Search

Oesophageal squamous cell carcinomas and oesophageal adenocarcinomas display distinct patterns of ErbB expression and dimers. The functional effects of specific ErbB homo- or heterodimers on oesophageal (cancer) cell behaviour, particularly invasion of early carcinogenesis remains unknown. Here, a new cellular model system for controlled activation of EGFR or HER2 and EGFR/HER2 or HER2/HER3 homo- and heterodimers was studied in non-neoplastic squamous oesophageal epithelial Het-1A cells. EGFR, HER2 and HER3 intracellular domains (ICDs) were fused to dimerization domains (DmrA / DmrA and DmrC), and transduced into Het-1A cells lacking ErbB expression. Dimerization of EGFR, HER2 or EGFR/HER2, HER2/HER3 ICDs was induced by synthetic ligands (A/A or A/C dimerizers). This was accompanied by phosphorylation of the respective EGFR, HER2 and HER3 ICDs and activation of distinct down-stream signalling pathways, such as PLCγ1, Akt, STAT and Src family kinases. Phenotypically, ErbB homo- and heterodimers caused cell rounding and non-apoptotic blebbing in EGFR/HER2 and HER2/HER3 heterodimer cells. In a Transwell assay, cell migration velocity was elevated in HER2-dimer as compared to empty vector cells. In addition, HER2-dimer cells showed in increased cell invasion, reaching significance for induced HER2/HER3 heterodimers (p=0.015). Importantly, in three-dimensional organotypic cultures, empty vector cells grew as a superficial cell layer, resembling oesophageal squamous epithelium. In contrast, induced HER2-dimer cells (HER2 homodimers) were highly invasive into the matrix and formed cell clusters. This was associated with partial loss of CK7 (when HER2 homodimers were modelled) and p63 (when EGFR/HER2 heterodimers were modelled), which suggests a change or loss of squamous cell differentiation. Controlled activation of specific EGFR, HER2 and HER3 homo- and heterodimers caused oesophageal squamous epithelial cell migration and/or invasion, especially in a three dimensional microenvironment, thereby functionally identifying ErbB homo- and heterodimers as important drivers of oesophageal carcinogenesis.

Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3(-/-) ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.

Background The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard‐to‐transfect cells remains challenging. Methods In a proof‐of‐principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high‐fidelity Cas9 variant was then transfected into patient‐derived BOECs using a ribonucleoprotein complex and a single‐strand DNA oligonucleotide. In addition, patient‐specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation. Results Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9‐induced mutations in predicted off‐target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1‐deficient BOECs displayed increased resistance to stress‐induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1‐deficient BOECs largely replaced corrected as well as heterozygous BOECs. Conclusion We here demonstrate that a non‐viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard‐to‐transfect endothelial cells (ECs). Comparing patient‐derived isogenic CCM1+/+, CCM1+/−, and CCM1−/− ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.

Objective Natural killer (NK) cells are the first defense against malignant cells, and their functions are severely impaired in individuals with obesity. However, it is not known whether functions can be re-activated after weight loss. The alterations of NK cell functions after fat mass reduction were investigated. Methods Thirty-two healthy adults with obesity were divided into control and experimental groups. Participants of the experimental group performed a 3-month program of exercise training and nutrition. Anthropometric, physiological, and metabolic parameters and plasma adipocytokines were determined. Peripheral blood mononuclear cells were analyzed by means of flow cytometry and Western blot assay for various NK cell-specific functional parameters and leptin signaling components. NK cell-mediated cytotoxicity assay with leptin stimulation was performed. Results Male participants significantly decreased their body fat mass (P

The double burden of under- and overnutrition profoundly affects human health globally. According to the World Health Organization, obesity and diabetes rates have almost doubled worldwide since 1980, and, in 2011, more than 40 million children under 5 years of age were overweight. Ecologic factors, parental genetics and fitness, and the intrauterine environment significantly influence the likelihood of offspring developing the dysmetabolic diathesis of obesity. This report examines the effects of these factors, including preconception, intrauterine and postnatal energy balance affecting programming of transgenerational transmission, and development of chronic diseases later in life-in particular, diabesity and its comorbidities.

Aurora A “over-”expression may induce supernumerary centrosomes, respective multipolar mitoses, and aneuploidy. Here, we examined Aurora A positive multipolar mitoses in aneuploid, microsatellite-stable (MSS, “CIN-type”) versus near-diploid, microsatellite-instable (MSI, “MIN-type”) colorectal carcinomas (CRC) and CRC cell lines as well as the effect of Aurora A inhibition in CRC cell lines. In situ, three-dimensional immunofluorescence (3D-IF) revealed Aurora A positive multipolar mitoses in both CIN- (n = 8) and MIN- (n = 10) type primary CRCs with similar frequencies (CIN: 27 ± 14%; MIN: 34 ± 14%, P = 0.224). In vitro, Aurora A positive multipolar mitoses were detected in asynchronized or thymidine synchronized CIN-type (HT29, CaCo-2), but not MIN-type (HCT116, DLD-1) CRC cells. Nocodazole treatment arrested mitotic cells with multiple centrosomal Aurora A signals in CIN- and MIN-type CRC cells, albeit to a lower extent in CaCo-2 cells. This was associated with concomitant activation of Aurora A (T288 phosphorylation) and Polo-like kinase 1 (PLK-1, T210 phosphorylation). Aurora A inhibition by siRNA resulted in increased apoptosis (>50%) in all cell lines, but did not abolish PLK-1 expression. Double 3D-IF revealed that Aurora A siRNA treated, still viable CIN-type (HT29, CaCo-2) CRC cells were Aurora A negative and mostly in prophase/(pro)metaphase with maintained phosphorylated PLK-1 T210 expression. Aurora A positive multipolar mitoses occur in both aneuploid, CIN- and near-diploid MIN-type CRCs. This appears to be largely independent of Aurora A expression alone. Although Aurora A inhibition causes apoptosis in both CIN- and MIN-type CRC cells, remaining PLK-1 activation by other factors may affect therapeutic Aurora inhibition. © 2011 Wiley Periodicals, Inc.

In diet-induced obese rats, leptin-mediated natural killer (NK) cell activation has been demonstrated to be impaired by abrogated intracellular JAK2-STAT3 signaling. The contribution of the obese microenvironment to this NK cell dysfunction and its reversibility remains elusive. In this study, the functions of NK cells from diet-induced obese rats after adoptive transfer into lean littermates were investigated using in vivo and in vitro approaches. Endogenous NK cells of normal-weight and diet-induced obese F344 rats were depleted in vivo. Then, NK cells from either normal-weight or obese donors were transferred. The numbers of peripheral blood NK cells were analyzed by fluorescence-activated cell sorting (FACS) and the distribution pattern of NK cells in lung and spleen by immunohistochemistry. Ob-R expression was evaluated by immunohistology and activation of intracellular target proteins of Ob-R by immunoblotting. The numbers of NK cells in blood and lung were significantly higher in obese animals compared to lean ones after transfer of NK cells from obese F344 rats. This was correlated with increased postreceptor signaling (JAK-2p, PKBpT308, ERK-2p) without altered Ob-R expression in those NK cells transferred to lean (ob--nw) vs. obese (ob--ob) animals. These results show for the first time that the altered phenotype of NK cells from obese rats can be normalized by generation of a physiological (metabolic) environment of lean rats.

The PGC-1α isoform referred to as PGC-1α4 has recently been shown to promote muscle hypertrophy and attenuate muscle wasting. Although it has been shown that PGC-1α4 can regulate the expression of ...

The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist, was characterized in DMSO-d6 by the combination of nmr and molecular modeling. The conformational space available to the peptide was explored using a distance geometry algorithm with distance constraints derived from 1H-nmr spectra. The dynamics of the peptide were examined by relaxation time measurements and low temperature studies. The results from the low temperature studies suggest that the peptide backbone does not exist in a single, well-defined conformation but undergoes exchange between multiple conformers. This result is consistent with the inability to find a single structure that satisfies all the nmr-derived constraints. The constraints could only be satisfied by considering pairs of conformers to represent the experimental data. The low energy conformers comprise type II' or type V beta-turns with distinct side-chain directionality. The Arg-Gly-Asp portion of the ring is flexible and can be described by amide-plane rotations of the Arg-Gly and Gly-Asp peptide bonds. Although some backbone flexibility is evident, the incorporation of beta,beta-dimethyl cysteine imparted greater conformational rigidity as compared to the previously studied cyclic pentapeptide, Ac-Cys-Arg-Gly-Asp-Cys-OH.

β-allenic hydrazones undergo hydrostannylation to afford cyclopentene derivatives and linear rearranged products depending on the substitution of the allenic and hydrazone moieties. A first example of asymmetric radical cyclization of a SAMP β-allenic hydrazone is described.