Resveratrol Decreases the Invasion Potential of Gastric Cancer Cells.

The cancer-preventive agent Resveratrol (RSV) [3,5,4'-trihydroxytrans-stilbene] is a widely recognized antioxidant molecule with antitumoral potential against several types of cancers, including prostate, hepatic, breast, skin, colorectal, and pancreatic. Herein, we studied the effect of RSV on the cell viability and invasion potential of gastric cancer cells. AGS and MKN45 cells were treated with different doses of RSV (0-200 µM) for 24 h. Cell viability was determined using the Sulphorhodamine B dye (SRB) assay. For invasion assays, gastric cells were pre-treated with RSV (5-25 µM) for 24 h and then seeded in a Transwell chamber with coating Matrigel. The results obtained showed that RSV inhibited invasion potential in both cell lines. Moreover, to elucidate the mechanism implicated in this process, we analyzed the effects of RSV on SOD, heparanase, and NF-κB transcriptional activity. The results indicated that RSV increased SOD activity in a dose-dependent manner. Conversely, RSV significantly reduced the DNA-binding activity of NF-κB and the enzymatic activity of heparanase in similar conditions, which was determined using ELISA-like assays. In summary, these results show that RSV increases SOD activity but decreases NF-kB transcriptional activity and heparanase enzymatic activity, which correlates with the attenuation of invasion potential in gastric cancer cells. To our knowledge, no previous study has described the effect of RSV on heparanase activity. This article proposes that heparanase could be a key effector in the invasive events occurring during gastric cancer metastasis.

Acellular derivatives of mesenchymal stem cells prevent peritoneal adhesions in an animal model.

BACKGROUND: Peritoneal adhesions are nonanatomical connections that bind organs to the abdominal wall or among them. They arise after peritoneal injury, which triggers an inflammatory response followed by a healing process that leads to fibrotic tissue formation. Mesenchymal stem cells and their secretion products, also referred to as acellular derivatives (ACDs), have anti-inflammatory, fibrinolytic, and antifibrogenic properties. The aim of this study was to determine the effect of intraoperative administration of ACD on the appearance, severity, and progression of peritoneal adhesions, in an animal model. MATERIALS AND METHODS: Cecal erosions were mechanically induced in adult mice. Before closure, the vehicle, ACD, or Seprafilm was administered. Seven days later, the presence and grade of peritoneal adhesions were assessed macroscopically. One, 3, and 7 d after intervention, molecular and cellular markers of inflammation, fibrinolysis, and fibrogenesis were evaluated both locally and systemically. RESULTS: ACDs avoided the appearance of clinically relevant peritoneal adhesions. The vehicle had no effect, and Seprafilm reduced them inconsistently. The antiadhesive effect of ACD was associated with an early reduction of proinflammatory cytokine (tumor necrosis factor-alpha and interferon-gamma) secretion and leukocyte (polymorphonuclears, mononuclears, and macrophages) infiltration. High levels of D-dimer, low fibrin deposits, low myofibroblasts infiltration, and less fibrosis were also observed. CONCLUSIONS: ACD administered at the end of abdominal surgeries prevents the formation of peritoneal adhesions due to the modulation of inflammatory, fibrinolytic, and fibrogenic processes.

PSF and MTF from a bar pattern in digital mammography.

Background.The MTF has difficulties being determined (according to the provisions of the IEC standards) in the hospital setting due to the lack of resources.Purpose.The objective of this work is to propose a quantitative method for obtaining the point spread function (PSF) and the modulation transfer function (MTF) of a digital mammography system from an image of a bar pattern.Methods.The method is based on the measurement of the contrast transfer function (CTF) of the system over the image of the bar pattern. In addition, a theoretical model for thePSFis proposed, from which the theoreticalCTFof the system is obtained by means of convolution with a square wave (mathematical simulation of the bar pattern). Through an iterative process, the free parameters of thePSFmodel are varied until the experimentalCTFcoincides with the one calculated by convolution. Once thePSFof the system is obtained, we calculate theMTFby means of its Fourier transform. TheMTFcalculated from the modelPSFhave been compared with those calculated from an image of a 65µm diameter gold wire using an oversampling process.Results.TheCTFhas been calculated for three digital mammographic systems (DMS 1, DMS 2 and DMS 3), no differences of more than 5 % were found with the CTF obtained with the PSF model. The comparison of theMTFshows us the goodness of thePSFmodel.Conclusions.The proposed method for obtainingPSFandMTFis a simple and accessible method, which does not require a complex configuration or the use of phantoms that are difficult to access in the hospital world. In addition, it can be used to calculate other magnitudes of interest such as the normalized noise power spectrum (NNPS) and the detection quantum efficiency (DQE).

Opinions Related to the Potential Application of Artificial Intelligence (AI) by the Responsible in Charge of the Administrative Management Related to the Logistics and Supply Chain of Medical Stock in Health Centers in North of Chile.

The research evaluated the opinion of those in charge of the administrative management of the logistics and supply chain of medical and pharmaceutical stocks of health care centers in the north of Chile and a potential improvement of their operations through the use of artificial intelligence (AI). The identification of the problem arose from the empirical analysis, where serious deficiencies in the manual handling and management of the stock of medicines and hospital supplies were evidenced. This deficiency does not allow a timely response to the demand of the logistics and supply chain, causing stock ruptures in health centers. Based on this finding, we asked ourselves how AI was observed as the most efficient tool to solve this difficulty. The results were obtained through surveys of personnel in charge of hospital and pharmacy supplies. The questions focused on the level of training, seniority in positions related to the problem, knowledge of regulations, degree of innovation in the procedures used in logistics and supply chain and procurement. However, a very striking fact was related to the importance of the use of AI, where, very surprisingly, 64.7% considered that it would not help to reduce human errors generated in the areas analyzed.

HIV-1 replication cycle.

The HIV-1 is a formidable pathogen with establishment of a persistent infection based on the ability to integrate the proviral genome into chronically infected cells, and by the rapid evolution made possible by a high mutation rate and frequent recombination during the viral replication. HIV-1 has a variety of novel genes that facilitate viral persistence and regulation of HIV replication, but this virus also usurps cellular machinery for HIV replication, particularly during gene expression and virion assembly and budding. Recent success with antiretroviral therapy may be limited by the emergence HIV drug resistance and by toxicities and other requirements for successful long-term therapy. Further investigation of HIV-1 replication may allow identification of novel targets of antiretroviral therapy that may allow continued virus suppression in patients of failing current regiments, particularly drugs that target HIV-1 entry and HIV-1 integration.

Pathway analysis reveals common pro-survival mechanisms of metyrapone and carbenoxolone after traumatic brain injury.

Developing new pharmacotherapies for traumatic brain injury (TBI) requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT) and carbenoxolone (CB), which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11ß hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks). We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that suppressing expression of potentially harmful genes, and also, surprisingly, reduced expression of pro-survival genes may be a hallmark of neuroprotective therapeutic effects.

Febrile infection-related epilepsy syndrome is not caused by SCN1A mutations.

Two distinctive epileptic encephalopathies, febrile infection-related epilepsy syndrome (FIRES) and Dravet syndrome (DS), present with febrile status epilepticus in a normal child followed by refractory focal seizures and cognitive decline although there are differentiating features. Abnormalities of the sodium channel gene SCN1A are found in 75% of DS patients. We found no SCN1A mutations or copy number variants in 10 patients with FIRES. Other genetic etiologies deserve consideration.

Influence of stochastic gene expression on the cell survival rheostat after traumatic brain injury.

Experimental evidence suggests that random, spontaneous (stochastic) fluctuations in gene expression have important biological consequences, including determination of cell fate and phenotypic variation within isogenic populations. We propose that fluctuations in gene expression represent a valuable tool to explore therapeutic strategies for patients who have suffered traumatic brain injury (TBI), for which there is no effective drug therapy. We have studied the effects of TBI on the hippocampus because TBI survivors commonly suffer cognitive problems that are associated with hippocampal damage. In our previous studies we separated dying and surviving hippocampal neurons by laser capture microdissection and observed unexplainable variations in post-TBI gene expression, even though dying and surviving neurons were adjacent and morphologically identical. We hypothesized that, in hippocampal neurons that subsequently are subjected to TBI, randomly increased pre-TBI expression of genes that are associated with neuroprotection predisposes neurons to survival; conversely, randomly decreased expression of these genes predisposes neurons to death. Thus, to identify genes that are associated with endogenous neuroprotection, we performed a comparative, high-resolution transcriptome analysis of dying and surviving hippocampal neurons in rats subjected to TBI. We found that surviving hippocampal neurons express a distinct molecular signature--increased expression of networks of genes that are associated with regeneration, cellular reprogramming, development, and synaptic plasticity. In dying neurons we found decreased expression of genes in those networks. Based on these data, we propose a hypothetical model in which hippocampal neuronal survival is determined by a rheostat that adds injury-induced genomic signals to expression of pro-survival genes, which pre-TBI varies randomly and spontaneously from neuron to neuron. We suggest that pharmacotherapeutic strategies that co-activate multiple survival signals and enhance self-repair mechanisms have the potential to shift the cell survival rheostat to favor survival and therefore improve functional outcome after TBI.

Impact of human immunodeficiency virus type 1 reverse transcriptase inhibitor drug resistance mutation interactions on phenotypic susceptibility.

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

A critical role for CD63 in HIV replication and infection of macrophages and cell lines.

HIV infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either CCR5 or CXCR4. Other cellular factors supporting HIV replication have also been characterized. We previously demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by anti-CD63 antibody pretreatment. To confirm the requirement for CD63 in HIV replication, we decreased CD63 expression using CD63-specific short interfering RNAs (siRNA), and showed inhibition of HIV replication in macrophages. Surprisingly, pretreatment with CD63 siRNA not only silenced CD63 expression by 90%, but also inhibited HIV-1 replication in a cultured cell line (U373-MAGI) which had been previously shown to be insensitive to CD63 monoclonal antibody inhibition. Although the anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, we now show a potential role for CD63 in later HIV replication events in macrophages and cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.

Delivery of double-stranded DNA thioaptamers into HIV-1 infected cells for antiviral activity.

Oligonucleotide agents (ODN) are emerging as attractive alternatives to chemical drugs. However, the clinical use of ODNs as therapeutics has been hindered by their susceptibility to degradation by cellular enzymes and their limited ability to penetrate intact cells. We have used various liposome-mediated transfection agents, for the in vitro delivery of DNA thioaptamers into U373-MAGI-CCR5 cells. Our lead thioaptamer, R12-2, targets the RNase H domain of the HIV-1 reverse transcriptase (RT) and inhibits viral infection in U373-MAGI-CCR5 cells. R12-2, a 62-base-pair, double-stranded DNA molecule with a monothio-phosphate modified backbone, was selected through a novel combinatorial selection method. We studied the use of oligofectamine (OF), TFX-20, Transmessenger (TM), and Gene Jammer (GJ) for transfection of the thio-modified DNA aptamers. OF-transfected U373-MAGI-CCR5 cells resulted in 68% inhibition of HIV infection in the treated cells compared to the untreated control. Inhibition was observed in a dose-dependent manner with maximal inhibition of 83%. In this report, we demonstrate that monothioate-modified DNA duplex oligonucleotides can be efficiently delivered into cells by liposome-based transfection agents to inhibit HIV replication.

Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase.

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi

Identification of a signal transduction switch in the chemokine receptor CXCR1.

Chemokine receptors belong to the superfamily of G protein-coupled receptors, which regulate the trafficking and activation of leukocytes, and operate as coreceptors in the entry of HIV-1. To investigate the early steps in the signal transmission from the chemokine-binding site to the G protein-coupling region we engineered metal ion-binding sites at putative extracellular sites in the chemokine receptor CXCR1. We introduced histidines into sites located in the second and third putative extracellular loops of CXCR1, creating single, double, and triple mutant receptors: R199H, R203H, D265H, R199H/R203H, R199H/D265H, R203H/D265H, R203H/H207Q, and R199H/R203H/D265H. Cells expressing the double mutants R199H/D265H and R203H/D265H and the triple mutant R199H/R203H/D265H failed to trigger interleukin 8-dependent calcium responses. Interestingly, calcium responses mediated by the single mutant R203H and the double mutants R199H/R203H and R203H/H207Q were blocked by Zn(II), indicating the creation of a functional metal ion-binding site. On the other hand, cells expressing all single, double, or triple histidine-substituted CXCR1 demonstrated high affinity binding to interleukin 8 in the presence and absence of metal ions. These findings indicate that occupation of the engineered metal-binding site uncouples the chemokine-binding site from the activation mechanism in CXCR1. Most importantly, we identify for the first time elements of an early signal transduction switch of chemokine receptors before the activation of cytoplasmic G proteins.

Potential role for CD63 in CCR5-mediated human immunodeficiency virus type 1 infection of macrophages.

Macrophages and CD4(+) lymphocytes are the principal target cells for human immunodeficiency virus type 1 (HIV-1) infection, but the molecular details of infection may differ between these cell types. During studies to identify cellular molecules that could be involved in macrophage infection, we observed inhibition of HIV-1 infection of macrophages by monoclonal antibody (MAb) to the tetraspan transmembrane glycoprotein CD63. Pretreatment of primary macrophages with anti-CD63 MAb, but not MAbs to other macrophage cell surface tetraspanins (CD9, CD81, and CD82), was shown to inhibit infection by several R5 and dualtropic strains, but not by X4 isolates. The block to productive infection was postfusion, as assessed by macrophage cell-cell fusion assays, but was prior to reverse transcription, as determined by quantitative PCR assay for new viral DNA formation. The inhibitory effects of anti-CD63 in primary macrophages could not be explained by changes in the levels of CD4, CCR5, or beta-chemokines. Infections of peripheral blood lymphocytes and certain cell lines were unaffected by treatment with anti-CD63, suggesting that the role of CD63 in HIV-1 infection may be specific for macrophages.

Structural biology of chemokine receptors

Chemokine receptors are G protein-coupled receptors that mediate migration and activation of leukocytes as an important part of a protective immune response to injury and infection. In addition, chemokine receptors are used by HIV-1 to infect CD4 positive cells. The structural bases of chemokine receptor recognition and signal transduction are currently being investigated. High-resolution X-ray diffraction and NMR spectroscopy of chemokines indicate that all these peptides exhibit a common folding pattern, in spite of its low degree of primary-sequence homology. Chemokines' functional motifs have been identified by mutagenesis studies, and a possible mechanism for receptor recognition and activation is proposed, but high-resolution structure data of chemokine receptors is not yet available. Studies with receptor chimeras have identified the putative extracellular domains as the major selectivity determinants. Single-amino acid substitutions in the extracellular domains produce profound changes in receptor specificity, suggesting that motifs in these domains operate as a restrictive barrier to a common activation motif. Similarly HIV-1 usage of chemokine receptors involves interaction of one or more extracellular domains of the receptor with conserved and variable domains on the viral envelope protein gp 120, indicating a highly complex interaction. Elucidating the structural requirements for receptor interaction with chemokines and with HIV-1 will provide important insights into understanding the mechanisms of chemokine recognition and receptor activation. In addition, this information can greatly facilitate the design of effective inmunomodulatory and anti-HIV-1 therapeutic agents