FLU, a negative feedback regulator of tetrapyrrole biosynthesis, is physically linked to the final steps of the Mg(++)-branch of this pathway

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control. Two effectors of feedback inhibition have been identified, heme and the FLU protein. Inhibition by heme implicates the Fe-branch via regulation of the initial step of tetrapyrrole synthesis. In the present work a FLU-containing chloroplast membrane complex was identified, that besides FLU comprises the four enzymes catalyzing the final steps of chlorophyll synthesis. The results support the notion that FLU links chlorophyll synthesis and the target of feedback control, glutamyl-tRNA reductase, thereby allowing also the Mg-branch to control the initial step of tetrapyrrole synthesis.     

HIV-1 Nef Inhibits Ruffles,Induces Filopodia,and Modulates Migration of Infected Lymphocytes

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.Human immunodeficiency virus type 1 (HIV-1) mostly replicates in T-cell areas of secondary lymphoid organs (SLOs) and induces pathological changes in their architecture. Such changes are likely due to a combination of events, including destruction of T cells, chronic immune activation, and alteration of T-cell motility toward and inside the SLOs (27, 37, 50, 53). Indeed, to fulfill their immune surveillance role, T cells continuously circulate in and out of blood, lymph nodes (LNs), and tissues (60).Lymphocyte recruitment from the bloodstream into LNs depends on three distinct processes, i.e., attachment to high endothelial venules (HEVs), extravasation, and cell migration (10, 60). Adhesion to the endothelium and extracellular matrix (ECM) is a crucial step, regulated in part by β1 integrins, α4β1 (VLA-4) and α5β1, that bind VCAM-1 and/or fibronectin (56). Chemokines and their Gαi-protein-coupled receptors are key regulators of lymphocyte trafficking (32). For instance, CCL19 and CCL21 are constitutively produced by HEVs and by fibroblastic reticular cells of T-cell areas of LNs (21, 28, 29). These two chemokines share the receptor CCR7, expressed by naïve T cells and a fraction of memory T cells (47). They play a major role in lymphocyte homing to LNs, in steady state as well as under conditions of inflammation, and may control T-cell positioning within defined functional compartments (1, 17, 18, 47). CXCR4 and its ligand CXCL12/SDF-1 also contribute to T-cell entry into LNs (5, 23, 40). In addition, effector and memory T cells express a broad range of receptors binding inflammatory chemokines, such as the CCR5 ligands CCL3 (MIP1α), CCL4 (MIP1β), and CCL5 (Rantes).Efficient accomplishment of lymphocyte migration and immune functions requires tight regulation of the cellular cytoskeleton (59). This is mediated by the small GTPases of the Rho subfamily, such as Rho, Rac, and Cdc42 (11, 58). They activate specific actin filament assembly factors to generate sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli) (6). These structures have different functions. Lamellipodia and ruffles are formed during crawling cell motility and spreading. Filopodia protrude from the leading edges of many motile cells. They appear to perform sensory and exploratory functions to steer cells, depending on cues from the environment (42). Moreover, filopodia, or other thin structures called tunneling nanotubes, have been shown to form intercellular bridges, allowing viruses to spread through remote contacts between infected cells and targets (44, 48, 49, 52).HIV-1 hijacks cytoskeleton dynamics in order to ensure viral entry and transport within and egress from target cells (34; reviewed in reference 13). In particular, the viral protein Nef modifies actin remodeling in various cell systems. In T cells, Nef alters actin rearrangements triggered by activation of T-cell (TCR) or chemokine receptors (22, 54). Nef inhibits immunological synapse formation, a dynamic process involving rapid actin modifications (57). Nef also affects plasma membrane plasticity, inducing secretion of microvesicle clusters (33). In macrophages, Nef induces the extension of long intercellular conduits allowing its own transfer to B cells (61). A number of studies have reported that Nef affects T-cell chemotaxis (generally to CXCL12) through the modulation of Rho-GTPase-regulated signaling pathways (7, 24, 39, 54). Migration studies have generally been performed using Nef-expressing cells, and rarely in the context of HIV-1 infection (54). From a molecular standpoint, it has recently been proposed that Nef acts in part by deregulating cofilin, an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (54).In the present study, our goal was to gain further insights into the effect of HIV-1 infection on cytoskeleton dynamics. We used a panel of innovative techniques allowing analysis of cell shape, adhesion, and motility. We report that in HIV-infected lymphocytes, Nef promotes filopodium-like formation while it inhibits membrane ruffling. Nef impairs cell adhesion on the extracellular matrix and decreases intrinsic cell motility. Lymphocyte migration toward various chemokines (CXCL12, CCL3, and CCL19) is also inhibited. Our results suggest that Nef may facilitate viral spread and contribute to AIDS pathogenesis by manipulating the migration of lymphocytes.     

Biological and molecular evidence for the transmission of aster yellows phytoplasma to French marigold (Tagetes patula) by the flatid planthopper Metcalfa pruinosa

Aster yellows (AY) phytoplasmas (Candidatus Phytoplasma asteris) are associated with a number of plant diseases throughout the world. Several insect vectors are responsible for spreading AY diseases resulting in wide distribution and low host specificity. Because the role of sucking insects as vectors of phytoplasmas is widely documented, and the citrus flatid planthopper Metcalfa pruinosa is a phloem feeder, it has been incriminated as a possible vector of phytoplasmas. However, its ability to transfer phytoplasma has not been confirmed. The present work shows that M. pruinosa (Hemiptera: Flatidae), a polyphagous planthopper, is able to vector Ca. P. asteris to French marigold (Tagetes patula). Transmission experiments were conducted in 2017 and 2018 in central Hungary by two approaches: (a) AY-infected M. pruinosa were collected from an area with severe incidence of the disease on T. patula and caged on test plants for an inoculation-access period of 2 weeks, and (b) presumably phytoplasma-free insects were collected from apparently healthy grapevines (Vitis vinifera L.) and fed on AY-infected T. patula plants for 2 weeks prior to being caged on test plants. AY disease symptoms developed on 4 out of 10 and 10 out of 15 test plants, respectively. All phytoplasma-positive marigold and M. pruinosa samples showed identical RFLP patterns and shared 100% 16S rDNA sequence identity with each other and with the aster yellows phytoplasma strain AJ33 (GenBank accession number MK992774). These results indicated that the phytoplasma belonged to the phytoplasma subgroup 16SrI-B Ca. P. asteris. Therefore, the work presented here provides experimental evidence that M. pruinosa is a vector of a 16SrI-B subgroup phytoplasma to T. patula.     

Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs

The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3′ uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.     

Effects of aging on thermoregulatory responses and hormonal changes in humans during the four seasons in Japan

Physiological functions are impaired in various organs in aged people, as manifest by, e.g., renal and cardiac dysfunction and muscle atrophy. The elderly are also at increased risk of both hypothermia and hyperthermia in extreme temperatures. The majority of those over 65 years old have elevated serum osmolality. Our hypothesis is that the elderly have suppressed osmolality control in different seasons compared to the young. Eight healthy young men and six healthy older men participated in this study. The experiments were performed during spring, summer, autumn and winter in Japan, with average atmospheric temperatures of 15–20°C in spring, 25–30°C in summer, 15–23°C in autumn and 5–10°C in winter. Each subject immersed his lower legs in warm water at 40°C for 30 min. Core (tympanic) temperature and sweat rate at chest were recorded continuously. Blood was taken pre-immersion to measure the concentrations of antidiuretic hormone, serum osmolality, plasma renin activity, angiotensin II, aldosterone, leptin, thyroid stimulating hormone, fT₃ and fT₄. The results suggested that the elderly have suppressed osmolality control compared to the young; osmolality was especially elevated in winter compared to the summer in elderly subjects. Therefore, particularly in the elderly, balancing fluid by drinking water should be encouraged to maintain euhydration status in winter.     

Rhizosphere disturbance influences fungal colonization and community development on dead fine roots

Little is known about the community dynamics of fungi on decomposing fine roots, despite the importance of fine roots as a source of carbon to detrital systems in forests. We examined fungal communities on dead roots in a sugar-maple dominated northern hardwood forest to test the hypothesis that community development is sensitive to rhizosphere disruption. We generated cohorts of dead fine roots in root windows and disturbed the rhizosphere microbial community in half of the windows by moving roots into sieved bulk soil. We sampled root fragments repeatedly over time and cultured fungi from these fragments to explore temporal patterns of fungal species composition. Disturbing the root rhizosphere prior to initiating decomposition changed the dominant fungal taxa, the distribution of dominant species within the community, and the temporal development in the culturable fungal community. Dominance in control roots shifted from Neonectria in early decay to Umbelopsis in later decay. Disturbance roots were more evenly dominated over time by Trichoderma, Neonectria, another species of Umbelopsis, and Pochonia. Our results suggest that species interactions are important in the ecology of fine root decay fungi, with the rhizosphere community of the living root influencing development of the decay community.     

Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman’s assay

Ellman’s assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1 M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman’s assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman’s method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman’s method into two successive steps that may be convenient for some applications.