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In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-glycomics analyses. The performance of the 3D system alone was benchmarked through the analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-glycomics analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.

The anti-CD52 monoclonal antibody alemtuzumab reduces disease activity in previously untreated patients with relapsing-remitting multiple sclerosis. We aimed to assess efficacy and safety of alemtuzumab compared with interferon beta 1a in patients who have relapsed despite first-line treatment.In our 2 year, rater-masked, randomised controlled phase 3 trial, we enrolled adults aged 18-55 years with relapsing-remitting multiple sclerosis and at least one relapse on interferon beta or glatiramer. Eligible participants were randomly allocated in a 1:2:2 ratio by an interactive voice response system, stratified by site, to receive subcutaneous interferon beta 1a 44 μg, intravenous alemtuzumab 12 mg per day, or intravenous alemtuzumab 24 mg per day. Interferon beta 1a was given three-times per week and alemtuzumab was given once per day for 5 days at baseline and for 3 days at 12 months. The 24 mg per day group was discontinued to aid recruitment, but data are included for safety assessments. Coprimary endpoints were relapse rate and time to 6 month sustained accumulation of disability, comparing alemtuzumab 12 mg and interferon beta 1a in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT00548405.202 (87%) of 231 patients randomly allocated interferon beta 1a and 426 (98%) of 436 patients randomly allocated alemtuzumab 12 mg were included in the primary analyses. 104 (51%) patients in the interferon beta 1a group relapsed (201 events) compared with 147 (35%) patients in the alemtuzumab group (236 events; rate ratio 0·51 [95% CI 0·39-0·65]; p0·0001), corresponding to a 49·4% improvement with alemtuzumab. 94 (47%) patients in the interferon beta 1a group were relapse-free at 2 years compared with 278 (65%) patients in the alemtuzumab group (p0·0001). 40 (20%) patients in the interferon beta 1a group had sustained accumulation of disability compared with 54 (13%) in the alemtuzumab group (hazard ratio 0·58 [95% CI 0·38-0·87]; p=0·008), corresponding to a 42% improvement in the alemtuzumab group. For 435 patients allocated alemtuzumab 12 mg, 393 (90%) had infusion-associated reactions, 334 (77%) had infections (compared with 134 [66%] of 202 patients in the interferon beta 1a group) that were mostly mild-moderate with none fatal, 69 (16%) had thyroid disorders, and three (1%) had immune thrombocytopenia.For patients with first-line treatment-refractory relapsing-remitting multiple sclerosis, alemtuzumab could be used to reduce relapse rates and sustained accumulation of disability. Suitable risk management strategies allow for early identification of alemtuzumab's main adverse effect of secondary autoimmunity.Genzyme (Sanofi) and Bayer Schering Pharma.

We report in situ studies on the shrinkage and expansion of graphene nanopores under electron beam irradiation at temperatures of 400–1200 °C using a thermal specimen holder in a transmission electron microscope. The shrinkage of nanopores of various sizes is observed. At all temperatures studied in the present work, nanopores with relatively small diameters could be fully closed. And at the low end of our temperature range, 400 °C, larger nanopores (those with a diameter of 10 nm) could also be closed completely. The nanopore shrinking process can be stopped by blocking the electron beam. We demonstrate an approach for tailoring the size of the graphene nanopore through a combination of electron beam irradiation and controlled heat.

The microstructure of natural graphite (NG) and highly ordered pyrolytic graphite (HOPG) blocks, and their mechanically exfoliated graphenes, prepared by only one stamping on a meshed Cu grid, was investigated using dual beam scanning electron microscope/focus ion beam (SEM/FIB) and transmission electron microscopy (TEM). Channeling contrast enhanced SEM/FIB images and cross section TEM micrographs showed that HOPGs have very shallow grains, less than 30 nm in thickness and 6–30 μm in size, while NGs have grains larger than mm in size with some folded layers. In order to understand the structure of exfoliated graphene, an observation along the z-axis of lattice on suspended membranes was conducted. Through the aid of diffraction pattern (DP) simulations, demonstrating the dependency of the intensity ratio between { 11 2 ¯ 0 } and { 1 1 ¯ 00 } on the number of layers and stacking sequences, a suspended monolayer could be distinguished from a multilayer. With the observations of unfolded graphene edges, we found that the graphene edges always have a roughness below a nanometer despite their straight feature in a low magnification view and have no preferential direction. DP and HREM analyses of folded edges in NG membranes also revealed that the folding direction is arbitrary and graphite is mostly stacked with AB sequence.

The skill of six well-known formulas developed for calculating the longshore sediment transport rate was evaluated in the present study. Formulas proposed by Bijker [Bijker, E.W., 1967. Some considerations about scales for coastal models with movable bed. Delft Hydraulics Laboratory, Publication 50, Delft, The Netherlands; Journal of the Waterways, Harbors and Coastal Engineering Division, 97 (4) (1971) 687.], Engelund–Hansen [Engelund, F., Hansen, E., 1967. A Monograph On Sediment Transport in Alluvial Streams. Teknisk Forlag, Copenhagen, Denmark], Ackers–White [Journal of Hydraulics Division, 99 (1) (1973) 2041], Bailard–Inman [Journal of Geophysical Research, 86 (C3) (1981) 2035], Van Rijn [Journal of Hydraulic Division, 110 (10) (1984) 1431; 110(11) (1984) 1613; 110(12) (1984) 1733], and Watanabe [Watanabe, A., 1992. Total rate and distribution of longshore sand transport. Proceedings of the 23rd Coastal Engineering Conference, ASCE, 2528–2541] were investigated because they are commonly employed in engineering studies to calculate the time-averaged net sediment transport rate in the surf zone. The predictive capability of these six formulas was examined by comparison to detailed, high-quality data on hydrodynamics and sediment transport from Duck, NC, collected during the DUCK85, SUPERDUCK, and SANDYDUCK field data collection projects. Measured hydrodynamics were employed as much as possible to reduce uncertainties in the calculations, and all formulas were applied with standard coefficient values without calibration to the data sets. Overall, the Van Rijn formula was found to yield the most reliable predictions over the range of swell and storm conditions covered by the field data set. The Engelund–Hansen formula worked reasonably well, although with large scatter for the storm cases, whereas the Bailard–Inman formula systematically overestimated the swell cases and underestimated the storm cases. The formulas by Watanabe and Ackers–White produced satisfactory results for most cases, although the former overestimated the transport rates for swell cases and the latter yielded considerable scatter for storm cases. Finally, the Bijker formula systematically overestimated the transport rates for all cases. It should be pointed out that the coefficient values in most of the employed formulas were based primarily on data from the laboratory or from the river environment. Thus, re-calibration of the coefficient values by reference to field data from the surf zone is expected to improve their predictive capability, although the limited amount of high-quality field data available at present makes it difficult to obtain values that would be applicable to a wide range of wave and beach conditions.

This paper presents an analysis of longshore sediment transport (LST) rates based on an accumulation of data obtained during five storms. Direct measurements of velocities and suspended sediment concentration were conducted at a minimum of nine positions across a barred profile in waves up to H m0 =3.5 m to provide a measure of the cross-shore distribution and total suspended-load sediment transport rates. The study was conducted at the US Army Engineer Waterways Experiment Station's Field Research Facility, located in Duck, NC. Measurements were made using the Sensor Insertion System (SIS) which provides an economical means to collect the required information. The largest LST rate computed from the measurements was 1780 m 3 h −1 . Although the cross-shore distribution of the LST varied, it most often had two peaks associated with wave shoaling and breaking at the bar and near the beach. Comparisons of measurement results with predictions using the `CERC' LST formula show the predicted rates were sometimes higher and other times lower; suggesting that additional terms may be required for short term predictions during storms. Comparisons to a `Bagnold' type formulation, which included a velocity term that could account for wind and other effects on LST, show better agreement for at least one of the storms. These results are intended to help fill a void of information documenting the cross-shore distribution and LST rates, particularly during storms.

Divinylbenzene (DVB) is a crosslinking monomer used primarily for copolymerization with styrene to produce ion-exchange resins. The toxicity of inhaled DVB was investigated because of the potential for worker exposure and the structural similarity of DVB to styrene, a potential carcinogen. Male and female B6C3F1 mice were exposed to 0, 25, 50, or 75 ppm DVB for 6 hr/day, 5 days/week for up to 2 weeks. Six mice/sex/dose group were killed after 3, 5, and 10 exposures and six mice/sex in the 75 ppm group were killed 7 days after 10 exposures. The most severe effects occurred in the nasal cavity and liver, with less severe effects occurring in the kidneys. In the nasal cavity olfactory epithelium acute necrosis and inflammation were present at early time points followed by regeneration, architectural reorganization, and focal respiratory metaplasia by 7 days after the last exposure. Olfactory epithelial changes were concentration-dependent with extensive involvement at 75 ppm and peripheral sparing at 25 ppm. There was also necrosis and regeneration of olfactory-associated Bowman's glands as well as the lateral nasal (Steno's) glands. Hepatocellular centrilobular (CL) necrosis was observed only in the 75 ppm dose group and was similar to that caused by styrene. A time-dependent progression was observed, characterized by CL degeneration after 1 exposure, necrosis after 3 and 5 exposures, and chronic inflammation with CL karyomegaly after 10 exposures and 7 days after the 10th exposure. Hepatic GSH levels were decreased in a dose-dependent manner. In the kidneys, transient tubular damage was observed in some male mice exposed to 75 ppm, and appeared to be a response to DVB-induced tubular epithelial injury.

Tax incidence is examined in an experimental general equilibrium production micro economy. Consistent with predictions, the imposition of a tax on corporate capital resulted in a flow of capital out of the corporate sector, decreased corporate usage of capital relative to labor, and tax-shifting through decreases in the relative prices of capital and non-corporate output. Although capital owners were able to shift some of the tax burden, the corporate tax resulted in a 35% drop in capital owner's share of payoffs. Implications for policy are discussed.

Styrene Inhalation Toxicity Studies in Mice. II. Sex Differences in Susceptibility of B6C3F1 Mice. Morgan, D. L., Mahler, J. F., Dill, J. A., Price, H. C., Jr., and Adkins, B., Jr. (1993). Fundam. Appl. Toxicol. 21, 317-325. Styrene is a commercially important chemical used in the production of plastics and resins. In initial short-term styrene inhalation studies, toxicity was significantly greater in male B6C3F1 mice than in females, suggesting that males may metabolize styrene more extensively and/or may be less able to detoxify reactive metabolites. In addition, a nonlinear dose-response was observed where toxicity and mortality were greater in mice exposed to 250 ppm than in those exposed to 500 ppm. These studies were conducted to investigate potential mechanism(s) for sex differences and the nonlinear dose-response in styrene toxicity by evaluating the effects of repeated styrene exposure on styrene oxide production, hepatic GSH availability, and hepatotoxicity in male and female B6C3F1 mice. Mice (36/sex/dose) were exposed to 0, 125, 250, or 500 ppm styrene 6 hr/day for up to 3 days. Styrene exposure caused increased mortality and hepatotoxicity (centrilobular necrosis, increased serum liver enzymes) in males and females after one or two exposures to 250 and 500 ppm. Hepatic GSH levels were decreased in a dose-dependent manner in males and females. After one exposure, GSH levels in males rebounded above controls in all dose groups. After three exposures to 125 or 250 ppm males appeared to maintain GSH levels; GSH was still decreased in the 500 ppm group. GSH levels in females were decreased after each exposure in all dose groups to lower levels than in males, and did not rebound above controls. Male mice had significantly greater blood styrene levels than females after one exposure to 500 ppm; however, there were no significant sex differences in blood styrene after subsequent exposures. Levels of SO in blood were not significantly greater in male mice than females within a dose group, and did not change significantly with repeated styrene exposures for 3 days. Blood styrene and SO levels were significantly higher at 500 ppm than at 250 ppm indicating that styrene uptake and metabolism are greater at 500 ppm than at 250 ppm. The higher incidence of mortality in male mice and the nonlinear dose-response to styrene cannot be explained by gender- or dose-related differences in hepatotoxicity, GSH depletion, or blood styrene or SO levels.

Studies were conducted to evaluate the toxic effects of short-term repeated styrene inhalation in B6C3F1 mice. Male and female mice were exposed to 0, 125, 250, or 500 ppm styrene, 6 hr/day, for up to 14 days. Styrene toxicity was characterized by severe centrilobular hepatic necrosis and deaths after one exposure to 500 ppm or two exposures to 250 ppm. Mortality and hepatotoxicity were not increased by additional exposures, and in surviving mice, regeneration and repair of initial hepatic injury occurred in spite of continued exposure for 14 days. A marked sex difference was observed, with male mice significantly more susceptible to styrene toxicity than females. A nonlinear dose response was observed where mortality in male and female mice was greater in the 250 ppm dose group than that in the 500 ppm dose group. Severe congestion and necrosis of the liver was present in moribund mice; hepatic congestion and serum alanine aminotransferase and sorbitol dehydrogenase were significantly greater in moribund animals.

The structures of three type I fructose-1, 6-bisphosphate aldolases have been determined and the common residues surrounding the Schiff base-forming Lys residue located. Armed with this information, it is now possible to propose a mechanism for this ubiquitous enzyme which is consistent with the recorded biochemical data. An interesting, but by no means mandatory, feature of the reaction mechanism is that catalysis can proceed without exchange with the solvent.

The structural gene (pgk) encoding 3-phosphoglycerate (PGK) from Bacillus stearothermophilus NCA1503, has been cloned in Escherichia coli and its complete nucleotide sequence determined. The gene consists of an open reading frame corresponding to a protein of 394 amino acids (aa) (calculated Mr 42,703) and, in common with other prokaryotic pgk genes, is preceded by the structural gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Constructs containing the B. stearothermophilus pgk gene and its flanking sequences in the high-copy plasmid, pUC9, co-express both PGK and GAPDH at high levels in transformed E. coli cells, typically producing PGK at levels of up to 30% of the soluble cell protein. The deduced aa sequence of B. stearothermophilus PGK is compared with those of the mesophilic (yeast) and extreme thermophilic (Thermus thermophilus) enzymes since the crystal structure of these PGKs are known or are in the process of being determined. Changes in the sequences of the three enzymes, as they appear to relate to the enhancement of thermal stability, are discussed.

The structure of the type I fructose 1,6-bisphosphate aldolase from human muscle has been extended from 3 A to 2 A resolution. The improvement in the resulting electron density map is such that the 20 or so C-terminal residues, known to be associated with activity and isozyme specificity, have been located. The side-chain of the Schiff's base-forming lysine 229 is located towards the centre of an eight-stranded β-barrel type structure. The C-terminal “tail” extends from the rim of the β-barrel towards lysine 229, thus forming part of the active site of the enzyme. This structural arrangement appears to explain the difference in activity and specificity of the three tissue-specific human aldolases and helps with our understanding of the type I aldolase reaction mechanism.

The carboxy propylium ions CH3CH2 C + HCOOH, CH3 C + HCH2COOH and (CH3)2 C + COOH have been generated in the gas phase by loss of I. from the respective ionized iodo acids. The straight chain isomers interconvert rapidly with themselves and with CH2CHCH2 C + (OH)2. The major unimolecular dissociation products are CH3CHCH C + O + H2O and CH3 C + O + CH3CHO, together with some CH3 C + HOH + CH2CO. Loss of H2O is proposed to occur directly from CH3CH2 C + HCOOH; formation of CH3 C + O and CH3 C + (H)OH involves rearrangement of CH3CH2 C + HCOOH to CH3CH(OH)CH2 C + O which, as shown by neutralization-reionization (NR) experiments collapses to the ion CH3CHO ţu H + ⋯ CH2CO, formally proton bound acetaldehyde/ketene. It is this species which is sampled in metastable ion (MI), collisional activation (CA) and NR experiments. Thus in the CA mass spectra of the carboxypropylium ions m/z 43, CH3 C + O, and m/z 45, CH3 C + (H)OH, are dominant peaks; upon neutralization the bridged condensate falls apart to CH3CHO and CH3O which is, thermodynamically and kinetically, the most favourable dissociation. Our work shows that insight into the mechanisms of fast isomerization reactions of molecular ions may greatly assist the confident interpretation of the mass spectra of unknowns. Thus for example the puzzling observation that m/z 43, CH3 C + O, is base peak in the normal electron impact mass spectrum of the hydroxy ester CH3CH2CH(OH)CH2COOCH3, methyl 3-hydroxy pentanoate, can now easily be rationalized.