Short-term effects of wake- and bright light therapy on sleep in depressed youth

Chronotherapeutics are well established for the treatment of depression and associated sleeping problems in adults. However, effects are still understudied in adolescents. Two pilot studies highlighted the crucial role of sleep when it comes to the treatment of depression, by means of chronotherapeutics, in adolescents. The aim of the present study was to investigate the role of adjunctive wake therapy (WT) in addition to bright light therapy (BLT) with respect to sleep behaviors. In the present study, 62 depressed inpatients (aged 13–18 years; diagnosed with Beck Depression Inventory Revision) were randomly assigned to two groups: BLT only (BLT-group) and a combination of BLT and WT (COMB-group). After one night of WT adolescents in the COMB-group revealed longer sleep durations, time in bed, advanced sleep onset, less wakes during night and an improved sleep efficiency. However, one night of WT plus BLT had no additional effect on sleep parameters compared with BLT-group in the long run. Therefore, future studies should assess whether more nights of WT might lead to more sustainable effects.     

Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.     

Propensity to Form Amyloid Fibrils Is Encoded as Excitations in the Free Energy Landscape of Monomeric Proteins

Protein aggregation, linked to many of diseases, is initiated when monomers access rogue conformations that are poised to form amyloid fibrils. We show, using simulations of src SH3 domain, that mechanical force enhances the population of the aggregation-prone (N^?) states, which are rarely populated under force free native conditions but are encoded in the spectrum of native fluctuations. The folding phase diagrams of SH3 as a function of denaturant concentration ([C]), mechanical force (f), and temperature exhibit an apparent two-state behavior, without revealing the presence of the elusive N^? states. Interestingly, the phase boundaries separating the folded and unfolded states at all [C] and f fall on a master curve, which can be quantitatively described using an analogy to superconductors in a magnetic field. The free energy profiles as a function of the molecular extension (R), which are accessible in pulling experiments, (R), reveal the presence of a native-like N^? with a disordered solvent-exposed amino-terminal β-strand. The structure of the N^? state is identical with that found in Fyn SH3 by NMR dispersion experiments. We show that the timescale for fibril formation can be estimated from the population of the N^? state, determined by the free energy gap separating the native structure and the N^? state, a finding that can be used to assess fibril forming tendencies of proteins. The structures of the N^? state are used to show that oligomer formation and likely route to fibrils occur by a domain-swap mechanism in SH3 domain.     

Beta-Blockers,Left and Right Ventricular Function,and In-Vivo Calcium Influx in Muscular Dystrophy Cardiomyopathy

Beta-blockers are used to treat acquired heart failure in adults, though their role in early muscular dystrophy cardiomyopathy is unclear. We treated 2 different dystrophic mouse models which have an associated cardiomyopathy (mdx: model for Duchenne Muscular Dystrophy, and Sgcd-/-: model for limb girdle muscular dystrophy type 2F) and wild type controls (C57 Bl10) with the beta blocker metoprolol or placebo for 8 weeks at an early stage in the development of the cardiomyopathy. Left and right ventricular function was assessed with cardiac magnetic resonance imaging (MRI) and in-vivo myocardial calcium influx with manganese enhanced MRI. In the mdx mice at baseline there was reduced stroke volume, cardiac index, and end-diastolic volume with preserved left ventricular ejection fraction. These abnormalities were no longer evident after treatment with beta-blockers. Right ventricular ejection fraction was reduced and right ventricular end-systolic volume increased in the mdx mice. With metoprolol there was an increase in right ventricular end-diastolic and end-systolic volumes. Left and right ventricular function was normal in the Sgcd-/- mice. Metroprolol had no significant effects on left and right ventricular function in these mice, though heart/body weight ratios increased after treatment. In-vivo myocardial calcium influx with MEMRI was significantly elevated in both models, though metoprolol had no significant effects on either. In conclusion, metoprolol treatment at an early stage in the development of cardiomyopathy has deleterious effects on right ventricular function in mdx mice and in both models no effect on increased in-vivo calcium influx. This suggests that clinical trials need to carefully monitor not just left ventricular function but also right ventricular function and other aspects of myocardial metabolism.     

CHARACTERIZATION OF TISSUE CULTURE INITIATION AND PLANT REGENERATION IN SPOROBOLUS VIRGINICUS (GRAMINEAE)

Tissue cultures of the halophytic saltmarsh grass Sporobolus virginicus were initiated from unemerged immature inflorescence tissue. Typical graminaceous embryogenic and nonembryogenic callus and cell types were noted. Embryogenic callus was compact golden yellow. Histological evidence indicated that proliferation of the ovary tissue of the immature pistil was the source for embryogenic callus. Plants regenerated after first reducing and then eliminating auxin from the culture medium. Regeneration was observed both through the concerted development of bipolar meristems from somatic embryos and by the formation of multiple shoot meristems that were either connected through callus tissue to root meristems or which later adventitiously rooted. The main mode of regeneration appeared to be somatic embryogenesis with additional multiple shoot formation probably due to precocious germination of somatic embryos. Plants recovered from culture were acclimated to soil, grown up in a greenhouse, and planted in field plots with saline irrigation to ensure stability of salt tolerance.     

Use of synthetic peptides to establish anti-human acetylcholine receptor CD4+ cell lines from myasthenia gravis patients

Acetylcholine receptor-(AcChR) specific T cell lines were propagated from the PBL of six myasthenia gravis (MG) patients by the use of a pool of synthetic peptides (alpha-pool) corresponding to the complete sequence of the alpha-subunit of the human AcChR. All the lines had CD4+ phenotype and strongly recognized the alpha-pool. Four lines cross-reacted with native Torpedo AcChR. Five lines showed, at certain stages of their propagation, some degree of reactivity to autologous or DR-matched APC. One of the CD4+ T lines was challenged with each one of the peptides present in the alpha-pool. Several peptides, corresponding to the sequence segments 48-67, 101-120, 304-322, 320-337, and 419-437 of the human alpha-subunit were recognized, indicating that different epitopes and multiple T cell clones are involved in the recognition of the autoantigen in MG. Human AcChR-specific CD4+ T cell lines will be useful to identify the repertoire of epitopes recognized by the autoreactive Th cells in MG, to investigate the TCR genes utilized by autoreactive Th cells and to develop specific immunosuppressive treatments using anti-T cell vaccination.     

Comparison of PCR and cell culture for detection of enteroviruses in sludge-amended field soils and determination of their transport.

PCR and cell culture assays for enteroviruses were conducted on soil samples collected from an experimental farm that had received mesophilic anaerobically digested sludge for the past 7 years. Of 24 samples assayed, 21 samples were positive by PCR, implying that at least some viral nucleic acid sequences remained intact. However, these viral particles were unable to infect the Buffalo Green Monkey cell line used in subsequent cell culture assays. It is significant that positive PCR detection of nucleic acid sequences occurred even though the most recent sludge application was 3 months prior to soil sampling. Viral nucleic acid sequences were detected by PCR at points vertically and laterally displaced from sludge injections, illustrating significant transport of viruses. Rainfall and irrigation events may have contributed to viral transport.     

Neuronal Regulation of Interleukin 6 Secretion in Murine Spleen: Adrenergic and Opioidergic Control

Abstract: The PNS was anticipated to be involved in the modulation of immune responses. To study aspects of this neuronal-immune communication, a recently developed tissue slice method was used to study the effects of adrenergic and opioidergic transmitters on interleukin 6 (IL-6) secretion in the spleen. The α₂-adrenergic agonist p -aminoclonidine (10⁻⁷ M ) inhibited IL-6 secretion (control vs. p -aminoclonidine, 100.0 ± 4.76 vs. 59.3 ± 6.6% of control values; p < 0.001). The α₁-adrenergic agonist methoxamine (10⁻⁸ M ) also inhibited IL-6 secretion (100.0 ± 4.8 vs. 71.5 ± 3.8%; p < 0.001). The endogenous opioids β-endorphin (10⁻¹⁰ M ), methionine-enkephalin (10⁻⁹ M ), and leucine-enkephalin (10⁻⁹ M ) inhibited IL-6 secretion as well ( p = 0.0051, p = 0.0337, and p = 0.0226, respectively). Electrical stimulation of spleen slices inhibited IL-6 secretion (100.0 ± 4.3 vs. 56.7 ± 4.6% of control values; p < 0.001). The involvement of α-adrenergic and opioidergic molecules in this electrically induced inhibition was shown by the use of antagonists. Electrical inhibition of IL-6 secretion was attenuated by phentolamine (10⁻⁷ M ; p = 0.0345), by naloxone (10⁻⁶ M ; p = 0.0046), by cyprodime (10⁻⁸ M ; p = 0.0014), and by the combination of cyprodime (10⁻⁷ M ) plus phentolamine (10⁻⁸ M ; p < 0.0001). We conclude from the complementary studies that the inhibition of IL-6 secretion induced by electrical pulses was mostly mediated by α-adrenergic and μ-opioidergic endogenous transmitters.     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.