Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

Carbamate derivatives of colchicine show potent activity towards primary acute lymphoblastic leukemia and primary breast cancer cells—in vitro and ex vivo study

Colchicine ( COL ) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N‐carbamates of N‐deacetyl‐4‐(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti‐proliferative activity than COL (IC₅₀ = 8.6 nM) towards primary ALL cells in cell viability assays (IC₅₀ range of 1.1‐6.4 nM), and several were more potent towards primary IDCG3 (IC₅₀ range of 0.1 to 10.3 nM) or MC (IC₅₀ range of 2.3‐9.1 nM) compared to COL (IC₅₀ of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non–small‐cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.     

The use of chromogenic bacteria as coloured substitutes for pathogens: A simple strategy during design and development of a new method for sample pretreatment

Aims: In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during set‐up and optimization of an isolation process of bacteria, designed for food samples. Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. Therefore, red‐coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique. Methods and Results: The chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g. centrifugal forces) and chemical (e.g. lysis buffer composition) conditions required during establishment of the new technique. Furthermore, the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar Typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods. Conclusion: Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalization of the method protocol. The presumptive bacterial targets can be allocated simply by visualization of their bright red colour silhouetted against the background sample matrix. Significance and Impact of Study: The use of pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. It saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession.     

Genome sequence of the fungus Glarea lozoyensis: the first genome sequence of a species from the Helotiaceae family

The anamorphic fungus Glarea lozoyensis mutant strain 74030 is an overproducer of pneumocandin B(0), which is chemically converted into Cancidas, a potent antibiotic against clinically important fungal pathogens. Pneumocandins are acylated, cyclic hexapeptides with unusual hydroxylated amino acids. With the Glarea lozoyensis genome, the first species from the large polyphyletic family Helotiaceae has been sequenced.     

Recoupling and decoupling of nuclear spin interactions at high MAS frequencies: numerical design of CN<Stack><Subscript><Emphasis Type="Italic">n</Emphasis></Subscript><Superscript>ν</Superscript></Stack> symmetry-based RF pulse schemes

The CN _n ^ν class of RF pulse schemes, commonly employed for recoupling and decoupling of nuclear spin interactions in magic angle spinning solid state NMR studies of biological systems, involves the application of a basic “C” element corresponding to an RF cycle with unity propagator. In this study, the design of CN _n ^ν symmetry-based RF pulse sequences for achieving ¹³C–¹³C double-quantum dipolar recoupling and through bond scalar coupling mediated ¹³C–¹³C chemical shift correlation has been examined at high MAS frequencies employing broadband, constant-amplitude, phase-modulated basic “C” elements. The basic elements were implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by an RF phase value. The phase-modulation profile of the “C” element was optimised numerically so as to generate efficient RF pulse sequences. The performances of the sequences were evaluated via numerical simulations and experimental measurements and are presented here. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

δ-Opioid receptor-stimulated Akt signaling in neuroblastoma × glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the “pro-survival” kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma × glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen^2,5]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G_i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G_q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.     

Troglitazone sensitizes tumor cells to TRAIL-induced apoptosis via down-regulation of FLIP and Survivin

Induction of apoptosis by the death ligand TRAIL might be a promising therapeutic approach in cancer therapy. However, since not all tumor cells are sensitive to TRAIL, there is a need for the development of strategies to overcome TRAIL-resistance. The results of the present study show that the anti-diabetic drug troglitazone sensitizes human glioma and neuroblastoma cells to TRAIL-induced apoptosis. This process is accompanied by a substantial increase of active caspase 8 and active caspase 3, but it is independent of troglitazone's effects on the nuclear receptor PPAR-γ. Troglitazone induces a pronounced reduction in protein expression levels of the anti-apoptotic FLICE-inhibitory protein (FLIP) without affecting FLIP mRNA levels. Further, protein and mRNA expression levels of the anti-apoptotic protein Survivin significantly decrease upon treatment with troglitazone. Moreover, sensitization to TRAIL is partly accompanied by an up-regulation of the TRAIL receptor, TRAIL-R2. A combined treatment with troglitazone and TRAIL might be a promising experimental therapy because troglitazone sensitizes tumor cells to TRAIL-induced apoptosis via various mechanisms, thereby minimizing the risk of acquired tumor cell resistance. This work was supported by a grant from the Deutsche Krebshilfe (German Cancer Aid, Max Eder Program).