Radiation techniques used in patients with breast cancer: Results of a survey in Spain

AimTo evaluate the resources and techniques used in the irradiation of patients with breast cancer after lumpectomy or mastectomy and the status of implementation of new techniques and therapeutic schedules in our country.BackgroundThe demand for cancer care has increased among the Spanish population, as long as cancer treatment innovations have proliferated. Radiation therapy in breast cancer has evolved exponentially in recent years with the implementation of three-dimensional conformal radiotherapy, intensity modulated radiotherapy, image guided radiotherapy and hypofractionation.Material and MethodsAn original survey questionnaire was sent to institutions participating in the SEOR-Mama group (GEORM). In total, the standards of practice in 969 patients with breast cancer after surgery were evaluated.ResultsThe response rate was 70% (28/40 centers). In 98.5% of cases 3D conformal treatment was used. All the institutions employed CT-based planning treatment. Boost was performed in 56.4% of patients: electrons in 59.8%, photons in 23.7% and HDR brachytherapy in 8.8%. Fractionation was standard in 93.1% of patients. Supine position was the most frequent. Only 3 centers used prone position. The common organs of risk delimited were: homolateral lung (80.8%) and heart (80.8%). In 84% histograms were used. An 80.8% of the centers used isocentric technique. In 62.5% asymmetric fields were employed. CTV was delimited in 46.2%, PTV in 65% and both in 38.5%. A 65% of the centers checked with portal films. IMRT and hypofractionation were used in 1% and in 5.5% respectively.ConclusionIn most of centers, 3D conformal treatment and CT-based planning treatment were used. IMRT and hypofractionation are currently poorly implemented in Spain.     

Interactions of the high-mobility-group-like Ceratitis capitata C1 proteins with DNA

We have studied the interactions of the high-mobility-group-like proteins (C1a1, C1a2 and C1b) from the fruit fly Ceratitis capitata with DNA. Nitrocellulose filter binding assays, thermal denaturation studies and spectrofluorimetry of the complexes revealed the existence of specific and nonspecific interactions. Thermal denaturation curves showed that the three proteins stabilized the DNA, thus suggesting a preferential binding to double-stranded DNA. The calculation of the thermodynamic parameters of the interactions showed that the nonspecific bindings were characterized by low association constants (Ka) with values ranging from 2.7 X 10(4) M-1 to 2.0 X 10(6) M-1. Also, the cooperativity of these interactions was relatively high (cooperativity factor, w, values ranging over 20-35), and the number of nucleotides involved was low (1-3 base pairs). On the other hand, the existence of specific interactions between C1 proteins and DNA was suggested by two facts: the retention of C. capitata [3H]DNA in nitrocellulose filters was only a low percentage of total input DNA and there was a marked size dependence of the binding (25% retention of a 40-kb DNA and only 3% retention with a DNA of 1 kb). The specific bindings had higher Ka values than the nonspecific ones, and they also were cooperative. Some differences were observed between C1b and the C1a proteins about the way they interact with C. capitata DNA.     

Chitosan influence on glucose and calcium availability from yogurt: In vitro comparative study with plants fibre

Since chitosan complies with the definition of dietary fibre is necessary to study the interaction of this biopolymer with nutrients. Yogurt with fortified chitosan and different types of plants fibres like wheat, bamboo, apple, psyllium and inulin was used as a food model. The availabilities of glucose and calcium in this model were studied by an in vitro gastrointestinal tract simulation. Results showed that the different fibres decreased both glucose and calcium availabilities whereas the effect of chitosan was more pronounced. (17.7 ± 2.1% and 21.0 ± 2.5% depress respectively). This work demonstrated that the addition of chitosan to yogurts influences the availability of nutrients.     

Expression and Functional Study of Extracellular BMP Antagonists during the Morphogenesis of the Digits and Their Associated Connective Tissues

The purpose of this study is to gain insight into the role of BMP signaling in the diversification of the embryonic limb mesodermal progenitors destined to form cartilage, joints, and tendons. Given the importance of extracellular BMP modulators in in vivo systems, we performed a systematic search of those expressed in the developing autopod during the formation of the digits. Here, we monitored the expression of extracellular BMP modulators including: Noggin, Chordin, Chordin-like 1, Chordin-like 2, Twisted gastrulation, Dan, BMPER, Sost, Sostdc1, Follistatin, Follistatin-like 1, Follistatin-like 5 and Tolloid. These factors show differential expression domains in cartilage, joints and tendons. Furthermore, they are induced in specific temporal patterns during the formation of an ectopic extra digit, preceding the appearance of changes that are identifiable by conventional histology. The analysis of gene regulation, cell proliferation and cell death that are induced by these factors in high density cultures of digit progenitors provides evidence of functional specialization in the control of mesodermal differentiation but not in cell proliferation or apoptosis. We further show that the expression of these factors is differentially controlled by the distinct signaling pathways acting in the developing limb at the stages covered by this study. In addition, our results provide evidence suggesting that TWISTED GASTRULATION cooperates with CHORDINS, BMPER, and NOGGIN in the establishment of tendons or cartilage in a fashion that is dependent on the presence or absence of TOLLOID.     

Does the morphological fit between flowers and pollinators affect pollen deposition? An experimental test in a buzz‐pollinated species with anther dimorphism

Some pollination systems, such as buzz‐pollination, are associated with floral morphologies that require a close physical interaction between floral sexual organs and insect visitors. In these systems, a pollinator's size relative to the flower may be an important feature determining whether the visitor touches both male and female sexual organs and thus transfers pollen between plants efficiently. To date, few studies have addressed whether in fact the “fit” between flower and pollinator influences pollen transfer, particularly among buzz‐pollinated species. Here we use Solanum rostratum, a buzz‐pollinated plant with dimorphic anthers and mirror‐image flowers, to investigate whether the morphological fit between the pollinator's body and floral morphology influences pollen deposition. We hypothesized that when the size of the pollinator matches the separation between the sexual organs in a flower, more pollen should be transferred to the stigma than when the visitor is either too small or too big relative to the flower. To test this hypothesis, we exposed flowers of S. rostratum with varying levels of separation between sexual organs, to bumblebees (Bombus terrestris) of different sizes. We recorded the number of visits received, pollen deposition, and fruit and seed production. We found higher pollen deposition when bees were the same size or bigger than the separation between anther and stigma within a flower. We found a similar, but not statistically significant pattern for fruit set. In contrast, seed set was more likely to occur when the size of the flower exceeded the size of the bee, suggesting that other postpollination processes may be important in translating pollen receipt to seed set. Our results suggest that the fit between flower and pollinator significantly influences pollen deposition in this buzz‐pollinated species. We speculate that in buzz‐pollinated species where floral morphology and pollinators interact closely, variation in the visitor's size may determine whether it acts mainly as a pollinator or as a pollen thief (i.e., removing pollen rewards but contributing little to pollen deposition and fertilization).     

Escherichia coli “TatExpress” strains export several g/L human growth hormone to the periplasm by the Tat pathway

Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 “TatExpress” strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.     

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Background

In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.

Methods

BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.

Results

AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4⁺ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.

Conclusion

Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.     

Comparative Genomic and Phylogenetic Analyses of Gammaproteobacterial glg Genes Traced the Origin of the Escherichia coli Glycogen glgBXCAP Operon to the Last Common Ancestor of the Sister Orders Enterobacteriales and Pasteurellales

Production of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria.     

Epidermal fatty acid‐binding protein protects nerve growth factor‐differentiated PC12 cells from lipotoxic injury

Epidermal fatty acid‐binding protein (E‐FABP/FABP5/DA11) binds and transport long‐chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E‐FABP protects nerve growth factor‐differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM‐induced lipotoxicity (PAM‐LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E‐FABP. Antioxidants MCI‐186 and N‐acetyl cysteine prevented E‐FABP's induction in expression by PAM‐LTx, while tert‐butyl hydroperoxide increased ROS and E‐FABP expression. Non‐metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E‐FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE‐FABP showed reduced E‐FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E‐FABP cellular levels by pre‐loading the cells with recombinant E‐FABP diminished the PAM‐induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E‐FABP expression and enhanced the resistance of NGFDPC12 cells to PAM‐LTx. We conclude that E‐FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS.