Biocide effect against SARS-CoV-2 and ESKAPE pathogens of a noncytotoxic silver-copper nanofilm.

Nanometric materials with biocidal properties effective against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and pathogenic bacteria could be used to modify surfaces, reducing the risk of touching transmission. In this work, we showed that a nanometric layer of bimetallic AgCu can be effectively deposited on polypropylene (PP) fibers. The virucidal properties of the AgCu nanofilm were evaluated by comparing the viral loads remaining on uncoated and coated PP after contact times between 2 and 24 h. Quantification of virion numbers for different initial concentrations indicated a reduction of more than 95% after 2 h of contact. The bactericidal action of the AgCu nanofilm was also confirmed by inoculating uncoated and coated PP with a pool of pathogenic bacteria associated with pneumonia (ESKAPE). Meanwhile, no cytotoxicity was observed for human fibroblasts and keratinocyte cells, indicating that the nanofilm could be in contact with human skin without threat. The deposition of the AgCu nanofilm on the nonwoven component of reusable cloth masks might help to prevent virus and bacterial infection while reducing the pollution burden related to the disposable masks. The possible mechanism of biocide contact action was studied by quantum chemistry calculations that show that the addition of Ag and/or Cu makes the polymeric fiber a better electron acceptor. This can promote the oxidation of the phospholipids present at both the virus and bacterial membranes. The rupture at the membrane exposes and damages the genetic material of the virus. More studies are needed to determine the mechanism of action, but the results reported here indicate that Cu and Ag ions are good allies, which can help protect us from the virus that has caused this disturbing pandemic.

UPD(14)mat and UPD(14)mat in concomitance with mosaic small supernumerary marker chromosome 14 in two new patients with Temple syndrome.

Temple syndrome (TS14) can be originated by maternal uniparental disomy (UPD(14)mat), paternal deletion, or epimutation, leading to disturbances in 14q32.2 imprinted region. The most frequent phenotypic manifestations are prenatal and postnatal growth failure, hypotonia, developmental delay, small hands/feet, precocious puberty, and truncal obesity. However, the diagnosis can be challenging due to the clinical overlap with other imprinting disorders such as Silver-Russell or Prader-Willi syndromes. Although rare, TS14 has been also reported in patients with concomitant UPD(14)mat and mosaic trisomy 14. In the present report, the clinical and genetic profiles of two new patients with TS14 are described. SNParray and MS-MLPA, allowed the determination of segmental UPD(14)mat and the hypomethylation of MEG3 gene. Additionally, in one of our patients we also observed by cytogenetics a small supernumerary marker chromosome that led to partial trisomy 14 in mosaic. Only few patients with concomitant UPD(14)mat and mosaic partial trisomy 14 have been reported. Our patients share cardinal TS14 phenotypic features that are associated to the genetic abnormalities detected; however, we also observed some clinical features such as fatty liver disease that had not previously been reported as part of this syndrome. The detailed clinical, cytogenetical and molecular description of these two new patients, contributes to a more accurately delineation of this syndrome.

Somatostatinergic modulation of firing pattern and calcium-activated potassium currents in medium spiny neostriatal neurons.

Somatostatin is synthesized and released by aspiny GABAergic interneurons of the neostriatum, some of them identified as low threshold spike generating neurons (LTS-interneurons). These neurons make synaptic contacts with spiny neostriatal projection neurons. However, very few somatostatin actions on projection neurons have been described. The present work reports that somatostatin modulates the Ca(2+) activated K(+) currents (K(Ca) currents) expressed by projection cells. These actions contribute in designing the firing pattern of the spiny projection neuron; which is the output of the neostriatum. Small conductance (SK) and large conductance (BK) K(Ca) currents represent between 30% and 50% of the sustained outward current in spiny cells. Somatostatin reduces SK-type K(+) currents and at the same time enhances BK-type K(+) currents. This dual effect enhances the fast component of the after hyperpolarizing potential while reducing the slow component. Somatostatin then modifies the firing pattern of spiny neurons which changed from a tonic regular pattern to an interrupted "stuttering"-like pattern. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) tissue expression analysis of dorsal striatal somatostatinergic receptors (SSTR) mRNA revealed that all five SSTR mRNAs are present. However, single cell RT-PCR profiling suggests that the most probable receptor in charge of this modulation is the SSTR2 receptor. Interestingly, aspiny interneurons may exhibit a "stuttering"-like firing pattern. Therefore, somatostatin actions appear to be the entrainment of projection neurons to the rhythms generated by some interneurons. Somatostatin is then capable of modifying the processing and output of the neostriatum.

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons.

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons possess both "big" and "small" types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM omega-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 microM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM omega-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 microM omega-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM omega-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.

Somatostatin modulates Ca2+ currents in neostriatal neurons.

Somatostatin is synthesized and released by aspiny interneurons of the neostriatum. This work investigates the actions of somatostatin on rat neostriatal neurons of medium size (ca. 6 pF). Somatostatin (1 microM) reduces both calcium action potentials (20 mM tetraethylammonium) by ca. 24% and calcium currents by ca. 35%, in all cells tested. This action was produced in the presence of tetrodotoxin and in dissociated cells and was blocked by cyclo(-7-aminoheptanoyl-phe-d-try-lys-O-benzyl-thr) acetate (CPP-1), a somatostatin receptor antagonist. Except for nitrendipine (5 microM), several calcium channel antagonists, 1 microM omega-conotoxin GVIA, 400 nM omega-agatoxin TK, and 1 microM omega-conotoxin MVIIC, partially occluded somatostatin action. According to the calcium channel types known to be blocked by these antagonists, P/Q-type channels appeared to be the channels mainly modulated by somatostatin, followed by N-type channels. Since these channel types generate the afterhyperpolarizing potential in spiny neurons, we investigated the action of somatostatin on this event. Somatostatin reduces the amplitude of the afterhyperpolarizing potential by ca. 39%. This action is occluded by omega-agatoxin TK and omega-conotoxin MVIIC but not by omega-conotoxin GVIA or nicardipine. Thus, the action of somatostatin on the afterhyperpolarizing potential is mainly mediated by P/Q-type calcium channels. The block of the slow afterhyperpolarizing potential made most neurons exhibit an irregular firing mode, suggesting that ion currents other than calcium may also be affected by somatostatin. We conclude that somatostatin exerts a direct postsynaptic effect on neostriatal neurons via the activation of somatostatin receptors. This action affects non-L-type calcium channels and therefore modifies the afterhyperpolarizing potential and the firing pattern. It is proposed that somatostatin and its analogues may have profound effects on the motor functions controlled by the basal ganglia.

Ca2+-activated outward currents in neostriatal neurons.

Whole-cell voltage-clamp recordings of outward currents were obtained from acutely dissociated neurons of the rat neostriatum in conditions in which inward Ca2+ current was not blocked and intracellular Ca2+ concentration was lightly buffered. Na+ currents were blocked with tetrodotoxin. In this situation, about 53 +/- 4% (mean +/- S.E.M.; n = 18) of the outward current evoked by a depolarization to 0 mV was sensitive to 400 microM Cd2+. A similar percentage was sensitive to high concentrations of intracellular chelators or to extracellular Ca2+ reduction (<500 microM); 35+/-4% (n=25) of the outward current was sensitive to 3.0 mM 4-aminopyridine. Most of the remaining current was blocked by 10 mM tetraethylammonium. The results suggest that about half of the outward current is activated by Ca2+ entry in the present conditions. The peptidic toxins charybdotoxin, iberotoxin and apamin confirmed these results, since 34 +/- 5% (n = 14), 29 5% (n= 14) and 28 +/- 6% (n=9) of the outward current was blocked by these peptides, respectively. The effects of charybdotoxin and iberotoxin added to that of apamin, but their effects largely occluded each other. There was additional Cd2+ block after the effect of any combination of toxins. Therefore, it is concluded that Ca2+-activated outward currents in neostriatal neurons comprise several components, including small and large conductance types. In addition, the present experiments demonstrate that Ca2+-activated K+ currents are a very important component of the outward current activated by depolarization in neostriatal neurons.

Characterization of [2-3H]deoxy-D-glucose uptake in retina and retinal pigment epithelium of normal and diabetic rats.

The outer blood-retinal barrier which results from the tight junctions between retinal pigment epithelial cells (RPE) restricts the flow of nutrients reaching the retina. We characterize the transport of [2-3H]deoxy-D-glucose (2-DG) across isolated mammalian neural retina and RPE in terms of their kinetics constants. In addition, the effect of insulin on glucose transport was studied by using streptozotocin-induced diabetic rats. RPE accumulates 2-DG by a temperature-sensitive and energy-dependent complex kinetics mechanism. The retina takes up 2-DG by an energy and Na(+)-dependent saturable system with an apparent Km of 2 mM. Insulin induced an increase of 2-DG uptake by normal retina. The retina of diabetic rats shows lower levels of 2-DG accumulation. These levels can be returned to the normal ones by exposure to insulin. Although insulin does not affect, significantly, 2-DG accumulation by RPE, 2-DG uptake of RPE from diabetic rats shows a normal saturable kinetics with an apparent Km of 20 mM. Those findings suggest the presence of different types of glucose transporters in retina and RPE. Insulin-sensitive glucose transport in retina might be involved in the manifestation of diabetic retinopathy.

Antimutagenicity of cyclohexanol towards 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone and N-nitrosodiethylamine in Salmonella typhimurium strain TA100.

The ability of cyclohexanol to inhibit the mutagenicity of tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and of N-nitrosodiethylamine (NDEA) was tested on Salmonella typhimurium strain TA100. Cyclohexanol produced a dose-dependent decrease in the number of revertants induced by a single dose of NNK (24 mumoles) or NDEA (59 mumoles). Nevertheless, this inhibitory effect was not observed with other premutagenic agents such as benzo[a]pyrene and 2-aminoanthracene nor with direct mutagens such as ethyl methanesulfonate and methyl methanesulfonate. These results suggest that cyclohexanol interferes with the 'bioactivation' of the tested nitrosamines in a similar way that other alcohols such as ethanol or isopropanol interfere with N-nitro-sodimethylamine and NDEA metabolism.

Effect of diabetes on levels and uptake of putative amino acid neurotransmitters in rat retina and retinal pigment epithelium.

Free amino acid levels and high affinity uptake of glutamate, aspartate, gamma-aminobutyrate, glycine and taurine were studied in retina and retinal pigment epithelium of streptozotocin diabetic rats. Results show that experimental diabetes produces a generalized fall in the content of free amino acids in both retina and retinal pigment epithelium. With regard to the high affinity uptake, in the two tissues of diabetic animals showed decreased aspartate uptake, enhanced taurine and gamma-aminobutyrate uptake, whereas that of glycine and glutamate was unchanged. These results might suggest that diabetes causes alterations of specific amino acid transport systems and/or alterations of some cell populations.

Changes in the redox state in the retina and brain during the onset of diabetes in rats.

Diabetic retinopathy is thought to result from chronic changes in the metabolic pathways of the retina. Hyperglycemia leads to increased intracellular glucose concentrations, alterations in glucose degradation and an increase in lactate/pyruvate ratio. We measured lactate content in retina and other ocular and non-ocular tissues from normal and diabetic rats in the early stages of streptozotocin-induced diabetes. The intracellular redox state was calculated from the cytoplasmic [lactate]/[pyruvate] ratio. Elevated lactate concentration were found in retina and cerebral cortex from diabetic rats. These concentrations led to a significant and progressive decrease in the NAD+/NADH ratio, suggesting that altered glucose metabolism is an initial step of retinopathy. It is thus possible that tissues such as cerebral cortex have mechanisms that prevent the damaging effect of lactate produced by hyperglycemia and/or alterations of the intracellular redox state.

Cholinergic modulation of neostriatal output: a functional antagonism between different types of muscarinic receptors.

It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (RN) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic RN increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M1,4 muscarinic receptor antagonist pirenzepine but not by M2 or M3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing RN and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M1- and M2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits.