Streptococcal quorum sensing peptide CSP-7 contributes to muscle inflammation and wasting.

Muscle wasting diseases, such as cancer cachexia and age-associated sarcopenia, have a profound and detrimental impact on functional independence, quality of life, and survival. Our understanding of the underlying mechanisms is currently limited, which has significantly hindered the development of targeted therapies. In this study, we explored the possibility that the streptococcal quorum sensing peptide Competence Stimulating Peptide 7 (CSP-7) might be a previously unidentified contributor to clinical muscle wasting. We found that CSP-7 selectively triggers muscle cell inflammation in vitro, specifically the release of IL-6. Furthermore, we demonstrated that CSP-7 can traverse the gastrointestinal barrier in vitro and is present in the systemic circulation in humans in vivo. Importantly, CSP-7 was associated with a muscle wasting phenotype in mice in vivo. Overall, our findings provide new mechanistic insights into the pathophysiology of muscle inflammation and wasting.

Evaluation of greenness and analytical performances of separative methods for chiral separation of novel lactam-based P2RX7-antagonists.

In this work, a preparative supercritical fluid chromatography (SFC) method was first developed to separate a series of chiral compounds evaluated as lactam-based P2RX7 antagonists. Subsequently, high-performance liquid chromatography, SFC, and capillary electrophoresis (CE) were comparatively investigated as QC tools to determine the enantiomeric purity of the separated isomers, including analytical performance and greenness. The screening of the best conditions was carried out in liquid and SFC on the nine derivatives and the amylose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase was found to be highly efficient. The same screening was carried out in CE and very different conditions, either in acidic or basic background electrolyte and different cyclodextrins used as chiral selectors, allowed the separation of six of the nine derivatives. 1-((3,4-Dichlorophenyl)carbamoyl)-5-oxopyrrolidine-2-carboxylic acid (compound 1) was chosen as a probe, and its semi-preparative separation by SFC and enantiomeric verification using the three techniques are presented. Its limit of detection and limit of quantification are calculated for each method. Finally, the greenness of each quality control method was evaluated.

BrainPepPass: A Framework Based on Supervised Dimensionality Reduction for Predicting Blood-Brain Barrier-Penetrating Peptides.

Peptides that pass through the blood-brain barrier (BBB) not only are implicated in brain-related pathologies but also are promising therapeutic tools for treating brain diseases, e.g., as shuttles carrying active medicines across the BBB. Computational prediction of BBB-penetrating peptides (B3PPs) has emerged as an interesting approach because of its ability to screen large peptide libraries in a cost-effective manner. In this study, we present BrainPepPass, a machine learning (ML) framework that utilizes supervised manifold dimensionality reduction and extreme gradient boosting (XGB) algorithms to predict natural and chemically modified B3PPs. The results indicate that the proposed tool outperforms other classifiers, with average accuracies exceeding 94% and 98% in 10-fold cross-validation and leave-one-out cross-validation (LOOCV), respectively. In addition, accuracy values ranging from 45% to 97.05% were achieved in the independent tests. The BrainPepPass tool is available in a public repository for academic use (https://github.com/ewerton-cristhian/BrainPepPass).

Emerging pharmaceutical therapies of Ascidian-derived natural products and derivatives.

In a growing multidrug-resistant environment, the identification of potential new drug candidates with an acceptable safety profile is a substantial crux in pharmaceutical discovery. This review discusses several aspects and properties of approved marine natural products derived from ascidian sources (phylum Chordata, subphylum Tunicata) and/or their deduced analogues including their biosynthetic origin, (bio)chemical preclinical assessments and known efficacy-safety profiles, clinical status in trials, but also translational developments, opportunities and final conclusions. The review also describes the preclinical assessments of a large number of other ascidian compounds that have not been involved in clinical trials yet. Finally, the emerging research on the connectivity of the ascidian hosts and their independent or obligate symbiotic guests is discussed. The review covers the latest information on the topic of ascidian-derived marine natural products over the last two decades including 2022, with the majority of publications published in the last decade.

Quantification and stability assessment of urinary phenolic and acidic biomarkers of non-persistent chemicals using the SPE-GC/MS/MS method.

Nowadays, people are exposed to numerous man-made chemicals, many of which are ubiquitously present in our daily lives, and some of which can be hazardous to human health. Human biomonitoring plays an important role in exposure assessment, but complex exposure evaluation requires suitable tools. Therefore, routine analytical methods are needed to determine several biomarkers simultaneously. The aim of this study was to develop an analytical method for quantification and stability testing of 26 phenolic and acidic biomarkers of selected environmental pollutants (e.g., bisphenols, parabens, pesticide metabolites) in human urine. For this purpose, a solid-phase extraction coupled with gas chromatography and tandem mass spectrometry (SPE-GC/MS/MS) method was developed and validated. After enzymatic hydrolysis, urine samples were extracted using Bond Elut Plexa sorbent, and prior to GC, the analytes were derivatized with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA). Matrix-matched calibration curves were linear in the range of 0.1-1000 ng mL-1 with R > 0.985. Satisfactory accuracy (78-118%), precision (< 17%), and limits of quantification (0.1-0.5 ng mL-1) were obtained for 22 biomarkers. The stability of the biomarkers in urine was assayed under different temperature and time conditions that included freezing and thawing cycles. All tested biomarkers were stable at room temperature for 24 h, at 4 °C for 7 days, and at -20 °C for 18 months. The total concentration of 1-naphthol decreased by 25% after the first freeze-thaw cycle. The method was successfully used for the quantification of target biomarkers in 38 urine samples.

Peptidomics: LC-MS operational parameters do matter.

The sensitive and specific detection of peptides at low levels in biofluids is critical to increase the lab-to-human translation of peptidomic research. An interesting group of peptides with increasing evidence for involvement in human diseases are quorum sensing peptides. To obtain more reliable conclusions on peptide measurands in biofluids, a selection of often neglected parts of the analytical process using LC-MS were investigated, with novel approaches recommended for each part. Quorum sensing peptides were used as the main model-peptides. The peptidomic parts investigated and discussed here are: Our work addresses aQbD-approached solutions to these challenges, encompassing sample stabilization measures, a suitable peptide anti-adsorption tool, judicious choice of injection solvent versus gradient system and optimal duty cycle parameters. Our recommendations will improve the peptidomics bio-analytics of not only quorum sensing peptides, but can also be of value for other measurands at low concentrations.

Bacterial Quorum-Sensing Peptides as Immune Modulators Present in Systemic Circulation.

Quorum-sensing peptides (QSPs) are bacterial peptides traditionally considered only as inter-bacterial communication molecules. Recently, their involvement in microbiome-host interactions influencing host diseases such as cancer and sarcopenia were explored. However, it is still unknown to what extent these peptides have the potential to modulate the immune system. In this proof-of-concept study, we screened 89 QSPs for their potential to induce IL-6 and TNFα in murine splenocytes and J774 macrophages. Confirmatory experiments on the positive screening-hits were conducted using murine splenocytes and human PBMCs of different ages. Finally, to investigate the biological relevance of immunomodulatory QSPs, we analysed plasma in a human cohort for the presence of the immunomodulatory QSP Q010. To do this, we used a newly developed UHPLC-MS/MS method. Our findings indicated that specific QSPs activate immune cells in vitro, with Q007, Q010, Q017 and Q212 being the top four screening hits. Q007 and Q010 were affirmed in subsequent confirmatory experiments using murine splenocytes and human PBMCs. Finally, Q010 was detected in human plasma, demonstrating for the first time the presence of an immunomodulatory QSP in human circulation. In conclusion, our data are the first evidence indicating the potential of biologically relevant quorum-sensing peptides to modulate the immune system.

Degradation kinetics of artesunate for the development of an ex-tempore intravenous injection.

BACKGROUND: Artesunate is recommended by the World Health Organization (WHO) for parenteral treatment of severe Plasmodium falciparum malaria. However, artesunate is inherently unstable in an aqueous solution and hydrolyses rapidly after its preparation for injection. Therefore, the aim of the study was to evaluate the stabilizing effects of phosphate buffer and mannitol against short-term (ex-tempore) artesunate hydrolysis. METHODS: A HPLC-UV isocratic method was developed using a reversed-phase fused core column (HALO RP-C18) and a mobile phase consisting of a mixture of 45% ammonium formate 10 mM in water (pH 4.5) and 55% methanol. Artesunate was formulated as aqueous solutions using a design of experiment (DOE) to investigate the artesunate stabilizing effects of pH (8-10), phosphate buffer strength (0.3-0.5 M), and mannitol (0-0.22 mmol/mL). The solutions were incubated at predefined temperatures (5, 25, and 40 °C) with subsequent analysis. Arrhenius equation was applied to model and evaluate the stability results. RESULTS: The developed HPLC-based method using fused-core stationary phase allowed to selectively quantify artesunate in the presence of its main hydrolysis degradants; namely ß-dihydroartemisinin (ß-DHA) and α-dihydroartemisinin (α-DHA) within 10 min. By applying the Arrhenius equation, the rate of hydrolysis of the drug increased approximately by 3.4 as the temperature raised by 10 °C. Buffer strength was found to be the main factor affecting the hydrolysis rate constants at 5 and 25 °C (p < 0.05), the activation energy (p = 0.009), and the frequency factor (p = 0.045). However, the effect of the buffer was predominant on the activation energy and hydrolysis rate constants, revealing its stabilizing effect on the drug at lower buffer strength (0.3 M). Within the investigated range (pH = 8-10), pH was found to influence the activation energy, with a positive stabilizing effect in the pH range of 8-9. The addition of mannitol as stabilizing agent into artesunate aqueous formulation did not show an improved response. CONCLUSION: Phosphate buffer was the main stability determining factor of artesunate in the aqueous intravenous (i.v.) formulation and was found to be more effective in stabilizing artesunate at a buffer strength of 0.3 M in pH 8-9, while mannitol lacked stabilizing effect.

The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction.

BACKGROUND: Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. RESULTS: Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. CONCLUSION: Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.

A fit-for-purpose LC-MS/MS method for the analysis of selected Streptococcal quorum sensing peptides in human saliva.

The development of analytical methods for the detection of peptides at the nanomolar level can be challenging. Peptides can suffer from adsorption, rendering the detection of peptides at these low levels difficult. A subset of peptides are the quorum sensing peptides, which are bacterial communication molecules demonstrating possible host effects as well. However, their direct presence in human biofluids has only rarely been reported. Therefore, a UHPLC-MS/MS method capable of detecting 15 selected Streptococcal competence stimulating quorum sensing peptides at the nanomolar level in human saliva was developed. This method, using an anti-adsorption diluent, was applied on saliva samples obtained from 38 healthy donors. Six donors did have a positive hit for at least one of three competence stimulating quorum sensing peptides using a triple quadrupole assay. These observations indicate that Streptococcus species produce quorum sensing peptides in the human oral cavity.

A bioanalytical screening method for Enterococcus faecalis RNPP-type quorum sensing peptides in murine feces.

Background: Bacteria coordinate their behavior as a group via communication with their peers, known as 'quorum sensing'. Enterococcus faecalis employs quorum sensing via RNPP-peptides which were not yet reported to be present in mammalian biofluids. Results: Solid phase extraction of murine feces was performed, followed by ultra high performance liquid chromatography (UHPLC-MS/MS) in multiple reaction monitoring (MRM) mode (in total <90 min/sample) for the nine known RNPP peptides. Limits of detection ranged between 0.045 and 52 nM. Adequate identification criteria allowed detection of RNPP quorum sensing peptides in 2/20 wild-type murine feces samples (i.e., cAM373 and cOB1). Conclusion: A fit-for-purpose UHPLC-MS/MS method detected these RNPP peptides in wild-type murine feces samples.

Statin Intake and All-Cause Mortality among Older Nursing Home Residents.

BACKGROUND: Statins are progressively accepted as being associated with reduced mortality. However, few real-world statin studies have been conducted on statin use in older people and especially the most frail, that is, the nursing home residents. OBJECTIVE: The aim of this study was to evaluate the impact of statin intake in nursing home residents on all-cause mortality. METHOD: This is a cross-sectional study of 1,094 older people residing in 6 nursing homes in Flanders (Belgium) between March 1, 2020 and May 30, 2020. We considered all residents who were taking statins for at least 5 days as statin users. All-cause mortality during the 3 months of data collection was the primary outcome. Propensity score overlap-weighted logistic regression models were applied with age, sex, functional status, diabetes, and cardiac failure/ischemia as potential confounders. RESULTS: 185 out of 1,094 residents were on statin therapy (17%). The statin intake was associated with decreased all-cause mortality: 4% absolute risk reduction; adjusted odds ratio 0.50; CI 0.31-0.81, p = 0.005. CONCLUSIONS: The statin intake was associated with decreased all-cause mortality in older people residing in nursing homes. More in-depth studies investigating the potential geroprotector effect of statins in this population are needed.

Acute sarcopenia changes following hospitalization: influence of pre-admission care dependency level.

INTRODUCTION: Hospitalization is associated with acute changes in sarcopenia status in older people, but the influencing factors are not fully understood. Pre-admission care dependency level as a risk factor has not yet been investigated. OBJECTIVE: Evaluate if pre-admission care dependency level is an independent predictor of sarcopenia changes following hospitalization. SETTING AND SUBJECTS: Data came from the Sarcopenia 9+ EAMA Project, a European prospective multi-centre study. For this study, 227 hospitalised older people were included from four different hospitals in Belgium, Spain and Poland, between 18 February 2019 and 5 September 2020. METHODS: Sarcopenia status at admission and discharge were calculated using a combined score (desirability value) based on muscle mass (calf circumference), strength (grip) and function (walking speed). Ratio of admission to discharge status was the outcome (desirability ratio; 1.00 meaning no difference). Predictor variable was the pre-admission care dependency level, classified into three groups: independent older people living at home, dependent older people living at home and older people living in a care home. Linear regression models were applied, considering potential confounders. RESULTS: Mean desirability ratio for dependent older people living at home ('middle dependent group') was lower (0.89) compared to independent older people (0.98; regression coefficient -0.09 [95% CI -0.16, -0.02]) and care home patients (1.05; -0.16 [95% CI -0.01, -0.31]). Adjusting for potential confounders or using another statistical approach did not affect the main results. CONCLUSION: Dependent older people living at home were at higher risk of deterioration in sarcopenia status following hospitalization. In-depth studies investigating causes and potential interventions of these findings are needed.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Hospital mortality in COVID-19 patients in Belgium treated with statins, ACE inhibitors and/or ARBs.

The COVID-19 pandemic has disrupted life throughout the world. Newly developed vaccines promise relief to people who live in high-income countries, although vaccines and expensive new treatments are unlikely to arrive in time to help people who live in low-and middle-income countries. The pathogenesis of COVID-19 is characterized by endothelial dysfunction. Several widely available drugs like statins, ACE inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) have immunometabolic activities that (among other things) maintain or restore endothelial cell function. For this reason, we undertook an observational study in four Belgian hospitals to determine whether in-hospital treatment with these drugs could improve survival in 959 COVID-19 patients. We found that treatment with statins and ACEIs/ARBs reduced 28-day mortality in hospitalized COVID-19 patients. Moreover, combination treatment with these drugs resulted in a 3-fold reduction in the odds of hospital mortality (OR = 0.33; 95% CI 0.17-0.69). These findings were in general agreement with other published studies. Additional observational studies and clinical trials are needed to convincingly show that in-hospital treatment with statins, ACEIs/ARBs, and especially their combination saves lives.

PapRIV, a BV-2 microglial cell activating quorum sensing peptide.

Quorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood-brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

BBPpred: Sequence-Based Prediction of Blood-Brain Barrier Peptides with Feature Representation Learning and Logistic Regression.

Blood-brain barrier peptides (BBPs) have a large range of biomedical applications since they can cross the blood-brain barrier based on different mechanisms. As experimental methods for the identification of BBPs are laborious and expensive, computational approaches are necessary to be developed for predicting BBPs. In this work, we describe a computational method, BBPpred (blood-brain barrier peptides prediction), that can efficiently identify BBPs using logistic regression. We investigate a wide variety of features from amino acid sequence information, and then a feature learning method is adopted to represent the informative features. To improve the prediction performance, seven informative features are selected for classification by eliminating redundant and irrelevant features. In addition, we specifically create two benchmark data sets (training and independent test), which contain a total of 119 BBPs from public databases and the literature. On the training data set, BBPpred shows promising performances with an AUC score of 0.8764 and an AUPR score of 0.8757 using the 10-fold cross-validation. We also test our new method on the independent test data set and obtain a favorable performance. We envision that BBPpred will be a useful tool for identifying, annotating, and characterizing BBPs. BBPpred is freely available at http://BBPpred.xialab.info.

Conformational stability as a quality attribute for the cell therapy raw material human serum albumin.

Although human serum albumin (HSA) has been used for many decades, there is still a lack of suitable quality control (QC) attributes. Its current use as a raw material in gene-, cell- and tissue-therapies requires more appropriate functionally-relevant quality attributes and methods. This study investigated the conformational stability of serum albumin using circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) for evaluating the thermal sensitivity, and quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) for assessing the adsorption behavior. Different serum albumin samples were used, encompassing plasma-derived HSA (pHSA), recombinant octanoate-stabilized HSA (rHSA) and bovine serum albumin (BSA). The melting temperature (T m) as well as the onset temperature (T onset) were obtained from the derivative curves of the temperature gradient CD data at 222 nm. The results from DLS, as well as from real-time QCM-D and LSPR silica-adsorption kinetic profiles confirmed the relatively higher conformational stability of the octanoate (fatty acid) containing rHSA, while the additional negative charge resulted in a lower amount adsorbed to the silica surface compared to the non-stabilized HSA and BSA. Adsorption studies further revealed that BSA has a lower conformational stability and undergoes more extensive adsorption-induced spreading compared to the non-stabilized HSA. Collectively, the temperature-based (CD and DLS) as well as adsorption-based biosensor (QCM-D and LSPR) approaches gave congruent and discriminatory information about the conformational stability of different serum albumins, indicating that these techniques provide information on valuable QC attributes.

Environmental risk assessment of the anthelmintic albendazole in Eastern Africa, based on a systematic review.

This study performs an environmental risk assessment (ERA) of the anthelmintic medicine albendazole (ABZ) in the eastern African region. A systematic literature search strategy was applied to obtain quantitative information on the physicochemical characteristics, the metabolization-fate, the ecotoxicity and the environmental occurrence in different countries worldwide serving as model regions. In addition, insilico tools were employed to obtain data on physicochemical characteristics and toxic hazards of ABZ and its metabolites. Moreover, ERA models were used to predict environmental concentrations in different compartments and compare them with the measured environmental concentrations. Finally, the environmental risk of ABZ in the eastern Africa was estimated by calculating the risk quotient (RQ), and its uncertainty estimated by Monte Carlo simulation. The predicted environmental concentrations of ABZ in surface water in the model region based on consumption (1.6-267 ng/L) were within the range of values obtained from the measured environmental concentrations of the same region (0.05-101,000 ng/L). Using these models with adapted input variables for eastern Africa, the predicted surface water concentration in that region was 19,600 ± 150 ng/L (95% CI). The calculated soil concentrations of ABZ in the model regions and the eastern Africa were found to be 0.057 ± 0.0 µg/kg and 0.022 ± 0.0 µg/kg, respectively. The environmental risk expressed as risk quotient of ABZ in eastern Africa estimated for the aquatic compartment (146 ± 1) indicated a significant environmental risk calling on appropriate actions from the competent authorities to reduce this risk in this region.