50 value of 2.45 and 0.71 µM, mean TGI value of 9.77 and 2.29 µM, and a mean LC50 of 26.92 and 11.48 µM for 1 and 2 respectively. Using the COMPARE algorithm, the new compounds were found to have similar NCI-60 response profiles to the known cardiac glycosides hyrcanoside and strophanthin. In addition, in an assay examining the viability and contractile function in human cardiomyocytes derived from induced pluripotent stem-cells, erythrofordins showed cardiotoxicity effects at concentrations as low as 0.03 µg/mL.

Mycobacterium tuberculosis transmission in an ethnically-diverse high incidence region in England, 2007-11.

BACKGROUND: Transmission patterns in high tuberculosis incidence areas in England are poorly understood but need elucidating to focus contact tracing. We study transmission within and between age, ethnic and immigrant groups using molecular data from the high incidence West Midlands region. METHODS: Isolates from culture-confirmed tuberculosis cases during 2007-2011 were typed using 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTR). We estimated the proportion of disease attributable to recent transmission, calculated the proportion of isolates matching those from the two preceding years ("retrospectively clustered"), and identified risk factors for retrospective clustering using multivariate analyses. We calculated the ratio (RCR) between the observed and expected proportion clustered retrospectively within or between age, ethnic and immigrant groups. RESULTS: Of the 2159 available genotypes (79% of culture-confirmed cases), 34% were attributed to recent transmission. The percentage retrospectively clustered decreased from 50 to 24% for 0-14 and ≥ 65 year olds respectively (p = 0.01) and was significantly lower for immigrants than the UK-born. Higher than expected clustering occurred within 15-24 year olds (RCR: 1.4 (95% CI: 1.1-1.8)), several ethnic groups, and between UK-born or long-term immigrants with the UK-born (RCR: 1.8 (95% CI: 1.1-2.4) and 1.6 (95% CI: 1.2-1.9) respectively). CONCLUSIONS: This study is the first to consider "who clusters with whom" in a high incidence area in England, laying the foundation for future whole-genome sequencing work. The higher than expected clustering seen here suggests that preferential mixing between some age, ethnic and immigrant groups occurs; prioritising contact tracing to groups with which cases are most likely to cluster retrospectively could improve TB control.

One-Pot Synthesis of Triazolobenzodiazepines Through Decarboxylative [3 + 2] Cycloaddition of Nonstabilized Azomethine Ylides and Cu-Free Click Reactions.

A one-pot synthesis of triazolobenzodiazepine-containing polycyclic compounds is introduced. The reaction process involves a decarboxylative three-component [3 + 2] cycloaddition of nonstabilized azomethine ylides, N-propargylation, and intramolecular click reactions.

One-Pot Double [3 + 2] Cycloadditions for Diastereoselective Synthesis of Pyrrolidine-Based Polycyclic Systems.

Sequential inter- and intramolecular [3 + 2] cycloadditions of azomethine ylides are developed for the one-pot and diastereoselective synthesis of highly condensed heterocyclic systems containing pyrrolidine and up to seven stereocenters. It has high synthetic efficiency and operational simplicity, and only water was generated as a byproduct.

The Impact of the Complexing Cation on the Sensitivity of the Collisional-Induced Dissociation Spectra to Fatty Acid Position for a Set of YXY/YYX-type Triglycerides.

RATIONAL: The development of an automated platform for the positional analysis of triglycerides based on electrospray tandem mass spectrometry continues to be pursued. This work compares the positional sensitivities of the collisional-induced dissociation spectra for a representative set of YXY/YYX triglycerides using ammonium, silver, sodium and lithium as complexing agents. METHODS: A set of triglycerides were synthesized and analyzed by electrospray tandem mass spectrometry using an ion trap mass spectrometer. Using different salt additives, the product ion spectra of the corresponding parent ions for twelve systems of the form YXY/YYX, where Y and X represent C16:0 , C18:1(c-9), C18:2(cc-9,12) and C20:4(cccc-5,8,11,14) , were collected. The data was used to prepare two-point calibration plots for each of the twelve positional isomer systems using each of the four complexing agents. RESULTS: The positional sensitivities for all twelve positional isomer systems were robust for both the sodium and lithium TAG adducts. The CID data for both the sodium and lithium TAG adducts are much less sensitive to the degree of unsaturation and double bond position of the fatty acids constituents than the CID data for the ammonium adducts. CONCLUSION: Using sodium or lithium TAG adducts may be advantageous for the development of an accurate predictive model for performing positional analysis of complex TAG mixtures based on electrospray tandem mass spectrometry. Ammonium adducts are likely complicated by the ability of the ammonium ion to provide extra stability to some parent ions through hydrogen bond-like interactions.

Unraveling complex molecular transformations of N-ß-alanyldopamine that account for brown coloration of insect cuticle.

RATIONALE: N-ß-Alanyldopamine (NBAD) and N-acetyldopamine (NADA) are catecholamines that are used by insects as sclerotizing precursors to harden their cuticle. They share a common pathway utilizing the same set of sclerotizing enzymes. Yet, cuticles using NBAD are brown, while cuticles using NADA are colorless. To identify the cause of this major unresolved color difference, molecular transformations of NBAD with cuticular enzymes were investigated. METHODS: Reactions of NBAD and NADA with native cuticle isolated from the wandering stages of Sarcophaga bullata larvae as well as the reactions of NBAD with cuticular sclerotization enzymes - phenoloxidase, quinone isomerase and quinone methide isomerase - were investigated using UV-Vis spectroscopy, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). In addition, the reactivity of enzymatically generated NBAD quinone was investigated by MS. RESULTS: Reactions of NBAD with sclerotizing enzymes isolated from Sarcophaga bullata larvae generate colorless products such as N-ß-alanylnorepinephrine, N-ß-alanylarterenone, dehydro NBAD, the benzodioxan dimers of dehydro NBAD and other minor adducts, the same kind of compounds generated by NADA reaction with cuticular enzymes. However, oxidation of NBAD produces colored quinone adducts, in addition. NADA, which lacks the amino group, did not produce these quinone adducts. CONCLUSIONS: LC/MS analysis of the reaction mixture of NBAD-cuticular enzyme reactions reveals the novel production of colored quinone adducts that are not possible for NADA. Therefore, our results suggest that the brown coloration of cuticle formed through NBAD crosslinking is likely due to the formation and accumulation of NBAD quinone and its adducts, while NADA quinone adducts tend not to form during NADA crosslinking, producing a nearly colorless cuticle.

A simple and economical strategy for obtaining calibration plots for relative quantification of positional isomers of YYX/YXY triglycerides using high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Positional analysis of intact triglycerides could provide greater insights into the link between fatty acid position and lipotoxic diseases. However, this methodology has been impeded by lack of commercial availability of positionally pure triglycerides. This work reports on a strategy for defining calibration plots for YXY/YYX triglyceride systems based on the product ion intensities in the collision-induced dissociation spectra of ammoniated precursor ions. METHODS: A set of triglycerides were synthesized and analyzed by electrospray ionization tandem mass spectrometry using an ion trap mass spectrometer. The product ion spectra of the ammoniated precursor ions were collected for 42 triglyceride systems of the form YXY/YYX, where Y represents C16:0 , C18:1(c-9) and C20:4(cccc-5,8,11,14) . Three-point calibration plots were prepared by plotting the relative abundance of the YY+ product ion vs. the relative abundance of the YYX positional isomer. RESULTS: The calibration plots were shown to give relative abundances of positional isomers accurate to within ±0.02 for most systems. Using an ion trap, under a controlled set of collision parameters, the slopes of the calibration plots can be used to compare the sensitivities of the product ion intensities to fatty acid position for various triglyceride systems. The average slopes of the calibration plots for the C16:0 , C18:1(c-9) and C20:4(cccc-5,8,11,14) systems were 0.29 ± 0.05, 0.21 ± 0.05 and 0.045 ± 0.005, respectively. CONCLUSIONS: While the presence of multiple unsaturated fatty acids tends to slightly decrease the slopes of the calibration plots, the data suggest that the sensitivities are sufficient for performing positional analysis of most triglyceride systems. However, the presence of unsaturated fatty acids that contain double bonds close to the carbonyl group, such as arachidonic acid, tends to dramatically decrease positional sensitivity.

Oxidative transformation of tunichromes - Model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine.

Tunichromes are 1,2-dehydrodopa containing bioactive peptidyl derivatives found in blood cells of several tunicates. They have been implicated in metal sequestering, tunic formation, wound healing and defense reaction. Earlier studies conducted on these compounds indicate their extreme liability, high reactivity and easy oxidative polymerization. Their reactions are also complicated by the presence of multiple dehydrodopyl units. Since they have been invoked in crosslinking and covalent binding, to understand the reactivities of these novel compounds, we have taken a simple model compound that possess the tunichrome reactive group viz., 1,2-dehydro-N-acetyldopamine (Dehydro NADA) and examined its reaction with N-acetylcysteine in presence of oxygen under both enzymatic and nonenzymatic conditions. Ultraviolet and visible spectral studies of reaction mixtures containing dehydro NADA and N-acetylcysteine in different molar ratios indicated the production of side chain and ring adducts of N-acetylcysteine to dehydro NADA. Liquid chromatography and mass spectral studies supported this contention and confirmed the production of several different products. Mass spectral analysis of these products show the potentials of dehydro NADA to form side chain adducts that can lead to polymeric products. This is the first report demonstrating the ability of dehydro dopyl units to form adducts and crosslinks with amino acid side chains.

Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes.

Tunichromes, small oligopeptides with dehydrodopa units isolated from the blood cells of ascidians, have been implicated in the defense reactions, metal binding, wound repair, or tunic formation. Their instability and high reactivity has severely hampered the assessment of their biological role. Experiments conducted with the model compound, 1,2-dehydro-N-acetyldopamine, indicated that the instability of tunichromes is due to this basic structure. Exposure of this catecholamine derivative to even mild alkaline condition such as pH 7.5 causes rapid nonenzymatic oxidation. High performance liquid chromatography and mass spectrometry studies confirmed the production of dimeric and other oligomeric products in the reaction mixture. The nonenzymatic reaction seemed to proceed through the intermediary formation of semiquinone free radical and superoxide anion. Ultraviolet and visible spectral studies associated with the oxidation of tunichromes isolated from Ascidia nigra by tyrosinase indicated the probable formation of oligomeric tunichrome products. Attempts to monitor the polymerization reaction by mass spectrometry ended in vain. Tunichrome also exhibited instability in mild alkaline conditions generating superoxide anions. Based on these studies, a possible role for oxidative transformation of tunichrome in defense reaction, tunic formation and wound healing is proposed.

Novel post-translational oligomerization of peptidyl dehydrodopa model compound, 1,2-dehydro-N-acetyldopa methyl ester.

Post-translational modification of peptidyl tyrosine to peptidyl dopa is widely observed in different marine organisms. While peptidyl dopas are oxidatively converted to dehydrodopa derivatives, nothing is known about the further fate of dehydrodopyl compounds. To fill this void, we studied the oxidation chemistry of a peptidyl dehydrodopa mimic, 1,2-dehydro-N-acetyldopa methyl ester with mushroom tyrosinase. We employed both routine biochemical studies and reversed phase liquid chromatography mass spectrometry to investigate the course of the reaction. Tyrosinase catalyzed the oxidation of 1,2-dehydro-N-acetyldopa methyl ester readily generating its typical o-quinone as the transient two-electron oxidation product. This quinone was extremely unstable and rapidly reacted with the parent compound forming benzodioxan type oligomeric products. Reaction mixture containing chemically made o-benzoquinone and 1,2-dehydro-N-acetyldopa methyl ester generated a mixed adduct of benzoquinone and 1,2-dehydro-N-acetyldopa methyl ester. Based on this finding, we propose that peptidyl dehydrodopa also exhibits a similar transformation accounting partially for the adhesive and cementing properties of dopyl proteins in nature.