Journal of Cellular Biotechnology

The multifunctional proteasome activator PA28γ is involved in proteolytic pathways. Not surprisingly, upregulation of PA28γ contributes to tumor growth and progression. Therefore, CRISPR/Cas9 technology was used to specifically knockout the PSME3 gene encoding PA28γ in S462 tumor cells and HEK 293 cells. RNA guide sequences specific for PSME3 were designed and cloned into the lentiCRISPRv2 expression vector. Initially, successful Cas9-mediated gene-editing was verified by T7 endonuclease I DNA-mismatch assay. After the clonal selection, several clones showed no detectable PA28γ expression on protein level. Subsequently, a complete knockout of PA28γ was confirmed on genomic level by sequencing.

Initial functional analysis of effector caspase activity in PA28γ knockout cells showed a higher basal activity and an increased sensitivity towards doxorubicin/ABT-737-induced apoptosis. Generally, this confirms the role of PA28γ as an anti-apoptotic regulator. To our knowledge, this is the first study addressing the role of proteasomal regulators in malignant peripheral nerve sheath tumor (MPNST) derived S462 cells.

Quantitative analysis of a target molecule in a microbead-based fluorescent assay requires a specific labeling procedure. For nucleic acid analysis the hybridization with florescent labeled oligonucleotides is the most common method. However, disadvantages are the necessity for direct labeling of probes and the sensitivity to detect low amounts of target molecules. In this study we established an alternative detection method for biomolecules on microbeads, the tyramide signal amplification (TSA). Hereby, biomolecules are detected by enzymatically activated and fluorophore-conjugated tyramides that bind to specific protein residues. This method has proven to be a versatile and robust enzyme amplification technique for sensitive immunohistochemical detection. Now, we present the feasibility of the TSA procedure to detect hybridized biotinylated oligonucleotide probes bound to protein coated microbead surfaces TSA was performed using fluorescent, size-encoded and streptavidin coated microbeads that were loaded with dual-biotinylated DNA capture probes, prepared from polymerase chain reaction. Beside streptavidin alone for surface coating of those microbeads, we applied different quantities of streptavidin in combination with bovine serum albumin, immunoglobulin G or Protein G/A, to check for positive effects on the resulting signal intensities through specific binding of tyramide molecules. For this method, streptavidin turned out as appropriate protein for the surface binding, without the need for further molecules. In comparison to a standard detection with common streptavidin-fluorophore-conjugates TSA showed its advantage in the detection of low probe amounts down to a concentration of 3.3·10^-4 ng/μL.

Microscopy plays a major role in the investigation of several diseases in human diagnostics and veterinary medicine. The microscopic analysis is mostly carried out in medical laboratories and requires specialised staff or expensive equipment. Therefore, providing results is time-consuming and far from the point-of-care where a fast diagnosis can be life-saving. Especially in infrastructural underdeveloped areas, with a lack of medical facilities and expert knowledge, this drawback is visible. Because of this, researchers started to develop “mobile microscopes” that can be used with a smartphone to enable everyone to be a specialist. This review is meant to give an overview about developed smartphone based mobile microscopes, their different construction methods, their technical advantages and disadvantages, their possible diagnostic applications and their limitations.

Chemotherapy and radiation therapy are used in malignant oncological diseases to increase the level of DNA double strand breaks (DSBs) in tumor cells. Unrepaired DSBs may either kill a cell or induce terminal arrest. Those DNA damages can be detected by fluorescence imaging of phosphorylated histone protein H2AX (γH2AX). Peripheral blood mononuclear cells (PBMCs) are easily available human material, which are usually stored by cryopreservation for repeated experiments or postponed analysis.

As DNA DSBs can be introduced through several different stressors, it was of interest to investigate the potential impact of cryopreservation, on the formation of DSBs in human PBMCs. The PBMCs were cryopreserved in four different media, containing different proportions of Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) as taken from the literature. Immunofluorescence staining of γH2AX was performed on freshly isolated and cryopreserved PBMCs.

A significant reduction on relative γH2AX level, after treatment with 100 μM etoposide (ETP), was observed for cryopreserved cells in medium containing DMEM, which seemed to be the limiting factor on DNA DSB formation. No change was observed for untreated PBMCs. Therefore, DSBs of cryopreserved human PBMCs could be used to investigate the influence of chemotherapeutic drugs or radiation therapy, assuming that the cryopreservation follows a standardized protocol with a tight control of potential DSB inducing stressors.

Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been found to be modulators of immune function. Additionally, they may affect the growth of colorectal cancer (CRC). With the advent of novel treatment approaches in oncology targeting immune checkpoint inhibition and aiming to boost the immune response against tumors the exact role of n-3 and n-6 PUFA in inflammation as well as in CRC needs to be re-evaluated in order to understand potential interactions with these new treatment options. Interestingly, for the cyclooxygenase (COX) inhibitor aspirin a possible synergistic effect together with an anti-programmed cell death protein-1 (anti-PD-1) antibody has been shown. However, could high n-3 PUFA be disadvantageous in the context of immune therapy due to an immune suppressive effect that has been described for these fatty acids in the past, or could they also enhance the effect of immune checkpoint inhibition?

In this paper, we explore this topic and show in a small experimental series that incubation of human peripheral blood mononuclear cells (PBMCs) with the n-3 PUFA docosahexaenoic acid (DHA) significantly decreases CRC-cell supernatant-triggered secretion of IL-10 and increases secretion TNF-α, while the n-6 PUFA arachidonic acid (AA) reduced TNF-α secretion. These changes in cytokine secretion upon incubation with DHA thus indicate a possible enhancing effect of n-3 PUFA on an anti-tumor immune response.