Mechanosensitivity of Cancer Cells in Contact with Soft Substrates Using AFM

Cancer cells are usually found to be softer than normal cells, but their stiffness changes when they are in contact with different environments because of mechanosensitivity. For example, they adhere to a given substrate by tuning their cytoskeleton, thus affecting their rheological properties. This mechanism could become efficient when cancer cells invade the surrounding tissues, and they have to remodel their cytoskeleton in order to achieve particular deformations. Here we use an atomic force microscope in force modulation mode to study how local rheological properties of cancer cells are affected by a change of the environment. Cancer cells were plated on functionalized polyacrylamide substrates of different stiffnesses as well as on an endothelium substrate. A new correction of the Hertz model was developed because measurements require one to account for the precise properties of the thin, layered viscoelastic substrates. The main results show the influence of local cell rheology (the nucleus, perinuclear region, and edge locations) and the role of invasiveness. A general mechanosensitive trend is found by which the cell elastic modulus and transition frequency increase with substrate elasticity, but this tendency breaks down with a real endothelium substrate. These effects are investigated further during cell transmigration, when the actin cytoskeleton undergoes a rapid reorganization process necessary to push through the endothelial gap, in agreement with the local viscoelastic changes measured by atomic force microscopy. Taken together, these results introduce a paradigm for a new—to our knowledge—possible extravasation mechanism.     

Triplex-forming oligonucleotides trigger conformation changes of a target hairpin sequence.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.     

Manual Manufacturing of Oligonucleotide, DNA, and Protein Microchips

A simple procedure for manufacturing microchips containing various gel-immobilized compounds is described. A gel photopolymerization technique is introduced to produce micromatrices of polyacrylamide gel pads (25 × 25 × 20 μm and larger) separated by a hydrophobic glass surface. A pin device for the manual application of a compound in solution onto the activated polyacrylamide gel pad for immobilization is described. Oligonucleotide, DNA, and protein microchips have been produced by this method and tested by hybridization and immunoanalysis monitored with a fluorescence microscope. The effect of the lengths of the immobilized oligonucleotides and the hybridized RNA and DNA on hybridization of the oligonucleotide microchips was evaluated. This method can also be used for manufacturing microchips containing a variety of other compounds.     

Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in Nocardia corallina: inactivation of mutB by chromosomal integration of a kanamycin resistance gene.

The gene encoding the large subunit of the methylmalonyl-coenzyme A (CoA) mutase in Nocardia corallina (mutBNc) was cloned. A 4.3-kbp BamHI fragment containing almost the entire mutBNc was identified by Southern hybridization experiments employing a digoxigenin-labeled probe deduced from mutB of Streptomyces cinnamonensis, mutBNc was interrupted by insertion of a kanamycin resistance gene block (mutB::kan or mutB::neo) and introduced into N. corallina to obtain mutB-negative strains by homologous recombination. Four of sixteen kanamycin-resistant clones occurred via double-crossover events and harbored only the interrupted mutBNc. These exhibited no growth on odd-chain fatty acids in the presence of kanamycin but exhibited wild-type growth on even-chain fatty acids, glucose, and succinate. Whereas the wild type of N. corallina accumulates a copolyester of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) containing more than 60 mol% 3HV from most carbon sources, mutB-negative strains accumulated poly(3HB-co-3HV) containing only 2 to 6 mol% 3HV. Methylmalonyl-CoA mutase activity was not found in these clones. Therefore, this study provides strong evidence that the majority of 3HV units in poly(3HB-co-3HV) accumulated by N. corallina are synthesized via the methylmalonyl-CoA pathway.     

Pollen grains ofAmbrosia in the air of Sofia,Bulgaria

Up to now,Ambrosia pollen has not been considered a cause of allergy symptoms in Bulgaria. However, the results of pollen monitoring suggest that such allergies will become inevitable in the near future. This conclusion is supported by allergometric data on 120 patients.     

Converging Climate Sensitivities of European Forests Between Observed Radial Tree Growth and Vegetation Models

The impacts of climate variability and trends on European forests are unevenly distributed across different bioclimatic zones and species. Extreme climate events are also becoming more frequent and it is unknown how they will affect feedbacks of CO₂ between forest ecosystems and the atmosphere. An improved understanding of species differences at the regional scale of the response of forest productivity to climate variation and extremes is thus important for forecasting forest dynamics. In this study, we evaluate the climate sensitivity of aboveground net primary production (NPP) simulated by two dynamic global vegetation models (DGVM; ORCHIDEE and LPJ-wsl) against tree ring width (TRW) observations from about 1000 sites distributed across Europe. In both the model simulations and the TRW observations, forests in northern Europe and the Alps respond positively to warmer spring and summer temperature, and their overall temperature sensitivity is larger than that of the soil-moisture-limited forests in central Europe and Mediterranean regions. Compared with TRW observations, simulated NPP from ORCHIDEE and LPJ-wsl appear to be overly-sensitive to climatic factors. Our results indicate that the models lack biological processes that control time lags, such as carbohydrate storage and remobilization, that delay the effects of radial growth dynamics to climate. Our study highlights the need for re-evaluating the physiological controls on the climate sensitivity of NPP simulated by DGVMs. In particular, DGVMs could be further enhanced by a more detailed representation of carbon reserves and allocation that control year-to-year variation in plant growth.     

Protective effects of a modified apelin-12 and dinitrosyl iron complexes in experimental cardioplegic ischemia and reperfusion

The maintenance of nitric oxide (NO) bioavailability has been recognized as an important component of myocardial protection during cardiac surgery. This study was designed to evaluate the efficacy of using two NO-donating compounds in cardioplegia and reperfusion: (i) a modified peptide apelin-12 (MA12) that activates endothelial NO synthase (eNOS) and (ii) dinitrosyl iron complexes with reduced glutathione (DNIC-GS), a natural NO vehicle. Isolated perfused working rat hearts were subjected to normothermic global ischemia and reperfusion. St. Thomas’ Hospital cardioplegic solution (STH) containing 140 μM MA12 or 100 μM DNIC-GS was used. In separate series, 140 μM MA12 or 100 μM DNIC-GS was administered at early reperfusion. Metabolic state of the hearts was evaluated by myocardial content of high-energy phosphates and lactate. Lactate dehydrogenase (LDH) activity in myocardial effluent was used as an index of cell membrane damage. Cardioplegia with MA12 or DNIC-GS improved recovery of coronary flow and cardiac function, and reduced LDH leakage in perfusate compared with STH without additives. Cardioplegic arrest with MA12 significantly enhanced preservation of high-energy phosphates and decreased accumulation of lactate in reperfused hearts. The overall protective effect of cardioplegia with MA12 was significantly greater than with DNIC-GS. The administration of MA12 or DNIC-GS at early reperfusion also increased metabolic and functional recovery of reperfused hearts. In this case, recovery of cardiac contractile and pump function indices was significantly higher if reperfusion was performed with DNIC-GS. The results show that MA12 and DNIC-GS are promising adjunct agents for protection of the heart during cardioplegic arrest and reperfusion.     

Blessed are the meek for they shall inherit the earth: quietness component of introversion is associated with single nucleotide polymorphisms in two circadian clock genes

Individual differences studied by chronobiologists and personality psychologists are usually shaped by polygenic selection occurring by small allele frequency shifts spreading across many loci. Therefore, the candidate gene association studies suffer from increased likelihood of false positive findings. We previously associated a PER3 single nucleotide polymorphism (SNP, rs228697) with self-ratings on personality-relevant nouns exemplifying personality dimension of Extraversion/Introversion in a sample of 88 female students. To replicate and extend this finding, we genotyped three more SNPs in three circadian clock genes. The results indicated that the minor alleles of PER3 rs228697 and PER2 rs934945 were rather similar in terms of their association with a personality type nicknamed “demure persona” (i.e. described by such nouns as “quietness”, “restraint”, “taciturnity”, “bashfulness”, “timidity”, “constraint”, and “reticence”). Analysis of data from populations of the 1000 Genomes Project suggested that, like frequencies of the minor alleles of many SNPs in circadian clock genes, the frequencies of these two SNPs were higher in populations of out-of-African ancestry compared to populations of African ancestry. We suggested that genetic candidates for Extraversion/Introversion can be prioritized in future association studies by means of identification of genetic signatures of polygenic selection imposed by out-of-Africa expansion of ancestral populations.