Binding of Per- and Polyfluoroalkyl Substances to ß-Lactoglobulin from Bovine Milk.

Per- and polyfluoroalkyl substances (PFAS) are known for their high environmental persistence and potential toxicity. The presence of PFAS has been reported in many dairy products. However, the mechanisms underlying the accumulation of PFAS in these products remain unclear. Here, we used native mass spectrometry and molecular dynamics simulations to probe the interactions between 19 PFAS of environmental concern and two isoforms of the major bovine whey protein ß-lactoglobulin (ß-LG). We observed that six of these PFAS bound to both protein isoforms with low- to mid-micromolar dissociation constants. Based on quantitative, competitive binding experiments with endogenous ligands, PFAS can bind orthosterically and preferentially to ß-LG's hydrophobic ligand-binding calyx. ß-Cyclodextrin can also suppress binding of PFAS to ß-LG owing to the ability of ß-cyclodextrin to directly sequester PFAS from solution. This research sheds light on PFAS-ß-LG binding, suggesting that such interactions could impact lipid-fatty acid transport in bovine mammary glands at high PFAS concentrations. Furthermore, our results highlight the potential use of ß-cyclodextrin in mitigating PFAS binding, providing insights toward the development of strategies to reduce PFAS accumulation in dairy products and other biological systems.

Protein-Small Molecule Interactions in Native Mass Spectrometry.

Small molecule drug discovery has been propelled by the continual development of novel scientific methodologies to occasion therapeutic advances. Although established biophysical methods can be used to obtain information regarding the molecular mechanisms underlying drug action, these approaches are often inefficient, low throughput, and ineffective in the analysis of heterogeneous systems including dynamic oligomeric assemblies and proteins that have undergone extensive post-translational modification. Native mass spectrometry can be used to probe protein-small molecule interactions with unprecedented speed and sensitivity, providing unique insights into polydisperse biomolecular systems that are commonly encountered during the drug discovery process. In this review, we describe potential and proven applications of native MS in the study of interactions between small, drug-like molecules and proteins, including large multiprotein complexes and membrane proteins. Approaches to quantify the thermodynamic and kinetic properties of ligand binding are discussed, alongside a summary of gas-phase ion activation techniques that have been used to interrogate the structure of protein-small molecule complexes. We additionally highlight some of the key areas in modern drug design for which native mass spectrometry has elicited significant advances. Future developments and applications of native mass spectrometry in drug discovery workflows are identified, including potential pathways toward studying protein-small molecule interactions on a whole-proteome scale.

Oligomeric Remodeling by Molecular Glues Revealed Using Native Mass Spectrometry and Mass Photometry.

Molecular glues stabilize interactions between E3 ligases and novel substrates to promote substrate degradation, thereby facilitating the inhibition of traditionally "undruggable" protein targets. However, most known molecular glues have been discovered fortuitously or are based on well-established chemical scaffolds. Efficient approaches for discovering and characterizing the effects of molecular glues on protein interactions are required to accelerate the discovery of novel agents. Here, we demonstrate that native mass spectrometry and mass photometry can provide unique insights into the physical mechanism of molecular glues, revealing previously unknown effects of such small molecules on the oligomeric organization of E3 ligases. When compared to well-established solution phase assays, native mass spectrometry provides accurate quantitative descriptions of molecular glue potency and efficacy while also enabling the binding specificity of E3 ligases to be determined in a single, rapid measurement. Such mechanistic insights should accelerate the rational development of molecular glues to afford powerful therapeutic agents.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Multiplexed Screening of Thousands of Natural Products for Protein-Ligand Binding in Native Mass Spectrometry.

The structural diversity of natural products offers unique opportunities for drug discovery, but challenges associated with their isolation and screening can hinder the identification of drug-like molecules from complex natural product extracts. Here we introduce a mass spectrometry-based approach that integrates untargeted metabolomics with multistage, high-resolution native mass spectrometry to rapidly identify natural products that bind to therapeutically relevant protein targets. By directly screening crude natural product extracts containing thousands of drug-like small molecules using a single, rapid measurement, we could identify novel natural product ligands of human drug targets without fractionation. This method should significantly increase the efficiency of target-based natural product drug discovery workflows.

Cyclisations and Strategies for Stereoselective Synthesis of Piperidine Iminosugars.

This personal account focuses on synthesis of polyhydroxylated piperidines, a subset of compounds within the iminosugar family. Cyclisations to form the piperidine ring include reductive amination, substitution via amines, iminium ions and cyclic nitrones, transamidification (N-acyl transfer), addition to alkenes, ring contraction and expansion, photoinduced electron transfer, multicomponent Ugi reaction and ring closing metathesis. Enantiomerically pure piperidines are obtained from chiral pool precursors (e. g. sugars, amino acids, Garner's aldehyde) or asymmetric reactions (e. g. epoxidation, dihydroxylation, aminohydroxylation, aldol, biotransformation). Our laboratory have contributed cascades based on reductive amination from glycosyl azide precursors as well as Huisgen azide-alkene cycloaddition. The latter's combination with allylic azide rearrangement has given substituted piperidines, including those with quaternary centres adjacent to nitrogen.

Delivering bioactive cyclic peptides that target Hsp90 as prodrugs.

The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell-permeable peptide, which theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilising acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.

Glastonbury Festival: Medical Care at the World's Largest Greenfield Music Festival.

INTRODUCTION: Music festivals have become an increasingly popular form of mass-gathering event, drawing an increasing number of attendees across the world each year. While festivals exist to provide guests with an enjoyable experience, there have been instances of serious illness, injury, and in some cases death. Large crowds, prolonged exposure to loud music, and high rates of drug and alcohol consumption can pose a dangerous environment for guests as well as those looking after them. METHODS: A retrospective review of electronic patient records (EPRs) at the 2022 Glastonbury Festival was undertaken. All patients who attended medical services on-site during the festival and immediately after were included. Patient demographics, diagnosis, treatment received, and discharge destination were obtained and analyzed. RESULTS: A total of 2,828 patients received on-site medical care. The patient presentation rate (PPR) was 13.47 and the transport-to-hospital rate (TTHR) was 0.30 per 1,000 guests. The most common diagnoses were joint injuries, gastrointestinal conditions, and blisters. Only 164 patients (5.48%) were diagnosed as being intoxicated. Overall, 552 patients (19.52%) were prescribed a medication to take away and 268 (9.48%) had a dressing for a minor wound. One patient (0.04%) underwent a general anesthetic and no patients required cardiopulmonary resuscitation. Most patients were discharged back to the festival site (2,563; 90.66%). DISCUSSION: Minor conditions were responsible for many presentations and most patients only required mild or non-invasive interventions, after which they could be safely discharged back to the festival. Older adults were diagnosed with a different frequency of conditions compared to the overall study population, something not reported previously. Intoxicated patients only accounted for a very small amount of the medical workload.

Flow Chemistry for Synthesis of 2-(C-Glycosyl)acetates from Pyranoses via Tandem Wittig and Michael Reactions.

C-Glycosyl compounds (C-glycosides) are a class of saccharide derivatives with improved stability over their O-linked counterparts. This paper reports the synthesis of several trans-2-(C-glycosyl)acetates via a tandem Wittig-Michael reaction from pyranoses (cyclic hemiacetals) using continuous flow processing, which gave improvements compared to reactions conducted in round-bottom flasks. Products were isolated in yields of >60% from reactions of benzyl-protected xylopyranoses, glucopyranoses, and galactopyranoses at higher temperatures and pressures, which were superior to yields from batch procedures. A two-step procedure involving the Wittig reaction followed by Michael reaction (intramolecular oxa-Michael) of the unsaturated ester obtained in the presence of DBU was developed. Reactions of protected mannopyranose gave low yields in corresponding reactions in flow due to competing C-2 epimerization.

Protein Interaction Kinetics Delimit the Performance of Phosphorylation-Driven Protein Switches.

Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can switch protein-protein interactions (PPIs) within signaling networks. Recently, de novo-designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation to modulate PPIs. PTM-driven protein switches are promising tools for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of the interaction modeling, we built and evaluated novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between "on" and "off" states. The response of the switches linearly correlated to the kinase concentration, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, the switches responded to specific kinase activity with an increase in the fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. The protein switch kinetics model developed here should enable PTM-driven switches to be designed with ideal performance for specific applications.

Phospholipids Differentially Regulate Ca2+ Binding to Synaptotagmin-1.

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca2+ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca2+ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca2+ with a KD ∼ 45 µM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca2+ binding affinity, we explored the extent that Ca2+ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and dioleoylphosphatidylserine (DOPS) positively modulated Ca2+ binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) was reduced with increasing [Ca2+]. Overall, we find that specific lipids differentially modulate Ca2+ binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca2+, and anionic phospholipids that may also control some aspects of vesicular exocytosis.

Flow chemistry based catalytic hydrogenation for improving the synthesis of 1-deoxynojirimycin (DNJ) from an l-sorbose derived precursor.

1-Deoxynojirimycin (1-DNJ) is a glycoprocessing inhibitor, and it serves as a synthetic precursor to two of three currently marketed iminosugar drugs, miglustat (N-butyl DNJ/Zavesca®) and miglitol (Glyset®). Herein a continuous flow procedure is presented that shortens a synthesis of 1-DNJ from an intermediate prepared from l-sorbose. Batch reactions involving an azide reduction, subsequent reductive amination-based cyclisation, and O-benzyl deprotection in a previous report required two steps and the use of an acid. Here, this sequence is achieved in one step using the H-Cube® MiniPlus continuous flow reactor. Subsequent reductive amination of 1-DNJ with butanal using the H-Cube® gave NB-DNJ.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

How weakness of the tensor fascia lata and gluteus maximus may contribute to ACL injury: A new theory.

Anterior cruciate ligament (ACL) injuries are common in sports including a significant failure rate following reconstruction. The iliotibial band (ITB) is an important stabilizer of the lateral portion of the knee and also an important lateral rotator of the tibia. Both the tensor fascia lata (TFL) and gluteus maximus (Gmax) muscles insert into the ITB proximally. This paper describes a theory that implicates weakened TFL and Gmax muscles as possible contributors to anterolateral rotatory instability. If the TFL and Gmax are important contributors to anterolateral rotatory instability, physical therapists can emphasize assessing for their weakness and developing a rehabilitation program to restore their strength.

THE RELATIONSHIP BETWEEN PASSIVE GLENOHUMERAL TOTAL ROTATION AND THE STRENGTH OF THE INTERNAL AND EXTERNAL ROTATOR MUSCLES, A PRELIMINARY STUDY.

BACKGROUND: There is little research on how the amount of shoulder joint range of motion, specifically glenohumeral rotation, may be related to the muscle strength of the rotator cuff muscles. A long held belief is that a joint with excessive range of motion needs sufficient muscular strength for stability. However, no studies have examined this concept. PURPOSE: The purpose of this study was to see if total arc of glenohumeral joint rotation (External rotation [ER]+Internal rotation [IR]) could predict peak isometric muscle strength of the IR or ER muscles of the shoulder. STUDY DESIGN: Cross-sectional study design. METHODS: Fifty-three participants (41 females, 12 males) participated in the study. Passive glenohumeral joint internal rotation and external rotation motion was measured for each participant with a standard goniometer. Isometric muscle force of the ER and IR muscles were tested using a handheld dynamometer in three positions: end range ER, neutral 0°, and end range IR. Data were analyzed using a non-parametric tree based regression method (CART) and then cross-validated. RESULTS: The results showed that those with an increased total arc of motion of glenohumeral rotation (greater than 165.0°) had less muscle isometric muscle strength in all tests positions than those with less glenohumeral rotation. CONCLUSION: Decreased force of the ER and IR muscles of the shoulder was noted in those with increased total arc glenohumeral rotation ( > 165.0°), specifically those with increased glenohumeral internal rotation ( > 80.0°) when compared to those with glenohumeral rotation ( < 165.0°) and glenohumeral internal rotation ( < 80.0°). Future studies should include more males and attempt to develop strategies to assist those with larger excursions of shoulder rotation who may be at risk of developing shoulder problems. LEVEL OF EVIDENCE: Level 2.

The influence of muscle length on one-joint shoulder internal and external rotator muscle strength.

BACKGROUND AND PURPOSE: Kendall suggests testing the rotator cuff muscles in their maximally shortened position, since one-joint muscles are thought to be strongest. We found little evidence to support this concept. The purpose of this study was to determine if the shoulder internal rotator (IR) and external rotator (ER) muscles are strongest when placed in their shortened length position. METHODS: Fifty-three subjects participated. Glenohumeral joint internal rotation and external rotation motion was measured. Muscle strength was then tested using a hand-held dynamometer in four positions: (1) end-range ER; (2) neutral 0°; (3) glenohumeral joint mid-range and (4) end-range IR. Data were analyzed using two repeated measures ANOVA's. RESULTS: The results suggest that rotator muscle strength is dependent on muscle length. IR strength was weakest at end-range IR in its shortest length; ER muscle strength was weakest at end-range ER in its shortest length. Muscle strength of the IR or ER was not significantly different when comparing neutral 0° to the mid-range position and at their most lengthened position. CONCLUSION: The IR and ER muscles were found to be weakest when placed in a position of shortest muscle length, while the neutral 0° and mid-range positions were the strongest positions.

A Physiological and Kinematic Comparison of two Different Lean Back Positions During Stationary Rowing on a Concept II Machine.

This study compared two different body positions at the finish of a stroke during stationary rowing exercise on physiological and kinematic measurements. Nine male and five female rowers volunteered for the study: mean age (± SD), body height and body mass were 27 ±9 yrs, 180.5 ±12.3 cm and 81.2 ±14.2 kg. The two body positions at the finish were controlled at an upright posture or a novel greater lean back position. All subjects completed 3 different experimental trials on a Concept IID rowing machine at 3 different exercise intensities and comparisons were made between the lean back position at the same stroke rate and the same power output as the upright trial. Power output, heart rate, oxygen uptake, energy expenditure and % efficiency were higher (p<0.05) with the greater lean back position at the same stroke rate compared to all other conditions. Range of motion at the hip, ankle, and elbow and the handle velocity and distance moved were greater (p<0.05) with the lean back position. In conclusion, a greater lean back posture at the finish during stationary rowing produces a higher power output and improved efficiency at the same stroke rate but at an elevated physiological cost compared to a more upright position. Despite the higher energy expenditure, the relative gain in power output and efficiency with no negative kinematic changes suggests that a greater lean back position at the finish will enhance performance during stationary rowing exercise.