Investigating DNA Radiation Damage Using X-Ray Absorption Spectroscopy

The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.     

Ecological validity of social interaction tests in rats and mice

Different rat and mouse models are used in studies of social interactions. Simple behavioral measures, which are commonly used in the laboratory, allow to perform relatively short experiments and to use multiple brain manipulation techniques. However, too much focus on the simplest behavioral models generates a serious risk of reducing ecological validity or even studying phenomena which would never happen outside of the laboratory. In this review, we discuss the suitability of mice and rats as model organisms for studying social behaviors, with focus on social transmission of fear paradigms. First, we briefly introduce the concept of domestication and what impact it had on laboratory rodents. Then, we present two aspects of social behaviors, sociability and dominance, which are crucial for social organization in these species. Finally, we present experimental models used for studying how animals transmit information about danger between each other, and how these models may reflect what happens in the natural environment. We discuss the difficulties that arise from our limited knowledge of rat and mouse ecology, especially their social life. We also explore the subject of balancing ecological validity and controllability in rodent models of social behaviors, the latter being particularly important for studying brain activity. Although it is very challenging, an efficient program for social neuroscience research should, in our opinion, aim at bridging the gap between laboratory and field studies.     

Fourier transform infrared spectroscopic signature of blood plasma in the progression of breast cancer with simultaneous metastasis to lungs

Despite advanced diagnostic techniques used for detecting cancer, this disease still remains a leading cause of death in the developed world. What is more, the greatest danger for patients is not related with growing of tumor but rather with metastasis of cancer cells to the distant organs. In this study, Fourier transform infrared (FTIR) spectroscopy was used to track chemical changes in blood plasma to find spectral markers of metastatic breast cancer during the disease progression. Plasma samples were taken 1‐5 weeks after orthotropic inoculation of 4T1 metastatic breast cancer cells to mice. The earliest changes detected by FTIR spectroscopy in plasma were correlated with unsaturation of phospholipids and secondary structures of proteins that appeared 2 and 3 weeks, respectively, after 4T1 cells inoculation (micrometastatic phase). Significant alternations in the content and structure of lipids and carbohydrates were identified in plasma at the later stages (macrometastatic phase). When large primary tumors in breast and macrometastases in lung were developed, all bands in FTIR spectra significantly differed from those at earlier phases of the cancer progression. In conclusion, we showed that each phase of the breast cancer progression and its pulmonary metastasis can be characterized by a specific panel of spectral markers.     

High Viral Loads of Epstein-Barr Virus DNA in Peripheral Blood of Patients with Chronic Lymphocytic Leukemia Associated with Unfavorable Prognosis

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. The potential involvement of EBV in the clinical course of chronic lymphocytic leukemia (CLL) remains unexplained. The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs) of CLL patients may influence heterogeneity in the course of the disease. The study included peripheral blood samples from 115 previously untreated patients with CLL (54 women and 61 men) and 40 healthy controls (16 women and 24 men). We analyzed the association between the EBV-DNA load in PBMCs and the stage of the disease, adverse prognostic factors, and clinical outcome. Detectable numbers of EBV-DNA copies in PBMCs were found in 62 out of 115 CLL patients (53.91%). The EBV-DNA copy number/μg DNA was significantly higher in patients who required early implementation of treatment, presented with lymphocyte count doubling time <12 months, displayed CD38-positive or ZAP-70-positive phenotype, and with the del(11q22.3) cytogenetic abnormality. Furthermore, the EBV-DNA copy number/μg DNA showed significant positive correlation with the concentrations of lactate dehydrogenase (LDH) and beta-2-microglobulin. We have shown that in CLL patients, higher EBV-DNA copy number predicted shorter survival and shorter time to disease progression, and it was associated with other established unfavorable prognostic factors. This suggests that EBV may negatively affect the outcome of CLL.     

Synthesis of New Styrylquinoline Cellular Dyes,Fluorescent Properties,Cellular Localization and Cytotoxic Behavior

New styrylquinoline derivatives with their photophysical constants are described. The synthesis was achieved via Sonogashira coupling using the newly developed heterogeneous nano-Pd/Cu catalyst system, which provides an efficient synthesis of high purity products. The compounds were tested in preliminary fluorescent microscopy studies to in order to identify their preferable cellular localization, which appeared to be in the lipid cellular organelles. The spectroscopic properties of the compounds were measured and theoretical TD-DFT calculations were performed. A biological analysis of the quinolines that were tested consisted of cytotoxicity assays against normal human fibroblasts and colon adenocarcinoma cells. All of the compounds that were studied appeared to be safe and indifferent to cells in a high concentration range. The presented results suggest that the quinoline compounds that were investigated in this study may be valuable structures for development as fluorescent dyes that could have biological applications.     

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols.     

Design,Synthesis and In Vitro Activity of Anticancer Styrylquinolines. The p53 Independent Mechanism of Action

A group of styrylquinolines were synthesized and tested for their anti-proliferative activity. Anti-proliferative activity was evaluated against the human colon carcinoma cell lines that had a normal expression of the p53 protein (HCT116 p53^+/+) and mutants with a disabled TP53 gene (HCT116 p53^-/-) and against the GM 07492 normal human fibroblast cell line. A SAR study revealed the importance of Cl and OH as substituents in the styryl moiety. Several of the compounds that were tested were found to have a marked anti-proliferative activity that was similar to or better than doxorubicin and were more active against the p53 null than the wild type cells. The cellular localization tests and caspase activity assays suggest a mechanism of action through the mitochondrial pathway of apoptosis in a p53-independent manner. The activity of the styrylquinoline compounds may be associated with their DNA intercalating ability.     

Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31∼P) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.     

Effect of tunicamycin on the biogenesis of hepatitis C virus glycoproteins

Hepatitis C virus (HCV) infects humans, with a prevalence around 3% of population, causing acute and chronic hepatitis and hepatocellular carcinoma. We studied the effect of inhibition of glycosylation on the assembly of the HCV particle. HCV possesses two envelope glycoproteins E1 and E2 that are highly modified by N-glycans. These glycan residues are crucial for viral entry and maturation of the progeny. Here, we examined the influence of inhibition of N-glycosylation on expression of E1 and E2. Since the propagation of HCV in cell culture is limited, we used a recombinant baculovirus producing viral-like particles in insect cells. Our data showed that blocking of N-glycan transfer to the nascent polypeptide chain with the antibiotic tunicamycin resulted in the loss of E1 and E2. We also found that a dose of tunicamycin that did not influence the cell viability significantly reduced the E2 level in infected cells. The results indicate that blocking of glycosylation at an early step efficiently reduces the assembly of HCV virions. Thus, we suggest that derivatives of tunicamycin that preferentially block glycosylation of viral proteins may become potential therapeutic agents against HCV.