DNA metabarcoding is increasingly being used to characterize the microbiological composition of both the indoor and outdoor environments of dwellings. Our study aimed to evaluate metabarcoding and ...bioinformatic analysis resulting from calibrated samples and samples collected by an electrostatic dust collector (EDC) in dwellings with no moisture problems. Thus, the fungal communities of 14 dwellings (eastern France, Franche-Comté region) were analyzed by Illumina MiSeq technology after amplification of the ITS2 region.
Using the standard samples of 11 species of yeasts and molds allowed us to validate the Operational taxonomic units (OTU) assignment. These calibrated samples also showed a low amplification bias, a low rate of sequencing errors and the semi-quantitative nature of the technique.
Only one species from the calibrated samples (Lichtheimia corymbifera) was less amplified probably due to the presence of two mismatches in its3 primer.
EDC analysis identified 3594OTU with 75% of reads corresponding to 30 genera. The main genera are those usually found by culture techniques (Penicillium, Aspergillus and Cladosporium), but findings also indicate others less commonly isolated in culture such as Epicoccum, the fourth detected genus in our study. The type of heating systems was correlated with fungal diversity. We found less diversity in the dwellings with wood heating and larger quantities of Epicoccum nigrum verified by qPCR.
DNA metabarcoding analysis applied to EDC seems promising. However, we think that it must be used along with qPCR, to obtain a more global view of microbial ecology and relative quantification of species of interest within communities.
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•Sampling by electrostatic dust collectors to assess fungal exposure•Evaluation of fungal metabarcoding with Illumina technology sequencing•Some fungal species less detected than others with metabarcoding•Metabarcoding must be accompanied by qPCR for accurate quantification.
•Nine bovine mastitis pathogens were tested for antimicrobial susceptibilities.•We report low resistance to antibiotics with breakpoints with few exceptions.•For antibiotics without CLSI-breakpoints, ...setting of breakpoints is important.•In this EU-wide monitoring survey prevalence of MRSA was very low.•Prevalence of ESBL/AmpC in Enterobacteriaceae was low.
VetPath is an ongoing pan-European antimicrobial susceptibility monitoring programme collecting pathogens from diseased cattle, pigs and poultry not recently treated with antibiotics. Non-duplicate isolates (n = 1244) were obtained from cows with acute clinical mastitis in eight countries during 2015–2016 for centrally antimicrobial susceptibility testing according CLSI standards.
Among Escherichia coli (n = 225), resistance was high to ampicillin and tetracycline, moderate to kanamycin and low to amoxicillin/clavulanic acid and cefazolin. The MIC50/90 of danofloxacin, enrofloxacin and marbofloxacin were 0.03 and 0.06 μg/mL. For Klebsiella spp. (n = 70), similar results were noted, except for ampicillin and kanamycin. We detected 3.7 % (11/295) Enterobacteriaceae isolates carrying an ESBL/AmpC gene. Staphylococcus aureus (n = 247) and coagulase-negative staphylococci (CoNS; n = 189) isolates were susceptible to most antimicrobials tested except to penicillin (25.1 and 29.1 % resistance). Two S. aureus and thirteen CoNS isolates harboured mecA gene. Streptococcus uberis isolates (n = 208) were susceptible to β-lactam antibiotics (87.1–94.7 % susceptibility), 23.9 % were resistant to erythromycin and 37.5 % to tetracycline. Resistance to pirlimycin was moderate. For Streptococcus dysgalactiae (n = 132) the latter figures were 10.6 and 43.2 %; pirlimycin resistance was low. MIC values for Streptococcus agalactiae, Trueperella pyogenes and Corynebacterium spp. were generally low.
This current VetPath study shows that mastitis pathogens were susceptible to most antimicrobials with exceptions of staphylococci against penicillin and streptococci against erythromycin or tetracycline. For most antimicrobials, the percentage resistance and MIC50/90 values among the major pathogens were comparable to that of the preceeding VetPath surveys. This work highlights the high need to set additional clinical breakpoints for antimicrobials frequently used to treat mastitis.
Bacterial typing is crucial to tackle the spread of bacterial pathogens but current methods are time-consuming and costly. Matrix-assisted laser desorption ionization–time of flight mass spectrometry ...(MALDI-TOF MS) has been recently integrated into the microbiology laboratory workflow for a quick and low-cost microbial species identification. Independent research groups have successfully redirected the original function of this technology from their primary purpose to discriminate subgroups within pathogen species. However, identical bacterial subgroups could be identified by unrelated peaks by independent methods, thus limiting their robustness and exportability. We propose several guidelines that could improve the performance of MALDI-TOF MS-based typing methods for use as a first-line epidemiological tool.
The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by ...interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the Vmax of the ACCase reaction without altering the Km for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.
Dwellings are increasingly well insulated to save energy and this leads to higher humidity and temperature, which improves conditions for mites.
Dermatophagoides
antigens are the main allergens ...involved and tested in atopic asthma. We developed three new species-specific quantitative PCR (qPCR) methods for house dust mites (
Dermatophagoides pteronyssinus
and
D. farinae
) and storages mites (
Acarus siro
,
Glycyphagus domesticus
,
Lepidoglyphus destructor
). We sampled dust with electrostatic dust collectors, in the bedrooms, under beds and in the kitchens of patients with allergies (n = 24) and healthy controls (n = 18). Mite quantification was carried out with the three new qPCRs and the qPCR previously described for the
Dermatophagoides
genus. The qPCRs were highly specific and efficient for house dust mite species and the storage mites. Storage mite concentrations were higher than house dust mite concentrations and were higher in dwellings of patients with allergies. Consequently, allergists should test more often patients against the storage mite antigens by prick tests or IgE serology. Dampness is a major factor in storage mite development and the presence of effective mechanical ventilation can reduce storage mite concentrations four-fold. In addition, to limit exposure to dust mites, treatments should be used throughout dwellings and not only in patients’ bedrooms.
X!TandemPipeline is a software designed to perform protein inference and to manage redundancy in the results of phosphosite identification by database search. It provides the minimal list of proteins ...or phosphosites that are present in a set of samples using grouping algorithms based on the principle of parsimony. Regarding proteins, a two-level classification is performed, where groups gather proteins sharing at least one peptide and subgroups gather proteins that are not distinguishable according to the identified peptides. Regarding phosphosites, an innovative approach based on the concept of phosphoisland is used to gather overlapping phosphopeptides. The graphical interface of X!TandemPipeline allows the users to launch X!tandem identification, to inspect spectra and to manually validate their assignment to peptides, to launch the grouping program, and to visualize elementary data as well as grouping and redundancy information. Identification results obtained from other search engines can also be processed. X!TandemPipeline results can be exported as ready-to-use tabulated files or as XML files that can be directly used by the PROTICdb database or by the MassChroQ quantification software. X!TandemPipeline runs fast, is easy to use, and can process hundreds of samples simultaneously. It is freely available under the GNU General Public License v3.0 at http://pappso.inra.fr/bioinfo/xtandempipeline/ .
Recent efforts have been made to review the state of the art on a variety of questions and targets in paleoparasitology, including protozoan taxa. Meanwhile, these efforts seemed to let aside
...Cryptosporidium
, and we then intended to review its paleoparasitological record to assess its past distribution and favored detection methods, and eventually highlight needed research trajectories. This review shows that contrary to other parasites, most of the positive results came from South-American sites and coprolites rather than sediment samples, highlighting the need to test this kind of material, notably in Europe where many negative results were reported in the published literature from sediment samples. Moreover, aDNA-based detections are nearly absent from the paleoparasitological record of this parasite, though punctually shown successful. With their potential to address the evolutionary history of
Cryptosporidium
species, notably through their 18S rRNA tree, aDNA-based approaches should be encouraged in the future. In sum, and though the limits of currently used methods and materials remain unclear, this review highlights the potential role of coprolites and aDNA for the study of
Cryptosporidium
species in the past and how this history shaped their current diversity and distribution, notably among human populations but also farm animals.
Platelet protease-activated receptor 1 (PAR1) is a cell surface G-protein-coupled receptor (GPCR) that acts as a thrombin receptor promoting platelet aggregation. Targeting the PAR1 pathway by ...vorapaxar, a PAR1 antagonist, leads to a reduction in ischemic events in cardiovascular patients with a history of myocardial infarction or with peripheral arterial disease. In platelets, specialized microdomains highly enriched in cholesterol act as modulators of the activity of several GPCRs and play a pivotal role in the signaling pathway. However, their involvement in platelet PAR1 function remains incompletely characterized. In this context, we aimed to investigate whether activation of PAR1 in human platelets requires its localization in the membrane cholesterol-rich microdomains. Using confocal microscopy, biochemical isolation, and proteomics approaches, we found that PAR1 was not localized in cholesterol-rich microdomains in resting platelets, and only a small fraction of the receptor relocated to the microdomains following its activation. Vorapaxar treatment increased the level of PAR1 at the platelet surface, possibly by reducing its endocytosis, while its colocalization with cholesterol-rich microdomains remained weak. Consistent with a cholesterol-dependent activation of Akt and p38 MAP kinase in thrombin receptor-activating peptide (TRAP)-activated platelets, the proteomic data of cholesterol-rich microdomains isolated from TRAP-activated platelets showed the recruitment of proteins contributing to these signaling pathways. In conclusion, contrary to endothelial cells, we found that PAR1 was only weakly present in cholesterol-rich microdomains in human platelets but used these microdomains for efficient activation of downstream signaling pathways following TRAP activation.
SNF1-related protein kinase-1 (SnRK1), the plant kinase homolog of mammalian AMP-activated protein kinase (AMPK), is a sensor that maintains cellular energy homeostasis via control of ...anabolism/catabolism balance. AMPK-dependent phosphorylation of p27(KIP1) affects cell-cycle progression, autophagy and apoptosis. Here, we show that SnRK1 phosphorylates the Arabidopsis thaliana cyclin-dependent kinase inhibitor p27(KIP1) homologs AtKRP6 and AtKRP7, thus extending the role of this kinase to regulation of cell-cycle progression. AtKRP6 and 7 were phosphorylated in vitro by a recombinant activated catalytic subunit of SnRK1 (AtSnRK1α1). Tandem mass spectrometry and site-specific mutagenesis identified Thr152 and Thr151 as the phosphorylated residues on AtKRP6- and AtKRP7, respectively. AtSnRK1 physically interacts with AtKRP6 in the nucleus of transformed BY-2 tobacco protoplasts, but, in contrast to mammals, the AtKRP6 Thr152 phosphorylation state alone did not modify its nuclear localization. Using a heterologous yeast system, consisting of a cdc28 yeast mutant complemented by A. thaliana CDKA;1, cell proliferation was shown to be abolished by AtKRP6(WT) and by the non-phosphorylatable form AtKRP6(T152A) , but not by the phosphorylation-mimetic form AtKRP6(T152D). Moreover, A. thaliana SnRK1α1/KRP6 double over-expressor plants showed an attenuated AtKRP6-associated phenotype (strongly serrated leaves and inability to undergo callogenesis). Furthermore, this severe phenotype was not observed in AtKRP6(T152D) over-expressor plants. Overall, these results establish that the energy sensor AtSnRK1 plays a cardinal role in the control of cell proliferation in A. thaliana plants through inhibition of AtKRP6 biological function by phosphorylation.
Recently, many software tools have been developed to perform quantification in LC‐MS analyses. However, most of them are specific to either a quantification strategy (e.g. label‐free or isotopic ...labelling) or a mass‐spectrometry system (e.g. high or low resolution). In this context, we have developed MassChroQ (Mass Chromatogram Quantification), a versatile software that performs LC‐MS data alignment and peptide quantification by peak area integration on extracted ion chromatograms. MassChroQ is suitable for quantification with or without labelling and is not limited to high‐resolution systems. Peptides of interest (for example all the identified peptides) can be determined automatically, or manually by providing targeted m/z and retention time values. It can handle large experiments that include protein or peptide fractionation (as SDS‐PAGE, 2‐D LC). It is fully configurable. Every processing step is traceable, the produced data are in open standard formats and its modularity allows easy integration into proteomic pipelines. The output results are ready for use in statistical analyses. Evaluation of MassChroQ on complex label‐free data obtained from low and high‐resolution mass spectrometers showed low CVs for technical reproducibility (1.4%) and high coefficients of correlation to protein quantity (0.98). MassChroQ is freely available under the GNU General Public Licence v3.0 at http://pappso.inra.fr/bioinfo/masschroq/.
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