Observation and quantification of nanoscale processes in lithium batteries by operando electrochemical (S)TEM.

An operando electrochemical stage for the transmission electron microscope has been configured to form a "Li battery" that is used to quantify the electrochemical processes that occur at the anode during charge/discharge cycling. Of particular importance for these observations is the identification of an image contrast reversal that originates from solid Li being less dense than the surrounding liquid electrolyte and electrode surface. This contrast allows Li to be identified from Li-containing compounds that make up the solid-electrolyte interphase (SEI) layer. By correlating images showing the sequence of Li electrodeposition and the evolution of the SEI layer with simultaneously acquired and calibrated cyclic voltammograms, electrodeposition, and electrolyte breakdown processes can be quantified directly on the nanoscale. This approach opens up intriguing new possibilities to rapidly visualize and test the electrochemical performance of a wide range of electrode/electrolyte combinations for next generation battery systems.

The evolution of ultrafast electron microscope instrumentation.

Extrapolating from a brief survey of the literature, we outline a vision for the future development of time-resolved electron probe instruments that could offer levels of performance and flexibility that push the limits of physical possibility. This includes a discussion of the electron beam parameters (brightness and emittance) that limit performance, the identification of a dimensionless invariant figure of merit for pulsed electron guns (the number of electrons per lateral coherence area, per pulse), and calculations of how this figure of merit determines the trade-off of spatial against temporal resolution for different imaging modes. Modern photonics' ability to control its fundamental particles at the quantum level, while enjoying extreme flexibility and a very large variety of operating modes, is held up as an example and a goal. We argue that this goal may be approached by combining ideas already in the literature, suggesting the need for large-scale collaborative development of next-generation time-resolved instruments.

A method for site-specific and cryogenic specimen fabrication of liquid/solid interfaces for atom probe tomography.

A site-specific, cryogenic, focused ion beam (FIB) method is presented for the preparation of atom probe tomography (APT) specimens from a frozen liquid/solid interface. As a practical example, the interface between water and a corroded boroaluminosilicate glass has been characterized by APT for the first time. The water/glass interface is preserved throughout specimen preparation by plunge freezing the corroding glass particles with the corrosion solution into slush nitrogen. Site-specific specimen preparation is enabled through a new approach to extract and mount a small volume of material using a cryogenically cooled FIB stage and micromanipulator. The prepared APT specimens are subsequently transferred from the FIB to APT under cryogenic and high-vacuum conditions using a novel FIB/APT transfer shuttle and home-built environmental transfer hub attached to the APT system. Particular focus is given to the technical methods for specimen fabrication under cryogenic conditions. Persistent challenges are discussed in addition to future opportunities for this new specimen preparation method.

Proteomic analysis of human meibomian gland secretions.

BACKGROUND/AIM: Human tears contain hundreds of proteins that may exert a significant influence on tear film stability, ocular surface integrity, and visual function. The authors hypothesise that many of these proteins originate from the meibomian gland. This study's aim was to begin to develop the proteomic methodology to permit the testing of their hypothesis. METHODS: Meibomian gland secretions were collected from the lower eyelids of adult volunteers and placed in a chloroform-methanol mixture. Samples were partitioned in a biphasic system and non-lipid phase materials were reduced, alkylated, and trypsin digested to obtain peptides for protein identification. This peptide mixture was separated by micro-capillary reverse phase chromatography and the effluent examined by nano-electrospray MS and data dependent MS/MS. SEQUEST software was used to identify proteins from the MS/MS spectra. RESULTS: The methodological approach to date has permitted the identification of more than 90 proteins in human meibomian gland secretions. Proteins include the alpha2-macroglobulin receptor, IgA alpha chain, farnesoid X activated receptor, interferon regulatory factor 3, lacritin precursor, lactotransferrin, lipocalin 1, lysozyme C precursor, potential phospholipid transporting ATPase IK, seven transmembrane helix receptor (also termed somatostatin receptor type 4), testes development related NYD-SP21 (also termed high affinity IgE receptor beta subunit), and TrkC tyrosine kinase. CONCLUSIONS: These findings indicate that the meibomian gland secretes a number of proteins into the tear film. It is quite possible that these proteins contribute to the dynamics of the tear film in both health and disease.

Inhibition of PM-2 DNA degradation by a human serum protein.

A unique DNA-binding protein was detected that inhibited DNA degradation induced by bleomycin and was decreased in sera of cancer patients. The protein from normal human serum was purified to homogeneity by ammonium sulfate precipitation and DEAE-cellulose and DNA-cellulose column chromatography. Two-dimensional isoelectric focusing gel electrophoresis revealed a single protein spot with a molecular weight of 64,000 and a pI at pH 5.9. The NH2 terminus was lysine, and the ratio of acidic to basic residues was 1.2. DNA binding was demonstrated by column chromatography, agarose gel electrophoresis, fluorescence quenching, and circular dichroism. The inhibitory activity was abolished by treatment with Pronase but not by RNase or DNase I. FeCl2 caused a partial loss of inhibitory activity. The inhibition of DNA degradation was more effective for breakage induced by bleomycin than neocarzinostatin, macromomycin, or DNase I. Evidence from DNA-binding studies suggests the inhibition is due to binding of the protein to sites on DNA preferred by bleomycin. Thus, the protein could be useful for studies on the mechanism of action of bleomycin and other antitumor agents, the cytotoxic effects of which are due primarily to damage of cellular DNA. The protein was decreased significantly in sera of cancer patients, and its potential use as a diagnostic tool for neoplasias is being investigated further.

Inhibition of bleomycin-induced DNA breakage by superoxide dismutase.

Inhibition of bleomycin (BLM)-induced DNA breakage by superoxide dismutase (SOD) has been reported and presumed to be due to its removal of the superoxide free radicals generated by BLM in the presence of iron(II). We have studied the possibility that the inhibitory effect might result from DNA-binding of SOD. The effect of copper-zinc SOD on BLM-induced DNA degradation was investigated using the PM-2 DNA fluorescence technique. PM-2 DNA was incubated with BLM in the presence or absence of native and heat-inactivated copper-zinc SOD as determined by the epinephrine autoxidation method. The concentrations of SOD required to inhibit 50% PM-2 DNA degradation for the native and the inactivated SOD were 100 and 120 microgram/ml, respectively. Analysis of the reaction mixture by agarose gel electrophoresis confirmed the absence of DNA degradation by BLM in the presence of either form of SOD. PM-2 DNA was shown to bind native or inactivated SOD by Sephadex G-100 column chromatography, fluorescence-quenching studies, and agarose gel electrophoresis. Thus, these results indicate that SOD is able to bind to PM-2 DNA and inhibit BLM-induced degradation independently of its free radical-scavenging activity. The inhibitor was more effective against BLM than other compounds which degrade PM-2 DNA. This suggests that SOD may bind to BLM-binding and/or BLM degradation sites in PM-2 DNA, and the observed inhibition is unrelated to its effects on free radicals.

Detection of a serum DNA-binding protein associated with cancer.

An accompanying report describes the purification and partial characterization of a unique DNA-binding protein (Mr 64,000; pI 5.9) that is present in human sera. This report gives the results of assays of sera from patients for the bleomycin inhibitor protein (BIP) using the Pseudomonas bacteriophage covalently closed circular DNA fluorescence technique standardized for DNA breakage induced by bleomycin. The results of the BIP assays were expressed by values of specific activity of inhibition. One arbitrary unit of inhibitory activity was defined as equivalent to the amount of serum protein required to cause 50% inhibition of DNA degradation using standard conditions of the DNA breakage assay. The mean values of specific activity of inhibition (SAI) for groups of healthy individuals (n = 26), patients with nonmalignant diseases (n = 33), and patients with malignant diseases (n = 83) were 12.60 +/- 4.69 (S.E.), 12.53 +/- 3.17, and 2.40 +/- 0.84 units/mg, respectively. Mean SAI values for patients with cancers of various types were: solid tumors (n = 46), 2.44 +/- 0.86; leukemias (n = 24), 2.59 +/- 0.96; and lymphomas (n = 18), 2.07 +/- 0.64. The decrease in BIP activity was not correlated with sex, age, or prior chemotherapy. Mean SAI values of male (n = 29) and female (n = 59) patients with cancer were 2.61 +/- 0.87 and 2.30 +/- 0.83 units/mg, respectively. Mean SAI values for different age groups were: 0 to 40 years (n = 21), 2.05 +/- 0.68 units/mg; 41 to 70 years (n = 56), 2.59 +/- 0.68 units/mg; and greater than 70 years (n = 11), 2.12 +/- 0.67 units/mg. Cancer patients with and without prior chemotherapy had mean SAI values of 2.97 +/- 0.85 (n = 23) and 2.20 +/- 0.86 units/mg (n = 65), respectively. Linear regression analysis comparing SAI values and serum protein levels showed no correlation (r = 0.21). These results suggest the decrease of the BIP is associated with malignant disease. Additional controlled studies are required before the significance of this association can be adequately assessed.

Location of double bonds in two unsaturated forms of phytanic acid from Refsum disease as determined by mass spectrometry.

A monounsaturated and a triunsaturated form of phytanic acid (3,7,11,15-tetramethylhexacosanoate) were isolated from plasma lipids of a patient with Refsum disease. Both were converted to their methyl esters, oxidized to polyhydroxy acids by treatment with OsO4 and converted to their vicinal trimethylsilyl ethers. These derivatives were analyzed by gas chromatography-mass spectrometry using both electron impact ionization (at 21 and 70 eV) and chemical ionization conditions to obtain clear evidence to establish the structure of the monounsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-15-monoenoic acid and that of the triunsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-6,10,14-trienoic acid. The possible metabolic and dietary sources for these novel fatty acids are discussed.

Occurrence of novel branched-chain fatty acids in Refsum's disease.

Two novel branched-chain fatty acids, which appear to be unsaturated analogs of phytanic acid, have been observed in sera and urine of patients with Refsum's disease. They occur in both phospholipids and neutral lipids, and have been isolated and characterized.

Subunit localizations of zinc(II) in DNA-dependent RNA polymerase from Escherichia coli B.

RNA Polymerase holoenzyme and core enzyme from Escherichia coli B have been shown to contain two zinc ions. Flameless atomic absorption spectroscopy of the isolated core subunits indicated that one zinc ion is localized on the beta subunit and the other is bound on the beta' subunit. Atomic fluorescence spectroscopy showed that prolonged dialysis of the metalloenzyme against 0.01 M o-phenanthroline resulted in the removal of both zinc(II) ions with accompanying loss of enzymatic activity. The activity of the apoenzyme was observed to be completely restored by readdition of zinc(II) and partially restored by cobalt(II).

Impact of antiandrogen treatment on the fatty acid profile of neutral lipids in human meibomian gland secretions.

The purpose of this study was to determine whether the use of antiandrogen medications is associated with significant alterations in the fatty acid (FA) profiles of neutral lipids in human meibomian gland secretions. Meibomian gland secretions were obtained from both eyes of patients receiving antiandrogen therapy and from age-related controls. Samples were processed for high-performance liquid chromatography/mass spectrometry and an evaluation of the mass/charge ratios of neutral lipid FA. Our results demonstrate that antiandrogen therapy is associated with significant and consistent alterations in the mass/charge ratios of neutral lipid fractions of meibomian gland secretions. Patients taking antiandrogen medications had significant changes in the occurrence of numerous diglyceride, triglyceride, and wax/cholesterol ester FA products, compared with age-matched controls. Statistical analyses of data within groups demonstrated very high correlation coefficients, and cross-correlation analyses revealed characteristic shifts in FA patterns between groups. Our findings show that antiandrogen use is paralleled by significant changes in the FA profiles of neutral lipid fractions in meibomian gland secretions.

Effect of androgen deficiency on the human meibomian gland and ocular surface.

The purpose of this study was to determine whether the chronic use of antiandrogen medications leads to meibomian gland dysfunction, altered lipid profiles in meibomian gland secretions, decreased tear film stability, and evaporative dry eye. Subjects taking antiandrogen therapy for prostatic indications, as well as age-related controls, were asked to complete a questionnaire that assessed dry eye symptoms and then were given a complete anterior segment examination. Moreover, meibomian gland secretions were obtained from each eye and analyzed by high-performance liquid chromatography/mass spectrometry for the relative content of cholesterol, cholesterol esters, wax esters, diglycerides, triglycerides, and specific molecular species in the diglyceride fraction. Our results demonstrate that patients taking antiandrogen treatment, compared with age-related controls, had a: 1) significant increase in the frequency of appearance of tear film debris, an abnormal tear film meniscus, irregular posterior lid margins, conjunctival tarsal injection, and orifice metaplasia of the meibomian glands; 2) significant increase in the degree of ocular surface vital dye staining; 3) significant decrease in the tear film breakup time and quality of meibomian gland secretions; and 4) significant increase in the frequency of light sensitivity, painful eyes, and blurred vision. In addition, the use of antiandrogen pharmaceuticals was associated with significant changes in the relative amounts of lipids in meibomian gland secretions. Our findings indicate that chronic androgen deficiency is associated with meibomian gland dysfunction and dry eye.

Embryonic and postnatal expression of four gamma-aminobutyric acid transporter mRNAs in the mouse brain and leptomeninges.

The distribution of gamma-aminobutyric acid (GABA) transporter mRNAs (mGATs) was studied in mouse brain during embryonic and postnatal development using in situ hybridization with radiolabeled oligonucleotide probes. Mouse GATs 1 and 4 were present in the ventricular and subventricular zones of the lateral ventricle from gestational day 13. During postnatal development, mGAT1 mRNA was distributed diffusely throughout the brain and spinal cord, with the highest expression present in the olfactory bulbs, hippocampus, and cerebellar cortex. The mGAT4 message was densely distributed throughout the central nervous system during postnatal week 1; however, the hybridization signal in the cerebral cortex and hippocampus decreased during postnatal weeks 2 and 3, and in adults, mGAT4 labeling was restricted largely to the olfactory bulbs, midbrain, deep cerebellar nuclei, medulla, and spinal cord. Mouse GAT2 mRNA was expressed only in proliferating and migrating cerebellar granule cells, whereas mGAT3 mRNA was absent from the brain and spinal cord throughout development. Each of the four mGATs was present to some degree in the leptomeninges. The expression of mGATs 2 and 3 was almost entirely restricted to the pia-arachnoid, whereas mGATs 1 and 4 were present only in specific regions of the membrane. Although mGATs 1 and 4 may subserve the classical purpose of terminating inhibitory GABAergic transmission through neuronal and glial uptake mechanisms, GABA transporters in the pia-arachnoid may help to regulate the amount of GABA available to proliferating and migrating neurons at the sub-pial surface during perinatal development.

Developmental changes in the expression of gamma-aminobutyric acidA/benzodiazepine receptor subunit mRNAs in the murine inferior olivary complex.

The pharmacological and physiological properties of ligand-gated ion channels are dependent on their subunit composition; spontaneously occurring changes in subunit composition during neuronal development may result in dramatic functional differences between embryonic and adult forms of the receptor complex. In the present study, in situ hybridization with antisense cRNA probes was used to examine the subunit composition of the gamma-aminobutyric acidA/benzodiazepine (GABAA/BZ) receptor in the developing inferior olivary complex. This receptor is thought to be a pentameric chloride channel comprised of selected alpha, beta, gamma, delta, and rho subunits, the majority of which have several isoforms: alpha 1-6, beta 1-4, gamma 1-4, and rho 1,2. Among the 13 subunit variants present in the mammalian central nervous system, alpha 2-5, beta 3, and gamma 1,2 mRNAs are expressed at significant levels in the inferior olivary complex. Two clearly different temporal patterns of GABAA/BZ receptor subunit mRNA expression were observed: The expression of alpha 3, alpha 5, beta 3, and gamma 2 mRNAs was at a peak during embryonic and early postnatal development followed by rapid down-regulation thereafter. Conversely, alpha 2, alpha 4, and gamma 1 mRNA expression was very low or absent during early development, and a pronounced increase was observed at the end of postnatal week 1. These studies suggest that there are developmental changes in the subunit composition of the GABAA/BZ receptor in inferior olivary neurons. These changes in subunit expression, which occur during a period of major alterations in afferent and efferent synaptic connections, may subserve a change in the role of GABA from its function as a neurotrophic factor to that of an inhibitory neurotransmitter.

Novel receptor protein tyrosine phosphatase (RPTPrho) and acidic fibroblast growth factor (FGF-1) transcripts delineate a rostrocaudal boundary in the granule cell layer of the murine cerebellar cortex.

We have identified a novel receptor-like protein tyrosine phosphatase (RPTPrho) transcript whose expression in the cerebellar cortex is restricted to the granule cell layer of lobules 1-6. Acidic fibroblast growth factor (FGF-1) mRNA follows a similar cerebellar expression pattern. Together, the two markers define a sharp boundary in lobule 6, slightly caudal to the primary fissure. Anterior and posterior compartments became discernible only during postnatal weeks two and six, for RPTPrho and FGF-1, respectively. A rostrocaudal boundary in lobule 6 of the murine cerebellar cortex has also been identified morphologically by the effects of the meander tail mutation. The position of the RPTPrho and FGF-1 boundary on the rostrocaudal axis of the cerebellar cortex was close to, but not coincident with, the caudal extent of the disorganized anterior lobe of meander tail and the rostral extent of Otx-2 expression. The restricted pattern of FGF-1 and RPTPrho implies that these molecules may have specific signaling roles in the tyrosine phosphorylation/dephosphorylation pathway in the anterior compartment of the adult cerebellar cortex.

Carbamazepine increases phenytoin serum concentration and reduces phenytoin clearance.

Addition of carbamazepine to phenytoin monotherapy resulted in the following significant (p less than 0.05) changes: (1) increased mean phenytoin serum concentration; (2) decreased phenytoin clearance, due to decreased production of phenytoin dihydrodiol and p-hydroxyphenyl-phenylhydantoin; (3) increased phenytoin elimination half-life; and (4) increased drug-related toxicity. Close monitoring is required after addition of carbamazepine to phenytoin.

N-acetyl-D-glucosamine is present in cysts and trophozoites of Giardia lamblia and serves as receptor for wheatgerm agglutinin.

Previously, on the basis of lectin binding and glycosidase digestion assays, we have suggested that N-acetyl-D-glucosamine residues (GlcNAc) are major structural components of both trophozoites and in vivo cysts of the intestinal parasite Giardia lamblia. In this report we confirm that GlcNAc is present both in trophozoites and in vitro cysts as assessed by lectin binding and glycosidase digestion assays, galactosyltransferase labeling, immunochemical analysis using antibodies specific for GlcNAc and its beta 1-4 oligomers, and by gas chromatography/mass spectrometry (GC/MS). The results show that wheatgerm agglutinin (WGA) binds specifically to intact trophozoites and in vitro cysts as well as to SDS-PAGE separated proteins. WGA binding to the separated proteins was markedly reduced after their digestion with N-acetyl-beta-D-glucosaminidase, supporting the conclusion that WGA is reacting with terminal beta-linked GlcNAc residues. Labeling of trophozoites and cysts by 3H-exogalactosylation with galactosyltransferase further confirmed the presence of terminal GlcNAc in both surface and intracellular glycoproteins. The presence of GlcNAc is also supported by microfluorometric analysis using antibodies to (GlcNAc)1, (GlcNAc)2, and (GlcNAc)3, which revealed a sugar-inhibitable binding of the antibody to live trophozoites. Finally, the presence of GlcNAc in both cysts and trophozoites was unequivocally confirmed by GC/MS analysis of detergent-extracted membranes and of glycoproteins isolated by affinity chromatography on WGA-agarose. GC/MS analysis also revealed mannose (Man), N-acetyl-D-galactosamine (GalNAc), fucose (Fuc), galactose (Gal), glucose (Glc) and N-acetylneuraminic acid (NANA) to be present in cysts. All these sugars were also present in trophozoites, except for GalNAc. The glycoproteins isolated by WGA affinity chromatography were 5- to 40-fold enriched in GlcNAc, further supporting the conclusion that WGA reacts with GlcNAc in Giardia. In summary, the data presented here provide biological and chemical evidence for GlcNAc in both cysts and trophozoites of G. lamblia and are consistent with previously published results from this and other laboratories.

Androgen influence on the meibomian gland.

PURPOSE: The hypothesis in the study was that androgens control meibomian gland function, regulate the quality and/or quantity of lipids produced by this tissue, and promote the formation of the tear film's lipid layer. To test this hypothesis, a study was conducted to determine whether androgen receptor protein exists in the epithelial cell nuclei of rat meibomian glands and, in addition, whether androgen deficiency and/or treatment influences the gross morphology, neutral lipid content, and fatty acid profile of the rabbit meibomian gland, as well as the appearance of the tear film lipid layer. METHODS: Rat lids were obtained and processed for immunohistochemistry. Meibomian glands from intact, androgen- and/or placebo-treated rabbits were analyzed by histology, and glandular lipids were evaluated by gas chromatography, high-performance liquid chromatography (HPLC), and mass spectrometry. The rabbit tear film lipid layer was assessed by interferometry. RESULTS: In the current study androgen receptor protein existed within acinar epithelial cell nuclei of rat meibomian glands; androgen deficiency was associated with alterations in the lipid content of the rabbit meibomian gland; 19-nortestosterone treatment modulated the fatty acid profile in the total and neutral lipid fractions of the rabbit meibomian gland; and androgens did not appear to influence the gross morphology of meibomian tissue or to exert a demonstrable effect on the rabbit tear film lipid layer. CONCLUSIONS: The findings show that the meibomian gland is an androgen target organ and that androgens influence the lipid profile within this tissue. However, the extent to which androgens regulate the production of these lipids and whether this action may impact tear film stability remain to be determined.

Expression and characterisation of the NS1 and NS2 proteins of respiratory syncytial virus.

The NS1 and NS2 proteins of human respiratory syncytial virus (RSV) were expressed using baculovirus. Antisera to these expressed proteins and to synthetic peptides were raised in rabbits and used to characterise the proteins. Multiple forms of both NS1 and NS2 proteins were detected in RSV infected cells by both immunoblotting and radioimmunoprecipitation when non-reducing, but not reducing, conditions were used. In pulse-labelling experiments the monomeric form of NS1 was stable, while that of NS2 was unstable with a half life of about 30 min. The NS1 protein associated with the matrix (M) protein and could be co-precipitated by a monoclonal antibody to M protein. The NS2 protein did not show any detectable association with RSV structural proteins. These results indicate that the NS1 and NS2 proteins have distinct roles in the viral life cycle.

Probes of eukaryotic DNA-dependent RNA polymerase II-II. Covalent binding of two purine nucleoside dialdehydes to the initiation subsite.

The catalytic center of wheat germ DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) as a model eukaryotic enzyme system was probed with two purine nucleoside dialdehydes, 6-methylthioinosinedicarboxaldehyde (MMPR-OP) and a derivative 6-[(acetylaminoethyl)-1-naphthylamine-5-sulfonyl]thioinosinedicarboxaldehyde (AMPR-OP). Both drugs gave noncompetitive inhibition with respect to [3H]UMP incorporations into RNA, and inhibitor bindings were reversed with initiation substrates. The Ki values for MMPR-OP and AMPR-OP were determined to be 0.64 mM and 1.0 muM respectively. The drugs were covalently bound to the catalytic center by NaBH4 reduction. Both were found bound to the largest enzyme subunit, IIa. It is tentatively concluded that MMPR-OP and AMPR-OP inhibit RNA polymerase II by binding to an essential lysine in the initiation subsite of the catalytic center located on the IIa subunit.