Prospective study of HDR brachytherapy (BT), external beam radiotherapy (EBRT) and androgen deprivation therapy (ADT): 10-years experience of an MRI-guided approach.

OBJECTIVES: To evaluate the predictive factors for biochemical failure and distant metastases in a prospective cohort of patients with localized prostate cancer treated with the combination of HDR BT and EBRT. METHODS AND MATERIALS: Patients with intermediate (IR) or high-risk (HR) prostate adenocarcinoma received a single fraction of HDR of 15 Gy combined with RT of 37.5 Gy in 15 fractions. ADT duration was used depending on risk-group. Descriptive analyses were performed. Univariate and multivariate Hazard Ratios were obtained. Finally, the Kaplan-Meier model was used to describe the survival of the events of interest. RESULTS: 309 patients were treated prospectively (199 were IR and 110 HR). Median age was 72 years; 58.3 % were MRI stage ≤ T2c, 34.1 % T3a and 7.6 % T3b; ISUP-grade 1-3 in 78.9 % and ISUP 4-5 in 21.1 %. 71.8 % of patients had ≤ 50 % positive-cores in biopsy and 28.2 % had > 50 %. Median pre-treatment PSA was 9.9 ng/mL. After a median follow-up of 88 months, 41 patients presented biochemical failure and 18 developed distant metastases. Multivariate cox-regression analyses found that MR-T3b Stage (HR 3.88, p = 0.001) and ADT use (HR 3.99, p = 0.03) were the only predictive factors for biochemical failure and the number of positive cores (>50 %) the only independent predictive factor of distant metastases (HR 4.36, p = 0.002). CONCLUSIONS: Patients with mpMRI evidence of invasion of the SV and involvement of more than 50% of the cores in the prostate biopsy are patients with a higher risk of presenting a biochemical recurrence or developing metastasis due to their prostate cancer, respectively.

The homozygous R504C mutation in MTO1 gene is responsible for ONCE syndrome.

We report clinical and biochemical finding from three unrelated patients presenting ONCE (Optic Neuropathy, Cardiomyopathy and Encephalopathy with lactic acidosis and combined oxidative phosphorylation deficiency) syndrome. Whole-exome sequencing (WES) of the three patients and the healthy sister of one of them was used to identify the carry gene. Clinical and biochemical findings were used to filter variants, and molecular, in silico and genetic studies were performed to characterize the candidate variants. Mitochondrial DNA (mtDNA) defects involving mutations, deletions or depletion were discarded, whereas WES uncovered a double homozygous mutation in the MTO1 gene (NM_001123226:c.1510C>T, p.R504C, and c.1669G>A, p.V557M) in two of the patients and the homozygous mutation p.R504C in the other. Therefore, our data confirm p.R504C as pathogenic mutation responsible of ONCE syndrome, and p.V557M as a rare polymorphic variant.

Prevalence of hepatitis C virus genotypes in a Spanish liver transplant unit.

INTRODUCTION: Among the at least six major identified genotypes of HCV, genotype 1b, the one associated with a poorer prognosis, is the most prevalent in Spain. We aimed to compare the distribution of hepatitis C virus genotypes in our liver transplant unit with that of the other HCV patients at our institution (n = 413) in order to assess whether genotype 1b is more prevalent among patients with more severe liver disease. PATIENTS AND METHODS: One hundred eight patients of mean age 56 years included 81 (75%) OLT recipients and 27 (25%) with HCV cirrhosis. Determination of HCV genotypes was made with the Inno-LiPA HCV III. RESULTS: The overall distribution of genotypes was: 1b, 93 patients (86.1%); 1a; eight patients (7.4%); 3, four patients (3.7%); 4; two patients (1.9%), and 2; one patient (0.9%). The distribution was similar among patients with cirrhosis and OLT. Genotype 1b patients were older. Eleven (78.6%) of 14 patients with hepatocellular carcinoma had genotype 1b. In the control group the distribution was: 1b, 287 patients (69.5%); 1a, 54 patients (12.1%); 3, 41 patients (9.9%); 4, 20 patients (4.8%), and genotype 2, 11 patients (2.7%). This differences in the distribution of genotypes between our population and the control group was statistically significant (P < .001). CONCLUSIONS: Genotype 1b, the most prevalent genotype in our liver transplant unit, included older patients in whom hepatocellular carcinoma was common, perhaps due to their higher prevalence of cirrhosis.

Adefovir dipivoxil therapy in liver transplant recipients with lamivudine-resistant hepatitis B virus.

Hepatitis B virus (HBV) infection is the leading cause of cirrhosis worldwide. One effective strategy to prevent recurrence or transmission of HBV infection after liver transplantation exists is prescription of Lamivudine, although it is associated with high resistance rates. Adefovir dipivoxil (AD) is a nucleotide analogue of adenosine that has achieved significant results in virologic, biochemical, and clinical parameters in lamivudine-resistant HBV-infected patients. Between 1990 and 2003 7 adult recipients of orthotopic liver transplants who experienced lamivudine-resistant HBV infection (pretransplantation or posttransplantation) were enrolled in a prospective study to administer AD for 48 weeks. At baseline they showed serum HBV DNA between 2.2 x 10(6) and 1.1 x 10(8) copies/mL. After 48 weeks of AD treatment, the median time-weighted average change in serum HBV DNA (log 10 copies/mL) was -3.19 (SD, 1.65). In 3 patients with HBV, DNA was undetectable (<400 copies/mL) at the end of the follow-up. HBe antigen seroconversion was observed in 1 patient. No significant adverse effects were recorded, except for renal functional impairment in 1 patient who had previous renal insufficiency. In our study, adefovir was an effective drug to suppression HBV replication in liver transplant recipients with lamivudine-resistant HBV. Excluding renal function abnormalities, tolerance of the drug was excellent. None of the patients developed resistance to adefovir. Therapy with AD in liver transplant recipients is effective and safe, although renal function should be monitored closely.

A study on the human mitochondrial RNA polymerase activity points to existence of a transcription factor B-like protein.

In the present work, the RNA polymerase activity of the human mitochondrial RNA polymerase mature protein (h-mtRPOLm) is shown, and its molecular activity calculated (2.1+/-0.9 min(-1)). An activity analysis of h-mtRPOLm and deleted versions of it has demonstrated that the entire recombinant protein is required for this activity. In addition, h-mtRPOLm alone or in presence of the known mitochondrial transcription factors (human mitochondrial transcription factor A and/or human mitochondrial transcription termination factor) is not able to initiate transcription from the specific human mitochondrial promoters pointing to the existence of a human mitochondrial transcription factor B-like protein.

Parallel-stranded hairpins containing 8-aminopurines. Novel efficient probes for triple-helix formation.

We describe novel oligomers with a greater propensity to form triplexes than oligomers containing only natural bases. They consist of a polypyrimidine sequence linked head-to-head with a polypurine sequence carrying one or several 8-aminoadenine or 8-aminoguanines. The presence of 8-aminopurines also stabilised the parallel-stranded duplex structure.

d(GA x TC)(n) microsatellite DNA sequences enhance homologous DNA recombination in SV40 minichromosomes.

The genomic distribution of the abundant eukaryotic d(GA x TC)(n) DNA microsatellite suggests that it could contribute to DNA recombination. Here, it is shown that this type of microsatellite DNA sequence enhances DNA recombination in SV40 minichromosomes, the rate of homologous DNA recombination increasing by as much as two orders of magnitude in the presence of a d(GA x TC)(22) sequence. This effect depends on the region of the SV40 genome at which the d(GA x TC)(22) sequence is cloned. It is high when the sequence is located proximal to the SV40 control region but no effect is observed when located 3.5 kb away from the SV40 ori. These results indicate that the recombination potential of d(GA x TC)(n) sequences is likely linked to DNA replication and/or transcription. The potential contribution of the structural properties of d(GA x TC)(n) sequences to this effect is discussed.

DDP1, a heterochromatin-associated multi-KH-domain protein of Drosophila melanogaster, interacts specifically with centromeric satellite DNA sequences.

DDP1 is a single-stranded nucleic acid binding protein of Drosophila melanogaster that associates with pericentric heterochromatin. DDP1 contains 15 consecutive KH domains and is homologous to the highly conserved vigilin proteins that, in Saccharomyces cerevisiae, are involved in the control of cell ploidy. DDP1 was identified and purified on the basis of its binding to the pyrimidine-rich C strand of the centromeric Drosophila dodeca-satellite. Here, the interaction of DDP1 with the dodeca-satellite C strand was analyzed in detail. This interaction is sequence specific. In particular, a guanine residue which is highly conserved in natural dodeca-satellite sequences was found to be essential for the efficient binding of DDP1. DDP1 binding was also found to be strongly influenced by the length and extent of secondary structure of the DNA substrate. Efficient DDP1 binding required a minimal length of about 75 to 100 nucleotides and was facilitated by the lack of secondary structure of the substrate. DDP1 also showed a significant affinity for the unstructured pyrimidine-rich strand of the most abundant centromeric Drosophila AAGAG satellite. The stoichiometry of the complexes formed with the dodeca-satellite C strand suggests that, in DDP1, the 15 consecutive KH domains are organized such that they define two nucleic acid binding surfaces. These results are discussed in the context of the possible contribution of DDP1 to heterochromatin organization and function.

Functional mapping of the GAGA factor assigns its transcriptional activity to the C-terminal glutamine-rich domain.

GAGA is a nuclear protein encoded by the Trithorax-like gene in Drosophila that is expressed in at least two isoforms generated by alternative splicing. By means of its specific interaction with DNA, GAGA has been involved in several nuclear transactions including regulation of gene expression. Here we have studied the GAGA(519) isoform as a transcription factor. In vitro, the transactivation domain has been assigned to the 93 C-terminal residues that correspond to a glutamine-rich domain (Q-domain). It presents an internal modular structure and acts independently of the rest of the protein. In vivo, in Drosophila SL2 cells, Q-domain can transactivate reporter genes either in the form of GAGA or Gal4BD-Q fusions, whereas a GAGA mutant deleted of the Q-domain cannot. Our results give support to the notion that GAGA can function as a transcription activating factor.

Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: first phosphodiesterase 7 inhibitors.

The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell-dependent disorders. The IC(50) values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 microM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC(50) = 25 microM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.

The GAGA factor of Drosophila interacts with SAP18, a Sin3-associated polypeptide.

SAP18, a polypeptide associated with the Sin3-HDAC co-repressor complex, was identified in a yeast two-hybrid screen as capable of interacting with the Drosophila GAGA factor. The interaction was confirmed in vitro by glutathione S-transferase pull-down assays using recombinant proteins and crude SL2 nuclear extracts. The first 245 residues of GAGA, including the POZ domain, are necessary and sufficient to bind dSAP18. In polytene chromosomes, dSAP18 and GAGA co-localize at a few discrete sites and, in particular, at the bithorax complex where GAGA binds some silenced polycomb response elements. When the dSAP18 dose is reduced, flies heterozygous for the GAGA mutation Trl67 show the homeotic transformation of segment A6 into A5, indicating that GAGA-dSAP18 interaction contributes to the functional regulation of the iab-6 element of the bithorax complex. These results suggest that, through recruitment of the Sin3-HDAC complex, GAGA might contribute to the regulation of homeotic gene expression.

The formation of triple-stranded DNA prevents spontaneous branch-migration.

Branch-migration is a fundamental step in the process of DNA recombination that determines the location, and extent, of the exchange between the recombining duplexes. Four-way Holliday junctions assembled in vitro can migrate spontaneously in an uncatalysed reaction that mimics some of the aspects involved in branch-migration. Here, we have analysed the effects of a d(GA.TC)22 and a d(CA.TG)30 sequence on the rate of spontaneous branch-migration. Under most of the experimental conditions assayed, no significant effect was observed. However, the d(GA.TC)22 sequence induces a very strong arrest when branch-migration is performed at low pH, under conditions where the repeated sequence is forming an intramolecular [C(+)T(GA.TC)] triplex. A similar arrest is observed when the recombining duplexes contain intermolecular triplexes arising from the annealing of a d(GA.TC)22 duplex and a d(TC)22 oligonucleotide, indicating that the formation of triplex DNA constitutes a strong barrier for the progression of the Holliday junction. These results are discussed in the context of the possible contribution of triplex DNA to DNA recombination.

Crystal structure of a DNA Holliday junction.

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 A resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.

The identification of nuclear proteins that bind the homopyrimidine strand of d(GA.TC)n DNA sequences, but not the homopurine strand.

Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences.

Pseudochylothorax in pleural effusion due to coronary artery bypass surgery.

This study describes a 64-yr-old male with a chronic left pleural effusion following a coronary artery bypass 3 yrs earlier. On thoracocentesis, turbid fluid was obtained with crystals of cholesterol on microscopic examination, establishing the diagnosis of pseudochylothorax. The pleural fluid cholesterol level was 207 mg x dL(-1) (5.36 mmol x L(-1)). This is the first report of pseudochylothorax in a chronic pleural effusion due to coronary artery bypass surgery.

DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy.

The centromeric dodeca-satellite of Drosophila forms altered DNA structures in vitro in which its purine-rich strand (G-strand) forms stable fold-back structures, while the complementary C-strand remains unstructured. In this paper, the purification and characterization of DDP1, a single-stranded DNA-binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca-satellite C-strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca-satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co-localizing with the heterochromatin protein HP1. DDP1 is a multi-KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a Deltascp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.

The N-terminal POZ domain of GAGA mediates the formation of oligomers that bind DNA with high affinity and specificity.

The Drosophila GAGA factor self-oligomerizes both in vivo and in vitro. GAGA oligomerization depends on the presence of the N-terminal POZ domain and the formation of dimers, tetramers, and oligomers of high stoichiometry is observed in vitro. GAGA oligomers bind DNA with high affinity and specificity. As a consequence of its multimeric character, the interaction of GAGA with DNA fragments carrying several GAGA binding sites is multivalent and of higher affinity than its interaction with fragments containing single short sites. A single GAGA oligomer is capable of binding adjacent GAGA binding sites spaced by as many as 20 base pairs. GAGA oligomers are functionally active, being transcriptionally competent in vitro. GAGA-dependent transcription activation depends strongly on the number of GAGA binding sites present in the promoter. The POZ domain is not necessary for in vitro transcription but, in its absence, no synergism is observed on increasing the number of binding sites contained within the promoter. These results are discussed in view of the distribution of GAGA binding sites that, most frequently, form clusters of relatively short sites spaced by small variable distances.