Aligned Fingolimod-Releasing Electrospun Fibers Increase Dorsal Root Ganglia Neurite Extension and Decrease Schwann Cell Expression of Promyelinating Factors.

Researchers are investigating the use of biomaterials with aligned guidance cues, like those provided by aligned electrospun fibers, to facilitate axonal growth across critical-length peripheral nerve defects. To enhance the regenerative outcomes further, these aligned fibers can be designed to provide local, sustained release of therapeutics. The drug fingolimod improved peripheral nerve regeneration in preclinical rodent models by stimulating a pro-regenerative Schwann cell phenotype and axonal growth. However, the systemic delivery of fingolimod for nerve repair can lead to adverse effects, so it is necessary to develop a means of providing sustained delivery of fingolimod local to the injury. Here we created aligned fingolimod-releasing electrospun fibers that provide directional guidance cues in combination with the local, sustained release of fingolimod to enhance neurite outgrowth and stimulate a pro-regenerative Schwann cell phenotype. Electrospun fiber scaffolds were created by blending fingolimod into poly(lactic-co-glycolic acid) (PLGA) at a w/w% (drug/polymer) of 0.0004, 0.02, or 0.04%. We examined the effectiveness of these scaffolds to stimulate neurite extension in vitro by measuring neurite outgrowth from whole and dissociated dorsal root ganglia (DRG). Subsequently, we characterized Schwann cell migration and gene expression in vitro. The results show that drug-loaded PLGA fibers released fingolimod for 28 days, which is the longest reported release of fingolimod from electrospun fibers. Furthermore, the 0.02% fingolimod-loaded fibers enhanced neurite outgrowth from whole and dissociated DRG neurons, increased Schwann cell migration, and reduced the Schwann cell expression of promyelinating factors. The in vitro findings show the potential of the aligned fingolimod-releasing electrospun fibers to enhance peripheral nerve regeneration and serve as a basis for future in vivo studies.

Exposure of Juncus effusus to seven perfluoroalkyl acids: Uptake, accumulation and phytotoxicity.

The extensive use of poly- and perfluoroalkyl substances (PFAS) has led to perfluoroalkyl acids (PFAAs) contamination in various environmental matrices. To remove PFAAs from contaminated water, this study investigated plant uptake of PFAAs by a native wetland plant species in the US, Juncus effusus. The results showed that J. effusus translocated selected PFAAs, including perfluoropentanoic acid (PFPA), perfluorobutanesulfonic acid (PFBS), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and perfluorooctanesulfonic acid (PFOS). During the 21-day experimental period, the uptake of PFAAs increased with increasing PFAAs exposure concentration and time. PFOS was largely accumulated in the roots with limited upward translocation. PFAAs with shorter carbon chain length were taken up by J. effusus roots and tended to accumulate in plant shoots. The highest removal efficiency (11.4%) of spiked PFAAs by J. effusus was achieved when it was exposed to PFAAs at around 4.6 mg/L for 21 days. The exposure to PFAAs stimulated the antioxidative defense system in J. effusus shoots but inhibited the superoxide dismutase (SOD) and catalase (CAT) activities and damaged the antioxidative defense system in J. effusus roots. These results warrant further studies to evaluate J. effusus's long-term performance in a PFAAs contaminated environment.

Production of pyranoanthocyanins using Escherichia coli co-cultures.

Hydroxyphenyl-pyranoanthocyanins are one of the pyranoanthocyanins found in red wines and some fruit juices. Since they have a fourth ring (pyran or ring D) which provides higher color intensity and exceptional stability toward pH variations in comparison to their anthocyanin precursors, these molecules are one of the most important candidates as natural colorants especially for low- and medium-acidic food and beverages. However, their isolation and characterization are difficult due to their very low concentration. In this study, we co-cultured recombinant E. coli strains to synthesize pyranoanthocyanins with improved titers and yields. To accomplish this task, firstly we engineered 4-vinylphenol and 4-vinylcatechol producer modules then we co-cultured each one of these strains with cyanidin-3-O-glucoside producer recombinant cells to obtain pyranocyanidin-3-O-glucoside-phenol (cyanidin-3-O-glucoside with vinylphenol adduct) and pyranocyanidin-3-O-glucoside-catechol (cyanidin-3-O-glucoside with vinylcatechol adduct). By optimizing the co-culture conditions, we were able to significantly increase final titers and yields, allowing our co-culture approach to easily outperform production of pyranoanthocyanins from red wine. Finally, we demonstrate that the produced pyranoanthocyanins are far more stable than the starting plant-produced cyanidin 3-O-glucoside.

Hydrogen sulfide inhibits amyloid formation.

Amyloid fibrils are large aggregates of misfolded proteins, which are often associated with various neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and vascular dementia. The amount of hydrogen sulfide (H2S) is known to be significantly reduced in the brain tissue of people diagnosed with Alzheimer's disease relative to that of healthy individuals. These findings prompted us to investigate the effects of H2S on the formation of amyloids in vitro using a model fibrillogenic protein hen egg white lysozyme (HEWL). HEWL forms typical ß-sheet rich fibrils during the course of 70 min at low pH and high temperatures. The addition of H2S completely inhibits the formation of ß-sheet and amyloid fibrils, as revealed by deep UV resonance Raman (DUVRR) spectroscopy and ThT fluorescence. Nonresonance Raman spectroscopy shows that disulfide bonds undergo significant rearrangements in the presence of H2S. Raman bands corresponding to disulfide (RSSR) vibrational modes in the 550-500 cm(-1) spectral range decrease in intensity and are accompanied by the appearance of a new 490 cm(-1) band assigned to the trisulfide group (RSSSR) based on the comparison with model compounds. The formation of RSSSR was proven further using a reaction with TCEP reduction agent and LC-MS analysis of the products. Intrinsic tryptophan fluorescence study shows a strong denaturation of HEWL containing trisulfide bonds. The presented evidence indicates that H2S causes the formation of trisulfide bridges, which destabilizes HEWL structure, preventing protein fibrillation. As a result, small spherical aggregates of unordered protein form, which exhibit no cytotoxicity by contrast with HEWL fibrils.

The detection of nicotine in a Late Mayan period flask by gas chromatography and liquid chromatography mass spectrometry methods.

Several ancient Mayan vessels from the Kislak Collection of the US Library of Congress were examined for the presence of alkaloids. One of them, a codex-style flask, bears a text that appears to read yo-'OTOT-ti 'u-MAY, spelling y-otoot 'u-may 'the home of its/his/her tobacco'. Samples extracted from this Late Classic period (600 to 900 AD) container were analyzed by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) methods. Nicotine was identified as the major component of the extracts. LC/MS analyses also yielded signals due to nicotine mono-oxides. The identities of the compounds were determined by comparison of the chromatographic and/or mass spectral characteristics with those from standards and literature data. High-resolution high mass accuracy tandem mass spectrometry (MS/MS) spectra of protonated nicotine and nicotine mono-oxides were measured to verify and to correct previous product ion assignments. These analyses provided positive evidence for nicotine from a Mayan vessel, indicating it as a likely holder of tobacco leafs. The result of this investigation is the first physical evidence of tobacco from a Mayan container, and only the second example where the vessel content recorded in a Mayan hieroglyphic text has been confirmed directly by chromatography/mass spectrometry trace analysis.

Progress in studies on the RNA world.

The montmorillonite-catalyzed reactions of D, L-ImpA with D, L-ImpU generates RNA-like oligomers. The structures of the dimers to pentamers were investigated and homochiral products were identified in greater amounts than would be expected if theoretical amounts of each were formed. The homochirality increased from 64% to 97% as the chain length increased from dimers to pentamers. Investigation of the effect of pH, occupancy of the interlayer space and the influence of various cations in the reaction provided further insight into physical process in the mechanism of the catalysis. A detailed analysis of dimers was carried out in view of there being key intermediates towards formation of higher oligomers. The study was extended to the synthesis of non-standard dimers including those formed with deoxy-ribonucleotides.

Analysis of E. coli K5 capsular polysaccharide heparosan.

Heparosan is the key precursor for the preparation of bioengineered heparin, a potential replacement for porcine intestinal heparin, an important anticoagulant drug. The molecular weight (MW) distribution of heparosan produced by the fermentation of E. coli K5 was investigated. Large-slab isocratic and mini-slab gradient polyacrylamide gel electrophoresis (PAGE) were used to analyze the MW and polydispersity of heparosan. A preparative method that allowed fractionation by continuous-elution PAGE was used to obtain heparosan MW standards. The MWs of the heparosan standards were determined by electrospray ionization Fourier-transform mass spectrometry (ESI-FT-MS). A ladder of the standards was then used to determine the MW properties of polydisperse heparosan samples. Unbleached and bleached heparosan produced by fermentation of E. coli K5 had similar number-averaged MWs (M(N)), weight-averaged MWs (M(W)), and MW ranges of 3,000 to 150,000 Da.

Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.

A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards 17alpha-methyl-T and 17alpha-methyl-DHT, were extracted from tissues and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in product ion spectra of HTP derivatives of both T and DHT that showed analyte-specific fragmentations; the latter fragmentations were characterized by the use of high-resolution Orbitrap MS/MS. These specific fragmentations enabled quantitation of T and DHT in the multiple-reaction monitoring (MRM) mode. The method was validated with charcoal-stripped serum as the matrix. The lower limit of quantitation (LLOQ) was 0.10ng/ml for T and 0.50ng/ml for DHT. The method was then used for determination of serum and tissue levels of T and DHT in transgenic mice carrying a hypomorphic NADPH-cytochrome P450 reductase gene (Cpr-low mice). Remarkably, ovarian T levels in Cpr-low mice were found to be 25-fold higher than those in wild-type mice, a finding that at least partly explains the female infertility seen in the Cpr-low mice. In conclusion, our method provides excellent sensitivity and selectivity for determination of endogenous levels of T and DHT in mouse tissues.

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis.

Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6h using continuous elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies.

Detection of in vivo matrix metalloproteinase activity using microdialysis sampling and liquid chromatography/mass spectrometry.

Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broad-spectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 microM was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 microM each) resulted in recovered product concentrations of approximately 2 microM. During a 50 microM GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling.

ENZYMATIC POLYMERIZATION OF PHENOLS IN ROOM TEMPERATURE IONIC LIQUIDS.

Soybean peroxidase (SBP) was used to catalyze the polymerization of phenols in room-temperature ionic liquids (RTILs). Phenolic polymers with number average molecular weights ranging from 1200 to 4100 D were obtained depending on the composition of the reaction medium and the nature of the phenol. Specifically, SBP was highly active in methylimidazolium-containing RTILs, including 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM(BF(4))), and 1-butyl-3-methylpyridinium tetrafluoroborate (BMPy(BF(4))) with the ionic liquid content as high as 90% (v/v); the balance being aqueous buffer. Gel permeation chromatography and MALDI-TOF analysis indicated that higher molecular weight polymers can be synthesized in the presence of higher RTIL concentrations, with selective control over polymer size achieved by varying the RTIL concentration. The resulting polyphenols exhibited high thermostability and possessed thermosetting properties.

Covalent binding of flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae.

Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na+-NQR complexes.

In situ and multisubstrate detection of elastase enzymatic activity external to microdialysis sampling probes using LC-ESI-MS.

Extracellular proteases play significant roles in mammalian development and disease. Enzymatic activity external to a microdialysis sampling probe can be determined by infusing judicious choices of substrates followed by collecting and measuring the products. Porcine pancreatic elastase was used as a model enzyme with two substrates possessing different cleavage sites, N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (FL-substrate) and N-succinyl-Ala-Ala-Ala-p-nitroanilide (UV-substrate). These substrates were infused through the microdialysis sampling probe to a solution containing elastase. The resulting four products and the remaining two substrates were collected into the dialysate and were subsequently analyzed off-line using liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). All analytes were identified using extracted ion chromatograms of m/z 628 (FL-substrate), m/z 452 (UV-substrate), m/z 471 (N-methoxysuccinyl-Ala-Ala-Pro-Val, FL-NTP), m/z 332 (N-succinyl-Ala-Ala-Ala, UV-NTP), m/z 176 (7-amino-4-methylcoumarin, AMC), and m/z 139 (p-nitroaniline, pNA). FL-NTP and FL-substrate exhibited 10-fold higher ion production as compared to AMC with equimolar standards. Microdialysis sampling combined with LC-ESI-MS detection allowed for in situ determination of the enzymatic activity of a protease external to the microdialysis probe when using different peptide-based substrates.

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition.

Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolution-alternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations.

Matrix-assisted laser desorption/ionization mass spectrometric analysis of uncomplexed highly sulfated oligosaccharides using ionic liquid matrices.

Direct UV matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis of uncomplexed, underivatized, highly sulfated oligosaccharides has been carried out using ionic liquids as matrices. Under conventionally used MALDI time-of-flight experimental conditions, uncomplexed polysulfated oligosaccharides do not produce any signal. We report that 1-methylimidazolium alpha-cyano-4-hydroxycinnamate and butylammonium 2,5-dihydroxybenzoate ionic liquid matrices allow the detection of picomole amounts of the sodium salts of a disaccharide, sucrose octasulfate, and an octasulfated pentasaccharide, Arixtra. The experimental results indicate that both analytes undergo some degree of thermal fragmentation with a mass loss corresponding to cleavage of O-SO3Na bonds in the matrix upon laser irradiation, reflecting lability of sulfo groups.

The generation of neutral derivatives of low-valence iron, RFe(I), by netralization-reionization mass spectrometry.

Neutralization-reionization mass spectrometry is applied for the generation of low-valence iron derivatives. Neutralization of Rfe(+) (R=H, F, Cl, Br, I, CN, OH, NH(2), acac, C(6)H(5)) ions resulted in the corresponding neutrals having lifetimes of at least 5 micros. Atom connectivities in RFe ions and neutrals were elucidated by a variety of tandem mass spectrometry techniques. The present study provides the first experimental evidence of the intrinsic stability of neutral IFe, NCFe, acacFe and C(6)H(5)Fe.