Sex-chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea).

Occasional XY recombination is a proposed explanation for the sex-chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, and a detailed survey of NW-European Hyla arborea populations identified male-specific alleles at sex-linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex-linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex-chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY-recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations).

Successful combined minimally invasive direct coronary artery bypass and transapical aortic valve implantation.

Transapical aortic valve implantation is indicated in high-risk patients with aortic stenosis and peripheral vascular disease requiring aortic valve replacement. Minimally invasive direct coronary artery bypass grafting is also a valid, minimally invasive option for myocardial revascularization in patients with critical stenosis on the anterior descending coronary artery. Both procedures are performed through a left minithoracotomy, without cardiopulmonary bypass, aortic cross-clamping, and cardioplegic arrest. We describe a successful combined transapical aortic valve implantation and minimally invasive direct coronary bypass in a high-risk patient with left anterior descending coronary artery occlusion and severe aortic valve stenosis.

A Mutation in CALM1 Encoding Calmodulin in Familial Idiopathic Ventricular Fibrillation in Childhood and Adolescence.

OBJECTIVES: This study aimed to identify the genetic defect in a family with idiopathic ventricular fibrillation (IVF) manifesting in childhood and adolescence. BACKGROUND: Although sudden cardiac death in the young is rare, it frequently presents as the first clinical manifestation of an underlying inherited arrhythmia syndrome. Gene discovery for IVF is important as it enables the identification of individuals at risk, because except for arrhythmia, IVF does not manifest with identifiable clinical abnormalities. METHODS: Exome sequencing was carried out on 2 family members who were both successfully resuscitated from a cardiac arrest. RESULTS: We characterized a family presenting with a history of ventricular fibrillation (VF) and sudden death without electrocardiographic or echocardiographic abnormalities at rest. Two siblings died suddenly at the ages of 9 and 10 years, and another 2 were resuscitated from out-of-hospital cardiac arrest with documented VF at ages 10 and 16 years, respectively. Exome sequencing identified a missense mutation affecting a highly conserved residue (p.F90L) in the CALM1 gene encoding calmodulin. This mutation was also carried by 1 of the siblings who died suddenly, from whom DNA was available. The mutation was present in the mother and in another sibling, both asymptomatic but displaying a marginally prolonged QT interval during exercise. CONCLUSIONS: We identified a mutation in CALM1 underlying IVF manifesting in childhood and adolescence. The causality of the mutation is supported by previous studies demonstrating that F90 mediates the direct interaction of CaM with target peptides. Our approach highlights the utility of exome sequencing in uncovering the genetic defect even in families with a small number of affected individuals.

P16 Mediated Suppression Of Telomerase In Normal And Malignant Human Breast Cells.

Summary The cyclin-dependent kinase inhibitor p16(INK4a) (CDKN2A) is an important tumor-suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal.

Mitotic figure counts are overrated in resection specimen of invasive breast carcinoma

In order for some patients to benefit from aggressive chemotherapy for invasive breast carcinoma, many patients are currently being treated without little or no benefit. Enormous effort is hence being directed towards the identification of those patients who will need chemotherapy and those who will not. Since chemotherapy targets proliferating cells pathologists focus on the proliferative activity of tumors, as assessed by mitotic figure counts or by cell cycle specific immunohistochemical markers, such as Ki-67 and H3 histone. As far as the tumor grade is concerned, many of these studies have reported a tendency to up-grade carcinomas in resection specimen when compared to the initial diagnosis on the biopsy material, and most studies have noted that the upgrade in resection specimen is due solely or to a large extent to an increase in the mitotic figure count. In the present study, we propose a different explanation for the divergence in mitotic figure counts between biopsy and resection material. We assessed the proliferative activity of 52 invasive ductal carcinomas and confirm that the number of mitotic figures significantly increased by a factor of more than 3 in resection specimen over the biopsy material, while at the same time the pan-cell cycle specific marker MIB-1 yieldes comparable results. we propose that the delayed formalin fixation of resection specimen allows cell cycle activities to continue for a long time, up to many hours, and that this leads to an arrest of mitoses in metaphase where they are readily identified by the pathologist. We propose that the mitotic figure count in the rapidly fixed biopsy cores better represent the tumor biology and should be used as a basis for chemotherapy therapeutic decisions.

Characterization of a MexAB-OprM efflux system necessary for productive metabolism of Pseudomonas azelaica HBP1 on 2-hydroxybiphenyl.

Pseudomonas azelaica HBP1 is one of the few bacteria known to completely mineralize the biocide and toxic compound 2-hydroxybiphenyl (2-HBP), but the mechanisms of its tolerance to the toxicity are unknown. By transposon mutant analysis and screening for absence of growth on water saturating concentrations of 2-HBP (2.7 mM) we preferentially found insertions in three genes with high homology to the mexA, mexB, and oprM efflux system. Mutants could grow at 2-HBP concentrations below 100 μM but at lower growth rates than the wild-type. Exposure of the wild-type to increasing 2-HBP concentrations resulted in acute cell growth arrest and loss of membrane potential, to which the cells adapt after a few hours. By using ethidium bromide (EB) as proxy we could show that the mutants are unable to expel EB effectively. Inclusion of a 2-HBP reporter plasmid revealed that the wild-type combines efflux with metabolism at all 2-HBP concentrations, whereas the mutants cannot remove the compound and arrest metabolism at concentrations above 24 μM. The analysis thus showed the importance of the MexAB-OprM system for productive metabolism of 2-HBP.

La tipicidad de las detenciones ilegales policiales. Revisión crítica de la aplicación jurisprudencial de los arts. 167 y 530 del Código Penal

Es difícil encontrar en el Código penal español una regulación tan deficiente y confusa de una serie de conductas delictivas tan relevantes para el funcionamiento del Estado de Derecho como es la relativa a las detenciones ilegales cometidas por funcionario público que se prevé en los arts. 167 y 530. Lejos de hallar criterios jurisprudenciales satisfactorios que reconduzcan, en la medida de lo posible, la situación se advierte que el caos se ha instalado en buena medida en las resoluciones de los tribunales. En este trabajo se describe la situación y se propone una revisión general de los criterios interpretativos de los tipos en cuestión con el fin de dotarlos de mayor coherencia y certeza en su aplicación.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

Sex-chromosome turnovers: the hot-potato model.

Abstract Sex-determining systems often undergo high rates of turnover but for reasons that remain largely obscure. Two recent evolutionary models assign key roles, respectively, to sex-antagonistic (SA) mutations occurring on autosomes and to deleterious mutations accumulating on sex chromosomes. These two models capture essential but distinct key features of sex-chromosome evolution; accordingly, they make different predictions and present distinct limitations. Here we show that a combination of features from the two models has the potential to generate endless cycles of sex-chromosome transitions: SA alleles accruing on a chromosome after it has been co-opted for sex induce an arrest of recombination; the ensuing accumulation of deleterious mutations will soon make a new transition ineluctable. The dynamics generated by these interactions share several important features with empirical data, namely, (i) that patterns of heterogamety tend to be conserved during transitions and (ii) that autosomes are not recruited randomly, with some chromosome pairs more likely than others to be co-opted for sex.

The G0/G1 switch gene 2 is a novel PPAR target gene.

PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma are a group of transcription factors that are involved in numerous processes, including lipid metabolism and adipogenesis. By comparing liver mRNAs of wild-type and PPARalpha-null mice using microarrays, a novel putative target gene of PPARalpha, G0S2 (G0/G1 switch gene 2), was identified. Hepatic expression of G0S2 was up-regulated by fasting and by the PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. Surprisingly, the G0S2 mRNA level was highest in brown and white adipose tissue and was greatly up-regulated during mouse 3T3-L1 and human SGBS (Simpson-Golabi-Behmel syndrome) adipogenesis. Transactivation, gel shift and chromatin immunoprecipitation assays indicated that G0S2 is a direct PPARgamma and probable PPARalpha target gene with a functional PPRE (PPAR-responsive element) in its promoter. Up-regulation of G0S2 mRNA seemed to be specific for adipogenesis, and was not observed during osteogenesis or myogenesis. In 3T3-L1 fibroblasts, expression of G0S2 was associated with growth arrest, which is required for 3T3-L1 adipogenesis. Together, these data indicate that G0S2 is a novel target gene of PPARs that may be involved in adipocyte differentiation.

A downstream mediator in the growth repression limb of the jasmonate pathway.

Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

Pacemaker therapy in patients with neurally mediated syncope and documented asystole: Third International Study on Syncope of Uncertain Etiology (ISSUE-3): a randomized trial.

BACKGROUND: The efficacy of cardiac pacing for prevention of syncopal recurrences in patients with neurally mediated syncope is controversial. We wanted to determine whether pacing therapy reduces syncopal recurrences in patients with severe asystolic neurally mediated syncope. METHODS AND RESULTS: Double-blind, randomized placebo-controlled study conducted in 29 centers in the Third International Study on Syncope of Uncertain Etiology (ISSUE-3) trial. Patients were ≥40 years, had experienced ≥3 syncopal episodes in the previous 2 years. Initially, 511 patients, received an implantable loop recorder; 89 of these had documentation of syncope with ≥3 s asystole or ≥6 s asystole without syncope within 12 ± 10 months and met criteria for pacemaker implantation; 77 of 89 patients were randomly assigned to dual-chamber pacing with rate drop response or to sensing only. The data were analyzed on intention-to-treat principle. There was syncope recurrence during follow-up in 27 patients, 19 of whom had been assigned to pacemaker OFF and 8 to pacemaker ON. The 2-year estimated syncope recurrence rate was 57% (95% CI, 40-74) with pacemaker OFF and 25% (95% CI, 13-45) with pacemaker ON (log rank: P=0.039 at the threshold of statistical significance of 0.04). The risk of recurrence was reduced by 57% (95% CI, 4-81). Five patients had procedural complications: lead dislodgment in 4 requiring correction and subclavian vein thrombosis in 1 patient. CONCLUSIONS: Dual-chamber permanent pacing is effective in reducing recurrence of syncope in patients ≥40 years with severe asystolic neurally mediated syncope. The observed 32% absolute and 57% relative reduction in syncope recurrence support this invasive treatment for the relatively benign neurally mediated syncope. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00359203.

Developmental changes in cardiac recovery from anoxia-reoxygenation.

The developing cardiovascular system is known to operate normally in a hypoxic environment. However, the functional and ultrastructural recovery of embryonic/fetal hearts subjected to anoxia lasting as long as hypoxia/ischemia performed in adult animal models remains to be investigated. Isolated spontaneously beating hearts from Hamburger-Hamilton developmental stages 14 (14HH), 20HH, 24HH, and 27HH chick embryos were subjected in vitro to 30 or 60 min of anoxia followed by 60 min of reoxygenation. Morphological alterations and apoptosis were assessed histologically and by transmission electron microscopy. Anoxia provoked an initial tachycardia followed by bradycardia leading to complete cardiac arrest, except for in the youngest heart, which kept beating. Complete atrioventricular block appeared after 9.4 +/- 1.1, 1.7 +/- 0.2, and 1.6 +/- 0.3 min at stages 20HH, 24HH, and 27HH, respectively. At reoxygenation, sinoatrial activity resumed first in the form of irregular bursts, and one-to-one atrioventricular conduction resumed after 8, 17, and 35 min at stages 20HH, 24HH, and 27HH, respectively. Ventricular shortening recovered within 30 min except at stage 27HH. After 60 min of anoxia, stage 27HH hearts did not retrieve their baseline activity. Whatever the stage and anoxia duration, nuclear and mitochondrial swelling observed at the end of anoxia were reversible with no apoptosis. Thus the embryonic heart is able to fully recover from anoxia/reoxygenation although its anoxic tolerance declines with age. Changes in cellular homeostatic mechanisms rather than in energy metabolism may account for these developmental variations.

FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

Diagnosis and management of blunt great vessel trauma.

Traditionally, thoracic aortic rupture, suspected after blunt thoracic trauma, is characterized by a chest radiograph showing a widened mediastinum. The diagnostic machinery consecutively activated still depends heavily on the pressure as additional traumatic lesions. A patient with additional cranio-cerebral trauma would typically undergo contrast-enhanced computed tomography or magnetic resonance imaging of head, chest, and other regions. In a number of patients these analyses would confirm the presence of blood in the mediastinum without formal proof of an aortic disruption. This is because mediastinal hematomas may be caused not only by an aortic rupture, but also by numerous other blood sources including fractures of the spine and other macro- and microvascular lesions providing similar images. Therefore, aortic angiography became our preferred diagnostic tool to identify or rule out acute traumatic lesions of not only the aorta but with great vessels. However recently, a number of traumatic aortic transsections have been identified by transoesophageal echocardiography (TEE). TEE has the additional advantage of being a bed-side procedure providing additional information about cardiac function. The latter analysis allows for identification and quantification of cardiac contusions, post-traumatic myocardial infarctions, and valvar lesions which are of prime importance to develop an adequate surgical strategy and to assess the risk of the numerous emergency procedures required in patients with polytrauma. The standard approach for repair of isthmic aortic rupture is through a lateral thoracotomy. Distal and proximal control of the aorta can be achieved in a substantial number of cases before complete aortic rupture occurs and a higher proportion of direct suture repair can be achieved under such circumstances. Most proximal descending aortic procedures are performed without cardiopulmonary bypass (clamp and go) but paraplegia may occur before, during, or after the procedure. Ascending aortic lesions and disruption of the aortic arch, the supra-aortic vessels, the main pulmonary arteries, the great veins as well as cardiac lesions are best approached through a sternotomy, which may have to be extended. Cardiopulmonary bypass allowing for deep hypothermia and circulatory arrest is often required and carries its own complications. It is not clear whether the increasing proportion of ascending aortic and cardiac lesions which are observed nowadays are due to a change in trauma mechanics (i.e., speed limits, seat belts, air-bags), an improvement of the diagnostic tools or both.