Tumor ablation with irreversible electroporation

We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 micros at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.     

Swine generated by somatic cell nuclear transfer have increased incidence of intrauterine growth restriction (IUGR)

While somatic cell nuclear transfer (SCNT) has been successful in several species, many pregnancies are lost and anomalies are found in fetal and perinatal stages. In this study SCNT and artificial inseminations (AI) populations were compared for litter size, average birth weight, piglets alive at birth, stillborn, mummies, dead at the first week, intrauterine growth restriction (IUGR) and large for gestational age (LGA). Twenty-three SCNT litters (143 individuals) were compared to 112 AI litters (1300 individuals). Litter size average was 11.5 for AI and 6.2 for SCNT. Litter weight and average birth weight adjusted by litter size were significantly (p < 0.05) higher in AI than in SCNT litters. The SCNT population had a significant (p < 0.01) increase in the number of IUGRs per litter with LSmeans 7.2 +/- 1.4 versus 19.4 +/- 3.5 and means 8.0 +/- 10.8 versus 15.5 +/- 24.5 for AI and SCNT, respectively. Additionally, there was a trend for higher postnatal mortality and stillbirths in the SCNT population. These findings demonstrate that there are some differences between SCNT-derived and AI litters. SCNT-derived pigs are excellent models to study epigenetic factors and genes involved in IUGRs, and to develop effective means to improve fetal growth in humans and animals.     

A balance index for phylogenetic trees based on rooted quartets

Journal of Mathematical Biology - We define a new balance index for rooted phylogenetic trees based on the symmetry of the evolutive history of every set of 4 leaves. This index makes sense for...     

Dysregulated expression of STAT1, miR-150, and miR-223 in peripheral blood mononuclear cells of coronary artery disease patients with significant or insignificant stenosis

Coronary artery disease (CAD) is a multicellular disease characterized by chronic inflammation. Peripheral blood-mononuclear cells (PBMCs), as a critical component of immune system, actively cross-talk with pathophysiological conditions induced by endothelial cell injury, reflecting in perturbed PBMC expression. STAT1 is believed to be relevant to CAD pathogenesis through regulating key inflammatory processes and modulating STAT1 expression play key roles in fine-tuning CAD-related inflammatory processes. This study evaluated PBMC expressions of STAT1, and its regulators (miR-150 and miR-223) in a cohort including 72 patients with CAD with significant ( ≥ 50%) stenosis, 30 patients with insignificant ( < 50%) coronary stenosis (ICAD), and 74 healthy controls, and assessed potential of PBMC expressions to discriminate between patients and controls. We designed quantitative real-time polymerase chain reaction (RT-qPCR) assays and identified stable reference genes for normalizing PBMC quantities of miR-150, miR-223, and STAT1 applying geNorm algorithm to six small RNAs and five mRNAs. There was no significant difference between CAD and ICAD patients regarding STAT1 expression. However, both groups of patients had higher levels of STAT1 than healthy controls. miR-150 and miR-223 were differently expressed across three groups of subjects and were downregulated in patients compared with healthy controls, with the lowest expression levels being observed in patients with ICAD. ROC curves suggested that PBMC expressions may separate between different groups of study subjects. PBMC expressions also discriminated different clinical manifestations of CAD from ICADs or healthy controls. In conclusion, the present study reported PBMC dysregulations of STAT1, miR-150, and miR-223, in patients with significant or insignificant coronary stenosis and suggested that these changes may have diagnostic implications.     

Differential interactions of antitumor antibiotics chromomycin A(3) and mithramycin with d(TATGCATA)(2) in presence of Mg(2+)

The antitumor antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) are known to inhibit macromolecular biosynthesis by reversibly binding to double stranded DNA with a GC base specificity via the minor groove in the presence of a divalent cation such as Mg(2+). Earlier reports from our laboratory showed that the antibiotics form two types of complexes with Mg(2+): complex I with 1:1 stoichiometry and complex II with 2:1 stoichiometry in terms of the antibiotic and Mg(2+). The binding potential of an octanucleotide, d(TATGCATA)(2), which contains one potential site of association with the above complexes of the two antibiotics, was examined using spectroscopic techniques such as absorption, fluorescence, and circular dichroism. We also evaluated thermodynamic parameters for the interaction. In spite of the presence of two structural moieties of the antibiotic in complex II, a major characteristic feature was the association of a single ligand molecule per molecule of octameric duplex in all cases. This indicated that the modes of association for the two types of complexes with the oligomeric DNA were different. The association was dependent on the nature of the antibiotics. Spectroscopic characterization along with analysis of binding and thermodynamic parameters showed that differences in the mode of recognition by complexes I and II of the antibiotics with polymeric DNA existed at the oligomeric level. Analysis of the thermodynamic parameters led us to propose a partial accommodation of the ligand in the groove without the displacement of bound water molecules and supported earlier results on the DNA structural transition from B --> A type geometry as an obligatory requirement for the accommodation of the bulkier complex II of the two drugs. The role of the carbohydrate moieties of the antibiotics in the DNA recognition process was indicated when we compared the DNA binding properties with the same type of Mg(2+) complex for the two antibiotics.     

Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.     

Mitochondrial requirement for endothelial responses to cyclic strain: implications for mechanotransduction

Mechanical strain triggers a variety of cellular responses, but the underlying mechanotransduction process has not been established. Endothelial cells (EC) respond to mechanical strain by upregulating adhesion molecule expression through a signaling process involving reactive oxygen species (ROS), but the site of their generation is unknown. Mitochondria anchor to the cytoskeleton and could function as mechanotransducers by releasing ROS during cytoskeletal strain. In human umbilical vein EC (HUVEC), ROS production increased 221 +/- 17% during 6 h of cyclic strain vs. unstrained controls. Mitochondrial inhibitors diphenylene iodonium or rotenone abrogated this response, whereas inhibitors of nitric oxide (NO) synthase (L-nitroarginine), xanthine oxidase (allopurinol), or NAD(P)H oxidase (apocynin) had no effect. The antioxidants ebselen and diethyldithiocarbamate inhibited the increase in ROS, but the NO scavenger Hb had no effect. Thus strain induces ROS release from mitochondria. In other studies, HUVEC were rendered mitochondria deficient (rho0 EC) to determine the requirement for electron transport in the response to strain. Strain-induced 2'7'-dichlorofluorescein fluorescence was attenuated by >80% in rho0 EC compared with HUVEC (43 +/- 7 vs. 221 +/- 17%). Treatment with cytochalasin D abrogated strain-induced ROS production, indicating a requirement for the actin cytoskeleton. Cyclic strain (6 h) increased VCAM-1 expression in wild-type but not rho0 EC. Increases in NF-kappaB activation and VCAM-1 mRNA expression during strain were prevented by antioxidants. These findings demonstrate that mitochondria function as mechanotransducers in endothelium by increasing ROS signaling, which is required for strain-induced increase in VCAM-1 expression via NF-kappaB.