Self-Limiting Robust Surface-Grafted Organic Nanofilms.

Robust surface-bound insulating polymer films with controlled thickness in <5 nm range are important for technological advances in diverse disciplines such as electrochemical sensors, molecular electronics, separations and anti-corrosive coatings. Creating these films by simple methods from readily available materials has been a significant challenge. Here we report a newly synthesized molecule combining a styrene and thiol moieties, joined via a short linker, that binds to the gold surface, polymerizes and crosslinks polymer chains to form robust films with uniform and controlled thickness and complete surface coverage. Additional layers can be deposited. These films that bridge two- and three-dimensional materials show excellent stability and insulating properties.

Women with hypoactive sexual desire disorder compared to normal females: a functional magnetic resonance imaging study.

Lack of sexual interest is the most common sexual complaint among women. However, factors affecting sexual desire in women have rarely been studied. While the role of the brain in integrating the sensory, attentional, motivational, and motor aspects of sexual response is commonly acknowledged as important, little is known about specific patterns of brain activation and sexual interest or response, particularly among women. We compared 20 females with no history of sexual dysfunction (NHSD) to 16 women with hypoactive sexual desire disorder (HSDD) in a functional magnetic resonance imaging (fMRI) study that included assessment of subjective sexual arousal, peripheral sexual response using a vaginal photoplethysmograph (VPP), as well as brain activation across three time points. Video stimuli included erotic, sports, and relaxing segments. Subjective arousal to erotic stimuli was significantly greater in NHSD participants compared with HSDD. In the erotic-sports contrast, NHSD women showed significantly greater activation in the bilateral entorhinal cortex than HSDD women. In the same contrast, HSDD females demonstrated higher activation than NHSD females in the medial frontal gyrus (Brodmann area (BA) 10), right inferior frontal gyrus (BA 47) and bilateral putamen. There were no between group differences in VPP-correlated brain activation and peripheral sexual response was not significantly associated with either subjective sexual response or brain activation patterns. Findings were consistent across the three experimental sessions. The results suggest differences between women with NHSD and HSDD in encoding arousing stimuli, retrieval of past erotic experiences, or both. The findings of greater activation in BA 10 and BA 47 among women with HSDD suggest that this group allocated significantly more attention to monitoring and/or evaluating their responses than NHSD participants, which may interfere with normal sexual response.

Controlled loading of building blocks into temporary self-assembled scaffolds for directed assembly of organic nanostructures.

Using temporary self-assembled scaffolds to preorganize building blocks is a potentially powerful method for the synthesis of organic nanostructures with programmed shapes. We examined the underlying phenomena governing the loading of hydrophobic monomers into lipid bilayer interior and demonstrated successful control of the amount and ratio of loaded monomers. When excess styrene derivatives or acrylates were added to the aqueous solution of unilamellar liposomes made from saturated phospholipids, most loading occurs within the first few hours. Dynamic light scattering and transmission electron microscopy revealed no evidence of aggregation caused by monomers. Bilayers appeared to have a certain capacity for accommodating monomers. The total volume of loaded monomers is independent of monomer structure. X-ray scattering showed the increase in bilayer thickness consistent with loading monomers into bilayer interior. Loading kinetics is inversely proportional to the hydrophobicity and size of monomers. Loading and extraction kinetic data suggest that crossing the polar heads region is the rate limiting step. Consideration of loading kinetics and multiple equilibria are important for achieving reproducible monomer loading. The total amount of monomers loaded into the bilayer can be controlled by the loading time or length of hydrophobic lipid tails. The ratio of loaded monomers can be varied by changing the ratio of monomers used for loading or by the time-controlled replacement of a preloaded monomer. Understanding and controlling the loading of monomers into bilayers contributes to the directed assembly of organic nanostructures.

IGF-I expression is decreased in LIF-deficient mice after peripheral nerve injury.

We investigated the regulation of insulin-like growth factor 1 (IGF-1) expression after sciatic nerve crush using leukemia inhibitory factor (LIF)-deficient mice. One day post-crush, IGF-1 mRNA levels were lower in the LIF-deficient mouse nerve than in the wild type nerve. IGF-1 protein, analyzed by immunohistochemistry, was also decreased 1 day post-crush in LIF-deficient nerves relative to wild type nerves. By 3 days post-crush, IGF-1 immunoreactivity was induced in Schwann cells to equivalent levels in both types of nerve. After crush, IGF-1 expression was also found in mast cells, and these were initially decreased in the LIF-deficient mice. Thus, LIF appears to regulate IGF-1 expression in the peripheral nerve basally and early in the regeneration response in vivo.

Leukaemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro.

The cytokine leukaemia inhibitory factor (LIF) is up-regulated in glial cells after injury to the peripheral and central nervous systems. In addition, LIF is required for the changes in neuropeptide expression that normally occur when the axons of sympathetic and sensory neurons are transected. We investigated whether LIF is also necessary for the initial inflammatory response that follows mechanical injury to the sciatic nerve and cerebral cortex of adult mice. We find that inflammatory cell infiltration into crushed sciatic nerve is significantly slower in LIF knock-out (KO) mice compared with wild-type (WT) mice. Similarly, the microglial and astroglial responses to surgical injury of the cortex are significantly slower in LIF KO mice compared with WT mice. Consistent with these in vivo results, LIF is chemotactic for peritoneal macrophages in a microchamber culture assay. Thus, LIF is a key regulator of neural injury in vivo, where it is produced by glia and can act directly on neurons, glia and inflammatory cells. We also find that the initial inflammatory response to cortical injury is diminished in interleukin (IL)-6 KO mice. Surprisingly, however, the inflammatory response in LIF-IL-6 double KO mice is very similar to that of the single KO mice, suggesting that these cytokines may act in series rather than in parallel in this response.

Relationship with Family of Origin Scale (REFAMOS). Interrater reliability and associations with childhood experiences.

BACKGROUND: Family relationships in the transition from childhood to adult life may be important mediators of risk and resilience for adult psychopathology. AIMS: To develop a reliable and valid measure of the quality of relationships with each parent in young adults. METHOD: Interrater reliability of the Relationship with Family of Origin Scale (REFAMOS) was assessed from audio-taped interviews with 59 subjects. Age-related trends and associations with recalled childhood relationships were examined in survivors of childhood cancer and their controls (n = 178). RESULTS: Intraclass correlation coefficients were in the range 0.69-0.95, and kappa values 0.80-0.82. Indices of current closeness to mothers were negatively correlated with age of subject and positively correlated with recalled maternal care in childhood. Negative qualities in current relationships were correlated with recalled overprotection. CONCLUSIONS: The REFAMOS has good interrater reliability, and shows the predicted age-related trends in scores and associations with recalled childhood relationships.

Leukemia inhibitory factor is an anti-inflammatory and analgesic cytokine.

The mRNA for leukemia inhibitory factor (LIF), a neuroimmune signaling molecule, is elevated during skin inflammation produced by intraplantar injection of complete Freund's adjuvant (CFA). Moreover, although LIF knock-out mice display normal sensitivity to cutaneous mechanical and thermal stimulation compared with wild-type mice, the degree of CFA-induced inflammation in mice lacking LIF is enhanced in spatial extent, amplitude, cellular infiltrate, and interleukin (IL)-1beta and nerve growth factor (NGF) expression. Conversely, local injection of low doses of recombinant LIF diminishes mechanical and thermal hypersensitivity as well as the IL-1beta and NGF expression induced by CFA. These data show that upregulation of LIF during peripheral inflammation serves a key, early anti-inflammatory role and that exogenous LIF can reduce inflammatory hyperalgesia.

Leukemia inhibitory factor is expressed in astrocytes following cortical brain injury.

The neuropoietic cytokine leukemia inhibitory factor (LIF) can act as a trophic factor, enhancing neuronal survival, and as a differentiation factor altering neuronal and glial gene expression. LIF also plays a role in the response to injury of the peripheral nervous system, as indicated by an increase in the amount of its mRNA within nonneuronal injury response in LIF knock-out mice. To determine if LIF is regulated after injury to the central nervous system, we surgically lesioned the cortex in adult rat brain. Using a quantitative RNAse protection assay, we find that LIF mRNA increases 30-fold following injury. The amount of this transcript goes up within 6 h after injury, reaches a peak at 24 h and returns to baseline by 7 days postlesion. In situ hybridization analysis reveals LIF transcript-containing cells scattered throughout the ipsilateral cortex close, but not immediately adjacent to the lesion site. Double-labeling with a variety of antibodies reveals that LIF mRNA is induced in GFAP-positive astrocytes as well as in a small number of microglial cells. The striking induction of LIF transcripts in glia suggests that this cytokine may play a key injury-response role in the CNS as it does in the PNS, where LIF has been demonstrated to regulate neuropeptide expression both in vivo and in vitro.

Normatology: a review and commentary with reference to abortion and physician-assisted suicide.

PIP: This article opens with a review of the concept of "normatology," which was developed by Sabshin and Offer in four books published over a period of 30 years. Normatology seeks to produce an "operational definition of normality and health" over the life cycle. Such a definition can be used as a guideline in the deliver of health care. The importance of this field of study is highlighted when considering issues such as abortion or physician-assisted suicide. Fortunately, the proclivity of Americans to conduct public opinion polls helps researchers determine what is considered "normal" at any given time. Gallup Polls, which have posed the same question about the legality of abortion from 1975 to 1995, indicate that about half of all Americans continuously occupy the middle ground on this issue despite a somewhat liberalizing trend. In general, public opinion holds that it is normal to want to avoid giving birth to a damaged child, to place the mother's health and safety above that of the fetus, and to terminate a pregnancy resulting from rape. It is less normal to abort a healthy fetus on demand. Thus, abortion will likely continue to be a source of controversy and confusion in our society and among psychiatric patients. In comparison, psychiatrists express attitudes about abortion that are more liberal than normal. In the case of physician-assisted suicide, public approval has increased since 1950 as scientific advancements have facilitated the prolongation of unproductive and painful life. If legalized, physician-assisted suicide may depend upon psychiatric assessment of an absence of mental disease. Such an assessment is required in the Northern Territory of Australia, where voluntary euthanasia is legal, but not in the Netherlands, where it is government-regulated. Psychiatrists must understand public opinion in order to influence it or deal with it competently.^ieng

Family relationships in survivors of childhood cancer: resource or restraint?

Family relationships may be an important mediator between childhood cancer and psychosocial adjustment in adult life. This paper focuses on the developmental task of the transition from adolescence to adulthood, with regard to the cancer survivor's family relationships. Theories, concepts and findings relevant to this specific focus are considered in relation to whether these relationships may prove a resource or a restraint in this process. Potential aspects of family relationships that may be of interest to research are discussed, and clinical implications drawn.

Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia.

The neuropoietic cytokine cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) acts as a trophic factor, enhancing neuronal survival, and as a differentiation factor, altering neuronal gene expression. There is also evidence that its plays a role in the response of adult neural tissue to injury. We have examined this possibility further in rats by analyzing changes in the levels of mRNAs for CDF/LIF and its two receptor subunits in response to peripheral nerve damage in culture and in vivo. Using a quantitative RNase protection assay, we find that CDF/LIF mRNA increases dramatically (176-fold) in adult, but not neonatal, sympathetic ganglia and in adult dorsal root ganglia and sciatic nerve after organ culture for 24 hr. This mRNA is clearly detectable by in situ hybridization only in the nonneuronal cells of these structures. When the sciatic nerve is transected in vivo, CDF/LIF mRNA increases significantly in the regions immediately proximal and distal to the lesion site. The mRNA for the ligand binding subunit of the CDF/LIF receptor complex decreases somewhat upon culture and nerve section. The dramatic rise in CDF/LIF mRNA after nerve injury is further evidence that this cytokine is involved in the response to damage, a function that overlaps with its postulated role in wounding or infection in several nonneural tissues.

Coronavirus mRNA transcription: UV light transcriptional mapping studies suggest an early requirement for a genomic-length template.

Mouse hepatitis virus (MHV) synthesizes seven to eight mRNAs, each of which contains a leader RNA derived from the 5' end of the genome. To understand the mechanism of synthesis of these mRNAs, we studied how the synthesis of each mRNA was affected by UV irradiation at different time points after infection. When MHV-infected cells were UV irradiated at a late time in infection (5 h postinfection), the syntheses of the various mRNAs were inhibited to different extents in proportion to the sizes of the mRNAs. Analysis of the UV inactivation kinetics revealed that the UV target size of each mRNA was equivalent to its own physical size. In contrast, when cells were irradiated at 2.5 or 3 h postinfection, there appeared to be two different kinetics of inhibition of mRNA synthesis: the synthesis of every mRNA was inhibited to the same extent by a small UV dose, but the remaining mRNA synthesis was inhibited by additional UV doses at different rates for different mRNAs in proportion to RNA size. The analysis of the UV inactivation kinetics indicated that the UV target sizes for the majority of mRNAs were equivalent to that of the genomic-size RNA early in the infection. These results suggest that MHV mRNA synthesis requires the presence of a genomic-length RNA template at least early in the infection. In contrast, later in the infection, the sizes of the templates used for mRNA synthesis were equivalent to the physical sizes of each mRNA. The possibility that the genomic-length RNA required early in the infection was used only for the synthesis of a polymerase rather than as a template for mRNA synthesis was ruled out by examining the UV sensitivity of a defective interfering (DI) RNA. We found that the UV target size for the DI RNA early in infection was much smaller than that for mRNAs 6 and 7, which are approximately equal to or smaller in size than the DI RNA. This result indicates that even though DI RNA and viral mRNAs are synthesized by the same polymerase, mRNAs are synthesized from a larger (genomic-length) template. We conclude that a genomic-length RNA template is required for MHV subgenomic mRNA synthesis at least early in infection. Several transcription models are proposed.

Random nature of coronavirus RNA recombination in the absence of selection pressure.

RNA-RNA recombination is thought to occur preferentially at certain selected sites and in only a few RNA viruses; the mechanism for these restrictions is unknown. In this paper we report the development of a recombination assay for coronavirus, using polymerase chain reaction, in the absence of selection pressure. Our results showed that within a 1-kb region of the peplomer gene, RNA recombination occurred at almost every potential crossover site. Thus, coronavirus RNA recombination appears to be more random than previously realized. However, after serial passages of the recombinant viruses in tissue culture, the recombination sites among the progeny viruses became clustered in the region which contains the previously reported "hot spot" for coronavirus recombination. These results suggest that RNA recombination is common and random in nature, but only certain recombinants can be selected. Thus, the presence of recombinational "hot spots" for coronavirus or other RNA viruses most likely resulted from selection of certain recombinant viruses and not restriction on the occurrence of RNA recombination. The failure to detect recombinants in other RNA viruses may therefore be due to unfavorable properties of recombinant viruses. This approach can be used to detect recombinants in these viruses.

Heterogeneity of gene expression of the hemagglutinin-esterase (HE) protein of murine coronaviruses.

The hemagglutinin-esterase (HE) membrane glycoprotein is present only in some members of the coronavirus family, including some strains of mouse hepatitis virus (MHV). In the JHM strain of MHV, expression of the HE gene is variable and corresponds to the number of copies of a UCUAA pentanucleotide sequence present at the 3'-end of the leader RNA. This copy number varies among MHV strains, depending on their passage history. The JHM isolates with two copies of UCUAA in their leader RNA showed a high level of HE expression, whereas the JHM isolate with three copies had a low-level expression. In this study, the analysis of HE gene expression was extended to other MHV strains. The synthesis of HE mRNA in these viruses also correlates with the copy number of UCUAA in the leader RNA and the particular intergenic sequence preceding the HE gene. In one MHV strain, MHV-1, no detectable HE mRNA was synthesized, despite the presence of a proper transcription initiation signal. This lack of HE mRNA expression was consistent with a leader RNA containing three UCUAA copies. However, mutations and deletions within the coding region of the MHV-1 HE gene have generated a stretch of sequence which resembled the transcriptional initiation motif, and was shown to initiate the synthesis of a novel smaller mRNA. These findings strengthened the theory that interactions between leader RNA and transcriptional initiation sequences regulate MHV subgenomic mRNA transcription. Sequence analysis revealed that most MHV strains, through extensive mutations, deletions, or insertions, have lost the complete HE open reading frame, thus turning HE into a pseudogene. This high degree of variation is unusual as the other three structural proteins (spike, membrane, and nucleocapsid) are well-maintained. In contrast to bovine coronavirus, which apparently requires HE for viral replication, the HE protein in MHV may be only an accessory protein which is not necessary for viral replication. JHM and MHV-S, however, have preserved the expression of HE protein.

Localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus JHM.

The intracellular RNA of two neurotropic variants of the JHM strain of mouse hepatitis virus (MHV) independently isolated from the brain and spinal cord of an infected Wistar Furth rat were compared with that of the parental virus. The mRNAs corresponding to the genes encoding the peplomer (S) and the hemagglutinin-esterase (HE) proteins of the variant viruses were found to be smaller in size. The possible sequence changes were studied by oligonucleotide fingerprinting and direct RNA sequencing. Both variants have a large deletion of 246 amino acids in the carboxy-terminal end of the HE protein. However, this truncated protein was not detected in the infected cells, suggesting either a translational regulation or rapid degradation of the truncated protein in these cells. The variant virus isolated from the spinal cord has a second deletion of 147 amino acids in the amino-terminal half of the S protein. This deletion site corresponds to a hypervariable region where deletions have been frequently noted among MHV variants with different biological properties. These findings suggest that the changes in pathogenic properties of the two neural isolates are associated with drastic alterations of the viral structural glycoproteins.

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris.

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.

A clustering of RNA recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus.

Coronaviruses undergo RNA recombination at a very high frequency. To understand the mechanism of recombination in murine coronavirus, we have performed RNA sequencing of viral genomic RNA to determine the precise sites of recombination in a series of recombinants which have crossovers within the gene encoding the peplomer protein. We found that all of the recombination sites are clustered within a region of 278 nucleotides in the 5'-half of the gene. This region in which all of the crossovers occurred represents a small fraction of the distance between the two selection markers used for the isolation of these recombinant viruses. This result suggests that this region may be a preferred site for RNA recombination. The crossover sites are located within and immediately adjacent to a hypervariable area of the gene. This area has undergone deletions of varying sizes in several virus strains which have been passaged either in vivo or in vitro. These results suggest that a similar RNA structure may be involved in the occurrence of both recombination and deletion events.

The use and effects of vital fluorescent dyes: observation of motor nerve terminals and satellite cells in living frog muscles.

Several different fluorescent mitochondrial dyes were tested as vital stains for motor nerve terminals and other cells in frog skeletal muscles. It was found that 3,3' diethyloxadicarbocyanine iodide and 4-(4-diethylaminostyryl)-N-methylpyridinium iodide were most useful. Both dyes labelled motor nerve terminals with high reliability. Electrophysiological and morphological control experiments showed that these dyes could be used to repeatedly observe neuromuscular junctions in living animals without affecting synaptic growth or remodelling. The importance of appropriate controls was emphasized by the finding that illumination, if excessively intense or prolonged, can cause physiological and structural damage to nerve terminals. Additional observations indicated that these dyes may be useful for determining the mitochondrial content, and therefore oxidative capacity, of living muscle fibres. It was also found that the fluorescent dyes labelled cells identified as muscle satellite cells, and that these myoblast precursors could be visualized in fixed whole mounts with a nitroblue tetrazolium stain. Both methods were used to study reactive cells that were closely associated with muscle fibres in lesioned muscles. Mitochondrial dyes also labelled the microvasculature, associated axons and other cells.

Repeated, in vivo observation of frog neuromuscular junctions: remodelling involves concurrent growth and retraction.

The fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide was used as a vital stain to study remodelling of motor nerve terminals in sartorius muscles of living frogs (Rana pipiens). Identified terminals were observed twice in vivo at intervals of 87-192 days. After the second observation, muscles were fixed and stained with the nitroblue tetrazolium method for nerve terminals and with cholinesterase stain. Observations were made of 243 junctions in 26 frogs. Most nerve terminals grew during the observation interval, with an average increase in total terminal length of 29%. This growth involved substantial remodelling. Within single junctions, the change in size was the net result of differing degrees of growth or shrinkage in individual nerve terminal branches. At least one new terminal branch appeared in 25% of the junctions. Terminal retraction was also common, with branch shortening seen in 60% of junctions and the complete disappearance of a branch in 12%. In one case the original axonal input retracted completely and the junction was partially reinnervated by a terminal sprout from a junction on an adjacent fibre. Some discrepancies between histological and in vivo observations of remodelling were noted. These observations confirm that frog neuromuscular junctions are highly dynamic synapses, subject to profound structural remodelling throughout adult life.