A compendium of multi-omics data illuminating host responses to lethal human virus infections.

Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology.

Current Concepts in Management of Acromioclavicular Joint Injury.

Background: The management of acromioclavicular joint injuries requires a thorough understanding of the anatomy and biomechanics of the joint, as well as knowledge of the pertinent physical exam findings and classification to determine an appropriate treatment approach, whether operative or nonoperative. In this article, we present a narrative review of the current state of understanding surrounding these issues. Although there are a large number of options for operative intervention, we additionally present our experience with anatomic coracoclavicular ligament reconstruction (ACCR) with imbrication of the deltoid fascia. Methods: A retrospective review of prospectively collected data on a total of 45 patients who had undergone ACCR between 2003 and 2016 were collected. Results: We found that improvements were seen in American Shoulder and Elbow Surgeons Score (ASES) (53 ± 19 to 81 ± 23), Simple Shoulder Test (SST) (6 ± 3 to 12 ± 13), Constant-Murley (CM) (60 ± 18 to 92 ± 8), and Rowe (67 ± 14 to 89 ± 11) and the mean post-operative SANE score was 86 ± 17. Conclusions: ACCR has the advantage of addressing both horizontal and vertical stability with good outcomes.

Computational tools and algorithms for ion mobility spectrometry-mass spectrometry.

Ion mobility spectrometry-mass spectrometry (IMS-MS or IM-MS) is a powerful analytical technique that combines the gas-phase separation capabilities of IM with the identification and quantification capabilities of MS. IM-MS can differentiate molecules with indistinguishable masses but different structures (e.g., isomers, isobars, molecular classes, and contaminant ions). The importance of this analytical technique is reflected by a staged increase in the number of applications for molecular characterization across a variety of fields, from different MS-based omics (proteomics, metabolomics, lipidomics, etc.) to the structural characterization of glycans, organic matter, proteins, and macromolecular complexes. With the increasing application of IM-MS there is a pressing need for effective and accessible computational tools. This article presents an overview of the most recent free and open-source software tools specifically tailored for the analysis and interpretation of data derived from IM-MS instrumentation. This review enumerates these tools and outlines their main algorithmic approaches, while highlighting representative applications across different fields. Finally, a discussion of current limitations and expectable improvements is presented.

Identification of a 2-Aminobenzimidazole Scaffold that Potentiates Gram-Positive Selective Antibiotics Against Gram-Negative Bacteria.

The development of novel therapeutic approaches is crucial in the fight against multi-drug resistant (MDR) bacteria, particularly gram-negative species. Small molecule adjuvants that enhance the activity of otherwise gram-positive selective antibiotics against gram-negative bacteria have the potential to expand current treatment options. We have previously reported adjuvants based upon a 2-aminoimidazole (2-AI) scaffold that potentiate macrolide antibiotics against several gram-negative pathogens. Herein, we report the discovery and structure-activity relationship (SAR) investigation of an additional class of macrolide adjuvants based upon a 2-aminobenzimidazole (2-ABI) scaffold. The lead compound lowers the minimum inhibitory concentration (MIC) of clarithromycin (CLR) from 512 to 2 µg/mL at 30 µM against Klebsiella pneumoniae 2146, and from 32 to 2 µg/mL at 5 µM, against Acinetobacter baumannii 5075. Preliminary investigation into the mechanism of action suggests that the compounds are binding to lipopolysaccharide (LPS) in K. pneumoniae, and modulating lipooligosaccharide (LOS) biosynthesis, assembly, or transport in A. baumannii.

Mobility Selective Ion Soft-Landing and Characterization Enabled Using Structures for Lossless Ion Manipulation.

While conventional ion-soft landing uses the mass-to-charge (m/z) ratio to achieve molecular selection for deposition, here we demonstrate the use of Structures for Lossless Ion Manipulation (SLIM) for mobility-based ion selection and deposition. The dynamic rerouting capabilities of SLIM were leveraged to enable the rerouting of a selected range of mobilities to a different SLIM path (rather than MS) that terminated at a deposition surface. A selected mobility range from a phosphazene ion mixture was rerouted and deposited with a current pulse (∼150 pA) resembling its mobility peak. In addition, from a mixture of tetra-alkyl ammonium (TAA) ions containing chain lengths of C5-C8, selected chains (C6, C7) were collected on a surface, reconstituted into solution-phase, and subsequently analyzed with a SLIM-qToF to obtain an IMS/MS spectrum, confirming the identity of the selected species. Further, this method was used to characterize triply charged tungsten-polyoxometalate anions, PW12O403- (WPOM). The arrival time distribution of the IMS/MS showed multiple peaks associated with the triply charged anion (PW12O403-), of which a selected ATD was deposited and imaged using TEM. Additionally, the identity of the deposited WPOM was ascertained using energy-dispersive (EDS) spectroscopy. Further, we present theory and computations that reveal ion landing energies, the ability to modulate the energies, and deposition spot sizes.

Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Accumulation of Large Ion Populations with High Ion Densities and Effects Due to Space Charge in Traveling Wave-Based Structures for Lossless Ion Manipulations (SLIM) IMS-MS.

The accumulation of very large ion populations in traveling wave (TW)-based Structures for Lossless ion Manipulations (SLIM) has been studied to better understand aspects of "in-SLIM" ion accumulation, and particularly its use in conjunction with ion mobility spectrometry (IMS). A linear SLIM ion path was implemented that had a "gate" for blocking and accumulating ions for arbitrary time periods. Removing the gate potential caused ions to exit, and the spatial distributions of accumulated ions examined. The ion populations for a set of peptides increased approximately linearly with increased accumulation times until space change effects became significant, after which the peptide precursor ion populations decreased due to growing space charge-related ion activation, reactions, and losses. Ion activation increased with added storage times and the TW amplitude. Lower amplitude TWs in the accumulation/storage region prevented or minimized ion losses or ion heating effects that can also lead to fragmentation. Our results supported the use of an accumulation region close to the SLIM entrance for speeding accumulation, minimizing ion heating, and avoiding ion population profiles that result in IMS peak tailing. Importantly, space charge-driven separations were observed for large populations of accumulated species and attributed to the opposing effects of space charge and the TW. In these separations, ion species form distributions or peaks, sometimes moving against the TW, and are ordered in the SLIM based on their mobilities. Only the highest mobility ions located closest to the gate in the trapped ion population (and where the highest ion densities were achieved) were significantly activated. The observed separations may offer utility for ion prefractionation of ions and increasing the dynamic range measurements, increasing the resolving power of IMS separations by decreasing peak widths for accumulated ion populations, and other purposes benefiting from separations of extremely large ion populations.

Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME).

Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.

Peripheral neuropathies after shoulder arthroscopy: a systematic review.

Purpose: Peripheral neuropathies after shoulder arthroscopy are rare, though likely under-reported. Many resolve spontaneously, but some patients are left with permanent neurological deficits. The purpose of this study was to review the literature to better characterize this patient population, diagnostic tests performed, the timing and type of surgical intervention, and report clinical outcomes. Methods: A systematic literature review was performed. Articles in English were identified from PubMed, EMBASE, and CINAHL in August 2021. Article titles and abstracts were screened for relevance by two authors and discordant abstracts were resolved by the senior author. Data were subsequently extracted from the included articles. Results: Seventeen articles were identified yielding a total of 91 patients. The average age was 53 ± 12 years, and most patients were male (72%). Rotator cuff repair (62%) was the most common procedure performed. A peripheral neuropathy was identified an average of 80 ± 81 days from the index procedure (range, 0-240 days). Most commonly, peripheral nerve injury presented as a mononeuropathy, with the median nerve (39%) and ulnar nerve (17%) affected predominantly. Seventeen percent of patients underwent a secondary surgery at an average of 232 ± 157 days after the index procedure. At the final follow-up, 55% of neuropathies had resolved, 14% partially improved, and 22% showed no clinical improvement. The most proposed etiologies were postoperative immobilization (29%) and intraoperative positioning (20%), but several possible etiologies have been suggested. Conclusions: Peripheral neuropathies after arthroscopic shoulder procedures are rare. While most spontaneously resolve, up to 1 in 5 patients may have persistent neuropathic symptoms. A high index of suspicion should be maintained throughout the postoperative period. When neurologic deficits are identified, patients should undergo a thorough diagnostic workup and be referred to a subspecialist in a timely manner.

A multi-country comparison of jurisdictions with and without mandatory nutrition labelling policies in restaurants: analysis of behaviours associated with menu labelling in the 2019 International Food Policy Study.

OBJECTIVE: To examine differences in noticing and use of nutrition information comparing jurisdictions with and without mandatory menu labelling policies and examine differences among sociodemographic groups. DESIGN: Cross-sectional data from the International Food Policy Study (IFPS) online survey. SETTING: IFPS participants from Australia, Canada, Mexico, United Kingdom and USA in 2019. PARTICIPANTS: Adults aged 18-99; n 19 393. RESULTS: Participants in jurisdictions with mandatory policies were significantly more likely to notice and use nutrition information, order something different, eat less of their order and change restaurants compared to jurisdictions without policies. For noticed nutrition information, the differences between policy groups were greatest comparing older to younger age groups and comparing high education (difference of 10·7 %, 95 % CI 8·9, 12·6) to low education (difference of 4·1 %, 95 % CI 1·8, 6·3). For used nutrition information, differences were greatest comparing high education (difference of 4·9 %, 95 % CI 3·5, 6·4) to low education (difference of 1·8 %, 95 % CI 0·2, 3·5). Mandatory labelling was associated with an increase in ordering something different among the majority ethnicity group and a decrease among the minority ethnicity group. For changed restaurant visited, differences were greater for medium and high education compared to low education, and differences were greater for higher compared to lower income adequacy. CONCLUSIONS: Participants living in jurisdictions with mandatory nutrition information in restaurants were more likely to report noticing and using nutrition information, as well as greater efforts to modify their consumption. However, the magnitudes of these differences were relatively small.

Myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia are indistinguishable by their cerebrospinal fluid proteomes.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and fibromyalgia have overlapping neurologic symptoms particularly disabling fatigue. This has given rise to the question whether they are distinct central nervous system (CNS) entities or is one an extension of the other. MATERIAL AND METHODS: To investigate this, we used unbiased quantitative mass spectrometry-based proteomics to examine the most proximal fluid to the brain, cerebrospinal fluid (CSF). This was to ascertain if the proteome profile of one was the same or different from the other. We examined two separate groups of ME/CFS, one with (n = 15) and one without (n = 15) fibromyalgia. RESULTS: We quantified a total of 2083 proteins using immunoaffinity depletion, tandem mass tag isobaric labelling and offline two-dimensional liquid chromatography coupled to tandem mass spectrometry, including 1789 that were quantified in all the CSF samples. ANOVA analysis did not yield any proteins with an adjusted p value <.05. CONCLUSION: This supports the notion that ME/CFS and fibromyalgia as currently defined are not distinct entities.Key messageME/CFS and fibromyalgia as currently defined are not distinct entities.Unbiased quantitative mass spectrometry-based proteomics can be used to discover cerebrospinal fluid proteins that are biomarkers for a condition such as we are studying.

Robust collection and processing for label-free single voxel proteomics.

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures important tissue heterogeneity, which make it impossible for proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single tissue voxel and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single tissue voxel dissected by LCM into the PCR tube cap and MS-compatible surfactant-assisted one-pot voxel processing in the collection cap. This convenient method allows reproducible label-free quantification of ∼900 and ∼4,600 proteins for single voxel from fresh frozen human spleen tissue at 20 µm × 20 µm × 10 µm (close to single cells) and 200 µm × 200 µm × 10 µm (∼100 cells), respectively. 100s-1000s of protein signatures with differential expression levels were identified to be spatially resolved between spleen red and white pulp regions depending on the voxel size. Region-specific signaling pathways were enriched from single voxel proteomics data. Antibody-based CODEX imaging was used to validate label-free MS quantitation for single voxel analysis. The wcSOP-MS method paves the way for routine robust single voxel proteomics and spatial proteomics.

Comparison of 3 diagnostic platforms for identification of bacteria and yeast from positive blood culture bottles.

Managing bloodstream infections requires fast and accurate diagnostics. Culture-based diagnostic methods for identification from positive blood culture require 24-hour subculture, potentially delaying time to appropriate therapy. Positive blood cultures were collected (n = 301) from September 2021 to August 2022 at the University of Maryland Medical Center. Platforms compared were BioFire® BCID2, Sepsityper®, and short-term culture. For monomicrobial cultures, FilmArray® BCID2 identified 88.3% (241/273) of pathogens. Rapid Sepsityper® identified 76.9% (210/273) of pathogens. Sepsityper® extraction identified 82.4% (225/273) of pathogens. Short-term culture identified 83.5% (228/273) of pathogens. For polymicrobial cultures, Sepsityper®, short-term culture, and BioFire® BCID2 had complete identifications at 10.7% (3/28), 0%, and 92.9% (26/28), respectively. Time-to-results for Rapid Sepsityper®, Sepsityper® extraction, BioFire® BCID2, and Short-term culture were 35, 52, 65, and 306 minutes, respectively. Performance of these platforms can reduce time-to-results and may help effectively treat bloodstream infections faster. Accuracy, time-to-result, and hands-on time are important factors when evaluation diagnostic platforms.

Plasma protein biomarkers predict the development of persistent autoantibodies and type 1 diabetes 6 months prior to the onset of autoimmunity.

Type 1 diabetes (T1D) results from autoimmune destruction of ß cells. Insufficient availability of biomarkers represents a significant gap in understanding the disease cause and progression. We conduct blinded, two-phase case-control plasma proteomics on the TEDDY study to identify biomarkers predictive of T1D development. Untargeted proteomics of 2,252 samples from 184 individuals identify 376 regulated proteins, showing alteration of complement, inflammatory signaling, and metabolic proteins even prior to autoimmunity onset. Extracellular matrix and antigen presentation proteins are differentially regulated in individuals who progress to T1D vs. those that remain in autoimmunity. Targeted proteomics measurements of 167 proteins in 6,426 samples from 990 individuals validate 83 biomarkers. A machine learning analysis predicts if individuals would remain in autoimmunity or develop T1D 6 months before autoantibody appearance, with areas under receiver operating characteristic curves of 0.871 and 0.918, respectively. Our study identifies and validates biomarkers, highlighting pathways affected during T1D development.

The Role of Ion Rotation in Ion Mobility: Ultrahigh-Precision Prediction of Ion Mobility Dependence on Ion Mass Distribution and Translational to Rotational Energy Transfer.

The role of ion rotation in determining ion mobilities is explored using the subtle gas phase ion mobility shifts based on differences in ion mass distributions between isotopomer ions that have been observed with ion mobility spectrometry (IMS) measurements. These mobility shifts become apparent for IMS resolving powers on the order of ∼1500 where relative mobilities (or alternatively momentum transfer collision cross sections; Ω) can be measured with a precision of ∼10 ppm. The isotopomer ions have identical structures and masses, differing only in their internal mass distributions, and their Ω differences cannot be predicted by widely used computational approaches, which ignore the dependence of Ω on the ion's rotational properties. Here, we investigate the rotational dependence of Ω, which includes changes to its collision frequency due to thermal rotation as well as the coupling of translational to rotational energy transfer. We show that differences in rotational energy transfer during ion-molecule collisions provide the major contribution to isotopomer ion separations, with only a minor contribution due to an increase in collision frequency due to ion rotation. Modeling including these factors allowed for differences in Ω to be calculated that precisely mirror the experimental separations. These findings also highlight the promise of pairing high-resolution IMS measurements with theory and computation for improved elucidation of subtle structural differences between ions.

A Dual-Gated Structures for Lossless Ion Manipulations-Ion Mobility Orbitrap Mass Spectrometry Platform for Combined Ultra-High-Resolution Molecular Analysis.

High-resolution ion mobility spectrometry-mass spectrometry (HR-IMS-MS) instruments have enormously advanced the ability to characterize complex biological mixtures. Unfortunately, HR-IMS and HR-MS measurements are typically performed independently due to mismatches in analysis time scales. Here, we overcome this limitation by using a dual-gated ion injection approach to couple an 11 m path length structures for lossless ion manipulations (SLIM) module to a Q-Exactive Plus Orbitrap MS platform. The dual-gate setup was implemented by placing one ion gate before the SLIM module and a second ion gate after the module. The dual-gated ion injection approach allowed the new SLIM-Orbitrap platform to simultaneously perform an 11 m SLIM separation, Orbitrap mass analysis using the highest selectable mass resolution setting (up to 140 k), and high-energy collision-induced dissociation (HCD) in ∼25 min over an m/z range of ∼1500 amu. The SLIM-Orbitrap platform was initially characterized using a mixture of standard phosphazene cations and demonstrated an average SLIM CCS resolving power (RpCCS) of ∼218 and an SLIM peak capacity of ∼156, while simultaneously obtaining high mass resolutions. SLIM-Orbitrap analysis with fragmentation was then performed on mixtures of standard peptides and two reverse peptides (SDGRG1+, GRGDS1+, and RpCCS = 305) to demonstrate the utility of combined HR-IMS-MS/MS measurements for peptide identification. Our new HR-IMS-MS/MS capability was further demonstrated by analyzing a complex lipid mixture and showcasing SLIM separations on isobaric lipids. This new SLIM-Orbitrap platform demonstrates a critical new capability for proteomics and lipidomics applications, and the high-resolution multimodal data obtained using this system establish the foundation for reference-free identification of unknown ion structures.

From bottle to bedside: Implementation considerations and antimicrobial stewardship considerations for bloodstream infection rapid diagnostic testing.

Antimicrobial stewardship (AMS) programs have been quick to adopt novel molecular rapid diagnostic technologies (mRDTs) for bloodstream infections (BSIs) to improve antimicrobial management. As such, most of the literature demonstrating the clinical and economic benefits of mRDTs for BSI is in the presence of active AMS intervention. Leveraging mRDTs to improve antimicrobial therapy for BSI is increasingly integral to AMS program activities. This narrative review discusses available and future mRDTs, the relationship between the clinical microbiology laboratory and AMS programs, and practical considerations for optimizing the use of these tools within a health system. Antimicrobial stewardship programs must work closely with their clinical microbiology laboratories to ensure that mRDTs are used to their fullest benefit while remaining cognizant of their limitations. As more mRDT instruments and panels become available and AMS programs continue to expand, future efforts must consider the expansion beyond traditional settings of large academic medical centers and how combinations of tools can further improve patient care.

Metatranscriptomics analysis reveals a novel transcriptional and translational landscape during Middle East respiratory syndrome coronavirus infection.

Among all RNA viruses, coronavirus RNA transcription is the most complex and involves a process termed "discontinuous transcription" that results in the production of a set of 3'-nested, co-terminal genomic and subgenomic RNAs during infection. While the expression of the classic canonical set of subgenomic RNAs depends on the recognition of a 6- to 7-nt transcription regulatory core sequence (TRS), here, we use deep sequence and metagenomics analysis strategies and show that the coronavirus transcriptome is even more vast and more complex than previously appreciated and involves the production of leader-containing transcripts that have canonical and noncanonical leader-body junctions. Moreover, by ribosome protection and proteomics analyses, we show that both positive- and negative-sense transcripts are translationally active. The data support the hypothesis that the coronavirus proteome is much vaster than previously noted in the literature.

Large Individual Ion FTICR Measurements from the Mid-1990s Using Reactions for Charge Determination Mass Spectrometry.

This article discusses from a personal and present-day perspective the first studies of large highly charged individual molecular ions that were conducted using electrospray ionization with Fourier transform ion cyclotron resonance MS in the mid-1990s. These studies are distinguished from Current Charge Detection Mass Spectrometry (CDMS) primarily by their use of individual ion charge state changes due to reactions for accurate charge determination. This work describes the key differences in technologies and methods with present CDMS and the likely implications of these differences. I comment on surprising individual ion behavior observed in some measurements involving increases in charge state, as well as their possible basis, and also briefly discuss the potential utility of the reaction-based mass measurement approach used in the context of what might more globally be referred to as "Charge Determination Mass Spectrometry".

Dimeric 2-aminoimidazoles are highly active adjuvants for gram-positive selective antibiotics against Acinetobacter baumannii.

The Centers for Disease Control and Prevention (CDC) reports that hospital acquired infections have increased by 65% since 2019. One of the main contributors is the gram-negative bacterium Acinetobacter baumannii. Previously, we reported aryl 2-aminoimidazole (2-AI) adjuvants that potentiate macrolide antibiotics against A. baumannii. Macrolide antibiotics are typically used to treat infections caused by gram-positive bacteria, but are ineffective against most gram-negative bacteria. We describe a new class of dimeric 2-AIs that are highly active macrolide adjuvants, with lead compounds lowering minimum inhibitory concentrations (MICs) to or below the gram-positive breakpoint level against A. baumannii. The parent dimer lowers the clarithromycin (CLR) MIC against A. baumannii 5075 from 32 µg/mL to 1 µg/mL at 7.5 µM (3.4 µg/mL), and a subsequent structure activity relationship (SAR) study identified several compounds with increased activity. The lead compound lowers the CLR MIC to 2 µg/mL at 1.5 µM (0.72 µg/mL), far exceeding the activity of both the parent dimer and the previous lead aryl 2-AI. Furthermore, these dimeric 2-AIs exhibit considerably reduced mammalian cell toxicity compared to aryl-2AI adjuvants, with IC50s of the two lead compounds against HepG2 cells of >200 µg/mL, giving therapeutic indices of >250.