The Soluble Guanylyl Cyclase Activator Bay 60-2770 Potently Relaxes The Pulmonary Artery On Congenital Diaphragmatic Hernia Rabbit Model.

Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage, and a significant cause of morbidity and mortality. In this study, we have used a rabbit model of CDH to evaluate the effects of BAY 60-2770 on the in vitro reactivity of left pulmonary artery. CDH was performed in New Zealand rabbit fetuses (n = 10 per group) and compared to controls. Measurements of body, total and left lung weights (BW, TLW, LLW) were done. Pulmonary artery rings were pre-contracted with phenylephrine (10 μM), after which cumulative concentration-response curves to glyceryl trinitrate (GTN; NO donor), tadalafil (PDE5 inhibitor) and BAY 60-2770 (sGC activator) were obtained as well as the levels of NO (NO3/NO2). LLW, TLW and LBR were decreased in CDH (p < 0.05). In left pulmonary artery, the potency (pEC50) for GTN was markedly lower in CDH (8.25 ± 0.02 versus 9.27 ± 0.03; p < 0.01). In contrast, the potency for BAY 60-2770 was markedly greater in CDH (11.7 ± 0.03 versus 10.5 ± 0.06; p < 0.01). The NO2/NO3 levels were 62 % higher in CDH (p < 0.05). BAY 60-2770 exhibits a greater potency to relax the pulmonary artery in CDH, indicating a potential use for pulmonary hypertension in this disease.

Bay 41-2272, A Soluble Guanylate Cyclase Stimulator, Relaxes Isolated Human Ureter In A Standardized In Vitro Model.

To characterize the relaxation induced by BAY 41-2272 in human ureteral segments. Ureter specimens (n = 17) from multiple organ human deceased donors (mean age 40 ± 3.2 years, male/female ratio 2:1) were used to characterize the relaxing response of BAY 41-2272. Immunohistochemical analysis for endothelial and neuronal nitric oxide synthase, guanylate cyclase stimulator (sGC) and type 5 phosphodiesterase was also performed. The potency values were determined as the negative log of the molar to produce 50% of the maximal relaxation in potassium chloride-precontracted specimens. The unpaired Student t test was used for the comparisons. Immunohistochemistry revealed the presence of endothelial nitric oxide synthase in vessel endothelia and neuronal nitric oxide synthase in urothelium and nerve structures. sGC was expressed in the smooth muscle and urothelium layer, and type 5 phosphodiesterase was present in the smooth muscle only. BAY 41-2272 (0.001-100 μM) relaxed the isolated ureter in a concentration dependent manner, with a potency and maximal relaxation value of 5.82 ± 0.14 and 84% ± 5%, respectively. The addition of nitric oxide synthase and sGC inhibitors reduced the maximal relaxation values by 21% and 45%, respectively. However, the presence of sildenafil (100 nM) significantly potentiated (6.47 ± 0.10, P <.05) this response. Neither glibenclamide or tetraethylammonium nor ureteral urothelium removal influenced the relaxation response by BAY 41-2272. BAY 41-2272 relaxes the human isolated ureter in a concentration-dependent manner, mainly by activating the sGC enzyme in smooth muscle cells rather than in the urothelium, although a cyclic guanosine monophosphate-independent mechanism might have a role. The potassium channels do not seem to be involved.

Immobilization Of Papain On Chitin And Chitosan And Recycling Of Soluble Enzyme For Deflocculation Of Saccharomyces Cerevisiae From Bioethanol Distilleries.

Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1-10% w·v(-1)), polyethyleneimine (0.5% v·v(-1)), and tripolyphosphate (1-10% w·v(-1)) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L(-1). Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Properties Of Bologna-type Sausages With Pork Back-fat Replaced With Pork Skin And Amorphous Cellulose.

Bologna-type sausages were produced with 50% of their pork back-fat content replaced with gels elaborated with different ratios of pork skin, water, and amorphous cellulose (1:1:0, 1:1:0.1, 1:1:0.2, 1:1:0.3, and 1:1:0.4). The impact of such replacement on the physico-chemical characteristics and the consumer sensory profiling was evaluated. The modified treatments had 42% less fat, 18% more protein, and 8% more moisture than the control group. Treatments with amorphous cellulose had a lower cooking loss and higher emulsion stability. High amorphous cellulose content (1:1:0.3 and 1:1:0.4) increased hardness, gumminess, and chewiness. The gel formulated with the ratio of 1:1:0.2 (pork skin: water: amorphous cellulose gel) provided a sensory sensation similar to that provided by fat and allowed products of good acceptance to be obtained. Therefore, a combination of pork skin and amorphous cellulose is useful in improving technological quality and producing healthier and sensory acceptable bologna-type sausages.

Pharmacological Characterization Of The Relaxation Induced By The Soluble Guanylate Cyclase Activator, Bay 60-2770 In Rabbit Corpus Cavernosum.

To characterize the relaxation induced by the soluble guanylate cyclase (sGC) activator, BAY 60-2770 in rabbit corpus cavernosum. Penis from male New Zealand rabbits were removed and fours strips of corpus cavernosum (CC) were obtained. Concentration-response curves to BAY 60-2770 were carried out in the absence and presence of inhibitors of nitric oxide synthase, L-NAME (100 μM), sGC, ODQ (10 μM) and phosphodiestarase type 5, tadalafil (0.1 μM). The potency (pEC50) and maximal response (Emax) values were determined. Second, electrical-field stimulation (EFS)-induced contraction or relaxation was realized in the absence and presence of BAY 60-2770 (0.1 or 1 μM) alone or in combination of ODQ (10 μM). In the case of EFS-induced relaxation two protocols were realized: 1) ODQ (10 μM) was first incubated for 20 min and then BAY 60-2770 (1 μM) was added for another 20 min (ODQ + BAY 60-2770). In different CC strips, BAY 60-2770 was incubated for 20 min followed by another 20 min with ODQ (BAY 60-2770 + ODQ). The intracellular levels of cyclic guanosine monophosphate (cGMP) were also determined. BAY 60-2770 potently relaxed rabbit CC with pEC50 and Emax values of 7.58 ± 0.19 and 81 ± 4%, respectively. The inhibitors ODQ (n=7) or tadalafil (n=7) produced 4.2- and 6.3-leftward shifts, respectively in BAY 60-2770-induced relaxation without interfering on the Emax values. The intracellular levels of cGMP were augmented after stimulation with BAY 60-2770 (1 μM) alone, whereas its co-incubation with ODQ produced even higher levels of cGMP. The EFS-induced contraction was reduced in the presence of BAY 60-2770 (1 μM) and this inhibition was even greater when BAY 60-2770 was co-incubated with ODQ. The nitrergic stimulation induced CC relaxation, which was abolished in the presence of ODQ. BAY 60-2770 alone increased the amplitude of relaxation. Co-incubation of ODQ and BAY 60-2770 did not alter the relaxation in comparison with ODQ alone. Interestingly, when BAY 60-2770 was incubated prior to ODQ, EFS-induced relaxation was partly restored in comparison with ODQ alone or ODQ + BAY 60-2770. Considering that the relaxation induced by the sGC activator, BAY 60-2770 was increased after sGC oxidation and unaltered in the absence of nitric oxide, these class of substances are advantageous over sGC stimulators or PDE5 inhibitors for the treatment in those patients with erectile dysfunction and high endothelial damage. This article is protected by copyright. All rights reserved.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Bleeding Complications In Dengue Are Not Associated With Significant Changes In The Modulators Of The Endothelial Barrier.

Bleeding complications in dengue may occur irrespective of the presence of plasma leakage. We compared plasma levels of modulators of the endothelial barrier among three dengue groups: bleedings without plasma leakage, dengue hemorrhagic fever, and non-complicated dengue. The aim was to evaluate whether the presence of subtle alterations in microvascular permeability could be detected in bleeding patients. Plasma levels of VEGF-A and its soluble receptors were not associated with the occurrence of bleeding in patients without plasma leakage. These results provide additional rationale for considering bleeding as a complication independent of endothelial barrier breakdown, as proposed by the 2009 WHO classification.

The Characterization Of The Endoglucanase Cel12a From Gloeophyllum Trabeum Reveals An Enzyme Highly Active On β-glucan.

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Oligomerization, Membrane Association, And In Vivo Phosphorylation Of Sugarcane Udp-glucose Pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

High-resolution Transcript Profiling Of The Atypical Biotrophic Interaction Between Theobroma Cacao And The Fungal Pathogen Moniliophthora Perniciosa.

Witches' broom disease (WBD), caused by the hemibiotrophic fungus Moniliophthora perniciosa, is one of the most devastating diseases of Theobroma cacao, the chocolate tree. In contrast to other hemibiotrophic interactions, the WBD biotrophic stage lasts for months and is responsible for the most distinctive symptoms of the disease, which comprise drastic morphological changes in the infected shoots. Here, we used the dual RNA-seq approach to simultaneously assess the transcriptomes of cacao and M. perniciosa during their peculiar biotrophic interaction. Infection with M. perniciosa triggers massive metabolic reprogramming in the diseased tissues. Although apparently vigorous, the infected shoots are energetically expensive structures characterized by the induction of ineffective defense responses and by a clear carbon deprivation signature. Remarkably, the infection culminates in the establishment of a senescence process in the host, which signals the end of the WBD biotrophic stage. We analyzed the pathogen's transcriptome in unprecedented detail and thereby characterized the fungal nutritional and infection strategies during WBD and identified putative virulence effectors. Interestingly, M. perniciosa biotrophic mycelia develop as long-term parasites that orchestrate changes in plant metabolism to increase the availability of soluble nutrients before plant death. Collectively, our results provide unique insight into an intriguing tropical disease and advance our understanding of the development of (hemi)biotrophic plant-pathogen interactions.

Apoplastic And Intracellular Plant Sugars Regulate Developmental Transitions In Witches' Broom Disease Of Cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

Brazilian Natural Fiber (jute) As Raw Material For Activated Carbon Production.

Jute fiber is the second most common natural cellulose fiber worldwide, especially in recent years, due to its excellent physical, chemical and structural properties. The objective of this paper was to investigate: the thermal degradation of in natura jute fiber, and the production and characterization of the generated activated carbon. The production consisted of carbonization of the jute fiber and activation with steam. During the activation step the amorphous carbon produced in the initial carbonization step reacted with oxidizing gas, forming new pores and opening closed pores, which enhanced the adsorptive capacity of the activated carbon. N2 gas adsorption at 77K was used in order to evaluate the effect of the carbonization and activation steps. The results of the adsorption indicate the possibility of producing a porous material with a combination of microporous and mesoporous structure, depending on the parameters used in the processes, with resulting specific surface area around 470 m2.g-1. The thermal analysis indicates that above 600°C there is no significant mass loss.

Lymphocyte Activation In Silica-exposed Workers.

Exposure to silica dust has been examined as a possible risk factor for autoimmune diseases, including systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus and ANCA-associated vasculitis. However, the underlying cellular and molecular mechanisms resulting in the increased prevalence of autoimmunity remain elusive. To clarify these mechanisms, we studied various markers of immune activation in individuals occupationally exposed to silica dust, i.e., serum levels of soluble IL-2 receptor (sIL-2R), levels of IL-2, other pro- and anti-inflammatory cytokines and lymphoproliferation. Our results demonstrate that silica-exposed individuals present important alterations in their immune response when compared to controls, as shown by increased serum sIL-2R levels, decreased production of IL-2 and increased levels of the pro-inflammatory (IFN-γ, IL-1α, TNF-α, IL-6) as well as anti-inflammatory (IL-10 and TGF-β) cytokines. Furthermore, silica-exposed individuals presented enhanced lymphoproliferative responses. Our findings provide evidence that the maintenance of immune homeostasis may be disturbed in silica-exposed individuals, possibly resulting in autoimmune disorders.

Removal Of Glyphosate Herbicide From Water Using Biopolymer Membranes.

Enormous amounts of pesticides are manufactured and used worldwide, some of which reach soils and aquatic systems. Glyphosate is a non-selective herbicide that is effective against all types of weeds and has been used for many years. It can therefore be found as a contaminant in water, and procedures are required for its removal. This work investigates the use of biopolymeric membranes prepared with chitosan (CS), alginate (AG), and a chitosan/alginate combination (CS/AG) for the adsorption of glyphosate present in water samples. The adsorption of glyphosate by the different membranes was investigated using the pseudo-first order and pseudo-second order kinetic models, as well as the Langmuir and Freundlich isotherm models. The membranes were characterized regarding membrane solubility, swelling, mechanical, chemical and morphological properties. The results of kinetics experiments showed that adsorption equilibrium was reached within 4 h and that the CS membrane presented the best adsorption (10.88 mg of glyphosate/g of membrane), followed by the CS/AG bilayer (8.70 mg of glyphosate/g of membrane). The AG membrane did not show any adsorption capacity for this herbicide. The pseudo-second order model provided good fits to the glyphosate adsorption data on CS and CS/AG membranes, with high correlation coefficient values. Glyphosate adsorption by the membranes could be fitted by the Freundlich isotherm model. There was a high affinity between glyphosate and the CS membrane and moderate affinity in the case of the CS/AG membrane. Physico-chemical characterization of the membranes showed low values of solubility in water, indicating that the membranes are stable and not soluble in water. The SEM and AFM analysis showed evidence of the presence of glyphosate on CS membranes and on chitosan face on CS/AG membranes. The results showed that the glyphosate herbicide can be adsorbed by chitosan membranes and the proposed membrane-based methodology was successfully used to treat a water sample contaminated with glyphosate. Biopolymer membranes therefore potentially offer a versatile method to eliminate agricultural chemicals from water supplies.

Elevated Hypercoagulability Markers In Hemoglobin Sc Disease.

Hemoglobin SC disease is a very prevalent hemoglobinopathy, however very little is known specifically about this condition. There appears to be an increased risk of thromboembolic events in hemoglobin SC disease, but studies evaluating the hemostatic alterations are lacking. We describe a cross-sectional observational study evaluating coagulation activation markers in adult hemoglobin SC patients, in comparison with sickle cell anemia patients and healthy controls. A total of 56 hemoglobin SC and 39 sickle cell anemia patients were included in the study, all in steady state, and 27 healthy controls. None of the patients were in use of hydroxyurea. Hemoglobin SC patients presented a significantly up-regulated relative expression of tissue factor, as well as elevations in thrombin-antithrombin complex and D-dimer, in comparison to controls (p<0.01). Hemoglobin SC patients presented lower tissue factor expression, and thrombin-antithrombin complex and D-dimer levels when compared to sickle cell anemia patients (p<0.05). Endothelial activation (soluble thrombomodulin and soluble vascular cell adhesion molecule-1), and inflammation (tumor necrosis factor-alpha) markers were both significantly elevated in hemoglobin SC patients when compared to controls, being as high as the levels seen in sickle cell anemia. Overall, in hemoglobin SC patients, higher hemolytic activity and inflammation were associated with a more intense activation of coagulation, and hemostatic activation was associated with two very prevalent chronic complications seen in hemoglobin SC disease: retinopathy and osteonecrosis. In summary, our results demonstrate that hemoglobin SC patients present a hypercoagulable state, although this manifestation was not as intense as that seen in sickle cell anemia.