Fiber-based biomaterial scaffolds for cell support towards the production of cultivated meat.

The in vitro production of animal-derived foods via cellular agriculture is emerging as a key solution to global food security challenges. Here, the potential for fiber-based scaffolds, including silk and cotton, in the cultivation of muscle cells for tissue formation was pursued. Mechanical properties and cytocompatibility with the mouse myoblast cell line C2C12 and immortalized bovine muscle satellite cells (iBSCs) were assessed, as well as pre-digestion options for the materials due to their resilience within the human digestive track.

A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies.

Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.

Confocal Raman characterization of different protein desorption behaviors from chromatographic particles.

Confocal Raman spectroscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure protein desorption from chromatographic particles. Monoclonal antibody was loaded onto the Fractogel EMD SO3 (M) cation exchanger at either pH 5 or pH 4.

Receptor tyrosine kinase inhibitor profiling using bead-based multiplex sandwich immunoassays.

Receptor tyrosine kinases (RTK) are important targets in drug discovery processes. Studying the phosphorylation pattern of RTKs enables the determination of their activation and inactivation states. Multiplex bead-based sandwich immunoassays are powerful tools for measuring the phosphorylation state of key regulators within cellular signalling networks.

Evaluation of a urinary kidney biomarker panel in rat models of acute and subchronic nephrotoxicity.

Several novel urinary kidney biomarkers were recently approved by the US-FDA and EMA for improved detection of nephrotoxicity, but few data regarding their performance are publicly available so far. In this study, we investigated the potential of some of the newly accepted makers (Kim-1, β-2-microglobulin, cystatin C, clusterin) along with six additional urinary key proteins of kidney injury (GST-α, Timp-1, VEGF, calbindin, NGAL/lipocalin-2, osteopontin) to detect proximal tubule damage in the rat model studying either acute drug-induced kidney injury or subchronic nephrotoxicity. Candidate proteins were measured in urine samples obtained from rats treated with gentamicin (0, 60 and 120 mg/kg bw for 7 days), BI-3 [3-pyrrolidineacetic acid, 5-[[[4'-[imino[(methoxycarbonyl) amino]methyl] [1,1'-biphenyl]-4-yl]oxy]methyl]-2-oxo-, methyl ester,(3S-trans)] (0, 100, and 1000 mg/kg bw for up to 14 days) or with the mycotoxin ochratoxin A (OTA) (0, 21, 70 and 210 μg/kg bw for up to 90 days) using a Luminex(®) xMAP(®) platform.

Sequential multiplex analyte capturing for phosphoprotein profiling.

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays.

Microsphere-based co-immunoprecipitation in multiplex.

Co-immunoprecipitation (co-IP) is a prominent technique for evaluating protein-protein interactions. Currently, large quantities of protein are required to perform co-IP followed by mass spectrometric or Western blot analyses of the interacting proteins. Here catenin-cadherin complexes were employed to establish a multiplexed microsphere-based co-immunoprecipitation (micro co-IP) protocol that allows the analysis of different complexes of a given protein with various interacting proteins within a single experiment using a limited amount of sample material.

Sphingolipid metabolism in neural cells.

Sphingolipids were discovered more than a century ago in the brain. Cerebrosides and sphingomyelins were named so because they were first isolated from neural tissue. Although glycosphingolipids and especially those containing sialic acid in their oligosaccharide moiety are particularly abundant in the brain, sphingolipids are ubiquitous cellular membrane components.

Host cell targets in HCV therapy: novel strategy or proven practice?

The development of novel antiviral drugs against hepatitis C is a challenging and competitive area of research. Progress of this research has been hampered due to the quasispecies nature of the hepatitis C virus, the absence of cellular infection models and the lack of easily accessible and highly representative animal models. The current combination therapy consisting of interferon-alpha and ribavirin mainly acts by supporting host cell defence.

Identification of inhibitors for a virally encoded protein kinase by 2 different screening systems: in vitro kinase assay and in-cell activity assay.

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates.

Expression of gastrointestinal glutathione peroxidase is inversely correlated to the presence of hepatitis C virus subgenomic RNA in human liver cells.

There is great medical need to develop novel therapies for treatment of human hepatitis C virus (HCV). By gene expression analysis of three HCV-subgenomic RNA replicon cell lines, we identified cellular proteins whose expression is affected by the presence of HCV and therefore may serve as drug targets. Data from cDNA array filter hybridization, as well as from Northern and Western blotting, revealed that the gastrointestinal-glutathione peroxidase (GI-GPx) was drastically down-regulated (up to 20-fold) in all replicon cell lines tested.

Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity.

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication.

Protein kinase C eta is associated with progression of renal cell carcinoma (RCC).

Background: The expression of all eleven PKC-isoforms in clear cell RCCs (ccRCC) and in the corresponding normal renal tissue was quantified. A possible association of PKC-isoforms with histopathological parameters was examined.

Materials And Methods: Proteins were isolated from tumor and normal tissue of 43 patients with ccRCC.

Exploiting features of adenovirus replication to support mammalian kinase production.

Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity.

The 3'-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR.

The expression of the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) is controlled by the stability of its mRNA. While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2 = 14 h), its half-life time is drastically reduced (t1/2 = 2 h) in cells treated with phorbol esters to activate protein kinase C (PKC) or treated with growth factors. In a first step to study the underlying mechanism we identified both a cis-element on the MARCKS mRNA and the corresponding trans-acting factors.

Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein.

The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A.

Loss of tumor suppressor protein PTEN during renal carcinogenesis.

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) encodes a dual specific protein and phospholipid phosphatase that affects cell proliferation, apoptosis and migration. In our study, we examined protein expression of PTEN in renal carcinogenesis. PTEN protein levels were examined in 42 clear cell renal cell carcinomas (ccRCC) and oncocytomas as well as in the corresponding normal renal tissue of the same patients using Western blot analysis.