Plasma levels of organic acids associated with the gut microbiome display significant alterations in neuroendocrine tumor patients.

Introduction: The gut microbiome, allegedly involved in both healthy homeostasis and development of disease, is found to be associated with several types of cancer. Short chain fatty acids (SCFAs), important metabolites derived from the gut microbiota, are described to carry both protective and promoting features in cancer development. Limited research exists on neuroendocrine tumors (NET) and their association with microbiota derived SCFAs.

Validation of two LCHRMS methods for large-scale untargeted metabolomics of serum samples: Strategy to establish method fitness-for-purpose.

Untargeted metabolomics by LCHRMS is a powerful tool to enhance our knowledge of pathophysiological processes. Whereas validation of a bioanalytical method is customary in most analytical chemistry fields, it is rarely performed for untargeted metabolomics. This study aimed to establish and validate an analytical platform for a long-term, clinical metabolomics study.

Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.

The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques.

Polyethylene glycol (PEG) as a broad applicability marker for LC-MS/MS-based biodistribution analysis of nanomedicines.

Polyethylene glycol (PEG) conjugation (PEGylation) is a well-established strategy to improve the pharmacokinetic and biocompatibility properties of a wide variety of nanomedicines and therapeutic peptides and proteins. This broad use makes PEG an attractive 'allround' candidate marker for the biodistribution of such PEGylated compounds. This paper presents the development of a novel strategy for PEG quantification in biological matrices.

Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.

Purine metabolism is essential for all known living creatures, including humans in whom elevated serum concentration of purine break-down product uric acid (UA) is probably an independent risk factor for mortality, type 2 diabetes and cardiovascular events. An automated multiplex assay that measures several purine metabolites could therefore prove useful in many areas of medical, veterinary and biological research. The aim of the present work was to develop a sensitive LC-MS/MS method for simultaneous quantitation of xanthine, hypoxanthine, UA, allantoin, and creatinine in biobanked urine samples.

Mucosal Metabolomic Profiling and Pathway Analysis Reveal the Metabolic Signature of Ulcerative Colitis.

The onset of ulcerative colitis (UC) is characterized by a dysregulated mucosal immune response triggered by several genetic and environmental factors in the context of host-microbe interaction. This complexity makes UC ideal for metabolomic studies to unravel the disease pathobiology and to improve the patient stratification strategies. This study aims to explore the mucosal metabolomic profile in UC patients, and to define the UC metabolic signature.

Lipidomics in Ulcerative Colitis Reveal Alteration in Mucosal Lipid Composition Associated With the Disease State.

Background: The onset of ulcerative colitis (UC) is associated with alterations in lipid metabolism and a disruption of the balance between pro- and anti-inflammatory molecules. Only a few studies describe the mucosal lipid biosignatures during active UC. Moreover, the dynamics of lipid metabolism in the remission state is poorly defined.

Lys-C/Trypsin Tandem-Digestion Protocol for Gel-Free Proteomic Analysis of Colon Biopsies.

The protocol presented was specifically optimized for in-depth analysis of the human colon mucosa proteome. After cell lysis in a sodium deoxycholate/urea buffer, a tandem digestion with Lys-C and trypsin was performed. Prior to LC-MS/MS analysis, peptides were TMT-labeled and fractionated by high pH reversed-phase spin columns.

Ulcerative colitis: functional analysis of the in-depth proteome.

Background: Ulcerative colitis (UC) is one major form of inflammatory bowel disease. The cause and the pathophysiology of the disease are not fully understood and we therefor aim in this study to identify important pathophysiological features in UC from proteomics data.

Methods: Colon mucosa biopsies from inflamed tissue of untreated UC patients at diagnosis and from healthy controls were obtained during colonoscopy.

A Quantitative Analysis of Colonic Mucosal Oxylipins and Endocannabinoids in Treatment-Naïve and Deep Remission Ulcerative Colitis Patients and the Potential Link With Cytokine Gene Expression.

Background: The bioactive metabolites of omega 3 and omega 6 polyunsaturated fatty acids (ω-3 and ω-6) are known as oxylipins and endocannabinoids (eCBs). These lipid metabolites are involved in prompting and resolving the inflammatory response that leads to the onset of inflammatory bowel disease (IBD). This study aims to quantify these bioactive lipids in the colonic mucosa and to evaluate the potential link to cytokine gene expression during inflammatory events in ulcerative colitis (UC).

Starvation induces rapid degradation of selective autophagy receptors by endosomal microautophagy.

It is not clear to what extent starvation-induced autophagy affects the proteome on a global scale and whether it is selective. In this study, we report based on quantitative proteomics that cells during the first 4 h of acute starvation elicit lysosomal degradation of up to 2-3% of the proteome. The most significant changes are caused by an immediate autophagic response elicited by shortage of amino acids but executed independently of mechanistic target of rapamycin and macroautophagy.

Straightforward Protocol for Gel-Free Proteomic Analysis of Adipose Tissue.

After conducting systematic and quantitative comparisons of different sample preparation techniques regarding their capability to efficiently and reproducibly recover proteins from biopsies, we present here our superior protocol for extracting proteins from low amounts of adipose tissue. Adipose tissue as a matrix in bottom-up proteomics is challenging due to the extremely high lipid content.The lysis buffer utilized contains the detergent sodium deoxycholate, which does not impair the activity of trypsin and therefore enables direct digestion without detergent removal steps.

The Proteome of Ulcerative Colitis in Colon Biopsies from Adults - Optimized Sample Preparation and Comparison with Healthy Controls.

Purpose: The purpose of the study was to optimize the sample preparation and to further use an improved sample preparation to identify proteome differences between inflamed ulcerative colitis tissue from untreated adults and healthy controls.

Experimental Design: To optimize the sample preparation, we studied the effect of adding different detergents to a urea containing lysis buffer for a Lys-C/trypsin tandem digestion. With the optimized method, we prepared clinical samples from six ulcerative colitis patients and six healthy controls and analysed them by LC-MS/MS.

The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release.

Unlabelled: The minor capsid protein of human BK polyomavirus (BKPyV), VP2, and its N-terminally truncated form, VP3, are both important for viral entry. The closely related simian virus 40 (SV40) reportedly produces an additional truncated form of VP2/3, denoted VP4, apparently functioning as a viroporin promoting progeny release. The VP4 open reading frame is conserved in some polyomaviruses, including BKPyV.

General Approach To Determine Disulfide Connectivity in Cysteine-Rich Peptides by Sequential Alkylation on Solid Phase and Mass Spectrometry.

Within the field of bioprospecting, disulfide-rich peptides are a promising group of compounds that has the potential to produce important leads for new pharmaceuticals. The disulfide bridges stabilize the tertiary structure of the peptides and often make them superior drug candidates to linear peptides. However, determination of disulfide connectivity in peptides with many disulfide bridges has proven to be laborious and general methods are lacking.

Proteomics of hydrophobic samples: Fast, robust and low-cost workflows for clinical approaches.

In a comparative study, we investigated the influence of nine sample preparation workflows and seven different lysis buffers for qualitative and quantitative analysis of the human adipose tissue proteome. Adipose tissue is not just a fat depot but also an endocrine organ, which cross-talks with other tissue types and organs throughout the body, like liver, muscle, pancreas, and brain. Its secreted molecules have an influence on the nervous, immune, and vascular system, thus adipose tissue plays an important role in the regulation of whole-body homeostasis.

β-CD-dextran polymer for efficient sequestration of cholesterol from phospholipid bilayers: Mechanistic and safe-toxicity investigations.

The aim of this work was to investigate the suitability of β-cyclodextrin-dextran (BCD-dextran) polymer as cholesterol sequestering agent in vitro. For this purpose, BCD-dextran-cholesterol complexation was studied by phase solubility studies as well as with a specifically designed in vitro model based on giant unilamellar vesicles (GUVs) to evaluate the ability of this polymer to sequestrate cholesterol from phospholipid bilayers. Cholesterol-sequestering ability of BCD-dextran was also investigated on different cell lines relevant for the hematopoietic system and results were correlated to cells toxicity.

New applications of phospholipid vesicle-based permeation assay: permeation model mimicking skin barrier.

The phospholipid vesicle-based permeation assay (PVPA), based on a tight barrier composed of liposomes mimicking cells, is providing an opportunity to predict passive drug permeability through biological membranes. Although it was originally developed to mimic the intestinal epithelia, this study focuses on its potential as a simple and affordable skin model for transdermal permeation of drug candidates and evaluation of various drugs and formulations at an early development stage. The changes induced in lipid composition of the lipid-based barriers to better mimic the in vivo stratum corneum lipid composition required optimization of liposomal properties and manufacturing conditions applied in barrier formation.

Adenylylation, MS, and proteomics--Introducing a "new" modification to bottom-up proteomics.

Although the addition of a 5'-adenosine phosphodiester group to proteins, called adenylylation, has been known for decades, the possibility that adenylylation could be a molecular switch in cellular signaling pathways has emerged recently. The distinct mass shift upon adenylation of threonine or tyrosine residues renders it a good target for MS detection and identification; however, the fragmentation of adenylylated peptides derived from proteolytic digestion of adenylylated proteins has not yet been systematically investigated. Here, we demonstrate that adenylylated peptides show loss of parts of the adenosine monophosphate (AMP) upon different fragmentation techniques.

Anticancer activity of small amphipathic β²,²-amino acid derivatives.

We report the anticancer activity from screening of a series of synthetic β(2,2)-amino acid derivatives that were prepared to confirm the pharmacophore model of short cationic antimicrobial peptides with high anti-Staphylococcal activity. The most potent derivatives against human Burkitt's lymphoma (Ramos) cells displayed IC(50) values below 8 μM, and low toxicity against human red blood cells (EC(50) > 200 μM). A more than 5-fold preference for Ramos cancer cells compared to human lung fibroblasts (MRC-5 cells) was also obtained for the most promising β(2,2)-amino acid derivative 3-amino-N-(2-aminoethyl)-2,2-bis(naphthalen-2-ylmethyl)propanamide (5c).

Anticancer mechanisms of action of two small amphipathic β(2,2)-amino acid derivatives derived from antimicrobial peptides.

We have recently discovered that small antimicrobial β(2,2)-amino acid derivatives (Mw<500) also display activity against cancer cells. To explore their drug potential, we have presently investigated the mechanisms of action of two derivatives BAA-1 (IC(50) 8.1μg/ml) and BAA-2 (IC(50) 3.

Metabolism of small antimicrobial β(2,2)-amino acid derivatives by murine liver microsomes.

We have investigated the in vitro metabolism of three small antimicrobial β(2,2)-amino acid derivatives (M (w) < 500) that are highly potent against methicillin resistant Staphylococcus aureus, and are among the first compounds designed from small cationic antimicrobial peptides with potential for oral administration. The β(2,2)-amino acid derivatives are virtually completely resistant against degradation by proteases, and to further explore their drug potential, we have investigated the hepatic Phase I metabolism of this class of antimicrobial compounds. The β(2,2)-amino acid derivatives were incubated with murine liver microsomes and the metabolites analyzed semi-quantitatively by HPLC-MS and qualitatively by ultra performance liquid chromatography coupled to a tandem mass spectrometer which enabled identification of the metabolites by careful interpretation of the collision activated dissociation spectra.

Synthesis of anticancer heptapeptides containing a unique lipophilic β(2,2) -amino acid building block.

We report a series of synthetic anticancer heptapeptides (H-KKWβ(2,2) WKK-NH(2)) containing eight different central lipophilic β(2,2) -amino acid building blocks, which have demonstrated high efficiency when used as scaffolds in small cationic antimicrobial peptides and peptidomimetics. The most potent peptides in the present study had IC(50) values of 9-23 µm against human Burkitt's lymphoma and murine B-cell lymphoma and were all nonhaemolytic (EC(50)  > 200 µm). The most promising peptide 10e also demonstrated low toxicity against human embryonic lung fibroblast cells and peripheral blood mononuclear cells and exceptional proteolytic stability.

Synthesis of cationic antimicrobial β(2,2)-amino acid derivatives with potential for oral administration.

We have prepared a series of highly potent achiral cationic β(2,2)-amino acid derivatives that fulfill the Lipinski's rule of five and that contain the basic structural requirements of short cationic antimicrobial peptides. Highest antimicrobial potency was observed for one of the smallest β(2,2)-amino acid derivatives (M(w) 423.6) exhibiting a MIC of 3.