Lipid A Structural Determination from a Single Colony.

We describe an innovative use for the recently reported fast lipid analysis technique (FLAT) that allows for the generation of MALDI tandem mass spectrometry data suitable for lipid A structure analysis directly from a single Gram-negative bacterial colony. We refer to this tandem MS version of FLAT as FLATn. Neither technique requires sophisticated sample preparation beyond the selection of a single bacterial colony, which significantly reduces overall analysis time (∼1 h), as compared to conventional methods. Moreover, the tandem mass spectra generated by FLATn provides comprehensive information on fragments of lipid A, for example, ester bonded acyl chain dissociations, cross-ring cleavages, and glycosidic bond dissociations, all of which allow the facile determination of novel lipid A structures or confirmation of expected structures. In addition to generating tandem mass spectra directly from single colonies, we also show that FLATn can be used to analyze lipid A structures taken directly from a complex biological clinical sample without the need for ex vivo growth. From a urine sample from a patient with an E. coli infection, FLATn identified the organism and demonstrated that this clinical isolate carried the mobile colistin resistance-1 gene (mcr-1) that results in the addition of a phosphoethanolamine moiety and subsequently resistance to the antimicrobial, colistin (polymyxin E). Moreover, FLATn allowed for the determination of the existence of a structural isomer in E. coli lipid A that had either a 1- or 4'-phosphate group modification by phosphoethanolamine generated by a change of bacterial culture conditions.

Detection of Volatile Organic Compounds by Using MEMS Sensors.

We report on the deployment of MEMS static bifurcation (DC) sensors for the detection of volatile organic compounds (VOCs): hydrogen sulfide and formaldehyde. We demonstrate a sensor that can detect as low as a few ppm of hydrogen sulfide. We also demonstrate a sensor array that can selectively detect formaldehyde in the presence of benzene, a closely related interferent. Toward that end, we investigate the sensitivity and selectivity of two detector polymers-polyaniline (PANI) and poly (2,5-dimethyl aniline) (P25DMA)-to both gases. A semiautomatic method is developed to functionalize individual sensors and sensor arrays with the detector polymers. We found that the sensor array can selectively sense 1 ppm of formaldehyde in the presence of benzene.

Partitioning of Seven Different Classes of Antibiotics into LPS Monolayers Supports Three Different Permeation Mechanisms through the Outer Bacterial Membrane.

The outer membrane (OM) of Gram-negative (G-) bacteria presents a barrier for many classes of antibacterial agents. Lipopolysaccharide (LPS), present in the outer leaflet of the OM, is stabilized by divalent cations and is considered to be the major impediment for antibacterial agent permeation. However, the actual affinities of major antibiotic classes toward LPS have not yet been determined. In the present work, we use Langmuir monolayers formed from E. coli Re and Rd types of LPS to record pressure-area isotherms in the presence of antimicrobial agents. Our observations suggest three general types of interactions. First, some antimicrobials demonstrated no measurable interactions with LPS. This lack of interaction in the case of cefsulodin, a third-generation cephalosporin antibiotic, correlates with its low efficacy against G- bacteria. Ampicillin and ciprofloxacin also show no interactions with LPS, but in contrast to cefsulodin, both exhibit good efficacy against G- bacteria, indicating permeation through common porins. Second, we observe substantial intercalation of the more hydrophobic antibiotics, novobiocin, rifampicin, azithromycin, and telithromycin, into relaxed LPS monolayers. These largely repartition back to the subphase with monolayer compression. We find that the hydrophobic area, charge, and dipole all show correlations with both the mole fraction of antibiotic retained in the monolayer at the monolayer-bilayer equivalence pressure and the efficacies of these antibiotics against G- bacteria. Third, amine-rich gentamicin and the cationic antimicrobial peptides polymyxin B and colistin show no hydrophobic insertion but are instead strongly driven into the polar LPS layer by electrostatic interactions in a pressure-independent manner. Their intercalation stably increases the area per molecule (by up to 20%), which indicates massive formation of defects in the LPS layer. These defects support a self-promoted permeation mechanism of these antibiotics through the OM, which explains the high efficacy and specificity of these antimicrobials against G- bacteria.

On-Tissue Derivatization of Lipopolysaccharide for Detection of Lipid A Using MALDI-MSI.

We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host-pathogen interactions. We previously demonstrated detection of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host-pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, Escherichia coli (Ec, m/z 1797) and Pseudomonas aeruginosa (Pa, m/z 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and O-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major Ec and Pa lipid A species (m/z 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host-pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host-pathogen interface.

Differential Interactions of Piscidins with Phospholipids and Lipopolysaccharides at Membrane Interfaces.

Piscidins 1 and 3 (P1 and P3) are potent antimicrobial peptides isolated from striped bass. Their mechanism of action involves formation of amphipathic α-helices on contact with phospholipids and destabilization of the microbial cytoplasmic membrane. The peptides are active against both Gram-positive and Gram-negative bacteria, suggesting easy passage across the outer membrane. Here, we performed a comparative study of these two piscidins at the air-water interface on lipopolysaccharide (LPS) monolayers modeling the outer bacterial surface of Gram-negative organisms and on phospholipid monolayers, which mimic the inner membrane. The results show that P1 and P3 are highly surface active (log KAW ∼ 6.8) and have similar affinities to phospholipid monolayers (log Klip ≈ 7.7). P1, which is more potent against Gram negatives, exhibits a much stronger partitioning into LPS monolayers (log KLPS = 8.3). Pressure-area isotherms indicate that under increasing lateral pressures, inserted P1 repartitions from phospholipid monolayers back to the subphase or to a more shallow position with in-plane areas of ∼170 Å2 per peptide, corresponding to fully folded amphipathic α-helices. In contrast, peptide expulsion from LPS occurs with areas of ∼35 Å2, suggesting that the peptides may not form the similarly oriented, rigid secondary structures when they avidly intercalate between LPS molecules. Patch-clamp experiments on Escherichia coli spheroplasts show that when P1 and P3 reach the outer surface of the bacterial cytoplasmic membrane, they produce fluctuating conductive structures at voltages above 80 mV. The data suggests that the strong activity of these piscidins against Gram-negative bacteria begins with the preferential accumulation of peptides in the outer LPS layer followed by penetration into the periplasm, where they form stable amphipathic α-helices upon contact with phospholipids and attack the energized inner membrane.

Host-based lipid inflammation drives pathogenesis in Francisella infection.

Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2-dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.

The TLR4 antagonist Eritoran protects mice from lethal influenza infection.

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4(-/-) mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)-a potent, well-tolerated, synthetic TLR4 antagonist-blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

In Vivo Intradermal Delivery of Bacteria by Using Microneedle Arrays.

Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida-coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant (F. novicida ΔlpxD1), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models.

Lipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship.

Strategies utilizing Toll-like receptor 4 (TLR4) agonists for treatment of cancer, infectious diseases, and other targets report promising results. Potent TLR4 antagonists are also gaining attention as therapeutic leads. Though some principles for TLR4 modulation by lipid A have been described, a thorough understanding of the structure-activity relationship (SAR) is lacking. Only through a complete definition of lipid A-TLR4 SAR is it possible to predict TLR4 signaling effects of discrete lipid A structures, rendering them more pharmacologically relevant. A limited 'toolbox' of lipid A-modifying enzymes has been defined and is largely composed of enzymes from mesophile human and zoonotic pathogens. Expansion of this 'toolbox' will result from extending the search into lipid A biosynthesis and modification by bacteria living at the extremes. Here, we review the fundamentals of lipid A structure, advances in lipid A uses in TLR4 modulation, and the search for novel lipid A-modifying systems in extremophile bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be relevant for other (+)RNA viruses as well.

LPS remodeling is an evolved survival strategy for bacteria.

Maintenance of membrane function is essential and regulated at the genomic, transcriptional, and translational levels. Bacterial pathogens have a variety of mechanisms to adapt their membrane in response to transmission between environment, vector, and human host. Using a well-characterized model of lipid A diversification (Francisella), we demonstrate temperature-regulated membrane remodeling directed by multiple alleles of the lipid A-modifying N-acyltransferase enzyme, LpxD. Structural analysis of the lipid A at environmental and host temperatures revealed that the LpxD1 enzyme added a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme added a 3-OH C16 acyl group at 18 °C (environment). Mutational analysis of either of the individual Francisella lpxD genes altered outer membrane (OM) permeability, antimicrobial peptide, and antibiotic susceptibility, whereas only the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. Additionally, growth-temperature analysis revealed transcriptional control of the lpxD genes and posttranslational control of the LpxD1 and LpxD2 enzymatic activities. These results suggest a direct mechanism for LPS/lipid A-level modifications resulting in alterations of membrane fluidity, as well as integrity and may represent a general paradigm for bacterial membrane adaptation and virulence-state adaptation.

Grandparent caregiving among rural African Americans in a community in the American South: challenges to health and wellbeing.

INTRODUCTION: An increasing number of grandparents in rural USA are serving as primary caregivers for their grandchildren because of parental incarceration, addiction, joblessness, or illness. Low-income, African American women from the South are overrepresented in this growing population. There is a paucity of research exploring the challenges faced by rural grandparent caregivers, and past studies have not explicitly addressed the potential consequences of rural grandparent caregiving for health. The purpose of this qualitative study was to explore grandparent caregiving among rural, low-income, African American grandmothers in a community in the American South, and to identify challenges to health that arose in that context. McLeroy's social ecological model (SEM) was used to examine these challenges at multiple levels of influence. METHODS: This qualitative interview-based study was conducted in a high-poverty community in rural Georgia. In-depth interviews were conducted with African American grandparent caregivers and key informants from local community-based organizations. A key informant assisted in identifying initial interview participants, and then snowball sampling was used to recruit additional participants. Interview questions were grouped under five domains (intrapersonal, interpersonal, community, organizational, and policy), according to the levels of the SEM. Iterative content analysis of interview transcripts was utilized. Transcripts were coded to identify text segments related to each domain of the SEM, which were grouped together for analysis by domain. Reflexive memo-writing aided in development of themes, and data quality was assessed using Lincoln and Guba's trustworthiness criteria. RESULTS: Rural African American grandparent caregivers faced a range of challenges to health. Direct physical challenges included chronic pain that interfered with sleep and daily functioning, mobility issues exacerbated by child care, and the pressure of managing their own medical conditions as well as their grandchildren's. Financial scarcity added to their vulnerability to poor health outcomes, especially when caregivers would forego purchase of medications or visits to the doctor because of expenses related to their grandchildren. In addition, lack of child care made health appointments and hospitalizations logistically difficult. Emotional strain was common as grandparent caregivers struggled to protect their grandchildren in communities where rates of drug use, HIV, and incarceration were high. Caregivers worried about their mortality and the related consequences for their grandchildren. Chronic stress, which is linked to a number of poor health outcomes, was self-reported by most rural grandparent caregivers. CONCLUSIONS: In this study, the challenges of rural grandparent caregiving among African American women posed multiple threats to health and wellbeing. Further research is needed, in different rural contexts and with different caregiver populations, to more thoroughly examine the health risks of grandparent caregiving. In addition, the development of multi-faceted interventions and programs will be critical to meeting the needs of rural grandparent caregivers. A few models for such programs exist, although resource shortfalls have often limited their impact.

Bulk Free Radical Terpolymerization of Butyl Acrylate, 2-Methylene-1,3-Dioxepane and Vinyl Acetate: Terpolymer Reactivity Ratio Estimation.

This investigation introduces the first estimation of ternary reactivity ratios for a butyl acrylate (BA), 2-methylene-1,3-dioxepane (MDO), and vinyl acetate (VAc) system at 50 °C, with an aim to develop biodegradable pressure-sensitive adhesives (PSAs). In this study, we applied the error-in-variables model (EVM) to estimate reactivity ratios. The ternary reactivity ratios were found to be r12 = 0.417, r21 = 0.071, r13 = 4.459, r31 = 0.198, r23 = 0.260, and r32 = 55.339 (BA/MDO/VAc 1/2/3), contrasting with their binary counterparts, which are significantly different, indicating the critical need for ternary system analysis to accurately model multicomponent polymerization systems. Through the application of a recast Alfrey-Goldfinger model, this investigation predicts the terpolymer's instantaneous and cumulative compositions at various conversion levels, based on the ternary reactivity ratios. These predictions not only provide crucial insights into the incorporation of MDO across different initial feed compositions but also offer estimates of the final terpolymer compositions and distributions, underscoring their potential in designing compostable or degradable polymers.

Sex differences in visceral sensitivity and brain activity in a rat model of comorbid pain: a longitudinal study.

ABSTRACT: Temporomandibular disorder (TMD) and irritable bowel syndrome (IBS) are 2 chronic overlapping pain conditions (COPCs) that present with significant comorbidity. Both conditions are more prevalent in women and are exacerbated by stress. While peripheral mechanisms might contribute to pain hypersensitivity for each individual condition, mechanisms underlying the comorbidity are poorly understood, complicating pain management when multiple conditions are involved. In this study, longitudinal behavioral and functional MRI-based brain changes have been identified in an animal model of TMD-like pain (masseter muscle inflammation followed by stress) that induces de novo IBS-like comorbid visceral pain hypersensitivity in rats. In particular, data indicate that increased activity in the insula and regions of the reward and limbic systems are associated with more pronounced and longer-lasting visceral pain behaviors in female rats, while the faster pain resolution in male rats may be due to increased activity in descending pain inhibitory pathways. These findings suggest the critical role of brain mechanisms in chronic pain conditions and that sex may be a risk factor of developing COPCs.

Characterization of spatial lipidomic signatures in tick-bitten guinea pig skin as a model for host-vector-pathogen interaction profiling.

IMPORTANCE: Here, we demonstrate the adaptability of spatial "omics" methods to identify interphylum processes regulated at the vector-host interface of ticks during a mammalian blood meal. This approach enables a better understanding of complex bipartite or tripartite molecular interactions between hosts, arthropod vectors and transmitted pathogens, and contributes toward the development of spatially aware therapeutic target discovery and description.

Spatial lipidomics reveals biased phospholipid remodeling in acute Pseudomonas lung infection.

Pseudomonas aeruginosa (Pa) is a pathogen causing chronic pulmonary infections in patients with cystic fibrosis (CF). Manipulation of lipids is an important feature of Pa infection and on a tissue-level scale is poorly understood. Using a mouse model of acute Pa pulmonary infection, we explored the whole-lung phospholipid response using mass spectrometry imaging (MSI) and spatial lipidomics. Using a histology-driven analysis, we isolated airways and parenchyma from both mock- and Pa-infected lungs and used systems biology tools to identify enriched metabolic pathways from the differential phospholipid identities. Infection was associated with a set of 26 ions, with 11 unique to parenchyma and 6 unique to airways. Acyl remodeling was differentially enriched in infected parenchyma as the predominant biological function. These functions correlated with markers of polymorphonuclear (PMN) cell influx, a defining feature of the lung response to Pa infection, implicating enzymes active in phospholipid remodeling.

Aß plaques do not protect against HSV-1 infection in a mouse model of familial Alzheimer's disease, and HSV-1 does not induce Aß pathology in a model of late onset Alzheimer's disease.

The possibility that the etiology of late onset Alzheimer's disease is linked to viral infections of the CNS has been actively debated in recent years. According to the antiviral protection hypothesis, viral pathogens trigger aggregation of Aß peptides that are produced as a defense mechanism in response to infection to entrap and neutralize pathogens. To test the causative relationship between viral infection and Aß aggregation, the current study examined whether Aß plaques protect the mouse brain against Herpes Simplex Virus 1 (HSV-1) infection introduced via a physiological route and whether HSV-1 infection triggers formation of Aß plaques in a mouse model of late-onset AD that does not develop Aß pathology spontaneously. In aged 5XFAD mice infected via eye scarification, high density of Aß aggregates did not improve survival time or rate when compared with wild type controls. In 5XFADs, viral replication sites were found in brain areas with a high density of extracellular Aß deposits, however, no association between HSV-1 and Aß aggregates could be found. To test whether HSV-1 triggers Aß aggregation in a mouse model that lacks spontaneous Aß pathology, 13-month-old hAß/APOE4/Trem2*R47H mice were infected with HSV-1 via eye scarification with the McKrae HSV-1 strain, intracranial inoculation with McKrae, intracranial inoculation after priming with LPS for 6 weeks, or intracranial inoculation with high doses of McKrae or 17syn + strains that represent different degrees of neurovirulence. No signs of Aß aggregation were found in any of the experimental groups. Instead, extensive infiltration of peripheral leukocytes was observed during the acute stage of HSV-1 infection, and phagocytic activity of myeloid cells was identified as the primary defense mechanism against HSV-1. The current results argue against a direct causative relationship between HSV-1 infection and Aß pathology.

Review of the Third Conference of the Imaging Mass Spectrometry Society (IMSS 3): Accounts of a Hybrid Virtual and In-Person Meeting and the State and Future of the Field.

The third annual conference of the Imaging Mass Spectrometry Society (IMSS3) was held October 3-6, 2021 in a hybrid format that included virtual and in-person attendance (Colorado Springs, CO). Here, we highlight many of the methods and applications presented, the state of the field, and some insights into the emerging areas in the field of imaging mass spectrometry. We also reflect upon the processes behind planning a hybrid conference and discuss the successes and challenges of the event in retrospect.

Characterization of an engineered mucus microenvironment for in vitro modeling of host-microbe interactions.

The human mucus layer plays a vital role in maintaining health by providing a physical barrier to pathogens. This biological hydrogel also provides the microenvironment for commensal bacteria. Common models used to study host-microbe interactions include gnotobiotic animals or mammalian-microbial co-culture platforms. Many of the current in vitro models lack a sufficient mucus layer to host these interactions. In this study, we engineered a mucus-like hydrogel Consisting of a mixed alginate-mucin (ALG-MUC) hydrogel network by using low concentration calcium chloride (CaCl2) as crosslinker. We demonstrated that the incorporation of ALG-MUC hydrogels into an aqueous two-phase system (ATPS) co-culture platform can support the growth of a mammalian monolayer and pathogenic bacteria. The ALG-MUC hydrogels displayed selective diffusivity against macromolecules and stability with ATPS microbial patterning. Additionally, we showed that the presence of mucin within hydrogels contributed to an increase in antimicrobial resistance in ATPS patterned microbial colonies. By using common laboratory chemicals to generate a mammalian-microbial co-culture system containing a representative mucus microenvironment, this model can be readily adopted by typical life science laboratories to study host-microbe interaction and drug discovery.

Benchmarking tools for detecting longitudinal differential expression in proteomics data allows establishing a robust reproducibility optimization regression approach.

Quantitative proteomics has matured into an established tool and longitudinal proteomics experiments have begun to emerge. However, no effective, simple-to-use differential expression method for longitudinal proteomics data has been released. Typically, such data is noisy, contains missing values, and has only few time points and biological replicates. To address this need, we provide a comprehensive evaluation of several existing differential expression methods for high-throughput longitudinal omics data and introduce a Robust longitudinal Differential Expression (RolDE) approach. The methods are evaluated using over 3000 semi-simulated spike-in proteomics datasets and three large experimental datasets. In the comparisons, RolDE performs overall best; it is most tolerant to missing values, displays good reproducibility and is the top method in ranking the results in a biologically meaningful way. Furthermore, RolDE is suitable for different types of data with typically unknown patterns in longitudinal expression and can be applied by non-experienced users.