COX-2 activation is associated with Akt phosphorylation and poor survival in ER-negative, HER2-positive breast cancer

<p>Abstract</p> <p>Background</p> <p>Inducible cyclooxgenase-2 (COX-2) is commonly overexpressed in breast tumors and is a target for cancer therapy. Here, we studied the association of COX-2 with breast cancer survival and how this association is influenced by tumor estrogen and HER2 receptor status and Akt pathway activation.</p> <p>Methods</p> <p>Tumor COX-2, HER2 and estrogen receptor α (ER) expression and phosphorylation of Akt, BAD, and caspase-9 were analyzed immunohistochemically in 248 cases of breast cancer. Spearman's correlation and multivariable logistic regression analyses were used to examine the relationship between COX-2 and tumor characteristics. Kaplan-Meier survival and multivariable Cox proportional hazards regression analyses were used to examine the relationship between COX-2 and disease-specific survival.</p> <p>Results</p> <p>COX-2 was significantly associated with breast cancer outcome in ER-negative [Hazard ratio (HR) = 2.72; 95% confidence interval (CI), 1.36-5.41; comparing high versus low COX-2] and HER2 overexpressing breast cancer (HR = 2.84; 95% CI, 1.07-7.52). However, the hazard of poor survival associated with increased COX-2 was highest among patients who were both ER-negative and HER2-positive (HR = 5.95; 95% CI, 1.01-34.9). Notably, COX-2 expression in the ER-negative and HER2-positive tumors correlated significantly with increased phosphorylation of Akt and of the two Akt targets, BAD at Ser136 and caspase-9 at Ser196.</p> <p>Conclusions</p> <p>Up-regulation of COX-2 in ER-negative and HER2-positive breast tumors is associated with Akt pathway activation and is a marker of poor outcome. The findings suggest that COX-2-specific inhibitors and inhibitors of the Akt pathway may act synergistically as anticancer drugs in the ER-negative and HER2-positive breast cancer subtype.</p

Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif

To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens

Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies

Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH4:H2 gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000±14,000 and 152,000±10,000 cells/cm2, respectively, compared to 113,000±10,000 cells/cm2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells

The smear layer created by scaling and root planing is physiologically eliminated in a biphasic process

Mechanical instrumentation of the root surface causes the formation of a smear layer, which is a physical barrier that can affect periodontal regeneration. Although different procedures have been proposed to remove the smear layer, there is no information concerning how long the smear layer persists on root surfaces after instrumentation in vivo. This study assessed the presence of the smear layer on root surfaces over a 28-day period after subgingival instrumentation with hand instruments. Fifty human teeth that were referred for extraction because of advanced periodontal disease were scaled and root planed (SRP) by a single experienced operator. Ten teeth were randomly assigned to be extracted 7, 14, 21, and 28 days after SRP. Another 10 teeth were extracted immediately after instrumentation (Day 0, control group). The subgingival area of the instrumented roots was evaluated with scanning electron microscopy. Representative photomicrographs were assessed by a blinded and calibrated examiner according to a scoring system. A rapid and significant (p < 0.05, Z test) initial reduction in the amount of smear layer was observed at 7 days, and a further significant (p < 0.05) decrease was observed 28 days after SRP. Interestingly, even 28 days after SRP, the smear layer was still present on root surfaces. This study showed that the physiological elimination of the smear layer occurred in a biphasic manner: a rapid initial reduction was observed 7 days after instrumentation, which was followed by a slow process leading to a significant decrease 28 days after instrumentation.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Estadual Paulista School of Dentistry at Araraquara Department of Diagnosis and SurgeryUniversidade Estadual Paulista School of Dentistry at Araraquara Department of Diagnosis and Surger

PALB2, CHEK2 and ATM rare variants and cancer risk: data from COGS

Background The rarity of mutations in PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study. Methods We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant. Results For European women, strong evidence of association with breast cancer risk was observed for PALB2 c.1592delT OR 3.44 (95% CI 1.39 to 8.52, p=7.1x10-5), PALB2 c.3113G>A OR 4.21 (95% CI 1.84 to 9.60, p=6.9x10-8) and ATM c.7271T>G OR 11.0 (95% CI 1.42 to 85.7, p=0.0012). We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p=0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03 (95% CI 1.53 to 6.03, p=0.0006) for African men and CHEK2 c.1312G>T OR 2.21 (95% CI 1.06 to 4.63, p=0.030) for European men. No evidence of association with ovarian cancer was found for any of these variants. Conclusions This report adds to accumulating evidence that at least some variants in these genes are associated with an increased risk of breast cancer that is clinically important