Feasibility of a deep hyperthermia and radiotherapy programme for advanced tumors: first Spanish experience.

BACKGROUND: Hyperthermia (HT) is used to increase the temperature of the tumor-sensitizing cells to the effects of radiation/chemotherapy. We aimed to assess the feasibility, tolerability and safety of hyperthermia treatment in a Radiation Oncology Department. METHODS: Between June 2015 and June 2017, 106 patients and a total of 159 tumor lesions were included in a prospective study (EudraCT 2018-001089-40) of HT concomitant with radiotherapy (RT). Systemic treatment was accepted. HT was given twice a week, 60 min per session, during RT treatment by a regional capacitive device (HY-DEEP 600WM system) at 13.56 MHz radiofrequency. RESULTS: Most lesions (138 cases, 86.8%) received all HT sessions planned. Thirteen lesions (12 patients) withdrew treatment due to grade ≥3 QMHT toxicity. All these 12 patients completed the prescribed radiotherapy and/or systemic treatment. CONCLUSIONS: Regional hyperthermia is a feasible and safe technique to be used in combination with radiotherapy and systemic treatment.

A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis.

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.

A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana.

We have used a modification of the classical ABA-insensitive screen (Koornneef et al. 1984) to isolate novel mutations in the ABA signal transduction pathway of Arabidopsis thaliana. In our screen, mutants were recovered on the basis of their growth-insensitivity to ABA (GIA) rather than germination-insensitivity. Here we present the isolation of the gia1 mutant as well as the identification of the gia1 gene by positional cloning and complementation studies. GIA1 is predicted to code for a bZIP transcription factor with high homology to previously characterized plant bZIP transcription factors (DPBF1, ABFs and TRAB1) known for their ability to bind ABA-responsive DNA elements. Our results provide in vivo evidence that a bZIP factor may indeed be involved in ABA signaling. Since GIA1 turned out to be identical to ABI5, we designated GIA1 as ABI5 in the present paper.

The transcription factor DBP affects circadian sleep consolidation and rhythmic EEG activity.

Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues. Mice lacking DBP display a shorter circadian period in locomotor activity and are less active. Thus, although DBP is not essential for circadian rhythm generation, it does modulate important clock outputs. We studied the role of DBP in the circadian and homeostatic aspects of sleep regulation by comparing DBP deficient mice (dbp-/-) with their isogenic controls (dbp+/+) under light-dark (LD) and constant-dark (DD) baseline conditions, as well as after sleep loss. Whereas total sleep duration was similar in both genotypes, the amplitude of the circadian modulation of sleep time, as well as the consolidation of sleep episodes, was reduced in dbp-/- under both LD and DD conditions. Quantitative EEG analysis demonstrated a marked reduction in the amplitude of the sleep-wake-dependent changes in slow-wave sleep delta power and an increase in hippocampal theta peak frequency in dbp-/- mice. The sleep deprivation-induced compensatory rebound of EEG delta power was similar in both genotypes. In contrast, the rebound in paradoxical sleep was significant in dbp+/+ mice only. It is concluded that the transcriptional regulatory protein DBP modulates circadian and homeostatic aspects of sleep regulation.

Circadian expression of the steroid 15 alpha-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP.

To study the molecular mechanisms of circadian gene expression, we have sought to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. Here, we report that the Cyp2a4 gene, encoding the cytochrome P450 steroid 15alpha-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver was significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouse liver.

Selective amplification via biotin- and restriction-mediated enrichment (SABRE), a novel selective amplification procedure for detection of differentially expressed mRNAs.

We present a novel subtractive enrichment protocol for the identification of differentially expressed mRNA species. This procedure, called SABRE (selective amplification via biotin- and restriction-mediated enrichment), uses selective streptavidin-biotin affinity and restriction enzyme site reconstitution to enrich for cDNA species more abundant in one population than in another. Analysis of liver cDNA from a mouse strain expressing the neomycin resistance gene demonstrated that this procedure is capable of identifying species present in one population but absent from another. Furthermore, experiments to identify genes with circadian expression patterns in mouse liver demonstrated that SABRE is capable of detecting even modest 2- to 10-fold differences in accumulation of moderately rare mRNA species, representing as little as 0.03% of total mRNA. These experiments identified the gene encoding coumarin 7-hydroxylase as displaying circadian expression in mouse liver.

The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior.

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. Here we report that DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP-null allele display less locomotor activity and free-run with a shorter period than otherwise isogenic wild-type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP-/- mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock.

Blockade of long-term potentiation and of NMDA receptors by the protein kinase C antagonist calphostin C.

We have studied the effects of calphostin C, an antagonist of the regulatory subunit of protein kinase C, on the induction and expression of long-term potentiation (LTP) and on responses mediated by activation of N-methyl-D-aspartate (NMDA) receptors in rat hippocampal slices. No effect of calphostin C was observed on pre-established LTP, even at concentrations of 2-3 mumol/l. In contrast, the drug was found to prevent LTP induction. This effect was concentration-dependent, although high concentrations were needed (1-2 mumol/l), and, at the lower concentrations, it could be partially antagonized by using coactivation of two pathways instead of single input activation. While calphostin C did not alter synaptic transmission mediated by activation of alpha-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA) receptors, it considerably interfered with the function of NMDA receptors. The drug blocked the NMDA receptor-mediated component of burst responses, significantly antagonized the NMDA receptor-mediated synaptic responses recorded in the presence of an AMPA receptor antagonist, and blocked the effect of iontophoretic application of NMDA on regular synaptic transmission. These results are consistent with the idea that calphostin C prevents the induction of long-term potentiation by interfering with the function of NMDA receptors.