Proteomic Analysis of Primary Graft Dysfunction in Porcine Lung Transplantation Reveals Alveolar-Capillary Barrier Changes Underlying the High Particle Flow Rate in Exhaled Breath.

Primary graft dysfunction (PGD) remains a challenge for lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for more detailed monitoring and understanding of the pathophysiology of PGD. The measurement of particle flow rate (PFR) in exhaled breath is a novel tool to monitor and understand the disease at the proteomic level. In total, 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on postoperative day 3. Exhaled breath particles (EBPs) were evaluated by mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Recipients with PGD had significantly higher PFR [686.4 (449.7-8,824.0) particles per minute (ppm)] compared to recipients without PGD [116.6 (79.7-307.4) ppm, p = 0.0005]. Porcine and human EBP proteins recapitulated proteins found in the BAL, demonstrating its utility instead of more invasive techniques. Furthermore, adherens and tight junction proteins were underexpressed in PGD tissue. Histological and proteomic analysis found significant changes to the alveolar-capillary barrier explaining the high PFR in PGD. Exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD.

Proteomic characteristics and diagnostic potential of exhaled breath particles in patients with COVID-19.

BACKGROUND: SARS-CoV-2 has been shown to predominantly infect the airways and the respiratory tract and too often have an unpredictable and different pathologic pattern compared to other respiratory diseases. Current clinical diagnostical tools in pulmonary medicine expose patients to harmful radiation, are too unspecific or even invasive. Proteomic analysis of exhaled breath particles (EBPs) in contrast, are non-invasive, sample directly from the pathological source and presents as a novel explorative and diagnostical tool. METHODS: Patients with PCR-verified COVID-19 infection (COV-POS, n = 20), and patients with respiratory symptoms but with > 2 negative polymerase chain reaction (PCR) tests (COV-NEG, n = 16) and healthy controls (HCO, n = 12) were prospectively recruited. EBPs were collected using a "particles in exhaled air" (PExA 2.0) device. Particle per exhaled volume (PEV) and size distribution profiles were compared. Proteins were analyzed using liquid chromatography-mass spectrometry. A random forest machine learning classification model was then trained and validated on EBP data achieving an accuracy of 0.92. RESULTS: Significant increases in PEV and changes in size distribution profiles of EBPs was seen in COV-POS and COV-NEG compared to healthy controls. We achieved a deep proteome profiling of EBP across the three groups with proteins involved in immune activation, acute phase response, cell adhesion, blood coagulation, and known components of the respiratory tract lining fluid, among others. We demonstrated promising results for the use of an integrated EBP biomarker panel together with particle concentration for diagnosis of COVID-19 as well as a robust method for protein identification in EBPs. CONCLUSION: Our results demonstrate the promising potential for the use of EBP fingerprints in biomarker discovery and for diagnosing pulmonary diseases, rapidly and non-invasively with minimal patient discomfort.

Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT.

P1 and Pk are glycosphingolipid antigens synthesized by the A4GALT-encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P1 (P1+Pk+) and P2 (P1-Pk+weak) phenotypes. A4GALT transcript levels are higher in P1 individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of A4GALT, particularly rs5751348, correlate with P1/P2 status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Krüppel-like factor 1 (KLF1) haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates A4GALT expression. Intriguingly, P1 -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain P1 -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of KLF1 did not affect A4GALT transcript levels. Instead, protein pull-down experiments using P1 but not P2 oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds P1 alleles selectively, and knockdown of RUNX1 significantly decreased A4GALT transcription. These data indicate that RUNX1 regulates A4GALT and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.

Disruption of a GATA1-binding motif upstream of XG/PBDX abolishes Xga expression and resolves the Xg blood group system.

The Xga blood group is differentially expressed on erythrocytes from men and women. The underlying gene, PBDX, was identified in 1994, but the molecular background for Xga expression remains undefined. This gene, now designated XG, partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated Xga allele frequencies in different populations with 2612 genetic variants in the XG region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on XG transcript levels in whole blood (P = 2.0 × 10-22). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid XG messenger RNA expression and causes the Xg(a-) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xga genotyping possible.

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry.

The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Δ1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1.

Structural and Biophysical Characterization of Human EXTL3: Domain Organization, Glycosylation, and Solution Structure.

Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of ß sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.

Elucidating the Molecular Composition of Cartilage by Proteomics.

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.

Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry.

Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234-1245. © 2016 Wiley Periodicals, Inc.

Galiellalactone is a direct inhibitor of the transcription factor STAT3 in prostate cancer cells.

The transcription factor STAT3 is constitutively active in several malignancies including castration-resistant prostate cancer and has been identified as a promising therapeutic target. The fungal metabolite galiellalactone, a STAT3 signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing active STAT3 and induces apoptosis of prostate cancer stem cell-like cells expressing phosphorylated STAT3 (pSTAT3). However, the molecular mechanism of this STAT3-inhibiting effect by galiellalactone has not been clarified. A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of galiellalactone target proteins. By adding streptavidin-Sepharose beads to GL-biot-treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Confocal microscopy revealed GL-biot in both the cytoplasm and the nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. Galiellalactone inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. Mass spectrometry analysis of recombinant STAT3 protein pretreated with galiellalactone revealed three modified cysteines (Cys-367, Cys-468, and Cys-542). Here we demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer.

Proteomic profiling reveals that ESR1 mutations enhance cyclin-dependent kinase signaling.

Three quarters of all breast cancers express the estrogen receptor (ER, ESR1 gene), which promotes tumor growth and constitutes a direct target for endocrine therapies. ESR1 mutations have been implicated in therapy resistance in metastatic breast cancer, in particular to aromatase inhibitors. ESR1 mutations promote constitutive ER activity and affect other signaling pathways, allowing cancer cells to proliferate by employing mechanisms within and without direct regulation by the ER. Although subjected to extensive genetic and transcriptomic analyses, understanding of protein alterations remains poorly investigated. Towards this, we employed an integrated mass spectrometry based proteomic approach to profile the protein and phosphoprotein differences in breast cancer cell lines expressing the frequent Y537N and Y537S ER mutations. Global proteome analysis revealed enrichment of mitotic and immune signaling pathways in ER mutant cells, while phosphoprotein analysis evidenced enriched activity of proliferation associated kinases, in particular CDKs and mTOR. Integration of protein expression and phosphorylation data revealed pathway-dependent discrepancies (motility vs proliferation) that were observed at varying degrees across mutant and wt ER cells. Additionally, protein expression and phosphorylation patterns, while under different regulation, still recapitulated the estrogen-independent phenotype of ER mutant cells. Our study is the first proteome-centric characterization of ESR1 mutant models, out of which we confirm estrogen independence of ER mutants and reveal the enrichment of immune signaling pathways at the proteomic level.

Fast surface acoustic wave-matrix-assisted laser desorption ionization mass spectrometry of cell response from islets of Langerhans.

A desire for higher speed and performance in molecular profiling analysis at a reduced cost is driving a trend in miniaturization and simplification of procedures. Here we report the use of a surface acoustic wave (SAW) atomizer for fast sample handling in matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) peptide and protein profiling of Islets of Langerhans, for future type 2 diabetes (T2D) studies. Here the SAW atomizer was used for ultrasound (acoustic) extraction of insulin and other peptide hormones released from freshly prepared islets, stimulated directly on a membrane. A high energy propagating SAW atomizes the membrane-bound liquid into approximately 2 µm diameter droplets, rich in cell-released molecules. Besides acting as a sample carrier, the membrane provides a purification step by entrapping cell clusters and other impurities within its fibers. A new SAW-based sample-matrix deposition method for MALDI MS was developed and characterized by a strong insulin signal, and a limit of detection (LOD) lower than 100 amol was achieved. Our results support previous work reporting the SAW atomizer as a fast and inexpensive tool for ultrasound, membrane-based sample extraction. When interfaced with MALDI MS, the SAW atomizer constitutes a valuable tool for rapid cell studies. Other biomedical applications of SAW-MALDI MS are currently being developed, aiming at fast profiling of biofluids. The membrane sampling is a simplistic and noninvasive collection method of limited volume biofluids such as the gingival fluid and the tearfilm.

Further insights into the catalytical properties of deglycosylated pyranose dehydrogenase from Agaricus meleagris recombinantly expressed in Pichia pastoris.

The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris . Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-(vinylimidazole))10Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N(75), N(175), and N(252) were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.

Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using 13C,15N-labeled peptides as internal standards.

Mucins are of great interest owing to their involvement in physiological and pathological processes in the airways. A method that allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic selected reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction, and alkylation prior to tryptic digestion. Solid phase extraction using C18 sorbent was used for sample cleanup prior to the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A cysteine-containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the quantification of MUC5B protein. Stable isotope-labeled synthetic peptides were used as internal standards, and linear calibration curves were constructed in the range of 0.3 to 40 pmol/L. Both mucins could be determined with a precision of 6 to 19% and an accuracy of 98 to 114%. The method is transferable to robotics and is suitable to be run in a 96-well format.

Selective protein aggregation confines and inhibits endotoxins in wounds: Linking host defense to amyloid formation.

Bacterial lipopolysaccharide (LPS) induces rapid protein aggregation in human wound fluid. We aimed to characterize these LPS-induced aggregates and their functional implications using a combination of mass spectrometry analyses, biochemical assays, biological imaging, cell experiments, and animal models. The wound-fluid aggregates encompass diverse protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. We identified proteins and peptides with a high aggregation propensity and verified selected components through Western blot analysis. Thioflavin T and Amytracker staining revealed amyloid-like aggregates formed after exposure to LPS in vitro in human wound fluid and in vivo in porcine wound models. Using NF-κB-reporter mice and IVIS bioimaging, we demonstrate that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation, further emphasizing the connection between host defense and amyloidogenesis.

Proteomic changes to immune and inflammatory processes underlie lung preservation using ex vivo cytokine adsorption.

Introduction: In recent years, the field of graft preservation has made considerable strides in improving outcomes related to solid organ restoration and regeneration. Ex vivo lung perfusion (EVLP) in line with the related devices and treatments has yielded promising results within preclinical and clinical studies, with the potential to improve graft quality. Its main benefit is to render marginal and declined donor lungs suitable for transplantation, ultimately increasing the donor pool available for transplantation. In addition, using such therapies in machine perfusion could also increase preservation time, facilitating logistical planning. Cytokine adsorption has been demonstrated as a potentially safe and effective therapy when applied to the EVLP circuit and post-transplantation. However, the mechanism by which this therapy improves the donor lung on a molecular basis is not yet fully understood. Methods: We hypothesized that there were characteristic inflammatory and immunomodulatory differences between the lungs treated with and without cytokine adsorption, reflecting proteomic changes in the gene ontology pathways and across inflammation-related proteins. In this study, we investigate the molecular mechanisms and signaling pathways of how cytokine adsorption impacts lung function when used during EVLP and post-transplantation as hemoperfusion in a porcine model. Lung tissues during EVLP and post-lung transplantation were analyzed for their proteomic profiles using mass spectrometry. Results: We found through gene set enrichment analysis that the inflammatory and immune processes and coagulation pathways were significantly affected by the cytokine treatment after EVLP and transplantation. Conclusion: In conclusion, we showed that the molecular mechanisms are using a proteomic approach behind the previously reported effects of cytokine adsorption when compared to the non-treated transplant recipients undergoing EVLP.

Serum proteome profiles in patients treated with targeted temperature management after out-of-hospital cardiac arrest.

BACKGROUND: Definition of temporal serum proteome profiles after out-of-hospital cardiac arrest may identify biological processes associated with severe hypoxia-ischaemia and reperfusion. It may further explore intervention effects for new mechanistic insights, identify candidate prognostic protein biomarkers and potential therapeutic targets. This pilot study aimed to investigate serum proteome profiles from unconscious patients admitted to hospital after out-of-hospital cardiac arrest according to temperature treatment and neurological outcome. METHODS: Serum samples at 24, 48, and 72 h after cardiac arrest at three centres included in the Target Temperature Management after out-of-hospital cardiac arrest trial underwent data-independent acquisition mass spectrometry analysis (DIA-MS) to find changes in serum protein concentrations associated with neurological outcome at 6-month follow-up and targeted temperature management (TTM) at 33 °C as compared to 36 °C. Neurological outcome was defined according to Cerebral Performance Category (CPC) scale as "good" (CPC 1-2, good cerebral performance or moderate disability) or "poor" (CPC 3-5, severe disability, unresponsive wakefulness syndrome, or death). RESULTS: Of 78 included patients [mean age 66 ± 12 years, 62 (80.0%) male], 37 (47.4%) were randomised to TTM at 36 °C. Six-month outcome was poor in 47 (60.3%) patients. The DIA-MS analysis identified and quantified 403 unique human proteins. Differential protein abundance testing comparing poor to good outcome showed 19 elevated proteins in patients with poor outcome (log2-fold change (FC) range 0.28-1.17) and 16 reduced proteins (log2(FC) between - 0.22 and - 0.68), involved in inflammatory/immune responses and apoptotic signalling pathways for poor outcome and proteolysis for good outcome. Analysis according to level of TTM showed a significant protein abundance difference for six proteins [five elevated proteins in TTM 36 °C (log2(FC) between 0.33 and 0.88), one reduced protein (log2(FC) - 0.6)] mainly involved in inflammatory/immune responses only at 48 h after cardiac arrest. CONCLUSIONS: Serum proteome profiling revealed an increase in inflammatory/immune responses and apoptosis in patients with poor outcome. In patients with good outcome, an increase in proteolysis was observed, whereas TTM-level only had a modest effect on the proteome profiles. Further validation of the differentially abundant proteins in response to neurological outcome is necessary to validate novel biomarker candidates that may predict prognosis after cardiac arrest.

Bioactive Suture with Added Innate Defense Functionality for the Reduction of Bacterial Infection and Inflammation.

Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.

Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs.

There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.

Peptimetric: Quantifying and Visualizing Differences in Peptidomic Data.

Finding new sustainable means of diagnosing and treating diseases is one of the most pressing issues of our time. In recent years, several endogenous peptides have been found to be both excellent biomarkers for many diseases and to possess important physiological roles which may be utilized in treatments. The detection of peptides has been facilitated by the rapid development of biological mass spectrometry and now the combination of fast and sensitive high resolution MS instruments and stable nano HP-LC equipment sequences thousands of peptides in one single experiment. In most research conducted with these advanced systems, proteolytically cleaved proteins are analyzed and the specific peptides are identified by software dedicated for protein quantification using different proteomics workflows. Analysis of endogenous peptides with peptidomics workflows also benefit from the novel sensitive and advanced instrumentation, however, the generated peptidomic data is vast and subsequently laborious to visualize and examine, creating a bottleneck in the analysis. Therefore, we have created Peptimetric, an application designed to allow researchers to investigate and discover differences between peptidomic samples. Peptimetric allows the user to dynamically and interactively investigate the proteins, peptides, and some general characteristics of multiple samples, and is available as a web application at https://peptimetric.herokuapp.com. To illustrate the utility of Peptimetric, we've applied it to a peptidomic dataset of 15 urine samples from diabetic patients and corresponding data from healthy subjects.

Method development and characterisation of the low-molecular-weight peptidome of human wound fluids.

The normal wound healing process is characterised by proteolytic events, whereas infection results in dysfunctional activations by endogenous and bacterial proteases. Peptides, downstream reporters of these proteolytic actions, could therefore serve as a promising tool for diagnosis of wounds. Using mass-spectrometry analyses, we here for the first time characterise the peptidome of human wound fluids. Sterile post-surgical wound fluids were found to contain a high degree of peptides in comparison to human plasma. Analyses of the peptidome from uninfected healing wounds and Staphylococcus aureus -infected wounds identify unique peptide patterns of various proteins, including coagulation and complement factors, proteases, and antiproteinases. Together, the work defines a workflow for analysis of peptides derived from wound fluids and demonstrates a proof-of-concept that such fluids can be used for analysis of qualitative differences of peptide patterns from larger patient cohorts, providing potential biomarkers for wound healing and infection.

Infected wounds and burns represent a serious risk to patients: they can delay healing and, if left untreated, can lead to generalised infection or sepsis, organ failure and death. Wounds and burns get infected when harmful micro-organisms, such as bacteria, enter the wound. Predicting the risk of infections, and detecting them early, could reduce their impact and make treating them easier. A way to distinguish between healing and infected wounds is to study how proteins are broken down in each situation. Proteases are the enzymes that break down proteins, and they are different in healing wounds and infected wounds that are failing to heal. This is because, while the body produces proteases, the bacteria that cause infection do so too. Each protease breaks down proteins in a specific way, resulting in a different set of protein fragments, known as peptides. Together, all the peptides in a wound are referred to as the wound's 'peptidome'. Studying the peptidome of a wound could show whether it is infected, and even what type of bacteria might be responsible, which could help identify suitable treatments. Van der Plas et al. used a technique called mass spectrometry to study the peptidome of wounds after surgery. Sterile post-surgical wounds showed high levels of peptides compared to plasma, the liquid component of blood, with up to 4,300 different peptides. Comparing healing wounds to ones infected with the bacterium Staphylococcus aureus revealed that infected wounds contained peptides from about 150 proteins not found in uninfected wounds, while peptides from 90 proteins were unique to uninfected wounds. The peptides exclusive to uninfected wounds included some linked to antimicrobial activity and immune system activity. Van der Plas et al.'s results suggest that analysing the peptidome may be an approach to tracking the healing status of wounds, making it easier to detect infection before symptoms are apparent. The next step will be to study more wounds and identify the reliable peptide markers to use them for diagnostic tests.