Large-scale asynchronous and distributed multidimensional replica exchange molecular simulations and efficiency analysis.

We describe methods to perform replica exchange molecular dynamics (REMD) simulations asynchronously (ASyncRE). The methods are designed to facilitate large scale REMD simulations on grid computing networks consisting of heterogeneous and distributed computing environments as well as on homogeneous high-performance clusters. We have implemented these methods on NSF (National Science Foundation) XSEDE (Extreme Science and Engineering Discovery Environment) clusters and BOINC (Berkeley Open Infrastructure for Network Computing) distributed computing networks at Temple University and Brooklyn College at CUNY (the City University of New York). They are also being implemented on the IBM World Community Grid. To illustrate the methods, we have performed extensive (more than 60 ms in aggregate) simulations for the beta-cyclodextrin-heptanoate host-guest system in the context of one- and two-dimensional ASyncRE, and we used the results to estimate absolute binding free energies using the binding energy distribution analysis method. We propose ways to improve the efficiency of REMD simulations: these include increasing the number of exchanges attempted after a specified molecular dynamics (MD) period up to the fast exchange limit and/or adjusting the MD period to allow sufficient internal relaxation within each thermodynamic state. Although ASyncRE simulations generally require long MD periods (>picoseconds) per replica exchange cycle to minimize the overhead imposed by heterogeneous computing networks, we found that it is possible to reach an efficiency similar to conventional synchronous REMD, by optimizing the combination of the MD period and the number of exchanges attempted per cycle.

An explanation for the difference in the percutaneous penetration behavior of tamsulosin induced by two different O-acylmenthol derivatives.

Using tamsulosin (TAL) as a model drug, the aim of this study was to investigate and compare the percutaneous permeation behavior of two menthol derivatives, 2-isopropyl-5-methylcyclohexyl heptanoate (M-HEP) and 2-isopropyl-5-methylcyclohexyl decanoate (M-DEC). In vitro transdermal permeation study was carried out using porcine skin. The residual amount of enhancers in the skin after permeation experiment was determined by gas chromatographic (GC) method. The penetration depths of fluorescein were visualized by two-photon confocal laser scanning microscopy (2P-LSM) after the skin being treated with different enhancers. Furthermore, changes in the stretching frequency of functional group of ceramide were investigated by using attenuated total reflectance Fourier transform infrared (ATR-FTIR) technique. After M-HEP addition, the cumulative amount of TAL permeated in 8 h (Q8) reached 20.57±0.54 µg/cm2 and the depth of fluorescein was 40 µm; the CH2 of ceramide symmetric stretching frequency was 4 cm−1 blue shifted. However, M-DEC has an opposite effect on TAL permeation compared with that of M-HEP. TAL is a crucial factor affecting permeation procedure, and microenvironment of lipid region determines promotion capability of the enhancers.

Structure elucidation and NMR assignments of an unusual aromatic monacolin analog from Monascus purpureus-fermented rice.

One unusual aromatic monacolin analog, aromonacolin A (1), was isolated from the ethanolic extract of Monascus purpureus-fermented rice. Its structure was elucidated by extensive spectroscopic (HRESIMS, (1)H NMR, (13)C NMR, HSQC, HMBC, and NOESY) and chemical methods. The absolute configuration of the C-6 secondary alcohol was deduced via the circular dichroism data of the in situ formed [Rh(2)(OCOCF(3))(4)] complex.

Safety assessment of stearyl heptanoate and related stearyl alkanoates as used in cosmetics.

Stearyl heptanoate is an ester of stearyl alcohol and heptanoic acid that functions in cosmetics as a skin conditioning agent and is in the general class of chemicals called stearyl alkanoates. Stearyl caprylate, stearyl palmitate, stearyl stearate, stearyl behenate, and stearyl olivate are stearyl alkanoates with similar chemical structures, toxicokinetics, and functions in cosmetics. These water-insoluble stearyl alkanoates, when metabolized, yield stearyl alcohol and a corresponding fatty acid. The available information supports the safety of all of the related stearyl alkanoates. The Expert Panel concluded that stearyl heptanoate, stearyl caprylate, stearyl palmitate, stearyl stearate, stearyl behenate, and stearyl olivate are safe in the present practices of use and concentration.

Interrelations between C4 ketogenesis, C5 ketogenesis, and anaplerosis in the perfused rat liver.

We investigated the interrelations between C(4) ketogenesis (production of beta-hydroxybutyrate + acetoacetate), C(5) ketogenesis (production of beta-hydroxypentanoate + beta-ketopentanoate), and anaplerosis in isolated rat livers perfused with (13)C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C(4) and C(5) ketone bodies and of related acyl-CoA esters reveal that C(4) and C(5) ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C(5) ketogenesis from heptanoate is much lower than the rate of C(4) ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C(5) ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of beta-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.

Assessing the DNA methylation status of single cells with the comet assay.

The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.

Structural determination of aspericins A-C, new furan and pyran derivates from the marine-derived fungus Rhizopus sp. 2-PDA-61, by 1D and 2D NMR spectroscopy.

Three new furan and pyran derivatives named aspericins A-C (1-3), as well as a known asperic acid (4), have been isolated from the marine-derived fungus Rhizopus sp. 2-PDA-61. The complete (1)H and (13)C NMR assignments for the new compounds were carried out using (1)H, (13)C, DEPT, COSY, HMQC, HMBC, and NOESY NMR experiments. Compounds 1-3 were evaluated for their cytotoxic activities on P388, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods.

Reaction strategies for the enzymatic synthesis of neopentyl glycol diheptanoate.

This work describes for the first time the green synthesis of neopentyl glycol diheptanoate in a solvent-free medium via an enzymatic pathway. The process has been carried out in an open-air reactor in order to ease water removal through evaporation and shift the chemical equilibrium towards product formation. The inhibiting effect of high concentrations of heptanoic acid has been put into evidence by a reduction of initial reaction rate when esterification was performed with stoichiometric amounts of substrates. Therefore, in this work different strategies for the stepwise addition of heptanoic acid are proposed, and best results were obtained when stoichiometric quantities of acid were divided in four equal amounts and added when previous batch was consumed. Biocatalyst Novozym® 435 concentration and temperature were optimised, giving yields of 90% in neopentyl glycol diheptanoate when 7.5% (w/w) and 70 °C were used. With a remaining 7% of heptanoic acid (probably caused by the alcohol evaporation) the addition of neopentyl glycol led to a conversion of 95%. Thus, product can be used in cosmetics without further purification and can be labelled as environmentally-friendly synthesized because of its enzymatic origin.

Solution structure of succinylacetone, an unsymmetrical beta-diketone, as studied by 13C NMR and GIAO-DFT calculations.

The enolization degrees of succinylacetone, an important heme biosynthesis inhibitor, have been determined in CDCl(3) and water solutions using (1)H NMR. The solution structures of SA have been investigated using a combined NMR/theoretical [GIAO DFT PBE1PBE/6-311++G(2d, p) PCM] approach. The populations of both enolic forms undergoing enol-enol equilibriums for SA and a series of unsymmetrical beta-diketones have been established by a quantitative comparison of the experimental (13)C NMR chemical shifts and calculated shielding constants. Moreover, using the same method and considering various trial structures differing in conformation and/or hydration of neutral SA molecule as well as its monoanion and dianion the structures of the most abundant species being present in the investigated water solutions have been deduced.

Influence of poly (lactide-co-glycolide) type and gamma irradiation on the betamethasone acetate release from the in situ forming systems.

In situ forming biodegradable polymeric systems were prepared from Poly (DL-lactide-co-glycolide), RG504H (50:50, lactide:glycolide), RG756 (75:25) and mixture of them. They were dissolved in N-methyl-2-pyrrolidone (33% w/w) and mixed with betamethasone acetate (BTMA, 5 and 10% w/w) and ethyl heptanoate (5% w/w, as an additive). The effects of gamma irradiation, drug loading, type of polymers and solvent removal were evaluated on release profiles. Scanning electron microscopy (SEM) of RG756 samples loaded by BTMA did not show any degradation until two weeks. Differential scanning calorimeter (DSC) experiments confirmed insignificant decrease in T(g), and consequently release rate. Declining T(g) of RG504H and RG756 after gamma irradiation was about 0.4 and 1.46 degrees C, respectively. High performance liquid chromatography (HPLC) revealed that BTMA release is more rapid from the formulations prepared using the RG504H with lower molecular weight. The formulations prepared by RG756 had lower burst release (2.5-41%) than the samples based on RG504H (60-67%) and mixture of them (30-33%). Regarding this research three different kinds of steriled in situ forming systems were developed which can release BTMA for 24, 90 and 60 days.

Improved method to determine succinylacetone in dried blood spots for diagnosis of tyrosinemia type 1 using UPLC-MS/MS.

We describe an improved diagnostic method for tyrosinemia type 1 based on quantifying succinylacetone in dried blood spots by ultra-performance liquid chromatography tandem mass spectrometry. Succinylacetone extracted from a single 3/16 inch disk of specimen collection paper containing a dried blood spot was derivatized with dansylhydrazine, separated on an Acquity UPLC BEH C(18) column (2.1 x 50 mm, 1.7 microm) and detected by electrospray ionization tandem mass spectrometry. Succinylacetone derivative eluted at 0.6 min with a complete run time of 1 min. Using a 13C4 labeled succinylacetone as an internal standard, the calibration plot was linear up to 100 micromol/L with a detection limit (S/N = 3) of 0.2 micromol/L. Intra-day (n = 13) and inter-day (n = 10) variations were better than 10%. The cutoff level of succinylacetone in dried blood spots from healthy infants obtained by the current method was 0.63 micromol/L (n = 151). In dried blood spots from patients with established tyrosinemia type 1 (n = 11), concentration of succinylacetone was 6.4-30.8 micromol/L.

Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride.

The current dietary treatment of long-chain fatty acid oxidation defects (high carbohydrate with medium-even-chain triglycerides and reduced amounts of long-chain fats) fails, in many cases, to prevent cardiomyopathy, rhabdomyolysis, and muscle weakness. We hypothesized that the apparent defect in energy production results from a depletion of the catalytic intermediates of the citric acid cycle via leakage through cell membranes (cataplerosis). We further hypothesized that replacing dietary medium-even-chain fatty acids (precursors of acetyl-CoA) by medium-odd-chain fatty acids (precursors of acetyl-CoA and anaplerotic propionyl-CoA) would restore energy production and improve cardiac and skeletal muscle function. We fed subjects with long-chain defects a controlled diet in which the fat component was switched from medium-even-chain triglycerides to triheptanoin. In three patients with very-long-chain acyl-CoA dehydrogenase deficiency, this treatment led rapidly to clinical improvement that included the permanent disappearance of chronic cardiomyopathy, rhabdomyolysis, and muscle weakness (for more than 2 years in one child), and of rhabdomyolysis and weakness in the others. There was no evidence of propionyl overload in these patients. The treatment has been well tolerated for up to 26 months and opens new avenues for the management of patients with mitochondrial fat oxidation disorders.

Comparison of a full systematic review versus rapid review approaches to assess a newborn screening test for tyrosinemia type 1.

BACKGROUND: Rapid reviews are increasingly used to replace/complement systematic reviews to support evidence-based decision-making. Little is known about how this expedited process affects results. OBJECTIVES: To assess differences between rapid and systematic review approaches for a case study of test accuracy of succinylacetone for detecting tyrosinemia type 1. METHODS: Two reviewers conducted an "enhanced" rapid review then a systematic review. The enhanced rapid review involved narrower searches, a single reviewer checking 20% of titles/abstracts and data extraction, and quality assessment using an unadjusted QUADAS-2. Two reviewers performed the systematic review with a tailored QUADAS-2. Post hoc analysis examined rapid reviewing with a single reviewer (basic rapid review). RESULTS: Ten papers were included. Basic rapid reviews would have missed 1 or 4 of these (dependent on which reviewer). Enhanced rapid and systematic reviews identified all 10 papers; one paper was only identified in the rapid review through reference checking. Two thousand one hundred seventy-six fewer title/abstracts and 129 fewer full texts were screened during the enhanced rapid review than the systematic review. The unadjusted QUADAS-2 generated more "unclear" ratings than the adjusted QUADAS-2 [29/70 (41.4%) versus 16/70 (22.9%)], and fewer "high" ratings [22/70 (31.4%) versus 42/70 (60.0%)]. Basic rapid reviews contained important inaccuracies in data extraction, which were detected by a second reviewer in the enhanced rapid and systematic reviews. CONCLUSIONS: Enhanced rapid reviews with 20% checking by a second reviewer may be an appropriate tool for policymakers to expeditiously assess evidence. Basic rapid reviews (single reviewer) have higher risks of important inaccuracies and omissions.

Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry.

Succinylacetone (SA) is a specific marker for the inherited metabolic disease, hepatorenal tyrosinemia. We developed a stable-isotope dilution liquid chromatography tandem mass spectrometry for the determination of SA in dried blood spots (DBS) and liquid urine using a (13)C(4)-SA as internal standard. SA was extracted, converted to the butyl ester and derivatized with dansylhydrazine (Dns-H). Calibration curves in DBS and urine calibrators were linear up to 100 and 30 microM, respectively. At a signal-to-noise ratio of 3, the limits of detection in DBS and urine were 0.2 and 0.005 microM, respectively. Total run time was 5 min. Intra- and inter-assay precision expressed as coefficient of variation were better than 9.1% with more than 96% recovery. The method was applied retrospectively and prospectively for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment.

The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors.

The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution. The ligands all bind by forming a covalent link with Lys263 at the active site. The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis. All of the inhibitors induce a significant ordering of the flap covering the active site. Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes. Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease. The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme.

An assessment of the safety of copper heptonate for parenteral therapy in sheep.

OBJECTIVE: To investigate any adverse effects of an intramuscular injection (i.m.) of copper heptonate (CuHep) in sheep. PROCEDURE: Merino wethers about 9 months old were retained in pens and given 1 or 2 mg Cu/kg body weight as CuHep or no Cu treatment. Sheep were weighed and samples of blood for haematology, Cu and enzyme assay and tissues for Cu and Fe assay were collected before and at intervals over 21 days after treatment. RESULTS: CuHep was removed from the injection site within 7 days of treatment and most of it was retained in the liver. Wethers had adequate liver Cu reserves before treatment and the higher dosage of CuHep raised liver Cu to values associated with Cu toxicity. No clinical signs of Cu toxicity were evident. Transient increases in plasma activity of the liver enzyme glutamate dehydrogenase suggested mild liver necrosis due to CuHep, but there was no histopathological evidence of liver necrosis 7 days after treatment. CONCLUSIONS: I.m. injection of Cu as CuHep appears to be readily transferred to the liver. No significant necrosis is caused at the injection site.

Mechanistic investigation and reversible effect of 2-isopropyl-5-methylcyclohexyl heptanoate on the in vitro percutaneous absorption of indomethacin.

In the present study, the enhancing effect of 2-isopropyl-5-methylcyclohexyl heptanoate (M-HEP) on the percutaneous absorption of indomethacin (IM) was evaluated by the in vitro penetration experiments using the rat abdominal skin as a barrier. Partition experiment, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrum and transepidermal water loss (TEWL), was employed to investigate the possible mechanisms of the action of M-HEP. Furthermore, the reversible effect of M-HEP on excised rat skin was also evaluated through in vitro permeation as a preliminary indicator of safety. The result of in vitro permeation experiment indicated that, 10% (w/w) M-HEP in combination with isopropyl palmitate (IPP) significantly increased (p < 0.05), the cumulative amount of IM in comparison with the control group (IPP only). The partition coefficient of IM between the stratum corneum (SC) and enhancer solution was also greater than that between the SC and IPP. A blue shift in the ATR-FTIR spectra of SC after treatment with M-HEP solution was observed at the CH(2) band, which indicating that M-HEP disrupted the intercellular lipid structure of the SC. In addition, both M-HEP/IPP and L-menthol (MT)/IPP improved the TEWL value of rat abdominal skin. After removal of M-HEP, the skin barrier function would be restored in 8 h. In conclusion, M-HEP could reversibly enhance the percutaneous absorption of IM by increasing the partitioning of IM into the SC from enhancer solution and disturbing the organized structure of SC lipids and the reversibility of M-HEP was better than MT.

Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process.

BACKGROUND: Succinylacetone (SUAC), a specific marker for tyrosinemia type I (Tyr I) cannot be detected by the routine LC-MS/MS screening of amino acids (AA) and acylcarnitines (AC) in newborns. The current derivatized methods require double extraction of newborn dried blood spots (DBS); one for AA and AC and the second for SUAC from the blood spot left after the first extraction. We have developed a method in which AA, AC and SUAC are extracted in a single extraction resulting in significant reduction in labor and assay time. METHODS: The 3.2 mm DBS were extracted by incubating at 45 °C for 45 min with 100 µl of acetonitrile (ACN)-water-formic acid mixture containing hydrazine and stable-isotope labeled internal standards of AA, AC and SUAC. The extract was derivatized with n-butanolic-HCl and analyzed by LC-MS/MS. RESULTS: The average inter-assay CVs for, AA, AC and SUAC were 10.1, 10.8 and 7.1% respectively. The extraction of analytes with ACN-water mixture showed no significant difference in their recovery compared to commonly used solvent MeOH. The concentration of hydrazine had considerable impact on SUAC extraction. CONCLUSION: We developed a new MS/MS derivatized method to detect AA/AC/SUAC in a single extraction process for screening Tyr I along with disorders of AA and AC.

Improved MS/MS analysis of succinylacetone extracted from dried blood spots when combined with amino acids and acylcarnitine butyl esters.

BACKGROUND: The utilization of succinylacetone (SUAC) as the primary metabolic marker for tyrosinemia Type I is now well known, thus new methods have been developed to analyze SUAC as a first tier test in newborn screening. One approach is to prepare a SUAC hydrazine derivative from the dried blood spots (DBS) previously utilized in the extraction of acylcarnitine (AC) and amino acids (AA). The final derivatized products of SUAC, AA and AC are combined in a single tandem mass spectrometric (MS/MS) analysis. However, butyl esterification techniques may result in contamination of underivatized acylcarnitines by as much as 20%. We have developed a simple wash step to improve the combined analysis of SUAC, AA and AC in DBS by MS/MS. METHODS: AA and AC were extracted with methanol containing labeled internal standard from 3.2mm punches taken from the DBS specimen. The previously extracted blood spot that remains after removal of the methanol extraction solvent was used in the preparation of SUAC with and without additional washing of the blood spot. The butyl ester eluates of AA and AC, and SUAC hydrazine derivatives were recombined and measured by MS/MS. RESULTS: Three additional methanol wash steps of the remaining DBS punches prior to SUAC derivatization reduced the presence of underivatized acylcarnitines, resulting in a 4-fold reduction of underivatized palmitoylcarnitine. Palmitoylcarnitine butyl ester is detected at m/z 456 while the underivatized species is detected at m/z 400, which is also the mass of dodecanoylcarnitine butyl ester. The linearity of the SUAC assay was unchanged by the additional wash steps. For butyl esterification methods, the preferred analytic procedure, the presence of AC can compromise the results of a newborn screen for the actual concentrations of acylcarnitines. It is essential to remove any underivatized acylcarnitines prior to SUAC analysis. CONCLUSION: The additional methanol wash steps did not alter SUAC assay results but did remove underivatized acylcarnitines which could result in the incorrect quantification of acylcarnitines.