Evidence that GZ proteins couple to hypothalamic 5-HT1A receptors in vivo

This is the publisher's version, also available electronically from "http://www.jneurosci.org".Using in situ hybridization and immunoblot analysis, the present studies identified Gz mRNA and Gz-protein in the hypothalamic paraventricular nucleus. The role of Gz-proteins in hypothalamic 5-HT1Areceptor signaling was examined in vivo. Activation of 5-HT1A receptors increases the secretion of oxytocin and ACTH, but not prolactin. Intracerebroventricular infusion (3–4 d) of Gz antisense oligodeoxynucleotides, with different sequences and different phosphorothioate modification patterns, reduced the levels of Gz-protein in the hypothalamic paraventricular nucleus, whereas missense oligodeoxynucleotides had no effect. Neither antisense nor missense oligodeoxynucleotide treatment altered basal plasma levels of ACTH, oxytocin, or prolactin, when compared with untreated controls. An antisense-induced decrease in hypothalamic Gz-protein levels was paralleled by a significant decrease in the oxytocin and ACTH responses to the 5-HT1A agonist 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT). In contrast, the prolactin response to 8-OH-DPAT (which cannot be blocked by 5-HT1A antagonists) was not inhibited by Gz antisense oligodeoxynucleotides. Gz-proteins are the only members of the Gi/Go-protein family that are not inactivated by pertussis toxin. In a control experiment, pertussis toxin treatment (1μg/5 μl, i.c.v.; 48 hr before the 8-OH-DPAT challenge) did not inhibit the ACTH response, potentiated the oxytocin response, and eliminated the prolactin response to 8-OH-DPAT. Thus, pertussis toxin-sensitive Gi/Go-proteins do not mediate the 5-HT1A receptor-mediated increase in ACTH and oxytocin secretion. Combined, these studies provide the firstin vivo evidence for a key role of Gz-proteins in coupling hypothalamic 5-HT1Areceptors to effector mechanisms

Characterization of the functional heterologous desensitization of hypothalamic 5-HT1A receptors after 5-HT2A receptor activation

This is the publisher's version, also available electronically from "www.jneurosci.org".Desensitization of 5-HT1A receptors could be involved in the long-term therapeutic effect of anxiolytic and antidepressant drugs. Pretreatment of rats with the 5-HT2A/2C agonist DOI induces an attenuation of hypothalamic 5-HT1Areceptor–Gz-protein signaling, measured as the ACTH and oxytocin responses to an injection of the 5-HT1A agonist 8-OH-DPAT. We characterized this functional heterologous desensitization of 5-HT1A receptors in rats and examined some of the mechanisms that are involved. A time course experiment revealed that DOI produces a delayed and reversible reduction of the ACTH and oxytocin responses to an 8-OH-DPAT challenge. The maximal desensitization occurred at 2 hr, and it disappeared 24 hr after DOI injection. The desensitization was dose-dependent, and it shifted the oxytocin and ACTH dose–response curves of 8-OH-DPAT to the right (increased ED50) with no change in their maximal responses (E max). The 5-HT2A receptor antagonist MDL 100,907 prevented the DOI-induced desensitization, indicating that 5-HT2Areceptors mediate the effect of DOI. Analysis of the components of the 5-HT1A receptor–Gz-protein signaling system showed that DOI did not alter the level of membrane-associated Gz-proteins in the hypothalamus. Additionally, DOI did not alter the binding of [3H]8-OH-DPAT or the inhibition by GTPγS of [3H]8-OH-DPAT binding in the hypothalamus. In conclusion, the activation of 5-HT2Areceptors induces a transient functional desensitization of 5-HT1A receptor signaling in the hypothalamus, which may occur distal to the 5-HT1A receptor–Gz-protein interface

Incidence of chromosome numerical changes in multiple myeloma: fluorescence in situ hybridization analysis using 15 chromosome-specific probes

Fue el primer trabajo precursor de los estudios con FISH en pacientes con Mieloma múltiple continuando la exitosa línea de investigación en mieloma del grupo de Salamanca, y por añadidura, del Grupo español de mieloma. La metodología FISH es hoy en día la principal herramienta de pronóstico en mieloma múltiple en todo el mundo.[EN]The presence of complex karotypes with frequent numerical and structural abnormalities has been reported in 20 to 50% of multiple myeloma (MM) patients. This variability is mainly due to the difficulty of conventional cytogenetics to obtain tumor metaphases representative of all possible neoplastic clones in MM. To gain insight into the real incidence of numerical chromosome changes in MM we have studied by fluorescence in situ hybridization technique 15 different human chromosomes, 1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, X, and Y, in a series of 52 MM patients. In all cases, the DNA index assessed by a propidium iodide/CD38 double-staining technique with flow cytometry was simultaneously investigated for correlation, with fluorescence in situ hybridization results. Additional aims of this study were 1) to analyze whether the abnormalities detected were common to all plasma cells or were present in only a subpopulation of tumor cells, 2) to explore changes caused by disease progression, and 3) to establish possible associations among the altered chromosomes. Although the overall incidence of numerical abnormalities was 67%, this frequency increased to 80% in the 41 cases in which 7 or more chromosomes were analyzed. Trisomies were significantly more common than monosomies (84% versus 16%). Chromosomes 9 and 15 were the most frequently altered (52% and 48% of cases, respectively), with all of their abnormalities corresponding to trisomies. The most frequent losses involved chromosomes 13 (26%) and X in females (32%). Other common numerical changes corresponded to chromosomes 1 (39%), 11 (37%), 6 (32%), 3 (31%), 18 (29%), 7 (28%), and 17 (22%). By contrast, chromosomes 8(13%), 10(8%), and 12(3%) were rarely altered. DNA aneuploidy by flow cytometry was detected in 67% of patients, and a high degree of correlation was observed between the DNA index obtained by flow cytometry and the chromosome index derived from fluorescence in situ hybridization studies, calculated according to two mathematical formulas (coefficient of correlation of 0.82 and 0.91 when at least 7 or 12 chromosomes were considered, respectively). The frequency of numeric chromosome aberrations was higher in those patients with progressive disease and, interestingly, trisomy of chromosome 8 was exclusively detected in this latter group of patients. Our study shows that, with the exception of chromosome 8, a possible marker of clonal evolution, the numeric chromosome changes are present in nearly all malignant plasma cells (r > 0.84). Finally, frequent associations between chromosomal aberrations were observed (ie, chromosomes 6, 7, 9, and 17; 7 and 15; and 11 and 17). By excluding them, it was found that two triple combinations of chromosome-specific probes, chromosomes 1 and 9 together with either chromosome 13 or 15, could be a useful marker for detection of residual disease, as it permits the identification of most MM patients displaying numerical changes.University Hosptial of Salamanca Universidad de SalamancaHospital Universitario de Salamanc

Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases

Automated array-CGH optimized for archival formalin-fixed, paraffin-embedded tumor material

BACKGROUND: Array Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH. METHODS: In this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use. RESULTS: Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method. CONCLUSION: This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility

A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion

NWOSupramolecular & Biomaterials Chemistr

Challenging compliance with international intellectual property norms in investor-state dispute settlement

Enforcing intellectual property (IP) rights abroad is not easy – not least because international IP treaties do not create global rights that can invoked in national courts. International investment law offers potential routes for overcoming these hurdles. Whenever investment treaties include IP rights as an investment and allow for investor-state dispute settlement (ISDS), investors can challenge host state measures affecting their IP rights in ISDS proceedings. As this article will show, this in turn offers a unique opportunity for invoking the standards of protection under international investment agreements (IIAs) to challenge host state compliance with international IP treaties. While challenging national IP regimes is an attractive option for right holders, these challenges potentially amount to a sea-change for the international IP regime and cause serious concern for host states. I however argue that most of the routes pursued by right holders under IIAs are unlikely to be successful. Investment protection standards such as fair and equitable treatment, umbrella clauses and most-favored nation treatment should not be construed to allow invoking alleged breaches of international IP norms in ISDS. Some IIAs however contain clauses that subject expropriation claims against compulsory licenses and other IP limitations to a test of consistency with the international IP rules governing these limitations. As they offer the only feasible route for investors to challenge host state compliance with international IP treaties, I review the implications of these clauses, recent reform proposals and suggest alternative mechanisms for aligning international IP and investment protection based on general international law.This is the author accepted manuscript. The final version is available from Oxford University Press via http://dx.doi.org/10.1093/jiel/jgw00

Hyperbaric oxygen therapy for late radiation-induced tissue toxicity: Prospectively patient-reported outcome measures in breast cancer patients

__Introduction:__ This study examines patient reported outcome measures of women undergoing hyperbaric oxygen treatment (HBOT) after breast-conserving therapy. __Method:__ Included were 57 women treated with HBOT for late radiation-induced tissue toxicity (LRITT) referred in the period January 2014-December 2015. HBOT consisted of (on average) 47 sessions. In total, 80 min of 100 % O2 was administered under increased pressure of 2.4 ATA. Quality of life was assessed before and after treatment using the European Organization for Research and Treatment of Cancer (EORTC) QLQ-BR23, and a NRS pain score. __Results:__ Fifty-seven women were available for evaluation before and after treatment. Before HBOT, patients had severe complaints of pain in the arm/shoulder (46 %), swollen arm/hand (14 %), difficulty to raise arm or move it sideways (45 %), pain in the area of the affected breast (67 %), swollen area of the affected breast (45 %), oversensitivity of the affected breast (54 %), and skin problems on/in the area of the affected breast (32 %); post HBOT, severe complaints were still experienced in 17, 7, 22, 15, 13, 15, and 11 % of the women, respectively. Differences were all significant. The NRS pain score improved at least 1 point (range 0-10) in 81 % of the patients (p < 0.05). __Conclusion:__ In these breast cancer patients treated with HBOT for LRITT, the patient-reported outcomes were positive and improvements were observed. HBOT was a well-tolerated treatment for LRITT and its side-effects were both minimal and reversible

Specific genomic aberrations in primary colorectal cancer are associated with liver metastases

Background: Accurate staging of colorectal cancer (CRC) with clinicopathological parameters is important for predicting prognosis and guiding treatment but provides no information about organ site of metastases. Patterns of genomic aberrations in primary colorectal tumors may reveal a chromosomal signature for organ specific metastases. Methods: Array Comparative Genomic Hybridization (aCGH) was employed to asses DNA copy number changes in primary colorectal tumors of three distinctive patient groups. This included formalin-fixed, paraffin-embedded tissue of patients who developed liver metastases (LM; n = 36), metastases (PM; n = 37) and a group that remained metastases-free (M0; n = 25). A novel statistical method for identifying recurrent copy number changes, KC-SMART, was used to find specific locations of genomic aberrations specific for various groups. We created a classifier for organ specific metastases based on the aCGH data using Prediction Analysis for Microarrays (PAM). Results: Specifically in the tumors of primary CRC patients who subsequently developed liver metastasis, KC-SMART analysis identified genomic aberrations on chromosome 20q. LM-PAM, a shrunken centroids classifier for liver metastases occurrence, was able to distinguish the LM group from the other groups (M0&PM) with 80% accuracy (78% sensitivity and 86% specificity). The classification is predominantly based on chromosome 20q aberrations. Conclusion: Liver specific CRC metastases may be predicted with a high accuracy based on specific genomic aberrations in the primary CRC tumor. The ability to predict the site of metastases is important for improvement of personalized patient management.MediamaticsElectrical Engineering, Mathematics and Computer Scienc

Urticaria and infections

Urticaria is a group of diseases that share a distinct skin reaction pattern. Triggering of urticaria by infections has been discussed for many years but the exact role and pathogenesis of mast cell activation by infectious processes is unclear. In spontaneous acute urticaria there is no doubt for a causal relationship to infections and all chronic urticaria must have started as acute. Whereas in physical or distinct urticaria subtypes the evidence for infections is sparse, remission of annoying spontaneous chronic urticaria has been reported after successful treatment of persistent infections. Current summarizing available studies that evaluated the course of the chronic urticaria after proven Helicobacter eradication demonstrate a statistically significant benefit compared to untreated patients or Helicobacter-negative controls without urticaria (p < 0.001). Since infections can be easily treated some diagnostic procedures should be included in the routine work-up, especially the search for Helicobacter pylori. This review will update the reader regarding the role of infections in different urticaria subtypes