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Intrinsically disordered proteins (IDPs) are important drug targets as they are key actors within cell signaling networks. However, the conformational plasticity of IDPs renders them challenging to characterize, which is a bottleneck in developing small molecule drugs that bind to IDPs and modulate their behavior. In relation to this, ion mobility mass spectrometry (IM-MS) is a useful tool to investigate IDPs, as it can reveal their conformational preferences. It can also offer important insights in drug discovery, as it can measure binding stoichiometry and unveil conformational shifts of IDPs exerted by the binding of small drug-like molecules. Herein, we have used IM-MS to investigate the effect of drug lead EPI-001 on the disordered N-terminal domain of the androgen receptor (AR-NTD). Despite structural heterogeneity rendering the NTD a challenging region of the protein to drug, this domain harbors most, if not all, of the transcriptional activity. We quantify the stoichiometry of EPI-001 binding to various constructs corresponding to functional domains of AR-NTD and show that it binds to separate constructs containing transactivation unit (TAU)-1 and TAU-5, respectively, and that 1-2 molecules bind to a larger construct containing both sequences. We also identify a conformational shift upon EPI-001 binding to the TAU-5, and to a much lesser extent with TAU-1 containing constructs. This work provides novel insight on the interactions of EPI-001 with the AR-NTD, and the structural alterations that it exerts, and positions IM-MS as an informative tool that will enhance the tractability of IDPs, potentially leading to better therapies., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

There were an estimated 249 million cases of malaria globally in 2022, causing approximately 608 000 deaths. Most of these are attributed to infection by P. falciparum . Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms, including P. falciparum . A panel of 25 S-MGBs was synthesised, including those bearing an amidine tail group, and their antiplasmodial activity against 3D7 and Dd2 strains was determined in vitro using an asexual P. falciparum imaging assay. Determination of activity against HEK293 cells allowed for selective cytotoxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. A comparison of 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant) potency showed no evidence of cross-resistance across the S-MGB set. S-MGB-356 , S-MGB-368 and S-MGB-359 , amidine tail containing S-MGBs, were identified as the most promising hit compounds based on their selectivity indices (HEK293/3D7) of >612.6, >335.8 and >264.8, respectively. S-MGB-356 , S-MGB-368 and S-MGB-359 were confirmed to bind to DNA as dimers, with gDNA thermal shifts (Δ T m ) of 12 °C, 3 °C and 16 °C, respectively. Together, these data demonstrate that amidine tail bearing S-MGBs are promising hit compounds against P. falciparum , and can be further optimised into lead compounds., Competing Interests: The authors declare the following competing financial interest(s): MP, LMCM, AIK, CJS and FJS are part of revenue sharing agreements with their University relating to the Strathclyde minor groove binder project. Additionally, CJS and FJS have financial interests through shares in the company, Rostra Therapeutics. All other authors: none to declare., (This journal is © The Royal Society of Chemistry.)

Background: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms., Objectives: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development., Methods: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays., Results and Discussion: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50 > 100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTm = 1 ± 0.1°C., Conclusions: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Hematopoietic stem cells (HSCs) reconstitute multilineage human hematopoiesis after clinical bone marrow (BM) transplantation and are the cells of origin of some hematological malignancies. Although HSCs provide multilineage engraftment, individual murine HSCs are lineage biased and contribute unequally to blood cell lineages. Here, we performed high-throughput single-cell RNA sequencing in mice after xenograft with molecularly barcoded adult human BM HSCs. We demonstrated that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identified platelet-biased and multilineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young and aged BM showed that both the proportion of platelet-biased HSCs and their level of transcriptional platelet priming increase with age. Therefore, platelet-biased HSCs and their increased prevalence and transcriptional platelet priming during aging are conserved features of mammalian evolution.

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearized plasmid DNA. Then, to develop EGFP - and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single-cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVVs that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies., Competing Interests: All listed authors are present or past employees of OXGENE, A WuXi Advanced Therapies Company. W.V. and D.C. are employees and hold stock or stock options within the company. R.P.-M., L.D., T.P., and R.C. are named co-inventors on a patent describing the molecular configuration of the packaging elements (US20200277629A1)., (© 2024 The Author(s).)

There is mounting evidence that crystal nucleation from supersaturated solution involves the formation and reorganization of prenucleation clusters, contradicting classical nucleation theory. One of the key unresolved issues pertains to the origin, composition, and structure of these clusters. Here, a range of amino acids and peptides is investigated using light scattering, mass spectrometry, and in situ terahertz Raman spectroscopy, showing that the presence of amorphous aggregates is a general phenomenon in supersaturated solutions. Significantly, these aggregates are found on a vast range of length scales from dimers to 30-mers to the nanometre and even micrometre scale, implying a continuous distribution throughout this range. Larger amorphous aggregates are sites of spontaneous crystal nucleation and act as intermediates for laser-induced crystal nucleation. These results are shown to be consistent with a nonclassical nucleation model in which barrierless (homogeneous) nucleation of amorphous aggregates is followed by the nucleation of crystals from solute-enriched aggregates. This provides a novel perspective on crystal nucleation and the role of nonclassical pathways., Competing Interests: The authors declare no conflicts of interests., (This journal is © The Royal Society of Chemistry.)

In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN : DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Ternary complex formation was also identified involving the DCAF15 : DDA1 : DDB1 E3 ligase in the presence of MG (E7820 or indisulam) and POI RBM39. Moreover, we uncovered that the DCAF15 : DDA1 : DDB1 E3 ligase self-associates into dimers and trimers when analysed alone at low salt concentrations (100 mM ammonium acetate) which dissociate into single copies of the complex at higher salt concentrations (500 mM ammonium acetate), or upon the addition of MG and POI, forming a 1 : 1 : 1 ternary complex. This work demonstrates the strength of nMS in TPD research, reveals novel binding mechanisms of the DCAF15 E3 ligase, and its self-association into dimers and trimers at reduced salt concentration during structural analysis.

Protein-protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs ( N ε -methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and phosphorylation at up to five sites were successfully prepared and isolated in high yield and purity. We further produced ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thus enables the systematic study of PTM function at a molecular level, and offers a convenient way for expedited screening of constructs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Rationale: Protein degraders are small molecules that promote cellular degradation of a target protein. Degraders simultaneously bind to their target and an E3 ligase, bringing them into close spatial proximity, but the formation of this ternary complex is difficult to measure with many biophysical techniques., Methods: Native mass spectrometry (nMS) is an effective label-free technique to identify the complexes formed by proteolysis-targeting chimeras (PROTACs). It can monitor the formation of ternary E3-PROTAC-target complexes and detect intermediate binary species. Experiments are described using a Synapt G2Si (Waters) equipped with a nano-electrospray ionisation source., Results: The protocol describes nMS experiments for measuring the complexes formed by PROTAC molecules. It also describes how to investigate differences in the affinity of PROTAC complexes, whether a PROTAC shows specificity for a given target and whether a PROTAC shows cooperative behaviour., Conclusions: Here, we provide step-by-step instructions for the sample preparation of PROTAC complexes and their nMS interrogation to obtain optimal information on their binding modes., (© 2023 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

A ReLU neural network functions as a continuous piecewise linear map from an input space to an output space. The weights in the neural network determine a partitioning of the input space into convex polytopes, where each polytope is associated with a distinct affine mapping. The structure of this partitioning, together with the affine map attached to each polytope, can be analyzed to investigate the behavior of the associated neural network. We investigate simple problems to build intuition on how these regions act and both how they can potentially be reduced in number and how similar structures occur across different networks. To validate these intuitions, we apply them to networks trained on MNIST to demonstrate similarity between those networks and the potential for them to be reduced in complexity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sattelberg, Cavalieri, Kirby, Peterson and Beveridge.)

Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8 + epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP 9-17 -B*27:05 CD8 + T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP 9-17 -B*27:05 and NP 105-113 -B*07:02 epitopes, respectively, increased CD8 + T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

Untreated psychosis in adolescents and young adults is associated with significant and progressive impairment. Early intervention to provide support and treatment for those at risk of psychosis is essential. Several early intervention models have been developed for those at-risk and those who are victims of a recent episode - including the Portland Identification and Early Referral model (PIER; McFarlane, 2001). This study extends previous work demonstrating a variety of positive treatment outcomes achieved by PIER in the context of a large-scale implementation across the state of Delaware. The sample included 108 youth and young adults who were either at risk for psychosis or had already experienced a first episode within the past two years. Participants received the PIER treatment model and were followed from baseline to six months after they were discharged from treatment. Researchers predicted that PIER participants would experience an increase in functioning and a decrease in positive psychosis symptoms. Change over time was examined through the lens of two analytic techniques: the Reliable Change Index (RCI) analyses and Growth Curve Modeling (GCM). Results show improvement on a number of outcomes over the course of the intervention as expected. Clinical implications, limitations, and suggestions for further research are discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remains difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions and their tendency to oligomerize. Here, we demonstrate that ion mobility mass spectrometry (IM-MS) overcomes these limitations. We used IM-MS to investigate the conformational states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which is driven by high-salt concentration and reversed by noncovalent interactions with ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as a mixture of monomers and dimers and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift toward extended conformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2/Ub complexes, which have compact geometries compared to free UBQLN2 dimers. Together, these results suggest that extended conformations of UBQLN2 are correlated with UBQLN2's ability to phase separate. Overall, delineating protein conformations that are implicit in LLPS will greatly increase understanding of the phase separation process, both in normal cell physiology and disease states.

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations., Competing Interests: Declaration of interests A.J.P. is chair of the DHSC Joint Committee on Vaccination and Immunisation (JCVI) but does not take part in the JCVI COVID-19 committee. Oxford University has entered into a partnership with AstraZeneca for development of a COVID-19 vaccine. A.J.P. has provided advice to Shionogi & Co., Ltd., on the development of a COVID-19 vaccine., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Mass spectrometry (MS) is now established as an analytical tool to interrogate the structure and dynamics of proteins and their assemblies. An array of MS-based technologies has been developed, with each providing unique information pertaining to protein structure, and forming the heart of integrative structural biology studies. This special issue includes a collection of review articles that discuss both established and emerging structural MS methodologies, along with examples of how these technologies are being deployed to interrogate protein structure and function. Combined, this collection highlights the immense potential of the structural MS toolkit in the study of molecular mechanisms underpinning cellular homeostasis and disease., (© 2023 The Author(s).)

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli . However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

The neglected tropical disease leishmaniasis, caused by Leishmania spp., is becoming more problematic due to the emergence of drug-resistant strains. Therefore, new drugs to treat leishmaniasis, with novel mechanisms of action, are urgently required. Strathclyde minor groove binders (S-MGBs) are an emerging class of anti-infective agent that have been shown to have potent activity against various bacteria, viruses, fungi and parasites. Herein, it is shown that S-MGBs have potent activity against L. donovani , and that an N-oxide derivation of the tertiary amine tail of typical S-MGBs leads to selective anti-leishmanial activity. Additionally, using S-MGB-219, the N-oxide derivation is shown to retain strong binding to DNA as a 2:1 dimer. These findings support the further study of anti-leishmanial S-MGBs as novel therapeutics., Competing Interests: C.J.S. and F.J.S. are part of revenue-sharing agreements with their university relating to the Strathclyde Minor Groove Binder project. Additionally, C.J.S. and F.J.S. have financial interests through shares in the company Rostra Therapeutics.

Quantitative drug imaging in live cells is a major challenge in drug discovery and development. Many drug screening techniques are performed in solution, and therefore do not consider the impact of the complex cellular environment in their result. As such, important features of drug-cell interactions may be overlooked. In this study, Raman microscopy is used as a powerful technique for semi-quantitative imaging of Strathclyde-minor groove binders (S-MGBs) in mammalian cells under biocompatible imaging conditions. Raman imaging determined the influence of the tail group of two novel minor groove binders (S-MGB-528 and S-MGB-529) in mammalian cell models. These novel S-MGBs contained alkyne moieties which enabled analysis in the cell-silent region of the Raman spectrum. The intracellular uptake concentration, distribution and mechanism were evaluated as a function of the p K a of the tail group, morpholine and amidine, for S-MGB-528 and S-MGB-529, respectively. Although S-MGB-529 had a higher binding affinity to the minor groove of DNA in solution-phase measurements, the Raman imaging data indicated that S-MGB-528 showed a greater degree of intracellular accumulation. Furthermore, using high resolution stimulated Raman scattering (SRS) microscopy, the initial localisation of S-MGB-528 was shown to be in the nucleus before accumulation in the lysosome, which was demonstrated using a multimodal imaging approach. This study highlights the potential of Raman spectroscopy for semi-quantitative drug imaging studies and highlights the importance of imaging techniques to investigate drug-cell interactions, to better inform the drug design process., Competing Interests: F. J. S. and C. J. S. are inventors on patents that pertain to S-MGBs, filed by the University of Strathclyde, and may benefit financially from commercial efforts relating to these patents., (This journal is © The Royal Society of Chemistry.)

Myoglobin (Mb) can react with hydrogen peroxide (H 2 O 2 ) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as pseudo-peroxidase activity, previous studies have focused on the modification of key amino acid residues of Mb or the heme cofactor. In this work, the Mb scaffold (apo-Mb) was systematically reconstituted with a set of cofactors based on six metal ions and two ligands. These Mb variants were fully characterised by UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and native mass spectrometry (nMS). The steady-state kinetics of guaiacol oxidation and 2,4,6-trichlorophenol (TCP) dehalogenation catalysed by Mb variants were determined. Mb variants with iron chlorin e6 (Fe-Ce6) and manganese chlorin e6 (Mn-Ce6) cofactors were found to have improved catalytic efficiency for both guaiacol and TCP substrates in comparison with wild-type Mb, i. e. Fe-protoporphyrin IX-Mb. Furthermore, the selected cofactors were incorporated into the scaffold of a Mb mutant, swMb H64D. Enhanced peroxidase activity for both substrates were found via the reconstitution of Fe-Ce6 into the mutant scaffold., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Background: Previously, we evaluated the intracellular mycobactericidal activity of the minor groove binder, S-MGB-364 against the clinical Mycobacterium tuberculosis (Mtb) strain HN878 in macrophages., Objectives: To assess the mycobactericidal activity of S-MGB-364 in Mtb-infected mice. Further, we investigated a plausible DNA binding mechanism of action of S-MGB-364., Methods: The anti-TB and host immune effects of intranasal S-MGB-364 or S-MGB-364 encapsulated in non-ionic surfactant vesicles (NIV) were assessed in Mtb-infected mice by cfu enumeration, ELISA, histology, and flow cytometry. DNA binding was examined using native mass spectrometry and UV-vis thermal melt determination. S-MGB interference with DNA-centric biological events was assessed using a representative panel of Mtb and human topoisomerase I, and gyrase assays., Results: S-MGB-364 bound strongly to DNA as a dimer, significantly increasing the stability of the DNA:S-MGB complex compared with DNA alone. Moreover, S-MGB-364 inhibited the relaxation of Mtb topoisomerase I but not the human form. In macrophages, S-MGB-364 or S-MGB-364-NIV did not cause DNA damage as shown by the low γ-H2AX expression. Importantly, in the lungs, the intranasal administration of S-MGB-364 or S-MGB-364-NIV formulation in Mtb-infected mice was non-toxic and resulted in a ∼1 log cfu reduction in mycobacterial burden, reduced the expression of proinflammatory cytokines/chemokines, altered immune cell recruitment, and importantly reduced recruitment of neutrophils., Conclusions: Together, these data provide proof of concept for S-MGBs as novel anti-TB therapeutics in vivo., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1 -/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

NP 105-113 -B*07:02-specific CD8 + T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP 105-113 -B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP 105-113 -B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP 105-113 -B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP 105-113 -B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design., (© 2021. The Author(s).)

Cyclic GMP-AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus-like particles (VLPs) containing HIV-1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV-G). cGAMP loading of VLPs augments CD4 and CD8 T-cell responses. It also increases VLP- and VSV-G-specific antibody titres in a STING-dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS-CoV-2 Spike protein enhances Spike-specific antibody titres. cGAMP-loaded VLPs are thus an attractive platform for vaccination., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A ( KMT2A ) gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor BCL2 and the proto-oncogene MYC However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses CASP9 , as well as KMT2A-AFF1-driven FFLs that regulate BCL2 and MYC through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response., (© 2021 Harman et al.; Published by Cold Spring Harbor Laboratory Press.)

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P) + , a typical oxidative co-enzyme. However, NAD(P) + here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDPs) and regions of intrinsic disorder (IDRs) are abundant in proteomes and are essential for many biological processes. Thus, they are often implicated in disease mechanisms, including neurodegeneration and cancer. The flexible nature of IDPs and IDRs provides many advantages, including (but not limited to) overcoming steric restrictions in binding, facilitating posttranslational modifications, and achieving high binding specificity with low affinity. IDPs adopt a heterogeneous structural ensemble, in contrast to typical folded proteins, making it challenging to interrogate their structure using conventional tools. Structural mass spectrometry (MS) methods are playing an increasingly important role in characterizing the structure and function of IDPs and IDRs, enabled by advances in the design of instrumentation and the development of new workflows, including in native MS, ion mobility MS, top-down MS, hydrogen-deuterium exchange MS, crosslinking MS, and covalent labeling. Here, we describe the advantages of these methods that make them ideal to study IDPs and highlight recent applications where these tools have underpinned new insights into IDP structure and function that would be difficult to elucidate using other methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Beveridge and Calabrese.)

This community-based participatory action research (CBPAR) study describes a method for evaluating an after-school resilience-focused intervention in a low-resource rural area of southern India. Communities Rising, a locally developed resilience and academic program, was evaluated in a cross-continent collaboration between a research team at a U.S. university and the local community. The CBPAR literature highlights the importance of cultural considerations, community considerations, and community participation in the research process. The present case study describes the CBPAR research process and considerations at every phase of the research project, providing a road map of how community engagement can strengthen research, empower the community, and provide valuable knowledge. This study was conducted in three phases that focused on inclusion of local voices in the development both of the resilience program and the evaluation data collection process. Youth surveyors were particularly key to the research process. Data on participant demographics, satisfaction with the program, and qualitative contributions are also provided. Strengths and limitations of this study process in a rural community are discussed., (© 2020 Wiley Periodicals LLC.)

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER., Competing Interests: MS, LP, AG, RB, ES, VS, AM, MC, YZ, CN, MM, ET, CL, BL, GD, MS, HC, AA, AS, KC, AD, IS, SA, LJ, KM, FI, TC, YD No competing interests declared, SM is member of the scientific advisory board of Casma Therapeutics, (© 2020, Stephani et al.)

Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that promote cellular degradation of a protein of interest (POI). PROTACs act as molecular mediators, bringing an E3 ligase and a POI into proximity, thus promoting ubiquitination and degradation of the targeted POI. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs., Competing Interests: The authors declare the following competing financial interest(s): D.K., K.R., and P.E. were employees of Boehringer Ingelheim at the time of this work., (Copyright © 2020 American Chemical Society.)

Collaboration among diverse stakeholders involved in the value transformation of health care requires consistent use of terminology. The objective of this study was to reach consensus definitions for the terms value-based care , value-based payment , and population health . A modified Delphi process was conducted from February 2017 to July 2017. An in-person panel meeting was followed by 3 rounds of surveys. Panelists anonymously rated individual components of definitions and full definitions on a 9-point Likert scale. Definitions were modified in an iterative process based on results of each survey round. Participants were a panel of 18 national leaders representing population health, health care delivery, academic medicine, payers, patient advocacy, and health care foundations. Main measures were survey ratings of definition components and definitions. At the conclusion of round 3, consensus was reached on the following definition for value-based payment, with 13 of 18 panelists (72.2%) assigning a high rating (7- 9) and 1 of 18 (5.6%) assigning a low rating (1-3): "Value-based payment aligns reimbursement with achievement of value-based care (health outcomes/cost) in a defined population with providers held accountable for achieving financial goals and health outcomes. Value-based payment encourages optimal care delivery, including coordination across healthcare disciplines and between the health care system and community resources, to improve health outcomes, for both individuals and populations." The iterative process elucidated specific areas of agreement and disagreement for value-based care and population health but did not reach consensus. Policy makers cannot assume uniform interpretation of other concepts underlying health care reform efforts.

Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions.

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.

Motion-onset visually evoked potentials (mVEPs) are neural potentials that are time-locked to the onset of motion of evoking stimuli. Due to their visually elegant properties, mVEP stimuli may be suited to video game control given gaming's inherent demand on the users' visual attention and the requirement to process rapidly changing visual information. Here, we investigate mVEPs associated with five different stimuli to control the position of a car in a visually rich 3D racing game in a group of 15 BCI naïve teenagers and compared with 19 BCI naive adults. Results from an additional 14 BCI experienced adults were compared with BCI naïve adults. Our results demonstrate that the game control accuracy is related to the number of trials used to make a decision on the users' chosen button/stimulus (76%, 62%, and 35% for 5, 3, and 1 trials, respectively) and information transfer rate (ITR) (13.4, 13.9, and 6.6 bits per minute (BPM)), although, even though accuracy decreases when using three compared to the commonly used five trial repetitions, ITR is maintained. A Kruskal-Wallis test suggests that BCI naïve adults do not outperform BCI naïve teenagers in the 3D racing game in the first and seconds laps (p > 0.05), but do outperform in the third lap (p < 0.05). A comparison between BCI naïve and BCI experienced adults indicates BCI experienced adults do not perform better than BCI naïve adults (p > 0.05).

The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27 Kip1 . We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.

Intrinsically disordered proteins have been reported to undergo disorder-to-order transitions upon binding to their partners in the cell. The extent of the ordering upon binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behavior for function, particularly in the C-terminal half of the protein. Herein, native ion mobility mass spectrometry (IM-MS) is employed to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. The trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity compared to Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N- and C-termini of p27 interact with Cdk2/cyclin A in multiple, closely associated states., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2-7 subcomplex of the replicative Cdc45-MCM-GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively., (© 2018 The Authors.)

In the version of this article initially published online, the authors used incorrectly defined restraints for specifying the distance between residues when using the HADDOCK portal. Following the publication of a Correspondence by the developers of the HADDOCK portal (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0017-6, 2018) and a Reply by the authors of the Protocol (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0018-5, 2018), the syntax in step 21 has been corrected. In addition, the input files (available as Supplementary Data 5-7) have been replaced.

Genomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated on the basis of a homologous template and primary sequence, and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data are further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6-12 d to complete, depending on the size of the proteins and the complexity of the cross-linking data.

I've been meeting with physicians nationwide about their planned, ongoing and potential transitions from fee-for-service to value-based reimbursement. It's certainly a work in progress.

Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1(Ubix), the cofactor confers structural stability to the enzyme. IM-MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM-MS data.

A brain-computer interface (BCI) offers movement-free control of a computer application and is achieved by reading and translating the cortical activity of the brain into semantic control signals. Motion-onset visual evoked potentials (mVEP) are neural potentials employed in BCIs and occur when motion-related stimuli are attended visually. mVEP dynamics are correlated with the position and timing of the moving stimuli. To investigate the feasibility of utilizing the mVEP paradigm with video games of various graphical complexities including those of commercial quality, we conducted three studies over four separate sessions comparing the performance of classifying five mVEP responses with variations in graphical complexity and style, in-game distractions, and display parameters surrounding mVEP stimuli. To investigate the feasibility of utilizing contemporary presentation modalities in neurogaming, one of the studies compared mVEP classification performance when stimuli were presented using the oculus rift virtual reality headset. Results from 31 independent subjects were analyzed offline. The results show classification performances ranging up to 90% with variations in conditions in graphical complexity having limited effect on mVEP performance; thus, demonstrating the feasibility of using the mVEP paradigm within BCI-based neurogaming., (© 2016 Elsevier B.V. All rights reserved.)

In recent years both mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS) have been developed as techniques with which to study proteins that lack a fixed tertiary structure but may contain regions that form secondary structure elements transiently, namely intrinsically disordered proteins (IDPs). IM-MS is a suitable method for the study of IDPs which provides an insight to conformations that are present in solution, potentially enabling the analysis of lowly populated structural forms. Here, we describe the IM-MS data of two IDPs; α-Synuclein (α-Syn) which is implicated in Parkinson's disease, and Apolipoprotein C-II (ApoC-II) which is involved in cardiovascular diseases. We report an apparent discrepancy in the way that ApoC-II behaves in the gas phase. While most IDPs, including α-Syn, present in many charge states and a wide range of rotationally averaged collision cross sections (CCSs), ApoC-II presents in just four charge states and a very narrow range of CCSs, independent of solution conditions. Here, we compare MS and IM-MS data of both proteins, and rationalise the differences between the proteins in terms of different ionisation processes which they may adhere to., (© 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.