Transcription factors Sp1 and Sp4 regulate TRPV1 gene expression in rat sensory neurons

<p>Abstract</p> <p>Background</p> <p>The capsaicin receptor, transient receptor potential vanilloid type -1 (TRPV1) directs complex roles in signal transduction including the detection of noxious stimuli arising from cellular injury and inflammation. Under pathophysiologic conditions, TRPV1 mRNA and receptor protein expression are elevated in dorsal root ganglion (DRG) neurons for weeks to months and is associated with hyperalgesia. Building on our previous isolation of a promoter system for the rat TRPV1 gene, we investigated the proximal TRPV1 P2-promoter by first identifying candidate Sp1-like transcription factors bound <it>in vivo </it>to the P2-promoter using chromatin immunoprecipitation (ChIP) assay. We then performed deletion analysis of GC-box binding sites, and quantified promoter activity under conditions of Sp1 / Sp4 over-expression versus inhibition/knockdown. mRNA encoding Sp1, Sp4 and TRPV1 were quantified by qRT-PCR under conditions of Sp1/Sp4 over-expression or siRNA mediated knockdown in cultured DRG neurons.</p> <p>Results</p> <p>Using ChIP analysis of DRG tissue, we demonstrated that Sp1 and Sp4 are bound to the candidate GC-box site region within the endogenous TRPV1 P2-promoter. Deletion of GC-box "a" or "a + b" within the P2- promoter resulted in a complete loss of transcriptional activity indicating that GC-box "a" was the critical site for promoter activation. Co-transfection of Sp1 increased P2-promoter activity in cultured DRG neurons whereas mithramycin-a, an inhibitor of Sp1-like function, dose dependently blocked NGF and Sp1-dependent promoter activity in PC12 cells. Co-transfection of siRNA directed against Sp1 or Sp4 decreased promoter activity in DRG neurons and NGF treated PC12 cells. Finally, electroporation of Sp1 or Sp4 cDNA into cultures of DRG neurons directed an increase in Sp1/Sp4 mRNA and importantly an increase in TRPV1 mRNA. Conversely, combined si-RNA directed knockdown of Sp1/Sp4 resulted in a decrease in TRPV1 mRNA.</p> <p>Conclusion</p> <p>Based on these studies, we now propose a model of TRPV1 expression that is dependent on Sp1-like transcription factors with Sp4 playing a predominant role in activating TRPV1 RNA transcription in DRG neurons. Given that increases of TRPV1 expression have been implicated in a wide range of pathophysiologic states including persistent painful conditions, blockade of Sp1-like transcription factors represents a novel direction in therapeutic strategies.</p

Assessment of fructooligosaccharides production from sucrose in aqueous and aqueous-organic systems using immobilized inulinase from Kluyveromyces marxianus NRRL Y-7571

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)This work investigated the fructooligosaccharides (FOS) synthesis by immobilized inulinase obtained from Kluyveromyces marxianus NRRL Y-7571 in aqueous and aqueous-organic systems using sucrose as substrate. The sequential strategy of experimental design was used to optimize the FOS conversion in both systems. For the aqueous-organic system, a 2(6-2) fractional design was carried out to evaluate the effects of temperature, sucrose concentration, pH, aqueous/organic ratio, enzyme activity, and polyethylene glycol concentration. For the aqueous system, a central composite design for the enzyme activity and the sucrose concentration was carried out. The highest fructooligosaccharides yield (Y-FOS) for the aqueous-organic system was 18.2 +/- S0.9 wt%, at 40 degrees C, pH 5.0, sucrose concentration of 60% (w/w), enzyme activity of 4 U.mL(-1) and aqueous/organic ratio of 25/75 wt%. The highest Y-FOS for the aqueous system was 14.6 +/- 0.9 wt% at 40 degrees C, pH 5.0, sucrose concentration of 60 wt%, and enzyme activity of 4.0 U.mL(-1).322245249Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca(2+) increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology

Produção de biogás por digestão em fase sólida de cama de frango.

RESUMO: A cama de frango é um resíduo da atividade avícola, que é gerado em grandes quantidades, e quando mal manuseado tem elevado potencial poluidor. No entanto, é possível aproveitar este resíduo para geração de biogás, contudo este substrato apresenta desafios devido a baixa umidade e composição química. Diante deste contexto, a digestão em fase sólida se destaca como um processo promissor pelo fato de evitar o manejo ou póstratamento da água derivada do processo. O objetivo do presente estudo foi avaliar a eficiência da produção de biogás de cama de frango em um reator anaeróbio de fase sólida (RDFS) operado em batelada. Ao realizar o processo de digestão em fase sólida, verificouse que este sistema apresentou um rendimento de biogás de 90,30 mLNbiogás.gSVadic -1 ,quando comparado ao valor de referência do teste do potencial bioquímico de biogás que foi de 281 mLNbiogás.gSVadic -1 , a eficiência foi de 32 % após 30 dias de digestão. No entanto, o teor de sólidos totais que o RDFS pode operar é de 30 %, enquanto que a digestão úmida a concentração de sólidos totais é em média de 10%. Neste caso utilizando o RDFS estaremos economizando líquido para diluição do substrato, e este é um sistema robusto, de fácil monitoramento que pode ser utilizado pelos avicultores, podendo ainda ser otimizado para melhoria do sistema e assim aumentar a eficiência de produção de biogás. ABSTRACT: The poultry litter is a residue from poultry activity, which is generated in large quantities and, when poorly handled, presents a high polluting potential. It is possible to take advantage of this residue to generate biogas, however this substrate consists in a challenging material due to its humidity and chemical composition. In this context, solid-state digestion stands out as a promising process because it prevents the handling or post-treatment of water derived from the process. The goal of the present study was to evaluate the efficiency of production of poultry litter biogas in a solid phase anaerobic reactor operated in batch. When carrying out the solid phase digestion process, it was verified that this system presented a biogas production of 90,30 mLNbiogas.gSVadd -1 . When comparing this to the reference value of the biochemical potential of biogas test, which is 281 mLNbiogas.gSVadd -1 , the efficiency attained was 32% at the 30-day of digestion However, the total solids content that the RDFS can operate is 30%, while the wet digestion at the total solids concentration is on average 10%. In this case using the RDFS we will be saving liquid for dilution of the substrate, and this is a robust system, easy to monitor that can be used by poultry farmers and can be optimized for system improvement and thus increase biogas production efficiency

Final report on the search for neutrinoless double-β decay of 76Ge from the Gotthard underground experiment

We report here on the final results of a search for Ge-76 double-beta decay conducted in the Gotthard underground laboratory. The detector consists of an array of eight high-purity natural germanium crystals totaling 1095 cm^3 fiducial volume. The accumulated data set represents a sensitivity of 10.0 kg yr. No indication of neutrinoless double-beta decay was found. The measured half-life limits are T1/2(0+ --> 0+) > 6.0(3.3) x 10^(23) yr for the transition to the ground state and T1/2(0+ --> 2+) > 1.4(0.65) x 10^(23) yr for the transition to the first excited state at 68% (90%) C.L. From these results we derive an upper limit for the Majorana mass of the neutrino in the range of 1.8 to 6.7 eV depending on matrix-element calculations. The same results allow limits to be set for the right-handed-current parameters: < 2.2 x 10^(-8)

Atmospheric Muon Flux at Sea Level, Underground, and Underwater

The vertical sea-level muon spectrum at energies above 1 GeV and the underground/underwater muon intensities at depths up to 18 km w.e. are calculated. The results are particularly collated with a great body of the ground-level, underground, and underwater muon data. In the hadron-cascade calculations, the growth with energy of inelastic cross sections and pion, kaon, and nucleon generation in pion-nucleus collisions are taken into account. For evaluating the prompt muon contribution to the muon flux, we apply two phenomenological approaches to the charm production problem: the recombination quark-parton model and the quark-gluon string model. To solve the muon transport equation at large depths of homogeneous medium, a semi-analytical method is used. The simple fitting formulas describing our numerical results are given. Our analysis shows that, at depths up to 6-7 km w. e., essentially all underground data on the muon intensity correlate with each other and with predicted depth-intensity relation for conventional muons to within 10%. However, the high-energy sea-level data as well as the data at large depths are contradictory and cannot be quantitatively decribed by a single nuclear-cascade model.Comment: 47 pages, REVTeX, 15 EPS figures included; recent experimental data and references added, typos correcte