Comparative Photoaffinity Profiling of Omega-3 Signaling Lipid Probes Reveals Prostaglandin Reductase 1 as a Metabolic Hub in Human Macrophages.

The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA).

Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na reabsorption via the mineralocorticoid receptor (MR).

Tuning the Transglycosylation Reaction of a GH11 Xylanase by a Delicate Enhancement of its Thumb Flexibility.

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution.

STA-55, an Easily Accessible, Broad-Spectrum, Activity-Based Aldehyde Dehydrogenase Probe.

Aldehyde dehydrogenases (ALDHs) convert aldehydes into carboxylic acids and are often upregulated in cancer. They have been linked to therapy resistance and are therefore potential therapeutic targets. However, only a few selective and potent inhibitors are currently available for this group of enzymes.

Development of a Retinal-Based Probe for the Profiling of Retinaldehyde Dehydrogenases in Cancer Cells.

Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs.

Systematic Dual Targeting of Dendritic Cell C-Type Lectin Receptor DC-SIGN and TLR7 Using a Trifunctional Mannosylated Antigen.

Dendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against invading pathogens. PRR ligands are frequently conjugated to tumor-associated antigens in a vaccination strategy to enhance the immune response toward such antigens. One of these PPRs, DC-SIGN, a member of the C-type lectin receptor (CLR) family, has been extensively targeted with Lewis structures and mannose glycans, often presented in multivalent fashion.

New Irreversible α-l-Iduronidase Inhibitors and Activity-Based Probes.

Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme ).

and comparative and competitive activity-based protein profiling of GH29 α-l-fucosidases.

GH29 α-l-fucosidases catalyze the hydrolysis of α-l-fucosidic linkages. Deficiency in human lysosomal α-l-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective and labeling of GH29 α-l-fucosidases from bacteria, mice and man.

Paenilamicin: structure and biosynthesis of a hybrid nonribosomal peptide/polyketide antibiotic from the bee pathogen Paenibacillus larvae.

The spore-forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae.

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS.

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described.

Novel activity-based probes for broad-spectrum profiling of retaining β-exoglucosidases in situ and in vivo.

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases (see picture). These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

Automated solid-phase synthesis of hyaluronan oligosaccharides.

Well-defined fragments of hyaluronic acid (HA) have been obtained through a fully automated solid-phase oligosaccharide synthesis. Disaccharide building blocks, featuring a disarmed glucuronic acid donor moiety and a di-tert-butylsilylidene-protected glucosamine part, were used in the rapid and efficient assembly of HA fragments up to the pentadecamer level, equipped with a conjugation-ready anomeric allyl function.

Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t.

Epithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered beta5t subunit that has an uncharted activity, if any. We synthesized fluorescent epoxomicin probes that were used in a chemical proteomics approach, entailing activity-based profiling, affinity purification, and LC-MS identification, to demonstrate that the beta5t subunit is catalytically active in the murine thymus.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer.

Background: The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice.

The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients.

Circulating tumour cells (CTC) and tumour-related methylated DNA in blood have been separately assessed for their utility as a marker for subclinical metastasis in breast cancer. However, no studies have looked into the relation between the both molecular markers in this type of cancer. In this study, we investigated the correlations between total/methylated DNA and CTC in the blood from metastatic breast cancer patients.

Relapse-free survival in breast cancer patients is associated with a gene expression signature characteristic for inflammatory breast cancer.

Purpose: We hypothesize that a gene expression profile characteristic for inflammatory breast cancer (IBC), an aggressive form of breast cancer associated with rapid cancer dissemination and poor survival, might be related to tumor aggressiveness in non-IBC (nIBC).

Experimental Design: RNA from 17 IBC samples and 40 nIBC samples was hybridized onto Affymetrix chips. A gene signature predictive of IBC was identified and applied onto 1,157 nIBC samples with survival data of 881 nIBC samples.

A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish.

Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development.

Chemical synthesis of picornaviral protein primers of RNA replication.

Naturally occurring nucleopeptidic replication primers (VPg-pUpU) of poliovirus and coxsackie virus were chemically synthesized. The synthesis was accomplished via block-coupling of two minimally protected fragments of the target structures: a short RNA-nucleopeptide and a longer peptide segment containing diverse side-chain functionalities. The synthetic VPg-pUpU of coxsackie virus was characterized by NMR spectroscopy.

Detection of circulating tumour cells in blood by quantitative real-time RT-PCR: effect of pre-analytical time.

Background: We and others have recently explored the use of quantitative real-time RT-PCR analysis for the detection of circulating tumour cells in blood of patients with breast cancer (BC). One major problem in these experiments is the in vitro instability of the cellular RNA. The copy number of mRNA can change during storage and transport at room temperature and this may hamper accurate quantitative measurements of specific transcripts, especially when working with small numbers of target mRNAs.