Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca²⁺]_i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca²⁺] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca²⁺]_i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca²⁺]_i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca²⁺] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca²⁺]_i oscillations and insulin release, probably by inducing Ca²⁺ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011. © 2010 Wiley‐Liss, Inc.     

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.     

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

BraX-Ray: An X-Ray of the Brazilian Computer Science Graduate Programs

Research productivity assessment is increasingly relevant for allocation of research funds. On one hand, this assessment is challenging because it involves both qualitative and quantitative analysis of several characteristics, most of them subjective in nature. On the other hand, current tools and academic social networks make bibliometric data web-available to everyone for free. Those tools, especially when combined with other data, are able to create a rich environment from which information on research productivity can be extracted. In this context, our work aims at characterizing the Brazilian Computer Science graduate programs and the relationship among themselves. We (i) present views of the programs from different perspectives, (ii) rank the programs according to each perspective and a combination of them, (iii) show correlation between assessment metrics, (iv) discuss how programs relate to another, and (v) infer aspects that boost programs'' research productivity. The results indicate that programs with a higher insertion in the coauthorship network topology also possess a higher research productivity between 2004 and 2009.     

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles⁷.     

Thrombospondin-1/HIV-1 tat protein interaction: modulation of the biological activity of extracellular Tat.

Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.     

Proteomic profiling of the hypothalamus in two mouse models of narcolepsy

Narcolepsy is a disabling neurological disorder of sleepiness linked to the loss of neurons producing orexin neuropeptides in the hypothalamus. Two well‐characterized phenotypic mouse models of narcolepsy, loss‐of‐function (orexin‐knockout), and progressive loss of orexin (orexin/ataxin‐3) exist. The open question is whether the proteomics signatures of the hypothalamus would be different between the two models. To address this gap, we utilized a label‐free proteomics approach and conducted a hypothalamic proteome analysis by comparing each disease model to that of wild type. Following data processing and statistical analysis, 14 484 peptides mapping to 2282 nonredundant proteins were identified, of which 39 proteins showed significant differences in protein expression across groups. Altered proteins in both models showed commonalties in pathways for mitochondrial dysfunction and neuronal degeneration, as well as altered proteins related to inflammatory demyelination, insulin resistance, metabolic responses, and the dopaminergic and monoaminergic systems. Model‐specific alterations in insulin degraded enzyme (IDE) and synaptosomal‐associated protein‐25 were unique to orexin‐KO and orexin/ataxin‐3, respectively. For both models, proteomics not only identified clinically suspected consequences of orexin loss on energy homeostasis and neurotransmitter systems, but also identified commonalities in inflammation and degeneration despite the entirely different genetic basis of the two mouse models.     

HIV-1 Tat causes apoptotic death and calcium homeostasis alterations in rat neurons

We investigated the role of the HIV-1 protein Tat in AIDS-associated dementia, by studying its toxicity on rat cortical and hippocampal neurons in vitro. We evaluated the involvement of astroglial cells and of caspase transduction pathway in determining Tat toxicity. Here we report that synthetic Tat(1-86) induced apoptotic death on cultured rat neurons in a time-dependent manner that was not influenced by glial coculture, and that was abolished by blocking caspase transduction pathway. A microfluorimetric analysis on the Tat excitatory properties on neurons, and its effect on intracellular calcium concentrations, revealed that Tat(1-86) induced increase in cytoplasmic free calcium concentrations in rat hippocampal and cortical neurons. This effect required extracellular calcium and was differently reduced by voltage dependent calcium channel blockers and both NMDA and non-NMDA glutamate receptors antagonists. Furthermore, we observed that Tat(1-86)-treated neurons showed increased sensitivity to the glutamate excitotoxicity. Thus, the Tat-induced neuronal injury seems to occur through a direct interaction of the protein with neurons, requires activation of caspases, and is likely to derive from Tat(1-86)-induced calcium loads and disruption of glutamatergic transmission.     

MALDI-TOF peptidomic analysis of serum and post-prostatic massage urine specimens to identify prostate cancer biomarkers

Background

Lower urinary tract symptoms (LUTS) and prostate specific antigen-based parameters seem to have only a limited utility for the differential diagnosis of prostate cancer (PCa). MALDI-TOF/MS peptidomic profiling could be a useful diagnostic tool for biomarker discovery, although reproducibility issues have limited its applicability until now. The current study aimed to evaluate a new MALDI-TOF/MS candidate biomarker.

Methods

Within- and between-subject variability of MALDI-TOF/MS-based peptidomic urine and serum analyses were evaluated in 20 and 15 healthy donors, respectively. Normalizations and approaches for accounting below limit of detection (LOD) values were utilized to enhance reproducibility, while Monte Carlo experiments were performed to verify whether measurement error can be dealt with LOD data. Post-prostatic massage urine and serum samples from 148 LUTS patients were analysed using MALDI-TOF/MS. Regression-calibration and simulation and extrapolation methods were used to derive the unbiased association between peptidomic features and PCa.

Results

Although the median normalized peptidomic variability was 24.9%, the within- and between-subject variability showed that median normalization, LOD adjustment, and log₂ data transformation were the best combination in terms of reliability; in measurement error conditions, intraclass correlation coefficient was a reliable estimate when the LOD/2 was substituted for below LOD values. In the patients studied, 43 peptides were shared by the urine and serum, and several features were found to be associated with PCa. Only few serum features, however, show statistical significance after the multiple testing procedures were completed. Two serum fragmentation patterns corresponded to the complement C4-A.

Conclusions

MALDI-TOF/MS serum peptidome profiling was more efficacious with respect to post-prostatic massage urine analysis in discriminating PCa.