Polyclonal antibodies towards abrin and ricin-design and potential application for mass spectrometry-based analysis of human biosamples.

The analysis of highly toxic proteins such as abrin and ricin is challenging but comprehensive analytical methods are essential for their unambiguous identification after ingestion. This study pursued three primary aims at detecting abrin and ricin in human biosamples while ensuring that laboratory staff remain protected from direct exposure to these toxic proteins. First, two polyclonal antibodies (pAB) against specific peptides of abrin-A and ricin should be produced.

Laminar Flow Alters EV Composition in HUVECs: A Study of Culture Medium Optimization and Molecular Profiling of Vesicle Cargo.

Endothelial cells (ECs) experience shear stress associated with blood flow. Such shear stress regulates endothelial function by altering cell physiology. Since most cell culture protocols and media compositions are designed for static cultures and experiments with ECs are predominantly conducted under these non-physiological conditions, a model for culturing ECs under flow conditions is developed, which more closely mimics their physiological environment.

The proteomic landscape of trophoblasts unravels calcium-dependent syncytialization processes and beta-chorionic gonadotropin (ß-hCG) production.

Background: The syncytiotrophoblast (STB) layer of the placenta is formed by cell fusion of cytotrophoblasts, acts as a feto-maternal barrier, is required for the production of pregnancy hormones such as chorionic gonadotropin, estradiol and progesterone and is also responsible for feto-maternal mineral exchange such as calcium. Adequate mineral supply and placental hormone production are essential for the maintenance of pregnancy, and disturbances in trophoblast integrity are associated with pregnancy complications. Since knowledge about the identity and expression levels of proteins in trophoblast and syncytiotrophoblast cells is limited so far, we analyzed the proteomes of trophoblast-like and syncytiotrophoblast-like BeWo cells under different calcium conditions.

Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca channel may modulate its β-adrenergic regulation.

Background: The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated Ca1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits.

Cavβ3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis.

Background: Tight control of cytoplasmic Ca concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca (Cav) channels, in modulating Ca signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier.

Methods: We investigated the function of Cavβ3 in BMECs by Ca imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3 (Cavβ3-deficient) mice as controls.

Experimental capture of miRNA targetomes: disease-specific 3'UTR library-based miRNA targetomics for Parkinson's disease.

The identification of targetomes remains a challenge given the pleiotropic effect of miRNAs, the limited effects of miRNAs on individual targets, and the sheer number of estimated miRNA-target gene interactions (MTIs), which is around 44,571,700. Currently, targetome identification for single miRNAs relies on computational evidence and functional studies covering smaller numbers of targets. To ensure that the targetome analysis could be experimentally verified by functional assays, we employed a systematic approach and explored the targetomes of four miRNAs (miR-129-5p, miR-129-1-3p, miR-133b, and miR-873-5p) by analyzing 410 predicted target genes, both of which were previously associated with Parkinson's disease (PD).

Protein profiling of conjunctival impression cytology samples of aniridia subjects.

Purpose: Congenital aniridia is a rare disease, which is in most cases related to PAX6 haploinsufficiency. Aniridia associated keratopathy (AAK) also belongs to ocular signs of congenital aniridia. In AAK, there is corneal epithelial thinning, corneal inflammation, vascularization and scarring.

Outside the limit: questioning the distance restrictions for cooperative miRNA binding sites.

Among the concepts in biology that are widely taken granted is a potentiated cooperative effect of multiple miRNAs on the same target. This strong hypothesis contrasts insufficient experimental evidence. The quantity as well as the quality of required side constraints of cooperative binding remain largely hidden.

Combining mass spectrometry and genetic labeling in mice to report TRP channel expression.

Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction.

Ca transport via TRPV6 is regulated by rapid internalization of the channel.

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane.

Additional data for the mouse TRPV6 expression atlas.

To identify TRPV6 expression in the whole mouse with a cellular resolution we took advantage of TRPV6-IRES-Cre knock-in mice crossed with the enhanced ROSA26-τGFP reporter line. In the resulting TRPV6-IC/eR26-τGFP animals, TRPV6-expressing cells are labeled with τGFP. Data were collected from organs prepared from fixed experimental adult and juvenile TRPV6-IC/eR26τGFP and Cre-negative eR26-τGFP control animals of both sexes.

Localization of the Priming Factors CAPS1 and CAPS2 in Mouse Sensory Neurons Is Determined by Their N-Termini.

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic.

Is the proteomic composition of the salivary pellicle dependent on the substrate material?

Purpose: The use of dental restorative materials is a routine task in clinical dentistry. Upon exposure to the oral cavity, continuous adsorption of salivary proteins and other macromolecules to all surfaces occurs, representing the first step in dental biofilm formation. Different physico-chemical properties of substrate materials potentially influence the composition of the initial biofilm, termed pellicle.

Mutations That Affect the Surface Expression of TRPV6 Are Associated with the Upregulation of Serine Proteases in the Placenta of an Infant.

Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the gene. One mutation results in an in frame stop codon (Rstop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (GR) that, surprisingly, does not affect the Ca permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 GR and Rstop mutants and combinations with wild type TRPV6.

A TRPV6 expression atlas for the mouse.

The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca-selective and has been implicated in mediating transcellular Ca transport and thus maintaining the Ca balance in the body. To characterize its physiological function(s), a detailed expression profile of the TRPV6 channel throughout the body is essential. Capitalizing on a recently established murine Trpv6-reporter strain, we identified primary TRPV6 channel-expressing cells in an organism-wide manner.

Cavβ3 Regulates Ca Signaling and Insulin Expression in Pancreatic β-Cells in a Cell-Autonomous Manner.

Voltage-gated Ca (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP). Similarly, Cavβ3 could affect cytosolic calcium concentration ([Ca ]) in pancreatic β-cells.

Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth.

Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto-maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of deficient placentae, and reduced Ca uptake of primary trophoblasts upon functional deletion of TRPV6. trophoblasts show a distinct calcium-dependent phenotype.

Roughness and wettability of titanium implant surfaces modify the salivary pellicle composition.

This study reports the differences in the protein composition of salivary pellicles formed under in situ conditions on two Titanium (Ti) surfaces, with different roughness and wettability. Smooth pretreatment Ti surfaces (Ti-PT) with an average roughness (Ra) of 0.45 μm and a water contact angle (WCA) of 92.

Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels.

Background/aims: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca influx into pancreatic beta cells by the opening of voltage-gated Ca channels. Transient Receptor Potential Melastatin 3 proteins build Ca permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca influx and increased insulin release.

Cathepsin A contributes to left ventricular remodeling by degrading extracellular superoxide dismutase in mice.

In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM remodeling and left ventricular (LV) dysfunction. Here, we aimed to evaluate the effects of CatA overexpression on LV remodeling.