Intramolecular Cyclization and a Retro-Ene Reaction Enable the Rapid Fragmentation of a Vitamin B1-derived Breslow Intermediate.

In solution, analogues of the Breslow intermediate formed during catalysis by benzoylformate decarboxylase (BFDC) undergo rapid, irreversible fragmentation. The ability of BFDC to prevent this reaction and preserve its' cofactor is a striking example of an enzyme 'steering' a reactive intermediate towards a productive pathway. To understand how BFDC suppresses the off-pathway reactivity of this Breslow intermediate, a clear mechanistic understanding of the fragmentation reaction is required. Here, DFT calculations reveal an unexpected mechanism for the solution-phase fragmentation that involves an intramolecular cyclization and a subsequent retro-ene reaction to release the final products. Free energy profiles demonstrate that this pathway is significantly more facile than the previously proposed mechanism that invoked Breslow intermediate enolates as intermediates. Additional computations have been performed to understand why related Breslow intermediates do not undergo analogous fragmentation reactions. Calculations performed with two closely related Breslow intermediates suggest that subtle differences in the relative values of ∆G‡ for protonation and fragmentation dictate whether a given intermediate will fragment or not. These differences and the fragmentation mechanism unveiled in this work may have ramifications for the mechanism of BFDC and other thiamin-dependent enzymes and could provide general lessons related to the control of reactive intermediates by enzymes.

A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

Top-Down Proteomics Identifies Plasma Proteoform Signatures of Liver Cirrhosis Progression.

Cirrhosis, advanced liver disease, affects 2-5 million Americans. While most patients have compensated cirrhosis and may be fairly asymptomatic, many decompensate and experience life-threatening complications such as gastrointestinal bleeding, confusion (hepatic encephalopathy), and ascites, reducing life expectancy from 12 to less than 2 years. Among patients with compensated cirrhosis, identifying patients at high risk of decompensation is critical to optimize care and reduce morbidity and mortality. Therefore, it is important to preferentially direct them towards specialty care which cannot be provided to all patients with cirrhosis. We used discovery Top-down Proteomics (TDP) to identify differentially expressed proteoforms (DEPs) in the plasma of patients with progressive stages of liver cirrhosis with the ultimate goal to identify candidate biomarkers of disease progression. In this pilot study, we identified 209 DEPs across three stages of cirrhosis (compensated, compensated with portal hypertension, and decompensated), of which 115 derived from proteins enriched in the liver at a transcriptional level and discriminated the three stages of cirrhosis. Enrichment analyses demonstrated DEPs are involved in several metabolic and immunological processes known to be impacted by cirrhosis progression. We have preliminarily defined the plasma proteoform signatures of cirrhosis patients, setting the stage for ongoing discovery and validation of biomarkers for early diagnosis, risk stratification, and disease monitoring.

Effectiveness of Autologous Skin Cell Suspension in Large Total Body Surface Area Burns: Analysis of Clinical Outcomes and Patient Charges.

Autologous skin cell suspension (ASCS) is an adjunct to conventional split-thickness skin grafts (STSG) for acute burns, enhancing healing and reducing donor site requirements. This study validates ASCS's predictive benefits in hospital stay reduction and cost savings by analyzing outcomes and real-world charges post-ASCS implementation at a single institution. A retrospective study (2018-2022) included burn patients with ≥10% TBSA. The study population comprised two groups: burns treated either with a combination of ASCS ± STSG or with STSG alone. Outcomes included LOS, surgeries, infection, complications, days on antibiotics, and adjusted charge per TBSA. The ASCS ± STSG group demonstrated significantly shorter LOS (Mdn: 16.0 days, IQR: 10-26) than the STSG group (Mdn: 20.0 days, IQR: 14-36; P = 0.017), and fewer surgeries (Mdn: 1.0, IQR: 1-2) versus the STSG group (Mdn: 1.0, IQR: 1-4; P = 0.020). Postoperative complications were significantly lower in ASCS ± STSG (11% vs. 36%; P < 0.001). The STSG group had a longer distribution of antibiotic days (IQR: 0-7.0, min-max: 0-76) than the ASCS ± STSG group (IQR: 0-0, min-max: 0-37; P = 0.014). Wound infection incidence did not differ (P = 0.843). ASCS ± STSG showed a lower distribution of adjusted charge per TBSA (IQR: $10,788.5 - $28,332.6) compared to the STSG group (IQR: $12,336.8 - $29,507.3; P = 0.602) with a lower mean adjusted charge per TBSA ($20,995.0 vs. $24,882.3), although this was not statistically significant. ASCS ± STSG utilization demonstrated significant reductions in LOS, surgeries, postoperative complications, antibiotics, and potential cost savings. These findings underscore the practicality of integrating ASCS in burn management, offering substantial benefits to patients and healthcare institutions.

Understanding noble gas incorporation in mantle minerals: an atomistic study.

Ab initio calculations in forsterite (Mg 2 SiO 4 ) are used to gain insight into the formation of point defects and incorporation of noble gases. We calculate the enthalpies of incorporation both at pre-existing vacancies in symmetrically non-equivalent sites, and at interstitial positions. At high pressure, most structural changes affect the MgO 6 units and the enthalpies of point defects increase, with those involving Mg and Si vacancies increasing more than those involving O sites. At 15 GPa Si vacancies and Mg interstitials have become the predominant intrinsic defects. We use these calculated enthalpies to estimate the total uptake of noble gases into the bulk crystal as a function of temperature and pressure both in the presence and absence of other heterovalent trace elements. For He and Ne our calculated solubilities point to atoms occupying mainly interstitial sites in agreement with previous experimental work. In contrast, Ar most likely substitutes for Mg due to its larger size and the deformation it causes within the crystal. Incorporation energies, as well as atomic distances suggest that the incorporation mainly depend on the size mismatch between host and guest atoms. Polarization effects arising from the polarizability of the noble gas atom or the presence of charged defects are minimal and do not contribute significantly to the uptake. Finally, the discrepancies between our results and recent experiments suggest that there are other incorporation mechanisms such as adsorption at internal and external interfaces, voids and grain boundaries which must play a major role in noble gas storage and solubility.

Enzyme-Polymer Conjugates with Photocleavable Linkers for Control Over Protein Activity.

Reversible conjugation of polymers to proteins is important for a variety of applications, for example to control protein activity. Light is often employed as an external trigger to allow for spatio and temporal control over release of a payload. In this report, we demonstrate preparation of photocleavable poly(polyethylene glycol) acrylate)-lysozyme (pPEGA-Lys) conjugates via ortho-nitrobenzyl linkages. The conjugates were made by both grafting-to and grafting-from in order to compare and contrast the two synthetic approaches. First, a lysine-reactive ortho-nitrobenzyl atom transfer radical polymerization (ATRP) initiator was synthesized. For the grafting-to strategy, the initiator was employed in the ATRP of PEGA, and the subsequent polymer was conjugated to the lysine residues of lysozyme. For the grafting-from strategy, lysozyme was modified first with the photocleavable initiator, and the purified macroinitiator was then subjected to polymerization conditions to synthesize the protein-polymer conjugate. The polymer was cleaved from the protein via UV light, and activity before and after polymer removal was evaluated, showing 83% recovery. This work provides evidence that reversing conjugation is successful for activity modulation for ortho-nitrobenzyl linked protein-polymer conjugates.

Air-plant interaction and air-soil exchange of polycyclic aromatic hydrocarbons in a large human-influenced reservoir in southwest China.

The Three Gorges Reservoir (TGR) is totally manmade, strongly influenced by anthropogenic activity, and lies on the upper reaches of Yangtze River. The periodic storage and discharge of water from the Three Gorges Dam could have altered the original air-plant/soil interactions of contaminants in TGR. Herein, paired atmospheric gas-particle, air-plant, and air-soil samples were collected to investigate the air-plant interaction and air-soil exchange of 16 USEPA priority polycyclic aromatic hydrocarbons (PAHs). The air-plant interaction based on McLachlan's framework to our datasets suggests that PAHs were absorbed via gaseous deposition that was restricted by the plant-gas dynamic equilibrium. The equilibrium indicates a dynamic balance between the gaseous phase and plant surface in PAH absorption. The main limiting factor influencing the PAH uptake was the plant species rather than the atmospheric PAH concentration. The air-soil exchange of PAHs exhibited a net volatilization flux of 16.71 ng/m2/d from the soil to the air based on annual average. There was more volatilization and less deposition in summer and more deposition and less volatilization in autumn and winter. The soil serves as a secondary source of atmospheric PAHs. As the first attempt on probing the multi-interface geochemical process of PAHs, this study highlights the influence of manual water level manipulation from the TGD and environmental factors (such as temperature, humidity, and soil properties) on the regional fate of PAHs in the TGR.

Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.

Multilevel quantum mechanical calculations show the role of promoter molecules in the dehydration of methanol to dimethyl ether in H-ZSM-5.

Methyl carboxylate esters promote the formation of dimethyl ether (DME) from the dehydration of methanol in H-ZSM-5 zeolite. We employ a multilevel quantum method to explore the possible associative and dissociative mechanisms in the presence, and absence, of six methyl ester promoters. This hybrid method combines density functional theory, with dispersion corrections (DFT-D3), for the full periodic system, with second-order Møller-Plesset perturbation theory (MP2) for small clusters representing the reaction site, and coupled cluster with single, double, and perturbative triple substitution (CCSD(T)) for the reacting molecules. The calculated adsorption enthalpy of methanol, and reaction enthalpies of the dehydration of methanol to DME within H-ZSM-5, agree with experiment to within chemical accuracy (∼4 kJ mol-1). For the promoters, a reaction pathway via an associative mechanism gives lower overall reaction enthalpies and barriers compared to the reaction with methanol only. Each stage of this mechanism is explored and related to experimental data. We provide evidence that suggests the promoter's adsorption to the Brønsted acid site is the most important factor dictating its efficiency.

MSModDetector: a tool for detecting mass shifts and post-translational modifications in individual ion mass spectrometry data.

MOTIVATION: Post-translational modifications (PTMs) on proteins regulate protein structures and functions. A single protein molecule can possess multiple modification sites that can accommodate various PTM types, leading to a variety of different patterns, or combinations of PTMs, on that protein. Different PTM patterns can give rise to distinct biological functions. To facilitate the study of multiple PTMs on the same protein molecule, top-down mass spectrometry (MS) has proven to be a useful tool to measure the mass of intact proteins, thereby enabling even PTMs at distant sites to be assigned to the same protein molecule and allowing determination of how many PTMs are attached to a single protein. RESULTS: We developed a Python module called MSModDetector that studies PTM patterns from individual ion mass spectrometry (I2MS) data. I2MS is an intact protein mass spectrometry approach that generates true mass spectra without the need to infer charge states. The algorithm first detects and quantifies mass shifts for a protein of interest and subsequently infers potential PTM patterns using linear programming. The algorithm is evaluated on simulated I2MS data and experimental I2MS data for the tumor suppressor protein p53. We show that MSModDetector is a useful tool for comparing a protein's PTM pattern landscape across different conditions. An improved analysis of PTM patterns will enable a deeper understanding of PTM-regulated cellular processes. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://github.com/marjanfaizi/MSModDetector.

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Probing the occurrence, sources and cancer risk assessment of polycyclic aromatic hydrocarbons in PM2.5 in a humid metropolitan city in China.

Fifty-two consecutive PM2.5 samples from December 2021 to February 2022 (the whole winter) were collected in the center of Chongqing, a humid metropolitan city in China. These samples were analysed for the 16 USEPA priority polycyclic aromatic hydrocarbons (16 PAHs) to explore their composition and sources, and to assess their cancer risks to humans. The total concentrations of the 16 PAHs (ng m-3) ranged from 16.45 to 174.15, with an average of 59.35 ± 21.45. Positive matrix factorization (PMF) indicated that traffic emissions were the major source (42.4%), followed by coal combustion/industrial emission (31.3%) and petroleum leakage/evaporation (26.3%). The contribution from traffic emission to the 16 PAHs increased from 40.0% in the non-episode days to as high as 46.2% in the air quality episode during the sampling period. The population attributable fraction (PAF) indicates that when the unit relative risk (URR) is 4.49, the number of lung cancer cases per million individuals under PAH exposure is 27 for adults and 38 for seniors, respectively. It was 5 for adults and 7 for seniors, when the URR is 1.3. The average incremental lifetime cancer risk (ILCR) for children, adolescents, adults and seniors was 0.25 × 10-6, 0.23 × 10-6, 0.71 × 10-6, and 1.26 × 10-6, respectively. The results of these two models complemented each other well, and both implied acceptable PAH exposure levels. Individual genetic susceptibility and exposure time were identified as the most sensitive parameters. The selection and use of parameters in risk assessment should be further deepened in subsequent studies to enhance the reliability of the assessment results.

PitSurgRT: real-time localization of critical anatomical structures in endoscopic pituitary surgery.

PURPOSE: Endoscopic pituitary surgery entails navigating through the nasal cavity and sphenoid sinus to access the sella using an endoscope. This procedure is intricate due to the proximity of crucial anatomical structures (e.g. carotid arteries and optic nerves) to pituitary tumours, and any unintended damage can lead to severe complications including blindness and death. Intraoperative guidance during this surgery could support improved localization of the critical structures leading to reducing the risk of complications. METHODS: A deep learning network PitSurgRT is proposed for real-time localization of critical structures in endoscopic pituitary surgery. The network uses high-resolution net (HRNet) as a backbone with a multi-head for jointly localizing critical anatomical structures while segmenting larger structures simultaneously. Moreover, the trained model is optimized and accelerated by using TensorRT. Finally, the model predictions are shown to neurosurgeons, to test their guidance capabilities. RESULTS: Compared with the state-of-the-art method, our model significantly reduces the mean error in landmark detection of the critical structures from 138.76 to 54.40 pixels in a 1280 × 720-pixel image. Furthermore, the semantic segmentation of the most critical structure, sella, is improved by 4.39% IoU. The inference speed of the accelerated model achieves 298 frames per second with floating-point-16 precision. In the study of 15 neurosurgeons, 88.67% of predictions are considered accurate enough for real-time guidance. CONCLUSION: The results from the quantitative evaluation, real-time acceleration, and neurosurgeon study demonstrate the proposed method is highly promising in providing real-time intraoperative guidance of the critical anatomical structures in endoscopic pituitary surgery.

Single Cell Analysis of Proteoforms.

We introduce single cell Proteoform imaging Mass Spectrometry (scPiMS), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single cells dropcast onto glass slides. Sampling and detection of whole proteoforms by individual ion mass spectrometry enable a scalable approach to single cell proteomics. This new scPiMS platform addresses the throughput bottleneck in single cell proteomics and boosts the cell processing rate by several fold while accessing protein composition with higher coverage.

Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry.

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a "bird's eye" view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0-4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

Does Size Matter? A Prospective Study on the Feasibility of Using a Handheld Ultrasound Device in Place of a Cart-Based System in the Evaluation of Trauma Patients.

BACKGROUND: As emergency physicians are looking at handheld devices as alternatives to the traditional, cart-based systems, concerns center around whether they are forsaking image quality for a lower price point and whether the handheld can be trusted for medical decision making. OBJECTIVE: We aimed to determine the feasibility of using a handheld ultrasound device in place of a cart-based system during the evaluation of trauma patients using the Focused Assessment with Sonography for Trauma (FAST) examination. METHODS: This was a prospective study of adult trauma patients who received a FAST examination as part of their evaluation. A FAST examination was performed using a cart-based machine and a handheld device. The results of the examinations were compared with computed tomography imaging. Images obtained from both ultrasound devices were reviewed by an expert for image quality. RESULTS: A total of 62 patients were enrolled in the study. The mean (SD) time to perform a FAST examination using the handheld device was 307.3 (65.3) s, which was significantly less (p = 0.002) than the 336.1 (86.8) s with the cart-based machine. There was strong agreement between the examination results of the handheld and cart-based devices and between the handheld and computed tomography. Image quality scores obtained with the handheld device were lower than those from the cart-based system. Most operators and reviewers agreed that the images obtained from the handheld were adequate for medical decision making. CONCLUSIONS: Data support that it is feasible to use the handheld ultrasound device for evaluation of the trauma patient in place of the cart-based system.

Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring.

Existing mass spectrometric assays used for sensitive and specific measurements of target proteins across multiple samples, such as selected/multiple reaction monitoring (SRM/MRM) or parallel reaction monitoring (PRM), are peptide-based methods for bottom-up proteomics. Here, we describe an approach based on the principle of PRM for the measurement of intact proteoforms by targeted top-down proteomics, termed proteoform reaction monitoring (PfRM). We explore the ability of our method to circumvent traditional limitations of top-down proteomics, such as sensitivity and reproducibility. We also introduce a new software program, Proteoform Finder (part of ProSight Native), specifically designed for the easy analysis of PfRM data. PfRM was initially benchmarked by quantifying three standard proteins. The linearity of the assay was shown over almost 3 orders of magnitude in the femtomole range, with limits of detection and quantification in the low femtomolar range. We later applied our multiplexed PfRM assay to complex samples to quantify biomarker candidates in peripheral blood mononuclear cells (PBMCs) from liver-transplanted patients, suggesting their possible translational applications. These results demonstrate that PfRM has the potential to contribute to the accurate quantification of protein biomarkers for diagnostic purposes and to improve our understanding of disease etiology at the proteoform level.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Short-chain fatty acids propionate and butyrate control growth and differentiation linked to cellular metabolism.

The short-chain fatty acids (SCFA) propionate and butyrate are produced in large amounts by microbial metabolism and have been identified as unique acyl lysine histone marks. In order to better understand the function of these modifications we used ChIP-seq to map the genome-wide location of four short-chain acyl histone marks H3K18pr/bu and H4K12pr/bu in treated and untreated colorectal cancer (CRC) and normal cells, as well as in mouse intestines in vivo. We correlate these marks with open chromatin regions along with gene expression to access the function of the target regions. Our data demonstrate that propionate and butyrate act as promoters of growth, differentiation as well as ion transport. We propose a mechanism involving direct modification of specific genomic regions, resulting in increased chromatin accessibility, and in case of butyrate, opposing effects on the proliferation of normal versus CRC cells.