Design of a rapid, multiplex, one-pot miRNA assay optimized by label-free analysis.

MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases. Detection and quantification of miRNAs is currently performed through complex and time consuming procedures. Herein we demonstrate a rapid, multiplex, one-pot detection method based on two-step amplification of the signal measured by Reflective Phantom Interface (RPI) label-free optical biosensor. We achieved sub-pM quantification of different miRNAs in about 1.5 h, through specific capture with surface DNA probes combined to a 35-fold mass amplification by an antibody targeting DNA-RNA hybrids and polyclonal secondary antibody, all performed without washing steps. The assay is the result of a modelling and optimization of the multi-step process that has been made possible by the RPI characterization of each individual interaction involved.

TMS Over the Cerebellum Interferes with Short-term Memory of Visual Sequences.

Growing evidence suggests that the cerebellum is not only involved in motor functions, but it significantly contributes to sensory and cognitive processing as well. In particular, it has been hypothesized that the cerebellum identifies recurrent serial events and recognizes their violations. Here we used transcranial magnetic stimulation (TMS) to shed light on the role of the cerebellum in short-term memory of visual sequences. In two experiments, we found that TMS over the right cerebellar hemisphere impaired participants' ability to recognize the correct order of appearance of geometrical stimuli varying in shape and/or size. In turn, cerebellar TMS did not affect recognition of highly familiar short sequences of letters or numbers. Overall, our data suggest that the cerebellum is involved in memorizing the order in which (concatenated) stimuli appear, this process being important for sequence learning.

Cerebral extract from morphine-tolerant rats shows antiopiate properties in guinea pig ileum bioassay.

The existence of an endogenous antiopiate system which counteracts endogenous opiate effects has been proposed. The present study set out to seek substance/s with morphine-antagonist activity in the brain and serum of morphine-tolerant rats. Cerebral extracts were partly purified on Sephadex G 25 and serum was ultrafiltered through membranes with pore diameter smaller than 0.005 micron. On the guinea pig ileum myenteric plexus longitudinal muscle a fraction of the cerebral extract and the serum ultrafiltrate in toto did increase electrically induced contractions, and antagonized the depressant effect of morphine. The serum ultrafiltrate also enhanced longitudinal smooth muscle tone. Preliminary findings suggest that levels of endogenous morphine-antagonist substance/s are higher in morphine-tolerant rats than in controls. Only cerebral extract, not serum ultrafiltrate, inhibited [3H]-naloxone binding to cerebral opiate receptors. In the guinea pig bioassay both the cerebral extract and serum ultrafiltrate antagonized, to some extent, the inhibition elicited by morphine, norepinephrine and adenosine. These observations support the existence of endogenous compound/s which may be functional antagonist/s of opiates and play a role in the development of tolerance and dependence.

Central pharmacological activities and opiate receptor binding studies of some dermorphin analogs.

A series of dermorphin-like compounds were injected intracerebroventricularly in the rat to assess in vivo their effects on intestinal motility and analgesia. In vitro they were tested by binding assay using 3H-naloxone as radioligand or by guinea pig ileum bioassay. The synthetic peptides were less potent than dermorphin in inhibiting intestinal transit and in producing analgesia, or even inactive up to doses 30 times the dermorphin ED50. This reduction in pharmacological activity was coupled with a decrease in binding potency. The 3H-naloxone binding studies in the absence or presence of Na+ indicated that Na+ reduced the interaction of dermorphin and its analogs with brain opiate receptors. Only the dibenzyl derivative was slightly affected by sodium, suggesting a dual action for this peptide, as confirmed by preliminary data from guinea pig ileum bioassay.

Dermorphin interaction with peripheral opioid receptors.

The interaction of dermorphin with different peripheral opioid receptor subtypes was investigated in vitro, using the guinea pig ileum as representative tissue for mu, the mouse vas deferens for delta, the rabbit vas deferens for kappa and the rat vas deferens for epsilon. The effect of dermorphin on each tissue preparation was compared with that of selective mu, delta, kappa epsilon agonists respectively morphine, met-enkephalinamide, ethylketocyclazocine and camel beta-endorphin. Antagonism with naloxone was also tested and calculated as Ke. It is concluded that dermorphin mainly interacts with the mu receptors, although it also binds to epsilon receptors; the interaction with delta receptors is questionable, and the kappa receptors are unaffected.

Loperamide: evidence of interaction with mu and delta opioid receptors.

Loperamide was tested on electrically-evoked contractions using a series of "in vitro" isolated preparations, in comparison with morphine, met-enkephalin, beta-endorphin, ethylketocyclazocine used as representative agonists of mu, delta, epsilon, kappa receptors respectively. The IC50 of loperamide on myenteric plexus longitudinal muscle of guinea pig ileum was found to be 1.90 X 10(-7)M and equal to that of morphine. The IC50 on mouse vas deferens was found to be 13.02 X 10(-7)M. In this tissue, loperamide resulted as active as morphine, but 54 times less active than met-enkephalin (IC50 0.24 X 10(-7)M). On the rat vas deferens where, as expected, beta-endorphin was strongly active (IC50 1.38 X 10(-7)M), morphine exerted a stimulatory action within the range 10(-5)M-10(-4)M and loperamide was only poorly depressive. The Ke value of naloxone, a specific mu receptor antagonist, against loperamide in the guinea pig ileum was 3.83 nM, and in the mouse vas deferens was 82.87 nM indicating that loperamide in the guinea pig ileum acts on mu receptors while in the mouse vas deferens on another opiate receptor.