Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The effect of CpG-ODN on antigen presenting cells of the foal

Background: Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. Methods: Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNα, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-κB p65 using a chemiluminescence assay. Results: The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNα (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells. Conclusion: CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNα cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment. © 2007 Flaminio et al; licensee BioMed Central Ltd.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Pancreatic Alpha-Cell Dysfunction Contributes to the Disruption of Glucose Homeostasis and Compensatory Insulin Hypersecretion in Glucocorticoid-Treated Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Long-term dexamethasone treatment alters the histomorphology of acinar cells in rat parotid and submandibular glands

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Different morphology, stage and treatment affect immune cell infiltration and long-term outcome in patients with non-small-cell lung carcinoma

Aims: Development of effective immune-based therapies for patients with non-small-cell lung carcinoma (NSCLC) depends on an accurate characterization of complex interactions that occur between immune cells and the tumour environment. Methods and results: Innate and adaptive immune responses were evaluated in relation to prognosis in 65 patients with surgically excised NSCLC. Immunohistochemistry and morphometry were used to determine the abundance and distribution of immune cells. We found low numbers of immune cells and levels of cytokines in the tumour environment when compared with surrounding parenchyma. Smoking was associated inversely with the adaptive immune response and directly with innate immunity. We observed a prominent adaptive immune response in squamous cell carcinomas (SCC) but greater innate immune responses in adenocarcinomas and large cell carcinomas. Cox model analysis showed a low risk of death for smoking <41 packs/year, N-0 tambour stage, squamous carcinoma, CD4(+) > 16.81% and macrophages/monocytes >4.5%. Collectively, the data indicate that in NSCLC there is not a substantive local immune cell infiltrate within the tumour. Conclusion: Although immune cell infiltration is limited in NSCLC it appears to have an impact on prognosis and this may be of relevance for new immunotherapeutic approaches.

Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25x10(6) cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10(6) or 2.5x10(6) cells from 13 weeks of age. A third, pre-symptomatic, group received 10(6) cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10(6) cells pre-symptomatically or 2.5x10(6) cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Abstract Background MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species. Results In this study, we have computationally identified 19 distinct sugarcane miRNA precursors, of which several are highly similar with their sorghum homologs at both nucleotide and secondary structure levels. The accumulation pattern of mature miRNAs varies in organs/tissues from the commercial sugarcane hybrid as well as in its corresponding founder species S. officinarum and S. spontaneum. Using sugarcane MIR827 as a query, we found a novel MIR827 precursor in the sorghum genome. Based on our computational tool, a total of 46 potential targets were identified for the 19 sugarcane miRNAs. Several targets for highly conserved miRNAs are transcription factors that play important roles in plant development. Conversely, target genes of lineage-specific miRNAs seem to play roles in diverse physiological processes, such as SsCBP1. SsCBP1 was experimentally confirmed to be a target for the monocot-specific miR528. Our findings support the notion that the regulation of SsCBP1 by miR528 is shared at least within graminaceous monocots, and this miRNA-based post-transcriptional regulation evolved exclusively within the monocots lineage after the divergence from eudicots. Conclusions Using publicly available nucleotide databases, 19 sugarcane miRNA precursors and one new sorghum miRNA precursor were identified and classified into 14 families. Comparative analyses between sugarcane and sorghum suggest that these two species retain homologous miRNAs and targets in their genomes. Such conservation may help to clarify specific aspects of miRNA regulation and evolution in the polyploid sugarcane. Finally, our dataset provides a framework for future studies on sugarcane RNAi-dependent regulatory mechanisms.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Abstract Background Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases.

Aktivierung der alpha-Sekretase ADAM10 als neuer therapeutischer Ansatz zur Behandlung der Alzheimer-Erkrankung

Die Stimulation der APP-prozessierenden α-Sekretase ADAM10 eröffnet eine vielversprechende Möglichkeit zur medizinischen Behandlung der Alzheimer-Krankheit. In dieser Arbeit wurden drei unterschiedliche Strategien zur therapeutischen Aktivierung von ADAM10 verfolgt: Die Aktivierung des G-Protein-gekoppelten Rezeptors PAC1 durch PACAP, die Gentherapie mit ADAM10-cDNA und die ADAM10-Promotorstimulation durch Retinoid-Rezeptor-Aktivierung. PACAP-38 stimuliert die α-Sekretase-vermittelte APPsα-Sekretion in humanen Neuroblastomzellen. Durch Aktivierung des PAC-1-Rezeptors via intranasal verabreichtem PACAP-38, konnte eine erhöhte α-sekretorische APP-Prozessierung bzw. verminderte Ablagerung von amyloiden Plaques in Mäusen gezeigt werden. Weiterhin sollte durch Immunoliposomen-basierte Transfektion die humane ADAM10-cDNA in den Neuronen der Maus überexprimiert werden. Hiefür wurde die DNA in Liposomen eingeschlossen, welche an ihrer Oberfläche mit anti-Transferrin-Antikörpern zur Überwindung der Blut-Hirn-Schranke gekoppelt waren. Für die Herstellung des DNA-Transportsystems wurden die Einzelschritte wie DNA-Einschluss mit einem Reportergen-Vektor, Konjugation mit verschiedenen Antikörpern und Größe der Liposomen erprobt und optimiert. Es konnte allerdings weder in vitro noch in vivo eine Immunoliposomen-vermittelte Transfektion nachgewiesen werden. In dieser Arbeit wurde zudem die Retinoid-basierte Expressionssteigerung von ADAM10 untersucht. Dafür wurden die beiden potentiellen Retinoid-Rezeptor-Bindestellen auf dem ADAM10-Promotor durch Verwendung selektiver nukleärer Rezeptor-Agonisten charakterisiert. Hierbei konnte erstmals gezeigt werden, dass der ADAM10-Promotor durch ein Dimer der nukleären Rezeptoren RAR und RXR aktiviert wird, wodurch eine erhöhte α-sekretorischen APP-Prozessierung in Neuroblastoma-Zellen resultiert. Weiterhin konnte gezeigt werden, dass die RAR/RXR-Heterodimeraktivierung sowohl auf dem humanen wie auf dem murinen ADAM10-Promotor identisch ist, so dass am Mausmodell entwickelte Retinoid-basierte Therapien auf den Menschen übertragbar sind. Für das Modell einer solchen Therapie wurde Acitretin verwendet, welches für die medizinische Behandlung humaner Hautkrankheiten seit Jahrzehnten eingesetzt wird. In dieser Arbeit konnte erstmals gezeigt werden, dass Acitretin in humanen und murinen Neuroblastoma-Zellen die Menge an ADAM10 erhöht, wodurch die α-sekretorische APP-Prozessierung gesteigert wird. Zudem wurden Mäuse mit Acitretin oral, subcutan und intranasal behandelt, wobei jedoch weder eine Veränderung in der APP-Prozessierung noch der Blut-Hirn-Transport von Acitretin eindeutig belegt werden konnten. Dennoch erschließt die α-Sekretase-erhöhende Eigenschaft von Acitretin einen neuen Therapieansatz, zur Behandlung von Demenzformen vom Typ des Morbus Alzheimer.

The cardio-renal axis. Non ST-segment elevation myocardial infarction risk stratification according to renal dysfunction

Background: Chronic kidney disease (CKD) is one of the strongest risk factor for myocardial infarction (MI) and mortality. The aim of this study was to assess the association between renal dysfunction severity, short-term outcomes and the use of in-hospital evidence-based therapies among patients with non–ST-segment elevation myocardial infarction (NSTEMI). Methods: We examined data on 320 patients presenting with NSTEMI to Maggiore’s Emergency Department from 1st Jan 2010 to 31st December 2011. The study patients were classified into two groups according to their baseline glomerular filtration rate (GFR): renal dysfunction (RD) (GFR<60) and non-RD (GFR≥60 ml/min). Patients were then classified into four groups according to their CKD stage (GFR≥60, GFR 59-30, GFR 29-15, GFR <15). Results: Of the 320 patients, 155 (48,4%) had a GFR<60 ml/min at baseline. Compared with patients with a GFR≥60 ml/min, this group was, more likely to be female, to have hypertension, a previous myocardial infarction, stroke or TIA, had higher levels of uric acid and C-reactive protein. They were less likely to receive immediate (first 24 hours) evidence-based therapies. The GFR of RD patients treated appropriately increases on average by 5.5 ml/min/1.73 m2. The length of stay (mean, SD) increased with increasing CKD stage, respectively 5,3 (4,1), 7.0 (6.1), 7.8 (7.0), 9.2 (5.8) (global p <.0001). Females had on average a longer hospitalization than males, regardless of RD. In hospital mortality was higher in RD group (3,25%). Conclusions: The in-hospital mortality not was statically difference among the patients with a GFR value ≥60 ml/min, and patients with a GFR value <60 ml/min. The length of stay increased with increasing CKD stages. Despite patients with RD have more comorbidities then without RD less frequently receive guideline –recommended therapy. The GFR of RD patients treated appropriately improves during hospitalization, but not a level as we expected.

Analysen zur molekularen Charakterisierung von Proteinen des humanen Usher-Syndroms und Evaluation genbasierter Therapiestrategien

Analysen zur molekularen Charakterisierung von Proteinen des humanen Usher-Syndroms und Evaluation genbasierter Therapiestrategien rnDas humane Usher Syndrom (USH) ist die häufigste Form vererbter Taub-Blindheit. In der vorliegenden Dissertation wurde diese komplexe Erkrankung auf verschiedenen Ebenen analysiert: in Arbeiten zur Expression und Lokalisation von USH-Proteinen, der Analyse der USH-Proteinnetzwerke und deren Funktionen sowie darauf aufbauend die Entwicklung von Therapiestrategien für USH.rnIm Rahmen der Arbeit wurde die Expression und (sub)-zelluläre Lokalisation des USH1D-Genproduktes CDH23 in der Retina und Cochlea analysiert. CDH23-Isoformen werden in der Maus zeitlich und räumlich differentiell exprimiert. In den Retinae von Mäusen, nicht humanen Primaten und Menschen zeigten Analysen eine unterschiedliche Expression und Lokalisation des Zell-Zelladhäsionsmoleküls CDH23, was auf Funktions-unterschiede der einzelnen Isoformen in den analysierten Spezies hindeutet.rnAnalysen zur Aufklärung der USH-Proteinnetzwerke ergaben eine potentielle Interaktion des USH1G-Gerüstproteins SANS mit dem Golgi- und Centrosom-assoziierten Protein Myomegalin. Die direkte Interaktion der Proteine konnte durch unabhängige Experimente verifiziert werden. Beide Interaktionspartner sind in den Retinae verschiedener Spezies partiell ko-lokalisiert und partizipieren im periciliären USH-Proteinnetzwerk. Die Assoziation von SANS und Myomegalin mit dem Mikrotubuli-Cytoskelett weist auf eine Funktion des Proteinkomplexes in gerichteten Transportprozessen innerhalb der Photorezeptoren hin und bekräftigt die Hypothese einer Rolle von SANS und assoziierten Netzwerken mit Transportprozessen.rnDas hier gewonnene erweiterte Verständnis der molekularen Grundlagen sowie die Aufklärung der zellulären Funktion der Proteinnetzwerke ermöglichen die Entwicklung therapeutischer Strategien für USH. Ein Fokus der vorliegenden Arbeit lag auf der Entwicklung genbasierter Therapiestrategien und deren Evaluation, wobei der Schwerpunkt auf der Therapiestrategie der Genreparatur lag. Die mit Hilfe von Zinkfinger-Nukleasen (ZFN) induzierte Homologe Rekombination für die Genkorrektur wurde exemplarisch an der 91C>T/p.R31X-Mutation im USH1C-Gen gezeigt. Effiziente ZFN wurden identifiziert, generiert und erfolgreich im Zellkulturmodellsystem eingesetzt. Die Analysen demonstrierten eine Reparatur der Mutation durch Homologe Rekombination auf genomischer Ebene und die Expression des wiederhergestellten Proteins. Durch die Genkorrektur im endogenen Lokus sind Größe des Gens, Isoformen oder die Art der Mutation keine limitierenden Faktoren für die Therapie. Die in der vorliegenden Arbeit durchgeführten Experimente unterstreichen das enorme Potential ZFN-basierter Therapiestrategien hin zu personalisierten Therapieformen nicht nur für USH sondern auch für andere erbliche Erkrankungen, deren genetische Grundlagen bekannt sind.rn

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

Presentation and outcome of patients with acute coronary syndromes in eastern Nepal

The burden of ischemic heart disease (IHD) in developing countries is on the rise, due to urbanisation, industrialisation and the low availability of evidence based therapies and interventions.

RNA interference in mammalian cell systems

In the last decade, few areas of biology have been transformed as thoroughly as RNA molecular biology. Without any doubt, one of the most significant advances has been the discovery of small (20-30 nucleotide) noncoding RNAs that regulate genes and genomes. The effects of small RNAs on gene expression and control are generally inhibitory, and the corresponding regulatory mechanisms are therefore collectively subsumed under the heading of RNA silencing and/or RNA interference. Two primary categories of these small RNAs - short interfering RNAs (siRNAs) and microRNAs (miRNAs) - act in both somatic and germline lineages of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA and miRNA-based regulation has direct implications for fundamental biology as well as disease aetiology and treatment as it is discussed in this review on 'new techniques in molecular biology'.