Treadmill Running and Tower Climbing Resistance Exercise Mitigate Disuse Bone Loss in Mice Equally Well

Ground-Based Model for Lunar Disuse Bone Loss During spaceflight, astronauts are susceptible to decrements in bone mineral density. This suscetibility leaves astronauts at an increased risk of fracture and compromises their likelihood of repeat missions. Our ground-based Lunar model simulates disuse bone loss. Through our novel exercise regimens, we were able to mitigate losses seen by group members not exercised

Proteomic analysis for bacterial characterisation using mass spectrometry

Grâce à la spectrométrie de masse de type MALDI-TOF, l'identification des bactéries est maintenant possible en quelques minutes. Mais le taux de mortalité des patients augmente lorsqu'une antibiothérapie inappropriée est utilisée et les instruments MALDI-TOF ne sont pas capables d'analyser rapidement et exhaustivement la résistance bactérienne. Actuellement, 6 à 24 heures sont nécessaires pour déterminer le phénotype de résistance. En couplant une chromatographie liquide et un spectromètre de masse à ionisation électrospray (LC-MS/MS), nous avons identifié les marqueurs de résistance en 1 à 2 heures. En 30 min, nous avons pu détecter les mécanismes de résistance aux β-lactamines, aux glycopeptides, à la méthicilline et aux fluoroquinolones, à l'aide de méthodes de type "Suivi de Réactions Sélectionnées", ou "Selected Reaction Monitoring" (SRM). Au cours de la même analyse multiplexée, des dizaines de protéines peuvent être détectées de façon hautement spécifique et sensible. Comme l'illustre l'étude de la résistance multifactorielle chez Acinetobacter baumannii, cette approche permet en outre une analyse quantitative d'un grand intérêt pour certains mécanismes de résistance. Cependant, malgré ces perspectives attrayantes, la LC-MS/MS reste, aujourd'hui, loin d'une possible implantation en routine dans les laboratoires de microbiologie. Les instruments sont trop coûteux et la technologie trop complexe pour un usage pour du diagnostic in vitro. La spectrométrie de masse pourrait déjà avantageusement compléter les technologies actuelles de biologie moléculaire. Aujourd'hui, le séquençage de nouvelle génération est la méthode de référence pour la caractérisation moléculaire des bactéries. Mais, comme démontré dans ce travail, l'annotation des gènes est perfectible. Pour quelques dizaines d'euros et quelques heures d'analyse, les peptides identifies par spectrométrie de masse facilitent l'assemblage des séquences (« scaffolds ») et la détection des gènes. De surcroît, la spectrométrie de masse permet une quantification précise des protéines. Elle apporte ainsi une nouvelle dimension analytique et une vision moléculaire plus proche du phénotype. En conclusion, la spectrométrie de masse LC-MS/MS peut être une technologie complémentaire attractive, voir une future alternative, à la biologie moléculaire pour la caractérisation des bactériesThanks to MALDI-TOF mass spectrometry, identification of isolated bacteria is now possible within a few minutes. But doctors also need to rapidly know the phenotype of resistance of the bacteria. Indeed, the patient mortality rate increases when the antibiotherapy is not appropriate. However, MALDI-TOF instruments are not able to analyze antibacterial resistance rapidly and comprehensively.Today, 6 to 24 hours are nedded for antibiotic susceptibility testing. When combining a liquid chromatography and a mass spectrometer with electrospray ionization (LC-MSMS), the detection of resistance biomarkers was possible within 1 to 2 hours. Using a Selected Reaction Monitoring (SRM) method, resistance mechanisms to beta-lactams, methicillin, glycopeptides and fluoroquinolones were detected in strains within 30 minutes. Tens of resistance determinants can be analyzed in a single multiplexed assay, with high specificity and sensitivity. Illustrated by the study of multifactorial resistance in Acinetobacter baumannii, the technology allows furthermore a quantitative analysis, which is of great value for some resistance mechanisms. Similarly, we identified virulent strains of enterohemorrhagic Escherichia coli by targeting toxins and serotype biomakers in the same assay. Mass spectrometry offered deeper bacterial characterization than conventional serotyping using polyclonal antibodies. However, despite all these favorable prospects, LC-MS/MS remains today far from reaching a routine use in microbiological hospital laboratories. Instruments are too expensive and the technology is too cumbersome for a daily in vitro diagnostic use. Waiting for a more suitable use, mass spectrometry could yet advantageously complement current molecular technologies. Today, the gold standard to study bacteria at molecular level is next generation sequencing. However, as demonstrated during this work, gene annotation remains imperfect. For tens of euros and few hours of analysis, peptides identified by mass spectrometry analysis of a bacteria might improve scaffold assembly and gene detection. Moreover, mass spectrometry gives an accurate protein quantitation and brings a new analytical dimension, potentially closer to the phenotype than molecular techniques. In conclusion, LC-MS/MS mass spectrometry could be an attractive complementary, or alternative technology in a near future, to conventional molecular biology techniques for deep characterization of bacteri

Direct and rapid identification of sepsis causative micro-organisms from blood cultures using SCOUT-MRM proteomics assay

International audienc

Fast and accurate identification and antibiotic resistance profiling of micro-organisms in blood cultures by SWATH-MS proteomics

International audienceToday, approximately 1.3 million people die each year worldwide from bacterial antimicrobial resistance (AMR). AMR is of great concern in the context of bloodstream infections (BSIs) when it evolves into sepsis. If MALDI-TOF has markedly shortened the delay of the pathogen identification step, AMR evaluation is still based on growth tests in the presence of antibiotics. The latter, negatively impacts the diagnosis turnaround time, thus the administration to the patient of active antimicrobial agents. With the aim of streamlining this turnaround time to less than an hour from a positive blood culture, the LC/SWATH-MS sensitivity and specificity performances were evaluated to concomitantly provide the identity and antibiotic resistance profile of pathogens related to BSIs based on peptide surrogates

Fast and accurate identification and antibiotic resistance profiling of micro-organisms in blood cultures by SWATH-MS proteomics

International audienceToday, approximately 1.3 million people die each year worldwide from bacterial antimicrobial resistance (AMR). AMR is of great concern in the context of bloodstream infections (BSIs) when it evolves into sepsis. If MALDI-TOF has markedly shortened the delay of the pathogen identification step, AMR evaluation is still based on growth tests in the presence of antibiotics. The latter, negatively impacts the diagnosis turnaround time, thus the administration to the patient of active antimicrobial agents. With the aim of streamlining this turnaround time to less than an hour from a positive blood culture, the LC/SWATH-MS sensitivity and specificity performances were evaluated to concomitantly provide the identity and antibiotic resistance profile of pathogens related to BSIs based on peptide surrogates

Fast and accurate identification and antibiotic resistance profiling of micro-organisms in blood cultures by SWATH-MS proteomics

International audienceIntroductionToday, approximately 1.3 million people die each year worldwide from bacterial antimicrobial resistance (AMR). AMR is of great concern in the context of bloodstream infections (BSIs) when it evolves into sepsis. If MALDI-TOF has markedly shortened the delay of the pathogen identification step, AMR evaluation is still based on growth tests in the presence of antibiotics. The latter, negatively impacts the diagnosis turnaround time, thus the administration to the patient of active antimicrobial agents. With the aim of streamlining this turnaround time to less than an hour from a positive blood culture, the LC/SWATH-MS sensitivity and specificity performances were evaluated to concomitantly provide the identity and antibiotic resistance profile of pathogens related to BSIs based on peptide surrogates. Methods Our methodology consists in a 10-min sample preparation procedure for pelleting the micro-organisms from a positive blood culture aliquot followed by 10-min concomitant ultrasonic bacterial lysis and tryptic digestion step. All LC/SWATH-MS runs were conducted on a Waters M-Class UPLC system connected to a 7600 ZenoTOF mass spectrometer. The peptides were separated on a C18 micro-LC column (300 µm × 150 mm) at a flow rate of 6 µL/min; 25min total chromatographic time). LC/SWATH-MS data was processed with DIA-NN (1.8) in library-free mode. Sensitivity and specificity performances were assessed with a collection of representative sensitive and resistant clinical strains inoculated in blood culture bottles.Preliminary dataDIA-NN’s deep-learning algorithm was used to generate an in silico spectral and retention time profiling library from a FASTA file containing the complete genus and species-specific protein sequences. This library was then used to analyze the raw LC/SWATH-MS data. A comprehensive quality by design approach has been selected to evaluate the key LC-SWATH-MS parameters having the most influence on the overall performance detection of peptide surrogates used for the identification of the pathogen and the detection of antibiotic resistance-related proteins. All optimization designs have been conducted on the same Staphylococcus aureus strain with the objective of identifying the optimal operating conditions for the detection of 7 proteotypic peptides of the Penicillin Binding Protein 2a (PBP2a). The latter, mediated through the mecA gene, confers resistance to methicillin. Initially, a screening design allowed us to determine which of the six parameters evaluated (gradient length, TOF accumulation time, number of SWATH windows, fixed/variable windows, MS m/z range, and MS/MS m/z range) had the highest impact on the number of PBP2a peptides detected. Subsequently, the two most critical parameters (number of SWATH windows and MS m/z range) have been thoroughly optimized using a response surface methodology. The other non-critical parameters were set at the level that produced the best response. Taking a step further, the sensitivity and specificity performances of the optimized LC-SWATH-MS method have been evaluated by analyzing a collection of epidemiologically relevant clinical strains of Gram-negative and Gram-positive bacteria inoculated in blood culture bottles. This study allowed the detection of some of the most common protein effectors involved in bacterial resistance mechanisms such as PBP2a, different types of β-lactamases (AmpC; TEM; SHV; GES; PER; OXA; CTX-M; KPC; NDM), porin and efflux pumps (OprD; MexA/MexB/OprM; MexX/MexY/OprM) as well as vancomycin resistance proteins (VanA; VanB; VanD) to mention a few. Novel aspect Concomitant identification and AMR profiling of pathogens in positive blood cultures by SWATH with a turnaround time of one hour

Deciphering multifactorial resistance phenotypes in acinetobacter baumannii by genomics and targeted label-free proteomics

Resistance to-lactams in Acinetobacter baumannii involves various mechanisms. To decipher them, whole genome sequencing (WGS) and real-time quantitative polymerase chain reaction (RT-qPCR) were complemented by mass spectrometry (MS) in selected reaction monitoring mode (SRM) in 39 clinical isolates. The targeted labelfree proteomic approach enabled, in one hour and using a single method, the quantitative detection of 16 proteins associated with antibiotic resistance: eight acquired-lactamases (i.e. GES, NDM-1, OXA-23, OXA-24, OXA-58, PER, TEM-1, and VEB), two resident-lactamases (i.e. ADC and OXA-51-like) and six components of the two major efflux systems (i.e. AdeABC and AdeIJK). Results were normalized using "bacterial quantotypic peptides," i.e. peptide markers of the bacterial quantity, to obtain precise protein quantitation (on average 8.93% coefficient of variation for three biological replicates). This allowed to correlate the levels of resistance to-lactam with those of the production of acquired as well as resident-lactamases or of efflux systems. SRM detected enhanced ADC or OXA-51-like production and absence or increased efflux pump production. Precise protein quantitation was particularly valuable to detect resistance mechanisms mediated by regulated genes or by overexpression of chromosomal genes. Combination of WGS and MS, two orthogonal and complementary techniques, allows thereby interpretation of the resistance phenotypes at the molecular level

Fast and accurate identification and antibiotic resistance profiling of micro-organisms in blood cultures by SWATH-MS proteomics

International audienceIntroductionToday, approximately 1.3 million people die each year worldwide from bacterial antimicrobial resistance (AMR). AMR is of great concern in the context of bloodstream infections (BSIs) when it evolves into sepsis. If MALDI-TOF has markedly shortened the delay of the pathogen identification step, AMR evaluation is still based on growth tests in the presence of antibiotics. The latter, negatively impacts the diagnosis turnaround time, thus the administration to the patient of active antimicrobial agents. With the aim of streamlining this turnaround time to less than an hour from a positive blood culture, the LC/SWATH-MS sensitivity and specificity performances were evaluated to concomitantly provide the identity and antibiotic resistance profile of pathogens related to BSIs based on peptide surrogates. Methods Our methodology consists in a 10-min sample preparation procedure for pelleting the micro-organisms from a positive blood culture aliquot followed by 10-min concomitant ultrasonic bacterial lysis and tryptic digestion step. All LC/SWATH-MS runs were conducted on a Waters M-Class UPLC system connected to a 7600 ZenoTOF mass spectrometer. The peptides were separated on a C18 micro-LC column (300 µm × 150 mm) at a flow rate of 6 µL/min; 25min total chromatographic time). LC/SWATH-MS data was processed with DIA-NN (1.8) in library-free mode. Sensitivity and specificity performances were assessed with a collection of representative sensitive and resistant clinical strains inoculated in blood culture bottles.Preliminary dataDIA-NN’s deep-learning algorithm was used to generate an in silico spectral and retention time profiling library from a FASTA file containing the complete genus and species-specific protein sequences. This library was then used to analyze the raw LC/SWATH-MS data. A comprehensive quality by design approach has been selected to evaluate the key LC-SWATH-MS parameters having the most influence on the overall performance detection of peptide surrogates used for the identification of the pathogen and the detection of antibiotic resistance-related proteins. All optimization designs have been conducted on the same Staphylococcus aureus strain with the objective of identifying the optimal operating conditions for the detection of 7 proteotypic peptides of the Penicillin Binding Protein 2a (PBP2a). The latter, mediated through the mecA gene, confers resistance to methicillin. Initially, a screening design allowed us to determine which of the six parameters evaluated (gradient length, TOF accumulation time, number of SWATH windows, fixed/variable windows, MS m/z range, and MS/MS m/z range) had the highest impact on the number of PBP2a peptides detected. Subsequently, the two most critical parameters (number of SWATH windows and MS m/z range) have been thoroughly optimized using a response surface methodology. The other non-critical parameters were set at the level that produced the best response. Taking a step further, the sensitivity and specificity performances of the optimized LC-SWATH-MS method have been evaluated by analyzing a collection of epidemiologically relevant clinical strains of Gram-negative and Gram-positive bacteria inoculated in blood culture bottles. This study allowed the detection of some of the most common protein effectors involved in bacterial resistance mechanisms such as PBP2a, different types of β-lactamases (AmpC; TEM; SHV; GES; PER; OXA; CTX-M; KPC; NDM), porin and efflux pumps (OprD; MexA/MexB/OprM; MexX/MexY/OprM) as well as vancomycin resistance proteins (VanA; VanB; VanD) to mention a few. Novel aspect Concomitant identification and AMR profiling of pathogens in positive blood cultures by SWATH with a turnaround time of one hour