Quinolobactin, a New Siderophore of Pseudomonas fluorescens ATCC 17400, the Production of Which Is Repressed by the Cognate Pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of ⁵⁹Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Loss of prion protein control of glucose metabolism promotes neurodegeneration in model of prion diseases

Corruption of cellular prion protein (PrP^C) function(s) at the plasma membrane of neurons is at the root of prion diseases, such as Creutzfeldt-Jakob disease and its variant in humans, and Bovine Spongiform Encephalopathies, better known as mad cow disease, in cattle. The roles exerted by PrP^C, however, remain poorly elucidated. With the perspective to grasp the molecular pathways of neurodegeneration occurring in prion diseases, and to identify therapeutic targets, achieving a better understanding of PrP^C roles is a priority. Based on global approaches that compare the proteome and metabolome of the PrP^C expressing 1C11 neuronal stem cell line to those of PrP^null-1C11 cells stably repressed for PrP^C expression, we here unravel that PrP^C contributes to the regulation of the energetic metabolism by orienting cells towards mitochondrial oxidative degradation of glucose. Through its coupling to cAMP/protein kinase A signaling, PrP^C tones down the expression of the pyruvate dehydrogenase kinase 4 (PDK4). Such an event favors the transfer of pyruvate into mitochondria and its conversion into acetyl-CoA by the pyruvate dehydrogenase complex and, thereby, limits fatty acids β-oxidation and subsequent onset of oxidative stress conditions. The corruption of PrP^C metabolic role by pathogenic prions PrP^Sc causes in the mouse hippocampus an imbalance between glucose oxidative degradation and fatty acids β-oxidation in a PDK4-dependent manner. The inhibition of PDK4 extends the survival of prion-infected mice, supporting that PrP^Sc-induced deregulation of PDK4 activity and subsequent metabolic derangements contribute to prion diseases. Our study posits PDK4 as a potential therapeutic target to fight against prion diseases.     

Building of an immunosensor: how can the composition and structure of the thiol attachment layer affect the immunosensor efficiency?

Immunosensors, based on the immobilization of a model rabbit antibody on mixed self-assembled monolayers and Protein A as a linking agent on gold transducers, were elaborated and characterized at each step by modulated polarization-infrared spectroscopy (PM-IRRAS) and occasionally by atomic force microscopy (AFM) and quartz crystal microbalance (QCM). By testing two different mixed SAMs comprising 11-mercaptoundecanoic acid (MUA), together with either decanethiol (C9CH3) or mercaptohexanol (C6OH), the role of the chemical composition and structure of the antibody attachment layer upon the sensor performance was demonstrated.     

Thymic nurse cells in culture: morphological and antigenic characterization

Epithelial monolayers were derived from thymic nurse cells (TNC), and were seeded onto collagen-coated dishes immediately after their isolation from young adult C3H-murine thymuses. Different media and supplements were tested in order to obtain cultures that were as pure as possible. Primary cultures were enriched in epithelial cells but always contained non-epithelial components among which fibroblasts predominated. Immunodetection of keratins, and repeated light- and electron-microscopic observations established the epithelial nature of the elongated cells derived from TNC; these elongated cells were cortical reticular cells, and were different from medullary globular cells that immediately adopted a mosaic pattern in vitro. At the beginning of the culture, the necrosis of cortical lymphocytes appeared to be toxic for epithelial cells; when epithelial cells survived, they showed a temporary lipid accumulation. After a 5-day culture, they still synthesized DNA but lost this capacity thereafter and dedifferentiated. The lympho-epithelial symbiosis appeared to be necessary to maintain some epithelial characteristics of the cultured cells, such as the clear vesicles and the expression of la antigens. In sub-cultures, the monolayers were almost purely epithelial in nature but growth was no longer observed. The cells remained reticular in shape, as they were in vivo, but their cytoplasm and their nucleus became larger and numerous cells were multinucleated. Confluence was not obtained with classical media even after mitogenic stimulation. The frequent observation of strongly keratinized areas suggested a process of terminal differentiation; this could not be avoided by using low serum concentration.     

Long-term effects of continuous direct seeding mulch-based cropping systems on soil nitrogen supply in the Cerrado region of Brazil

In the Cerrado region of Brazil conventional soybean monoculture is since the 1980s being replaced by direct seeding mulch-based cropping (DMC) with two crops per year and absence of tillage practices. The objective of this study was to assess the long-term impact of DMC on soil organic matter accumulation and nitrogen (N) mineralization. Measurements of soil organic carbon (C) content, soil total N content and soil N mineralization, both under laboratory conditions using disturbed soil samples and under field conditions using intact soil cores were conducted on a chronosequence of 2-, 6-, 9- and 14-year-old DMC fields (DMC-2, DMC-6, DMC-9 and DMC-14, respectively). The average increase of organic C in the 0–30 cm topsoil layer under DMC was 1.91 Mg C ha⁻¹ year⁻¹. Soil total N increased with 103 kg N ha⁻¹ year⁻¹ (0–30 cm). The potential N mineralization rate under laboratory conditions (28°C, 75% of soil moisture at field capacity) was 0.27, 0.28, 0.39 and 0.36 mg N kg soil⁻¹ day⁻¹ for, respectively, the DMC-2, DMC-6, DMC-9 and DMC-14 soils. The corresponding specific N mineralization rates were 0.16, 0.15, 0.22 and 0.17 mg N g N⁻¹ day⁻¹. There was no obvious explanation for the higher specific N mineralization rate of soils under DMC-9, given the similar soil conditions and land-use history before DMC was introduced. Results from the in situ N incubation experiments were in good agreement with those from the laboratory incubations. We estimated that soil N mineralization increases with about 2.0 kg N ha⁻¹ year⁻¹ under DMC. The increase was mainly attributed to the larger soil total N content. These results indicate that even in the medium term (10 years), continuous DMC cropping has limited implications for N fertilization recommendations, since the extra soil N supply represents less than 20% of the common N fertilization dose for maize in the region.     

Outbreak of Cutaneous Leishmaniasis among military personnel in French Guiana, 2020: Clinical,phylogenetic, individual and environmental aspects

BackgroundCutaneous Leishmaniasis (CL) is endemic in French Guiana but cases are usually sporadic. An outbreak signal was issued on May 15^th 2020 with 15 suspected cases after a military training course in the rainforest. An outbreak investigation was carried out.Methodology/Principal findingsThirty cases were confirmed. Leishmania guyanensis was the most frequent species (90%). The most frequent presentation was ulcerative (90%). Lesions on the face and hands were frequent (40% each). Eight cases (26%) presented a poor outcome after treatment with pentamidine and required a second line with amphotericin B. Three of them required further treatments with meglumine antimoniate or miltefosine. Two spots within the training area were deemed as likely sites of contamination, due to illegal logging. The isolated Leishmania strains did not form a separate cluster. Participation in Week 13 of year 2020 was associated with infection (OR = 4.59 [1.10–19.83]; p = 0.016) while undergoing only the “Fighting” exercise was protective (OR = 0.1 [0–0.74]; p = 0.021). There was no association between infection and other risk factors at the individual level. The attack rate of Regiment B (14/105 = 13.3%) was significantly higher (OR = 4.22 [1.84–9.53], p = 0.0001) compared to Regiment A (16/507 = 3.2%). The attack rate during this training course (30/858 = 3.5%) was significantly higher (OR 2.29 [1.28–4.13]; p = 0.002) than for other missions in French Guiana during the same period (22/1427 = 1.5%).ConclusionsThis outbreak could be explained by a combination of factors: climatic conditions around week 13, at-risk activities including night trainings, absence of impregnation, a lesser experience of rainforest duties in Regiment B and illegal logging attracting sandflies on military training grounds.     

A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer’s disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.     

Effector memory CD8 T cell response elicits Hepatitis E Virus genotype 3 pathogenesis in the elderly

Genotype 3 Hepatitis E virus (HEV-3) is an emerging threat for aging population. More than one third of older infected patients develops clinical symptoms with severe liver damage, while others remain asymptomatic. The origin of this discrepancy is still elusive although HEV-3 pathogenesis appears to be immune-mediated. Therefore, we investigated the role of CD8 T cells in the outcome of the infection in immunocompetent elderly subjects. We enrolled twenty two HEV-3-infected patients displaying similar viral determinants and fifteen healthy donors. Among the infected group, sixteen patients experienced clinical symptoms related to liver disease while six remained asymptomatic. Here we report that symptomatic infection is characterized by an expansion of highly activated effector memory CD8 T (EM) cells, regardless of antigen specificity. This robust activation is associated with key features of early T cell exhaustion including a loss in polyfunctional type-1 cytokine production and partial commitment to type-2 cells. In addition, we show that bystander activation of EM cells seems to be dependent on the inflammatory cytokines IL-15 and IL-18, and is supported by an upregulation of the activating receptor NKG2D and an exuberant expression of T-Bet and T-Bet-regulated genes including granzyme B and CXCR3. We also show that the inflammatory chemokines CXCL9-10 are increased in symptomatic patients thereby fostering the recruitment of highly cytotoxic EM cells into the liver in a CXCR3-dependent manner. Finally, we find that the EM-biased immune response returns to homeostasis following viral clearance and disease resolution, further linking the EM cells response to viral burden. Conversely, asymptomatic patients are endowed with low-to-moderate EM cell response. In summary, our findings define immune correlates that contribute to HEV-3 pathogenesis and emphasize the central role of EM cells in governing the outcome of the infection.     

Neural Androgen Receptor Deletion Impairs the Temporal Processing of Objects and Hippocampal CA1-Dependent Mechanisms

We studied the role of testosterone, mediated by the androgen receptor (AR), in modulating temporal order memory for visual objects. For this purpose, we used male mice lacking AR specifically in the nervous system. Control and mutant males were gonadectomized at adulthood and supplemented with equivalent amounts of testosterone in order to normalize their hormonal levels. We found that neural AR deletion selectively impaired the processing of temporal information for visual objects, without affecting classical object recognition or anxiety-like behavior and circulating corticosterone levels, which remained similar to those in control males. Thus, mutant males were unable to discriminate between the most recently seen object and previously seen objects, whereas their control littermates showed more interest in exploring previously seen objects. Because the hippocampal CA1 area has been associated with temporal memory for visual objects, we investigated whether neural AR deletion altered the functionality of this region. Electrophysiological analysis showed that neural AR deletion affected basal glutamate synaptic transmission and decreased the magnitude of N-methyl-D-aspartate receptor (NMDAR) activation and high-frequency stimulation-induced long-term potentiation. The impairment of NMDAR function was not due to changes in protein levels of receptor. These results provide the first evidence for the modulation of temporal processing of information for visual objects by androgens, via AR activation, possibly through regulation of NMDAR signaling in the CA1 area in male mice.     

Mutant loxP vectors for selectable marker recycle and conditional knock-outs

Background  

Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP.