Plant and microbial toxins such as ricin, staphylococcal enterotoxin B (SEB), and the botulinum neurotoxins (BoNT) are considered as potential biological warfare agents. Specific screening methods ...are, therefore, required that enable unambiguous and sensitive identification of these biohazards, particularly for the occurrence of the toxins in complex sample matrixes. The present study describes a combination of a multiplex-immunoaffinity purification approach, followed by matrix-assisted laser desorption/ionization (MALDI)-based detection for the simultaneous identification of ricin, SEB, BoNT/A, and BoNT/B. The method comprises an affinity enrichment step, using specific monoclonal antibodies for each of the four toxins which have been selected from a pool of antibodies. The selected antibodies allow for specific and simultaneous capture of ricin, SEB, BoNT/A, BoNT/B, and the corresponding BoNT complexes. These were subsequently identified by MALDI time-of-flight (TOF) mass spectrometry (MS), following tryptic digest. The sensitivity of the technique was approximately 500 fmol for each of the toxins. These toxins were detectable within 8 h, even when present in complex matrixes such as milk or juice. Furthermore, the MALDI-based multiplex assay allowed for the discrimination of closely related BoNT sero- and subtypes, including a real case of food-borne botulism in Germany.
Seventeen bacterial strains able to suppress plant pathogens have been isolated from healthy Vietnamese crop plants and taxonomically assigned as members of the Bacillus cereus group. In order to ...prove their potential as biocontrol agents, we perform a comprehensive analysis that included the whole-genome sequencing of selected strains and the mining for genes and gene clusters involved in the synthesis of endo- and exotoxins and secondary metabolites, such as antimicrobial peptides (AMPs). Kurstakin, thumolycin, and other AMPs were detected and characterized by different mass spectrometric methods, such as MALDI-TOF-MS and LIFT-MALDI-TOF/TOF fragment analysis. Based on their whole-genome sequences, the plant-associated isolates were assigned to the following species and subspecies: B. cereus subsp. cereus (6), B. cereus subsp. bombysepticus (5), Bacillus tropicus (2), and Bacillus pacificus. These three isolates represent novel genomospecies. Genes encoding entomopathogenic crystal and vegetative proteins were detected in B. cereus subsp. bombysepticus TK1. The in vitro assays revealed that many plant-associated isolates enhanced plant growth and suppressed plant pathogens. Our findings indicate that the plant-associated representatives of the B. cereus group are a rich source of putative antimicrobial compounds with potential in sustainable agriculture. However, the presence of virulence genes might restrict their application as biologicals in agriculture.
Figure 6 of the original publication 1 contained an error in the Wavenumber in panels B and C. The wavenumbers 1616 (Cm-1) in panels B and C should have been 1516 (cm-1). The updated figure has been ...published in this correction article; the original article has been updated.
Methanogenic archaea from Siberian permafrost are suitable model organisms that meet many of the preconditions for survival on the martian subsurface. These microorganisms have proven to be highly ...resistant when exposed to diverse stress factors such as desiccation, radiation and other thermo-physical martian conditions. In addition, the metabolic requirements of methanogenic archaea are in principle compatible with the environmental conditions of the Red Planet.
The ExoMars mission will deploy a rover carrying a Raman spectrometer among the analytical instruments in order to search for signatures of life and to investigate the martian geochemistry. Raman spectroscopy is known as a powerful nondestructive optical technique for biosignature detection that requires only little sample preparation. In this study, we describe the use of confocal Raman microspectroscopy (CRM) as a rapid and sensitive technique for characterization of the methanogenic archaeon Methanosarcina soligelidi SMA-21 at the single cell level. These studies involved acquisition of Raman spectra from individual cells isolated from microbial cultures at different stages of growth. Spectral analyses indicated a high degree of heterogeneity between cells of individual cultures and also demonstrated the existence of growth-phase specific Raman patterns. For example, besides common Raman patterns of microbial cells, CRM additionally revealed the presence of lipid vesicles and CaCO3 particles in microbial preparations of M. soligelidi SMA-21, a finding that could be confirmed by electron microscopy. The results of this study suggest that heterogeneity and diversity of microorganisms have to be considered when using Raman-based technologies in future space exploration missions.
•Raman investigation of terrestrial extremophiles as model for astrobiology.•Biosignature identification by means of confocal Raman microspectroscopy on the single cell level.•Growth-dependent biosignatures in methanogenic archaeon from Siberian permafrost.•Diversity in the chemical composition of cell populations.•Influence of the phenotypic heterogeneity on the construction of Raman biosignature databases.
Critical Coronavirus disease 2019 (COVID-19) developed in a 7-year-old girl with a history of dystrophy, microcephaly, and central hypothyroidism. Starting with gastrointestinal symptoms, the patient ...developed severe myocarditis followed by progressive multiple organ failure complicated by Pseudomonas aeruginosa bloodstream infection. Intensive care treatment consisting of invasive ventilation, drainage of pleural effusion, and high catecholamine therapy could not prevent the progression of heart failure, leading to the implantation of venoarterial extracorporeal life support (VA-ECLS) and additional left ventricle support catheter (Impella® pump). Continuous venovenous hemofiltration (CVVH) and extracorporeal hemadsorption therapy (CytoSorb®) were initiated. Whole exome sequencing revealed a mutation of unknown significance in DExH-BOX helicase 30 (DHX30), a gene encoding a RNA helicase. COVID-19 specific antiviral and immunomodulatory treatment did not lead to viral clearance or control of hyperinflammation resulting in the patient’s death on extracorporeal life support-(ECLS)-day 20. This fatal case illustrates the potential severity of pediatric COVID-19 and suggests further evaluation of antiviral treatment strategies and vaccination programs for children.
Over the past decade, confocal Raman microspectroscopic (CRM) imaging has matured into a useful analytical tool to obtain spatially resolved chemical information on the molecular composition of ...biological samples and has found its way into histopathology, cytology, and microbiology. A CRM imaging data set is a hyperspectral image in which Raman intensities are represented as a function of three coordinates: a spectral coordinate λ encoding the wavelength and two spatial coordinates x and y. Understanding CRM imaging data is challenging because of its complexity, size, and moderate signal-to-noise ratio. Spatial segmentation of CRM imaging data is a way to reveal regions of interest and is traditionally performed using nonsupervised clustering which relies on spectral domain-only information with the main drawback being the high sensitivity to noise. We present a new pipeline for spatial segmentation of CRM imaging data which combines preprocessing in the spectral and spatial domains with k-means clustering. Its core is the preprocessing routine in the spatial domain, edge-preserving denoising (EPD), which exploits the spatial relationships between Raman intensities acquired at neighboring pixels. Additionally, we propose to use both spatial correlation to identify Raman spectral features colocalized with defined spatial regions and confidence maps to assess the quality of spatial segmentation. For CRM data acquired from midsagittal Syrian hamster (Mesocricetus auratus) brain cryosections, we show how our pipeline benefits from the complex spatial-spectral relationships inherent in the CRM imaging data. EPD significantly improves the quality of spatial segmentation that allows us to extract the underlying structural and compositional information contained in the Raman microspectra.
In silico spectral library prediction of all possible peptides from whole organisms has a great potential for improving proteome profiling by data-independent acquisition (DIA) and extending its ...scope of application. In combination with other recent improvements in the field of mass spectrometry (MS)-based proteomics, including sample preparation, peptide separation, and data analysis, we aimed to uncover the full potential of such an advanced DIA strategy by optimization of the data acquisition. The results demonstrate that the combination of high-quality in silico libraries, reproducible and high-resolution peptide separation using micropillar array columns, as well as neural network supported data analysis enables the use of long MS scan cycles without impairing the quantification performance. The study demonstrates that mean coefficient of variations of 4% were obtained even at only 1.5 data points per peak (full width at half-maximum) across different gradient lengths, which in turn improved proteome coverage up to more than 8000 proteins from HeLa cells using empirically corrected libraries and more than 7000 proteins using a whole human in silico predicted library. These data were obtained using a Q Exactive orbitrap mass spectrometer with moderate scanning speed (12 Hz) and perform very well in comparison to recent studies using more advanced MS instruments, which underline the high potential of this optimization strategy for various applications in clinical proteomics, microbiology, and molecular biology.
The combination of short liquid chromatography (LC) gradients and data-independent acquisition (DIA) by mass spectrometry (MS) has proven its huge potential for high-throughput proteomics. However, ...the optimization of isolation window schemes resulting in a certain number of data points per peak (DPPP) is understudied, although it is one of the most important parameters for the outcome of this methodology. In this study, we show that substantially reducing the number of DPPP for short-gradient DIA massively increases protein identifications while maintaining quantitative precision. This is due to a large increase in the number of precursors identified, which keeps the number of data points per protein almost constant even at long cycle times. When proteins are inferred from its precursors, quantitative precision is maintained at low DPPP while greatly increasing proteomic depth. This strategy enabled us to quantify 6018 HeLa proteins (>80 000 precursor identifications) with coefficients of variation below 20% in 30 min using a Q Exactive HF, which corresponds to a throughput of 29 samples per day. This indicates that the potential of high-throughput DIA-MS has not been fully exploited yet. Data are available via ProteomeXchange with identifier PXD036451.
The last years have witnessed substantial progress toward the application of mass spectrometry in microbiology. Although MALDI-TOF MS has already revolutionized clinical microbiology, substantial ...progress has been achieved also in the development of LC-MS for microbiological applications. Here we suggest LC-MS1 fingerprinting as a rapid and accurate microbial identification technique. Experimental MS1 data are tested against a library of strain-specific in silico mass profiles generated from publicly available proteome resources. The results highlight the potentials of LC-MS1 in microbiology.
Display omitted
Highlights•Rapid identification of bacteria by LC-MS1 and in silico databases.•Pattern analysis-based strategy for identifying bacteria by experimental LC-MS1 data.•Compilation of an in silico database of taxon-specific tryptic peptide mass profiles.•Tests of the identification pipeline with LC-MS1 data from bacteria.
Over the past decade, modern methods of MS (MS) have emerged that allow reliable, fast and cost-effective identification of pathogenic microorganisms. Although MALDI-TOF MS has already revolutionized the way microorganisms are identified, recent years have witnessed also substantial progress in the development of liquid chromatography (LC)-MS based proteomics for microbiological applications. For example, LC-tandem MS (LC-MS2) has been proposed for microbial characterization by means of multiple discriminative peptides that enable identification at the species, or sometimes at the strain level. However, such investigations can be laborious and time-consuming, especially if the experimental LC-MS2 data are tested against sequence databases covering a broad panel of different microbiological taxa. In this proof of concept study, we present an alternative bottom-up proteomics method for microbial identification. The proposed approach involves efficient extraction of proteins from cultivated microbial cells, digestion by trypsin and LC–MS measurements. Peptide masses are then extracted from MS1 data and systematically tested against an in silico library of all possible peptide mass data compiled in-house. The library has been computed from the UniProt Knowledgebase covering Swiss-Prot and TrEMBL databases and comprises more than 12,000 strain-specific in silico profiles, each containing tens of thousands of peptide mass entries. Identification analysis involves computation of score values derived from correlation coefficients between experimental and strain-specific in silico peptide mass profiles and compilation of score ranking lists. The taxonomic positions of the microbial samples are then determined by using the best-matching database entries. The suggested method is computationally efficient – less than 2 mins per sample - and has been successfully tested by a test set of 39 LC-MS1 peak lists obtained from 19 different microbial pathogens. The proposed method is rapid, simple and automatable and we foresee wide application potential for future microbiological applications.
Purpose: This phase I trial investigating the vascular targeting agent NGR-hTNF aimed to determine the ( a ) dose-limiting toxicities, ( b ) maximum tolerated dose (MTD), ( c ) pharmacokinetics and ...pharmacodynamics, ( d ) vascular response by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and ( e ) preliminary clinical activity in solid tumors.
Experimental Design: NGR-hTNF was administered once every 3 weeks by a 20- to 60-minute i.v. infusion to cohorts of three to six patients with
solid tumors in escalating doses. Pharmacokinetic and pharmacodynamic analyses in blood were done during the first four cycles.
DCE-MRI was done in cycle 1 at baseline and 2 hours after the start of the infusion.
Results: Sixty-nine patients received a total of 201 cycles of NGR-hTNF (0.2-60 μg/m 2 ). Rigors and fever were the most frequently observed toxicities. Four dose-limiting toxicities were observed (at doses of
1.3, 8.1, and 60 μg/m 2 ), of which three were infusion related. The MTD was 45 μg/m 2 . The mean apparent terminal half-life ranged from 0.963 to 2.08 hours. DCE-MRI results of tumors showed a vascular response
to NGR-hTNF. No objective responses were observed, but 27 patients showed stable disease with a median duration of 12 weeks.
Conclusions: NGR-hTNF was well tolerated. The MTD was 45 μg/m 2 administered in 1 hour once every 3 weeks. DCE-MRI results showed the antivascular effect of NGR-hTNF. These findings call
for further research for defining the optimal biological dose and clinical activity of NGR-hTNF as a single agent or in combination
with cytotoxic drugs. Clin Cancer Res; 16(4); 1315–23
PDF
