Membrane proteins govern many important functions in cells via dynamic oligomerization into active complexes. However, analytical methods to study their distribution and functional state in relation to the cellular structure are currently limited. Here, we introduce a technique for studying single-membrane proteins within their native context of the intact plasma membrane. SKBR3 breast cancer cells were grown on silicon microchips with thin silicon nitride windows. The cells were fixed, and the epidermal growth factor receptor ErbB2 was specifically labeled with quantum dot (QD) nanoparticles. For correlative fluorescence- and liquid-phase electron microscopy, we enclosed the liquid samples by chemical vapor deposited (CVD) graphene films. Depending on the local cell thickness, QD labels were imaged with a spatial resolution of 2 nm at a low electron dose. The distribution and stoichiometric assembly of ErbB2 receptors were determined at several different cellular locations, including tunneling nanotubes, where we found higher levels of homodimerization at the connecting sites. This experimental approach is applicable to a wide range of cell lines and membrane proteins and particularly suitable for studies involving both inter- and intracellular heterogeneity in protein distribution and expression.

There is evidence of blood-borne miRNA signatures for various human diseases. To dissect the origin of disease-specific miRNA expression in human blood, we separately analyzed the miRNome of different immune cell subtypes, each in lung cancer patients and healthy individuals. Each immune cell type revealed a specific miRNA expression pattern also dependinging on the cell origin, line of defense, and function. The overall expression pattern of each leukocyte subtype showed great similarities between patients and controls. However, for each cell subtype we identified miRNAs that were deregulated in lung cancer patients including hsa-miR-21, a well-known oncomiR associated with poor lung cancer prognosis that was up-regulated in all leukocyte subtype comparisons of cancer versus controls. While the miRNome of cells of the adaptive immune system allowed only a weak separation between patients and controls, cells of the innate immune system allowed perfect or nearly perfect classification. Leukocytes of lung cancer patients show a cancer-specific miRNA expression profile. Our data also show that cancer specific miRNA expression pattern of whole blood samples are not determined by a single cell type. The data indicate that additional blood components, like erythrocytes, platelets, or exosomes might contribute to the disease specificity of a miRNA signature.

Consolidated knowledge is accumulating as to the role of epigenetic regulatory mechanisms in the physiology of vascular development and vascular tone as well as in the pathogenesis of cardiovascular disease. The modulation of gene expression through modification of the epigenome by structural changes of the chromatin architecture without alterations of the associated genomic DNA sequence is part of the cellular response to environmental changes. Such environmental conditions, which are finally being translated into adaptations of the cardiovascular system, also comprise pathological conditions such as atherosclerosis or myocardial infarction. This review summarizes recent findings on the epigenetics of vascular regulation and disease and presents nutritional and pharmacological approaches as novel epigenetic strategies in the prevention and treatment of cardiovascular disease.

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.

Inflammatory endothelial processes are regulated by the nuclear factor-κB (NF-κB) pathway, which involves phosphorylation of p65. Because p65 is a substrate of CK2, we herein investigated, whether this pleiotropic protein kinase may be a beneficial anti-inflammatory target. For this purpose, we analyzed in human dermal microvascular endothelial cells (HDMEC) the effect of CK2 inhibition by quinalizarin and CX-4945 on cell viability, adhesion molecule expression and NF-κB pathway activation. Leukocyte binding to HDMEC was assessed in an in vitro adhesion assay. Dorsal skinfold chambers in BALB/c mice were used to study leukocyte-endothelial cell interaction and leukocyte transmigration by means of repetitive intravital fluorescence microscopy and immunohistochemistry. We found that quinalizarin and CX-4945 effectively suppressed the activity of CK2 in HDMEC without affecting their viability. This was associated with a significant down-regulation of tumor necrosis factor (TNF)-α-induced E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression due to a reduction of shuttling, phosphorylation and transcriptional activity of the NF-κB complex. In consequence, leukocyte binding to quinalizarin- and CX-4945-treated HDMEC was diminished. Finally, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in dorsal skinfold chambers exposed to TNF-α in vivo. These findings indicate that CK2 is a key regulator of leukocyte-endothelial cell interaction in inflammation by regulating the expression of E-selectin, ICAM-1 and VCAM-1 via affecting the transcriptional activity of the NF-κB complex. Accordingly, CK2 represents a promising target for the development of novel anti-inflammatory drugs.