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Here we used native mass spectrometry (native MS) to probe a SARS-CoV protease, PLpro, which plays critical roles in coronavirus disease by affecting viral protein production and antagonizing host antiviral responses. Ultraviolet photodissociation (UVPD) and variable temperature electrospray ionization (vT ESI) were used to localize binding sites of PLpro inhibitors and revealed the stabilizing effects of inhibitors on protein tertiary structure. We compared PLpro from SARS-CoV-1 and SARS-CoV-2 in terms of inhibitor and ISG15 interactions to discern possible differences in protease function. A PLpro mutant lacking a single cysteine was used to localize inhibitor binding, and thermodynamic measurements revealed that inhibitor PR-619 stabilized the folded PLpro structure. These results will inform further development of PLpro as a therapeutic target against SARS-CoV-2 and other emerging coronaviruses.

The main protease (M pro ) of SARS-CoV-2 is an essential enzyme for coronaviral maturation and is the target of Paxlovid, which is currently the standard-of-care treatment for COVID-19. There remains a need to identify new inhibitors of M pro as viral resistance to Paxlovid emerges. Here, we report the use of native mass spectrometry coupled with 193 nm ultraviolet photodissociation (UVPD) and integrated with other biophysical tools to structurally characterize M pro and its interactions with potential covalent inhibitors. The overall energy landscape was obtained using variable temperature nanoelectrospray ionization (vT-nESI), thus providing quantitative evaluation of inhibitor binding on the stability of M pro . Thermodynamic parameters extracted from van't Hoff plots revealed that the dimeric complexes containing each inhibitor showed enhanced stability through increased melting temperatures as well as overall lower average charge states, giving insight into the basis for inhibition mechanisms.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans. The most comprehensive characterization of intact glycoproteins requires integration of a suitable separation method and high performance tandem mass spectrometry to provide both protein sequence information and glycosite localization. Here, we couple ultraviolet photodissociation and hydrophilic interaction chromatography with high resolution mass spectrometry to advance the characterization of intact glycoproteins ranging from 15 to 34 kDa, offering site localization of glycans, providing sequence coverages up to 93%, and affording relative quantitation of individual glycoforms.

Gram-negative bacteria develop and exhibit resistance to antibiotics, owing to their highly asymmetric outer membrane maintained by a group of six proteins comprising the Mla (maintenance of lipid asymmetry) pathway. Here, we investigate the lipid binding preferences of one Mla protein, MlaC, which transports lipids through the periplasm. We used ultraviolet photodissociation (UVPD) to identify and characterize modifications of lipids endogenously bound to MlaC expressed in three different bacteria strains. UVPD was also used to localize lipid binding to MlaC residues 130-140, consistent with the crystal structure reported for lipid-bound MlaC. The impact of removing the bound lipid from MlaC on its structure was monitored based on collision cross section measurements, revealing that the protein unfolded prior to release of the lipid. The lipid selectivity of MlaC was evaluated based on titrimetric experiments, indicating that MlaC-bound lipids in various classes (sphingolipids, glycerophospholipids, and fatty acids) as long as they possessed no more than two acyl chains.

Background: Transcatheter renal denervation (RDN) has had inconsistent efficacy and concerns for durability of denervation. We aimed to investigate long-term safety and efficacy of transcatheter microwave RDN in vivo in normotensive sheep in comparison to conventional radiofrequency ablation., Methods and Results: Sheep underwent bilateral RDN, receiving 1 to 2 microwave ablations (maximum power of 80-120 W for 240 s-480 s) and 12 to 16 radiofrequency ablations (180 s-240 s) in the main renal artery in a paired fashion, alternating the side of treatment, euthanized at 2 weeks (acute N=15) or 5.5 months (chronic N=15), and compared with undenervated controls (N=4). Microwave RDN produced substantial circumferential perivascular injury compared with radiofrequency at both 2 weeks [area 239.8 (interquartile range [IQR] 152.0-343.4) mm 2 versus 50.1 (IQR, 32.0-74.6) mm 2 , P <0.001; depth 16.4 (IQR, 13.9-18.9) mm versus 7.5 (IQR, 6.0-8.9) mm P <0.001] and 5.5 months [area 20.0 (IQR, 3.4-31.8) mm 2 versus 5.0 (IQR, 1.4-7.3) mm 2 , P =0.025; depth 5.9 (IQR, 1.9-8.8) mm versus 3.1 (IQR, 1.2-4.1) mm, P =0.005] using mixed models. Renal denervation resulted in significant long-term reductions in viability of renal sympathetic nerves [58.9% reduction with microwave ( P =0.01) and 45% reduction with radiofrequency ( P =0.017)] and median cortical norepinephrine levels [71% reduction with microwave ( P <0.001) and 72.9% reduction with radiofrequency ( P <0.001)] at 5.5 months compared with undenervated controls., Conclusions: Transcatheter microwave RDN produces deep circumferential perivascular ablations without significant arterial injury to provide effective and durable RDN at 5.5 months compared with radiofrequency RDN.

GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal β-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual β-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack β-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients., Competing Interests: Conflict of interest The author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The measurement of collision cross sections (CCS, σ) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the modified hard collision model, thus deviating from the prior FT-MS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision induced unfolding and tandem mass spectrometry experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.

Objective: To investigate clinically meaningful change for ROADS and ALSFRS-R using a patient-defined approach., Methods: Data were reviewed from participants assessed at the Emory ALS Center from 2019-2022 with two assessments using both ROADS and ALSFRS-R and a completed patient-reported global impression of change scale at the second visit. Minimal important difference (MID), or the smallest amount of change that is clinically relevant, was assessed based on patient reported impression of change for ROADS and ALSFRS-R. Minimal detectable change (MDC), the smallest amount of change exceeding the threshold for measurement error, was assessed for ROADS and ALSFRS-R using standard deviations for participants self-rated as "unchanged"., Results: Data were included from 162 participants. For ROADS (total possible normed score = 146), MID = 5.81 and MDC = 2.83 points. For ALSFRS-R (total possible sum-score = 48), MID = 3.24 and MDC = 1.59 points. Clinically meaningful decline during the assessment period was observed in 98/162 (60.49%) participants on ROADS and 75/162 (46.30) participants on ALSFRS-R (OR = 1.63, 95% CI [1.0009, 2.66])., Conclusions: Changes that are on average less than 5.81 points (3.98%) on the normed ROADS score or less than 3.24 points (6.75%) on the ALSFRS-R sum-score may not be clinically meaningful according to a patient-defined approach. Understanding the clinical and statistical limitations of these scales is crucial when designing and interpreting ALS research studies.

Measurement of collision cross section (CCS), a parameter reflecting an ion's size and shape, alongside high-resolution mass analysis extends the depth of molecular analysis by providing structural information beyond molecular mass alone. Although these measurements are most commonly undertaken using a dedicated ion mobility cell coupled to a mass spectrometer, alternative methods have emerged to extract CCSs directly by analysis of the decay rates of either time-domain transient signals or the FWHM of frequency domain peaks in FT mass analyzers. This information is also accessible from FTMS mass spectra obtained in commonly used workflows directly without the explicit access to transient or complex Fourier spectra. Previously, these experiments required isolation of individual charge states of ions prior to CCS analysis, limiting throughput. Here we advance Orbitrap CCS measurements to more users and applications by determining CCSs from commonly available mass spectra files as well as estimating CCS for multiple charge states simultaneously and showcase these methods by the measurement of CCSs of fragment ions produced from collisional activation of proteins.

The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.

The structural diversity of phospholipids plays a critical role in cellular membrane dynamics, energy storage, and cellular signaling. Despite its importance, the extent of this diversity has only recently come into focus, largely owing to advances in separation science and mass spectrometry methodology and instrumentation. Characterization of glycerophospholipid (GP) isomers differing only in their acyl chain configurations and locations of carbon-carbon double bonds (C═C) remains challenging due to the need for both effective separation of isomers and advanced tandem mass spectrometry (MS/MS) technologies capable of double-bond localization. Drift tube ion mobility spectrometry (DTIMS) coupled with MS can provide both fast separation and accurate determination of collision cross section (CCS) of molecules but typically lacks the resolving power needed to separate phospholipid isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous double-bond localization but is challenging to implement on the timescales of modern commercial drift tube time-of-flight mass spectrometers. Here, we present a novel method for coupling DTIMS with a UVPD-enabled Orbitrap mass spectrometer using absorption mode Fourier transform multiplexing that affords simultaneous localization of double bonds and accurate CCS measurements even when isomers cannot be fully resolved in the mobility dimension. This method is demonstrated on two- and three-component mixtures and shown to provide CCS measurements that differ from those obtained by individual analysis of each component by less than 1%.

Glycerophospholipids (GPLs), one of the main components of bacterial cell membranes, exhibit high levels of structural complexity that are directly correlated with biophysical membrane properties such as permeability and fluidity. This structural complexity arises from the substantial variability in the individual GPL structural components such as the acyl chain length and headgroup type and is further amplified by the presence of modifications such as double bonds and cyclopropane rings. Here we use liquid chromatography coupled to high-resolution and high-mass-accuracy ultraviolet photodissociation mass spectrometry for the most in-depth study of bacterial GPL modifications to date. In doing so, we unravel a diverse array of unexplored GPL modifications, ranging from acyl chain hydroxyl groups to novel headgroup structures. Along with characterizing these modifications, we elucidate general trends in bacterial GPL unsaturation elements and thus aim to decipher some of the biochemical pathways of unsaturation incorporation in bacterial GPLs. Finally, we discover aminoacyl-PGs not only in Gram-positive bacteria but also in Gram-negative C. jejuni , advancing our knowledge of the methods of surface charge modulation that Gram-negative organisms may adopt for antibiotic resistance.

Importance: A new outcome measure for overall disability level with improved responsiveness is needed for amyotrophic lateral sclerosis (ALS) clinical trials., Objective: To describe the creation and development of a new self-reported ALS disability scale with improved item targeting and psychometric properties that used a mathematically rigorous Rasch methodology., Design, Setting, and Participants: A preliminary ALS disability questionnaire with 119 questions was created based on literature review, clinical judgement of an expert panel, and patient input. Patients with ALS were recruited from January 2017 to June 2019 from the Emory University and Atlanta VA Medical Center ALS clinics, both in Atlanta, Georgia, during regularly scheduled clinic appointments to complete the draft questionnaire and standard ALS outcome measures. All consecutive patients seen at the Emory University and Atlanta VA Medical Center ALS clinics during the recruitment period with a diagnosis of ALS who were able to provide informed consent were invited to participate in the study. Rasch analyses were performed, and items were systematically removed based on missing data, model fit, disordered thresholds, item bias, and clinical judgment. A total of 509 patients with ALS were seen at the 2 sites during the recruitment period, and 264 patients provided informed consent., Interventions: Participants completed the draft Rasch questionnaire and the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R)., Main Outcomes and Measures: Rasch analyses and standard scale metrics were performed to create the new scale, and Rasch analyses were performed on the ALSFRS-R for comparison., Results: Overall, 243 participants with ALS completed the draft questionnaire, and 230 participants were included for Rasch analyses. The mean (SD) age for study participants was 61.9 (11.1) years, 146 (60.1%) were men, and site of onset was 23.0% bulbar (n = 56), 36.2% upper extremity (n = 88), and 39.5% lower extremity (n = 96). A 28-question Rasch-Built Overall ALS Disability Scale (ROADS) was constructed with each item scored 0, 1, or 2. The ROADS fulfilled Rasch model requirements, demonstrated improved item targeting compared with the ALSFRS-R, and had test-retest reliability of 0.97. Individual question fit statistics demonstrated infit values from 0.68 to 1.37 and outfit values from 0.66 to 1.43. The difference between the empirical variance explained by the measures and the modeled variance was 0.1%. The ALSFRS-R violated Rasch model expectations and demonstrated disordered thresholds for 9 of 12 questions; 13 of 48 answer choices on the ALSFRS-R were never the most probable answer choice for any overall disability level., Conclusions and Relevance: In this study, the 28-question, self-reported ROADS, which is linearly weighted, had improved item targeting compared with the ALSFRS-R, had high test-retest reliability, and was validated. ROADS may serve as a valuable and easily accessible outcome measure for use in ALS trials and in the clinic with improved responsiveness compared with the ALSFRS-R.

Background: Clinical studies of transcatheter radiofrequency renal denervation for treating hypertension have been hampered by the lack of consistent denervation efficacy. We aimed to demonstrate the short-term efficacy and safety of transcatheter microwave renal denervation., Methods: A novel 7F microwave system was validated in a sheep model of unilateral renal denervation. Up to two microwave ablations were delivered to each artery with maximum power at 100-110 W for 480 s., Results: Catheter deployment and ablation was successful in all 19 targeted vessel segments, and ablation produced substantial circumferential perivascular injury; median ablation lesion area greater than 395 [interquartile range (IQR) 251-437] mm, depth 17.1 (IQR 15.8-18.4) mm, length 16 (IQR 12-20) mm, without collateral visceral injury. Limiting power to 100 W minimized arterial injury, while maintaining a deep circumferential perivascular ablation. Microwave denervation reduced median functional sympathetic nerve surface area at the renal hilum on antityrosine hydroxylase staining by 100% (IQR 87-100%, P = 0.0039), and median renal cortical norepinephrine content by 83% (IQR 76-92%, P = 0.0078), compared to the paired control kidney at 2-3 weeks postprocedure., Conclusion: Transcatheter microwave ablation can produce deep circumferential perivascular ablations over a long segment of the renal artery without significant arterial or collateral visceral injury to provide effective renal denervation.

Objectives: This study sought to develop a method to assess renal sympathetic nerve function through localization and pacing of aorticorenal ganglia (ARG)., Background: Transcatheter renal denervation procedures often fail to produce complete renal denervation because of the lack of a physiological procedural endpoint., Methods: High-frequency pacing was performed in the inferior vena cava and aorta in sheep (n = 19) to identify ARG pace-capture sites. Group A (n = 5) underwent injection at the ARG pace-capture site for histological verification, group B (n = 6) underwent unilateral irrigated radiofrequency ablation of ARG pace-capture sites and assessment of renal innervation at 1 week post-procedure; and group C (n = 8) underwent ARG pacing before and 2 to 3 weeks after unilateral microwave renal denervation., Results: ARG pace-capture responses were observed at paired discrete sites above the ipsilateral renal artery eliciting a change in mean arterial blood pressure of 22.2 (interquartile range [IQR]: 15.5 to 34.3 mm Hg; p < 0.001) with concurrent ipsilateral renal arterial vasoconstriction, change in main renal artery diameter of -0.42 mm (IQR: -0.64 to -0.24 mm; p < 0.0001), and without consistent contralateral renal vasoconstriction. Sympathetic ganglionic tissue was observed at ARG pace-capture sites, and ganglion ablation led to significant ipsilateral renal denervation. Circumferential renal denervation resulted in immediate and sustained abolition of ARP pacing-induced renal vasoconstriction and significant ipsilateral renal denervation., Conclusions: Transvascular ARG pace-capture is feasible and recognized by concurrent hypertensive and ipsilateral renal arterial vasoconstrictive responses. Abolition of ARG pacing-induced vasoconstriction may indicate successful renal sympathetic denervation and serve as a physiological procedural endpoint to guide transcatheter renal denervation., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Aims: Trials of transcatheter renal artery denervation (RDN) have failed to show consistent antihypertensive efficacy. Procedural factors and limitations of radiofrequency ablation can lead to incomplete denervation. The aim of the study was to show that non-contact microwave catheter ablation could produce deep circumferential perivascular heating while avoiding injury to the renal artery intima and media., Methods and Results: A novel microwave catheter was designed and tested in a renal artery model consisting of layers of phantom materials embedded with a thermochromic liquid crystal sheet, colour range 50-78°C. Ablations were performed at 140 W for 180 sec and 120 W for 210 sec, delivering 25,200 J with renal arterial flow at 0.5 L/min and 0.1 L/min. Transcatheter microwave ablations 100-160 W for 180 sec were then performed in the renal arteries of five sheep. In vitro, ablations at 140 W and 0.5 L/min flow produced circumferential lesions 5.9±0.2 mm deep and 19.2±1.5 mm long with subendothelial sparing depth of 1.0±0.1 mm. In vivo, transcatheter microwave ablation was feasible with no collateral visceral thermal injury. There was histological evidence of preferential outer media and adventitial ablation., Conclusions: Transcatheter microwave ablation for RDN appears feasible and provides a heating pattern that may enable more complete denervation while sparing the renal arterial intima and media.

The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103(+) dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3(+) dermal DCs (thought to be equivalent to murine CD103(+) dermal DCs) and DC-SIGN(+) DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3(+) dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3(+) dermal DCs and DC-SIGN(+) cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.