Sixty years of research on bracken fern (Pteridium spp.) toxins: Environmental exposure, health risks and recommendations for bracken fern control.

Bracken fern (Pteridium spp.) is a highly problematic plant worldwide due to its toxicity in combination with invasive properties on former farmland, in deforested areas and on disturbed natural habitats. The carcinogenic potential of bracken ferns has caused scientific and public concern for six decades. Its genotoxic effects are linked to illudane-type glycosides (ITGs), their aglycons and derivatives. Ptaquiloside is considered the dominating ITG, but with significant contributions from other ITGs. The present review aims to compile evidence regarding environmental pollution by bracken fern ITGs, in the context of their human and animal health implications. The ITG content in bracken fern exhibits substantial spatial, temporal, and chemotaxonomic variation. Consumption of bracken fern as food is linked to human gastric cancer but also causes urinary bladder cancers in bovines browsing on bracken. Genotoxic metabolites are found in milk and meat from bracken fed animals. ITG exposure may also take place via contaminated water with recent data pointing to concentrations at microgram/L-level following rain events. Airborne ITG-exposure from spores and dust has also been documented. ITGs may synergize with major biological and environmental carcinogens like papillomaviruses and Helicobacter pylori to induce cancer, revealing novel instances of chemical and biological co-carcinogenesis. Thus, the emerging landscape from six decades of bracken research points towards a global environmental problem with increasingly complex health implications.

Label-Free In Situ Chemical Characterization of Amyloid Plaques in Human Brain Tissues.

The accumulation of amyloid plaques and increased brain redox burdens are neuropathological hallmarks of Alzheimer's disease. Altered metabolism of essential biometals is another feature of Alzheimer's, with amyloid plaques representing sites of disturbed metal homeostasis. Despite these observations, metal-targeting disease treatments have not been therapeutically effective to date. A better understanding of amyloid plaque composition and the role of the metals associated with them is critical. To establish this knowledge, the ability to resolve chemical variations at nanometer length scales relevant to biology is essential. Here, we present a methodology for the label-free, nanoscale chemical characterization of amyloid plaques within human Alzheimer's disease tissue using synchrotron X-ray spectromicroscopy. Our approach exploits a C-H carbon absorption feature, consistent with the presence of lipids, to visualize amyloid plaques selectively against the tissue background, allowing chemical analysis to be performed without the addition of amyloid dyes that alter the native sample chemistry. Using this approach, we show that amyloid plaques contain elevated levels of calcium, carbonates, and iron compared to the surrounding brain tissue. Chemical analysis of iron within plaques revealed the presence of chemically reduced, low-oxidation-state phases, including ferromagnetic metallic iron. The zero-oxidation state of ferromagnetic iron determines its high chemical reactivity and so may contribute to the redox burden in the Alzheimer's brain and thus drive neurodegeneration. Ferromagnetic metallic iron has no established physiological function in the brain and may represent a target for therapies designed to lower redox burdens in Alzheimer's disease. Additionally, ferromagnetic metallic iron has magnetic properties that are distinct from the iron oxide forms predominant in tissue, which might be exploitable for the in vivo detection of amyloid pathologies using magnetically sensitive imaging. We anticipate that this label-free X-ray imaging approach will provide further insights into the chemical composition of amyloid plaques, facilitating better understanding of how plaques influence the course of Alzheimer's disease.

Advancing PROTAC Characterization: Structural Insights through Adducts and Multimodal Tandem-MS Strategies.

Proteolysis targeting chimeras (PROTACs) are specialized molecules that bind to a target protein and a ubiquitin ligase to facilitate protein degradation. Despite their significance, native PROTACs have not undergone tandem mass spectrometry (MS) analysis. To address this gap, we conducted a pioneering investigation on the fragmentation patterns of two PROTACs in development, dBET1 and VZ185. Employing diverse cations (sodium, lithium, and silver) and multiple tandem-MS techniques, we enhanced their structural characterization. Notably, lithium cations facilitated comprehensive positive-mode coverage for dBET1, while negative polarity mode offered richer insights. Employing de novo structure determination on 2DMS data from degradation studies yielded crucial insights. In the case of VZ185, various charge states were observed, with [M + 2H]2+ revealing fewer moieties than [M + H]+ due to charge-related factors. Augmenting structural details through silver adducts suggested both charge-directed and charge-remote fragmentation. This comprehensive investigation identifies frequently dissociated bonds across multiple fragmentation techniques, pinpointing optimal approaches for elucidating PROTAC structures. The findings contribute to advancing our understanding of PROTACs, pivotal for their continued development as promising therapeutic agents.

Towards detection of structurally-diverse glycated epitopes in native proteins: Single-chain antibody directed to non-A1c epitope in human haemoglobin.

Over 500 million people worldwide are affected by diabetes mellitus, a chronic disease that leads to high blood glucose levels and causes severe side effects. The predominant biological marker for diagnosis of diabetes is glycated haemoglobin (GHb). In human blood the predominant reducing sugar, glucose, irreversibly conjugates onto accessible amine groups within Hb. Most methods for diagnosis and monitoring of diabetes selectively detect N-terminal glycation at Val-1 on the ß-globin chain, but not glycation at other sites. Detection of other glycated epitopes of GHb has the potential to provide new information on the extent, duration and timing of elevated glucose, facilitating personalised diagnosis and intelligent diabetic control. In this work, a new anti-GHb Fab antibody (Fab-1) specific for haemoglobin A1c (HbA1c) with nanomolar affinity was discovered via epitope-directed immunisation and phage display. A single chain variable fragment (scFv) antibody derived from Fab-1 retained affinity and specificity for HbA1c, and affinity was enhanced tenfold upon addition of an enhanced green fluorescent protein tag. Both the scFv and Fab-1 recognised an epitope within HbA1c that was distinct from ß-Val-1, and our data suggest that this epitope may include glycation at Lys-66 in the ß-globin chain. To our knowledge, this is the first report of an scFv/Fab anti-glycated epitope antibody that recognises a non-A1c epitope in GHb, and confirms that fructosamine attached to different, discrete glycation sites within the same protein can be resolved from one another by immunoassay.

Characterizing lignins from various sources and treatment processes after optimized sample preparation techniques and analysis via ESI-HRMS and custom mass defect software tools.

Sample preparation of complex, natural mixtures such as lignin prior to mass spectrometry analysis, however minimal, is a critical step in ensuring accurate and interference-free results. Modern shotgun-MS techniques, where samples are directly injected into a high-resolution mass spectrometer (HRMS) with no prior separation, usually still require basic sample pretreatment such as filtration and appropriate solvents for full dissolution and compatibility with atmospheric pressure ionization interfaces. In this study, sample preparation protocols have been established for a unique sample set consisting of a wide variety of degraded lignin samples from numerous sources and treatment processes. The samples were analyzed via electrospray (ESI)-HRMS in negative and positive ionization modes. The resulting information-rich HRMS datasets were then transformed into the mass defect space with custom R scripts as well as the open-source Constellation software as an effective way to visualize changes between the samples due to the sample preparation and ionization conditions as well as a starting point for comprehensive characterization of these varied sample sets. Optimized conditions for the four investigated lignins are proposed for ESI-HRMS analysis for the first time, giving an excellent starting point for future studies seeking to better characterize and understand these complex mixtures.

In Silico Demonstration of Two-Dimensional Mass Spectrometry Using Spatially Dependent Fragmentation.

Two-dimensional mass spectrometry (2DMS) allows for the analysis of complex mixtures of all kinds at high speed and resolution without data loss from isolation or biased acquisition, effectively generating tandem mass spectrometry information for all ions at once. Currently, this technique is limited to instruments utilizing an ion trap such as the Fourier transform ion cyclotron resonance or linear ion traps. To overcome this limitation, new fragmentation waveforms were used in either a temporal or spatial configuration, allowing for the application of 2DMS on a much wider array of instruments. A simulated example of a time-of-flight-based instrument is shown with the new waveforms, which allowed for the correlation of fragment ions to their respective precursors through the processing of the modulation of fragmentation intensity with a Fourier transform. This application indicated that 2D modulation and Fourier precursor/fragment intensity correlation are possible in any case where separation, either temporally or spatially, can be achieved, allowing 2DMS to be applied to almost every type of mass spectrometry instrument.

Resourcing the arts for youth well-being: challenges in Aotearoa New Zealand.

BACKGROUND: This paper synthesises findings from two research projects with organisations involved in arts for youth well-being. Since 2017, Aotearoa New Zealand's government has recognised the importance of the arts for well-being. However, the sector in Aotearoa has historically lacked recognition and support and this paper identifies a number of challenges that remain entrenched in the funding system. METHODS: Study One used an online survey to understand the approaches, aspirations and challenges of 19 organisations involved in youth arts for well-being. Study Two used ethnographic methods with three youth arts organisations to explore their experiences of the funding and policy context. RESULTS: Specific aspects of the funding system in Tamaki Makaurau, Auckland, hinder the sustainable development of creatively rich, culturally responsive, inclusive and strengths-based practice that takes youth participation seriously. CONCLUSIONS: New approaches to resourcing youth arts for well-being are needed to better support good practice and sector development.

Enhancing Biomolecule Analysis and 2DMS Experiments by Implementation of (Activated Ion) 193 nm UVPD on a FT-ICR Mass Spectrometer.

Ultraviolet photodissociation is a fast, photon-mediated fragmentation method that yields high sequence coverage and informative cleavages of biomolecules. In this work, 193 nm UVPD was coupled with a 12 Tesla FT-ICR mass spectrometer and 10.6 µm infrared multi-photon dissociation to provide gentle slow-heating of UV-irradiated ions. No internal instrument hardware modifications were required. Adjusting the timing of laser pulses to the ion motion within the ICR cell provided consistent fragmentation yield shot-to-shot and may also be used to monitor ion positions within the ICR cell. Single-pulse UVPD of the native-like 5+ charge state of ubiquitin resulted in 86.6% cleavage coverage. Additionally, IR activation post UVPD doubled the overall fragmentation yield and boosted the intensity of UVPD-specific x-type fragments up to 4-fold. This increased yield effect was also observed for the 6+ charge state of ubiquitin, albeit less pronounced. This indicates that gentle slow-heating serves to sever tethered fragments originating from non-covalently linked compact structures and makes activation post UVPD an attractive option to boost fragmentation efficiency for top-down studies. Lastly, UVPD was implemented and optimized as a fragmentation method for 2DMS, a data-independent acquisition method. UVPD-2DMS was demonstrated to be a viable method using BSA digest peptides as a model system.

Local Effect of Ballistic Fragments Embedded Along the Carotid Sheath of a Porcine Animal Model.

INTRODUCTION: Energized ballistic fragments from improvised explosive devices were the most common cause of injury to coalition service personnel during conflicts in Iraq and Afghanistan. Surgical excision of retained fragments is not routinely performed unless there is a concern for injury to vital structures. However, no clear guidelines dictate when or if a fragment should be removed, reflecting a lack of objective evidence of their long-term effects. Using a porcine model, we aimed to evaluate changes to the carotid artery produced by retained fragments over time. MATERIALS AND METHODS: Institutional Animal Care and Use Committee approval for all experiments was obtained before commencement of the study. Eighteen female swine (mean mass 62.0 ± 3.4 kg) were randomized into three study groups corresponding to the time of survival after implantation of ballistic fragments: 1, 6, and 12 weeks. Two animals from each group were randomly assigned to have one of the three different fragments implanted within the right carotid sheath in zones 1-3 of the neck. The left carotid served as the control. The vascular flow rate and arterial diameter were measured at each level before implantation and again after the survival interval. Baseline and interval angiograms were performed to identify gross vascular changes. RESULTS: No abnormalities were identified on baseline or interval angiograms. No significant difference was found when the baseline was compared to interval measurements or when compared to the control side for all gross and physiological measures at 1 and 6 weeks (P = .053-.855). After 12 weeks, the flow and diameter changed significantly (P < .001-.03), but this significant change was found in both the control and affected carotid. CONCLUSIONS: The lack of significant gross anatomical and physiological changes at 6 weeks postimplantation lends evidence toward the current policy that early removal of retained ballistic fragments around cervical vessels is not required. Changes were significant after 12 weeks which suggest that surveillance may be required; however, such changes could be explained by physiological animal growth.

Stochasticity of poly(2-oxazoline) oligomer hydrolysis determined by tandem mass spectrometry.

Understanding modification of synthetic polymer structures is necessary for their accurate synthesis and potential applications. In this contribution, a series of partially hydrolyzed poly(2-oxazoline) species were produced forming poly[(2-polyoxazoline)-co-(ethylenimine)] (P(EtOx-co-EI)) copolymers; EI being the hydrolyzed product of Ox. Bulk mass spectrometry (MS) measurements accurately measured the EI content. Tandem mass spectrometry analysis of the EI content in the copolymer samples determined the distribution of each monomer within the copolymer and corresponded to a theoretically modelled random distribution. The EI distribution across the polymers was shown to be effected by the choice of terminus, with a permanent hydrolysis event observed at an OH terminus. A neighbouring group effect wasn't observed at the polymer length analysed (approximately 25-mer species), suggesting that previously observed neighbouring group effects require a larger polymer chain. Although clearly useful for random polymer distribution this approach may be applied to many systems containing non-specific modifications to determine if they are directed or random locations across peptides, proteins, polymers, and nucleic acids.

Fine Structure in Isotopic Peak Distributions Measured Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: A Comparison between an Infinity ICR Cell and a Dynamically Harmonized ICR Cell.

The fine structure of isotopic peak distributions of glutathione in mass spectra is measured using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) at 12 and 15 T magnetic field, with an infinity cell and a dynamically harmonized cell (DHC) respectively. The resolved peaks in the fine structure of glutathione consist of 2H, 13C, 15N, 17O, 18O, 33S, 34S, 36S, and combinations of them. The positions of the measured fine structure peaks agree with the simulated isotopic distributions with the mass error less than 250 ppb in broadband mode for the infinity cell and no more than 125 ppb with the DHC after internal calibration. The 15 T FT-ICR MS with DHC cell also resolved around 30 isotopic peaks in broadband with a resolving power (RP) of 2 M. In narrowband (m/z 307-313), our current highest RP of 13.9 M in magnitude mode was observed with a 36 s transient length by the 15 T FT-ICR MS with the DHC and 2ω detection on the 15 T offers slightly higher RP (14.8 M) in only 18 s. For the 12 T FT-ICR MS with the infinity cell, the highest RP achieved was 15.6 M in magnitude mode with a transient length of 45 s. Peak decay was observed for low abundance peaks, which could be due to the suppression effects from the most abundant peak, as result of ion cloud Coulombic interactions (space-charge).

Revealing the Reactivity of Individual Chemical Entities in Complex Mixtures: the Chemistry Behind Bio-Oil Upgrading.

Bio-oils are precursors for biofuels but are highly corrosive necessitating further upgrading. Furthermore, bio-oil samples are highly complex and represent a broad range of chemistries. They are complex mixtures not simply because of the large number of poly-oxygenated compounds but because each composition can comprise many isomers with multiple functional groups. The use of hyphenated ultrahigh-resolution mass spectrometry affords the ability to separate isomeric species of complex mixtures. Here, we present for the first time, the use of this powerful analytical technique combined with chemical reactivity to gain greater insights into the reactivity of the individual isomeric species of bio-oils. A pyrolysis bio-oils and its esterified bio-oil were analyzed using gas chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry, and in-house software (KairosMS) was used for fast comparison of the hyphenated data sets. The data revealed a total of 10,368 isomers in the pyrolysis bio-oil and an increase to 18,827 isomers after esterification conditions. Furthermore, the comparison of the isomeric distribution before and after esterification provide new light on the reactivities within these complex mixtures; these reactivities would be expected to correspond with carboxylic acid, aldehyde, and ketone functional groups. Using this approach, it was possible to reveal the increased chemical complexity of bio-oils after upgrading and target detection of valuable compounds within the bio-oils. The combination of chemical reactions alongside with in-depth molecular characterization opens a new window for the understanding of the chemistry and reactivity of complex mixtures.

Differentiation of Dihydroxylated Vitamin D3 Isomers Using Tandem Mass Spectrometry.

Vitamin D compounds are a group of secosteroids derived from cholesterol that are vital for maintaining bone health in humans. Recent studies have shown extraskeletal effects of vitamin D, involving vitamin D metabolites such as the dihydroxylated vitamin D3 compounds 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Differentiation and characterization of these isomers by mass spectrometry can be challenging due to the zero-mass difference and minor structural differences between them. The isomers usually require separation by liquid chromatography (LC) prior to mass spectrometry, which adds extra complexity to the analysis. Herein, we investigated and revisited the use of fragmentation methods such as collisional induced dissociation (CID), infrared multiphoton dissociation (IRMPD), electron induced dissociation (EID), and ultraviolet photodissociation (UVPD), available on a 12T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) to generate characteristic fragments for the dihydroxylated vitamin D3 isomers that can be used to distinguish between them. Isomer-specific fragments were observed for the 1,25-dihydroxyvitamin D3, which were clearly absent in the 24,25-dihydroxyvitamin D3 MS/MS spectra using all fragmentation methods mentioned above. The fragments generated due to cleavage of the C-6/C-7 bond in the 1,25-dihydroxyvitamin D3 compound demonstrate that the fragile OH groups were retained during fragmentation, thus enabling differentiation between the two dihydroxylated vitamin D3 isomers without the need for prior chromatographic separation or derivatization.

Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides.

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.

Characterization Across a Dispersity: Polymer Mass Spectrometry in the Second Dimension.

Due to the natural dispersity that is present in synthetic polymers, an added complexity is always present in the analysis of polymeric species. Tandem mass spectrometry analysis requires the isolation of individual precursors before a fragmentation event to allow the unambiguous characterization of these species and is not viable at certain levels of complexity due to achievable isolation widths. Two-dimensional mass spectrometry (2DMS) fragments ions and correlates fragments with their corresponding precursors without the need for isolation. In this study, 2DMS electron capture dissociation (ECD) fragmentation of a polyoxazoline and polyacrylamide species was carried out, resulting in the analysis of byproducts and individual polymer species without the use of chromatographic techniques. This study shows that 2DMS ECD is a powerful tool for the analysis of polyacrylamide and polyoxazoline species and offers a new dimension in the characterization of polymers.

Facile protein conjugation of platinum for light-activated cytotoxic payload release.

The novel Pt(iv) complex trans,trans-[Pt(N3)2(Py)2(OH)(OCO-(PEG)2-NHCSNH-Ph-NCS)] (Pt4) conjugates to the side chain of lysine amino acids in proteins under mild conditions. Reaction with myoglobin generated a bioconjugate that was stable in the dark, but released a Pt(iv) prodrug upon visible light irradiation. A similar procedure was used to conjugate Pt4 to the antibody trastuzumab, resulting in the first photoactivatable Pt(iv)-antibody conjugate, demonstrating potential for highly selective cancer phototherapy.

Combining Ultraviolet Photodissociation and Two-Dimensional Mass Spectrometry: A Contemporary Approach for Characterizing Singly Charged Agrochemicals.

Ultraviolet photodissociation (UVPD) has been shown to produce extensive structurally informative data for a variety of chemically diverse compounds. Herein, we demonstrate the performance of the 193 nm UVPD fragmentation technique on structural/moiety characterization of 14 singly charged agrochemicals. Two-dimensional mass spectrometry (2DMS) using infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID) have previously been applied to a select range of singly charged pesticides. The ≥80% moiety coverage achieved for the majority of the species by the UVPD and 2D-UVPD methods was on par with and, in some cases, superior to the data obtained by other fragmentation techniques in previous studies, demonstrating that UVPD is viable for these types of species. A three-dimensional (3D) peak picking method was implemented to extract the data from the 2DMS spectrum, overcoming the limitations of the line extraction method used in previous studies, successfully separating precursor specific fragments with milli-Dalton accuracy. Whole spectrum internal calibration combined with 3D peak picking obtained sub-part-per-million (ppm) to part-per-billion (ppb) mass accuracies across the entire 2DMS spectrum.

Two-Dimensional Mass Spectrometry Analysis of IgG1 Antibodies.

Two-dimensional mass spectrometry (2DMS) is a new, and theoretically ideal, data-independent analysis tool, which allows the characterization of a complex mixture and was used in the bottom-up analysis of IgG1 for the identification of post-translational modifications. The new peak picking algorithm allows the distinction between chimeric peaks in proteomics. In this application, the processing of 2DMS data correlates fragments to their corresponding precursors, with fragments from precursors which are <0.1 m/z at m/z 840 easily resolved, without the need for quadrupole or chromatographic separation.

Biogenic metallic elements in the human brain?

The chemistry of copper and iron plays a critical role in normal brain function. A variety of enzymes and proteins containing positively charged Cu+, Cu2+, Fe2+, and Fe3+ control key processes, catalyzing oxidative metabolism and neurotransmitter and neuropeptide production. Here, we report the discovery of elemental (zero-oxidation state) metallic Cu0 accompanying ferromagnetic elemental Fe0 in the human brain. These nanoscale biometal deposits were identified within amyloid plaque cores isolated from Alzheimer's disease subjects, using synchrotron x-ray spectromicroscopy. The surfaces of nanodeposits of metallic copper and iron are highly reactive, with distinctly different chemical and magnetic properties from their predominant oxide counterparts. The discovery of metals in their elemental form in the brain raises new questions regarding their generation and their role in neurochemistry, neurobiology, and the etiology of neurodegenerative disease.

Cu(III)-bis-thiolato complex forms an unusual mono-thiolato Cu(III)-peroxido adduct.

The stable complex [bis(toluene-3,4-dithiolato)copper(iii)][NEt3H] has been synthesised and characterised as a square-planar Cu(iii) complex by X-ray photoelectron spectroscopy, cyclic voltammetry and DFT calculations. Intriguingly, when fragmented in FTICR-MS, an unusual [(toluene-3,4-dithiolate)Cu(iii)(peroxide)]- complex is formed by reaction with oxygen. Natural 1,2-dithiolenes known to bind molybdenum might stabilise Cu(iii) in vivo.