Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

Multidimensional Cross-Linking and Real-Time Informatics for Multiprotein Interaction Studies.

Chemical cross-linking combined with mass spectrometry is a technique used to study protein structures and identify protein complexes. Traditionally, chemical cross-linkers contain two reactive groups, allowing them to covalently bond a pair of proximal residues, either within a protein or between two proteins. The output of a cross-linking experiment is a list of interacting site pairs that provide structural constraints for modeling of new structures and complexes. Due to the binary reactive nature of cross-linking reagents, only pairs of interacting sites can be directly observed, and assembly of higher-order structures typically requires prior knowledge of complex composition or iterative docking to produce a putative model. Here, we describe a new tetrameric cross-linker bearing four amine-reactive groups, allowing it to covalently link up to four proteins simultaneously and a real-time instrument method to facilitate the identification of these tetrameric cross-links. We applied this new cross-linker to isolated mitochondria and identified a number of higher-order cross-links in various OXPHOS complexes and ATP synthase, demonstrating its utility in characterizing complex interfaces. We also show that higher-order cross-links can be used to effectively filter models of large protein assemblies generated by using Alphafold. Higher-dimensional cross-linking provides a new avenue for characterizing multiple protein interfaces, even in complex samples such as intact mitochondria.

In-Cell Labeling and Mass Spectrometry for Systems-Level Structural Biology.

Biological systems have evolved to utilize proteins to accomplish nearly all functional roles needed to sustain life. A majority of biological functions occur within the crowded environment inside cells and subcellular compartments where proteins exist in a densely packed complex network of protein-protein interactions. The structural biology field has experienced a renaissance with recent advances in crystallography, NMR, and CryoEM that now produce stunning models of large and complex structures previously unimaginable. Nevertheless, measurements of such structural detail within cellular environments remain elusive. This review will highlight how advances in mass spectrometry, chemical labeling, and informatics capabilities are merging to provide structural insights on proteins, complexes, and networks that exist inside cells. Because of the molecular detection specificity provided by mass spectrometry and proteomics, these approaches provide systems-level information that not only benefits from conventional structural analysis, but also is highly complementary. Although far from comprehensive in their current form, these approaches are currently providing systems structural biology information that can uniquely reveal how conformations and interactions involving many proteins change inside cells with perturbations such as disease, drug treatment, or phenotypic differences. With continued advancements and more widespread adaptation, systems structural biology based on in-cell labeling and mass spectrometry will provide an even greater wealth of structural knowledge.

Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p.

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential crosslinking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture crosslinking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine-lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein-protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how crosslinking MS will be an essential tool for understanding complex phenotypes.

The use of large language models like ChatGPT on delivering patient information relating to surgery.

not applicable.

Assessing the quality and readability of online patient information: ENT UK patient information e-leaflets vs responses by a Generative Artificial Intelligence.

BACKGROUND: The evolution of artificial intelligence has introduced new ways to disseminate health information, including natural language processing models like ChatGPT. However, the quality and readability of such digitally-generated information remains understudied. This study is the first to compare the quality and readability of digitally-generated health information against leaflets produced by professionals. METHODOLOGY: Patient information leaflets for five ENT UK leaflets and their corresponding ChatGPT responses were extracted from the Internet. Assessors with various degree of medical knowledge evaluated the content using the Ensuring Quality Information for Patients (EQIP) tool and readability tools including the Flesch-Kincaid Grade Level (FKGL). Statistical analysis was performed to identify differences between leaflets, assessors, and sources of information. RESULTS: ENT UK leaflets were of moderate quality, scoring a median EQIP of 23. Statistically significant differences in overall EQIP score were identified between ENT UK leaflets but ChatGPT responses were of uniform quality. Non-specialist doctors rated the highest EQIP scores while medical students scored the lowest. The mean readability of ENT UK leaflets was higher than ChatGPT responses. The information metrics of ENT UK leaflets were moderate and varied between topics. Equivalent ChatGPT information provided comparable content quality, but with reduced readability. CONCLUSIONS: ChatGPT patient information and professionally-produced leaflets had comparable content, but LLM content were required a higher reading age. With the increasing use of online health resources, this study highlights the need for a balanced approach that considers optimises both the quality and readability of patient education materials.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges. Here, we report the combination of protein-denaturation principles with quantitative cross-linking mass spectrometry using isobaric quantitative protein interaction reporter technologies. This method enables the evaluation of ligand-induced protein engagement through analysis of cross-link relative ratios across chemical denaturation. As a proof of concept, we found ligand-stabilized cross-linked lysine pairs in well-studied bovine serum albumin and ligand bilirubin. These links map to the known binding sites Sudlow Site I and subdomain IB. We propose that protein denaturation and qXL-MS can be combined with similar peptide-level quantification approaches, like SPROX, to increase the coverage information profiled for facilitating protein-ligand engagement efforts.

Perturbing HIV-1 Ribosomal Frameshifting Frequency Reveals a cis Preference for Gag-Pol Incorporation into Assembling Virions.

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks" scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a "weighted Goldilocks" scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.

ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses.

Previously, we reported that cellular transcription factor ZASC1 facilitates DNA-dependent/RNA-independent recruitment of HIV-1 TAT and the cellular elongation factor P-TEFb to the HIV-1 promoter and is a critical factor in regulating HIV-1 transcriptional elongation (PLoS Path e1003712). Here we report that cellular transcription factor ZBTB2 is a novel repressor of HIV-1 gene expression. ZBTB2 strongly co-immunoprecipitated with ZASC1 and was dramatically relocalized by ZASC1 from the cytoplasm to the nucleus. Mutations abolishing ZASC1/ZBTB2 interaction prevented ZBTB2 nuclear relocalization. We show that ZBTB2-induced repression depends on interaction of cellular histone deacetylases (HDACs) with the ZBTB2 POZ domain. Further, ZASC1 interaction specifically recruited ZBTB2 to the HIV-1 promoter, resulting in histone deacetylation and transcription repression. Depleting ZBTB2 by siRNA knockdown or CRISPR/CAS9 knockout in T cell lines enhanced transcription from HIV-1 vectors lacking Vpr, but not from these vectors expressing Vpr. Since HIV-1 Vpr activates the viral LTR by inducing the ATR kinase/DNA damage response pathway, we investigated ZBTB2 response to Vpr and DNA damaging agents. Expressing Vpr or stimulating the ATR pathway with DNA damaging agents impaired ZASC1's ability to localize ZBTB2 to the nucleus. Moreover, the effects of DNA damaging agents and Vpr on ZBTB2 localization could be blocked by ATR kinase inhibitors. Critically, Vpr and DNA damaging agents decreased ZBTB2 binding to the HIV-1 promoter and increased promoter histone acetylation. Thus, ZBTB2 is recruited to the HIV-1 promoter by ZASC1 and represses transcription, but ATR pathway activation leads to ZBTB2 removal from the promoter, cytoplasmic sequestration and activation of viral transcription. Together, our data show that ZASC1/ZBTB2 integrate the functions of TAT and Vpr to maximize HIV-1 gene expression.

Applications and advancements of FT-ICR-MS for interactome studies.

The set of all intra- and intermolecular interactions, collectively known as the interactome, is currently an unmet challenge for any analytical method, but if measured, could provide unparalleled insight on molecular function in living systems. Developments and applications of chemical cross-linking and high-performance mass spectrometry technologies are beginning to reveal details on how proteins interact in cells and how protein conformations and interactions inside cells change with phenotype or during drug treatment or other perturbations. A major contributor to these advances is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) technology and its implementation with accurate mass measurements on cross-linked peptide-pair precursor and fragment ions to enable improved identification methods. However, these applications place increased demands on mass spectrometer performance in terms of high-resolution spectral acquisition rates for on-line MSn experiments. Moreover, FT-ICR-MS also offers unique opportunities to develop and implement parallel ICR cells for multiplexed signal acquisition and the potential to greatly advance accurate mass acquisition rates for interactome studies. This review highlights our efforts to exploit accurate mass FT-ICR-MS technologies with chemical cross-linking and developments being pursued to realize parallel MS array capabilities that will further advance visualization of the interactome.

A planar quadrupole device for transmitting and trapping ions in high vacuum.

RATIONALE: Hybrid mass spectrometers combine multiple mass analyzers to achieve optimal performance in terms of tandem mass spectrometry, high mass resolving power, and mass measurement accuracy for studying highly complex samples. As a result, the need for transport, trapping, and control of ion kinetic energies is critical for the successful integration of multiple mass analyzers and hybrid instrument operation. In addition, transportation of ion populations between two physically distinct locations can result in time-of-flight (TOF) discrimination against ions with widely disparate m/z values, compromising full mass spectral performance. In this work, we demonstrated a new ion guide, referred to as a planar quadrupole (PQ) ion guide, composed of two parallel printed circuit boards (PCB) that allow radiofrequency (RF) and direct current (DC) voltages to be combined to enable both axial transport and trapping of ion populations in the ultrahigh vacuum region of the mass spectrometer. As compared with a conventional multipole ion guide, the PQ ion guide showed comparable performance in ion m/z values, signal-to-noise, and intensity and effectively reduced mass discrimination caused by TOF effects. METHODS: A PQ device was developed with two PCBs and simulated with SIMION 8.1. Electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry instrumentation were used for the testing of PQ performance. RESULTS: .In this work, we demonstrated a planar quadrupole (PQ) ion guide composed of two parallel PCB plates. The PQ enables both axial ion transport and trapping of ion populations throughout the ion transfer process from a LTQ to an ICR cell. As compared with a conventional multipole ion guide, the PQ showed comparable ion transmission efficiency and effectively reduced mass discrimination caused by TOF effects. CONCLUSIONS: The PQ is a simple design that can be implemented for ion transmission and trapping on virtually any mass spectrometer.

Mitochondrial protein interaction landscape of SS-31.

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.

Improved Interpretation of Protein Conformational Differences and Ligand Occupancy in Large-Scale Cross-Link Data.

Chemical cross-linking of proteins in complex samples, cells, or even tissues is emerging to provide unique structural information on proteins and complexes that exist within native or nativelike environments. The public database XLinkDB automatically maps cross-links to available structures based on sequence homology. Structures most likely to reflect protein conformations in the cross-linked sample are routinely identified by having cross-linked residues separated by Euclidean distances within the maximum span of the applied cross-linker. Solvent accessible surface distance (SASD), which considers the accessibility of the cross-linked residues and the path connecting them, is a better predictor of consistency than the Euclidean distance. However, SASDs of structures are not publicly available, and their calculation is computationally intensive. Here, we describe in XLinkDB version 4.0 the automatic calculation of SASDs using Jwalk for all cross-links mapped to structures, both with and without regard to ligands, and derive empirical maximum SASD spans for BDP-NHP and DSSO cross-linkers of 51 and 43 Å, respectively. We document ligands proximal to cross-links in structures and demonstrate how SASDs can be used to help infer sample protein conformations and ligand occupancy, highlighting cross-links sensitive to ADP binding in mitochondria isolated from HEK293 cells.

Multiplexed Isobaric Quantitative Cross-Linking Reveals Drug-Induced Interactome Changes in Breast Cancer Cells.

The study of protein structures and interactions is critical to understand their function. Chemical cross-linking of proteins with mass spectrometry (XL-MS) is a rapidly developing structural biology technique able to provide valuable insight into protein conformations and interactions, even as they exist within their native cellular environment. Quantitative analysis of cross-links can reveal protein conformational and interaction changes that occur as a result of altered biological states, environmental conditions, or pharmacological perturbations. Our laboratory recently developed an isobaric quantitative protein interaction reporter (iqPIR) cross-linking strategy for comparative interactome studies. This strategy relies on isotope encoded chemical cross-linkers that have the same molecular mass yet produce unique and specific isotope signatures upon fragmentation in the mass spectrometer which can be used for quantitative analysis of cross-linked peptides. The initial set of iqPIR molecules allowed for binary comparisons. Here, we describe the in vivo application of an extended set of six iqPIR reagents (6-plex iqPIR), allowing multiplexed quantitative interactome analysis of up to six biological samples in a single LC-MS acquisition. Multiplexed iqPIR is demonstrated on MCF-7 breast cancer cells treated with five different Hsp90 inhibitors revealing large scale protein conformational and interaction changes specific to the molecular class of the inhibitors.

A systematic review of endotracheal stenting in patients with locally advanced thyroid cancer.

OBJECTIVE: Locally aggressive thyroid cancer can result in airway obstruction secondary to tracheal compression or vocal cord palsy. A tracheal stent provides an alternative to surgical resection, tracheostomy or conservative management in patients with compressive symptoms. This systematic review synthesises the current evidence associated with tracheal stenting in locally advanced thyroid cancer. DESIGN, SETTING AND PARTICIPANTS: We conducted a systematic review of tracheal stenting in locally advanced thyroid cancers. We searched MEDLINE, Embase and Web of Science for studies until 22 September 2020. Inclusion criteria were studies involving patients who had received tracheal stents to treat laryngotracheal stenosis secondary to locally advanced thyroid cancer. Single case reports or single cases were not included. MAIN OUTCOME MEASURES: We assessed studies for data on the performance of tracheal stenting; defined as symptomatic relief, spirometry data, complication rates and mortality. We also extracted data pertaining to the use of different types of stent. RESULTS: We identified eight full-text articles from 325 titles found in our search. These were all single-centre retrospective studies that lacked homogeneity of thyroid cancer histotypes. The number of patients in each study ranged from 4 to 35 patients. Stenting improved performance status (two of two studies), symptoms (five of five studies) and spirometry (two of three studies). The most common complications were tracheal granulation, tumour overgrowth, stent migration and sputum retention. CONCLUSION: There is a lack of evidence in the literature of tracheal stents in locally advanced thyroid cancer. However, the evidence available suggests tracheal stenting may be a useful treatment adjunct in advanced thyroid cancer-causing symptomatic airway obstruction.

A high-definition, low cost endoscope to video record head and neck surgery- our experience.

Head and neck surgery is a challenging speciality to video-record due to its open, small and sometimes deep operative field. Consequently current commercial technologies yield a high financial cost. This study explores how a low-cost, commercially available endoscope, called a borescope, may be used to overcome these challenges. It was hypothesised that due to its size, versatility and low-cost, it may be an accessible tool to circumnavigate the pitfalls of previously trialled recording devices. We report two cases in which a borescope was used intra-operatively. We found that the borescope can capture images suitable for teaching and training purposes but not when mounted as a headcam. As such the borescope is unable to provide a surgeons point of view.

Leveraging the Entirety of the Protein Data Bank to Enable Improved Structure Prediction Based on Cross-Link Data.

XLinkDB is a fast-expanding public database now storing more than 100 000 distinct identified cross-linked protein residue pairs acquired by chemical cross-linking with mass spectrometry from samples of 12 species (J. Proteome Res.2019, 18 (2), 753-758). Mapping identified cross-links to protein structures, when available, provides valuable guidance on protein conformations detected in the cross-linked samples. As more and more structures become available in the Protein Data Bank (Nucleic Acids Res.2000, 28 (1), 235-242), we sought to leverage their utility for cross-link studies by automatically mapping identified cross-links to structures based on sequence homology of the cross-linked proteins with those within structures. This enables use of structures derived from organisms different from those of samples, including large multiprotein complexes and complexes in alternative states. We demonstrate utility of mapping to orthologous structures, highlighting a cross-link between two subunits of mouse mitochondrial Complex I that was mapped to 15 structures derived from five mammals, its distances there of 16.2 ± 0.4 Å indicating strong conservation of the protein interaction. We also show how multimeric structures enable reassessment of cross-links presumed to be intraprotein as potentially homodimeric interprotein in origin.