Activation of the MAPK network provides a survival advantage during the course of COVID-19-induced sepsis: a real-world evidence analysis of a multicenter COVID-19 Sepsis Cohort.

PURPOSE: There is evidence that lower activity of the RAF/MEK/ERK network is associated with positive outcomes in mild and moderate courses of COVID-19. The effect of this cascade in COVID-19 sepsis is still undetermined. Therefore, we tested the hypothesis that activity of the RAF/MEK/ERK network in COVID-19-induced sepsis is associated with an impact on 30-day survival. METHODS: We used biomaterial from 81 prospectively recruited patients from the multicentric CovidDataNet.NRW-study cohort (German clinical trial registry: DRKS00026184) with their collected medical history, vital signs, laboratory parameters, microbiological findings and patient outcome. ERK activity was measured by evaluating ERK phosphorylation using a Proximity Ligation Assay. RESULTS: An increased ERK activity at 4 days after diagnosis of COVID-19-induced sepsis was associated with a more than threefold increased chance of survival in an adjusted Cox regression model. ERK activity was independent of other confounders such as Charlson Comorbidity Index or SOFA score (HR 0.28, 95% CI 0.10-0.84, p = 0.02). CONCLUSION: High activity of the RAF/MEK/ERK network during the course of COVID-19 sepsis is a protective factor and may indicate recovery of the immune system. Further studies are needed to confirm these results.

Plasma Proteomics Enable Differentiation of Lung Adenocarcinoma from Chronic Obstructive Pulmonary Disease (COPD).

Chronic obstructive pulmonary disease (COPD) is a major risk factor for the development of lung adenocarcinoma (AC). AC often develops on underlying COPD; thus, the differentiation of both entities by biomarker is challenging. Although survival of AC patients strongly depends on early diagnosis, a biomarker panel for AC detection and differentiation from COPD is still missing. Plasma samples from 176 patients with AC with or without underlying COPD, COPD patients, and hospital controls were analyzed using mass-spectrometry-based proteomics. We performed univariate statistics and additionally evaluated machine learning algorithms regarding the differentiation of AC vs. COPD and AC with COPD vs. COPD. Univariate statistics revealed significantly regulated proteins that were significantly regulated between the patient groups. Furthermore, random forest classification yielded the best performance for differentiation of AC vs. COPD (area under the curve (AUC) 0.935) and AC with COPD vs. COPD (AUC 0.916). The most influential proteins were identified by permutation feature importance and compared to those identified by univariate testing. We demonstrate the great potential of machine learning for differentiation of highly similar disease entities and present a panel of biomarker candidates that should be considered for the development of a future biomarker panel.

The hypertrehalosaemic neuropeptide conformational twins of cicadas consist of only L-amino acids: are they cis-trans isomers?

It is known for almost 25 years that the corpora cardiaca (neurosecretory glands) of cicadas synthesize two isobaric peptides with hypertrehalosaemic activity denominated Placa-HrTH-I and II. Both decapeptides have the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn amide) and mass, but differ in their chromatographic retention time. The slightly more hydrophobic peptide, Placa-HrTH-II, co-elutes with the synthetic peptide of the same sequence and is less active in biological assays than Placa-HrTH-I. Ion mobility separation in conjunction with high-resolution mass spectrometry detected the differing structural feature between both peptides in the region Pro6-Ser7-Trp8. Here, it was shown that Placa-HrTH-I co-eluted with a synthetic peptide containing D-Pro in position 6, while dextrorotatory amino acid residues in positions 7 and 8 could be excluded in this way. Amino acid hydrolysis followed by chiral analysis using a relative of Marfey's reagent was then used to validate the presence of D-Pro in Placa-HrTH-I. Interestingly, this experiment unambiguously proved both the absence of D-Pro and the presence of L-Pro in Placa-HrTH-I. Racemization as a reason for the structural differences of the twin adipokinetic hormones was hence ruled out and cis-trans isomerism as the likely alternative came into focus. It remains to be investigated if Pro6 in cis-conformation is indeed present and responsible for the increased bioactivity of Placa-HrTH-I.

Pyrylium-based dye and charge tagging in proteomics.

The pyrylium group is a selective reagent for ε-amino groups in proteins. In particular, for fluorescence labeling, a number of advantages over traditional N-hydroxysuccinimidyl ester chemistry were recognized such as the rapid prestaining procedure. Here, we have investigated the labeling reaction for the fluorogenic pyrylium dye Py-1 using liquid chromatography coupled to MS with the aim of determining its specificity and possible side products. Peptides containing no, one, and two lysine residue and a choice of no or one cysteine residue were labeled with Py-1 at yields > 30%. Gas phase fragmentation proved both labeling of lysine residues as well as that of the N-terminus also in peptides that contained a lysine residue. Evidence for cysteine labeling was not found, but several other products were detected such as the results of rearrangements with adjacent acidic amino acids. Apart from the use as a fluorogenic label, Py-1 recommends itself for N-terminal charge tagging as alternative to the commonly used quaternary ammonium salts. Predominantly a- and b-type ion series were observed for N-terminally labeled peptides. Further applications include chromophore tagging since the labeled product is not only fluorescent but also colored red.

Exploring the relationship between HCMV serostatus and outcomes in COVID-19 sepsis.

Background: Sepsis, a life-threatening condition caused by the dysregulated host response to infection, is a major global health concern. Understanding the impact of viral or bacterial pathogens in sepsis is crucial for improving patient outcomes. This study aimed to investigate the human cytomegalovirus (HCMV) seropositivity as a risk factor for development of sepsis in patients with COVID-19. Methods: A multicenter observational study enrolled 95 intensive care patients with COVID-19-induced sepsis and 80 post-surgery individuals as controls. HCMV serostatus was determined using an ELISA test. Comprehensive clinical data, including demographics, comorbidities, and 30-day mortality, were collected. Statistical analyses evaluated the association between HCMV seropositivity and COVID-19 induced sepsis. Results: The prevalence of HCMV seropositivity did not significantly differ between COVID-19-induced sepsis patients (78%) and controls (71%, p = 0.382) in the entire cohort. However, among patients aged ≤60 years, HCMV seropositivity was significantly higher in COVID-19 sepsis patients compared to controls (86% vs 61%, respectively; p = 0.030). Nevertheless, HCMV serostatus did not affect 30-day survival. Discussion: These findings confirm the association between HCMV seropositivity and COVID-19 sepsis in non-geriatric patients. However, the lack of an independent effect on 30-day survival can be explained by the cross-reactivity of HCMV specific CD8+ T-cells towards SARS-CoV-2 peptides, which might confer some protection to HCMV seropositive patients. The inclusion of a post-surgery control group strengthens the generalizability of the findings. Further research is needed to elucidate the underlying mechanisms of this association, explore different patient populations, and identify interventions for optimizing patient management. Conclusion: This study validates the association between HCMV seropositivity and severe COVID-19-induced sepsis in non-geriatric patients, contributing to the growing body of evidence on viral pathogens in sepsis. Although HCMV serostatus did not independently influence 30-day survival, future investigations should focus on unraveling the intricate interplay between HCMV, immune responses, and COVID-19. These insights will aid in risk stratification and the development of targeted interventions for viral sepsis.

Substance P Serum Degradation in Complex Regional Pain Syndrome - Another Piece of the Puzzle?

In a previous study, we demonstrated that the serum peptidase system might be less efficient in complex regional pain syndrome (CRPS). Since the neuropeptide substanc P (SP) contributes to inflammation in CRPS, we now investigated the metabolism of SP in CRPS specifically. An SP metabolism assay was performed in 24 CRPS patients, which constitute a subgroup of our previous investigation on BK degradation. In addition, we included 26 healthy controls (24 newly recruited plus 2 from our previous investigation), and 13 patients after limb trauma, who did not fulfil the CRPS diagnostic criteria (trauma controls, TC) were included. We adapted a thin layer chromatography assay (TLC) to quantify SP disappearance after incubation with 7.5 µL of serum. These results were compared with bradykinin (BK) metabolization to BK1-8 and BK1-5 fragments from our previous study. In addition, TC were clinically and quantitative sensory testing (QST) phenotyped; the phenotyping of CRPS patients was retrieved from our existing database. SP metabolism was less efficient in CRPS and TC patient serum vs human control (HC) serum (P < .03) suggesting reduced activity of the neutral endopeptidase (NEP) and/or the angiotensin converting enzyme (ACE). Together with the decreased occurrence of BK1-5 fragment in CRPS and TC, this suggests a reduced activation of the angiotensin converting enzyme (ACE). There was no clear clinical phenotype related to impaired SP degradation; duration of disease and gender were also not associated. Most importantly, results in TC did not differ from CRPS. Collectively, our current and previous experimental results suggest that limb trauma reduces serum peptidase metabolism of SP ex vivo, specifically serum ACE activity. However, this finding is not CRPS-specific and seems to be rather a long-term consequence of the trauma itself. PERSPECTIVE: The experimental data from this study further support the hypothesis that impaired metabolism of inflammatory peptides potentially contribute to the development of posttraumatic pain in CRPS or limb trauma patients.

Validation data for the use of bradykinin and substance P protease activity assays with capillary blood and blood cards.

In the associated main paper ("Labeled substance P as a neuropeptide reporter substance for enzyme activity" (http://doi.org/10.1016/j.jpba.2019.112953)), substance P was shown to be a valuable neuropeptide reporter substance to monitor the protease activity of serum. The assay was developed based on the predecessor assay using bradykinin ("A vote for robustness: Monitoring serum enzyme activity by thin-layer chromatography of dabsylated bradykinin products", http://doi.org/10.1016/j.jpba.2017.06.007). Both neuropeptides are of interest in inflammation and pain research and were thus explored for use with capillary blood and blood cards (see associated MethodX paper "Neuropeptide reporter assay for serum, capillary blood and blood cards"). Here, we present validation data for the assay when sampling with blood cards as well as data on the use of fresh capillary blood.

Labeled substance P as a neuropeptide reporter substance for enzyme activity.

Recently, we developed a bradykinin reporter assay and demonstrated the differing protease activity in Complex Regional Pain Syndrome patients vs. controls. In order to further characterize CRPS pathophysiology, the neuropeptide substance P was evaluated as possible reporter substance, here. It was labeled with a chromophore at the lysine residue and generated two major fragments following incubation with serum (amino acid residues 3-8 and 3-11) which were reproducibly separated by thin-layer chromatography. Dabsylated substance P was shown to be a substrate of angiotensin-converting enzyme. The combination of both bradykinin and substance P reporter substances with specific enzyme inhibitors will shed more light on biochemical pathways in inflammatory processes and pain. Comparative clinical studies are now needed to define the application range of both assays in more detail.

Are formalin-fixed and paraffin-embedded tissues fit for proteomic analysis?

Formalin-fixed and paraffin-embedded (FFPE)-tissue archives are potential treasure troves in the search for clinically interesting specimens. However, while the FFPE-treatment provides excellent conservation of the three-dimensional structure of the tissue and prevents degradation over decades, it also introduces numerous nonspecific and irreversible protein modifications. In this study, we have evaluated several published workflows for FFPE-tissue by fit-for-purpose proteomics technologies. We demonstrate that many protein modifications and cross-links remain after treatment and conclude that the proteomics of FFPE-tissue is of value, but clear-cut limitations must be kept in mind. The analysis of abundant proteins in FFPE is straightforward, but confident identification of low-level proteins and/or biologically relevant modifications is seriously hampered by the FFPE-treatment. Peptide assignment should only be performed on high-quality spectra, even if this is at the cost of lower numbers of protein IDs. As Yergey and Coorssen stated in 2015: "Data quality is considered the primary criterion, and we thus emphasize that the standards of Analytical Chemistry must apply throughout any proteomic analysis."

The serum protease network-one key to understand complex regional pain syndrome pathophysiology.

Complex regional pain syndrome (CRPS) develops after fracture. The acute CRPS phenotype resembles exaggerated inflammation, which is explained by local and systemic activation of a proinflammatory network including peptides and cytokines. Epidemiologic data suggest that inactivation of the peptidase angiotensin-converting enzyme in patients treated for hypertension increases the odds to develop CRPS. This hint leads us to investigate the serum protease network activity in patients with CRPS vs respective controls. For this purpose, we developed a dabsyl-bradykinin (DBK)-based assay and used it to investigate patients with CRPS, as well as healthy and pain (painful diabetic neuropathy [dPNP]) controls. The major result is that the degradation of DBK to fragments 1-8 and 1-5 in healthy control and dPNP is shifted to higher values for DBK1-8 and lower values for DBK1-5 at 1 hour of incubation in patients with CRPS. Using this novel reporter peptide assay, we have been able to show that the resolving protease network for mediators such as BK might be different in patients with CRPS; having a look at the clinical signs, which resemble inflammation, this resolving protease network is probably less effective in CRPS.

A vote for robustness: Monitoring serum enzyme activity by thin-layer chromatography of dabsylated bradykinin products.

High-end analytical methods provide excellent data but may lack the robustness required in large analytical studies. In particular complex chemical matrices may cause difficulties and increase the need for extensive sample preparation. For screening of patients we thus developed a low-tech assay to monitor bradykinin degradation by serum proteases. The bradykinin concentration mirrors the activity of angiotensin-converting enzyme (ACE). Dabsylated bradykinin (DBK) and its labeled fragments DBK1-8 and DBK1-5 were visualized by thin-layer chromatography using only 3µL of serum. Lower DBK1-5 levels indicated reduced ACE activity due to medication (ACE-inhibitors) or disease. Provided that purified DBK is available, the assay protocol itself is very simple and does not require any expensive high-end equipment.