Franco Basile

Geodermatophilus obscurus G20 is a multi-resistant extremophile observed to survive significant dosages of ultraviolet (UV) radiation. Despite previous work having measured G. obscurus G20’s proteome in relation to its genome sequence, its proteomic response to stressors has yet to be explored. This study utilizes bottom-up proteomics with data independent acquisition to derive comprehensive proteomic measurements in both healthy and two UV-stressed conditions induced through 254 nm UV radiation administered at a median lethal dose. The first, an acute response treatment group, was recovered and flash-frozen immediately after UV radiation. The second, a recovery response treatment group, was allowed to incubate for 1 hour following UV radiation. Gene enrichment analysis revealed an increase in NADH dehydrogenase activity among both stressed treatment groups, however the recovery response treatment group experienced additional changes including reduced peptidoglycan synthesis and increased translational activity. Results suggest that the repair process undergone by G. obscurus G20 persists well after initial UV exposure and is likely to be inducing noteworthy energetic demands to facilitate UV induced genomic repair. Proteomic analyses revealed patterns that advance our mechanistic and temporal understanding of the G. obscurus metabolic response to UV irradiation.

A novel Gram-stain-positive, black-pigmented bacterium, designated as WL48A ^T, was isolated from the surface of badland sedimentary rock in the Red Desert of Wyoming and characterized using a polyphasic taxonomic approach. Good growth occurred at 28–32 °C, pH 7–9, and NaCl less than 1% (w/v). Colonies, growing well on International Streptomyces Project media (ISP) 3 and ISP 7, were black and adhering to the agar. Phylogenetic analyses based on 16S rRNA gene and draft genome sequences showed that strain WL48A^T belongs to the family Geodermatophilaceae, forming a distinct sub-branch with Geodermatophilus bullaregiensis DSM 46841^T. The organism showed 16S rRNA gene sequence similarity of 98.8% with G. bullaregiensis DSM 46841^T. Digital DNA–DNA hybridization value between the genome sequences of strain WL48A ^T and G. bullaregiensis DSM 46841^T was 51.8%, below the threshold of 70% for prokaryotic species delineation. The chemotaxonomic investigation revealed the presence of galactose, glucose, mannose, xylose and ribose as well as meso-DAP in the peptidoglycan layer. The polar lipid profiles contained phosphatidylcholine (PC), phosphatidylinositol (PI), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) phosphoglycolipid, phospholipids and an unidentified lipid. The menaquinone profile consisted of MK-9(H₄) (98.2%) and MK-9(H₂) (10.8%). The major fatty acid profile (>15%) comprised iso-C_15 : 0 and iso-C_16 : 0. Based on phenotypic, genetic and genomic data, strain WL48A^T (=DSM 116197^T = NCIMB 15483^T=NCCB 100957^T =ATCC TSD-376^T) merits to be considered as a novel species for which the name Geodermatophilus maliterrae sp. nov. is proposed.

Coal-derived carbon nanofibers (CCNFs) have been recently found to be a promising and low-cost electrode material for high-performance supercapacitors. However, the knowledge gap still exists between holistic understanding of coal precursors derived from different solvents and resulting CCNFs’ properties, prohibiting further optimization of their electrochemical performance. In this paper, assisted by laser desorption/ionization (LDI) and gas chromatography–mass spectrometry (GC–MS) technologies, a systematic study was performed to holistically characterize mass distribution and chemical composition of coal precursors derived from various ionic liquids (ILs) as extractants. Sequentially, X-ray photoelectron spectroscopy (XPS) revealed that the differences in chemical properties of various coal products significantly affected the surface oxygen concentrations and certain species distributions on the CCNFs, which, in turn, determined the electrochemical performances of CCNFs as electrode materials. We report that the CCNF that was produced by an oxygen-rich coal fragment from C₆mimCl ionic liquid extraction showed the highest concentrations of quinone and ester groups on the surface. Consequentially, C₆mimCl-CCNF achieved the highest specific capacitance and lowest ion diffusion resistance. Finally, a symmetric carbon/carbon supercapacitor fabricated with such CCNF as electrode delivered an energy density of 21.1 Wh/kg at the power density of 0.6 kW/kg, which is comparable to commercial active carbon supercapacitors.

Aims The identification and differentiation of antibiotic‐resistant bacteria by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) profiling remains a challenge due to the difficulty in detecting unique protein biomarkers associated with this trait. To expand the detectable proteome in antibiotic‐resistant bacteria, we describe a method implementing offline LC protein separation/fractionation prior to MALDI‐ToF‐MS and top‐down MALDI‐ToF/ToF‐MS (tandem MS or MS/MS) for the analysis of several antibiotic‐resistant Escherichia coli isolates. Methods and Results Coupling offline LC with MALDI‐ToF‐MS increased the number of detected protein signals in the typically analyzed mass regions (m/z 3000–20 000) by a factor of 13. Using the developed LC‐MALDI‐ToF‐MS protocol in conjunction with supervised principal components analysis, we detected a protein biomarker at m/z 9355 which correlated to β‐lactam resistance among the E. coli bacteria tested. Implementing a top‐down MALDI‐ToF/ToF‐MS approach, the prefractionated protein biomarker was inferred as a DNA‐binding HU protein, likely translated from the bla_CMY‐2 gene (encoding AmpC‐type β‐lactamase) in the incompatibility plasmid complex A/C (IncA/C). Conclusions Our results demonstrate the utility of LC‐MALDI‐MS and MS/MS to extend the number of proteins detected and perform MALDI‐accessible protein biomarker discovery in microorganisms. Significance and Impact of the Study This outcome is significant since it expands the detectable bacterial proteome via MALDI‐ToF‐MS.

A method based on plasmon surface resonance absorption and heating was developed to perform a rapid on-surface protein thermal decomposition and digestion suitable for imaging mass spectrometry (MS) and/or profiling. This photothermal process or plasmonic thermal decomposition/digestion (plasmonic-TDD) method incorporates a continuous wave (CW) laser excitation and gold nanoparticles (Au-NPs) to induce known thermal decomposition reactions that cleave peptides and proteins specifically at the C-terminus of aspartic acid and at the N-terminus of cysteine. These thermal decomposition reactions are induced by heating a solid protein sample to temperatures between 200 and 270 °C for a short period of time (10–50 s per 200 μm segment) and are reagentless and solventless, and thus are devoid of sample product delocalization. In the plasmonic-TDD setup the sample is coated with Au-NPs and irradiated with 532 nm laser radiation to induce thermoplasmonic heating and bring about site-specific thermal decomposition on solid peptide/protein samples. In this manner the Au-NPs act as nanoheaters that result in a highly localized thermal decomposition and digestion of the protein sample that is independent of the absorption properties of the protein, making the method universally applicable to all types of proteinaceous samples (e.g., tissues or protein arrays). Several experimental variables were optimized to maximize product yield, and they include heating time, laser intensity, size of Au-NPs, and surface coverage of Au-NPs. Using optimized parameters, proof-of-principle experiments confirmed the ability of the plasmonic-TDD method to induce both C-cleavage and D-cleavage on several peptide standards and the protein lysozyme by detecting their thermal decomposition products with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The high spatial specificity of the plasmonic-TDD method was demonstrated by using a mask to digest designated sections of the sample surface with the heating laser and MALDI-MS imaging to map the resulting products. The solventless nature of the plasmonic-TDD method enabled the nonenzymatic on-surface digestion of proteins to proceed with undetectable delocalization of the resulting products from their precursor protein location. The advantages of this novel plasmonic-TDD method include short reaction times (30 kDa) proteins, and the rapid analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples.