Dual Targeted Therapy with p53 siRNA and Epigallocatechingallate in a Triple Negative Breast Cancer Cell Model

Triple-negative breast cancer (TNBC) is a highly aggressive phenotype that is resistant to standard therapy. Thus, the development of alternative therapeutic strategies for TNBC is essential. The purpose of our in vitro study was to evaluate the impact of p53 gene silencing in conjunction with the administration of a natural compound, epigallocatechingallate (EGCG). RT²Profiler PCR Array technology was used to evaluate the impact of dual treatment on the main genes involved in apoptosis in the Hs578T cell culture model of TNBC. Gene expression analysis revealed 28 genes were significantly altered (16 upregulated and 12 downregulated) in response to combined p53 siRNA and EGCG treatment. Further analysis revealed that p53 siRNA and EGCG dual therapy leads to the activation of pro-apoptotic genes and the inhibition of pro-survival genes, autophagy, and cell network formation. These results indicate that this dual therapy targets both the apoptotic and angiogenic pathways, which may improve treatment effectiveness for tumors resistant to conventional treatment.     

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10⁻⁶, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5–10, p = 3.3 x 10⁻⁶, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.     

Risk Factors for Plasmodium falciparum Gametocyte Positivity in a Longitudinal Cohort

Malaria transmission intensity is highly heterogeneous even at a very small scale. Implementing targeted intervention in malaria transmission hotspots offers the potential to reduce the burden of disease both locally and in adjacent areas. Transmission of malaria parasites from man to mosquito requires the production of gametocyte stage parasites. Cluster analysis of a 19-year long cohort study for gametocyte carriage revealed spatially defined gametocyte hotspots that occurred during the time when chloroquine was the drug used for clinical case treatment. In addition to known risk factors for gametocyte carriage, notably young age (<15 years old) and associated with a clinical episode, blood groups B and O increased risk compared to groups A and AB. A hotspot of clinical P. falciparum clinical episodes that overlapped the gametocyte hotspots was also identified. Gametocyte positivity was found to be increased in individuals who had been treated with chloroquine, as opposed to other drug treatment regimens, for a clinical P. falciparum episode up to 30 days previously. It seems likely the hotspots were generated by a vicious circle of ineffective treatment of clinical cases and concomitant gametocyte production in a sub-population characterized by an increased prevalence of all the identified risk factors. While rapid access to treatment with an effective anti-malarial can reduce the duration of gametocyte carriage and onward parasite transmission, localised hotspots represent a challenge to malaria control and eventual eradication.     

The Impact of Heat on an Emergency Department in Italy: Attributable Visits among Children,Adults, and the Elderly during the Warm Season

Introduction

Recent studies suggest that heat is associated with an increase in the number of ambulance calls and emergency department visits. We investigated the association between heat and daily number of emergency department visits at the University Hospital of Verona during the warm seasons 2011–2012 and we assessed the magnitude of the impact in terms of attributable events, focusing on the role of age and triage codification.

Materials and methods

We used a Poisson model to analyse the association between daily number of visits and daily mean apparent temperature, accounting for air pollution level and seasonality. The analyses were stratified by age group and were performed both on the total number of emergency department visits and on the subsample of high-priority visits. Impact estimates were obtained only for this subsample, using a Monte Carlo approach to account for sampling variability. Number of attributable events and attributable community rate were calculated.

Results

We found a positive and immediate association between event occurrence and mean apparent temperatures exceeding a threshold located around 28–29°C. The estimated percent change in the total number of visits per 1°C increase of exposure above the threshold was equal to 3.75 (90% CI: 3.01; 4.49). Focusing only on high-priority visits, the estimated percent change was larger and the greatest effect was among children. We estimated that apparent temperatures above the threshold were responsible for 1177 high-priority visits during the study period. Due to the record high temperatures observed in 2012 in Italy and in Europe, the impact in 2012 was much larger than in 2011, and consisted in 34 high-priority visits every 10000 children, 30 every 10000 people aged 15–64, and 38 every 10000 people aged 65 and over.

Discussion

Our results indicate that heat affects not only the elderly, but also children and non-elderly adults, stressing the need for developing public health preparedness plans for the entire community.     

Therapeutic antibody engineering by high efficiency cell screening

In recent years, several cell-based screening technologies for the isolation of antibodies with prescribed properties emerged. They rely on the multi-copy display of antibodies or antibody fragments on a cell surface in functional form followed by high through put screening and isolation of cell clones that carry an antibody variant with the desired affinity, specificity, and stability. Particularly yeast surface display in combination with high-throughput fluorescence-activated cell sorting has proven successful in the last fifteen years as a very powerful technology that has some advantages over classical generation of monoclonals using the hybridoma technology or bacteriophage-based antibody display and screening. Cell-based screening harbours the benefit of single-cell online and real-time analysis and characterisation of individual library candidates. Moreover, when using eukaryotic expression hosts, intrinsic quality control machineries for proper protein folding and stability exist that allow for co-selection of high-level expression and stability simultaneously to the binding functionality. Recently, promising technologies emerged that directly rely on antibody display on higher eukaryotic cell lines using lentiviral transfection or direct screening on B-cells. The combination of immunisation, B-cell screening and next generation sequencing may open new avenues for the isolation of therapeutic antibodies with prescribed physicochemical and functional characteristics.     

S-nitrosothiols increases cystic fibrosis transmembrane regulator expression and maturation in the cell surface

S-nitrosothiols (SNOs) are endogenous signaling molecules with a broad spectrum of beneficial airway effects. SNOs are normally present in the airway, but levels tend to be low in cystic fibrosis (CF) patients. We and others have demonstrated that S-nitrosoglutathione (GSNO) increases the expression, maturation, and function of wild-type and mutant F508del cystic fibrosis transmembrane conductance regulator (CFTR) in human bronchial airway epithelial (HBAE) cells. We hypothesized that membrane permeable SNOs, such as S-nitrosoglutathione diethyl ester (GNODE) and S-nitroso-N-acetyl cysteine (SNOAC) may be more efficient in increasing the maturation of CFTR. HBAE cells expressing F508del CFTR were exposed to GNODE and SNOAC. The effects of these SNOs on the expression and maturation of F508del CFTR were determined by cell surface biotinylation and Western blot analysis. We also found for the first time that GNODE and SNOAC were effective at increasing CFTR maturation at the cell surface. Furthermore, we found that cells maintained at low temperature increased cell surface stability of F508del CFTR whereas the combination of low temperature and SNO treatment significantly extended the half-life of CFTR. Finally, we showed that SNO decreased the internalization rate of F508del CFTR in HBAE cells. We anticipate identifying the novel mechanisms, optimal SNOs, and lowest effective doses which could benefit cystic fibrosis patients.     

Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: A real-time study

α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale—both in situ and in real-time—the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.