PCR‐based detection of Aspergillus fumigatus and absence of azole resistance due to TR34/L98H in a french multicenter cohort of 137 patients with fungal rhinosinusitis

Fungal rhinosinusitis (FRS) has a worldwide distribution, comprises distinct clinical entities but is mostly due to Aspergillus among which Aspergillus fumigatus plays a major role in European countries. Although, there is accumulating evidence for the emergence of environmentally acquired‐azole resistance in A. fumigatus (such as TR₃₄/L98H) in various clinical settings, there is few data for patients with FRS. In this study, we aimed to investigate the prevalence of A. fumigatus azole resistance due to TR₃₄/L98H in a multicentre cohort of patients with FRS. One hundred and thirty‐seven patients with FRS admitted between 2002 and 2016 at four French medical centres were retrospectively enrolled. Clinical and mycological findings were collected. Aspergillus fumigatus and the TR₃₄/L98H alteration conferring azole resistance were investigated directly from clinical samples using the commercial CE‐IVD marked MycoGENIE^® A. fumigatus real‐time PCR assay. Fungal ball was the more frequent clinical form (n = 118). Despite the presence of fungal hyphae at direct microscopic examination, mycological cultures remained negative for 83 out of the 137 patients (60.6%). The PCR assay proved to be useful allowing the identification of A. fumigatus and etiological diagnosis in 106 patients (77.4%) compared with 44 patients (32.1%) when using culture as the reference method. Importantly, neither TR₃₄ nor L98H alterations were evidenced.     

Effect of CRP value on <Superscript>18</Superscript>F–FDG PET vascular positivity in Takayasu arteritis: a systematic review and per-patient based meta-analysis

Purpose

The aim of this study was to quantify the association between the CRP value and ¹⁸F–FDG PET vascular positivity in Takayasu arteritis (TAK) through a structured dedicated systematic review and meta-analysis.

Methods

From January 2000 to December 2016, the PubMed/MEDLINE database was searched for articles specifically dealing with the assessment of vascular inflammation using ¹⁸F–FDG PET and CRP biomarkers in TAK. Inclusion criteria for the qualitative analysis were (1) ¹⁸F–FDG PET used to assess the disease activity, (2) The use of the ACR criteria for the diagnosis of TAK, (3) No case mixed vasculitis (i.e., no giant cell arteritis), and (4) CRP concentration and clinical disease activity available. For the meta-analysis, PET-positive and PET-negative subgroups with the corresponding CRP concentrations were generated based on per patient data. The standard mean difference, which represents the effect of the CRP concentrations on the ¹⁸F–FDG PET vascular uptake, was computed for all studies, and then the results were pooled together.

Results

Among the 33 initial citations, nine complete articles including 210 patients fulfilled the inclusion criteria. Five studies found a significant correlation between the ¹⁸F–FDG PET and CRP concentration, one provided a trend towards association and three did not find any association between the two biomarkers. Six studies found a significant association between ¹⁸F–FDG PET and clinical disease activity, one found a trend towards association and the last two studies did not evaluate this correlation. The meta-analysis (121 patients) provided the following results: Standard Mean Deviation = 0.54 [0.15;0.92]; Chi² = 3.35; I² = 0%; Test for overall effect: Z = 2.70 (P = 0.007).

Conclusion

The CRP concentration only moderately reflects the ¹⁸F–FDG PET vascular positivity in TAK, suggesting dissociated information. Standardized longitudinal prospective studies are necessary to assess the value of ¹⁸F–FDG PET as an independent biomarker for subtle vascular wall inflammation detection.

     

Primary care physician management of tick bites in the Franche-Comté region (Eastern France, 2013)

Introduction

Tick bites, which may lead to Lyme disease, often prompt patients to consult their primary care physicians (PCPs). The aim of the present study was to assess how and how often PCPs in the Franche-Comté region of France manage tick bites.

Reduced nighttime transpiration is a relevant breeding target for high water-use efficiency in grapevine

Increasing water scarcity challenges crop sustainability in many regions. As a consequence, the enhancement of transpiration efficiency (TE)—that is, the biomass produced per unit of water transpired—has become crucial in breeding programs. This could be achieved by reducing plant transpiration through a better closure of the stomatal pores at the leaf surface. However, this strategy generally also lowers growth, as stomatal opening is necessary for the capture of atmospheric CO₂ that feeds daytime photosynthesis. Here, we considered the reduction in transpiration rate at night (E_n) as a possible strategy to limit water use without altering growth. For this purpose, we carried out a genetic analysis for E_n and TE in grapevine, a major crop in drought-prone areas. Using recently developed phenotyping facilities, potted plants of a cross between Syrah and Grenache cultivars were screened for 2 y under well-watered and moderate soil water deficit scenarios. High genetic variability was found for E_n under both scenarios and was primarily associated with residual diffusion through the stomata. Five quantitative trait loci (QTLs) were detected that underlay genetic variability in E_n. Interestingly, four of them colocalized with QTLs for TE. Moreover, genotypes with favorable alleles on these common QTLs exhibited reduced E_n without altered growth. These results demonstrate the interest of breeding grapevine for lower water loss at night and pave the way to breeding other crops with this underexploited trait for higher TE.Understanding how plants make efficient use of water has become a priority in the context of climate change and reduced water availability (1, 2). Plants inevitably lose water vapor by transpiration while capturing atmospheric CO₂ for photosynthesis through stomatal pores at the leaf surface. Photosynthesis and transpiration covary with the aperture and density of stomatal pores depending on genotypes and environmental conditions (3, 4). In addition, photosynthesis and transpiration rates are codetermined by the leaf area, which is involved in light capture and is subjected to evaporation. Thus, plant productivity is positively coupled with transpirational water losses through stomatal characteristics and shoot development. This coupling has prompted plant scientists to define transpiration efficiency (TE) as the amount of biomass produced per unit of water used through transpiration (5). Because TE shows significant variations across species and varieties, it has been proposed as a relevant target in breeding programs for areas with restricted water availability (2).Breeders have substantially improved TE by selecting plants with more efficient photosynthesis or higher allocation of photosynthates to harvested organs (6). Another way to improve TE is to decrease the amount of water lost by transpiration. However, the tight coupling between transpiration and photosynthesis during the day in C₃ and C₄ species makes it challenging to decrease water loss without reducing crop yield: genotypes that save the most water often show the lowest photosynthesis rate and yield (7). This drawback may be circumvented by selecting alleles or manipulating genes that uncouple transpiration from photosynthesis. Such a selection has been facilitated by the widespread use of δ¹³C, a proxy for daytime TE based on the carbon isotope discrimination that occurs during photosynthesis (8). This strategy has been successfully implemented in both model plants and crops to detect genomic regions associated with the control of TE, which are being exploited in breeding programs (9, 10), or serve as starting points to investigate the genetic determinism and ecological significance of the variation in TE (11–13).An alternative yet unexplored strategy to improve TE (14) could be to select plants with a reduced rate of water loss at night (E_n), when photosynthesis is not operating due to the absence of light. Although plants actively close their stomata in the dark, stomatal closure is largely incomplete and nighttime transpiration can be substantial, accounting for up to 30% of the plant’s daytime water loss (15). Furthermore, variation in E_n has been identified across (15–17) and within (15, 18–20) plant species, but little is known about the underlying genetic determinisms. In the model plant Arabidopsis thaliana, natural variation in nighttime transpiration was detected (21, 22) and found to contribute significantly to total transpiration (23). However, genotypes with reduced E_n also exhibited lower photosynthesis rate and growth (21), hindering further developments in breeding programs. More recently, A. thaliana mutants were isolated, showing impaired nighttime transpiration and intact growth (24). This suggests that genomic regions may be identified with beneficial alleles, allowing plants to save water at night without altering growth. However, no study so far has attempted to jointly analyze the genetic determinisms of E_n, TE, and growth at the whole genome scale of a plant species.Here we show that TE can be bred by reducing E_n independently of daytime stomatal behavior in grapevine (Vitis vinifera L.), a cultivated perennial species of high economic importance for drought-prone areas. Genetic variations in TE (25) and E_n (20, 26, 27) have been independently reported in V. vinifera, but the genetic basis of a possible link between these two traits has never been examined. In our study, we jointly examine TE and E_n in a mapping population obtained from the cross between two widespread cultivars, Syrah and Grenache (S×G), previously described as exhibiting different daytime water use (28, 29). We highlight that part of the genetic variation in TE is tightly linked to the variation in E_n. Incomplete stomatal closure and, to a lesser extent, water loss through the cuticle account for a large part of the genetic variability in E_n. Several genomic loci underlying the variation in E_n are identified under well-watered (WW) conditions and soil water deficit (WD). Based on genetic information on these loci, offspring genotypes could be selected with favorable alleles that enhance TE by reducing E_n without altering plant growth.     

Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes

Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.In the last decade communication between cells via secretion of membrane vesicles (collectively called extracellular vesicles or EVs) has become the focus of increasing interest (reviewed in refs. 1–3). EVs are composed of a lipid bilayer containing transmembrane proteins and enclosing soluble proteins and RNA. The communication via EVs might be mediated by their multiple components. The surface receptors of EVs allow their targeting and capture by recipient cells, which can then incorporate proteic, lipidic, and even genetic messages carried by the vesicles, resulting in modifications of their physiological state (1, 4, 5). Consequently, the possibility of using these vesicles as biomarkers in various diseases, as vehicles of immunotherapies, or as targets to be eliminated to improve patients’ health is currently being explored (2, 6).It has become clear, however, in the past few years, that cells can release EVs of different types. Some EVs are directly formed and released from the cells’ plasma membrane (PM), and are often called microparticles, (shed) microvesicles, or ectosomes: these EVs display sizes ranging from a few dozens of nanometers to a few micrometers. Internal vesicles generated within multivesicular endosomal compartments (MVB) are secreted when these compartments fuse with the PM, thus releasing their internal vesicles in the extracellular milieu: these EVs are termed exosomes (3, 7). Exosomes are classically defined by their size, similar to the size of intraluminal vesicles of MVBs (i.e., below 150 nm in diameter), and their content of endosome-associated proteins. However, the term exosome is often used in the literature for EVs of small size, passing through 220-nm pore filters or recovered by high-speed ultracentrifugation (8), in the absence of demonstration of their intracellular origin. Such isolation procedures coisolate mixed EV populations, which we will call “small EVs” (sEVs), for lack of better term, in the rest of this article. Because EVs of different intracellular origins probably have different functional properties (9, 10), the mixed nature of EV preparations has made the growing literature increasingly confusing, with contradictory proposed functions and clinical uses of vesicles being regularly published. The lack of specific purification and characterization tools prevents a clear understanding of the specific versus shared functional properties of the different EVs. There is, therefore, a critical need for the identification of specific markers defining the different subtypes of EVs.To achieve this goal, we have performed here an extensive comparative analysis of the protein composition of all EVs recovered in the different steps of the differential ultracentrifugation (DUC) protocol classically used to isolate sEVs (11). As a source of EVs, we used human primary monocyte-derived dendritic cells (DCs), whose exosomes and EVs have been described to promote immune responses (1, 12).We found that several proteins often used in the literature as “exosome-markers,” such as flotillin-1, heat-shock 70-kDa proteins (HSC70/HSP73, HSP70/HSP72), or major histocompatibility complex (MHC) class I and class II proteins are present, to various extents, not only in the sEVs pelleted at high speed, but also in the larger/heavier EVs pelleted at medium or low speed. We further separated the pelleted EVs by floatation into a density gradient or by immuno-isolation with antibodies specific for the three tetraspanins classically used as exosome markers (CD9, CD63, CD81), and performed a label-free quantitative proteomic analysis of their respective composition. We thus identified more specific protein markers of either the sEVs, among which a subpopulation bearing all tetraspanins correspond to endosome-derived exosomes, or the larger/heavier vesicles.     

Does computer-assisted surgery influence survivorship of cementless total knee arthroplasty in patients with primary osteoarthritis? A 10-year follow-up study

Purpose

Computer-assisted surgery (CAS) has been proposed to improve the performance of total knee arthroplasty (TKA) by reducing implant mal-position and mechanical axis mal-alignment. However, no clinical study has been performed to evaluate whether CAS improves survivorship of cementless TKA at long-term follow-up. This prospective and comparative study evaluated the outcome and survivorship of a cementless rotating mobile-bearing TKA performed with or without CAS at a minimum 10-year follow-up.

Methods

A continuous series of 138 TKA (SCORE^®, Amplitude, Valence, France) comparing 87 CAS TKA versus 51 conventional mechanical technique (MECA) TKA was prospectively included in our total joint registry.

Results

At 10.5 years after implantation, 95 TKA (59 CAS and 36 MECA TKA) were evaluated. No significant difference was detected in the clinical outcome and mechanical axis between the two groups. The overall 10-year survivorship using revision for any reason as end-point was 91 ± 5 % without significant difference detected between the two groups [86 ± 10 % in the MECA group and 94 ± 5 % in the CAS group (n.s.)]. Using aseptic loosening as end-point, the 10-year survivorship was 100 % in both groups. Using secondary patellar resurfacing as end-point, the 10-year survivorship was significantly higher in the CAS than in MECA group (100 and 85 ± 15 %, respectively; p = 0.0039).

Conclusion

With no implant aseptic loosening or mechanical failure at 10.5 years after implantation, the cementless rotating mobile-bearing SCORE^® TKA demonstrated favourable survivorship without influence of CAS. However, CAS might influence TKA survivorship by limiting secondary patellar resurfacing.

Level of evidence

Therapeutic, Level II.