The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

Immediate and delayed effects of in vitro ischemia on glutamate efflux from guinea-pig cerebral cortex slices

Immediate and delayed effects of glucose deprivation, oxygen deprivation (hypoxia) and both oxygen and glucose deprivation (in vitro ischemia) on glutamate efflux from guinea pig cerebral cortex slices were studied. Immediate effects were evaluated by measuring changes of glutamate efflux during the metabolic insults. Delayed effects were evaluated by measuring the response of the tissue to a 50 mM KCI pulse applied 60 min after the metabolic insults. Deprivation of glucose in the medium did not induce either immediate or delayed effects, while hypoxic condition produced an immediate slight stimulation of glutamate efflux without any delayed effect. Conversely, in vitro ischemia produced both immediate and delayed effects on glutamate efflux. During in vitro ischemia glutamate efflux dramatically increased in a calcium-independent and tetrodotoxin-sensitive manner; this effect was potentiated by a low sodium containing medium. The blockade of the sodium/potassium ATPase exchanger by ouabain caused a glutamate outflow similar to that induced by in vitro ischemia. On the whole, these data demonstrate the central role played by the sodium electrochemical gradient and by the membrane glutamate uptake system in the glutamate overflow induced by in vitro ischemia. Moreover, in slices previously exposed to both oxygen and glucose deprivation the effect of KCI on glutamate efflux was potentiated. This in vitro ischemia-induced delayed potentiation of neurotransmitter efflux, until now unreported in the literature, was found to be selectively restricted to glutamatergic structures and to be mainly due to an enhancement of the exocytotic component of glutamate release

The nicotinic modulation of [(3)H]D-aspartate outflow in primary cultures of neocortical neurons: effect of acute and long term nicotine treatment.

The effect of nicotine 1 nM-10 microM on the efflux of [(3)H]D-aspartate was tested in primary cultures of rat cortical neurons kept at rest and subjected to electrical field stimulation. Two trains of pulses at 20 Hz for 20 s were applied at the 60th (St(1)) and 90th (St(2)) min of perfusion. The drug slightly and transiently increased the efflux of resting cells while, when given during St(2), it greatly enhanced the electrically evoked efflux estimated as St(2)/St(1) ratio, EC(50) being 107 nM. The nicotinic receptors (nAChR) giving rise to this positive modulation were partly mecamylamine- and partly alpha-bungarotoxin-sensitive. They appeared to be located at the nerve endings since nicotine facilitation was only slightly prevented by tetrodotoxin during depolarisation with 15 mM KCl. Pretreatment with glutamate antagonists did not reveal any interaction between nAChR and ionotropic glutamate receptors. Membrane glutamate carrier involvement in the nicotine effect was ruled out. Long-term treatment with nicotine 1 microM (from the 3rd-4th to the 8th-9th day in vitro) reduced the maximal response to the drug but shifted its threshold concentration to the left (from 10 nM to 1 nM), leaving the contribution of the two receptor subtypes unchanged. Reduced responsiveness to nicotine was also evident in long-term treated cerebellar granule cells. In conclusion, presynaptic nAChR's, both containing and lacking alpha(7) subunits, can contribute to enhance the glutamatergic secretion in primary cultures of rat cortical neurons, chiefly during electrical stimulation

Reversal of clonidine effect on acetylcholine release during morphine tolerance.

Effect of clonidine on acetylcholine release during morphine tolerance