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.

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.

Factors influencing quantitative liquid (scanning) transmission electron microscopy.

One of the experimental challenges in the study of nanomaterials in liquids in the (scanning) transmission electron microscope ((S)TEM) is gaining quantitative information. A successful experiment in the fluid stage will depend upon the ability to plan for sensitive factors such as the electron dose applied, imaging mode, acceleration voltage, beam-induced solution chemistry changes, and the specifics of solution reactivity. In this paper, we make use of a visual approach to show the extent of damage of different instrumental and experimental factors in liquid samples imaged in the (S)TEM. Previous results as well as new insights are presented to create an overview of beam-sample interactions identified for changing imaging and experimental conditions. This work establishes procedures to understand the effect of the electron beam on a solution, provides information to allow for a deliberate choice of the optimal experimental conditions to enable quantification, and identifies the experimental factors that require further analysis for achieving fully quantitative results in the liquid (S)TEM.

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.

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.

Extracts of retina and brain that excite afferent fibers innervating hair cells contain a compound related to hydroxyphenylglycine-N-carbamoyl.

To identify new neurotransmitter and modulator candidates that might be important in transmission from sensory hair cells to afferent nerves, we examined extracts of neural tissue for compounds that excite afferent fibers innervating hair cells. Here, we describe the extraction and purification from retina and brain of a potent, unstable, excitatory compound with pharmacological activity similar to glutamate on afferent fibers innervating hair cells. This compound, however, was clearly distinguished from glutamate, other common amino acids, and known endogenous glutamate-receptor agonists. After derivatization and analysis by gas chromatography-mass spectrometry, the major compound found in highly purified neuroactive chromatographic fractions had the same gas chromatographic elution time and mass spectrum as the compound formed by derivatization of L-p-hydroxyphenylglycine-N-carbamoyl. Hydroxyphenylglycine-N-carbamoyl, however, did not copurify with the neuroactive compound and was not neuroactive. We thus hypothesize that the detected compound was produced from a precursor, structurally related to L-p-hydroxyphenylglycine-N-carbamoyl, that was a major component of the neuroactive chromatographic fractions. Because several compounds related to hydroxyphenylglycine are known to act on glutamate receptors, such a compound is an interesting candidate to be an endogenous glutamate-receptor ligand in the mammalian nervous system.

Multilevel investigation of variation in HoNOS ratings by mental health professionals: a naturalistic study of consecutive referrals.

Episodes of mental healthcare in specialist psychiatric services often begin with the assessment of clinical and psychosocial needs of patients by healthcare professionals. Particularly for patients with complex needs or severe problems, ratings of clinical and social functioning at the start of each episode of care may serve as a baseline against which subsequent measures can be compared. Currently, little is known about service variations in such assessments on referrals from primary care. We set out to quantify variability in initial assessments performed by healthcare professionals in three CMHTs in Bristol (UK) using the Health of the Nation Outcome Scales (HoNOS). We tested the hypothesis that variations in HoNOS total and sub-scale scores are related to referral source (general practices), healthcare assessor (in CMHTs) and the assessor's professional group. Statistical analysis was performed using multilevel variance components models with cross-classified random effects. We found that variation due to assessor substantially exceeded that due to referral source (general practices). Furthermore, patient variance differed by assessor profession for the HoNOS--Impairment scores. Assessor variance differed by assessor profession for the HoNOS--Social scores. As HoNOS total and subscale scores show much larger variation by assessor than by referral source, investigations of HoNOS scores must take assessors into account. Services should implement and evaluate interdisciplinary training to improve consistency in use of rating thresholds; such initiatives could be evaluated using these extensions of multilevel models. Future research should aim to integrate routine diagnostic data with continuous outcomes to address selection effects (of patients to assessors) better.

Executive control of cognitive processes in task switching.

In 4 experiments, participants alternated between different tasks or performed the same task repeatedly. The tasks for 2 of the experiments required responding to geometric objects in terms of alternative classification rules, and the tasks for the other 2 experiments required solving arithmetic problems in terms of alternative numerical operations. Performance was measured as a function of whether the tasks were familiar or unfamiliar, the rules were simple or complex, and visual cues were present or absent about which tasks should be performed. Task alternation yielded switching-time costs that increased with rule complexity but decreased with task cuing. These factor effects were additive, supporting a model of executive control that has goal-shifting and rule-activation stages for task switching. It appears that rule activation takes more time for switching from familiar to unfamiliar tasks than for switching in the opposite direction.

Identity of nuclear high-mobility-group protein, HMG-1, and sulfoglucuronyl carbohydrate-binding protein, SBP-1, in brain.

High-mobility-group (HMG) proteins are a family of non-histone chromosomal proteins which bind to DNA. They have been implicated in multiple aspects of gene regulation and cellular differentiation. Sulfoglucuronyl carbohydrate binding protein, SBP-1, which is also localized in the neuronal nuclei, was shown to be required for neurite outgrowth and neuronal migration during development of the nervous system. In order to establish relationship between SBP-1 and HMG family proteins, two HMG proteins were isolated and purified from developing rat cerebellum by heparin-sepharose and sulfatide-octyl-sepharose affinity column chromatography and their biochemical and biological properties were compared with those of SBP-1. Characterization by high performance liquid chromatography--mass spectrometry (HPLC-MS), partial peptide sequencing and western blot analysis showed the isolated HMG proteins to be HMG-1 and HMG-2. Isoelectric focusing, HPLC-MS and peptide sequencing data also suggested that HMG-1 and SBP-1 were identical. Similar to SBP-1, both HMG proteins bound specifically to sulfated glycolipids, sulfoglucuronylglycolipids (SGGLs), sulfatide and seminolipid in HPTLC-immuno-overlay and solid-phase binding assays. The HMG proteins promoted neurite outgrowth in dissociated cerebellar cells, which was inhibited by SGGLs, anti-Leu7 hybridoma (HNK-1) and anti-SBP-1 peptide antibodies, similar to SBP-1. The proteins also promoted neurite outgrowth in explant cultures of cerebellum. The results showed that the cerebellar HMG-1 and -2 proteins have similar biochemical and biological properties and HMG-1 is most likely identical to SBP-1.

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.

Variable absorption of carbidopa affects both peripheral and central levodopa metabolism.

Carbidopa (CD), a competitive inhibitor of aromatic l-amino acid decarboxylase that does not cross the blood-brain barrier, is routinely administered with levodopa (LD) to patients with Parkinson disease (PD) to reduce the peripheral decarboxylation of LD to dopamine. Using a stable isotope-labeled form of LD, the authors examined in 9 PD patients the effects of variable CD absorption on peripheral and central LD metabolism. Subjects were administered orally 50 mg of CD followed in 1 hour by a slow bolus intravenous infusion of 150 mg stable isotope-labeled LD (ring 1',2',3',4',5',6'-13C). Eight patients underwent a lumbar puncture 6 hours following the infusion. Blood and cerebrospinal fluid (CSF) samples were analyzed for labeled and unlabeled metabolites using a combination of high-performance liquid chromatography and mass spectrometry. When patients were divided into "slow" and "rapid" CD absorption groups, significantly greater peripheral LD decarboxylation (as measured by area under the curve [AUC]-labeled serum HVA) was noted in the poor absorbers (p = 0.05, Mann-Whitney U test). Elimination half-lives for serum LD did not differ between groups, suggesting a further capacity for decarboxylation inhibition in the "rapid" absorbers. A significant correlation between AUC serum CD and percent-labeled HVA in CSF was found for all patients (R = 0.786, p = 0.02). "Rapid" as compared to "slow" CD absorbers had significantly more percent-labeled CSF HVA (60 vs. 49, p = 0.02, Mann-Whitney U test), indicating greater central-labeled DA production in the better CD absorbers. The data suggest that peripheral aromatic l-amino acid decarboxylase activity is not saturated at CD doses used in current practice. The authors believe that future studies to better examine a dose dependence of CD on peripheral LD decarboxylation and LD brain uptake are warranted.

Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases.

Linoleic acid diol glucuronides have been isolated previously from urine of patients suffering from generalized peroxisomal disorders. Glucuronidation of linoleic acid and linoleic acid diols by human liver microsomes was studied to investigate the role of glucuronide conjugation in the metabolism of linoleic acid diols. Glucuronide products were isolated and analyzed by TLC and HPLC-MS. HPLC-MS showed ions with (m/z) corresponding to singly glucuronidated linoleic acid diols while TLC revealed that the glucuronidation was at a hydroxyl position. Kinetic analysis gave apparent K(m) values in the range of 50-200 microM and V(max) rates from 5 to 12 nmol/mg x min. These rates are substantially higher than activities seen for most endogenous hydroxylated substrates. Assays using each of the four individually purified linoleic acid diol enantiomers suggest that glucuronidation occurs at only one of the two hydroxyl groups of each enantiomer. These results show for the first time that hydroxylated fatty acids are actively glucuronidated by human liver microsomes and suggest that glucuronidation may play a significant role in the biotransformation of linoleic acid diols in humans.

Modulation of GABA(A) receptor subunit mRNA levels in olivocerebellar neurons of purkinje cell degeneration and weaver mutant mice.

In olivocerebellar circuits, changes in the subunit composition of GABA(A) receptors occur at a time of extensive synaptic remodeling. In the deep cerebellar nuclei, GABA(A) receptor alpha1, beta2, and gamma2 subunit mRNA expression increases throughout neonatal development, whereas in the inferior olivary complex, the perinatal combination of alpha3, alpha5, beta3, and gamma2 mRNAs switches to the adult combination of alpha2, alpha4, beta3 and gamma1 during postnatal week 2. In situ hybridization was used to examine changes in subunit expression in the olivocerebellar nuclei of Purkinje cell degeneration and weaver mutant mice. In Purkinje cell degeneration, subunit transcripts decreased below control levels in olivary neurons; however, alpha1, beta2, and gamma2 transcript levels were slightly increased in the medial nucleus of the deep cerebellar nuclei. In weaver olivary neurons, although the switch from early- to late-onset subunit mRNAs occurred as in normal mice, transcript levels were differentially modulated by the mutation. Our studies indicate that major alterations in synaptic connectivity do not prevent developmentally programmed switches in GABA(A) receptor gene expression but can modulate the timing and level of transcript expression in afferent and efferent neurons.

Harmaline-induced changes in gamma aminobutyric acid(A) receptor subunit mRNA expression in murine olivocerebellar nuclei.

Increased CNS activity in the form of electrically or chemically induced seizures is known to alter the properties of GABA(A) receptors. The tremorgen, harmaline, causes a bursting pattern of activity in inferior olivary neurons, the effects of which are transmitted throughout the olivocerebellar circuit to other regions of the CNS. In situ hybridization was used to determine the effect of this increased activity on gamma aminobutyric acid(A) (GABA(A)) receptor subunit gene expression in the cerebellar Purkinje cell layer, deep cerebellar nuclei and inferior olivary complex of adult mice. In Purkinje cells, the expression of alpha(1), beta(2), and gamma(2) mRNAs was increased only slightly (<5%) by harmaline administration, while in deep cerebellar neurons, beta(2) transcript levels were initially elevated (26%), but dropped to control levels immediately thereafter. The expression of alpha(2), alpha(4), beta(3) and gamma(1) mRNAs in olivary neurons was affected differentially by harmaline administration. The alpha(4) transcript was increased, reaching >60% above control levels at 6 h post-injection. A smaller increase was observed for alpha(2) mRNA, while beta(3) and gamma(1) transcripts dropped below control levels during the same period. The expression of corticotropin-releasing factor mRNA was also elevated in the olivary complex. These data indicate that while Purkinje cells and deep cerebellar neurons are only minimally affected, harmaline induced changes in cellular properties may result in increased numbers of alpha(4)-containing, diazepam-insensitive, GABA(A) receptors in olivary neurons.

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.

Synthesis of 8-epi-prostaglandin F2alpha by human endothelial cells: role of prostaglandin H2 synthase.

The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F(2alpha) (8-epi-PGF(2alpha)) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF(2alpha) in the medium was determined by ELISA. The production of 8-epi-PGF(2alpha) decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF(2alpha) production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF(2alpha) production. However, 8-epi-PGF(2alpha) production was inhibited by catalase. The production of 8-epi-PGF(2alpha) was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF(2alpha) production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF(2alpha) production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF(2alpha) synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF(2alpha) synthesis is mediated by prostaglandin H(2) synthase (PGHS). To verify the role of PGHS in 8-epi-PGF(2alpha) synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF(2alpha). In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE(2) and 8-epi-PGF(2alpha). These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF(2alpha) and that 8-epi-PGF(2alpha) synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.