Protein complexes associated with β-catenin differentially influence the differentiation profile of neonatal and adult CD8+ T cells

The canonical Wnt signaling pathway is a master cell regulator involved in CD8⁺ T cell proliferation and differentiation. In human CD8⁺ T cells, this pathway induces differentiation into memory cells or a “stem cell memory like” population, which is preferentially present in cord blood. To better understand the role of canonical Wnt signals in neonatal or adult blood, we compared the proteins associated with β-catenin, in nonstimulated and Wnt3a-stimulated human neonatal and adult naive CD8⁺ T cells. Differentially recruited proteins established different complexes in adult and neonatal cells. In the former, β-catenin-associated proteins were linked to cell signaling and immunological functions, whereas those of neonates were linked to proliferation and metabolism. Wnt3a stimulation led to the recruitment and overexpression of Wnt11 in adult cells and Wnt5a in neonatal cells, suggesting a differential connexion with planar polarity and Wnt/Ca²⁺ noncanonical pathways, respectively. The chromatin immunoprecipitation polymerase chain reaction β-catenin was recruited to a higher level on the promoters of cell renewal genes in neonatal cells and of differentiation genes in those of adults. We found a preferential association of β-catenin with CBP in neonatal cells and with p300 in the adult samples, which could be involved in a higher self-renewal capacity of the neonatal cells and memory commitment in those of adults. Altogether, our results show that different proteins associated with β-catenin during Wnt3a activation mediate a differential response of neonatal and adult human CD8⁺ T cells.     

A field portable method for the semi‐quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (F_v/fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt‐solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50–200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial F_v/fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi‐quantitative expression of DHT that facilitates comparisons of species with different morpho‐physiological traits and/or ecological attributes.     

Effect of stirring speed on the production of phenolic secondary metabolites and growth of Buddleja cordata cells cultured in mechanically agitated bioreactor

Plant Cell, Tissue and Organ Culture (PCTOC) - Shake-flask in vitro culture of Buddleja cordata cells produces large amounts of biomass and synthetizes verbascoside (VB), linarin and...     

Improvement of almond production using Bombus terrestris (Hymenoptera: Apidae) in Mediterranean conditions

Almond trees are one of the most important crops in the Balearic Islands. The pollination of almonds is limited to the activity of insects, and cross‐pollination is necessary for fruit development. Currently, honey bees and wild bee populations are declining considerably due to multiple causes, such as the use of pesticides, diseases and habitat loss. An alternative to increase the almond production is the use of commercial pollinators. In this long‐term (3 years) study, the effect of the introduction of Bombus terrestris colonies on almond production was evaluated in two orchards. Two experimental designs were carried out to study the best management of this pollinator. For 2 years, all bumble bee colonies were placed in the middle of the plot and during the last year, the bumble bee colonies were distributed homogenously in the plot. Fruit set and the foraging behaviour of bumble bees during the blossoming period was determined, and the effect of different environmental variables on the visitation rate of bumble bees was assessed by means of a generalized linear mixed model (GLMM). Moreover, for the first time, the spatial distribution of fruit set was evaluated. Our results show that fruit set was significantly higher in the fields where B. terrestris had been introduced than in the control plots. This increased production resulted in a positive economic balance for the farmer. Moreover, bumble bees showed to prefer trees in a southwest orientation that were close to their colony. The activity of bumble bees showed to be significantly influenced by wind speed (the higher the speed the more flowers are visited by B. terrestris) and time after flowering (visitation rate decreased with days after flowering). In order to improve its management and obtain the highest possible almond production, it is important to understand the activity and behaviour of this pollinator.     

Deeplasmid: deep learning accurately separates plasmids from bacterial chromosomes

Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC–ROC of over 89%, and it was more accurate than five other plasmid classification methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 kb long plasmid, demonstrating Deeplasmid''s ability to detect novel plasmids.     

FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg²⁺ at the subunit interface, a K⁺ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Bacterial cell division critically relies on the tubulin homolog FtsZ, which assembles into filaments that treadmill, fuelled by GTP hydrolysis. This structural and biochemical study of FtsZ from Staphylocuccus aureus reveals the mechanism of GTP hydrolysis and its connection with filament dynamics.     

Neuronal and Glial Expression of the Multidrug Resistance Gene Product in an Experimental Epilepsy Model

1. Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood. It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy. 2. Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy. Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p. for either 4 days (MP-4) or 7 days (MP-7). Control rats received an equivalent volume of vehicle. One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170. As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group. 3. Increased Pgp-170 immunoreactivity was detected in MP-treated animals. Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats. Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution. MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling. 4. Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures. 5. The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.