Analysis of ECs and related compounds in plasma: artifactual isomerization and ex vivo enzymatic generation of 2-MGs

The analysis of peripheral endocannabinoids (ECs) is a good biomarker of the EC system. Their concentrations, from clinical studies, strongly depend on sample collection and time processing conditions taking place in clinical and laboratory settings. The analysis of 2-monoacylglycerols (MGs) (i.e., 2-arachidonoylglycerol or 2-oleoylglycerol) is a particularly challenging issue because of their ex vivo formation and chemical isomerization that occur after blood sample collection. We provide evidence that their ex vivo formation can be minimized by adding Orlistat, an enzymatic lipase inhibitor, to plasma. Taking into consideration the low cost of Orlistat, we recommend its addition to plasma collecting tubes while maintaining sample cold chain until storage. We have validated a method for the determination of the EC profile of a range of MGs and N-acylethanolamides in plasma that preserves the original isomer ratio of MGs. Nevertheless, the chemical isomerization of 2-MGs can only be avoided by an immediate processing and analysis of samples due to their instability during conservation. We believe that this new methodology can aid in the harmonization of the measurement of ECs and related compounds in clinical samples.     

Reconfigurable Boolean Logic Using Magnetic Single-Electron Transistors

We propose a novel hybrid single-electron device for reprogrammable low-power logic operations, the magnetic single-electron transistor (MSET). The device consists of an aluminium single-electron transistor with a GaMnAs magnetic back-gate. Changing between different logic gate functions is realized by reorienting the magnetic moments of the magnetic layer, which induces a voltage shift on the Coulomb blockade oscillations of the MSET. We show that we can arbitrarily reprogram the function of the device from an n-type SET for in-plane magnetization of the GaMnAs layer to p-type SET for out-of-plane magnetization orientation. Moreover, we demonstrate a set of reprogrammable Boolean gates and its logical complement at the single device level. Finally, we propose two sets of reconfigurable binary gates using combinations of two MSETs in a pull-down network.     

Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants

Transgenic Research - Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of...     

A single column method for the assay of adenylate cyclase

An improved, one-step method for the separation of cyclic AMP from other nucleotides on disposable columns of neutral aluminum oxide is described. The method consists of several modifications of an established assay for adenylate cyclase. These modifications were designed to increase the sensitivity of the method, to decrease the time required for column preparation, and to eliminate the variable elution patterns for cyclic AMP that are obtained when using aluminum oxide from different commercial sources. Uniform elution patterns and high recoveries (approximately 80%) of cyclic AMP were obtained when 0.1 M ammonium acetate was used to elute cyclic AMP instead of Tris-HCl buffer. Prior to column chromatography, the adenylate cyclase reactions were terminated with the addition of hydrochloric acid and the mixtures were heated to degrade acid-labile nucleotides that would otherwise elute with cyclic AMP from aluminum oxide columns. Disposable polypropylene columns, fabricated with a reservoir and fast-flow filters, were used for column chromatography. Low blank values, generally less than 15 dpm/assay tube, were obtained when the acidified reaction mixtures were applied directly to aluminum oxide columns without prior neutralization. The proposed method should be useful for the routine assay of adenylate cyclase activity.     

Behavioral thermoregulation of Bulla gouldiana (Gastropoda: Opisthobranchia: Cephalaspidea)

1. 1. The preferred temperature of Bulla gouldiana is 26.7–28.7°C.

2. 2. In constant scotophase, photophase, and light and dark photoperiod the organisms do not have a diel cycle of thermoregulation.

3. 3. It takes the animal 6–16 h to reach the preferred temperature.

4. 4. The lowest and highest temperatures visited were 11 and 33°C.

5. 5. Spawning of the species occurred in the thermal gradient between 27 and 28.5°C.

     

Climate oscillations and species interactions: large‐scale congruence but regional differences in the phylogeographic structures of an alpine plant and its monophagous insect

Aim To predict the fate of alpine interactions involving specialized species, using a monophagous beetle and its host plant as a case study. Location The Alps. Methods We investigated genetic structuring of the herbivorous beetle Oreina gloriosa and its specific host‐plant Peucedanum ostruthium. We used genome fingerprinting (in the insect and the plant) and sequence data (in the insect) to compare the distribution of the main gene pools in the two associated species and to estimate divergence time in the insect, a proxy for the temporal origin of the interaction. We quantified the similarity in spatial genetic structures by performing a Procrustes analysis, a tool from shape theory. Finally, we simulated recolonization of an empty space analogous to the deglaciated Alps just after ice retreat by two lineages from two species showing unbalanced dependence, to examine how timing of the recolonization process, as well as dispersal capacities of associated species, could explain the observed pattern. Results Contrasting with expectations based on their asymmetrical dependence, patterns in the beetle and plant were congruent at a large scale. Exceptions occurred at a regional scale in areas of admixture, matching known suture zones in Alpine plants. Simulations using a lattice‐based model suggested these empirical patterns arose during or soon after recolonization, long after the estimated origin of the interaction c. 0.5 million years ago. Main conclusions Species‐specific interactions are scarce in alpine habitats because glacial cycles have limited the opportunities for co‐evolution. Their fate, however, remains uncertain under climate change. Here we show that whereas most dispersal routes are paralleled at a large scale, regional incongruence implies that the destinies of the species might differ under changing climate. This may be a consequence of the host dependence of the beetle, which locally limits the establishment of dispersing insects.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Polarized Cell Migration during Cell-to-Cell Transmission of Herpes Simplex Virus in Human Skin Keratinocytes

In addition to transmission involving extracellular free particles, a generally accepted model of virus propagation is one wherein virus replicates in one cell, producing infectious particles that transmit to the next cell via cell junctions or induced polarized contacts. This mechanism of spread is especially important in the presence of neutralizing antibody, and the concept underpins analysis of virus spread, plaque size, viral and host functions, and general mechanisms of virus propagation. Here, we demonstrate a novel process involved in cell-to-cell transmission of herpes simplex virus (HSV) in human skin cells that has not previously been appreciated. Using time-lapse microscopy of fluorescent viruses, we show that HSV infection induces the polarized migration of skin cells into the site of infection. In the presence of neutralizing antibody, uninfected skin cells migrate to the initial site of infection and spread over infected cells to become infected in a spatially confined cluster containing hundreds of cells. The cells in this cluster do not undergo cytocidal cell lysis but harbor abundant enveloped particles within cells and cell-free virus within interstitial regions below the cluster surface. Cells at the base and outside the cluster were generally negative for virus immediate-early expression. We further show, using spatially separated monolayer assays, that at least one component of this induced migration is the paracrine stimulation of a cytotactic response from infected cells to uninfected cells. The existence of this process changes our concept of virus transmission and the potential functions, virus, and host factors involved.     

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

Tempol Moderately Extends Survival in a hSOD1G93A ALS Rat Model by Inhibiting Neuronal Cell Loss,Oxidative Damage and Levels of Non-Native hSOD1G93A Forms

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1^G93A . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat) in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week) extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS.