Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus.

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here, we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa protein without functional annotation. The protein comprises two characteristic PepSY domains, which is why we termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications toward the sustainable purification and separation of rare-earth elements.

Determination of carbon in microplastics and single cells by total consumption microdroplet ICP-TOFMS.

Recently, spICP-MS analyses of microplastics have demonstrated that the detection capabilities of ICP-MS are sufficient to determine the size and composition of such materials. However, solution nebulization or microdroplet generation limits the sizes of droplets, microparticles, or cells that can be efficiently introduced into commonly used horizontal ICP-MS configurations. Therefore, we introduced the development of a downward-pointing ICP coupled to a time-of-flight mass spectrometer (ICP-TOFMS), which enables quantitative transport of large microdroplets (diameters up to 90 µm) into the ICP. Here, we report the capabilities of downward ICP-TOFMS for the quantitative analysis of single cells and microplastic particles. For calibration of element mass amount per particle or cell, microdroplets (70 µm diameter) composed of multielement solutions were measured by ICP-TOFMS. Microplastic beads (polystyrene) and spleenocyte cells were then also embedded in microdroplets and measured by ICP-TOFMS with ion optics optimized to determine the signals from 12C+ and other isotopes of interest, including 140Ce, 153Eu, 165Ho, and 175Lu from the REE beads and 31P for the cells. The results achieved using the prototype instrument of a vertical downward-pointing ICP-TOFMS demonstrate that such a plasma configuration is well suited to analyze microplastics and single cells. For single microbead and cell analyses, the critical mass for carbon was 4.8 pg, and the mean determined carbon mass amounts were 14 and 23 pg, respectively. For the microbead analysis, the detected carbon mass corresponds to a particle diameter of 2.93 ± 0.24 µm, which is consistent with the scanning (transmission) electron microscopy-determined diameter of 2.97 ± 0.04 µm. Furthermore, in the analysis of spleenocyte cells, carbon and phosphorus masses were determined to be correlated.

New Orientation: A Downward-pointing Vertical Inductively Coupled Plasma Mass Spectrometer for the Analysis of Microsamples.

We present a prototype of a vertical-downward configuration of an inductively coupled plasma mass spectrometer (ICPMS) allowing the sample introduction from the top. With this novel approach to orient the ICP downward, we aim to expand the sample transport capabilities in ICPMS especially for the transport of droplets or particles with a final goal to analyze individual cells. Because of this gravity-assisted sampling approach, the transport of larger sized droplets, that is, droplets that would be difficult to transport into a horizontally oriented ICPMS, becomes possible and, furthermore, becomes independent of the droplets' size or size distribution. We demonstrate that droplets of an initial size of 70 µm can be successfully transported into the plasma at dispensing frequencies up to 1 kHz without the need for a desolvation device. In addition, we observed that the implementation of a desolvation device, that is, a gas-exchange device (GED), can improve the detection efficiencies (DEs). Compared to operating conditions that are commonly reported for ICPMS experiments, significantly different optimization parameters (radio frequency power and gas flow rates) were tested in the presented experiments here while instrument type-specific DEs were obtained.

Fundamental studies on droplet throughput and the analysis of single cells using a downward-pointing ICP-time-of-flight mass spectrometer.

Capabilities of the downwardly oriented inductively coupled plasma mass spectrometer (ICP-MS) recently reported (Vonderach et al. 2021) were studied using a time-of-flight mass spectrometer (TOFMS) yielding benefits for the fast detection of short transient signals containing multi-element information. The previously reported sample inlet configuration for the analysis of microdroplets was equipped with two extra gas inlets for the supply of argon and helium, which enabled a more precise optimization of the sample introduction and operating conditions of the plasma. Furthermore, the sample supply system was operated at elevated temperatures to enhance the desolvation of the droplets prior to their introduction into the plasma. Transient droplet signals with frequencies of up to 1000 Hz were recorded for 74 µm (diameter) sized droplets. The upper detectable droplet size was limited by the droplet generator used and was measured at 93 µm (diameter). The droplets served as the transporter for biological cells so that the described setup could be used to analyze single cells. Mouse lung cells embedded into droplets were detected successfully according to their Cs droplet tracer, Ir nucleus marker, surface markers and the phosphorus content. Transient signals were recorded at a time resolution of 33 µs in order to investigate the signal structure of single droplet-cell events containing multiple elements. Signals between 200-400 µs (FW base) and ≤100 µs (FWHM) in duration were measured. To ensure that the droplet formation process did not affect the sampled cells, different types of cells were localized within the droplets using optical inspection directly after droplet formation and it was possible to observe that cells remained intact with random sampling. The results indicate that a downward-pointing ICP-MS in combination with the microdroplet-based approach can be considered as an alternative to commonly used ICP-MS systems for single cell analysis, and might be suitable for online coupling to flow cytometry.

Use of rare-earth elements in the phyllosphere colonizer Methylobacterium extorquens PA1.

Until recently, rare-earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase XoxF that strictly relies on REEs for its activity. Some methylotrophs only contain xoxF, while others, including the model phyllosphere colonizer Methylobacterium extorquens PA1, harbor this gene in addition to mxaFI encoding a Ca2+ -dependent enzyme. Here we found that REEs induce the expression of xoxF in M. extorquens PA1, while repressing mxaFI, suggesting that XoxF is the preferred methanol dehydrogenase in the presence of sufficient amounts of REE. Using reporter assays and a suppressor screen, we found that lanthanum (La3+ ) is sensed both in a XoxF-dependent and independent manner. Furthermore, we investigated the role of REEs during Arabidopsis thaliana colonization. Element analysis of the phyllosphere revealed the presence of several REEs at concentrations up to 10 µg per g dry weight. Complementary proteome analyses of M. extorquens PA1 identified XoxF as a top induced protein in planta and a core set of La3+ -regulated proteins under defined artificial media conditions. Among these was a REE-binding protein that is encoded next to a gene for a TonB-dependent transporter. The latter was essential for REE-dependent growth on methanol indicating chelator-assisted uptake of REEs.

Peptide-Coated Platinum Nanoparticles with Selective Toxicity against Liver Cancer Cells.

Peptide-stabilized platinum nanoparticles (PtNPs) were developed that have significantly greater toxicity against hepatic cancer cells (HepG2) than against other cancer cells and non-cancerous liver cells. The peptide H-Lys-Pro-Gly-dLys-NH2 was identified by a combinatorial screening and further optimized to enable the formation of water-soluble, monodisperse PtNPs with average diameters of 2.5 nm that are stable for years. In comparison to cisplatin, the peptide-coated PtNPs are not only more toxic against hepatic cancer cells but have a significantly higher tumor cell selectivity. Cell viability and uptake studies revealed that high cellular uptake and an oxidative environment are key for the selective cytotoxicity of the peptide-coated PtNPs.

Mass spectrometry-based approaches to study lanthanides and lanthanide-dependent proteins in the phyllosphere.

Rare-earth elements (REEs) were recently discovered to be biologically significant. The finding was originally made with the methanol dehydrogenase XoxF, which depends on REEs for its activity, and reports of lanthanide-utilizing bacteria have since expanded. Environmental proteomics allows the identification of proteins specifically induced by the presence of lanthanides or can provide insights into the preferred use of lanthanide-dependent and -independent isoenzymes, for example. Here we describe protocols for the growth and subsequent mass spectrometry-based proteome analysis of bacteria obtained from controlled artificial media and from the phyllosphere of the model plant Arabidopsis thaliana. In addition, the use of inductively coupled plasma mass spectrometry (ICP-MS) is described for the quantification of REEs in biological samples.

Lone-Pair-Induced Structural Ordering in the Mixed-Valent 0D Metal-Halides Rb23BiIII x SbIII 7-x SbV 2Cl54 (0 ≤ x ≤ 7).

Mixed-valent metal-halides containing ns2 lone pairs may exhibit intense visible absorption, while zero-dimensional (0D) ns2-based metal-chlorides are generally colorless but have demonstrated promising optoelectronic properties suitable for thermometry and radiation detection. Here, we report solvothermally synthesized mixed-valent 0D metal-halides Rb23BiIII x SbIII 7-x SbV 2Cl54 (0 ≤ x ≤ 7). Rb23SbIII 7SbV 2Cl54 crystallizes in an orthorhombic space group (Cmcm) with a unique, layered 0D structure driven by the arrangement of the 5s2 lone pairs of the SbIIICl6 octahedra. This red material is likely the true structure of a previously reported monoclinic "Rb2.67SbCl6" phase, the structure of which was not determined. Partially or fully substituting SbIII with isoelectronic BiIII yields the series Rb23BiIII x SbIII 7-x SbV 2Cl54 (0 < x ≤ 7), which exhibits a similar layered 0D structure but with additional disorder that yields a trigonal crystal system with an enantiomorphic space group (R32). Second harmonic generation of 532 nm light from a 1064 nm laser using Rb23BiIII 7SbV 2Cl54 powder confirms the noncentrosymmetry of this space group. As with the prototypical mixed-valent pnictogen halides, the visible absorption bands of the Rb23BiIII x SbIII 7-x SbV 2Cl54 family are the result of intervalent SbIII-SbV and mixed-valent BiIII-SbV charge transfer bands (CTB), with a blueshift of the absorption edge as BiIII substitution increases. No PL is observed from this family of semiconductors, but a crystal of Rb23BiIII 7SbV 2Cl54 exhibits a high resistivity of 1.0 × 1010 Ω·cm and X-ray photoconductivity with a promising µτ product of 8.0 × 10-5 cm2 s-1 V-1. The unique 0D layered structures of the Rb23BiIII x SbIII 7-x SbV 2Cl54 family highlight the versatility of the ns2 lone pair in semiconducting metal-halides, pointing the way toward new functional 0D metal-halide compounds.

Untargeted metabolomics links glutathione to bacterial cell cycle progression.

Cell cycle progression requires the coordination of cell growth, chromosome replication, and division. Consequently, a functional cell cycle must be coupled with metabolism. However, direct measurements of metabolome dynamics remained scarce, in particular in bacteria. Here, we describe an untargeted metabolomics approach with synchronized Caulobacter crescentus cells to monitor the relative abundance changes of ~400 putative metabolites as a function of the cell cycle. While the majority of metabolite pools remains homeostatic, ~14% respond to cell cycle progression. In particular, sulfur metabolism is redirected during the G1-S transition, and glutathione levels periodically change over the cell cycle with a peak in late S phase. A lack of glutathione perturbs cell size by uncoupling cell growth and division through dysregulation of KefB, a K+/H+ antiporter. Overall, we here describe the impact of the C. crescentus cell cycle progression on metabolism, and in turn relate glutathione and potassium homeostasis to timely cell division.

Unraveling the Surface Chemistry and Structure in Highly Active Sputtered Pt3Y Catalyst Films for the Oxygen Reduction Reaction.

Platinum is the most widely used and best performing sole element for catalyzing the oxygen reduction reaction (ORR) in low-temperature fuel cells. Although recyclable, there is a need to reduce the amount used in current fuel cells for their extensive uptake in society. Alloying platinum with rare-earth elements such as yttrium can provide an increase in activity of more than seven times, reducing the amount of platinum and the total amount of catalyst material required for the ORR. As yttrium is easily oxidized, exposure of the Pt-Y catalyst layer to air causes the formation of an oxide layer that can be removed during acid treatment, leaving behind a highly active pure platinum overlayer. This paper presents an investigation of the overlayer composition and quality of Pt3Y films sputtered from an alloy target. The Pt3Y catalyst surface is investigated using synchrotron radiation X-ray photoelectron spectroscopy before and after acid treatment. A new substoichiometric oxide component is identified. The oxide layer extends into the alloy surface, and although it is not completely removed with acid treatment, the catalyst still achieves the expected high ORR activity. Other surface-sensitive techniques show that the sputtered films are smooth and bulk X-ray diffraction reveals many defects and high microstrain. Nevertheless, sputtered Pt3Y exhibits a very high activity regardless of the film's oxide content and imperfections, highlighting Pt3Y as a promising catalyst. The obtained results will help to support its integration into fuel cell systems.