Triple-negative breast cancer (TNBC) represents a significant therapeutic challenge owing to the scarcity of targeted medicines and elevated recurrence rates. We previously reported the development of the nuclease-resistant RNA sTN58 aptamer, which selectively targets TNBC cells. Here, sTN58 aptamer was employed to capture and purify its binding target from the membrane protein fraction of cisplatin-resistant mesenchymal stem-like TNBC cells. Mass spectrometry in conjunction with aptamer binding assays across various cancer cell lines identified CD44 as the cellular target of sTN58. By binding to CD44, sTN58 inhibits the invasive growth and hyaluronic acid-dependent tube formation in chemoresistant TNBC cells, where CD44 serves as a key driver of tumor cell aggressiveness and stem-like plasticity. Moreover, in vivo studies demonstrated the aptamer's high tumor targeting efficacy and its capacity to significantly inhibit tumor growth and lung metastases following intravenous administration in mice with orthotopic TNBC. Overall, our findings reveal the striking potential of sTN58 as a targeting reagent for the recognition and therapy of cancers overexpressing CD44.

The kinetic properties of four columns packed with fully porous particles and three with superficially porous particles were characterized for possible application in proteomic bottom-up analyses. All columns provided an attachment of hydrophobic C18 chains at the surface of the stationary phase. However, they differed in the additional attachment of polar groups and/or endcapping procedure. We have used the retention modeling protocol to explore the separation efficiency and maximal achievable peak capacity on tested columns. Almost all columns provided comparable maximal peak capacity in the range of 500 - 700 for the eight-hour gradient run. This confirms that the family of the stationary phases used in the bottom-up proteomics can be extended. In the case of fully porous particles, we found that the higher the column peak capacity, the higher the number of identified peptides in the simple proteomic sample, with approximately one identified peptide per peak capacity unit. On the contrary, in the case of the superficially porous particles, the number of identified peptides in the sample decreased with the higher column peak capacity. This trend can be overturned only when the lower amount of the sample is injected. Hence, when bottom-up proteomics is considered, the lower loadability of the superficially porous particles still needs to be addressed. Most stationary phases tested can be successfully used in the bottom-up analyses. However, the stationary phases with incorporated polar functional groups reduced the undesirable contribution of free silanol groups to peptide peak tailing and increased the information provided by LC-MS analysis.

The mammalian suprachiasmatic nucleus (SCN) orchestrates daily (circadian) rhythms of physiology and behavior by broadcasting timing cues generated autonomously by its mutually reinforcing network of ~10,000 neurons and ~3000 astrocytes. Although astrocytic control of extracellular glutamate and GABA has been implicated in driving circadian oscillations in SCN gene expression and neuronal activity, the full scale of the network-level signaling mechanisms is unknown. To understand better how this astrocyte-neuron network operates, we adopted a multi-omics approach, first using SILAC-based mass spectrometry to generate an SCN proteome where ~7% of identified proteins were circadian. This circadian proteome was analyzed bioinformatically alongside existing single-cell RNAseq transcriptomic data to identify the cell-types and processes to which they contribute. This highlighted "S100 protein binding," tracked to astrocytes, and revealed annexin-A2 (Anxa2) as an astrocyte-enriched circadian protein for further investigation. We show that Anxa2 and its partner S100a10 are co-expressed and enriched in SCN astrocytes. We also show that pharmacological disruption of their association acutely and reversibly dysregulated the circadian cycle of astrocytic calcium levels and progressively compromised SCN neuronal oscillations. Anxa2 and S100a10 interaction therefore constitutes an astrocytic cellular signaling axis that regulates circadian neuronal excitability and ultimately SCN network coherence necessary for circadian timekeeping.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide, driven by elevated intraocular pressure (IOP) due to trabecular meshwork (TM) fibrosis, extracellular matrix (ECM) accumulation, and increased aqueous humor outflow resistance. Transforming growth factor-beta 2 (TGF-β2) promotes the expression of fibrosis-related genes, exacerbating these effects. Salidroside, a bioactive compound, has been shown to inhibit TGF-β2-induced ECM expression and alleviate ocular hypertension. However, its underlying molecular mechanisms remain unclear. This study explores the transcriptional, proteomic, and metabolic changes in human TM cells treated with TGF-β2 and salidroside. Human TM cells were treated with TGF-β2 (5 ng/mL) for 48 h, followed by salidroside (30 μM) for 24 h. Multi-omics analyses, including transcriptomics, label-free proteomics, and non-targeted metabolomics, were performed to identify differentially expressed genes (DEGs), proteins (DEPs), and metabolites. The results revealed that TGF-β2 inhibited HTM cell metabolism, affecting pathways like the TCA cycle. Salidroside restores balance by regulating 15 key biomolecules, including MELTF and SLC25A10, through dual-level and post-translation mechanisms. ROC and docking analyses highlight salidroside's role in enhancing metabolic transport and energy activity, with SLC25A10 also linked to RNA processing, showcasing its therapeutic potential. These findings provide valuable insights into POAG pathogenesis and the therapeutic potential of salidroside, offering a foundation for the future development of novel treatment strategies targeting transcriptional, translational, and metabolic dysregulation in POAG.

Chronic ionizing radiation causes elevated levels of DNA damage and reactive oxygen species in plants. Aquatic ecosystems in Chornobyl zone, a major radiological disaster site, are contaminated by harmful radionuclides. We focused on explaining the biochemical mechanisms responsible for the susceptibility of a wild aquatic plant (common reed, Phragmites australis) grown in Chornobyl zone to biotic stress. The fungal infection assay indicated that life in a radionuclide-contaminated environment might compromise plant immunity. Proteome profiling identified 1,867 proteins and we selected several dozen proteins with consistently higher and lower abundance in the samples from the littoral of contaminated lakes by hierarchical clustering. Discordant expression of coding genes pointed to posttranscriptional regulation. Proteins that accumulated in reed upon chronic irradiation suggested a biochemically stable phenotype with effective protection against reactive carbonyls. Simultaneously, proteins that depleted in plants collected from the littoral of radiologically contaminated lakes indicated worse stress resilience and enhanced susceptibility to biotic agents. Furthermore, the quantification of antioxidant enzyme activities and carbonylated proteins rebutted the idea about substantial oxidative stress in chronically irradiated plants. We advocate the necessity to consider increased pathogen sensitivity while developing policies for the management of radionuclide-contaminated areas.

Mass spectrometry (MS) based proteomics provides unbiased quantification of all proteins in plasma, which can dynamically reflect individual health states in real time. However, large-scale proteomics studies are constrained by the excessive dynamic range of plasma proteome and low throughput. Herein, two kinds of magnetic metal-organic frameworks (MOFs) modified with ion exchange functional groups (denoted as MHP-UiO-66-SAX and MHP-HKUST-1-SCX) were designed and fabricated to exhibit large protein adsorption capability, which were combined with an automated Liquid-handling System, thus realizing in-depth, high-throughput and automated proteomics studies. The constructed workflow could automatically complete the sample preparation before MS within only six hours and nearly a thousand protein groups per sample could be quantified. In the cohort study of nearly one hundred breast cancer neoadjuvant chemotherapy (NC) plasma samples, two differentially expressed proteins previously reported as biomarkers were related with the pathological complete response (PCR) of the breast cancer, demonstrating the feasibility of the developed technology for preparing large-scale clinical samples and exhibiting the potential application in monitoring the effect of chemotherapy.

Atherosclerosis, one of the main causes of cardiovascular disease, is driven by complex interactions between lipid metabolism and immune mechanisms in the vascular system. Regulatory molecules, particularly protein fragments derived from cytokines, chemokines and other immune-related proteins, play a central role in modulating inflammation and immune responses in atherosclerotic plaques.

Recent advances in peptidomics have revealed the dual role of immune system-derived peptides as indicators and effectors of atherosclerotic cardiovascular disease (ASCVD). Certain subsets of immune cells, such as pro-inflammatory monocytes and regulatory T cells, contribute to this peptide-mediated regulation. New findings suggest that these peptides may serve as diagnostic biomarkers and therapeutic targets in atherosclerosis.

This review highlights the translational relevance of immune-mediated peptides in ASCVD and emphasizes their diagnostic and therapeutic potential. By integrating peptidomics with immunology research, a new framework for understanding and targeting inflammation in atherosclerosis is proposed, opening new avenues for precision medicine in cardiovascular care.

Therapeutic options for aggressive cancer types such as breast and lung remain limited; disease relapse and death occur in 30-60% of non-small cell lung cancer (NSCLC) patients, whereas in triple-negative breast cancer or TNBC, recurrence-free survival occurs in less than 30% patients. The kinases, MNK and p70S6K have been proposed as targets for the potential treatment of breast cancer (BC) and lung cancer but currently, no drug that was purposely designed to inhibit these kinases have been approved by the FDA for the treatment of BC or NSCLC. In this study, we have identified HSND80 (a morpholino nicotinamide analog of ponatinib) as a potent MNK/p70S6K inhibitor that has excellent activity against TNBC and NSCLC cell lines. HSND80 has a longer target residence time (τ) of 45 mins and 58 mins against MNK1 and MNK2 respectively, compared to τ of eFT508 (tomivosertib) against MNK1 and MNK2 (τ = 1 min and 5 min, respectively). Molecular dynamics simulation was used to provide some insights into the binding of HSND80 to MNK and p70S6K kinases. Western blotting analysis and phosphoproteomics analysis of the TNBC cell line, MDA-MB-231, revealed that phosphorylations of elF4E (MNK target) and elF4B and S6 (p70S6K targets) were reduced upon compound treatment, which is in line with the proposed mechanism of action; dual MNK/p70S6K targeting. HSND80 could be dosed orally at 15 and 30 mg/kg and at such doses, could reduce tumor volume in a syngeneic NSCLC mouse model.

A positive association between antithrombin activity and selenium level has been reported. Selenoprotein P, the most important selenium carrier, was identified within human plasma fibrin clots.

To investigate the relationship between selenoprotein P and antithrombin and its role in modulation of fibrin clot properties in antithrombin-deficient patients.

Proteomic analysis of plasma fibrin clots was performed with mass spectrometry. In 108 patients with genetically confirmed type I (57%) or type II (43%) antithrombin deficiency and in healthy controls (n = 50), we assessed plasma selenoprotein P levels and thiobarbituric acid-reactive substances by enzyme-linked immunosorbent assay, along with fibrin clot permeability, clot lysis time, and thrombin generation.

Clot-bound antithrombin concentration was 0.46 ± 0.32 mg/g protein, while selenoprotein P level was 30-fold lower (0.015 ± 0.012 mg/g). Type I compared to type II antithrombin-deficient patients had higher clot-bound antithrombin and selenoprotein P levels (both P < .001), associated together (ρ = 0.93, P < .001). Individuals with type I compared to type II antithrombin deficiency or controls had about 40% lower plasma selenoprotein P levels (P < .001). In antithrombin-deficient patients, plasma selenoprotein P was associated with antithrombin antigen (ρ = 0.35, P < .001) and thiobarbituric acid-reactive substances (ρ = 0.42, P < .001). Plasma selenoprotein P also correlated with endogenous thrombin potential (r = -0.33, P < .001), fibrin clot permeability (r = 0.43, P < .001), and clot lysis time (r = -0.40, P < .001) in antithrombin-deficient patients but not in controls.

Patients with type I antithrombin deficiency had higher clot-bound selenoprotein P and reduced plasma selenoprotein P levels. Plasma selenoprotein P was associated with prothrombotic fibrin clot phenotype and enhanced thrombin generation.

Chamuangone, a natural compound extracted from Garcinia cowa leaves, has demonstrated potential in cancer therapeutics, but its effects on lung cancer cells remain unclear. This study investigates the apoptotic effects of Chamuangone on human lung adenocarcinoma cells (A549). The A549 cells were treated with Chamuangone, and the cytotoxic effects were evaluated using an MTT assay, revealing a dose-dependent inhibition of cell proliferation with an IC50 value of 19.43 μM. Annexin V assays further confirmed that Chamuangone induces apoptosis in A549 cells, showing increased levels of late apoptosis with higher concentrations. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) microscopy provided insights into macromolecular changes, highlighting significant alterations in proteins, lipids, and nucleic acids. These structural changes in key cellular macromolecules were supported by proteomic analysis, which identified the upregulation of apoptosis-related proteins, including Peroxiredoxin-2 and Na+/H+ exchange regulatory cofactor NHE-RF1. Canonical pathway analysis indicated that Chamuangone affects oxidative phosphorylation and mitochondrial dysfunction, both crucial pathways for apoptosis. Additionally, upstream regulator analysis demonstrated significant inhibition of the epidermal growth factor receptor (EGFR), a key player in lung cancer progression. These findings suggest that Chamuangone triggers apoptosis through mitochondrial pathways and EGFR inhibition, positioning it as a promising therapeutic candidate for lung cancer treatment.

Environmental stressors in the modern world can fundamentally affect human physiology and health. Exposure to stressors like air pollution, heat, and traffic noise has been linked to a pronounced increase in non-communicable diseases. Specifically, aircraft noise has been identified as a risk factor for cardiovascular and metabolic diseases, such as arteriosclerosis, heart failure, stroke, and diabetes. Noise stress leads to neuronal activation with subsequent stress hormone release that ultimately activates the renin-angiotensin-aldosterone system, increases inflammation and oxidative stress thus substantially affecting the cardiovascular system. However, despite the epidemiological evidence of a link between noise stress and metabolic dysfunction, the consequences of exposure at the molecular, metabolic level of the cardiovascular system are largely unknown. Here, we use a murine model system of short-term aircraft noise exposure to show that noise stress profoundly alters heart metabolism. Within 4 days of noise exposure, the heart proteome and metabolome bear the hallmarks of reduced potential for generating ATP from fatty-acid beta-oxidation, the tricarboxylic acid cycle, and the electron transport chain. This is accompanied by the increased expression of glycolytic metabolites, including the end-product, lactate, suggesting a compensatory shift of energy production towards anaerobic glycolysis. Intriguingly, the metabolic shift is reminiscent of what is observed in failing and ischaemic hearts. Mechanistically, we further show that the metabolic rewiring is likely driven by reactive oxygen species (ROS), as we can rescue the phenotype by knocking out NOX-2/gp91phox, a ROS inducer, in mice. Our results suggest that within a short exposure time, the cardiovascular system undergoes a fundamental metabolic shift that bears the hallmarks of cardiovascular disease. These findings underscore the urgent need to comprehend the molecular consequences of environmental stressors, paving the way for targeted interventions to mitigate health risks associated with chronic noise exposure in modern, environments heavily disturbed by noise pollution.

Biomolecules preserved in dental pulp are increasingly being used to identify individuals in the context of forensics and archaeology. Despite the vast amount of research into host and pathogen DNA, the potential use of physiologically informative proteins preserved in dental pulp has rarely been studied. Here, we hypothesized that pregnancy-specific proteins circulating in the blood could be identified from the dental pulp of postpartum individuals and this was investigated using eight human third molars extracted from four postpartum and three control individuals during clinical treatment. A total of 885 proteins were identified from these eight dental pulp samples using liquid chromatography coupled tandem mass spectrometry, whose gene ontology compositions were similar to previous studies. However, despite our hypothesis, pregnancy-specific proteins were not identified from the dental pulp of postpartum individuals (n = 5, 4-12 months postpartum). Although the dental pulp proteomes obtained from three individuals postpartum ≤6 months were distinct from those of other individuals by principal component analysis (PCA), their driving proteins were less evident. Although our hypothesis was not supported, sample collection, protein extraction, and mass spectrometry analysis could be improved to explore the forensic application of detecting pregnancy-specific proteins in dental pulp.

High royal jelly production is an adaptive reproductive investment syndrome in honey bees that enhances their nursing ability to queen bee larvae. However, the biological basis of this reproduction investment at the multi-organ level remains elusive. In this study, proteome across 11 organs of two bee stocks: high royal jelly production bees (RJBs) and Italian bees (ITBs) was compared. Our analysis revealed significant differences in protein expression profiles in brain, fat body, mandibular gland, and Malpighian tubule, highlighting their crucial roles in regulating royal jelly secretion in RJBs. The increased energy turnover, protein synthesis, and lipid synthesis observed in RJBs compared to ITBs highlight their enhanced metabolic activity, which is essential for the robust secretion of royal jelly in RJBs. The elevated abundance of major royal jelly proteins (MRJPs), hexamerins, and vitellogenin suggests their critical contributions to the nutritional and material requirement necessary for royal jelly secretion. Furthermore, the high level of vitellogenin and juvenile hormone esterase may suppress juvenile hormones, which contribute to a strong royal jelly secretion and sensitivity of RJBs to larval pheromones relative to ITBs. This comprehensive dataset contributes to a better understanding of nursing behavior and reproductive investment in honey bees. Significiance. The royal jelly secretion syndrome is a colony level social trait dominated by the intricate interplay of multiple organs. However, previous studies have primarily focused on individual organs. In this study, the proteome of 11 organs was compared between high royal jelly production bees (RJBs) and Italian bees (ITBs) to provide knowledge on how multiple organs cooperate to boost the elevated royal jelly production by RJBs. Nutrition supply was sufficient at multiple organs of RJBs when compared to ITBs, indicating that nutrition plays an essential role in boosting energy metabolism, protein and lipid synthesis, and directly contributes to the amount of royal jelly secretion. The high level of secretion of storage proteins, such as MRJPs, hex, and vitellogenin, provides sufficient nutrition and material for royal jelly secretion. Moreover, the higher levels of vitellogenin and juvenile hormone esterase may suppress juvenile hormone synthesis, and contributing to stronger sense of RJBs to larval pheromone relative to ITBs. This suggests that nutrition can influence the hormone levels and sensory abilities of RJBs nurse bees to promote their royal jelly secretion ability. The reported data provide insights into the systematic regulation strategy of honeybee nursing behavior and reproductive investment.

The sting of the scorpion Centruroides bicolor causes a large morbidity in Panama. To characterize its venom, transcriptomic and proteomic analyses of the venom glands and the crude venom were performed. These two approaches utilized high-throughput sequencing to enhance the likelihood of detecting a wide range of venom proteins correlated with the venom proteome. After RNA venom gland extraction, a cDNA library was constructed and sequenced by RNA-seq. Also, the crude venom was digested using trypsin and chymotrypsin, and the resulting peptides were analyzed using a nano-LC-MS/MS. Notably, transcriptomic and proteomic venom approaches identified a hyaluronidase, alpha- and beta-neurotoxins that affect Na+ channels, CRISP proteins, metalloproteinases, transferrin, monooxygenase alpha-peptidyl-glycine, serine proteases, alpha pancreatic amylase, lysozyme, neurotoxins targeting K+ channels, neprilysin, scorpine, angiotensin-converting enzyme, insulin-like growth factor-binding domain proteins, nucleobindin-like proteins, and uncharacterized proteins. Interestingly, some of the venom proteins such as nucleobindin and angiotensin-converting enzymes have been not reported in the proteome, their predicted presence has only been previously derived from the genomic sequence of Centruroides sculpturatus and C. vittatus. These newly identified components enhance the understanding of the venomous nature of C. bicolor. SIGNIFICANCE: The proteins and peptides found in Centruroides bicolor venom by transcriptomic and proteomic analyses were assessed according to the protein and toxin databases available on public domains. Notably, some of the venom proteins such as nucleobindin and angiotensin-converting enzymes have been not reported in the proteome, their predicted presence has only been previously derived from the genomic sequence of Centruroides sculpturatus and C. vittatus. Moreover, enzymatic assays, including hyaluronidase, phospholipase A2, and proteolytic activity were conducted to confirm the presence or absence of those enzymes. Interestingly, neurotoxins from C. limbatus, a related species in the region, were found in the proteome but no mRNAs were identified in the transcriptome. These newly identified components enhance the understanding of the venomous nature of Centruroides bicolor.

Palaeoproteomics leverages the persistence, diversity, and biological import of ancient proteins to explore the past, and answer fundamental questions about phylogeny, environment, diet, and disease. These insights are largely gleaned from hard tissues like bone and teeth, as well-established protocols exist for extracting ancient proteins from mineralised tissues. No such method, however, exists for the soft tissues, which are underexplored in palaeoproteomics given permission for destructive analysis routinely depends on a proven methodology. Considering less than one-tenth of all human proteins are expressed in bone, compared to three-quarters in the internal organs, the amount of biological information presently inaccessible is substantial. We address this omission with an optimised LC-FAIMS-MS/MS workflow yielding the largest, most diverse palaeoproteome yet described. Using archaeological human brains, we test ten protocols with varied chemistries and find that urea lysis effectively disrupts preserved membrane regions to expose low-abundant, intracellular analytes. Further, we show that ion mobility spectrometry improves unique protein identification by as much as 40%, and represents a means of "cleaning" dirty archaeological samples. Our methodology will be useful for improving protein recovery from a range of ancient tissues and depositional environments.

Sprint interval training (SIT) is a time-efficient type of endurance training that involves large type 2 muscle fibre recruitment. Effective antioxidant supplementation may mitigate positive training adaptations by limiting the oxidant challenge. Our aim was to test whether SIT affects type 2 more than type 1 muscle fibres, and whether the muscular training response is mitigated by antioxidant treatment. Young men performed three weekly SIT sessions (4-6 × 30 s all-out cycling) for 3 weeks while treated with antioxidants (vitamin C, 1 g day-1; vitamin E, 235 mg day-1) or placebo. Vastus lateralis biopsies were taken to measure (i) activation of genes for reactive oxygen/nitrogen species (ROS) sensors and inflammatory mediators with quantitative RT-PCR and (ii) fibre type-specific proteome adaptations using MS-based proteomics. Vitamin treatment decreased the upregulation of genes for ROS sensors and inflammatory regulators during the first SIT session. The 3 weeks of SIT caused generally larger proteome adaptations in type 2 than in type 1 fibres, and this included larger increases in abundance of proteins involved in mitochondrial energy production. Vitamin treatment blunted the SIT-induced proteome adaptations, whereas it did not affect the training-induced improvement in maximal cycling performance. In conclusion, (i) the large type 2 fibre recruitment and resulting proteome adaptations are instrumental to the effectiveness of SIT and (ii) antioxidant supplementation counteracts positive muscular adaptations to SIT, which would blunt any improvement in submaximal endurance performance, whereas it does not affect the improvement in maximal cycling performance, where O2 delivery to muscle would be limiting. KEY POINTS: Sprint interval training (SIT) is a time-efficient type of endurance training that involves large recruitment of fast-twitch muscle fibres. Treatment with antioxidants may mitigate the positive effects of endurance training. Fibre type-specific proteomics performed on muscle biopsies obtained from young men before and after 3 weeks of SIT showed larger training effects in fast- than in slow-twitch fibres. Antioxidant treatment in the form of vitamin C and E pills counteracted the positive muscular adaptations to the 3 weeks of SIT. These results increase our understanding of why SIT is an effective endurance training regime and provide further evidence against the common belief that antioxidant supplements are beneficial in a physical exercise context.

Persistent bacterial airway infection is a hallmark feature of cystic fibrosis (CF). Achromobacter spp. are gram-negative rods that can cause persistent airway infection in people with CF (pwCF), but the knowledge of host immune responses to these bacteria is limited. The aim of this study was to investigate if patients develop antibodies against Achromobacter xylosoxidans, the most common Achromobacter species, and to identify the bacterial antigens that induce specific IgG responses. Seven serum samples from pwCF with Achromobacter infection were screened for antibodies against bacteria in an ELISA coated with A. xylosoxidans, A. insuavis, or Pseudomonas aeruginosa. Sera from pwCF with or without P. aeruginosa infection (n = 22 and 20, respectively) and healthy donors (n = 4) were included for comparison. Serum with high titers to A. xylosoxidans was selected for affinity purification of bacterial antigens using serum IgGs bound to protein G beads. The resulting IgG-antigen complexes were then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Selected antigens of interest were produced in recombinant form and used in an ELISA to confirm the results. Four of the seven patients with Achromobacter infection had serum antibodies against Achromobacter. Using patient serum-IgG for affinity purification of A. xylosoxidans proteins, we identified eight antigens. Three of these, which were not targeted by anti-P. aeruginosa antibodies, were expressed recombinantly for further validation: dihydrolipoyl dehydrogenase (DLD), type I secretion C-terminal target domain-containing protein, and domain of uncharacterized function 336 (DUF336). While specific IgG against all three recombinant antigens was confirmed in the patient serum with high titers against Achromobacter, DLD and DUF336 showed the least binding to serum IgG from pwCF without Achromobacter spp. infection. Using serum IgG affinity purification in combination with LC-MS/MS and confirming the results using ELISA against recombinant proteins, we have identified bacterial antigens from A. xylosoxidans.IMPORTANCEAchromobacter species are opportunistic pathogens that can cause airway infections in people with cystic fibrosis. In this patient population, persistent Achromobacter infection is associated with low lung function, but the knowledge about bacterial interactions with the host is currently limited. In this study, we identify protein antigens that induce specific antibody responses in the host. The identified antigens may potentially be useful in serological assays, serving as a complement to culturing methods for the diagnosis and surveillance of Achromobacter infection.

Diminished bioavailability of nitric oxide (NO) contributes to the pathogenesis of cardiometabolic disorders. However, the alterations in signaling under NO deficiency remain mostly unknown. We combined genetics and proteomics to quantify changes in the heart proteome, phosphoproteome, and S-nitrosocysteine proteome in mice lacking nitric oxide synthases (NOS1, NOS2, NOS3), lacking all three enzymes (tNOS), or the alpha 1-regulatory subunit of the soluble guanylate cyclase (sGCα1). Modest changes of less than 1% in the proteome and 4% in the phosphoproteome in single NOS gene or sGCα1 null mouse hearts indicate sufficient biological compensation. In contrast, the number of S-nitrosylated proteins declined by 80%, 57%, and 35% in NOS3, NOS1, and NOS2 null mice, respectively. A 21% remodeling of the proteome and 9% of the phosphoproteome in the tNOS null mice included integral kinases that provide adaptive rewiring of signaling. The data revealed the emergence of enhanced mitogen-activated-kinases Mapk3/Mapk1 signaling, documented by increased phosphorylation of these kinases and their downstream targets. The data highlight that adaptive compensation of signaling prevents overt phenotypes during NO signaling deficits. In contrast, maladaptive signaling via Mapk3/Mapk1 may promote pathological cardiomyopathy that progressively develops in the tNOS null mice.

Microphysiological systems, such as multicellular spheroids, hold great promise for drug screening experiments. Spheroids may be dosed statically, where the drug is introduced to the growing chamber at one time point, or dynamically, where the drug is introduced via a fluidic component. Dynamic dosing can generate pharmacokinetic curves that more closely represent those seen in vivo than static dosing. In this work, we demonstrate the dynamic dosing of colorectal cancer spheroids in a 3D printed fluidic device with liposomal doxorubicin. Spheroids are valuable models to evaluate dynamic dosing, as they recapitulate the nutrient, oxygen, and pH gradients of solid tumors. Spheroids feature distinct cellular populations with a necrotic core, quiescent middle layer, and proliferative outer layer. Drug and liposome penetration are tracked with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and fluorescence imaging, showing that liposomal doxorubicin is stable to fluidic dosing and penetrates spheroids after 48 h. To provide a comprehensive pharmacodynamic profile of the distinct cellular regions within spheroids, we employ spatially stable isotopic labeling by amino acids in cell culture (spatial SILAC) proteomics to isotopically label the core and outer layers. Proteomic analysis reveals 714 upregulated proteins in the core upon treatment and 30 in the outer layers, as well as 103 downregulated proteins in the core and 1276 in the outer layers. Spatial SILAC uncovers the differential regulation of proteins associated with glycolysis, the TCA cycle, and lipid synthesis upon drug treatment between the spheroid core and outer layers. Using MALDI MSI and spatial SILAC proteomics, we interrogate the effects of dynamic dosing with liposomal doxorubicin on spheroid regions that would be overlooked by bulk analysis.

Human blood contains proteins secreted by various organs, but there is no consensus on whether serum or plasma is preferable for proteome studies. Mass spectrometry employing data-independent acquisition has emerged as a transformative methodology in proteomics, enabling reproducible large-scale quantification of proteomes during one LC-MS/MS analytical run and facilitating identification of potential markers and elucidation of biological processes. Here, we profiled the proteome data of ten paired plasma and serum samples in the initial sample set. Functional analysis revealed similarities and differences in biological functions and the preference for different organs between serum and plasma. Furthermore, comparative proteomic analysis highlighted the different proteomic characteristics. Plasma-overrepresented pathways were related to the phagosome and immune, while serum-overrepresented pathways were associated with amino acid metabolism, which were further validated by the follow-up sample set composed of eight paired plasma and serum samples. We have detected potential markers in plasma and serum for various cancers and explored their association with prognosis using data from the TCGA pan-cancer cohort and HPA database. Further assessment is required to validate the reproducibility of the quantification for these markers. Overall, this study highlights the commonality and specificity of plasma and serum at the molecular level, underscoring their respective utility in biological exploration and clinical applications.

Phenylbutazone (PBZ) can potentially induce gastrointestinal ulceration, and early detection of PBZ-induced gastroenteropathy will be useful for the diagnosis, treatment, and prevention of PBZ toxicity.

To identify putative proteins associated with equine gastric ulcer syndrome after clinical dose (4.4 mg/kg) administration of PBZ by proteomic study.

In vivo experiments.

Proteomic analysis using LC-MS/MS compared protein expression in serum and faeces of seven PBZ-treated horses with seven placebo-treated controls, and a novel putative biomarker was validated via enzyme-linked immunosorbent assay.

Differentially expressed proteins (DEPs) analysis on 5298 serum annotated proteins and 3538 faecal annotated proteins using the DESeq2 were performed between the control and treatment of EGUS groups. The results showed a list of 226 and 181 significant proteins in serum and faecal samples, respectively with a p adjust value <0.05. The proteomic serum and faeces samples were integrated into STITCH to illustrate PBZ interaction with bile acid homeostasis. FABP6 was significantly increased in PBZ-treated horses. The serum FABP6 concentration in the treatment group on Day 8 (1.80 ± 0.37 ng/mL) was higher than on Day 0 (1.15 ± 0.33 ng/mL, p = 0.01, 95% CI [-1.07, -0.25]). On Day 8, the serum FABP6 concentration in the treatment group was also higher than the control group (1.20 ± 0.48 ng/mL; p = 0.02, 95% CI [-1.10, -0.11]).

Validation of all expressed proteins is a main limitation.

Administration of PBZ at a clinical dose of 4.4 mg/kg twice daily for 7 days may cause gastric mucosal damage. PBZ treatment increased the expression of SLC10A1 and FABP6, suggesting that early gastric mucosal injury may be linked to the bile acid pathway. Bile acids could potentially exacerbate PBZ-induced EGUS.

Heterologous expression in well-studied model strains is a routinely applied method to investigate biosynthetic pathways. Here, we pursue a comparative approach of large-scale DNA-affinity-capturing assays (DACAs) coupled with semi-quantitative mass spectrometry (MS) to identify putative regulatory proteins from Streptomyces coelicolor M512, which bind to the heterologously expressed biosynthetic gene clusters (BGCs) of the liponucleoside antibiotics caprazamycin and liposidomycin. Both gene clusters share an almost identical genetic arrangement, including the location of promoter regions, as detected by RNA sequencing. A total of 2,214 proteins were trapped at the predicted promoter regions, with only three binding to corresponding promoters in both gene clusters. Among these, the overexpression of a yet uncharacterized TetR-family regulator (TFR), Sco4385, increased caprazamycin but not liposidomycin production. Protein-DNA interaction experiments using biolayer interferometry confirmed the binding of Sco4385 to Pcpz10 and PlpmH at different locations within both promoter regions, which might explain its functional variance. Sequence alignment allowed the determination of a consensus sequence present in both promoter regions, to which Sco4385 was experimentally shown to bind. Furthermore, we found that the overexpression of the Crp regulator, Sco3571, leads to a threefold increase in caprazamycin and liposidomycin production yields, possibly due to an increased expression of a precursor pathway.IMPORTANCEStreptomycetes are well-studied model organisms for the biosynthesis of pharmaceutically, industrially, and biotechnologically valuable metabolites. Their naturally broad repertoire of natural products can be further exploited by heterologous expression of biosynthetic gene clusters (BGCs) in non-native host strains. This approach forces the host to adapt to a new regulatory and metabolic environment. In our study, we demonstrate that a host regulator not only interacts with newly incorporated gene clusters but also regulates precursor supply for the produced compounds. We present a comprehensive study of regulatory proteins that interact with two genetically similar gene clusters for the biosynthesis of liponucleoside antibiotics. Thereby, we identified regulators of the heterologous host that influence the production of the corresponding antibiotic. Surprisingly, the regulatory interaction is highly specific for each biosynthetic gene cluster, even though they encode largely structurally similar metabolites.

The increasing prevalence of tick-borne arboviral infections worldwide necessitates advanced control strategies, particularly those targeting vectors, to mitigate the disease burden. However, the cellular interactions between arboviruses and ticks, especially for negative-strand RNA viruses, remain largely unexplored. Here, we employ a proteomics informed by transcriptomics approach to elucidate the cellular response of the Rhipicephalus microplus-derived BME/CTVM6 cell line to severe fever with thrombocytopenia syndrome virus (SFTSV) infection. We generate the de novo transcriptomes and proteomes of SFTSV- and mock-infected tick cells, identifying key host responses and regulatory pathways. Additionally, interactome analysis of the viral nucleoprotein (N) integrated host responses with viral replication and dsRNA-mediated gene silencing screen reveals two anti-SFTSV effectors: the N interacting RNA helicases DHX9 and UPF1. Collectively, our results provide insights into the antiviral responses of R. microplus vector cells and highlight critical SFTSV restriction factors, while enriching transcriptomic and proteomic resources for future research.

In the field of proteomics, generating biologically relevant results from mass spectrometry (MS) signals remains a challenging task. This is partly due to the fact that the computational strategies for converting MS signals into biologically interpretable data depend heavily on the MS acquisition method. Additionally, the processing and the analysis of these data vary depending on whether the proteomic experiment was performed with or without labeling, and with or without fractionation. Several R packages have been developed for processing and analyzing MS data, but they only incorporate identification and quantification data; none of them takes into account other invaluable information collected during MS runs. To address this limitation, we introduce MCQR, an alternative R package for the in-depth exploration, processing, and analysis of quantitative proteomics data generated from either data-dependent or data-independent acquisition methods. MCQR leverages experimental retention time measurements for quality control, data filtering, and processing. Its modular architecture offers flexibility to accommodate various types of proteomics experiments, including label-free, label-based, fractionated, or those enriched for specific post-translational modifications. Its functions, designed as simple building blocks, are user-friendly, making it easy to test parameters and methods, and to construct customized analysis scenarios. These unique features position MCQR as a comprehensive toolbox, perfectly suited to the specific needs of MS-based proteomics experiments.

Oligoadenylate synthetase (OAS) proteins are immune sensors for double-stranded RNA and are critical for restricting viruses. OAS2 comprises two OAS domains, only one of which can synthesize 2'-5'-oligoadenylates for RNase L activation. Existing structures of OAS1 provide a model for enzyme activation, but they do not explain how multiple OAS domains discriminate RNA length. Here, we discover that human OAS2 exists in an auto-inhibited state as a zinc-mediated dimer and present a mechanism for RNA length discrimination: the catalytically deficient domain acts as a molecular ruler that prevents autoreactivity to short RNAs. We demonstrate that dimerization and myristoylation localize OAS2 to Golgi membranes and that this is required for OAS2 activation and the restriction of viruses that exploit the endomembrane system for replication, e.g., coronaviruses. Finally, our results highlight the non-redundant role of OAS proteins and emphasize the clinical relevance of OAS2 by identifying a patient with a loss-of-function mutation associated with autoimmune disease.

The timing of flowering is a critical agronomic trait governed by an extensive and sophisticated regulatory network. To date, limited understanding of how posttranslational modifications regulate flowering time exists. Here, using Arabidopsis, we resolve a role for the B4 Raf-like MAPKKK protein kinase RAF24 in regulating flowering time. Loss of RAPIDLY ACCELERATED FIBROSARCOMA24 (RAF24) led to premature flowering time through altered expression of FLC and FT. Comparative phosphoproteomic analysis of raf24 and wild-type plants revealed a list of known flowering-related phosphoproteins from distinct flowering pathways displaying downregulated phosphorylation. Of these, the RING-type ubiquitin ligase HISTONE MONO-UBIQUITINATION 2 (HUB2) lacked phosphorylation in the absence of RAF24. Absence of RAF24 induced H2Bub1 overaccumulation, with protein-protein interactome analysis of HUB2 in the presence and absence of RAF24 influencing HUB2 protein interaction partners, such as H2B. HUB2 was also found to physically interact with SUCROSE NONFERMENTING KINASE 2.4 (SnRK2.4) and SnRK2.6, known substrates of RAF24. Using phospho-mimetic and phospho-ablative plant lines, we then validated the importance of HUB2 phosphorylation at serine 314 (S314) in maintaining appropriate flowering time. Our findings uncovered a novel biological role of RAF24, as a higher-order flowering regulator, while further implicating HUB2 as a centerpiece of flowering time regulation.

The integration of microbiology and nanotechnology offers a novel strategy for cancer treatment. In this study, we innovatively propose the use of Pseudomonas aeruginosa bacterial membranes as nanocarriers. These membranes possess a simple and unique self-enriching property for iron, which, in addition to the inherent immune effects of the membrane itself, can facilitate tumor chemodynamic therapy through Fenton reactions. The system encapsulates the anticancer drug β-Lapachone, which can generate a large amount of hydrogen peroxide within cells, further serving as a substrate for the Fenton reaction, leading to a cascade reaction that achieves a synergistic effect of three therapeutic modalities in tumor treatment. Moreover, the aptamer AS1411 is used to enhance tumor targeting and optimize drug delivery within the tumor microenvironment. This investigation presents a multimodal antitumor strategy that demonstrates enhanced antitumor effects both in vitro and in vivo, providing a new paradigm for the antitumor application of bacterial membrane nanocarriers.

Trivalent arsenic, i.e., As(III), is the main form of arsenic species in the environment. Prolonged exposure to arsenicals through ingesting contaminated food and water has been implicated in the development of cancer and diabetes as well as cardiovascular and neurodegenerative diseases. A number of studies have been conducted to examine the mechanisms underlying the toxic effects of arsenite exposure, where As(III) was shown to displace Zn(II) and impair the functions of zinc-binding proteins. Considering that many zinc-binding proteins can bind to nucleic acids, we reason that systematic identification of arsenite-binding proteins in the nucleus may provide additional insights into the molecular targets of arsenite, thereby improving our understanding of the mechanisms of arsenic toxicity. Here, we conducted a quantitative proteomics experiment relying on affinity pull-down from nuclear protein lysate with a biotin-As(III) probe to identify nuclear arsenite-binding proteins. We uncovered a number of candidate As(III)-binding proteins that are involved in mRNA splicing, DNA repair, and replication. We also found that As(III) could bind to splicing factor 1 (SF1) and that this binding perturbs mRNA splicing in human cells. Together, our work provided insights into the mechanisms of As(III) toxicity by revealing new nuclear protein targets of As(III).

Cholangiocarcinoma (CCA), a neoplasm arising from biliary epithelial cells, is particularly widespread in Southeast Asia, with northeastern Thailand exhibiting the greatest prevalence attributed to Opisthorchis viverrini infection. This malignancy exhibits considerable molecular heterogeneity, leading to therapeutic resistance and recurrence. Comprehending its molecular mechanisms is essential for enhancing diagnostic and treatment approaches. Our research utilized multi-region LC-MS/MS proteomic analysis to investigate intratumor heterogeneity (ITH) in CCA. We examined 52 tumor areas and 13 neighboring tissues from 13 patients, concentrating on protein profiling, pathway analysis, differential protein expression, and the identification of shared and unique protein signatures. The findings indicated considerable inter-patient proteome variability, characterized by markedly distinct protein expressions among individuals, aligning with prior cancer research. Intra-tumor heterogeneity was apparent, with merely 18 proteins common to all tumor areas and patients, underscoring the intricacy of CCA. Significantly, the common proteins were associated with metabolic reprogramming and oxidative stress pathways, indicating possible indicators and therapeutic targets. This work highlights the significant proteome variability in CCA at both intra-tumor and inter-patient levels, underscoring the necessity for customized therapeutic approaches to tackle the disease's complexity and improve treatment outcomes.

Coronavirus 2019 (COVID-19) leads to substantial morbidity and excess mortality all over the world which may be aggravated by the propensity of Severe Acute Respiratory Syndrome Coronavirus 2 to mutate. Mechanisms for development of severe COVID-19 are poorly understood. The air we exhale contains endogenous proteins and represents a highly accessible yet unexploited biological sample that can be collected without use of invasive procedures. We collected exhaled breath condensate samples from 28 patients hospitalised due to COVID-19 at admission and discharge using RTubes™. Bottom-up proteomic analysis of tandem mass-tag-labelled single exhaled breath samples was performed in 25 exhaled breath samples collected at admission and 13 samples collected at discharge using discovery-based nano-liquid chromatography-tandem mass spectrometry. In total, 232 proteins were identified in the exhaled breath samples after stringent data filtering. Most of the exhaled proteins were related to the immune systems function and regulation. The levels of four proteins, KRT77, DCD, CASP14 and SERPINB12 decreased from admission to discharge as patients generally recovered from the infection. These proteins are expressed in lung epithelium or macrophages and are associated with the regulation of inflammation drivers in COVID-19. In particular, the alarmins S100A8 and S100A9 accounted for 8% of the exhaled breath proteins. In conclusion, our study demonstrates that analysis of the exhaled breath protein composition can give insights into mechanisms related to inflammation and response to infections, and hereby also of severe COVID-19.Clinical Trial No: NCT04598620.

LRP1 is a multifunctional endocytosis receptor involved in the regulation of cancer cell aggressiveness, fibroblast phenotype and angiogenesis. In breast cancer microenvironment, cancer-associated fibroblasts (CAFs) play a crucial role in matrix remodeling and tumor niche composition. LRP1 expression was described in fibroblasts and CAFs but remains poorly understood regarding its impact on endothelial cell behavior and angiocrine signaling. We analyzed the angio-modulatory effect of LRP1 expression in murine embryonic fibroblasts (MEFs) and breast cancer-educated CAF2 cells. We employed conditioned media and fibroblast-derived matrices to model fibroblastic cells angiogenic effects on human umbilical vein endothelial cells (HUVEC). Neither the extracellular matrix assembled by MEFs knock-out for LRP1 (PEA-13) nor their secretome modify the migration of HUVEC as compared to wild-type. Conversely, LRP1-deficient CAF2 secretome and matrices stimulate endothelial cell migration. Using spheroids, we demonstrate that PEA-13 secretome does not affect HUVEC angio-invasion. By contrast, CAF2 secretome invalidated for LRP1 stimulates endothelial sprouting as compared to controls. In addition, it specifically stabilized peripheral VE-cadherin-mediated endothelial cell junctions. A global proteomic analysis revealed that LRP1 expression in CAFs orchestrates a specific mobilization of secreted matricial components, surface receptors and membrane-associated proteins at the endothelial cell surface, thereby illustrating the deep influence exerted by LRP1 in angiogenic signals emitted by activated fibroblasts.

Chronic inflammation plays a central role in the pathogenesis of lung diseases including asthma, long COVID, chronic obstructive pulmonary disease (COPD), and lung cancer. Lipopolysaccharide (LPS) is a potent inflammatory agent produced by Gram-negative bacteria and also found in cigarette smoke. Our earlier study revealed that the intranasal exposure of A/J mice to LPS for 7 days altered gene expression levels in alveolar Type II epithelial cells (AECIIs), which serve as precursors to lung adenocarcinoma and are also preferentially targeted by SARS-CoV-2. In the present work, we employed a comprehensive multi-omics approach to characterize changes in DNA methylation/hydroxymethylation, gene expression, and global protein abundances in the AECIIs of A/J mice following the sub-chronic exposure to LPS and after a 4-week recovery period. Exposure to LPS led to hypermethylation at regulatory elements within the genome such as enhancer regions and expression changes in genes known to play a role in lung cancer tumorigenesis. Changes in protein abundance were consistent with an inflammatory phenotype and also included tumor suppressor proteins. Integration of the multi-omics data resulted in a model where LPS-driven inflammation in AECIIs triggers epigenetic changes that, along with genetic mutations, may contribute to lung cancer development.

Metaproteomics analyzes the functional dynamics of microbial communities by identifying peptides and mapping them to the most likely proteins and taxa. One challenge in this field lies in seamlessly integrating taxonomic and functional annotations to accurately represent the contributions of individual microbial taxa to functional diversity. We introduce MetaX, a comprehensive tool for analyzing taxon-function relationships in metaproteomics by mapping peptides to their lowest common ancestors and assigning functions based on proportional thresholds, ensuring accurate peptide-level mappings. Importantly, MetaX introduces the Operational Taxon-Function (OTF), a new conceptual unit for exploring microbial roles and interactions within ecosystems. Additionally, MetaX includes extensive statistical and visualization tools, establishing it as a robust platform for metaproteomics analysis. We validated MetaX by reanalyzing ex vivo gut microbiome metaproteomic data exposed to various sweeteners, yielding more detailed results than traditional protein analysis. Furthermore, using the peptide-centric approach and OTF, we observed that Parabacteroides distasonis significantly responds to certain sweeteners, highlighting its role in modifying specific metabolic functions. With its intuitive, user-friendly interface, MetaX facilitates a detailed study of the complex interactions between microbial taxa and their functions in metaproteomics. It enhances our understanding of microbial roles in ecosystems and health.

Plant-beneficial fungi play an important role in enhancing plant health and resistance against biotic and abiotic stresses. Although extensive research has focused on their role in eliciting plant defences against pathogens, their contribution to induced resistance against herbivorous insects and the underlying mechanisms remain poorly understood. In this study, we used insect bioassays and untargeted metabolomics to investigate the impact of root inoculation of sweet pepper with the plant-beneficial fungus Trichoderma harzianum T22 on direct defence responses against the insect herbivore Nezara viridula.

We observed reduced relative growth rate of N. viridula on leaves of fungus-inoculated plants, with no change in mortality. Untargeted metabolomic analyses revealed that inoculation with T. harzianum did not affect the leaf metabolome in the absence of herbivory five weeks after inoculation. However, compared to non-inoculated plants, inoculated plants exhibited significant metabolic alterations in herbivore-damaged leaves following N. viridula feeding, while changes in the metabolic profile of distant leaves were less pronounced. Notably, metabolites involved in the shikimate-phenylpropanoid pathway, known to be involved in plant defence responses, displayed higher accumulation in damaged leaves of inoculated plants compared to non-inoculated plants.

Our results indicate that root inoculation with T. harzianum T22 affects plant defences against N. viridula, leading to reduced insect performance. Metabolite-level effects were primarily observed in damaged leaves, suggesting that the priming effect mainly results in localized metabolite accumulation at the site of attack. Future research should focus on identifying the detected compounds and determining their role in impairing N. viridula performance.

The hijacking of CRM1 export is an important step of the retroviral replication cycle. Here, we investigated the consequences of this hijacking for the host. During HTLV-1 infection, we identified that this hijacking by the viral protein Rex favours the association between CRM1 and the RNA helicase UPF1, leading to a decreased affinity of UPF1 for cellular RNA and its nuclear retention. As a consequence, we found that the nonsense-mediated mRNA decay (NMD), known to have an antiviral function, was inhibited. Corroborating these results, we described a similar process with Rev, the functional homolog of Rex from HIV-1. Unexpectedly, we also found that, for HTLV-1, this process is coupled with the specific loading of UPF1 onto vRNA, independently of NMD. In this latter context, UPF1 positively regulates several steps of the viral replication cycle, from the nuclear export of vRNA to the production of mature viral particles.

Because limited oxygen and glucose supply to tissues is a serious challenge that cells must properly measure to decide between surviving or triggering cell death, organisms have developed accurate mechanisms for sensing and signaling these conditions. In recent years, signaling through posttranslational modification of proteins by covalent attachment of the Small Ubiquitin-like Modifier (SUMO) is gaining notoriety. Enhanced sumoylation in response to oxygen and glucose deprivation (OGD) constitutes a safeguard mechanism for cells and a new avenue for therapeutic intervention. However, indiscriminate global sumoylation can limit the therapeutic potential that a more precise action on selected targets would have. To clear up this, we have conducted a proteomic approach in P19 cells to identify specific SUMO targets responding to OGD and to investigate the potential that these targets and their sumoylation have in preserving cells from death. Proteins undergoing sumoylation in response to OGD are mostly related to transcription and RNA processing, and the majority of them are rapidly desumoylated when restoring oxygen and glucose (ROG), confirming the high dynamics of this modification. Since OGD is linked to brain ischemia, we have also studied cells differentiated into neurons. However, no major differences have been observed between the SUMO-proteomes of proliferating and differentiated cells. We show that the overexpression of the transcription factor SOX2 or the SUMO ligase PIAS4 has a manifest cell protective effect largely depending on their sumoylation, and that maintaining the sumoylation capacity of the coregulator NAB2 is also important to face OGD. Conversely, sumoylation of the pluripotency factor OCT4, which is sumoylated under OGD, and is a target of the SUMO protease SENP7 for desumoylation after ROG, seems to block its cell survival-promoting capacity. Thus, better outcomes in cell protection would rely on the appropriate combination of sumoylated and non-sumoylated forms of selected factors.

SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES (SERKs), which are subfamily II of leucine-rich repeat receptor-like kinases (LRR-RLKs), play diverse roles in development and immunity in the angiosperm Arabidopsis thaliana. AtSERKs act as co-receptors for many LRR-RLKs, including BRASSINOSTEROID INSENSITIVE 1 (BRI1) and FLAGELLIN SENSITIVE 2 (FLS2).1,2,3,4 The conserved tyrosine (Y) residue in AtSERK3 is crucial for signaling specificity in differentiating BRI1- and FLS2-mediated pathways.5 BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1)-INTERACTING RECEPTOR-LIKE KINASES (BIRs) interact with SERKs under resting conditions, negatively regulating SERK-mediated pathways.6,7 SERK and BIR are highly conserved in land plants, whereas BRI1 and FLS2 homologs are absent or poorly conserved in bryophyte lineages.8,9 The biological functions of SERK homologs in non-flowering plants are largely unknown. The genome of the liverwort Marchantia polymorpha encodes single homologs for SERK and BIR, namely MpSERK and MpBIR.9 We here show that Mpserk disruptants display growth and developmental defects with no observable sexual or vegetative reproduction. Complementation analysis revealed a contribution of the conserved Y residue of MpSERK to growth. Proximity-labeling-based interactomics identified MpBIR as a MpSERK interactor. Mpbir disruptants displayed defects in reproductive organ development. Patterns of development- and immunity-related gene expression in Mpserk and Mpbir were antagonistic, suggesting that MpBIR functions as an MpSERK repressor. The pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 grew poorly on Mpbir, indicating a significant role of the MpSERK-MpBIR module in immunity. Taken together, we propose that the SERK-BIR functional module was already regulating both development and immunity in the last common ancestor of land plants.