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Vapor-phase film deposition of metal-organic frameworks (MOFs) would facilitate the integration of these materials into electronic devices. We studied the vapor-phase layer-by-layer deposition of zeolitic imidazolate framework 8 (ZIF-8) by consecutive, self-saturating reactions of diethyl zinc, water, and 2-methylimidazole on a substrate. Two approaches were compared: (1) Direct ZIF-8 "molecular layer deposition" (MLD), which enables a nanometer-resolution thickness control and employs only self-saturating reactions, resulting in smooth films that are crystalline as deposited, and (2) two-step ZIF-8 MLD, in which crystallization occurs during a postdeposition treatment with additional linker vapor. The latter approach resulted in a reduced deposition time and an improved MOF quality, i.e., increased crystallinity and probe molecule uptake, although the smoothness and thickness control were partially lost. Both approaches were developed in a modified atomic layer deposition reactor to ensure cleanroom compatibility.

Infrared ion spectroscopy is increasingly recognized as a method to identify mass spectrometry-detected analytes in many (bio)chemical areas and its integration in analytical laboratories is now on the horizon. Commercially available quadrupole ion trap mass spectrometers are attractive ion spectroscopy platforms but operate at relatively high pressures. This promotes collisional deactivation which directly interferes with the multiple-photon excitation process required for ion spectroscopy. To overcome this, infrared lasers having a high instantaneous power are required and therefore a majority of analytical studies have been performed at infrared free electron laser facilities. Proliferation of the technique to routine use in analytical laboratories requires table-top infrared lasers and optical parametric oscillators (OPOs) are the most suitable candidates, offering both relatively high intensities and reasonable spectral tuning ranges. Here, we explore the potential of a range of commercially available high-power OPOs for ion spectroscopy, comparing systems with repetition rates of 10 Hz, 20 kHz, 80 MHz and a continuous-wave (cw) system. We compare the performance for various molecular ions and show that the kHz and MHz repetition-rate systems outperform cw and 10 Hz systems in photodissociation efficiency and offer several advantages in terms of cost-effectiveness and practical implementation in an analytical laboratory not specialized in laser spectroscopy., Evaluation of four table-top IR lasers for ion spectroscopy in ion trap mass spectrometers shows high rep-rate lasers offer better photodissociation efficiency and are more cost-effective and practical compared to low rep-rate or cw alternatives.

Sodiated complexes of the aliphatic amino acids, Gly, Ala, Val, Leu, and Ile, were examined with infrared multiple-photon dissociation action spectroscopy utilizing light from a free-electron laser. To identify structures, the experimental spectra were compared to linear spectra calculated at the B3LYP/6-311+G(d,p) level of theory. Relative energetics of all complexes were calculated at B3LYP, B3P86, MP2(full), B3LYP-GD3BJ, and M06-2X levels using a 6-311+G(2d,2p) basis set. Spectral comparison for all complexes indicates that the dominant conformation, [N, CO], binds to the amino nitrogen and carbonyl oxygen. For all complexes except Gly, contributions are observed from [CO2-] structures, where the sodium cation binds to both oxygens of the carboxylate group in the zwitterionic form of the amino acid. The semiquantitative distribution between these two structures appears to be best-predicted by the B3LYP and MP2(full) levels of theory, with predictions from the other three levels inconsistent with the experiment.

In vitro (geno)toxicity assessment of electronic vapour products (EVPs), relative to conventional cigarette, currently uses assays, including the micronucleus and Ames tests. Whilst informative on induction of a finite endpoint and relative risk posed by test articles, such assays could benefit from mechanistic supplementation. The ToxTracker and Aneugen Clastogen Evaluation analysis can indicate the activation of reporters associated with (geno)toxicity, including DNA damage, oxidative stress, the p53-related stress response and protein damage. Here, we tested for the different effects of a selection of neat e-liquids, EVP aerosols and Kentucky reference 1R6F cigarette smoke samples in the ToxTracker assay. The assay was initially validated to assess whether a mixture of e-liquid base components, propylene glycol (PG) and vegetable glycerine (VG) had interfering effects within the system. This was achieved by spiking three positive controls into the system with neat PG/VG or phosphate-buffered saline bubbled (bPBS) PG/VG aerosol (nicotine and flavour free). PG/VG did not greatly affect responses induced by the compounds. Next, when compared to cigarette smoke samples, neat e-liquids and bPBS aerosols (tobacco flavour; 1.6% freebase nicotine, 1.6% nicotine salt or 0% nicotine) exhibited reduced and less complex responses. Tested up to a 10% concentration, EVP aerosol bPBS did not induce any ToxTracker reporters. Neat e-liquids, tested up to 1%, induced oxidative stress reporters, thought to be due to their effects on osmolarity in vitro. E-liquid nicotine content did not affect responses induced. Additionally, spiking nicotine alone only induced an oxidative stress response at a supraphysiological level. In conclusion, the ToxTracker assay is a quick, informative screen for genotoxic potential and mechanisms of a variety of (compositionally complex) samples, derived from cigarettes and EVPs. This assay has the potential for future application in the assessment battery for next-generation (smoking alternative) products, including EVPs.

Breslow intermediates (BIs) are the crucial nucleophilic amino enol intermediates formed from electrophilic aldehydes in the course of N‐heterocyclic carbene (NHC)‐catalyzed umpolung reactions. Both in organocatalytic and enzymatic umpolung, the question whether the Breslow intermediate exists as the nucleophilic enol or in the form of its electrophilic keto tautomer is of utmost importance for its reactivity and function. Herein, the preparation of charge‐tagged Breslow intermediates/keto tautomers derived from three different types of NHCs (imidazolidin‐2‐ylidenes, 1,2,4‐triazolin‐5‐ylidenes, thiazolin‐2‐ylidenes) and aldehydes is reported. An ammonium charge tag is introduced through the aldehyde unit or the NHC. ESI‐MS IR ion spectroscopy allowed the unambiguous conclusion that in the gas phase, the imidazolidin‐2‐ylidene‐derived BI indeed exists as a diamino enol, while both 1,2,4‐triazolin‐5‐ylidenes and thiazolin‐2‐ylidenes give the keto tautomer. This result coincides with the tautomeric states observed for the BIs in solution (NMR) and in the crystalline state (XRD), and is in line with our earlier calculations on the energetics of BI keto–enol equilibria., Breslow intermediates in the gas phase: In N‐heterocyclic carbene (NHC)‐catalyzed Umpolung, the reaction of the substrate aldehyde with the NHC gives the Breslow intermediate (BI) as pivotal species. The combination of IR ion spectroscopy with quantum chemical computations can determine whether the BI exists as a nucleophilic amino enol or as its keto tautomer in the gas phase, which is decisive for its reactivity both in enzymatic catalysis and in organocatalysis.

The capability of an ion trap mass spectrometer to store ions for an arbitrary amount of time allows the use of a single infrared (IR) laser to perform two-color double resonance IR–IR spectroscopic experiments on mass-to-charge (m/z) selected ions. In this single-laser IR2MS3 scheme, one IR laser frequency is used to remove a selected set of isomers from the total trapped ion population and the second IR laser frequency, from the same laser, is used to record the IR spectrum of the remaining precursor ions. This yields isomer-specific vibrational spectra of the m/z-selected ions, which can reveal the structure and identity of the initially co-isolated isomeric species. The use of a single laser greatly reduces the experimental complexity of two-color IR2MS3 and enhances its application in fields employing analytical MS. In this work, we demonstrate the methodology by acquiring single-laser IR2MS3 spectra in a forensic context, identifying two previously unidentified isomeric novel psychoactive substances (NPS) from a sample that was confiscated by the Amsterdam Police.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut. ispartof: VIRULENCE vol:12 issue:1 pages:329-345 ispartof: location:United States status: published

Deamidation of asparaginyl (Asn) peptides is a spontaneous post-translational modification that plays a significant role in degenerative diseases and other biological processes under physiological conditions. In the gas phase, deamidation of protonated peptides is a major fragmentation channel upon activation by collision-induced dissociation. Here, we present a full description of the deamidation process from protonated asparagine-serine, [AsnSer+H]+, -via infrared (IR) action spectroscopy and threshold collision-induced dissociation (TCID) experiments in combination with theoretical calculations. The IR results demonstrate that deamidation proceeds via bifurcating reaction pathways leading to furanone- and succinimide-type product ion structures, with a population analysis indicating the latter product dominates. Theory demonstrates that nucleophilic attack of the peptidyl amide oxygen onto the Asn side chain leads to furanone formation, whereas nudeophilic attack by the peptidyl amide nitrogen onto the Asn side-chain carbonyl carbon leads to the formation of the succinimide product structure. TCID experiments find that furanone formation has a threshold energy of 145 +/- 12 kJ/mol and succinimide formation occurs with a threshold energy of 131 +/- 12 kJ/mol, consistent with theoretical energies and with the spectroscopic results indicating that succinimide dominates. The results provide information regarding the inductive and steric effects of the Ser side chain on the deamidation process. The other major channel observed in the TCID experiments of [AsnSer+H]+ is dehydration, where a threshold energy of 104 +/- 10 kJ/mol is determined. A complete IR and theoretical analysis of this pathway is also provided. As for deamidation, a bifurcating pathway is found with both dominant oxazoline and minor diketopiperazine products identified. Here, the Ser side chain is directly involved in both pathways.

Brown carbon (BrC) organic molecules absorb solar radiation in the visible range and thus can influence the optical properties of atmospheric aerosol particles and cloud droplets. The absorption pr...

The hydration of carbon–carbon triple bonds is an important and atom economic synthetic transformation. Herein, we report a mild and selective method for the catalytic Markovnikov hydration of (E)-aryl enynes to the corresponding enones, mediated through the bench-stable aminium salt, tris(4-bromophenyl)ammoniumyl hexachloroantimonate (TBPA). The chemoselective and diastereoselective method proceeds under neutral metal-free conditions, delivering excellent product yields from terminal and internal alkyne units. The synthesis of biologically important (E)-3-styrylisocoumarins, including a formal synthesis of the natural product achlisocoumarin III, demonstrates the utility of this novel transformation.

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge" in the utilization of this approach is metabolite identification-confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of alpha-aminoadipic semialdehyde (alpha-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While alpha-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

Iron-sulfur (Fe-S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe-S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe-S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe-S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe-S clusters assemble, and has a cytosolic localization in most eukaryotes studied thus far. Unexpectedly, we found that the NBP35 homolog of the apicomplexan Toxoplasma gondii (TgNBP35) localizes to the outer mitochondrial membrane, with mitochondrial targeting mediated by an N-terminal transmembrane domain. We demonstrate that TgNBP35 is critical for parasite proliferation, but that, despite its mitochondrial localization, it is not required for Fe-S cluster synthesis in the mitochondrion. Instead, we establish that TgNBP35 is important for the biogenesis of cytosolic Fe-S proteins. Our data are consistent with TgNBP35 playing a central and specific role in cytosolic Fe-S cluster biosynthesis, and imply that the assembly of cytosolic Fe-S clusters occurs on the cytosolic face of the outer mitochondrial membrane in these parasites.

The product of a revealed transformation-NHC-ethynyl coupling-was observed as a catalyst transformation pathway in the Sonogashira cross-coupling, catalyzed by Pd/NHC complexes. The 2-ethynylated azolium salt was isolated in individual form and fully characterized, including X-ray analysis. A number of possible intermediates of this transformation with common formulae (NHC)n Pd(C2 Ph) (n=1,2) were observed and subjected to collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) experiments to elucidate their structure. Measured bond dissociation energies (BDEs) and IRMPD spectra were in an excellent agreement with quantum calculations for coupling product π-complexes with Pd0 . Molecular dynamics simulations confirmed the observed multiple CID fragmentation pathways. An unconventional methodology to study catalyst evolution suggests the reported transformation to be considered in the development of new catalytic systems for alkyne functionalization reactions.

Understanding the mode-of-action (MOA) of genotoxic compounds and differentiating between direct DNA interaction and indirect genotoxicity is crucial for their reliable safety assessment. ToxTracker is a stem cell-based reporter assay that detects activation of various cellular responses that are associated with genotoxicity and cancer. ToxTracker consists of 6 different GFP reporter cell lines that can detect the induction of DNA damage, oxidative stress, and protein damage in a single test. The assay can thereby provide insight into the MOA of compounds. Genotoxicity is detected in ToxTracker by activation of 2 independent GFP reporters. Activation of the Bscl2-GFP reporter is associated with induction of DNA adducts and subsequent inhibition of DNA replication and the Rtkn-GFP reporter is activated following the formation of DNA double-strand breaks. Here, we show that the differential activation of these 2 genotoxicity reporters could be used to further differentiate between a DNA reactive and clastogenic or a non-DNA-reactive aneugenic MOA of genotoxic compounds. For further classification of aneugenic and clastogenic compounds, the ToxTracker assay was extended with cell cycle analysis and aneuploidy assessment. The extension was validated using a selection of 16 (genotoxic) compounds with a well-established MOA. Furthermore, indirect genotoxicity related to the production of reactive oxygen species was investigated using the DNA damage and oxidative stress ToxTracker reporters in combination with different reactive oxygen species scavengers. With these new extensions, ToxTracker was able to accurately classify compounds as genotoxic or nongenotoxic and could discriminate between DNA-reactive compounds, aneugens, and indirect genotoxicity caused by oxidative stress.

Diversity Oriented Clicking (DOC) is a unified click-approach for the modular synthesis of lead-like structures through application of the wide family of click transformations. DOC evolved from the concept of achieving "diversity with ease", by combining classic C-C π-bond click chemistry with recent developments in connective SuFEx-technologies. We showcase 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs) as a new class of connective hub in concert with a diverse selection of click-cycloaddition processes. Through the selective DOC of SASFs with a range of dipoles and cyclic dienes, we report a diverse click-library of 173 unique functional molecules in minimal synthetic steps. The SuFExable library comprises 10 discrete heterocyclic core structures derived from 1,3- and 1,5-dipoles; while reaction with cyclic dienes yields several three-dimensional bicyclic Diels-Alder adducts. Growing the library to 278 discrete compounds through late-stage modification was made possible through SuFEx click derivatization of the pendant sulfonyl fluoride group in 96 well-plates-demonstrating the versatility of the DOC approach for the rapid synthesis of diverse functional structures. Screening for function against MRSA (USA300) revealed several lead hits with improved activity over methicillin.

The B1A' ← X1A' electronic spectra of the 1-butyn-3-yl cation (H3CCHCCH+) and the H3CCHCCH+-Ne and H3CCHCCH+-Ar complexes are measured using resonance enhanced photodissociation over the 245-285 nm range, with origin transitions occurring at 35936, 35930, and 35928 cm-1, respectively. Vibronic bands are assigned based on quantum chemical calculations and comparison of the spectra with those of the related linear methyl propargyl (H3C4H2+) and propargyl (H2C3H+) cations. The photofragment ions are C2H3+ (major) and C4H3+ (minor), with the preference for C2H3+ consistent with master equation simulations for a mechanism that involves rapid electronic deactivation and dissociation on the ground state potential energy surface.

Experimental IR spectra in the 500-1850 cm-1 fingerprint frequency range are presented for the isolated, gaseous redox pair ions [Ru(bpy)3]2+, and [Ru(bpy)3]+, where bpy = 2,2'-bipyridine. Spectra are obtained using the FELIX free-electron laser and a quadrupole ion trap mass spectrometer. The 2+ complex is generated by electrospray ionization and the charge-reduced radical cation is produced by gas-phase one-electron reduction in an ion-ion reaction with the fluoranthene radical anion. Experimental spectra are compared against computed spectra predicted by density functional theory (DFT) using different levels of theory. For the closed-shell [Ru(bpy)3]2+ ion, the match between experimental and computed IR spectra is very good; however, this is not the case for the charge-reduced [Ru(bpy)3]+ ion, which demands additional theoretical investigation. When using the hybrid B3LYP functional, we observe that better agreement with experiment is obtained upon reduction of the Hartree-Fock exact-exchange contribution from 204 calculations using the M06 functional appear to be promising in terms of the prediction of IR spectra; however, it is unclear if the correct electronic structure is obtained. The M06 and B3LYP functionals indicate that the added electron in [Ru(bpy)3)]+ is delocalized over the three bpy ligands, while the long-range corrected LC-BLYP and the CAM-B3LYP functionals show it to be more localized on a single bpy ligand. Although these latter levels of theory fail to reproduce the experimentally observed IR frequencies, one may argue that the unusually large bandwidths observed in the spectrum are due to the fluxional character of a complex with the added electron not symmetrically distributed over the ligands. The experimental IR spectra presented here can serve as benchmark for further theoretical investigations.

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.

Distinguishing positional isomers, such as compounds having different substitution patterns on an aromatic ring, presents a significant challenge for mass spectrometric analyses and is a frequently encountered difficulty in, for example, drug metabolism research. Here, we demonstrate infrared ion spectroscopy (IRIS) as a promising new mass spectrometry-based technique that easily differentiates between positional isomers of disubstituted phenyl-containing compounds. By analyzing different substitution patterns over several sets of isomeric compounds, we show that IRIS produces a highly consistent and distinct pattern of IR bands, especially in the range between 650 and 900 cm-1, that are mostly independent of the specific chemical functionality contained in the substituent group. These patterns are accurately predicted by quantum-chemically computed IR spectra and correspond well with tabulated IR group-frequencies known from conventional absorption spectroscopy. Therefore, we foresee that this method will be generally applicable to disubstituted phenyl-containing compounds and that direct interpretation of experimental IRIS spectra in terms of ortho-, meta- or para-substitution is possible, even without comparison to experimental or computationally predicted reference spectra. Strategies for the analysis of larger compounds having more congested IR spectra as well as of compounds having low (electrospray) ionization efficiencies are presented in order to demonstrate the broad applicability of this methodology.

Microspheres containing radioactive 166holmium-acetylacetonate are employed in emerging radionuclide therapies for the treatment of malignancies. At the molecular level, details on the coordination geometries of the Ho complexes are however elusive. Infrared ion spectroscopy (IRIS) was used to characterize several 165Ho-acetylacetonate complexes derived from non-radioactive microspheres. The coordination geometry of four distinct ionic complexes were fully assigned by comparison of their measured IR spectra with spectra calculated at the density functional theory (DFT) level. The coordination of each acetylacetonate ligand is dependent on the presence of other ligands, revealing an asymmetric chelation motif in some of the complexes. A fifth, previously unknown constituent of the microspheres was identified as a coordination complex containing an acetic acid ligand. These results pave the way for IRIS-based identification of microsphere constituents upon neutron activation of the metal center.

The boom in growth of 1,4-disubstituted triazole products, in particular, since the early 2000's, can be largely attributed to the birth of click chemistry and the discovery of the CuI -catalyzed azide-alkyne cycloaddition (CuAAC). Yet the synthesis of relatively simple, albeit important, 1-substituted-1,2,3-triazoles has been surprisingly more challenging. Reported here is a straightforward and scalable click-inspired protocol for the synthesis of 1-substituted-1,2,3-triazoles from organic azides and the bench stable acetylene surrogate ethenesulfonyl fluoride (ESF). The new transformation tolerates a wide selection of substrates and proceeds smoothly under metal-free conditions to give the products in excellent yield. Under controlled acidic conditions, the 1-substituted-1,2,3-triazole products undergo a Michael addition reaction with a second equivalent of ESF to give the unprecedented 1-substituted triazolium sulfonyl fluoride salts.

Film deposition and high-resolution patterning of ionic liquids (ILs) remain a challenge, despite a broad range of applications that would benefit from this type of processing. Here, we demonstrate for the first time the chemical vapor deposition (CVD) of ILs. The IL-CVD method is based on the formation of a non-volatile IL through the reaction of two vaporized precursors. Ionogel micropatterns can be easily obtained via the combination of IL-CVD and standard photolithography, and the resulting microdrop arrays can be used as microreactors. The IL-CVD approach will facilitate leveraging the properties of ILs in a range of applications and microfabricated devices. ispartof: ANGEWANDTE CHEMIE-INTERNATIONAL EDITION vol:60 issue:49 pages:25668-25673 ispartof: location:Germany status: published

Welding fumes induce lung toxicity and are carcinogenic to humans but the molecular mechanisms have yet to be clarified. The aim of this study was to evaluate the toxicity of stainless and mild steel particles generated via gas–metal arc welding using primary human small airway epithelial cells (hSAEC) and ToxTracker reporter murine stem cells, which track activation of six cancer-related pathways. Metal content (Fe, Mn, Ni, Cr) of the particles was relatively homogenous across particle size. The particles were not cytotoxic in reporter stem cells but stainless steel particles activated the Nrf2-dependent oxidative stress pathway. In hSAEC, both particle types induced time- and dose-dependent cytotoxicity, and stainless steel particles also increased generation of reactive oxygen species. The cellular metal content was higher for hSAEC compared to the reporter stem cells exposed to the same nominal dose. This was, in part, related to differences in particle agglomeration/sedimentation in the different cell media. Overall, our study showed differences in cytotoxicity and activation of cancer-related pathways between stainless and mild steel welding particles. Moreover, our data emphasizes the need for careful assessment of the cellular dose when comparing studies using different in vitro models.

IntroductionAlthough associations of total plasma N-glycome (TPNG) with type 2 diabetes have been reported, little is known on the role of TPNG in type 2 diabetes complications, a major cause of type 2 diabetes-related morbidity and mortality. Here, we assessed TPNG in relation to type 2 diabetes complications in subsamples of two Dutch cohorts using mass spectrometry (n=1815 in DiaGene and n=1518 in Hoorn Diabetes Care System).Research design and methodsBlood plasma samples and technical replicates were pipetted into 96-well plates in a randomized manner. Peptide:N-glycosidase F (PNGase F) was used to release N-glycans, whereafter sialic acids were derivatized for stabilization and linkage differentiation. After total area normalization, 68 individual glycan compositions were quantified in total and were used to calculate 45 derived traits which reflect structural features of glycosylation. Associations of glycan features with prevalent and incident microvascular or macrovascular complications were tested in logistic and Cox regression in both independent cohorts and the results were meta-analyzed.ResultsOur results demonstrated similarities between incident and prevalent complications. The strongest association for prevalent cardiovascular disease was a high level of bisection on a group of diantennary glycans (A2FS0B; OR=1.38, p=1.34×10−11), while for prevalent nephropathy the increase in 2,6-sialylation on triantennary glycans was most pronounced (A3E; OR=1.28, p=9.70×10−6). Several other TPNG features, including fucosylation, galactosylation, and sialylation, firmly demonstrated associations with prevalent and incident complications of type 2 diabetes.ConclusionsThese findings may provide a glance on how TPNG patterns change before complications emerge, paving the way for future studies on prediction biomarkers and potentially disease mechanisms.

Increasing efforts are directed towards the development of sustainable alternative protein sources among which microbial protein (MP) is one of the most promising. Especially when waste streams are used as substrates, the case for MP could become environmentally favorable. The risks of using organic waste streams for MP production–the presence of pathogens or toxicants–can be mitigated by their anaerobic digestion and subsequent aerobic assimilation of the (filter-sterilized) biogas. Even though methane and hydrogen oxidizing bacteria (MOB and HOB) have been intensively studied for MP production, the potential benefits of their co-cultivation remain elusive. Here, we isolated a diverse group of novel HOB (that were capable of autotrophic metabolism), and co-cultured them with a defined set of MOB, which could be grown on a mixture of biogas and H2/O2. The combination of MOB and HOB, apart from the CH4 and CO2 contained in biogas, can also enable the valorization of the CO2 that results from the oxidation of methane by the MOB. Different MOB and HOB combinations were grown in serum vials to identify the best-performing ones. We observed synergistic effects on growth for several combinations, and in all combinations a co-culture consisting out of both HOB and MOB could be maintained during five days of cultivation. Relative to the axenic growth, five out of the ten co-cultures exhibited 1.1–3.8 times higher protein concentration and two combinations presented 2.4–6.1 times higher essential amino acid content. The MP produced in this study generally contained lower amounts of the essential amino acids histidine, lysine and threonine, compared to tofu and fishmeal. The most promising combination in terms of protein concentration and essential amino acid profile was Methyloparacoccus murrelli LMG 27482 with Cupriavidus necator LMG 1201. Microbial protein from M. murrelli and C. necator requires 27–67% less quantity than chicken, whole egg and tofu, while it only requires 15% more quantity than the amino acid-dense soybean to cover the needs of an average adult. In conclusion, while limitations still exist, the co-cultivation of MOB and HOB creates an alternative route for MP production leveraging safe and sustainably-produced gaseous substrates.

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is ‘trans-stimulated’ by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites., Author summary The causative agent of toxoplasmosis, Toxoplasma gondii, is a versatile intracellular parasite that can proliferate within nucleated cells of warm-blooded organisms. In order to survive, T. gondii parasites must scavenge the cationic amino acids lysine and arginine from their hosts. In a previous study, we demonstrated that a plasma membrane-localized protein called TgApiAT1 facilitates the uptake of arginine into the parasite. We found that parasites lacking TgApiAT1 could proliferate when cultured in medium containing high concentrations of arginine, suggesting the existence of an additional uptake pathway for arginine. In the present study, we demonstrate that this second uptake pathway is mediated by TgApiAT6-1, a protein belonging to the same solute transporter family as TgApiAT1. We show that TgApiAT6-1 is the major lysine transporter of the parasite, and that it is critical for parasite proliferation. Furthermore, we demonstrate that TgApiAT6-1 can transport arginine into parasites under conditions in which arginine concentrations are high and lysine concentrations are comparatively lower. These data support a model for the finely-tuned acquisition of essential cationic amino acids that involves multiple transporters, and which likely contributes to these parasites being able to survive and proliferate within a wide variety of host cell types.

Introduction Autosomal dominant polycystic kidney disease (ADPKD) causes kidney failure typically in adulthood, but the disease starts in utero. Copeptin, epidermal growth factor (EGF), and monocyte chemoattractant protein-1 (MCP-1) are associated with severity and hold prognostic value in adults but remain unstudied in the early disease stage. Kidneys from adults with ADPKD exhibit macrophage infiltration, and a prominent role of MCP-1 secretion by tubular epithelial cells is suggested from rodent models. Methods In a cross-sectional study, plasma copeptin, urinary EGF, and urinary MCP-1 were evaluated in a pediatric ADPKD cohort and compared with age-, sex-, and body mass index (BMI)-matched healthy controls. MCP-1 was studied in mouse collecting duct cells, human proximal tubular cells, and fetal kidney tissue. Results Fifty-three genotyped ADPKD patients and 53 controls were included. The mean (SD) age was 10.4 (5.9) versus 10.5 (6.1) years (P = 0.543), and the estimated glomerular filtration rate (eGFR) was 122.7 (39.8) versus 114.5 (23.1) ml/min per 1.73 m2 (P = 0.177) in patients versus controls, respectively. Plasma copeptin and EGF secretion were comparable between groups. The median (interquartile range) urinary MCP-1 (pg/mg creatinine) was significantly higher in ADPKD patients (185.4 [213.8]) compared with controls (154.7 [98.0], P = 0.010). Human proximal tubular cells with a heterozygous PKD1 mutation and mouse collecting duct cells with a PKD1 knockout exhibited increased MCP-1 secretion. Human fetal ADPKD kidneys displayed prominent MCP-1 immunoreactivity and M2 macrophage infiltration. Conclusion An increase in tubular MCP-1 secretion is an early event in ADPKD. MCP-1 is an early disease severity marker and a potential treatment target., Graphical abstract

Recently, small compound-based therapies have provided new insights into the treatment of glioblastoma multiforme (GBM) by inducing oxidative impairment. Kinetin riboside (KR) and newly designed derivatives (8-azaKR, 7-deazaKR) selectively affect the molecular pathways crucial for cell growth by interfering with the redox status of cancer cells. Thus, these compounds might serve as potential alternatives in the oxidative therapy of GBM. The increased basal levels of reactive oxygen species (ROS) in GBM support the survival of cancer cells and cause drug resistance. The simplest approach to induce cell death is to achieve the redox threshold and circumvent the antioxidant defense mechanisms. Consequently, cells become more sensitive to oxidative stress (OS) caused by exogenous agents. Here, we investigated the effect of KR and its derivatives on the redox status of T98G cells in 2D and 3D cell culture. The use of spheroids of T98G cells enabled the selection of one derivative—7-deazaKR—with comparable antitumor activity to KR. Both compounds induced ROS generation and genotoxic OS, resulting in lipid peroxidation and leading to apoptosis. Taken together, these results demonstrated that KR and 7-deazaKR modulate the cellular redox environment of T98G cells, and vulnerability of these cells is dependent on their antioxidant capacity.

Chemical vapor deposition of metal-organic frameworks (MOF-CVD) will facilitate the integration of porous and crystalline coatings in electronic devices. In the two-step MOF-CVD process, a precursor layer is first deposited and subsequently converted to a MOF through exposure to linker vapor. We herein report the impact of different metal oxide and metalcone layers as precursors for zeolitic imidazolate framework ZIF-8 films. ispartof: DALTON TRANSACTIONS vol:50 issue:20 pages:6784-6788 ispartof: location:England status: published

Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inheritable kidney disease characterized by the development of fluid-filled cysts in all nephron segments, leading to loss of renal function. Mutations in PKD1 or PKD2, which encode polycystin-1 and polycystin-2, are the most common cause of ADPKD. The molecular mechanisms underlying cystogenesis are poorly characterized but it is postulated that disturbed calcium homeostasis is a primary event in cystogenesis. The precise molecular players that cause this disturbance are still a poorly explored area, especially in relevant human cell types. We therefore aim to characterize the profile of calcium-coupled receptors and channels in a human renal epithelial cell model, to identify which receptors and channels are present and whether their function is affected in ADPKD. Method Human urine-derived conditionally immortalized proximal tubule epithelial cells (ciPTECs) of ADPKD patients and healthy controls were screened for calcium-coupled GPCRs, using a GPCR agonist library on Fura-2 loaded cell populations seeded in 96-well format using the Flexstation3 (Molecular Devices). Validation of specific hits was done using single-cell measurements with a fluorescence microscope and built-in perfusion system. The expression of TRP channels and STIM/Orai proteins was determined via qPCR. Results From a library of 418 GPCR agonists a selective amount of calcium-coupled GPCRs was found functionally active in ciPTECs. ciPTECs from both healthy controls and ADPKD patients were found to functionally express purinergic -, histamine -, serotonin and dopamine receptors. Through qPCR we found expression of various TRP channels, including TRPML1, TRPC1/3, TRPM3/4/7, TRPV4 and TRPA1, as well as high expression of STIM1/2 and Orai1/2/3. Conclusion We describe the first thorough characterization of molecular players involved in calcium signalling mechanisms in human renal epithelial cells, including the profile of calcium-coupled GPCRs and the expression of TRP channels and STIM/Orai proteins, of further interest to investigate disturbed calcium dynamics in ADPKD.

Liquid chromatography-mass spectrometry (LC-MS) is, due to its high sensitivity and selectivity, currently the method of choice in (bio)analytical studies involving the (comprehensive) profiling of metabolites in body fluids. However, as closely related isomers are often hard to distinguish on the basis of LC-MS(MS) and identification is often dependent on the availability of reference standards, the identification of the chemical structures of detected mass spectral features remains the primary limitation. Infrared ion spectroscopy (IRIS) aids identification of MS-detected ions by providing an infrared (IR) spectrum containing structural information for a detected MS-feature. Moreover, IR spectra can be routinely and reliably predicted for many types of molecular structures using quantum-chemical calculations, potentially avoiding the need for reference standards. In this work, we demonstrate a workflow for reference-free metabolite identification that combines experiments based on high-pressure liquid chromatography (HPLC), MS and IRIS with quantum-chemical calculations that efficiently generate IR spectra and give the potential to enable reference-standard free metabolite identification. Additionally, a scoring procedure is employed which shows the potential for automated structure assignment of unknowns. Via a simple, illustrative example where we identify lysine in the plasma of a hyperlysinemia patient, we show that this approach allows the efficient assignment of a database-derived molecular structure to an unknown.

Electronic spectra of mass-selected HC4H+-Arn (n = 1-3) and HC4H+-(N2)n (n = 1-2) complexes are measured over the 290-530 nm range using resonance-enhanced photodissociation spectroscopy in a tandem mass spectrometer. Vibronic transitions in the visible region are compared with previous experimental and theoretical results for the A2Πu ← X2Πg band system of HC4H+. Hole burning experiments confirm that transitions over the 290-340 nm range involve the diacetylene cation (HC4H+). On the basis of previous experiments and comparison with spectra of isoelectronic molecules the peaks are assigned to the 22Πu ← X2Πg band system, with the origin transition for HC4H+-Ar occurring at 29723 cm-1. The main progression has a spacing of 906 cm-1 and is assigned to the symmetric C-C stretch vibrational mode (ν3). The assignment of additional bands is complicated by spectral congestion, the possible presence of energetically close-lying electronic states, vibronic coupling effects, and by the fact that HC4H+ possibly becomes nonlinear in the 22Πu state.

Gas-phase conformations of the sodium-cationized forms of the 2′-deoxycytidine and cytidine mononucleotides, [pdCyd+Na]+ and [pCyd+Na]+, are examined by infrared multiple photon dissociation action spectroscopy. Complimentary electronic structure calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory provide candidate conformations and their respective predicted IR spectra for comparison across the IR fingerprint and hydrogen-stretching regions. Comparisons of the predicted IR spectra and the measured infrared multiple photon dissociation action spectra provide insight into the impact of sodium cationization on intrinsic mononucleotide structure. Further, comparison of present results with those reported for the sodium-cationized cytidine nucleoside analogues elucidates the impact of the phosphate moiety on gas-phase structure. Across the neutral, protonated, and sodium-cationized cytidine mononucleotides, a preference for stabilization of the phosphate moiety and nucleobase orientation is observed, although the details of this stabilization differ with the state of cationization. Several low-energy conformations of [pdCyd+Na]+ and [pCyd+Na]+ involving several different orientations of the phosphate moiety and sugar puckering modes are observed experimentally.

L-Thymidine (L-dThd) is the enantiomer of D-thymidine (dThd), a naturally-occurring pyrimidine nucleoside found within DNA nucleic acids. L-dThd, also known as Telbivudine, does not occur naturally, but in the last decade has found successful application as an antiviral medication for hepatitis B virus infection. In this work, the gas-phase conformers of the protonated and sodium cationized forms of L-dThd, [L-dThd+H]+ and [L-dThd + Na]+, are investigated using infrared multiple photon dissociation (IRMPD) action spectroscopy complemented by electronic structure calculations performed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory. Comparisons between the experimental IRMPD spectra and theoretical linear IR spectra elucidate the stable low-energy conformations adopted by these L-dThd complexes generated by electrospray ionization. Minor 2,4-dihydroxy tautomers (T) and O2 protonated conformers contribute to the experimental [L-dThd+H]+ population, whereas conformers involving tridentate binding of Na+ to the O2, O4′, and O5′ atoms primarily contribute to the experimental [L-dThd + Na]+ population. Theory predicts a tautomer as the protonated ground conformer of [L-dThd+H]+ with thymine in an anti orientation and a tridentate (O2O4′O5′) sodium cationized ground conformer with a syn thymine orientation, consistent with theoretical predictions for [dThd+H]+ and [dThd + Na]+, respectively. Both protonated and sodium cationized L-dThd and dThd illustrate highly parallel IRMPD spectral features as expected. Survival yield analyses of data from energy-resolved collision-induced dissociation experiments elucidate the relative stabilities of [L-dThd+H]+ and [L-dThd + Na]+ as compared to the corresponding enantiomeric systems. Identical results are exhibited in the survival yield analyses as anticipated for enantiomeric complexes to simple cations. This work employs the same robust methodology that has provided structural characterization and energetic insight for similar systems preceding it to validate the parallel theoretical and experimental behaviors expected for enantiomers.

Infrared multiple photon dissociation action spectroscopy was performed on the AlaOrn b(2)(+) and AlaAlaOrn b(3)(+) fragment ions from ornithine-containing tetrapeptides. Infrared spectra were obtained in the fingerprint region (1000 – 2000 cm(−1)) using the infrared free electron lasers at the Centre Laser Infrarouge d’Orsay (CLIO) facility in Orsay, France and the Free Electron Lasers for Infrared eXperiments (FELIX) facility in Nijmegen, the Netherlands. A novel terminal ornithine lactam AO(+) b2(+) structure was synthesized for experimental comparison and spectroscopy confirms that the b2(+) fragment ion from AOAA forms a lactam structure. Comparison of experimental spectra with scaled harmonic frequencies at the B3LYP/6-31+G(d,p) level of theory shows that AO(+) b(2)(+) forms a terminal lactam protonated either on the lactam carbonyl oxygen or the N-terminal nitrogen atom. Several low-lying conformers of these isomers are likely populated following IRMPD dissociation. Similarly, a comparison of the experimental IRMPD spectrum with calculated spectra shows that AAO(+) b(3)(+)-ions also adopt a lactam structure, again with multiple different protonation sites, during fragmentation. This study provides spectroscopic confirmation for the lactam cyclization proposed for the “ornithine effect” and represents an alternative b(n)(+) structure to the oxazolone and diketopiperazine/macrocycle structures most often formed.