Enhancement of Th1 immune response by CD8α dendritic cells loaded with heat shock proteins enriched tumor extract in tumor-bearing mice

The discovery of dendritic cells (DCs) as professional antigen presenting cells has opened up new possibilities for their use in the development of tumor vaccines. We investigated the effect of the CD8α⁺ DCs loaded with heat-treated tumor lysate (HTL) as a vaccine in tumor immunotherapy. The HTL loaded CD8α⁺ DCs, TL loaded CD8α⁺ DCs and unloaded CD8α⁺ DCs were subcutaneously injected in the fibrosarcoma-bearing mice. The splenocyte proliferation and the shifting of Th1/Th2 response were measured. The results indicated a significant increase in the lymphocytes proliferation and the IFN-γ production in the test group of mouse splenocytes. According to the results, HTL loaded CD8α⁺ DCs vaccine significantly decreased tumor growth and longer survival than the other immunized animals. These findings show that anti-tumor immune response against the fibrosarcoma can be induced by HTL loaded CD8α⁺ DCs and may provide a useful therapeutic model for development of approaches to tumor treatments.     

Interleukin-18 gene promoter and serum level in women with ovarian cancer

IL-18, initially defined as a potent inducer of IFN- γ production, is a systemic, multifunctional cytokine with both pro-cancerous and anti-cancer activities. The contribution of the IL-18 promoter polymorphisms at positions −607 (C/A) and −137 (G/C) to cancer development has been reported. We sought to examine IL-18 serum level and its polymorphisms in Iranian women with ovarian cancer. Single nucleotide polymorphisms (SNPs) at positions −607 (C/A) and −137 (G/C) were analyzed by allele-specific polymerase chain reaction in 85 women with ovarian cancer and 158 healthy controls. IL-18 serum level was determined using ELISA method. No significant association was found between the allele, genotype, and haplotype distributions of the SNPs and ovarian cancer. Mean IL-18 serum level was significantly higher in patients than in controls (P = 0.008). Comparing IL-18 serum levels with genotypes at positions −607 and −137 revealed no significant difference. No association was also found between IL-18 levels and the disease stage. In conclusion, our results indicate that IL-18 promoter polymorphisms at positions −607 (C/A) and −137 (G/C) appear not to confer susceptibility to ovarian cancer in Iranian population; however, IL-18 serum level increases in ovarian cancer patients.     

Lack of association between interleukin-13 gene polymorphisms (−1055 C/T and +2044 G/A) in Iranian patients with lung cancer

Lung cancer is one of the leading causes of death from cancer. Both immune cells and tumor cells play a key role in lung cancer immunity by secretion of cytokines and developing type-2 cell-mediated immune response. IL-13 is an immunoregulatory cytokine affecting tumor immunosurveillance by deviation of immune response from Th1 to Th2. In the present study we sought to determine the association of single nucleotide polymorphisms (SNPs) of IL-13 gene at positions +2044 (G/A) and −1055 (C/T) and lung cancer. One hundred forty one patients and 113 controls were recruited; control group was subdivided into smoker and nonsmoker individuals for serum detection. Genotyping was carried out by PCR-RFLP assay and IL-13 detection by ELISA method. No statistically significant difference was found in the frequency of genotypes, alleles, and haplotypes at positions +2044 (G/A) and −1055 (C/T) of IL-13 gene between lung cancer patients and controls. Serum level of IL-13 was not detectable in both groups. The results of this study reveal that although +2044 (G/A) and −1055 (C/T) SNPs in IL-13 are implicated in some pulmonary processes, they do not confer susceptibility to lung cancer in Iranian population.     

Assembly,Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit. The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided. Common errors in assembly and instrument tuning are discussed. A representative data set that shows the secondary structure of an amyloid fibril is presented.     

Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins

Mitoxantrone is a potent antitumor drug, widely used in the treatment of various cancers. In the present study, we have investigated and compared the affinity of anticancer drug, mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), DNA and histones employing UV/Vis, fluorescence, CD spectroscopy, gel electrophoresis and equilibrium dialysis techniques. The results showed that the interaction of mitoxantrone with SE-chromatin proceeds into compaction/aggregation as revealed by reduction in the absorbencies at 608 and 260 nm (hypochromicity) and disappearance of both histones and DNA on the gels. Mitoxantrone interacts strongly with histone proteins in solution making structural changes in the molecule as shown by CD and fluorescence analysis. The binding isotherms demonstrate a positive cooperative binding pattern for the chromatin- mitoxantrone interaction. It is suggested higher binding affinity of mitoxantrone to chromatin compared to DNA implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process.     

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.The pathology of influenza A virus infection usually arises from acute lymphopenia and inflammation of the lungs and airway columnar epithelial cells (23, 38). Influenza A virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells, and apoptosis is a source of tissue damage during infection (3, 22, 33) and increased susceptibility to bacterial pathogens postinfection (31). While the induction of apoptosis by influenza A virus has been well documented (4, 19-21, 28, 33, 37), the mechanisms of this interaction are not well understood. Two viral proteins, NS1 and PB1-F2, have been associated with viral killing of cells. NS1, originally characterized as being proapoptotic (34), was later identified as being an interferon antagonist, inhibiting the activation of several key antiviral responses and restricting the apoptotic response to infection (1, 10, 15, 18, 35, 39, 46). In contrast, PB1-F2 induces apoptosis primarily by localizing to the outer mitochondrial membrane, promoting cytochrome c release, and triggering the apoptotic cascade (43). This effect, however, is typically restricted to infected monocytes, leading to the hypothesis that PB1-F2 induces apoptosis specifically to clear the landscape of immune responders (5, 44). Although PB1-F2 activity does not directly manipulate virus replication or virus-induced apoptosis, PB1-F2 localization to the mitochondrial membrane during infection potentiates the apoptotic response in epithelial and fibroblastic cells through tBID signaling with proapoptotic Bcl-2 family protein members Bax and Bak (22, 43, 44).The Bcl-2 protein family consists of both pro- and antiapoptotic members that regulate cytochrome c release during mitochondrion-mediated apoptosis through the formation of pore-like channels in the outer mitochondrial membrane (12, 16). During the initiation of mitochondrion-mediated apoptosis, cytoplasmic Bid is cleaved to form tBID. This, in turn, activates proapoptotic Bax and Bak (40), which drive cytochrome c release and subsequent caspase activation. Bak is constitutively associated with the mitochondrial membrane, whereas inactive Bax is primarily cytosolic, translocating to the outer mitochondrial membrane only after activation (6). The activation of Bax and Bak results in homo- and heterodimer formation at the outer mitochondrial membrane, generating pores that facilitate mitochondrial membrane permeabilization and cytochrome c release (14, 17), leading to caspase activation and the apoptotic cascade (8). Antiapoptotic members of the Bcl-2 protein family, including Bcl-2, inhibit the activation of proapoptotic Bax and Bak primarily by sequestering inactive Bax and Bak monomers via interactions between their BH3 homology domains (7).Bcl-2 expression has been linked to decreased viral replication rates (26). Bcl-2 overexpression inhibits influenza A virus-induced cell death and reduces the titer and spread of newly formed virions (29). The activation of caspase-3 in the absence of sufficient Bcl-2 is critical to the influenza A virus life cycle. Both Bcl-2 expression and the lack of caspase activation during infection lead to the nuclear accumulation of influenza virus ribonucleoprotein (RNP) complexes, thereby leading to the improper assembly of progeny virions and a marked reduction in titers of infectious virus (26, 41, 42, 45).Here we show that influenza A virus induces mitochondrion-mediated (intrinsic-pathway) apoptosis signaled specifically through Bax and that this Bax signaling is essential for the maximum efficiency of virus propagation. In contrast, Bak expression is strongly downregulated during infection. Cells lacking Bak (while expressing Bax) display a much more severe apoptotic phenotype in response to infection and produce infectious virions at a higher rate than the wild type (WT), suggesting that Bak, which can suppress viral replication, is potentially downregulated by the virus. Our results indicate essential and opposing roles for Bax and Bak in both the response of cells to influenza A virus infection and the ability of the virus to maximize its own replicative potential.     

Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Kavosh: a new algorithm for finding network motifs

Background  

Complex networks are studied across many fields of science and are particularly important to understand biological processes. Motifs in networks are small connected sub-graphs that occur significantly in higher frequencies than in random networks. They have recently gathered much attention as a useful concept to uncover structural design principles of complex networks. Existing algorithms for finding network motifs are extremely costly in CPU time and memory consumption and have practically restrictions on the size of motifs.     

Chromatin restoration following nucleotide excision repair involves the incorporation of ubiquitinated H2A at damaged genomic sites

Restoration of functionally intact chromatin structure following DNA damage processing is crucial for maintaining genetic and epigenetic information in human cells. Here, we show the UV-induced uH2A foci formation in cells lacking XPC, DDB2, CSA or CSB, but not in cells lacking XPA, XPG or XPF indicating that uH2A incorporation relied on successful damage repair occurring through either GGR or TCR sub-pathway. In contrast, XPA, XPG or XPF were not required for formation of γH2AX foci in asynchronous cells. Notably, the H2A ubiquitin ligase Ring1B, a component of Polycomb repressor complex 1, did not localize at DNA damage sites. However, histone chaperone CAF-1 showed distinct localization to the damage sites. Knockdown of CAF-1 p60 abolished CAF-1 as well as uH2A foci formation. CAF-1 p150 was found to associate with NER factors TFIIH, RPA p70 and PCNA in chromatin. These data demonstrate that successful NER of genomic lesions and prompt CAF-1-mediated chromatin restoration link uH2A incorporation at the sites of damage repair within chromatin.