Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.     

Performance characteristics of a rapid new immunochromatographic test for detection of antibodies to human immunodeficiency virus

A new immunochromatographic rapid test (Rapid Check HIV 1 and 2; Núcleo de Doen?as Infecciosas) for the detection of antibodies to human immunodeficiency virus type 1 and type 2 in human samples (whole blood, serum, and plasma) was evaluated and compared to the commercially available Determine (Abbott Laboratories). When whole-blood samples were evaluated, the specificity and sensitivity of both tests were 100%. However, when plasma samples were used, sensitivity for the Rapid Check HIV 1&2 and the Determine tests were 100 and 98.58%, respectively. The observed specificity for plasma samples was 98.94% for the Rapid Check HIV 1&2 and 96.97% for the Determine test. The results presented here are encouraging and support the adoption of both tests as an alternative to enzyme-lined immunosorbent assay and/or Western blots in regions where laboratorial infrastructure is not available or for use in the management of occupational accidents for healthcare workers.     

Responses to natural scenes in cat V1

Studies on processing in primary visual areas often use artificial stimuli such as bars or gratings. As a result, little is known about the properties of activity patterns for the natural stimuli processed by the visual system on a daily basis. Furthermore, in the cat, a well-studied model system for visual processing, most results are obtained from anesthetized subjects and little is known about neuronal activations in the alert animal. Addressing these issues, we measure local field potentials (lfp) and multiunit spikes in the primary visual cortex of awake cats. We compare changes in the lfp power spectra and multiunit firing rates for natural movies, movies with modified spatio-temporal correlations as well as gratings. The activity patterns elicited by drifting gratings are qualitatively and quantitatively different from those elicited by natural stimuli and this difference arises from both spatial as well as temporal properties of the stimuli. Furthermore, both local field potentials and multiunit firing rates are most sensitive to the second-order statistics of the stimuli and not to their higher-order properties. Finally, responses to natural movies show a large variability over time because of activity fluctuations induced by rapid stimulus motion. We show that these fluctuations are not dependent on the detailed spatial properties of the stimuli but depend on their temporal jitter. These fluctuations are important characteristics of visual activity under natural conditions and impose limitations on the readout of possible differences in mean activity levels.     

Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle fibres upon electrical stimulation of the innervated skeletal muscle of the frog

The independent release of adenosine and adenine nucleotides upon electrical stimulation was studied in the innervated sartorius muscle of the frog after blockade of the extracellular catabolism of adenosine monophosphate (AMP) through exo-AMP deaminase and ecto-5-nucleotidase. Nerve stimulation (30 min, 0.2Hz) induced the release of both adenosine (19±3 pmol) and adenine nucleotides (101±7 pmol). Experiments performed in the presence of tubocurarine (5 M) to prevent purine release due to nerve-evoked muscle twitching, or under direct stimulation of the muscle in low calcium solutions to prevent pre-synaptic release of purines, showed that there was an evoked release of adenosine and adenine nucleotides both from the nerve endings and from the twitching muscle fibres. Removal of ecto-5-nucleotidase inhibition shows that the catabolism of adenine nucleotides released during stimulation contributes in about 50% to the amount of endogenous extracellular adenosine. When only one of the enzymes catabolizing AMP (ecto-5-nucleotidase or exo-AMP deaminase) was inhibited, the evoked release of adenine nucleotides was undetectable, suggesting that each enzyme is able to catabolize all the AMP formed from adenine nucleotides released upon stimulation. It is concluded that the concentration of endogenous extracellular adenosine is under the control of the relative activities of exo-AMP deaminase and ecto-5-nucleotidase.Brief accounts of some of the results in this study have been published previously (refs. [6, 7]).     

Synthetic MRI improves radiomics-based glioblastoma survival prediction

Glioblastoma is an aggressive and fast-growing brain tumor with poor prognosis. Predicting the expected survival of patients with glioblastoma is a key task for efficient treatment and surgery planning. Survival predictions could be enhanced by means of a radiomic system. However, these systems demand high numbers of multicontrast images, the acquisitions of which are time consuming, giving rise to patient discomfort and low healthcare system efficiency. Synthetic MRI could favor deployment of radiomic systems in the clinic by allowing practitioners not only to reduce acquisition time, but also to retrospectively complete databases or to replace artifacted images. In this work we analyze the replacement of an actually acquired MR weighted image by a synthesized version to predict survival of glioblastoma patients with a radiomic system. Each synthesized version was realistically generated from two acquired images with a deep learning synthetic MRI approach based on a convolutional neural network. Specifically, two weighted images were considered for the replacement one at a time, a T2w and a FLAIR, which were synthesized from the pairs T1w and FLAIR, and T1w and T2w, respectively. Furthermore, a radiomic system for survival prediction, which can classify patients into two groups (survival >480 days and $\le$ 480 days), was built. Results show that the radiomic system fed with the synthesized image achieves similar performance compared with using the acquired one, and better performance than a model that does not include this image. Hence, our results confirm that synthetic MRI does add to glioblastoma survival prediction within a radiomics-based approach.     

Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.     

Expression and function of alpha 4/beta 7 integrin on human natural killer cells.

In this report we have analysed the expression and function of the alpha 4/beta 7 heterodimer in human natural killer (NK) cells. The expression of alpha 4 beta 7 is induced in NK cells upon activation as the anti alpha 4 beta 2 ACT-1 monoclonal antibody (mAb) family stained a minority of peripheral blood NK cells, whereas it strongly reacted with in vitro long-term interleukin-2 (IL-2)-activated NK cells. Incubation with ACT-1 on its F(ab) fragments induced a strong homotypic adhesion of NK cells, comparable to than stimulated by the anti-alpha 4 HPI 7 mAb. Cell cell interaction induced by the ACT-1 mAb was only prevented by another anti-alpha 4 mAb (HP2.1) that recognizes a different epitope. In alpha 4 beta 7-mediated cell aggregation the alpha 4 beta 7 heterodimer was redistributed to intercellular contact sites thus, suggesting a direct involvement of this integrin in the formation of cell clusters. In NK cells attached to Fibronectin (FN38) or vascular cell adhesion molecule-1 (VCAM-1), both alpha 4 beta 7 and alpha 4 beta 7 integrins were redistributed at the ventral cellular membrane forming discrete contact sites. The ACT-1 mAb only partially blocked NK cell binding to FN38, but in combination with the anti-beta 7 mAb LIAI 2, NK cell binding to FN38 was completely inhibited. In contrast. ACT-1 did not modify NK cell adhesion to VCAM-1 thus supporting the theory that the alpha 4 beta 7 binding sites for both ligands appear to be different. Our results indicate that upon IL-2-activation, expression of functional alpha 4 beta-integrin is induced on NK cells potentially participating in their interaction with both extracellular matrix and endothelial cells.     

The release of acetylcholine from carotid body tissues. Further study on the effects of acetylcholine and cholinergic blocking agents on the chemosensory discharge

1. Both carotid bodies were removed from cats and placed in a small Perspex channel through which Locke solution was allowed to flow under a layer of paraffin oil. Stimulation of the upstream (;donor') organ elicited an increased sensory discharge in the downstream (;detector') preparation (Loewi effect).2. This effect was enhanced by eserine, depressed by hexamethonium and blocked by either mecamylamine or acetylcholinesterase. The Loewi effect did not disappear when chronically denervated (4 days) ;donor' carotid bodies were stimulated.3. These experiments led to the conclusion that ACh is released by the carotid body tissues (probably from the glomus cells) during stimulation and that this substance is responsible for the initiation of the chemosensory discharges.4. In single preparations the chemosensory endings proved to be very sensitive to ACh especially in the presence of eserine which, in all probability, inactivated the tissue cholinesterase. Curarizing agents such as mecamylamine, hexamethonium, (+)-tubocurarine and atropine blocked the response of the sensory endings to applied ACh in what appeared to be ;surmountable' antagonism.     

A sodium-activated potassium current in intact ventricular myocytes isolated from the guinea-pig heart

A current generated by the Na-activated K channel has been identified in whole cell currents recorded from isolated guinea-pig ventricular myocytes. A partial activation of this current can be achieved near to the physiological range of intracellular sodium concentration [( Na+]i) when it contributes significantly to the global outward current. The decline of the Na-activated K current, the lengthening of the action potential duration and the recovery of [Na+]i occur with a similar time course during recovery from Na loading.     

The Na+-activated K+ channel contributes to K+ efflux in Na+-loaded guinea-pig but not rat ventricular myocytes

Activation of the Na+-activated K+ channels (KNa channels) has been suggested to contribute to the ischaemia-induced accumulation of extracellular K+ (K+e) in the mammalian myocardium. Recent evidence shows that these channels are not present in rat ventricular myocytes [9]. We have therefore investigated the effect of raised intracellular Na+ activity (aiNa) on intracellular K+ activity (aiK) in guinea-pig myocytes, which possess the channels, and on rat ventricular myocytes which do not. The Na+-activated K+ current was activated by an increase in aiNa induced by removing extracellular Ca2+ and Mg2+ and inhibiting the Na-pump. The aiNa increased and the aiK decreased in both guinea-pig and rat myocytes superfused with Ca2+- and Mg2+-free Tyrode. The new steady-state increase in aiNa and decline in aiK were similar in both species. Inhibition of the Na-pump resulted in an additional increase in aiNa and decrease in aiK in both species. However, both the increase in aiNa and decrease in aiK were greater in guinea-pig myocytes and the decline in aiK in guinea-pig myocytes followed the development of a large Na+-activated K+ current. When Li+ replaced Na+ in the superfusate the Na+-activated K+ current did not develop and the fall in aiK was reduced. In Na+-loaded rat myocytes, which do not have a Na+-activated K+ current, the decline in aiK was reduced and blocked by 2 mM Mg2+ suggesting that a Mg2+-sensitive non-specific cation channel may be involved in the K+ efflux from rat myocytes [12]. These data suggest that KNa channels are a major route for K+ efflux from Na+-loaded guinea-pig myocytes.