Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Centrosome-declustering drugs mediate a two-pronged attack on interphase and mitosis in supercentrosomal cancer cells

Classical anti-mitotic drugs have failed to translate their preclinical efficacy into clinical response in human trials. Their clinical failure has challenged the notion that tumor cells divide frequently at rates comparable to those of cancer cells in vitro and in xenograft models. Given the preponderance of interphase cells in clinical tumors, we asked whether targeting amplified centrosomes, which cancer cells carefully preserve in a tightly clustered conformation throughout interphase, presents a superior chemotherapeutic strategy that sabotages interphase-specific cellular activities, such as migration. Herein we have utilized supercentrosomal N1E-115 murine neuroblastoma cells as a test-bed to study interphase centrosome declustering induced by putative declustering agents, such as Reduced-9-bromonoscapine (RedBr-Nos), Griseofulvin and PJ-34. We found tight ‘supercentrosomal'' clusters in the interphase and mitosis of ~80% of patients'' tumor cells with excess centrosomes. RedBr-Nos was the strongest declustering agent with a declustering index of 0.36 and completely dispersed interphase centrosome clusters in N1E-115 cells. Interphase centrosome declustering caused inhibition of neurite formation, impairment of cell polarization and Golgi organization, disrupted cellular protrusions and focal adhesion contacts—factors that are crucial prerequisites for directional migration. Thus our data illustrate an interphase-specific potential anti-migratory role of centrosome-declustering agents in addition to their previously acknowledged ability to induce spindle multipolarity and mitotic catastrophe. Centrosome-declustering agents counter centrosome clustering to inhibit directional cell migration in interphase cells and set up multipolar mitotic catastrophe, suggesting that disbanding the nuclear–centrosome–Golgi axis is a potential anti-metastasis strategy.Unlike in vitro cell cultures, cancer cells in patients'' tumor tissues have low mitotic indices and proliferation rates.¹ Consequently, drugs targeting mitosis demonstrate limited clinical efficacy, which exposes a fundamental weakness in the rationale underlying their clinical development. By contrast, classical microtubule-targeting agents (MTAs), largely believed to act by perturbing mitosis, remain the mainstay of chemotherapy in the clinic. Given the miniscule population of mitotic cells in patient tumors,^{2, 3} it stands to reason that MTAs must target interphase.⁴ This paradigm shift has spurred an intense search for novel interphase targets that combine the ‘ideal'' attributes of cancer-cell selectivity and the ability to confer vulnerability on a large proportion of tumor cells.Centrosomes, the major microtubule-organizing centers (MTOCs) of cells, are required for accurate cell division, cell motility and cilia formation.⁵ The number of centrosomes within a cell is strictly controlled, and their duplication occurs only once per cell cycle. Nearly all types of cancer cells have abnormal numbers of centrosomes,^{6, 7, 8} which correlates with chromosomal instability during tumorigenesis.^{9, 10, 11} Supernumerary centrosomes in cancer cells can cause spindle multipolarity and thus non-viable progeny. Cancer cells avoid this outcome by clustering centrosomes to assemble a pseudo-bipolar mitotic spindle, which yields viable daughter cells.¹² Thus disrupting centrosome clustering may selectively drive cancer cells with amplified centrosomes to mitotic catastrophe and apoptosis without affecting normal cells.The fate and interphase role of the supercentrosomal cluster inherited by each daughter cell at the end of a pseudobipolar mitosis is unknown. This is an important research question, because a majority of cells within tumors are in interphase and the centrosomes'' command over microtubule nucleation is crucial for the cellular organization and motility in interphase. If cancer cells cluster centrosomes in interphase, then disrupting the cluster could impact interphase-specific processes, opening up a vital therapeutic avenue. We envision that centrosome declustering would (a) derail interphase-specific polarization and migration processes and (b) precipitate multipolar mitosis culminating in apoptosis. This two-pronged strategy would impact a significantly larger proportion of tumor cells and consign them to death. Our study herein establishes that centrosome-declustering drugs (RedBr-Nos, Griseofulvin and PJ-34) achieve this two-pronged attack as a unique class of agents that exhibit multiple cellular activities.     

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli.

We have isolated an Escherichia coli gene which, when overexpressed, is able to complement the permeability defects of a vancomycin-susceptible mutant. This gene, designated sanA, is located at min 47 of the E. coli chromosome and codes for a 20-kDa protein with a highly hydrophobic amino-terminal segment. A strain carrying a null mutation of the sanA gene, transferred to the E. coli chromosome by homologous recombination, is perfectly viable, but after two generations at high temperature (43 degrees C), the barrier function of its envelope towards vancomycin is defective.     

Cdc42-dependent localization of polarisome component Spa2 to the incipient bud site is independent of the GDP/GTP exchange factor Cdc24

Cdc42, a member of the Rho subfamily of small GTPases, is highly conserved in both sequence and function across eukaryotic species. In budding yeast, Cdc42 triggers polarized growth necessary for bud emergence via rearrangement of the actin cytoskeleton. It has been shown that the role of Cdc42 in bud emergence requires both Cdc28-Cln (G1) kinase and the passage through START. In this report, we show that Cdc42 also serves an essential function in the establishment of bud site prior to START by catalyzing the translocation of bud-site components such as Spa2 to the cell cortex. Our analysis of various conditional alleles of CDC42 suggests that these two functions (bud site establishment and bud emergence) are genetically separable. Surprisingly, the role of Cdc42 in the cortical localization of Spa2 appears to be independent of its well known GTP/GDP exchange factor Cdc24. We also provide evidence that this role of Cdc42 requires the function of the COPI coatomer complex.     

Serotonin,Eating Behavior,and Fat Intake

There is an intimate relationship between nutritional intake (eating) and serotonin activity. Experimental manipulations (mainly neuropharmacological) of serotonin influence the pattern of eating behavior, subjective feelings of appetite motivation, and the response to nutritional challenges. Similarly, nutritional manipulations (food restriction, dieting, or altered nutrient supply) change the sensitivity of the serotonin network. Traditionally, serotonin has been linked to the macronutrient carbohydrate via the intermediary step of plasma amino acid ratios. However, it has also been demonstrated that 5-HT drugs will reduce energy intake and reverse body weight gain in rats exposed to weight increasing high fat diets. 5-HT drugs can also reduce food intake and block weight gain of rats on a high fat cafeteria diet. Some diet selection studies in rats indicate that the most prominent reduction of macronutrient intake is for fat. These data indicate that 5-HT activity can bring about a reduction in fat consumption. In turn, different types of dietary fat can alter brain 5-HT activity. In human studies the methodology of food choice experiments has often precluded the detection of an effect of 5-HT manipulation on fat intake. However, there is evidence that in obese and lean subjects some 5-HT drugs can readily reduce the intake of high fat foods. Data also suggest that 5-HT activation can lead to a selective avoidance of fat in the diet. These effects of 5-HT on the intake of dietary fat may involve a pre-absorptive mechanism and there is evidence that 5-HT is linked to cholecystokinin and enterostatin. These proposals have theoretical and practical implications and suggest possible strategies to intensify or advance fat-induced satiety signals.     

Diarrhea-associated biofilm formed by enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis> and aggregative <Emphasis Type="Italic">Citrobacter freundii</Emphasis>: a consortium mediated by putative F pili

Background  

Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea.     

Noninvasive high resolution mechanical strain maps of the spine intervertebral disc using nonrigid registration of magnetic resonance images

High resolution strain measurements are of particular interest in load bearing tissues such as the intervertebral disc (IVD), permitting characterization of biomechanical conditions which could lead to injury and degenerative outcomes. Magnetic resonance (MR) imaging produces excellent image contrast in cartilaginous tissues, allowing for image-based strain determination. Nonrigid registration (NRR) of MR images has previously demonstrated sub-voxel registration accuracy although its accuracy and precision in determining strain has not been evaluated. Accuracy and precision of NRR-derived strain measurements were evaluated using computer generated deformations applied to both computer generated images and MR images. Two different measures of registration similarity--the cost function which drives the registration algorithm--were compared: Mutual Information (MI) and Least Squares (LS). Strain error was evaluated with respect to signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and strain heterogeneity. Additionally, the creep strain response from an in vitro loaded porcine IVD is shown and comparisons between similarity measures are presented. MI showed a decrease in strain precision with increasing CNR and decreasing SNR while LS was insensitive to both. Both similarity measures showed a decrease in strain precision with increasing strain heterogeneity. When computer generated heterogeneous strains were applied to MR images of the IVD, LS showed substantially lower strain error in comparison to MI. Results suggest that LS-driven NRR provides a more accurate image-based method for mapping large and heterogeneous strain fields and this method can be applied to studies of the IVD and, potentially, other soft tissues which present sufficient image texture.     

Cryopreservation of Teucrium polium L. shoot-tips by vitrification and encapsulation-dehydration

Teucrium polium L. with the common name of Felty Germander is one of the plants flora that is widely used in folk medicine in many Middle East countries, it is an endangered plant species and must be highly considered for preservation. Cryopreservation of T. polium by vitrification and encapsulation-dehydration was successfully achieved in this study. Shoot-tips were excised aseptically from in vitro grown plants and incubated for 3?days on solid hormone free-Murashige and Skoog (HF-MS) media supplemented with 0.3?M sucrose under complete darkness at 24?±?1?°C. In vitrification, shoot-tips were loaded in 0.4?M sucrose and 2?M glycerol for 20?min followed by desiccation with different combinations and concentrations of plant vittrification solution 2 (PVS2), before immersion in Liquid Nitrogen (LN). Whereas for the encapsulation-dehydration; shoot-tips were encapsulated in calcium alginate and dehydrated under laminar air flow cabinet for 0, 3, 6, or 9?h. A total of 60?% of the cryopreserved vitrified shoot-tips survived when desiccated in concentrated PVS2 solution for 20?min, whereas, 28?% of the cryopreserved vitrified shoot-tips were regrown after 20?min of desiccation by two step increase in PVS2 concentration. Complete survival were obtained for the non-cryopreserved encapsulated shoot-tips treated for 3?days in 0.5?M sucrose with MS media without or with 3?h of dehydration, whereas, only 20?% of the cryopreserved encapsulated shoot-tips were regrown. The procedures developed in this study are easy to handle and produced a high levels of shoot formation.