Modeling and biological investigations of an unusual behavior of novel synthesized acridine-based polyamine ligands in the binding of double helix and G-quadruplex DNA

Three novel 2,7-substituted acridine derivatives were designed and synthesized to investigate the effect of this functionalization on their interaction with double-stranded and G-quadruplex DNA. Detailed investigations of their ability to bind both forms of DNA were carried out by using spectrophotometric, electrophoretic, and computational approaches. The ligands in this study are characterized by an open-chain (L1) or a macrocyclic (L2, L3) framework. The aliphatic amine groups in the macrocycles are joined by ethylene (L2) or propylene chains (L3). L1 behaved similarly to the lead compound m-AMSA, efficiently intercalating into dsDNA, but stabilizing G-quadruplex structures poorly, probably due to the modest stabilization effect exerted by its protonated polyamine chains. L2 and L3, containing small polyamine macrocyclic frameworks, are known to adopt a rather bent and rigid conformation; thus they are generally expected to be sterically impeded from recognizing dsDNA according to an intercalative binding mode. This was confirmed to be true for L3. Nevertheless, we show that L2 can give rise to efficient π-π and H-bonding interactions with dsDNA. Additionally, stacking interactions allowed L2 to stabilize the G-quadruplex structure: using the human telomeric sequence, we observed the preferential induction of tetrameric G-quadruplex forms. Thus, the presence of short ethylene spacers seems to be essential for obtaining a correct match between the binding sites of L2 and the nucleobases on both DNA forms investigated. Furthermore, current modeling methodologies, including docking and MD simulations and free energy calculations, provide structural evidence of an interaction mode for L2 that is different from that of L3; this could explain the unusual stabilizing ability of the ligands (L2>L3>L1) toward G-quadruplex that was observed in this study.     

Dynamic helical CT of breast tumors

Expression of liver-enriched trans-acting hepatocyte nuclear factors 1alpha (HNF1alpha) and 4 (HNF4) is correlated with the hepatic phenotype in cultured rat hepatoma cells. We have used a hepatoma variant cell line, H11, that specifically lacks the HNF4 --> HNF1alpha pathway as a model to understand mechanisms controlling hepatic gene expression. We have introduced randomly marked human chromosomes into H11 cells and have isolated a number of microcell hybrids that have rescued hepatic gene expression, including HNF4, HNF1alpha, and alpha1-antitrypsin. Chromosomal analysis of cell hybrids showed that the rescued hepatic phenotype correlated closely with the presence of human chromosome 12p sequences. Although the gene encoding HNF1alpha is located on chromosome 12q24, its retention was not required to rescue the hepatic phenotype. Thus, we suggest that a locus on human chromosome 12p plays an important role in maintenance of hepatic gene expression through activation of the HNF4 --> HNF1alpha pathway.     

Computer-aided analysis of the forging process

This paper presents computer simulation of the forging process using the finite volume method (FVM). The process of forging is highly non-linear, where both large deformations and continuously changing boundary conditions occur. In most practical cases, the initial billet shape is relatively simple, but the final shape of the end product is often geometrically complex, to the extent that it is commonly obtained using multiple forming stages.Examples of the numerical simulation of the forged pieces provided were created using Msc/SuperForge computer code. The main results of the analysis are deformed shape, temperature, pressure, effective plastic strain, effective stress and forces acting on the die.Nomenclature C material constant - M strain rate hardening exponent - N strain hardening exponent - S coefficient of the microstructure - T temperature - u _i velocity component - x _j Cartesian coordinates - ̄ effective strain tensor - effective strain rate - ̇ proportionality factor in flow rules - _ij Cauchy stress tensor - _i deviator stress tensor - ̄ effective stress tensor - _y flow stress     

Supramolecular Polymorphism of (G4C2)n Repeats Associated with ALS and FTD

Guanine-rich DNA sequences self-assemble into highly stable fourfold structures known as DNA-quadruplexes (or G-quadruplexes). G-quadruplexes have furthermore the tendency to associate into one-dimensional supramolecular aggregates termed G-wires. We studied the formation of G-wires in solutions of the sequences d(G₄C₂)_n with n = 1, 2, and 4. The d(G₄C₂)_n repeats, which are associated with some fatal neurological disorders, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), represent a challenging research topic due to their extensive structural polymorphism. We used dynamic light scattering (DLS) to measure translational diffusion coefficients and consequently resolve the length of the larger aggregates formed in solution. We found that all three sequences assemble into longer structures than previously reported. The d(G₄C₂) formed extremely long G-wires with lengths beyond 80 nm. The d(G₄C₂)₂ formed a relatively short stacked dimeric quadruplex, while d(G₄C₂)₄ formed multimers corresponding to seven stacked intramolecular quadruplexes. Profound differences between the multimerization properties of the investigated sequences were also confirmed by the AFM imaging of surface films. We propose that π-π stacking of the basic G-quadruplex units plays a vital role in the multimerization mechanism, which might be relevant for transformation from the regular medium-length to disease-related long d(G₄C₂)_n repeats.     

MicroRNA-199b Deregulation Shows Oncogenic Properties and Promising Clinical Value as Circulating Marker in Locally Advanced Rectal Cancer Patients

The identification of robust prognostic markers still represents a need in locally advanced rectal cancer (LARC). MicroRNAs (miRs) have progressively emerged as promising circulating markers, overcoming some limitations that traditional biopsy comprises. Tissue miR-199b deregulation has been reported to predict outcome and response to neoadjuvant chemoradiotherapy (nCRT) in LARC, and was also found to be associated with disease progression in colorectal cancer. However, its biological and clinical relevance remains to be fully clarified. Thus, we observed here that miR-199b regulates cell migration, aggressiveness, and cell growth, and inhibits colonosphere formation and induces caspase-dependent apoptosis. Moreover, miR-199b expression was quantified by real-time PCR in plasma samples from LARC patients and its downregulation was observed in 22.7% of cases. This alteration was found to be associated with higher tumor size (p = 0.002) and pathological stage (p = 0.020) after nCRT. Notably, we observed substantially lower global miR-199b expression associated with patient downstaging (p = 0.009), as well as in non-responders compared to those cases who responded to nCRT in both pre- (p = 0.003) and post-treatment samples (p = 0.038). In concordance, we found that miR-199b served as a predictor marker of response to neoadjuvant therapy in our cohort (p = 0.011). Altogether, our findings here demonstrate the functional relevance of miR-199b in this disease and its potential value as a novel circulating marker in LARC.     

GABAA Receptor Modulators with a Pyrazolo[1,5-a]quinazoline Core: Synthesis,Molecular Modelling Studies and Electrophysiological Assays

As a continuation of our study in the GABA_A receptor modulators field, we report the design and synthesis of new 8-chloropyrazolo[1,5-a]quinazoline derivatives. Molecular docking studies and the evaluation of the ‘Proximity Frequencies’ (exploiting our reported model) were performed on all the final compounds (3, 4, 6a–c, 7a,b, 8, 9, 12a–c, 13a,b, 14–19) to predict their profile on the α1β2γ2-GABA_AR subtype. Furthermore, to verify whether the information coming from this virtual model was valid and, at the same time, to complete the study on this series, we evaluated the effects of compounds (1–100 µM) on the modulation of GABA_A receptor function through electrophysiological techniques on recombinant α1β2γ2L-GABA_A receptors expressed in Xenopus laevis oocytes. The matching between the virtual prediction and the electrophysiological tests makes our model a useful tool for the study of GABA_A receptor modulators.     

Simulation analysis of mini-load multi-shuttle automated storage and retrieval systems

Technological developments in warehouses have changed processes of storage operations, which reflect in short response times of the storage or retrieval of goods, the reduction of stocks and the volume of storage work as well as the automation of the entire warehouse management. Many companies are replacing traditional warehouses with automated storage and retrieval systems, which can be classified into unit-load and mini-load systems. In this paper, the simulation analysis of mini-load multi-shuttle systems is discussed and evaluated. Multi-shuttle systems are based on the quadruple and sextuple command cycle and could therefore achieve higher throughput capacities due to single-shuttle systems. Different analytical models are used by practitioners for designing multi-shuttle systems. The problem arises with the selection of the appropriate analytical model for which the condition of minimal differences with actual circumstances in practice is fulfilled. For the evaluation of the two well-known analytical models, the discrete event simulations have been used. Beside the evaluation of analytical models, the results of simulation analyses showed throughput improvements for triple-shuttle systems according to dual-shuttle systems. The main objective of this paper is to determine the performance of presented models (analytical and simulation models) of multi-shuttle systems, which represents the main share and support in design process of mini-load multi-shuttle automated storage and retrieval systems.     

Immunotherapy of experimental metastases by vaccination with interleukin gene-transduced adenocarcinoma cells sharing tumor-associated antigens. Comparison between IL-12 and IL-2 gene-transduced tumor cell vaccines

We have compared the therapeutic activity and characterized the antitumor response induced by IL-12 and IL-2 gene-transduced tumor cell vaccines. Mice bearing lung metastases of the BALB/c colon carcinoma C51 were treated with syngenic, histologically related, and antigenically cross-reacting irradiated IL-12 (C26/IL12) or IL-2 (C26/IL2) gene-transduced C26 tumor cells given s.c. Vaccination with C26/IL12 cells cured 40% of mice, while vaccination with C26/IL2 cells reduced the number of metastatic nodules without affecting survival. Despite this difference, similar antitumor CTL activation was shown in mice treated with C26/IL12 or C26/IL2 cells. The lytic pattern of CTL was shown to be directed to tumor-associated Ags (TAA) shared between the colon carcinomas C51, C26, and CC36 as well as with other syngenic tumors. Both treatments induced anti-TAA Abs, but only sera from mice treated with C26/IL12 contained Ab that lysed tumor cells in a C-dependent cytotoxicity assay. Early infiltration of activated T cells was found in the lungs of mice vaccinated with C26/IL12. CD4+ lymphocytes purified from the lymph nodes draining the vaccination site or from the spleen showed a higher production of IFN-gamma in response to anti-CD3 mAb in C26/IL12 vaccinated mice, while a higher production of IL-4 was shown in mice vaccinated with C26/IL2 cells. These results indicate that the better therapeutic efficacy of vaccination with C26/IL12 is associated with the production of C-binding Ab, an early infiltration of the metastatic lungs by activated T lymphocytes and a predominant systemic activation of Th1 more than Th2 cells.