Low back pain scores correlate with the cytokine mRNA level in lumbar disc biopsies: a study of inflammatory markers in patients undergoing lumbar spinal fusion.

PURPOSE: The molecular mechanism behind pain in degenerative disc disease (DDD) and chronic low back pain (LBP) patients is largely unknown. This present study examines the association of LBP and disability to mediators of the inflammatory cascade, as indexed by mRNA gene expression of pro-inflammatory cytokine markers in the intervertebral disc (IVD). METHODS: Biopsies of the annulus fibrosus (AF) and the nucleus pulposes (NP) from patients with DDD undergoing 1-2 level fusion surgery at L4/L5 or L5/S1 were obtained from total of 34 patients [9 M, 25 F] with average age of 53 [32-63]. The mRNA expression of TNF-α, IL-1ß, and IL-6 in the AF and NP was analyzed using quantitative real-time polymerase chain reaction (RT-qPCR), and the expression level of these markers was correlated to the visual analogue scale (VAS) and Oswestry Disability Index (ODI) scores (0-100) for pain and disability. RESULTS: We report a statistically significant positive correlation between pain intensity (VAS score) and the expression of TNF-α in both the AF (r = 0.54, p = 0.001) and NP (r = 0.40, p = 0.02), similarly with IL-1ß in AF (r = 0.37, p = 0.02) and IL-6 in NP (r = 0.40, p = 0.02). In addition, we found significant positive correlation observed between disability score (ODI) and expression of IL-6 in both AF (r = 0.36, p = 0.03) and NP (r = 0.41, p = 0.01). CONCLUSION: We conclude that the intensity of LBP and disability is associated with the level of inflammation in the disc.

Polarization-insensitive silicon nitride Mach-Zehnder lattice wavelength demultiplexers for CWDM in the O-band.

Polarization-insensitive silicon nitride (SiN) 4-channel wavelength (de)multiplexers based on Mach-Zehnder interferometer lattice filters for coarse wavelength division multiplexing (CWDM) in the O-band are demonstrated in a SiN-on-silicon photonic platform. For the best-performing device, the insertion loss was < 2.8 dB, the inter-channel crosstalk was < -11.5 dB for a polarization scrambled input, and the passband shift between the orthogonal polarizations was < 1.5 nm. Across the 200mm wafer, the die-averaged insertion loss and maximum crosstalk were 3.1 dB and -10.6 dB, respectively. The higher-than-expected crosstalk was due to dimensional variations. This work shows the potential of SiN photonic circuits for CWDM without polarization diversity.

Recombinant PAPP-A resistant insulin-like growth factor binding protein 4 (dBP4) inhibits angiogenesis and metastasis in a murine model of breast cancer.

BACKGROUND: The Insulin-like growth factor (IGF) pathway plays a role in tumour development and progression. In vivo, IGF1 activity is regulated by the IGF binding proteins (IGFBPs). IGFBP4 inhibits the activity of IGF1 but proteolytic cleavage by pregnancy-associated plasma protein-A (PAPP-A) releases active IGF1. A modified IGFBP4, dBP4, which was resistant to PAPP-A cleavage but retained IGF1 binding capacity, was engineered, expressed in Human Embryonic Kidney (HEK) 293 cells and purified. This study examined the effects of dBP4 on IGF1-induced cell migration, invasion and angiogenesis in vitro. The effect of intra-tumour injections of dBP4 on tumour angiogenesis and metastasis was examined using the 4T1.2luc orthotopic model of breast cancer. METHODS: PAPP-A resistance and IGF binding capacity of dBP4 were characterized by Western blot and surface plasmon resonance, respectively. 4T1.2luc are mouse mammary adenocarcinoma cells transfected with luciferase to allow in vivo imaging. The effect of dBP4 on IGF1-induced Akt activation in 4T1.2luc cells was assessed by Western blot. Cell migration and invasion assays were performed using 4T1.2luc cells. Angiokit™ assays and Matrigel® implants were used to assess the effects of dBP4 on angiogenesis in vitro and in vivo, respectively. An orthotopic breast cancer model - 4T1.2luc cells implanted in the mammary fat pad of BALB/c mice - was used to assess the effect of intra tumour injection of purified dBP4 on tumour angiogenesis and metastasis. Tumour growth and lung metastasis were examined by in vivo imaging and tumour angiogenesis was evaluated by CD31 immunohistochemistry. RESULTS: Our engineered, PAPP-A resistant IGFBP4 (dBP4) retained IGF1 binding capacity and inhibited IGF1 activation of Akt as well as IGF1-induced migration and invasion by 4T1.2 mammary adenocarcinoma cells. dBP4 inhibited IGF1-induced angiogenesis in vitro and in Matrigel implants in vivo. Direct intra-tumour injection of soluble dBP4 reduced angiogenesis in 4T1.2 luc mammary tumours tumour and reduced lung metastasis. CONCLUSION: A PAPP-A resistant IGFBP4, dBP4, inhibits angiogenesis and metastasis in 4T1.2 mammary fat pad tumours. This study highlights the therapeutic potential of dBP4 as an approach to block the tumour-promoting actions of IGF1.

Defective RNA sensing by RIG-I in severe influenza virus infection.

Influenza virus infection causes worldwide seasonal epidemics. Although influenza is usually a mild disease, a minority of patients experience very severe fulminating disease courses. Previous studies have demonstrated a role for type I interferon (IFN) in anti-viral responses during influenza. So far, however, IFN regulatory factor (IRF)7 deficiency is the only genetic cause of severe influenza described in humans. In this study we present a patient with severe influenza A virus (IAV) H1N1 infection during the 2009 swine flu pandemic. By whole exome sequencing we identified two variants, p.R71H and p.P885S, located in the caspase activation and recruitment domain (CARD) and RNA binding domains, respectively, of DExD/H-box helicase 58 (DDX58) encoding the RNA sensor retinoic acid inducible gene 1 (RIG-I). These variants significantly impair the signalling activity of RIG-I. Similarly, patient cells demonstrate decreased antiviral responses to RIG-I ligands as well as increased proinflammatory responses to IAV, suggesting dysregulation of the innate immune response with increased immunopathology. We suggest that these RIG-I variants may have contributed to severe influenza in this patient and advocate that RIG-I variants should be sought in future studies of genetic factors influencing single-stranded RNA virus infections.

IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP.

Innate immune activation by macrophages is an essential part of host defence against infection. Cytosolic recognition of microbial DNA in macrophages leads to induction of interferons and cytokines through activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Other host factors, including interferon-gamma inducible factor 16 (IFI16), have been proposed to contribute to immune activation by DNA. However, their relation to the cGAS-STING pathway is not clear. Here, we show that IFI16 functions in the cGAS-STING pathway on two distinct levels. Depletion of IFI16 in macrophages impairs cGAMP production on DNA stimulation, whereas overexpression of IFI16 amplifies the function of cGAS. Furthermore, IFI16 is vital for the downstream signalling stimulated by cGAMP, facilitating recruitment and activation of TANK-binding kinase 1 in STING complex. Collectively, our results suggest that IFI16 is essential for efficient sensing and signalling upon DNA challenge in macrophages to promote interferons and antiviral responses.

Positive allosteric modulators of the α7 nicotinic acetylcholine receptor potentiate glutamate release in the prefrontal cortex of freely-moving rats.

Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (α7nAChRs) exhibit pro-cognitive effects in animal models of schizophrenia and are targets for the discovery of cognition-enhancing drugs. However, little is known about their in vivo mechanism of action because such studies have been performed in vitro. Here we test the hypothesis that PAMs' potentiation of glutamate release in prefrontal cortex depends upon the level of endogenous cholinergic activity. NMDA stimulation of the nucleus accumbens shell (0.05-0.30 µg in 0.5 µL) increased extracellular choline (0.87 ± 0.15 - 1.73 ± 0.31 µM) and glutamate (0.15 µg, 3.79 ± 0.87 µM) in medial prefrontal cortex, and the glutamate release was prevented by local infusions of MLA (6.75 µg, 0.19 ± 0.06 µM). The lower dose (1 mg/kg) of AVL3288 (type I) potentiated the glutamate release to a greater degree after the high dose of NMDA (0.30 µg; 84.7% increase vs AVL vehicle) versus the low dose of NMDA (0.05 µg; 24.2% increase), whereas glutamate release was inhibited when the high dose of NMDA was combined with the high dose of AVL3288 (64.2% decrease). In contrast, PNU120596 (type II) only potentiated glutamate release when the high dose (9 mg/kg) was combined with the low dose of NMDA (0.05 µg; 211% increase from PNU vehicle). Collectively, the results suggest a potential in vivo mechanism for the pro-cognitive effects of PAMs and provide the proof-of-concept for the continued focus on allosteric modulation of cortical α7nAChRs for cognition-enhancing drug development.

Rapid quantification of casein in skim milk using Fourier transform infrared spectroscopy, enzymatic perturbation, and multiway partial least squares regression: Monitoring chymosin at work.

In this study, we introduce enzymatic perturbation combined with Fourier transform infrared (FTIR) spectroscopy as a concept for quantifying casein in subcritical heated skim milk using chemometric multiway analysis. Chymosin is a protease that cleaves specifically caseins. As a result of hydrolysis, all casein proteins clot to form a creamy precipitate, and whey proteins remain in the supernatant. We monitored the cheese-clotting reaction in real time using FTIR and analyzed the resulting evolution profiles to establish calibration models using parallel factor analysis and multiway partial least squares regression. Because we observed casein-specific kinetic changes, the retrieved models were independent of the chemical background matrix and were therefore robust against possible covariance effects. We tested the robustness of the models by spiking the milk solutions with whey, calcium, and cream. This method can be used at different stages in the dairy production chain to ensure the quality of the delivered milk. In particular, the cheese-making industry can benefit from such methods to optimize production control.

Lentivirus pre-packed with Cas9 protein for safer gene editing.

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.

Encenicline, an α7 Nicotinic Acetylcholine Receptor Partial Agonist, Reduces Immune Cell Infiltration in the Colon and Improves Experimental Colitis in Mice.

The α7 pentamer nicotinic acetylcholine receptors (nAChRs) are a target in transduction of anti-inflammatory signals from the central nervous system to the gastrointestinal (GI) tract. The aim of this study was to investigate the anti-inflammatory action of the novel α7 nAChR partial agonist encenicline and to determine the mechanism underlying its activity. Anti-inflammatory activity of encenicline was evaluated using trinitrobenzenesulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced models of colitis. Macroscopic score, ulcer score, colon length and thickness, as well as myeloperoxidase (MPO) activity were recorded. Immunohistochemistry (IHC) was used to measure the infiltration of immune cells in the colon. Furthermore, we employed flow cytometry to determine the effect of encenicline on frequencies of FoxP3(+) and interleukin (IL)-17A(+) T cells in the mouse colon. Encenicline attenuated TNBS- and DSS-induced colitis in mice via α7 nAChRs, as indicated by significantly reduced macroscopic parameters and MPO activity. Treatment with encenicline significantly reduced the infiltration of macrophages, neutrophils, and B cells in the colon of TNBS-treated animals, as indicated by IHC. In the TNBS model encenicline reduced the frequency of FoxP3(+) IL-17A(+) T cells in the colon. In the DSS-model treatment encenicline increased the frequency of FoxP3(+) T cells and reduced IL-17A(+) T cells. Stimulation of α7 nAChR with partial agonist encenicline alleviates colitis via alteration of the number and/or activation status of the immune cells in the gut, emphasizing a potential role of α7 nAChRs as a target for anticolitic drugs.

Regional brain volumes, diffusivity, and metabolite changes after electroconvulsive therapy for severe depression.

OBJECTIVE: To investigate the role of hippocampal plasticity in the antidepressant effect of electroconvulsive therapy (ECT). METHOD: We used magnetic resonance (MR) imaging including diffusion tensor imaging (DTI) and proton MR spectroscopy (1 H-MRS) to investigate hippocampal volume, diffusivity, and metabolite changes in 19 patients receiving ECT for severe depression. Other regions of interest included the amygdala, dorsolateral prefrontal cortex (DLPFC), orbitofrontal cortex, and hypothalamus. Patients received a 3T MR scan before ECT (TP1), 1 week (TP2), and 4 weeks (TP3) after ECT. RESULTS: Hippocampal and amygdala volume increased significantly at TP2 and continued to be increased at TP3. DLPFC exhibited a transient volume reduction at TP2. DTI revealed a reduced anisotropy and diffusivity of the hippocampus at TP2. We found no significant post-ECT changes in brain metabolite concentrations, and we were unable to identify a spectral signature at ≈1.30 ppm previously suggested to reflect neurogenesis induced by ECT. None of the brain imaging measures correlated to the clinical response. CONCLUSION: Our findings show that ECT causes a remodeling of brain structures involved in affective regulation, but due to their lack of correlation with the antidepressant effect, this remodeling does not appear to be directly underlying the antidepressant action of ECT.

High plasma triglyceride levels strongly correlate with low kisspeptin in the arcuate nucleus of male rats.

OBJECTIVE: It is well known that reproductive capacity is lower in obese individuals, but what mediators and signals are involved is unclear. Kisspeptin is a potent stimulator of GnRH release, and it has been suggested that kisspeptin neurons located in the arcuate nucleus transmit metabolic signals to the GnRH neurons. METHODS: In this study, we measured body weight and plasma concentrations of leptin, insulin, testosterone, and triglycerides after high fat diet exposure and correlated these parameters with the number of kisspeptin-immunoreactive neurons in the arcuate nucleus of male rats. In this model, a high fat diet (45% or 60% energy from fat, respectively) or a control diet (10% energy from fat) was provided after weaning for three months. RESULTS: We find a significant increase in body weight and plasma leptin concentration, but no change in the number of kisspeptin-immunoreactive cells with increased fat in the diet. Kisspeptin-immunoreactive cells are not correlated with body weight, testosterone, leptin or insulin. However, we find that the number of kisspeptin-immunoreactive cells is strongly and negatively correlated with the level of plasma triglycerides (R2=0.49, p=0.004). CONCLUSION: We find a strong negative correlation between plasma triglyceride concentrations and the number of kisspeptin neurons in the rat arcuate nucleus regardless of the percentage of fat in the diet. In line with the lipotoxicity hypothesis, our results suggest that it is the level of hypertriglyceridemia per se that is a detrimental factor for kisspeptin expression in the arcuate nucleus.

Can laccases catalyze bond cleavage in lignin?

Modification of lignin is recognized as an important aspect of the successful refining of lignocellulosic biomass, and enzyme-assisted processing and upcycling of lignin is receiving significant attention in the literature. Laccases (EC 1.10.3.2) are taking the centerstage of this attention, since these enzymes may help degrading lignin, using oxygen as the oxidant. Laccases can catalyze polymerization of lignin, but the question is whether and how laccases can directly catalyze modification of lignin via catalytic bond cleavage. Via a thorough review of the available literature and detailed illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin model compounds; ii) For laccases to catalyze inter-unit bond cleavage in lignin substrates, the presence of a mediator system is required. Clearly, the higher the redox potential of the laccase enzyme, the broader the range of substrates, including o- and p-diphenols, aminophenols, methoxy-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin is proposed.

Mutants of Micromonospora viridifaciens sialidase have highly variable activities on natural and non-natural substrates.

This study aimed to improve the hydrolase activity of the well-characterised bacterial sialidase from Micromonospora viridifaciens. The enzyme and its mutated versions were produced in Bacillus subtilis and secreted to the growth medium. Twenty amino acid positions in or near the active site were subjected to site-saturation mutagenesis and evaluated on the artificial sialidase substrate 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid and on the natural substrate casein glycomacropeptide. A considerably higher fraction of the mutants exhibited increased activity on the artificial substrate compared with the natural one, with the most proficient mutant showing a 13-fold improvement in kcat/Km. In contrast, no mutants displayed more than a 2-fold increase in activity on the natural substrate. To gain further insight into this important discrepancy, we analysed the stability of mutants using the PoPMuSiC software, a property that also correlates with the potential for introducing chemical variation, after validating the method with a set of experimental stability estimates. We found a significant correlation between improved hydrolase activity on the artificial substrate and reduced apparent stability. Together with the minor improvement on the natural substrate this shows an important difference between naturally evolved functionality and new laboratory functionality. Our results suggest that when engineering sialidases and potentially other proteins towards non-natural substrates that are not optimized by natural evolution, major changes in chemical properties are advantageous, and these changes tend to correlate with decreased stability, partly explaining commonly observed trade-offs between stability and proficiency.

Photoperiodic co-regulation of kisseptin, neurokinin B and dynorphin in the hypothalamus of a seasonal rodent.

In many species, sexual activity varies on a seasonal basis. Kisspeptin (Kp), a hypothalamic neuropeptide acting as a strong activator of gonadotrophin-releasing hormone neurones, plays a critical role in this adaptive process. Recent studies report that two other neuropeptides, namely neurokinin B (NKB) and dynorphin (DYN), are co-expressed with Kp (and therefore termed KNDy neurones) in the arcuate nucleus and that these peptides are also considered to influence GnRH secretion. The present study aimed to establish whether hypothalamic NKB and DYN expression is photoperiod-dependent in a seasonal rodent, the Syrian hamster, which exhibits robust seasonal rhythms in reproductive activity. The majority of Kp neurones in the arcuate nucleus co-express NKB and DYN and the expression of all three peptides is decreased under a short (compared to long) photoperiod, leading to a 60% decrease in the number of KNDy neurones under photo-inhibitory conditions. In seasonal rodents, RFamide-related peptide (RFRP) neurones of the dorsomedial hypothalamus are also critical for seasonal reproduction. Interestingly, NKB and DYN are also expressed in the dorsomedial hypothalamus but do not co-localise with RFRP-immunoreactive neurones, and the expression of both NKB and DYN is higher under a short photoperiod, which is opposite to the short-day inhibition of RFRP expression. In conclusion, the present study shows that NKB and DYN display different photoperiodic variations in the Syrian hamster hypothalamus. In the arcuate nucleus, NKB and DYN, together with Kp, are down-regulated under a short photoperiod, whereas, in the dorsomedial hypothalamus, NKB and DYN are up-regulated under a short photoperiod.

Transient inactivation of the ventral hippocampus in neonatal rats impairs the mesolimbic regulation of prefrontal glutamate release in adulthood.

Cognitive deficits in schizophrenia (SZ) reflect maturational disruptions within a neural system that includes the ventral hippocampus (VH), nucleus accumbens (NAc), basal forebrain, and prefrontal cortex (PFC). A better understanding of these changes may reveal drug targets for more efficacious cognition enhancers. We have utilized an animal model in which the above distributed system is altered, during a sensitive period of development, by transiently inactivating the VH and its efferent projections. We determined the ability of NAc shell activation to evoke prefrontal glutamate release in adult male Wistar rats that had received saline (Sal) or tetrodotoxin (TTX) as neonates (PD7) or as adolescents (PD32). The nucleus accumbens shell (NAcSh) was activated by NMDA infusions (0.05-0.30 µg/0.5 µL). Basal and evoked glutamate levels were measured amperometrically using a glutamate-sensitive microelectrode. There were no differences in basal glutamate levels among the groups tested (overall 1.41 ± 0.26 uM). However, the dose-related stimulation of prefrontal glutamate levels seen in control rats treated with saline on PD7 (4.31 ± 0.22 µM after 0.15 µg) was markedly attenuated in rats treated with TTX on PD7 (0.45 ± 0.12 µM after 0.15 µg). This effect was age-dependent as infusions of TTX on PD32 did not alter the NMDA-induced increases in glutamate release (4.10 ± 0.37 µM after 0.15 µg). Collectively, these findings reveal that transient inactivation of VH transmission, during a sensitive period of development, leads to a functional mesolimbic-cortical disconnection that produces neurochemical and ultimately cognitive impairments resembling those seen in SZ.

5-HT(2A) and mGlu2 receptor binding levels are related to differences in impulsive behavior in the Roman Low- (RLA) and High- (RHA) avoidance rat strains.

The Roman Low- and High-Avoidance rat strains (RLA-I vs RHA-I) have been bidirectionally selected and bred according to their performance in the two-way active avoidance response in the shuttle-box test. Numerous studies have reported a pronounced divergence in emotionality between the two rat strains including differences in novelty seeking, anxiety, stress coping, and susceptibility to addictive substances. However, the underlying molecular mechanisms behind these divergent phenotypes are not known. Here, we determined impulsivity using the 5-choice serial reaction time task and levels of serotonin transporter (SERT), 5-HT(2A) and 5-HT(1A) receptor binding using highly specific radioligands ((3)H-escitalopram, (3)H-MDL100907 and (3)H-WAY100635) and mGlu2/3 receptor binding ((3)H-LY341495) using receptor autoradiography in fronto-cortical sections from RLA-I (n=8) and RHA-I (n=8) male rats. In the more impulsive RHA-I rats, 5-HT(2A), 5-HT(1A) and SERT binding in the frontal cortex was significantly higher compared to RLA-I rats. In contrast, mGlu2/3 receptor binding was decreased by 40% in RHA-I rats compared to RLA-I rats. To differentiate between mGlu2 and mGlu3 receptor protein levels, these were further studied using western blotting, which showed non-detectable levels of mGlu2 receptor protein in RHA rats, while no differences were observed for mGlu3 receptor protein levels. Collectively, these data show general congenital differences in the serotonergic system and a pronounced difference in mGlu2 receptor protein levels. We suggest that the differences in the serotonergic system may mediate some of the phenotypic characteristics in this strain such as hyper-impulsivity and susceptibility to drug addiction.

Localized infusions of the partial alpha 7 nicotinic receptor agonist SSR180711 evoke rapid and transient increases in prefrontal glutamate release.

The ability of local infusions of the alpha 7 nicotinic acetycholine receptor (α7 nAChR) partial agonist SSR180711 to evoke glutamate release in prefrontal cortex was determined in awake rats using a microelectrode array. Infusions of SSR180711 produced dose-dependent increases in glutamate levels. The lower dose (1.0µg in 0.4µL) evoked a rapid rise (∼1.0s) in glutamate (1.41±0.30µM above baseline). The higher dose (5.0µg) produced a similarly rapid, yet larger increase (3.51±0.36µM above baseline). After each dose, the glutamate signal was cleared to basal levels within 7-18s. SSR180711-evoked glutamate was mediated by the α7 nAChR as co-infusion of the selective α7 nAChR antagonist α-bungarotoxin (10.0µM)+SSR1808711 (5.0µg) reduced the effect of 5.0µg alone by 87% (2.62 vs. 0.35µM). Finally, the clearance of the SSR180711 (5.0µg)-evoked glutamate was bidirectionally affected by drugs that inhibited (threo-beta-benzyl-oxy-aspartate (TßOA), 100.0µM) or facilitated (ceftriaxalone, 200mg/kg, i.p.) excitatory amino acid transporters. TßOA slowed both the clearance (s) and rate of clearance (µM/s) by 10-fold, particularly at the mid-late stages of the return to baseline. Ceftriaxone reduced the magnitude of the SSR180711-evoked increase by 65%. These results demonstrate that pharmacological stimulation of α7 nAChRs within the prefrontal cortex is sufficient to evoke rapid yet transient increases in glutamate levels. Such increases may underlie the cognition-enhancing effects of the drug in animals; further justifying studies on the use of α7 nAChR-positive modulators in treating cognition-impairing disorders in humans.

Enzymatic depolymerization of gum tragacanth: bifidogenic potential of low molecular weight oligosaccharides.

Gum tragacanth derived from the plant "goat's horn" (Astragalus sp.) has a long history of use as a stabilizing, viscosity-enhancing agent in food emulsions. The gum contains pectinaceous arabinogalactans and fucose-substituted xylogalacturonans. In this work, gum tragacanth from Astragalus gossypinus was enzymatically depolymerized using Aspergillus niger pectinases (Pectinex BE Color). The enzymatically degraded products were divided into three molecular weight fractions via membrane separation: HAG1 < 2 kDa; 2 kDa < HAG2 < 10 kDa; HAG3 > 10 kDa. Compositional and linkage analyses showed that these three fractions also varied with respect to composition and structural elements: HAG1 and HAG2 were enriched in arabinose, galactose, and galacturonic acid, but low in fucose and xylose, whereas HAG3 was high in (terminal) xylose, fucose, and 1,4-bonded galacturonic acid, but low in arabinose and galactose content. The growth-stimulating potential of the three enzymatically produced gum tragacanth fractions was evaluated via growth assessment on seven different probiotic strains in single-culture fermentations on Bifidobacterium longum subsp. longum (two strains), B. longum subsp. infantis (three strains), Lactobacillus acidophilus , B. lactis, and on one pathogenic strain of Clostridium perfringens . The fractions HAG1 and HAG2 consistently promoted higher growth of the probiotic strains than HAG3, especially of the three B. longum subsp. infantis strains, and the growth promotion on HAG1 and HAG2 was better than that on galactan (control). HAG3 completely inhibited the growth of the C. perfringens strain. Tragacanth gum is thus a potential source of prebiotic carbohydrates that exert no viscosity effects and which may find use as natural functional food ingredients.

Distribution and seasonal variation in hypothalamic RF-amide peptides in a semi-desert rodent, the jerboa.

The jerboa is a semi-desert rodent, in which reproductive activity depends on the seasons, being sexually active in the spring-summer. The present study aimed to determine whether the expression of two RF-amide peptides recently described to regulate gonadotrophin-releasing hormone neurone activity, kisspeptin (Kp) and RF-amide-related peptide (RFRP)-3, displays seasonal variation in jerboa. Kp and/or RFRP-3 immunoreactivity was investigated in the hypothalamus of jerboas captured in the field of the Middle Atlas mountain (Morocco), either in the spring or autumn. As in other rodents, the Kp-immunoreactive (-IR) neurones were found in the anteroventro-periventricular and arcuate nuclei. RFRP-3 neurones were noted within the dorso/ventromedial hypothalamus. A marked sexual dimorphism in the expression of Kp (but not RFRP-3) was observed. The number of Kp-IR neurones was nine-fold higher, and the density of Kp-IR fibres and terminal-like elements in the median eminence was two-fold higher in females than in males. Furthermore, a significant seasonal variation in peptide expression was obtained with an increase in both Kp- and RFRP-3-IR cell bodies in sexually active male jerboas captured in the spring compared to sexually inactive autumn animals. In the arcuate nucleus, the level of Kp-IR cells and fibres was significant higher during the sexually active period in the spring than during the autumnal sexual quiescence. Similarly, the number of RFRP-3-IR neurones in the ventro/dorsomedial hypothalamus was approximately three-fold higher in sexually active jerboa captured in the spring compared to sexually inactive autumn animals. Altogether, the present study reports the distribution of Kp and RFRP-3 neurones in the hypothalamus of a desert species and reveals a seasonal difference in their expression that correlates with sexual activity. These findings suggest that these two RF-amide peptides may act in concert to synchronise the gonadotrophic activity of jerboas with the seasons.

Enhanced enzymatic cellulose degradation by cellobiohydrolases via product removal.

Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5-6.5, and CBHII: 46 °C, pH 4.8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion in the parallel batch reaction was only ~5 % by weight. Also, a synergistic effect, achieving ~27 % substrate conversion, was obtained by addition of endo-1,4-ß-D-glucanase. The synergistic effect was only obtained with product removal. By using pure, monoactive enzymes, the work illustrates the profound gains achievable by intermittent product removal during cellulose hydrolysis.