A comprehensive assessment of the applicability of RoboColumn as a chromatography scale-down model for use in biopharmaceutical process validation.

High-throughput process development has become a standard practice in the biopharmaceutical industry to enable time, cost, and material savings. In downstream biopharmaceutical process development, miniaturized, parallelized chromatography columns, known as RoboColumn, have become the standard for process development, as RoboColumn have shown generally comparable performance to bench and manufacturing scale columns. However, RoboColumn have yet to be widely implemented in process validation and characterization, where many multifactor experiments are typically executed, and there is a strong value proposition for performing high-throughput experiments.

Depletion of pyruvate kinase (PK) activity causes glycolytic intermediate imbalances and reveals a PK-TXNIP regulatory axis.

Objective: Cancer cells convert more glucose into lactate than healthy cells, what contributes to their growth advantage. Pyruvate kinase (PK) is a key rate limiting enzyme in this process, what makes it a promising potential therapeutic target. However, currently it is still unclear what consequences the inhibition of PK has on cellular processes.

Purification of hydrophobic complex antibody formats using a moderately hydrophobic mixed mode cation exchange resin.

Immunoglobulins of complex formats possess great potential for increased biopharmaceutical efficacy. However, challenges arise during their purification as the removal of numerous product-related impurities typically requires several expensive chromatographic steps. Additionally, many complex antibody formats have a high hydrophobicity which impairs the use of conventional mixed mode chromatography.

Isoxazole Nucleosides as Building Blocks for a Plausible Proto-RNA.

The question of how RNA, as the principal carrier of genetic information evolved is fundamentally important for our understanding of the origin of life. The RNA molecule is far too complex to have formed in one evolutionary step, suggesting that ancestral proto-RNAs (first ancestor of RNA) may have existed, which evolved over time into the RNA of today. Here we show that isoxazole nucleosides, which are quickly formed from hydroxylamine, cyanoacetylene, urea and ribose, are plausible precursors for RNA.

Isoform-specific and ubiquitination dependent recruitment of Tet1 to replicating heterochromatin modulates methylcytosine oxidation.

Oxidation of the epigenetic DNA mark 5-methylcytosine by Tet dioxygenases is an established route to diversify the epigenetic information, modulate gene expression and overall cellular (patho-)physiology. Here, we demonstrate that Tet1 and its short isoform Tet1s exhibit distinct nuclear localization during DNA replication resulting in aberrant cytosine modification levels in human and mouse cells. We show that Tet1 is tethered away from heterochromatin via its zinc finger domain, which is missing in Tet1s allowing its targeting to these regions.

Drug screen in iPSC-Neurons identifies nucleoside analogs as inhibitors of (GC) expression in C9orf72 ALS/FTD.

An intronic (GC) expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent.

5-Hydroxymethyl-, 5-Formyl- and 5-Carboxydeoxycytidines as Oxidative Lesions and Epigenetic Marks.

The four non-canonical nucleotides in the human genome 5-methyl-, 5-hydroxymethyl-, 5-formyl- and 5-carboxydeoxycytidine (mdC, hmdC, fdC and cadC) form a second layer of epigenetic information that contributes to the regulation of gene expression. Formation of the oxidized nucleotides hmdC, fdC and cadC requires oxidation of mdC by ten-eleven translocation (Tet) enzymes that require oxygen, Fe(II) and α-ketoglutarate as cosubstrates. Although these oxidized forms of mdC are widespread in mammalian genomes, experimental evidence for their presence in fungi and plants is ambiguous.

Analysis of an Active Deformylation Mechanism of 5-Formyl-deoxycytidine (fdC) in Stem Cells.

The removal of 5-methyl-deoxycytidine (mdC) from promoter elements is associated with reactivation of the silenced corresponding genes. It takes place through an active demethylation process involving the oxidation of mdC to 5-hydroxymethyl-deoxycytidine (hmdC) and further on to 5-formyl-deoxycytidine (fdC) and 5-carboxy-deoxycytidine (cadC) with the help of α-ketoglutarate-dependent Tet oxygenases. The next step can occur through the action of a glycosylase (TDG), which cleaves fdC out of the genome for replacement by dC.

Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice.

Unlabelled: Due to its ability to inhibit prometastatic matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP)-1 has been thought to suppress tumor metastasis. However, elevated systemic levels of TIMP-1 correlate with poor prognosis in cancer patients, suggesting a metastasis-stimulating role of TIMP-1. In colorectal cancer patients, tumor as well as plasma TIMP-1 levels were correlated with synchronous liver metastasis or distant metastasis-associated disease relapse.

The MET Oncogene as a Therapeutical Target in Cancer Invasive Growth.

The MET proto-oncogene, encoding the tyrosine kinase receptor for Hepatocyte Growth Factor (HGF) regulates invasive growth, a genetic program that associates control of cell proliferation with invasion of the extracellular matrix and protection from apoptosis. Physiologically, invasive growth takes place during embryonic development, and, in post-natal life, in wound healing and regeneration of several tissues. The MET oncogene is overexpressed and/or genetically mutated in many tumors, thereby sustaining pathological invasive growth, a prerequisite for metastasis.

Tumor cell-derived Timp-1 is necessary for maintaining metastasis-promoting Met-signaling via inhibition of Adam-10.

In many different tumor entities, increased expression of tissue inhibitor of metalloproteinases-1 (Timp-1) is associated with poor prognosis. We previously reported in mouse models that elevated systemic levels of Timp-1 induce a gene expression signature in the liver microenvironment increasing the susceptibility of this organ to tumor cells. This host effect was dependent on increased activity of the hepatocyte growth factor (Hgf)/hepatocyte growth factor receptor (Met) signaling pathway.

Full-length L1CAM and not its Δ2Δ27 splice variant promotes metastasis through induction of gelatinase expression.

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform.

Identification of a survival-independent metastasis-enhancing role of hypoxia-inducible factor-1alpha with a hypoxia-tolerant tumor cell line.

During tumor progression, malignant cells must repeatedly survive microenvironmental stress. Hypoxia-inducible factor-1 (HIF-1) signaling has emerged as one major pathway allowing cellular adaptation to stress. Recent findings led to the hypothesis that HIF-1alpha may enhance the metastatic potential of tumor cells by a survival-independent mechanism.

A disintegrin and metalloproteinase-10 (ADAM-10) mediates DN30 antibody-induced shedding of the met surface receptor.

Met, the tyrosine kinase receptor for the hepatocyte growth factor is a prominent regulator of cancer cell invasiveness and has emerged as a promising therapeutic target. Binding of the anti-Met monoclonal antibody DN30 to its epitope induces the proteolytic cleavage of Met, thereby impairing the invasive growth of tumors. The molecular mechanism controlling this therapeutic shedding process has so far been unknown.