Brainstem Circuitry Regulating Phasic Activation of Trigeminal Motoneurons during REM Sleep

Background

Rapid eye movement sleep (REMS) is characterized by activation of the cortical and hippocampal electroencephalogram (EEG) and atonia of non-respiratory muscles with superimposed phasic activity or twitching, particularly of cranial muscles such as those of the eye, tongue, face and jaw. While phasic activity is a characteristic feature of REMS, the neural substrates driving this activity remain unresolved. Here we investigated the neural circuits underlying masseter (jaw) phasic activity during REMS. The trigeminal motor nucleus (Mo5), which controls masseter motor function, receives glutamatergic inputs mainly from the parvocellular reticular formation (PCRt), but also from the adjacent paramedian reticular area (PMnR). On the other hand, the Mo5 and PCRt do not receive direct input from the sublaterodorsal (SLD) nucleus, a brainstem region critical for REMS atonia of postural muscles. We hypothesized that the PCRt-PMnR, but not the SLD, regulates masseter phasic activity during REMS.

Methodology/Principal Findings

To test our hypothesis, we measured masseter electromyogram (EMG), neck muscle EMG, electrooculogram (EOG) and EEG in rats with cell-body specific lesions of the SLD, PMnR, and PCRt. Bilateral lesions of the PMnR and rostral PCRt (rPCRt), but not the caudal PCRt or SLD, reduced and eliminated REMS phasic activity of the masseter, respectively. Lesions of the PMnR and rPCRt did not, however, alter the neck EMG or EOG. To determine if rPCRt neurons use glutamate to control masseter phasic movements, we selectively blocked glutamate release by rPCRt neurons using a Cre-lox mouse system. Genetic disruption of glutamate neurotransmission by rPCRt neurons blocked masseter phasic activity during REMS.

Conclusions/Significance

These results indicate that (1) premotor glutamatergic neurons in the medullary rPCRt and PMnR are involved in generating phasic activity in the masseter muscles, but not phasic eye movements, during REMS; and (2) separate brainstem neural circuits control postural and cranial muscle phasic activity during REMS.     

Global responses of Aliivibrio salmonicida to hydrogen peroxide as revealed by microarray analysis

Aliivibrio salmonicida causes "cold-water vibriosis" (or "Hitra disease") in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and microarray to study how A. salmonicida responds to hydrogen peroxide (H(2)O(2)). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H(2)O(2) response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis and microarray analysis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down regulated genes are distributed among all functional gene categories, but a striking number of ≥2 fold up regulated genes encode proteins involved in detoxification and DNA repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADPH. Our predictions and -omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.     

Distribution of the UV filter 3-benzylidene camphor in rat following topical application

A straightforward analytical method for determination of 3-benzylidene camphor (3-BC) in rat adipose tissue, brain, liver, muscle, plasma and testis following topical application was developed and validated. Three exposure levels (60, 180 and 540 mg kg(-1) day(-1)) were tested for 65 days in male Sprague-Dawley rats (24 days postnatal). Sample preparation involving homogenization and n-heptane or methanol extraction of the tissue was applied before injection into the LC-ESI-MS-MS system. The response was linear from 2 to 100 microg l(-1) for the qualifier and the quantifier MRM transitions (R(2) (quantifier) > 0.994). Detection limit of the method corresponded to 0.005 microg g(-1) tissue and 12.5 microg l(-1) plasma, respectively. Recovery was determined for all tissues (adipose tissue: 40%; all other tissues: 80-100%) at three individual levels. 3-(4-Methyl benzylidene camphor) (4-MBC) was used throughout the study as internal standard. 3-Benzylidene camphor was detected in all tissues at all exposure levels at concentrations between 0.05 microg g(-1) (liver) and 36 microg g(-1) (adipose tissue) and in plasma at 16-89 microg l(-1). The method allowed for the quantification of 3-benzylidene camphor in all tested tissues following topical application. Furthermore, it was shown that 3-benzylidene camphor can be found in various tissues in the rat following topical application. These findings may suggest that following use of 3-benzylidene camphor containing sunscreen, similar disposition and distribution may occur in humans.     

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.     

Recrafting the neighbor-joining method

Background  

The neighbor-joining method by Saitou and Nei is a widely used method for constructing phylogenetic trees. The formulation of the method gives rise to a canonical Θ(n ³) algorithm upon which all existing implementations are based.     

Synthesis of twisted intercalating nucleic acids possessing acridine derivatives. Thermal stability studies

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5'-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.     

Differential regulation of IL-6 and TNF-alpha via calcineurin in human skeletal muscle cells

Interleukin-6 increases in skeletal muscle during exercise, and evidence points to Ca2+ as an initiator of IL-6 production. However, the signalling pathway whereby this occurs is unknown. One candidate for Ca2+ -mediated IL-6 induction is calcineurin, an activator of NF-AT. Here we investigated whether skeletal myocytes produce IL-6 in a Ca2+/calcineurin-dependent manner, and whether TNF-alpha, an inducer of IL-6, is affected by these stimuli. Human skeletal muscle cell cultures were stimulated with ionomycin time-and dose-dependently to elevate intracellular Ca2+ levels, with or without addition of cyclosporin A (CSA); a calcineurin inhibitor. mRNA was extracted from myocytes and analysed for IL-6 and TNF-alpha gene expression. IL-6 mRNA increased time- and dose-dependently with ionomycin stimulation, an effect that was blunted by approximately 75% in the presence of CSA. In contrast, TNF-alpha gene expression was decreased by approximately 70% in response to ionomycin treatment, but increased in response to addition of CSA. These data demonstrate that IL-6 and TNF-alpha are regulated differentially in skeletal muscle cells in response to a Ca2+ stimulus. Blocking the calcineurin pathway resulted in inhibition of the IL-6 response to ionomycin, whereas TNF-alpha increased by addition of CSA, further indicating a differential regulation of IL-6 and TNF-alpha in human skeletal myocytes.     

Environmental risk factors differ between rheumatoid arthritis with and without auto-antibodies against cyclic citrullinated peptides

The aim of this study was to evaluate new and previously hypothesised non-genetic risk factors for serologic subtypes of rheumatoid arthritis (RA) defined by the presence or absence of auto-antibodies to cyclic citrullinated peptides (CCP). In a national case-control study, we included 515 patients recently diagnosed with RA according to the American College of Rheumatology 1987 classification criteria and 769 gender- and age-matched population controls. Telephone interviews provided information about non-genetic exposures, and serum samples for patients were tested for anti-CCP-antibodies. Associations between exposure variables and risk of anti-CCP-positive and anti-CCP-negative RA were evaluated using logistic regression. A series of RA subtype-specific risk factors were identified. Tobacco smoking (odds ratio [OR] = 1.65; 95% confidence interval: 1.03–2.64, for >20 versus 0 pack-years) was selectively associated with risk of anti-CCP-positive RA, whereas alcohol consumption exhibited an inverse dose-response association with this RA subtype (OR = 1.98, 1.22–3.19, for 0 versus >0–5 drinks per week). Furthermore, coffee consumption (OR = 2.18; 1.07–4.42, for >10 versus 0 cups per day), ever use of oral contraceptives (OR = 1.65; 1.06–2.57) and having a first-degree relative with schizophrenia (OR = 4.18; 1.54–11.3) appeared more strongly associated with risk of anti-CCP-positive RA. Obesity was selectively associated with risk of anti-CCP-negative RA, with obese individuals being at more than 3-fold increased risk of this subtype compared with normal-weight individuals (OR = 3.45; 1.73–6.87). Age at menarche was the only examined factor that was significantly associated with both serologic subtypes of RA (p-trends = 0.01); women with menarche at age ≥ 15 years had about twice the risk of either RA subtype compared with women with menarche at age ≤ 12 years. Major differences in risk factor profiles suggest distinct etiologies for anti-CCP-positive and anti-CCP-negative RA.     

The fungal metabolite culmorin and related compounds

This paper reviews the toxicology of culmorins, a family of compounds found in grains contaminated by Fusarium graminearum and related fungi. We include the results of an Ames test and studies based on Quantitative Structure-Activity Relationships. Culmorin has low toxicity in several in vitro assays and in one study in swine and is Ames test negative. Culmorin is moderately antifungal. QSAR analysis suggested that the plant compound longifolene was similar. Longifolene is a GRAS compound used in cosmetics and is also moderately antifungal.     

Directed evolution of a fungal peroxidase

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.