Output from VIP cells of the mammalian central clock regulates daily physiological rhythms

The suprachiasmatic nucleus (SCN) circadian clock is critical for optimising daily cycles in mammalian physiology and behaviour. The roles of the various SCN cell types in communicating timing information to downstream physiological systems remain incompletely understood, however. In particular, while vasoactive intestinal polypeptide (VIP) signalling is essential for SCN function and whole animal circadian rhythmicity, the specific contributions of VIP cell output to physiological control remains uncertain. Here we reveal a key role for SCN VIP cells in central clock output. Using multielectrode recording and optogenetic manipulations, we show that VIP neurons provide coordinated daily waves of GABAergic input to target cells across the paraventricular hypothalamus and ventral thalamus, supressing their activity during the mid to late day. Using chemogenetic manipulation, we further demonstrate specific roles for this circuitry in the daily control of heart rate and corticosterone secretion, collectively establishing SCN VIP cells as influential regulators of physiological timing

Efferent Projections of Prokineticin 2 Expressing Neurons in the Mouse Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Prokineticin 2 (PK2) is one of the candidate output molecules from the SCN. In this study, we investigated the efferent projections of PK2-expressing neurons in the SCN through a transgenic reporter approach. Using a bacterial artificial chromosome (BAC) transgenic mouse line, in which the enhanced green fluorescence protein (EGFP) reporter gene expression was driven by the PK2 promoter, we were able to obtain an efferent projections map from the EGFP-expressing neurons in the SCN. Our data revealed that EGFP-expressing neurons in the SCN, hence representing some of the PK2-expressing neurons, projected to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus. The efferent projections of PK2-expressing neurons supported the role of PK2 as an output molecule of the SCN

Circadian Integration of Glutamatergic Signals by Little SAAS in Novel Suprachiasmatic Circuits

Neuropeptides are critical integrative elements within the central circadian clock in the suprachiasmatic nucleus (SCN), where they mediate both cell-to-cell synchronization and phase adjustments that cause light entrainment. Forward peptidomics identified little SAAS, derived from the proSAAS prohormone, among novel SCN peptides, but its role in the SCN is poorly understood.Little SAAS localization and co-expression with established SCN neuropeptides were evaluated by immunohistochemistry using highly specific antisera and stereological analysis. Functional context was assessed relative to c-FOS induction in light-stimulated animals and on neuronal circadian rhythms in glutamate-stimulated brain slices. We found that little SAAS-expressing neurons comprise the third most abundant neuropeptidergic class (16.4%) with unusual functional circuit contexts. Little SAAS is localized within the densely retinorecipient central SCN of both rat and mouse, but not the retinohypothalamic tract (RHT). Some little SAAS colocalizes with vasoactive intestinal polypeptide (VIP) or gastrin-releasing peptide (GRP), known mediators of light signals, but not arginine vasopressin (AVP). Nearly 50% of little SAAS neurons express c-FOS in response to light exposure in early night. Blockade of signals that relay light information, via NMDA receptors or VIP- and GRP-cognate receptors, has no effect on phase delays of circadian rhythms induced by little SAAS.Little SAAS relays signals downstream of light/glutamatergic signaling from eye to SCN, and independent of VIP and GRP action. These findings suggest that little SAAS forms a third SCN neuropeptidergic system, processing light information and activating phase-shifts within novel circuits of the central circadian clock

A new chemical formulation for control of dental unit water line contamination: An 'in vitro' and clinical 'study'

BACKGROUND: Water delivered by dental units during routine dental practice is highly contaminated. The aim of this study is to evaluate the efficacy of a new chemical solution flushed through Dental Unit Water Lines (DUWL) for the control of contamination inside dental units. MATERIALS AND METHODS: Six old dental units equipped with a device designed to automatically flush disinfecting solutions through the water system (Castellini Autosteril) were selected. Water samples from DUWL effluents were collected in each dental unit for 10 randomly selected days, before and after a 5 minute DUWL disinfecting cycle with TetraAcetylEthileneDiamine (TAED) and persalt (Ster4spray produced by Farmec spa, and distributed by Castellini spa). Water samples were plated in R2A Agar and cultured at room temperature for 7 days, and the total number of heterotrophic microorganisms counted and expressed in Log(10) CFU/mL A general linear model was fitted and multiple regression ANOVA for repeated measures was used for the statistical analysis. RESULTS: The mean contamination in DUWL effluent at baseline was 5.45 ± 0.35 CFU/mL (range 4.79 to 5.93 CFU/mL). When water samples were tested "in vitro" against the chemical, no growth of heterotrophic bacteria was detected after a 5 minute contact in any of the water samples tested. After undergoing a 5 minute disinfecting cycle with the chemical, DUWL mean contamination in water effluents was 2.01 ± 0.32 CFU/mL (range 1.30 to 2.74 CFU/mL) (significant difference with respect to baseline). CONCLUSIONS: An inbetween patient disinfecting procedure consisting of flushing DUWL with TAED and persalt equivalent to 0.26% peracetic acid could be useful in routine dental practice for cross-contamination control

A Novel Screening System for Claudin Binder Using Baculoviral Display

Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator

Telomere Shortening Impairs Regeneration of the Olfactory Epithelium in Response to Injury but Not Under Homeostatic Conditions

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc−/−) with short telomeres compared to wild type mice (mTerc+/+) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc−/− mice compared to mTerc+/+ mice. Seven days after chemical induced damage, G3 mTerc−/− mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc+/+ mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people

Multilocus Bayesian Estimates of Intra-Oceanic Genetic Differentiation, Connectivity, and Admixture in Atlantic Swordfish (Xiphias gladius L.)

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