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Competing Interests: Nil.

Pyoderma gangrenosum (PG) is a rare neutrophilic dermatosis. Half of the cases are associated with an immune dysfunction and are frequently triggered by pathergy such as a tissular aggression via surgery or burn wounds. A patient with ulcerative colitis presented a PG at the site of an iontophoresis patch for tendinopathy. Treatment in a specialized burn center, corticosteroid therapy and adapted local care contributed to a favourable evolution. PG remains a diagnosis of exclusion and inflammatory phenomena must be differentiated from infectious causes such as necrotizing fasciitis to initiate immunosuppressive treatment. Being rare and difficult to diagnose and to treat as well as associated with potentially severe sequelae, a multidisciplinary team is required for the management of PG.

There are substantial concerns about impaired honey bee health and colony losses due to several poorly understood factors. We used MALDI profiling (MALDI BeeTyping®) analysis to investigate how some environmental and management factors under field conditions across Europe affected the honey bee haemolymph peptidome (all peptides in the circulatory fluid), as a profile of molecular markers representing the immune status of Apis mellifera. Honey bees were exposed to a range of environmental stressors in 128 agricultural sites across eight European countries in four biogeographic zones, with each country contributing eight sites each for two different cropping systems: oilseed rape (OSR) and apple (APP). The full haemolymph peptide profiles, including the presence and levels of three key immunity markers, namely the antimicrobial peptides (AMPs) Apidaecin, Abaecin and Defensin-1, allowed the honey bee responses to environmental variables to be discriminated by country, crop type and site. When considering just the AMPs, it was not possible to distinguish between countries by the prevalence of each AMP in the samples. However, it was possible to discriminate between countries on the amounts of the AMPs, with the Swedish samples in particular expressing high amounts of all AMPs. A machine learning model was developed to discriminate the haemolymphs of bees from APP and OSR sites. The model was 90.6 % accurate in identifying the crop type from the samples used to build the model. Overall, MALDI BeeTyping® of bee haemolymph represents a promising and cost-effective "blood test" for simultaneously monitoring dozens of peptide markers affected by environmental stressors at the landscape scale, thus providing policymakers with new diagnostic and regulatory tools for monitoring bee health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Importance: Pain is a common out-of-hospital symptom among patients, and opioids are often prescribed. Research suggests that overprescribing for acute traumatic pain is still prevalent, even when limits restricting opioid prescriptions have been implemented. Ketamine hydrochloride is an alternative to opioids in adults with out-of-hospital traumatic pain., Objective: To assess the noninferiority of intravenous ketamine compared with intravenous morphine sulfate to provide pain relief in adults with out-of-hospital traumatic pain., Design, Setting, and Participants: The Intravenous Subdissociative-Dose Ketamine Versus Morphine for Prehospital Analgesia (KETAMORPH) study was a multicenter, single-blind, noninferiority randomized clinical trial comparing ketamine hydrochloride (20 mg, followed by 10 mg every 5 minutes) with morphine sulfate (2 or 3 mg every 5 minutes) in adult patients with out-of-hospital trauma and a verbal pain score equal to or greater than 5. Enrollment occurred from November 23, 2017, to November 26, 2022, in 11 French out-of-hospital emergency medical units., Interventions: Patients were randomly assigned to ketamine (n = 128) or morphine (n = 123)., Main Outcomes and Measures: The primary outcome was the between-group difference in mean change in verbal rating scale pain scores measured from the time before administration of the study drug to 30 minutes later. A noninferiority margin of 1.3 was chosen., Results: A total of 251 patients were randomized (median age, 51 [IQR, 34-69] years; 111 women [44.9%] and 140 men [55.1%] among the 247 with data available) and were included in the intention-to-treat population. The mean pain score change was -3.7 (95% CI, -4.2 to -3.2) in the ketamine group compared with -3.8 (95% CI, -4.2 to -3.4) in the morphine group. The difference in mean pain score change was 0.1 (95% CI, -0.7 to 0.9) points. There were no clinically meaningful differences for vital signs between the 2 groups. The intravenous morphine group had 19 of 113 (16.8% [95% CI, 10.4%-25.0%]) adverse effects reported (most commonly nausea [12 of 113 (10.6%)]) compared with 49 of 120 (40.8% [95% CI, 32.0%-49.6%]) in the ketamine group (most commonly emergence phenomenon [24 of 120 (20.0%)]). No adverse events required intervention., Conclusions and Relevance: In the KETAMORPH study of patients with out-of-hospital traumatic pain, the use of intravenous ketamine compared with morphine showed noninferiority for pain reduction. In the ongoing opioid crisis, ketamine administered alone is an alternative to opioids in adults with out-of-hospital traumatic pain., Trial Registration: ClinicalTrials.gov Identifier: NCT03236805.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections. For this study, the susceptibility profiles to antipseudomonal antibiotics and a quaternary ammonium compound, didecyldimethylammonium chloride (DDAC), widely used as a disinfectant, were established for 180 selected human and environmental hospital strains isolated between 2011 and 2020. Furthermore, a genomic study determined resistome and clonal putative relatedness for 77 of them. During the ten-year study period, it was estimated that 9.5% of patients' strains were resistant to carbapenems, 11.9% were multidrug-resistant (MDR), and 0.7% were extensively drug-resistant (XDR). Decreased susceptibility (DS) to DDAC was observed for 28.0% of strains, a phenotype significantly associated with MDR/XDR profiles and from hospital environmental samples (p < 0.0001). According to genomic analyses, the P. aeruginosa population unsusceptible to carbapenems and/or to DDAC was diverse but mainly belonged to top ten high-risk clones described worldwide by del Barrio-Tofiño et al. The carbapenem resistance appeared mainly due to the production of the VIM-2 carbapenemase (39.3%) and DS to DDAC mediated by MexAB-OprM pump efflux overexpression. This study highlights the diversity of MDR/XDR populations of P. aeruginosa which are unsusceptible to compounds that are widely used in medicine and hospital disinfection and are probably distributed in hospitals worldwide., (© 2023. The Author(s).)

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections in humans. This bacterium is less represented in veterinary medicine, despite causing difficult-to-treat infections due to its capacity to acquire antimicrobial resistance, produce biofilms, and persist in the environment, along with its limited number of veterinary antibiotic therapies. Here, we explored susceptibility profiles to antibiotics and to didecyldimethylammonium chloride (DDAC), a quaternary ammonium widely used as a disinfectant, in 168 P. aeruginosa strains isolated from animals, mainly Equidae. A genomic study was performed on 41 of these strains to determine their serotype, sequence type (ST), relatedness, and resistome. Overall, 7.7% of animal strains were resistant to carbapenems, 10.1% presented a multidrug-resistant (MDR) profile, and 11.3% showed decreased susceptibility (DS) to DDAC. Genomic analyses revealed that the study population was diverse, and 4.9% were ST235, which is considered the most relevant human high-risk clone worldwide. This study found P. aeruginosa populations with carbapenem resistance, multidrug resistance, and DS to DDAC in equine and canine isolates. These strains, which are not susceptible to antibiotics used in veterinary and human medicine, warrant close the setting up of a clone monitoring, based on that already in place in human medicine, in a one-health approach.

Transition metals are essential for a wealth of metabolic reactions, but their concentrations need to be tightly controlled across cells and cell compartments, as metal excess or imbalance has deleterious effects. Metal homeostasis is achieved by a combination of metal transport across membranes and metal binding to a variety of molecules. Gene duplication is a key process in evolution, as emergence of advantageous mutations on one of the copies can confer a new function. Here, we report that the poplar genome contains two paralogues encoding NRAMP3 metal transporters localized in tandem. All Populus species analyzed had two copies of NRAMP3, whereas only one could be identified in Salix species indicating that duplication occurred when the two genera separated. Both copies are under purifying selection and encode functional transporters, as shown by expression in the yeast heterologous expression system. However, genetic complementation revealed that only one of the paralogues has retained the original function in release of metals stored in the vacuole previously characterized in A. thaliana. Confocal imaging showed that the other copy has acquired a distinct localization to the Trans Golgi Network (TGN). Expression in poplar suggested that the copy of NRAMP3 localized on the TGN has a novel function in the control of cell-to-cell transport of manganese. This work provides a clear case of neo-functionalization through change in the subcellular localization of a metal transporter as well as evidence for the involvement of the secretory pathway in cell-to-cell transport of manganese., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The epidemiology of severe PAD, as characterized by short-distance intermittent claudication (IC) and chronic limb-threatening ischemia (CLTI), remains undefined in New Zealand (NZ). This was a retrospective observational cohort study of the Midland region in NZ, including all lower limb PAD-related surgical and percutaneous interventions between the 1st of January 2010 and the 31st of December 2021. Overall, 2541 patients were included. The mean annual incidence of short-distance IC was 15.8 per 100,000, and of CLTI was 36.2 per 100,000 population. The annual incidence of both conditions was greater in men. Women presented 3 years older with PAD (p < 0.001). Patients with short-distance IC had lower ipsilateral major limb amputation at 30 days compared to CLTI (IC 2, 0.3% vs. CLTI 298, 16.7%, p < 0.001). The 30-day mortality was greater in elderly patients (<65 years 2.7% vs. ≥65 years 4.4%, p = 0.049), but did not differ depending on sex (females 36, 3.7% vs. males 64, 4.1%, p = 0.787). Elderly age was associated with a worse survival for both short-distance IC and CLTI. There was a worse survival for females with CLTI. In conclusion, PAD imposes a significant burden in NZ, and further research is required in order to reduce this disparity.

During multicellularization, plants evolved unique cell-cell connections, the plasmodesmata (PD). PD of angiosperms are complex cellular domains, embedded in the cell wall and consisting of multiple membranes and a large number of proteins. From the beginning, it had been assumed that PD provide passage for a wide range of molecules, from ions to metabolites and hormones, to RNAs and even proteins. In the context of assimilate allocation, it has been hypothesized that sucrose produced in mesophyll cells is transported via PD from cell to cell down a concentration gradient towards the phloem. Entry into the sieve element companion cell complex (SECCC) is then mediated on three potential routes, depending on the species and conditions, - either via diffusion across PD, after conversion to raffinose via PD using a polymer trap mechanism, or via a set of transporters which secrete sucrose from one cell and secondary active uptake into the SECCC. Multiple loading mechanisms can likely coexist. We here review the current knowledge regarding photoassimilate transport across PD between cells as a prerequisite for translocation from leaves to recipient organs, in particular roots and developing seeds. We summarize the state-of-the-art in protein composition, structure, transport mechanism and regulation of PD to apprehend their functions in carbohydrate allocation. Since many aspects of PD biology remain elusive, we highlight areas that require new approaches and technologies to advance our understanding of these enigmatic and important cell-cell connections., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus that emerges as an important opportunistic pathogen. However, little is known about the regulation underlying the transition from commensal to virulent state. Based on knowledge of S. aureus virulence, we suspected that the agr quorum sensing system may be an important determinant for the pathogenicity of S. lugdunensis . We investigated the functions of the transcriptional regulator AgrA using the agrA deletion mutant. AgrA played a role in cell pigmentation: Δ argA mutant colonies were white while the parental strains were slightly yellow. Compared with the wild-type strain, the Δ argA mutant was affected in its ability to form biofilm and was less able to survive in mice macrophages. Moreover, the growth of Δ agrA was significantly reduced by the addition of 10% NaCl or 0.4 mM H 2 O 2 and its survival after 2 h in the presence of 1 mM H 2 O 2 was more than 10-fold reduced. To explore the mechanisms involved beyond these phenotypes, the Δ agrA proteome and transcriptome were characterized by mass spectrometry and RNA-Seq. We found that AgrA controlled several virulence factors as well as stress-response factors, which are well correlated with the reduced resistance of the Δ agrA mutant to osmotic and oxidative stresses. These results were not the consequence of the deregulation of RNAIII of the agr system, since no phenotype or alteration of the proteomic profile has been observed for the ΔRNAIII mutant. Altogether, our results highlighted that the AgrA regulator of S. lugdunensis played a key role in its ability to become pathogenic. IMPORTANCE Although belonging to the natural human skin flora, Staphylococcus lugdunensis is recognized as a particularly aggressive and destructive pathogen. This study aimed to characterize the role of the response regulator AgrA, which is a component of the quorum-sensing agr system and known to be a major element in the regulation of pathogenicity and biofilm formation in Staphylococcus aureus. In the present study, we showed that, contrary to S. aureus, the agrA deletion mutant produced less biofilm. Inactivation of agrA conferred a white colony phenotype and impacted S. lugdunensis in its ability to survive in mice macrophages and to cope with osmotic and oxidative stresses. By global proteomic and transcriptomic approaches, we identified the AgrA regulon, bringing molecular bases underlying the observed phenotypes. Together, our data showed the importance of AgrA in the opportunistic pathogenic behavior of S. lugdunensis allowing it to be considered as an interesting therapeutic target.

Trapeziometacarpal prostheses have been used in the treatment of first carpometacarpal joint osteoarthritis for many years. No studies have demonstrated statistical superiority over gold-standard trapeziectomy, but they have been proved to enable shorter convalescence, better pain relief and faster functional recovery. The aims of the present study were to report functional results in a large cohort treated with the Touch® new-generation dual mobility trapeziometacarpal prosthesis, with comparison to results in the literature. A retrospective study included 92 Touch® prostheses. Assessment comprised pre- and post-operative pain, QuickDASH score and satisfaction rate. Mean follow-up was 1.33 ± 0.4 years. Pain significantly improved after surgery. Functional QuickDASH scores did not significantly differ from those reported in the age-matched general population. Return to work was fast, at 2.6 months. Satisfaction scores were high. There were no major complications such as dislocation, fracture or loosening, but the rate of De Quervain's tenosynovitis was higher than in other studies. The Touch® prosthesis appeared to be a safe and stable implant, providing good satisfaction and very good functional scores and fast return to work and leisure activity. Considering the high rate of postoperative De Quervain's tenosynovitis, we suggest opening the first sheath of the extensors tendons while positioning the prosthesis., (Copyright © 2021 SFCM. Published by Elsevier Masson SAS. All rights reserved.)

The adherent-invasive Escherichia coli (AIEC) pathotype has been implicated in the pathogenesis of inflammatory bowel diseases in general and in Crohn's disease (CD) in particular. AIEC strains are primarily characterized by their ability to adhere to and invade intestinal epithelial cells. However, the genetic and phenotypic features of AIEC isolates vary greatly as a function of the strain's clonality, host factors, and the gut microenvironment. It is thus essential to identify the determinants of AIEC pathogenicity and understand their role in intestinal epithelial barrier dysfunction and inflammation. We reasoned that soil nematode Caenorhabditis elegans (a simple but powerful model of host-bacterium interactions) could be used to study the virulence of AIEC vs. non- AIEC E. coli strains. Indeed, we found that the colonization of C. elegans (strain N2) by E. coli impacted survival in a strain-specific manner. Moreover, the AIEC strains' ability to invade cells in vitro was linked to the median lifespan in C. elegans (strain PX627). However, neither the E. coli intrinsic invasiveness (i.e., the fact for an individual strain to be characterized as invasive or not) nor AIEC's virulence levels (i.e., the intensity of invasion, established in % from the infectious inoculum) in intestinal epithelial cells was correlated with C. elegans ' lifespan in the killing assay. Nevertheless, AIEC longevity of C. elegans might be a relevant model for screening anti-adhesion drugs and anti-invasive probiotics.

Background: Staphylococcus lugdunensis is a coagulase-negative Staphylococcus part of the commensal skin flora but emerge as an important opportunistic pathogen. Because iron limitation is a crucial stress during infectious process, we performed phenotypic study and compared proteomic profiles of this species incubated in absence and in presence of the iron chelator 2,2'-dipyridyl (DIP)., Results: No modification of cell morphology nor cell wall thickness were observed in presence of DIP. However iron-limitation condition promoted biofilm formation and reduced the ability to cope with oxidative stress (1 mM H 2 O 2 ). In addition, S. lugdunensis N920143 cultured with DIP was significantly less virulent in the larvae of Galleria mellonella model of infection than that grown under standard conditions. We verified that these phenotypes were due to an iron limitation by complementation experiments with FeSO 4 . By mass spectrometry after trypsin digestion, we characterized the first iron-limitation stress proteome in S. lugdunensis. Among 1426 proteins identified, 349 polypeptides were differentially expressed. 222 were more and 127 less abundant in S. lugdunensis incubated in iron-limitation condition, and by RT-qPCR, some of the corresponding genes have been shown to be transcriptionally regulated. Our data revealed that proteins involved in iron metabolism and carriers were over-expressed, as well as several ABC transporters and polypeptides linked to cell wall metabolism. Conversely, enzymes playing a role in the oxidative stress response (especially catalase) were repressed., Conclusions: This phenotypic and global proteomic study allowed characterization of the response of S. lugdunensis to iron-limitation. We showed that iron-limitation promoted biofilm formation, but decrease the oxidative stress resistance that may, at least in part, explained the reduced virulence of S. lugdunensis observed under low iron condition.

MAIN CONCLUSION: pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were identified and their strength and specificity were compared to those of previously described promoters in this species. Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and derivatives in tall glandular trichomes, which actively synthesize and secrete specialized metabolites. However, implementation of an entire biosynthetic pathway in glandular trichomes requires the identification of trichome-specific promoters to appropriately drive the expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR analysis was carried out on wild-type N. tabacum plants to compare the expression pattern and gene expression level of NtRbcS-T1 and NtMALD1, two newly identified genes expressed in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which were reported in the literature to be specifically expressed in glandular trichomes. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 is also expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by Agrobacterium-mediated transformation. Almost all transgenic lines displayed GUS activity in tall glandular trichomes, indicating that the appropriate cis regulatory elements were included in the selected promoter regions. However, unlike for the other promoters, no trichome-specific line was obtained for pNtLTP1:GUS-VENUS, in agreement with the RT-qPCR data. These data thus provide two new transcription promoters that could be used in metabolic engineering of glandular trichomes.

Purpose: To assess the clinical efficacy of endovascular angiosome-oriented wound-targeted revascularization (WTR) vs indirect (wound-indifferent) revascularization (IR) in diabetic patients with neuroischemic foot ulcers. Materials and Methods: Between April 2009 and July 2017, 167 diabetic patients (mean age 72.8 years; 137 men) with chronic limb-threatening ischemia (Rutherford category 5) and foot wounds (Wagner 2-4) in 194 limbs were prospectively registered and scheduled for primary infragenicular endovascular treatment. Specific angiosome source artery reperfusion sustained by patent foot arches or arterial-arterial connections was attempted initially. If this approach failed, topographic revascularization via available collaterals (WTRc) and IR were sequentially attempted. Results: Reperfusion was successful in 176 (91%) of 194 limbs (113 with WTR, 28 with WTRc, and 35 with IR); the global angiosome-oriented technical success (WTR and WTRc) was 73% (141/194). The mean follow-up was 10.9±0.7 months (range 3-12.5). Over 1 year, 102 (58%) of the 176 successfully treated limbs experienced wound healing [79/113 (70%) in the WTR group, 15/28 (54%) in the WTRc group, and 7/35 (20%) in the IR group; p=0.011]. The mean time to healing was 6.8±0.4 months in the WTR group, 7.9±0.6 months in the WTRc group, and 9.8±0.7 months in the IR group (p=0.001). Relapses were noted in 18 (16%) WTR limbs, 5 (18%) WTRc limbs, and 6 (17%) IR limbs. Comparison between WTR and IR and WTRc vs IR showed improved cicatrization in the angiosome-oriented groups (p<0.05). Major adverse limb events (MALE) and limb salvage were different between WTR and WTRc and between WTR and IR groups (p<0.05), while WTRc vs IR was not. Amputation-free survival was not influenced by the revascularization strategy (p=0.093). Conclusion: Wound healing in diabetic patients with chronic limb-threatening ischemia appeared to be improved by intentional wound-targeted revascularization, but no uniform benefit concerning MALE or limb preservation was observed. IR still represents an alternative for limb salvage in cases in which angiosome-guided revascularization fails.

Nutrient recycling and mobilization from organ to organ all along the plant lifespan is essential for plant survival under changing environments. Nutrient remobilization to the seeds is also essential for good seed production. In this review, we summarize the recent advances made to understand how plants manage nutrient remobilization from senescing organs to sink tissues and what is the contribution of autophagy in this process. Plant engineering manipulating autophagy for better yield and plant tolerance to stresses will be presented., Competing Interests: The authors declare no conflict of interest.

An original immuno-regulatory strategy against inflammatory bowel diseases based on the use of 28 kDa glutathione S-transferase (P28GST), a unique schistosome protein, was recently proposed. Improvement of intestinal inflammation occurs through restoration of the immunological balance between pro-inflammatory T-helper 1 (Th1) responses and both T-helper 2 (Th2) and regulatory responses. However, detailed mechanisms explaining how P28GST prevents colitis and promotes gut homeostasis remain unknown. Considering the complex interplay between the adaptive and innate immune system and the intestinal microbiota, we raised the question of the possible role of the microbial ecosystem in the anti-inflammatory effects mediated by the helminth-derived P28GST protein. We first analyzed, by 16S rRNA sequencing, the bacterial profiles of mice fecal microbiota at several time points of the P28GST-immunomodulation period prior to trinitrobenzene sulfonic acid (TNBS)-colitis. The influence of gut microbiota in the P28GST-mediated anti-inflammatory effects was then assessed by fecal microbiota transplantation experiments from P28GST-immunized mice to either conventional or microbiota depleted naïve recipient mice. Finally, the experimental data were supplemented by the temporal fecal microbiota compositions of P28GST-treated Crohn's disease patients from a pilot clinical study (NCT02281916). The P28GST administration slightly modulated the diversity and composition of mouse fecal microbiota while it significantly reduced experimental colitis in mice. Fecal microbiota transplantation experiments failed to restore the P28GST-induced anti-inflammatory effects. In Crohn's disease patients, P28GST also induced slight changes in their overall fecal bacterial composition. Collectively, these results provide key elements in both the anti-inflammatory mechanisms and the safe therapeutic use of immunomodulation with such promising helminth-derived molecules., Competing Interests: None of the authors of this manuscript have a financial interest related to this work.

Micronutrient deficiencies affect a large part of the world's population. These deficiencies are mostly due to the consumption of grains with insufficient content of iron (Fe) or zinc (Zn). Both de novo uptake by roots and recycling from leaves may provide seeds with nutrients. Autophagy, which is a conserved mechanism for nutrient recycling in eukaryotes, was shown to be involved in nitrogen remobilization to seeds. Here, we have investigated the role of this mechanism in micronutrient translocation to seeds. We found that Arabidopsis thaliana plants impaired in autophagy display defects in nutrient remobilization to seeds. In the atg5-1 mutant, which is completely defective in autophagy, the efficiency of Fe translocation from vegetative organs to seeds was severely decreased even when Fe was provided during seed formation. Combining atg5-1 with the sid2 mutation that counteracts premature senescence associated with autophagy deficiency and using 57Fe pulse labeling, we propose a two-step mechanism in which Fe taken up de novo during seed formation is first accumulated in vegetative organs and subsequently remobilized to seeds. Finally, we show that translocation of Zn and manganese (Mn) to seeds is also dependent on autophagy. Fine-tuning autophagy during seed formation opens up new possibilities to improve micronutrient remobilization to seeds.

Histopathological diagnosis of biopsy samples and margin assessment of surgical specimens are challenging aspects in sarcoma. Using dog patient tissues, we assessed the performance of a recently developed technology for fast ex vivo molecular lipid-based diagnosis of sarcomas. The instrument is based on mass spectrometry (MS) molecular analysis through a laser microprobe operating under ambient conditions using excitation of endogenous water molecules. Classification models based on cancer/normal/necrotic, tumor grade, and subtypes showed a minimum of 97.63% correct classification. Specific markers of normal, cancer, and necrotic regions were identified by tandem MS and validated by MS imaging. Real-time detection capabilities were demonstrated by ex vivo analysis with direct interrogation of classification models., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Although iron is present in large amounts in the soil, its poor solubility means that plants have to use various strategies to facilitate its uptake. In this study, we show that expression of NtPDR3/NtABCG3, a Nicotiana tabacum plasma-membrane ABC transporter in the pleiotropic drug resistance (PDR) subfamily, is strongly induced in the root epidermis under iron deficiency conditions. Prevention of NtPDR3 expression resulted in N. tabacum plants that were less tolerant to iron-deficient conditions, displaying stronger chlorosis and slower growth than those of the wild-type when not supplied with iron. Metabolic profiling of roots and root exudates revealed that, upon iron deficiency, secretion of catechol-bearing O-methylated coumarins such as fraxetin, hydroxyfraxetin, and methoxyfraxetin to the rhizosphere was compromised in NtPDR3-silenced plants. However, exudation of flavins such as riboflavin was not markedly affected by NtPDR3-silencing. Expression of NtPDR3 in N. tabacum Bright Yellow-2 (BY-2) cells resulted in altered intra- and extracellular coumarin pools, supporting coumarin transport by this transporter. The results demonstrate that N. tabacum secretes both coumarins and flavins in response to iron deficiency and that NtPDR3 plays an essential role in the plant response to iron deficiency by mediating secretion of O-methylated coumarins to the rhizosphere.

Pneumocystis organisms are airborne-transmitted fungal parasites that infect the lungs of numerous mammalian species with strong host specificity. In this study, we investigated the genetic diversity and host specificity of Pneumocystis organisms infecting Southeast Asian murid rodents through PCR amplification of two mitochondrial genes and tested the co-phylogeny hypothesis among these fungi and their rodent hosts. Pneumocystis DNA was detected in 215 of 445 wild rodents belonging to 18 Southeast Asian murid species. Three of the Pneumocystis lineages retrieved in our phylogenetic trees correspond to known Pneumocystis species, but some of the remaining lineages may correspond to new undescribed species. Most of these Pneumocystis species infect several rodent species or genera and some sequence types are shared among several host species and genera. These results indicated a weaker host specificity of Pneumocystis species infecting rodents than previously thought. Our co-phylogenetic analyses revealed a complex evolutionary history among Pneumocystis and their rodent hosts. Even if a significant global signal of co-speciation has been detected, co-speciation alone is not sufficient to explain the observed co-phylogenetic pattern and several host switches are inferred. These findings conflict with the traditional view of a prolonged process of co-evolution and co-speciation of Pneumocystis and their hosts.

Background: Acute pain is a common condition among prehospital patients and prompt management is pivotal. Opioids are the most frequently analgesics used in the prehospital setting. However, opioids are highly addictive, and some patients may develop opioid dependence, even when they are exposed to brief opioid treatments. Therefore, alternative non-opioid analgesia should be developed to manage pain in the prehospital setting. Used at subdissociative doses, ketamine, a noncompetitive N-methyl-D-aspartate and glutamate receptor antagonist, provides analgesic effects accompanied by preservation of protective airway reflexes. In this context, we will carry out a randomized controlled, open-label, multicenter trial to compare a subdissociative dose of ketamine to morphine to provide pain relief in the prehospital setting, in patients with traumatic and non-traumatic pain., Methods/design: This will be a multicenter, single-blind, randomized controlled trial. Consecutive adults will be enrolled in the prehospital setting if they experience moderate to severe, acute, non-traumatic and traumatic pain, defined as a numeric rating scale score greater or equal to 5. Patients will be randomized to receive ketamine or morphine by intravenous push. The primary outcome will be the between-group difference in mean change in numeric rating scale pain scores measured from the time before administration of the study medication to 30 min later., Discussion: This upcoming randomized clinical trial was design to assess the efficacy and safety of ketamine, an alternative non-opiate analgesia, to manage non-traumatic and traumatic pain in the prehospital setting. We aim to provide evidence to change prescribing practices to reduce exposition to opioids and the subsequent risk of addiction., Trial Registration: ClinicalTrials.gov, ID: NCT03236805 . Registered on 2 August 2017.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fixes atmospheric CO 2 into organic compounds and is composed of eight copies each of a large subunit (RbcL) and a small subunit (RbcS). Recent reports have revealed unusual RbcS, which are expressed in particular tissues and confer higher catalytic rate, lesser affinity for CO 2 , and a more acidic profile of the activity versus pH. The resulting Rubisco was proposed to be adapted to a high CO 2 environment and recycle CO 2 generated by the metabolism. These RbcS belong to a cluster named T (for trichome), phylogenetically distant from cluster M, which gathers well-characterized RbcS expressed in mesophyll or bundle-sheath tissues. Cluster T is largely represented in different plant phyla, including pteridophytes and bryophytes, indicating an ancient origin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pneumocystis pneumonia is a serious complication that may affect immunosuppressed patients. The absence of reliable and safe therapeutic alternatives to trimethoprim-sulfamethoxazole (TMP/SMX) justifies the search for more effective and less toxic agents. In this study, the in vitro and in vivo anti-Pneumocystis jirovecii activity of iclaprim, a diaminopyrimidine compound that exerts its antimicrobial activity through the inhibition of dihydrofolate reductase (DHFR), as does TMP, was evaluated alone or in combination with SMX. The antimicrobial activity of iclaprim was tested in vitro using an efficient axenic culture system, and in vivo using P. carinii endotracheally inoculated corticosteroid-treated rats. Animals were orally administered iclaprim (5, 25, 50 mg/kg/day), iclaprim/SMX (5/25, 25/125, 50/250 mg/kg/day), TMP (50 mg/kg/day), or TMP/SMX (50/250 mg/kg/day) once a day for ten consecutive days. The in vitro maximum effect (E max ) and the drug concentrations needed to reach 50% of E max (EC 50 ) were determined, and the slope of the dose-response curve was estimated by the Hill equation (E max sigmoid model). The iclaprim EC 50 value was 20.3 μg/mL. This effect was enhanced when iclaprim was combined with SMX (EC 50 : 13.2/66 μg/mL) (p = 0.002). The TMP/SMX EC 50 value was 51.4/257 μg/mL. In vivo, the iclaprim/SMX combination resulted in 98.1% of inhibition compared to TMP/SMX, which resulted in 86.6% of inhibition (p = 0.048). Thus, overall, the iclaprim/SMX combination was more effective than TMP/SMX both in vitro and in vivo, suggesting that it could be an alternative therapy to the TMP/SMX combination for the treatment of Pneumocystis pneumonia.

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO 2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco ( Nicotiana tabacum ) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher V max values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO K m values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO 2 is released by the active specialized metabolism., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Background: The purpose of this study was to describe the sleep structure (especially slow wave sleep) in adults with congenital central hypoventilation syndrome (CCHS), a rare genetic disease due to mutations in the PHOX2B gene. Fourteen patients aged 23 (19.0; 24.8) years old (median [1 rst -3rd quartiles]) with CCHS underwent a sleep interview and night-time attended polysomnography with their ventilatory support. Their sleep variables were compared to those collected in 15 healthy control subjects matched for age, sex and body mass index., Results: The latency to N3 sleep was shorter in patients (26.3 min [24.0; 30.1]) than in controls (49.5 min [34.3; 66.9]; P = 0.005), and sleep onset latency tended to be shorter in patients (14.0 min [7.0; 20.5]) than in controls (33.0 min [18.0; 49.0]; P = 0.052). Total sleep time, sleep stage percentages, sleep fragmentation as well as respiratory and movement index were within normal ranges and not different between groups., Conclusions: Normal sleep in adult patients with CCHS and adequate ventilator support indicates that the PHOX2 gene mutations do not affect brain sleep networks. Consequently, any complaint of disrupted sleep should prompt clinicians to look for the usual causes of sleep disorders, primarily inadequate mechanical ventilation. Shorter N3 latency may indicate a higher need for slow wave sleep, to compensate for the abnormal respiratory-related cortical activity during awake quiet breathing observed in patients with CCH.

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

To quantitatively assess the risk of contamination by Pneumocystis depending on the degree of immunosuppression (ID) of the exposed rat hosts, we developed an animal model, where rats went through different doses of dexamethasone. Then, natural and aerial transmission of Pneumocystis carinii occurred during cohousing of the rats undergoing gradual ID levels (receivers) with nude rats developing pneumocystosis (seeders). Following contact between receiver and seeder rats, the P. carinii burden of receiver rats was determined by toluidine blue ortho staining and by qPCR targeting the dhfr monocopy gene of this fungus. In this rat model, the level of circulating CD4(+) and CD8(+) T lymphocytes remained significantly stable and different for each dose of dexamethasone tested, thus reaching the goal of a new stable and gradual ID rat model. In addition, an inverse relationship between the P. carinii burden and the level of circulating CD4(+) or CD8(+) T lymphocytes was evidenced. This rat model may be used to study other opportunistic pathogens or even co-infections in a context of gradual ID., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)

Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as cadmium (Cd(2+) ), which is highly toxic. However, transporters of the natural resistance-associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast-based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd(2+) sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd(2+) sensitivity while maintaining the ability of AtNRAMP4 to transport Fe(2+) in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn(2+) transport, whereas only one, E401K, impairs Mn(2+) transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd(2+) and Zn(2+) accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd(2+) transport by other NRAMP transporters from animals or plants., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Cryptosporidium, a protozoan parasite that can cause severe diarrhea in a wide range of vertebrates including humans, is increasingly recognized as a parasite of a diverse range of wildlife species. However, little data are available regarding the identification of Cryptosporidium species and genotypes in wild aquatic environments, and more particularly in edible freshwater fish. To evaluate the prevalence of Cryptosporidiumspp. in fish from Lake Geneva (Lac Léman) in France, 41 entire fish and 100 fillets (cuts of fish flesh) were collected from fishery suppliers around the lake. Nested PCR using degenerate primers followed by sequence analysis was used. Five fish species were identified as potential hosts of Cryptosporidium: Salvelinus alpinus, Esox lucius, Coregonus lavaretus, Perca fluviatilis, and Rutilus rutilus. The presence of Cryptosporidium spp. was found in 15 out of 41 fish (37%), distributed as follows: 13 (87%) C. parvum, 1 (7%) C. molnari, and 1 (7%) mixed infection (C. parvum and C. molnari). C. molnari was identified in the stomach, while C. parvum was found in the stomach and intestine. C. molnari was also detected in 1 out of 100 analyzed fillets. In order to identify Cryptosporidium subtypes, sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) was performed. Among the C. parvum positive samples, three gp60 subtypes were identified: IIaA15G2R1, IIaA16G2R1, and IIaA17G2R1. Histological examination confirmed the presence of potential developmental stages of C. parvum within digestive epithelial cells. These observations suggest that C. parvum is infecting fish, rather than being passively carried. Since C. parvum is a zoonotic species, fish potentially contaminated by the same subtypes found in terrestrial mammals would be an additional source of infection for humans and animals, and may also contribute to the contamination of the environment with this parasite. Moreover, the risk of human transmission is strengthened by the observation of edible fillet contamination.

While Pneumocystis pneumonia (PcP) still impacts the AIDS patients, it has a growing importance in immunosuppressed HIV-negative patients. To determine the anti-Pneumocystis therapeutic efficacy of new compounds, animal and in vitro models have been developed. Indeed, well-designed mouse or rat experimental models of pneumocystosis can be used to describe the in vivo anti-Pneumocystis activity of new drugs. In vitro models, which enable the screening of a large panel of new molecules, have been developed using axenic cultures or co-culture with feeder cells; but no universally accepted standard method is currently available to evaluate anti-Pneumocystis molecules in vitro. Thus, we chose to explore the use of the SYTO-13 dye, as a new indicator of Pneumocystis viability. In the present work, we established the experimental conditions to define the in vitro pharmacodynamic parameters (EC50, Emax) of marketed compounds (trimethoprim/sulfamethoxazole, pentamidine, atovaquone) in order to specifically measure the intrinsic activity of these anti-P. carinii molecules using the SYTO-13 dye for the first time. Co-labelling the fungal organisms with anti-P. carinii specific antibodies enabled the measurement of viability of Pneumocystis organisms while excluding host debris from the analysis. Moreover, contrary to microscopic observation, large numbers of fungal cells can be analyzed by flow cytometry, thus increasing statistical significance and avoiding misreading during fastidious quantitation of stained organisms. In conclusion, the SYTO-13 dye allowed us to show a reproducible dose/effect relationship for the tested anti-Pneumocystis drugs.

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.

Poplar is commonly used for phytoremediation of metal polluted soils. However, the high concentrations of trace elements present in leaves may return to soil upon leaf abscission. To investigate the mechanisms controlling leaf metal content, metal concentrations and expression levels of genes involved in metal transport were monitored at different developmental stages on leaves from different poplar genotypes growing on a contaminated field. Large differences in leaf metal concentrations were observed among genotypes. Whereas Mg was remobilized during senescence, Zn and Cd accumulation continued until leaf abscission in all genotypes. A positive correlation between Natural Resistance Associated Macrophage Protein 1 (NRAMP1) expression levels and Zn bio-concentration factors was observed. Principal component analyses of metal concentrations and gene expression levels clearly discriminated poplar genotypes. This study highlights a general absence of trace element remobilization from poplar leaves despite genotype specificities in the control of leaf metal homeostasis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO(®) expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD. Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS)., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Uridine diphosphate UDP-glycosyltransferases (UGTs) are detoxification enzymes widely distributed within living organisms. They are involved in the biotransformation of various lipophilic endogenous compounds and xenobiotics, including odorants. Several UGTs have been reported in the olfactory organs of mammals and involved in olfactory processing and detoxification within the olfactory mucosa but, in insects, this enzyme family is still poorly studied. Despite recent transcriptomic analyses, the diversity of antennal UGTs in insects has not been investigated. To date, only three UGT cDNAs have been shown to be expressed in insect olfactory organs. In the present study, we report the identification of eleven putative UGTs expressed in the antennae of the model pest insect Spodoptera littoralis. Phylogenetic analysis revealed that these UGTs belong to five different families, highlighting their structural diversity. In addition, two genes, UGT40R3 and UGT46A6, were either specifically expressed or overexpressed in the antennae, suggesting specific roles in this sensory organ. Exposure of male moths to the sex pheromone and to a plant odorant differentially downregulated the transcription levels of these two genes, revealing for the first time the regulation of insect UGTs by odorant exposure. Moreover, the specific antennal gene UGT46A6 was upregulated by insecticide topical application on antennae, suggesting its role in the protection of the olfactory organ towards xenobiotics. This work highlights the structural and functional diversity of UGTs within this highly specialized tissue., (© 2014 The Royal Entomological Society.)

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. (1)H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or "closed chain" structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.

Background: Histoplasma capsulatum and Pneumocystis organisms cause host infections primarily affecting the lung tissue. H. capsulatum is endemic in the United States of America and Latin American countries. In special environments, H. capsulatum is commonly associated with bat and bird droppings. Pneumocystis-host specificity has been primarily studied in laboratory animals, and its ability to be harboured by wild animals remains as an important issue for understanding the spread of this pathogen in nature. Bats infected with H. capsulatum or Pneumocystis spp. have been found, with this mammal serving as a probable reservoir and disperser; however, the co-infection of bats with both of these microorganisms has never been explored. To evaluate the impact of H. capsulatum and Pneumocystis spp. infections in this flying mammal, 21 bat lungs from Argentina (AR), 13 from French Guyana (FG), and 88 from Mexico (MX) were screened using nested-PCR of the fragments, employing the Hcp100 locus for H. capsulatum and the mtLSUrRNA and mtSSUrRNA loci for Pneumocystis organisms., Results: Of the 122 bats studied, 98 revealed H. capsulatum infections in which 55 of these bats exhibited this infection alone. In addition, 51 bats revealed Pneumocystis spp. infection of which eight bats exhibited a Pneumocystis infection alone. A total of 43 bats (eight from AR, one from FG, and 34 from MX) were found co-infected with both fungi, representing a co-infection rate of 35.2% (95% CI = 26.8-43.6%)., Conclusion: The data highlights the H. capsulatum and Pneumocystis spp.co-infection in bat population's suggesting interplay with this wild host.

Seed formation is an important step of plant development which depends on nutrient allocation. Uptake from soil is an obvious source of nutrients which mainly occurs during vegetative stage. Because seed filling and leaf senescence are synchronized, subsequent mobilization of nutrients from vegetative organs also play an essential role in nutrient use efficiency, providing source-sink relationships. However, nutrient accumulation during the formation of seeds may be limited by their availability in source tissues. While several mechanisms contributing to make leaf macronutrients available were already described, little is known regarding micronutrients such as metals. Autophagy, which is involved in nutrient recycling, was already shown to play a critical role in nitrogen remobilization to seeds during leaf senescence. Because it is a non-specific mechanism, it could also control remobilization of metals. This article reviews actors and processes involved in metal remobilization with emphasis on autophagy and methodology to study metal fluxes inside the plant. A better understanding of metal remobilization is needed to improve metal use efficiency in the context of biofortification.

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.

Screening of the antifungal activities of ten Guadeloupean plants was undertaken to find new extracts and formulations against superficial mycoses such as onychomycosis, athlete's foot, Pityriasis versicolor, as well as the deep fungal infection Pneumocystis pneumonia. For the first time, the CMI of these plant extracts [cyclohexane, ethanol and ethanol/water (1:1, v/v)] was determined against five dermatophytes, five Candida species, Scytalidium dimidiatum, a Malassezia sp. strain and Pneumocystis carinii. Cytotoxicity tests of the most active extracts were also performed on an HaCat keratinocyte cell line. Results suggest that the extracts of Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum and Pluchea carolinensis have interesting activities and could be good candidates for developing antifungal formulations., (Copyright © 2012 John Wiley & Sons, Ltd.)

Some compounds articulated around a piperazine or an ethylenediamine linker have been evaluated in vitro to determine their activity in the presence of a 3T6 fibroblast cell line and an axenic culture of Pneumocystis carinii, respectively. The most efficient antifungal derivatives, namely N,N'-bis(benzamidine-4-yl)ethane-1,2-diamine (compound 6, a diamidine) and N-(benzamidine-4-yl)-N'-phenylethane-1,2-diamine (compound 7, a monoamidine), exhibited no cytotoxicity and were evaluated in vivo in a rat model. Only the diamidine 6 emerged as a promising hit for further studies.

To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.

Cytochrome P450 enzymes (P450s) are involved in many physiological functions in insects, such as the metabolism of signal molecules, adaptation to host plants and insecticide resistance. Several P450s have been reported in the olfactory organs of insects, the antennae, and have been proposed to play a role in odorant processing and/or xenobiotic metabolism. Despite recent transcriptomic analyses in several species, the diversity of antennal P450s in insects has not yet been investigated. Here, we report the identification of 37 putative P450s expressed in the antennae of the pest moth Spodoptera littoralis, as well as the characterization of a redox partner, cytochrome P450 reductase (CPR). Phylogenetic analysis revealed that S. littoralis P450s belong to four clades defined by their conservation with vertebrate P450s and their cellular localization. Interestingly, the CYP3 and CYP4 clans, which have been described to be mainly involved in the metabolism of plant compounds and xenobiotics, were largely predominant. More surprisingly, two P450s related to ecdysteroid metabolism were also identified. Expression patterns in adult and larval tissues were studied. Eight P450s appeared to be specific to the chemosensory organs, ie the antennae and proboscis, suggesting a specific role in odorant and tastant processing. Moreover, exposure of males to a plant odorant down-regulated the transcript level of CPR, revealing for the first time the regulation of this gene by odorants within insect antennae. This work suggests that the antennae of insects are a key site for P450-mediated metabolism of a large range of exogenous and endogenous molecules., (© 2012 Royal Entomological Society.)

Bats belong to a wide variety of species and occupy diversified habitats, from cities to the countryside. Their different diets (i.e., nectarivore, frugivore, insectivore, hematophage) lead Chiroptera to colonize a range of ecological niches. These flying mammals exert an undisputable impact on both ecosystems and circulation of pathogens that they harbor. Pneumocystis species are recognized as major opportunistic fungal pathogens which cause life-threatening pneumonia in severely immunocompromised or weakened mammals. Pneumocystis consists of a heterogeneous group of highly adapted host-specific fungal parasites that colonize a wide range of mammalian hosts. In the present study, 216 lungs of 19 bat species, sampled from diverse biotopes in the New and Old Worlds, were examined. Each bat species may be harboring a specific Pneumocystis species. We report 32.9% of Pneumocystis carriage in wild bats (41.9% in Microchiroptera). Ecological and behavioral factors (elevation, crowding, migration) seemed to influence the Pneumocystis carriage. This study suggests that Pneumocystis-host association may yield much information on Pneumocystis transmission, phylogeny, and biology in mammals. Moreover, the link between genetic variability of Pneumocystis isolated from populations of the same bat species and their geographic area could be exploited in terms of phylogeography.

Cloistered monks and nuns adhere to a 10-century-old strict schedule with a common zeitgeber of a night split by a 2- to 3-h-long Office (Matins). The authors evaluated how the circadian core body temperature rhythm and sleep adapt in cloistered monks and nuns in two monasteries. Five monks and five nuns following the split-sleep night schedule for 5 to 46 yrs without interruption and 10 controls underwent interviews, sleep scales, and physical examination and produced a week-long sleep diary and actigraphy, plus 48-h recordings of core body temperature. The circadian rhythm of temperature was described by partial Fourier time-series analysis (with 12- and 24-h harmonics). The temperature peak and trough values and clock times did not differ between groups. However, the temperature rhythm was biphasic in monks and nuns, with an early decrease at 19:39 ± 4:30 h (median ± 95% interval), plateau or rise of temperature at 22:35 ± 00:23 h (while asleep) lasting 296 ± 39 min, followed by a second decrease after the Matins Office, and a classical morning rise. Although they required alarm clocks to wake-up for Matins at midnight, the body temperature rise anticipated the nocturnal awakening by 85 ± 15 min. Compared to the controls, the monks and nuns had an earlier sleep onset (20:05 ± 00:59 h vs. 00:00 ± 00:54 h, median ± 95% confidence interval, p= .0001) and offset (06:27 ± 0:22 h, vs. 07:37 ± 0:33 h, p= .0001), as well as a shorter sleep time (6.5 ± 0.6 vs. 7.6 ± 0.7 h, p= .05). They reported difficulties with sleep latency, sleep duration, and daytime function, and more frequent hypnagogic hallucinations. In contrast to their daytime silence, they experienced conversations (and occasionally prayers) in dreams. The biphasic temperature profile in monks and nuns suggests the human clock adapts to and even anticipates nocturnal awakenings. It resembles the biphasic sleep and rhythm of healthy volunteers transferred to a short (10-h) photoperiod and provides a living glance into the sleep pattern of medieval time.

Rationale: Fluid accumulation in the legs and its overnight redistribution into the neck appears to play a causative role in obstructive sleep apnea (OSA) in sedentary men. Chronic venous insufficiency (CVI) promotes fluid accumulation in the legs that can be counteracted by compression stockings., Objectives: To test the hypotheses that, in nonobese subjects with CVI and OSA, wearing compression stockings during the day will attenuate OSA by reducing the amount of fluid displaced into the neck overnight., Methods: Nonobese subjects with CVI and OSA were randomly assigned to 1 week of wearing compression stockings or to a 1-week control period without compression stockings, after which they crossed over to the other arm. Polysomnography and measurement of overnight changes in leg fluid volume and neck circumference were performed at baseline and at the end of compression stockings and control periods., Measurements and Main Results: Twelve subjects participated. Compared with the end of the control period, at the end of the compression stockings period there was a 62% reduction in the overnight leg fluid volume change (P = 0.001) and a 60% reduction in the overnight neck circumference increase (P = 0.001) in association with a 36% reduction in the number of apneas and hypopneas per hour of sleep (from 48.4 ± 26.9 to 31.3 ± 20.2, P = 0.002)., Conclusions: Redistribution of fluid from the legs into the neck at night contributes to the pathogenesis of OSA in subjects with CVI. Prevention of fluid accumulation in the legs during the day, and its nocturnal displacement into the neck, attenuates OSA in such subjects.

The characteristics of residual excessive sleepiness (RES), defined by an Epworth score >10 in adequately treated apnoeic patients, are unknown. 40 apnoeic patients, with (n = 20) and without (n = 20) RES, and 20 healthy controls underwent clinical interviews, cognitive and biological tests, polysomnography, a multiple sleep latency test, and 24-h sleep monitoring. The marked subjective sleepiness in the RES group (mean ± sd score 16.4 ± 3) contrasted with moderately abnormal objective measures of sleepiness (90% of patients with RES had daytime sleep latencies >8 min). Compared with patients without RES, the patients with RES had more fatigue, lower stage N3 percentages, more periodic leg movements (without arousals), lower mean sleep latencies and longer daytime sleep periods. Most neuropsychological dimensions (morning headaches, memory complaints, spatial memory, inattention, apathy, depression, anxiety and lack of self-confidence) were not different between patients with and without RES, but gradually altered from controls to apnoeic patients without and then with RES. RES in apnoeic patients differs markedly from sleepiness in central hypersomnia. The association between RES, periodic leg movements, apathy and depressive mood parallels the post-hypoxic lesions in noradrenaline, dopamine and serotonin systems in animals exposed to intermittent hypoxia.

The amount of fluid displaced overnight from the legs into the neck as a consequence of lying recumbent correlates with the number of apneas and hypopneas per hour of sleep (AHI). Sedentary living promotes dependent fluid accumulation in the legs that can be counteracted by venous compression of the legs (compression stockings). We hypothesized that, in non-obese sedentary men with obstructive sleep apnea (OSA), wearing compression stockings during daytime will reduce the AHI by reducing the amount of fluid available for the displacement into the neck overnight. Polysomnography and measurement of overnight changes in leg fluid volume and neck circumference were performed at baseline and after one day of legs venous compression. The median AHI decreased from 30.9 (interquartile range 19.6-60.4) to 23.4 (12.9-31.8) (P=0.016) in association with a median 40% reduction in the change in leg fluid volume (P=0.016) and a median 42% reduction in the increase in neck circumference (P=0.016). These results provide proof-of-principle that overnight fluid displacement into the neck plays a causative role in OSA., (Copyright © 2011 Elsevier B.V. All rights reserved.)