Isolation of intermediate valence hybrids between ferrous and methemoglobin at subzero temperatures.

Quenching a hemoglobin solution partially saturated with carbon monoxide into a hydro-organic solvent containing ferricyanide will produce under suitable conditions a population of partially oxidized and CO-bound hemoglobin molecules. Since each Fe3+ heme carries one extra charge, it should be possible, in theory, to resolve the spectrum of intermediate compounds between hemoglobin and carbon monoxide, which was originally present in solution. In this study we report: 1) the development of a simple and rapid method to quench aqueous hemoglobin solutions into a hydro-organic solvent at subzero temperatures; 2) the determination of suitable experimental conditions to isolate valence hybrids between carbonmonoxy- and methemoglobin by isoelectric focusing at temperatures as low as -25 degrees C; and 3) the identification and isolation of all valence hybrids of different charge between carbonmonoxy- and methemoglobin

Galectin-3: An early predictive biomarker of modulation of airway remodeling in patients with severe asthma treated with omalizumab for 36 months

Background: Bronchial asthma is a heterogeneous disease characterized by three cardinal features: chronic inflammation, variable airflow obstruction, and airway hyperresponsiveness. Asthma has traditionally been defined using nonspecific clinical and physiologic variables that encompass multiple phenotypes and are treated with nonspecific anti-inflammatory therapies. Based on the modulation of airway remodeling after 12 months of anti-immunoglobulin E (IgE) treatment, we identified two phenotypes (omalizumab responder, OR; and non-omalizumab responder, NOR) and performed morphometric analysis of bronchial biopsy specimens. We also found that these two phenotypes were correlated with the presence/absence of galectin-3 (Gal-3) at baseline (i.e., before treatment). The aims of the present study were to investigate the histological and molecular effects of long-term treatment (36 months) with anti-IgE and to analyze the behavior of OR and NOR patients. Methods: All patients were treated with the monoclonal antibody anti-IgE omalizumab for 36 months. The bronchial biopsy specimens were evaluated using morphometric, eosinophilic, and proteomic analysis (MudPIT). New data were compared with previous data, and unsupervised cluster analysis of protein profiles was performed. Results: After 36 months of treatment with omalizumab, reduction of reticular basement membrane (RBM) thickness was confirmed in OR patients (Gal-3-positive at baseline); similarly, the protein profiles (over 500 proteins identified) revealed that, in the OR group, levels of proteins specifically related to fibrosis and inflammation (e.g., smooth muscle and extracellular matrix proteins (including periostin), Gal-3, and keratins decreased by between 5- and 50-fold. Eosinophil levels were consistent with molecular data and decreased by about tenfold less in ORs and increased by twofold to tenfold more in NORs. This tendency was confirmed (p < 0.05) based on both fold change and DAVE algorithms, thus indicating a clear response to anti-IgE treatment in Gal-3-positive patients. Conclusions: Our results showed that omalizumab can be considered a disease-modifying treatment in OR. The proteomic signatures confirmed the presence of Gal-3 at baseline to be a biomarker of long-term reduction in bronchial RBM thickness, eosinophilic inflammation, and muscular and fibrotic components in omalizumab-treated patients with severe asthma. Our findings suggest a possible relationship between Gal-3 positivity and improved pulmonary function

BISCOITO ENRIQUECIDO DA FARINHA DA CASCA DO MARACUJÁ E SABORIZADO DO CHÁ DA CASCA DE LARANJA

A utilização dos subprodutos (resíduos agroindustriais) nas formulações de produtos alimentícios, exploram ao máximo o aproveitamento das partes comestíveis não convencionais dos alimentos. A casca, bem como as sementes do maracujá, quando adicionadas aos produtos, incorporam componentes ricos em pectina e fibras alimentares. A casca quando triturada, com foco na obtenção da farinha, agrega na sua estrutura, propriedades antioxidantes, que auxiliam na eliminação das toxinas do sistema gástrico, prevenindo refluxos, incontinência fecal, azia, constipação e cólicas abdominais. O chá da casca de laranja, quando adicionado aos biscoitos, realça o sabor e o aroma, auxiliando na sua percepção sensorial, e promovendo, quando possível, a aceitabilidade dos produtos. Estes compostos antioxidantes e vitaminas do complexo A, quando acrescidos aos produtos alimentícios, tornam-se de grande valia, e agregam expressivo valor nutricional, despertando interesse por parte do público idoso. Com base no exposto acima, o objetivo deste trabalho foi elaborar uma formulação de biscoito acrescido de farinha da casca do maracujá e chá da casca de laranja, com foco em um público específico, com idade avançada, os idosos, devido às propriedades nutricionais que estes subprodutos acrescem à formulação supracitada deste trabalho. Destaca-se também que as sementes do maracujá, são ricas em piceatannol, um composto químico que atua como antioxidante no organismo, a qual também foram utilizadas vislumbrando desenvolver uma crocância aceitável aos biscoitos. Neste trabalho foram desenvolvidas duas formulações, uma com subproduto da farinha da casca do maracujá e chá da casca de laranja, e outra formulação considerada controle, sem a adição destes subprodutos. Foram realizadas análises microbiológicas para verificar o crescimento de bolores e leveduras, bem como análises sensoriais com teste de preferência para verificar qual amostra possui maior preferência. O resultado da análise microbiológica do biscoito sem subproduto foi de 6.10² UFC/g, já a amostra com subproduto obteve o resultado de 5.10¹ UFC/g. Tais valores indicam adequação da matéria-prima e do processamento, garantindo uma maior segurança alimentar para o consumidor, sendo considerado como um nível baixo de contaminação. As análises sensoriais apontaram que a formulação sem subproduto obteve maior preferência, no entanto, a respeito das formulações preparadas com adição de subproduto, foram pontuados o sabor amargo decorrente da farinha de casca de maracujá e chá da casca de laranja, a não formação de crosta e consistência endurecida. Conclui-se portanto, levando em consideração os aspectos supracitados, que será necessária a diminuição completa ou parcial do chá da casca de laranja, para suavizar o amargor e destacar o gosto da farinha, visando assim uma maior aceitabilidade sensorial. Acerca das competências de técnico em alimentos, o biscoito concretiza seu objetivo de ser um alimento rico em fibras, e de grande aporte nutricional, evocando novas alternativas de consumo

BISCOITO ENRIQUECIDO DA FARINHA DA CASCA DO MARACUJÁ E SABORIZADO DO CHÁ DA CASCA DE LARANJA

A utilização dos subprodutos (resíduos agroindustriais) nas formulações de produtos alimentícios, explora ao máximo o aproveitamento das partes comestíveis não convencionais dos alimentos. A casca, bem como as sementes do maracujá, quando adicionadas aos produtos, incorporam componentes ricos em pectina e fibras alimentares. A casca quando triturada, com foco na obtenção da farinha, agrega na sua estrutura, propriedades antioxidantes, que auxiliam na eliminação das toxinas do sistema gástrico, prevenindo refluxos, incontinência fecal, azia, constipação e cólicas abdominais. O chá da casca de laranja, quando adicionado aos biscoitos, realça o sabor e o aroma, auxiliando na sua percepção sensorial, e promovendo, quando possível, a aceitabilidade dos produtos. Estes compostos antioxidantes e vitaminas do complexo A, quando acrescidos aos produtos alimentícios, tornam-se de grande valia, e agregam expressivo valor nutricional, despertando interesse por parte do público idoso. Com base no exposto acima, o objetivo deste trabalho foi elaborar uma formulação de biscoito acrescido de farinha da casca do maracujá e chá da casca de laranja, que pode atender inclusive um público específico, com idade avançada, os idosos, devido às propriedades nutricionais que estes subprodutos acrescem à formulação supracitada deste trabalho. Destaca-se também que as sementes do maracujá, são ricas em piceatannol, um composto químico que atua como antioxidante no organismo, as quais também foram utilizadas vislumbrando desenvolver uma crocância aceitável aos biscoitos. Neste trabalho foram desenvolvidas duas formulações, uma com subproduto da farinha da casca do maracujá e chá da casca de laranja, e outra formulação considerada controle, sem a adição destes subprodutos. Foram realizadas análises microbiológicas para verificar o crescimento de bolores e leveduras, bem como análises sensoriais com teste de preferência para verificar qual amostra possui maior preferência. O resultado da análise microbiológica do biscoito sem subproduto foi de 6.10² UFC/g, já a amostra com subproduto obteve o resultado de 5.10¹ UFC/g. Tais valores indicam adequação da matéria-prima e do processamento, garantindo uma maior segurança alimentar para o consumidor, sendo considerado como um nível baixo de contaminação. As análises sensoriais apontaram que a formulação sem subproduto obteve maior preferência, no entanto, a respeito das formulações preparadas com adição de subproduto, foram pontuados o sabor amargo decorrente da farinha de casca de maracujá e chá da casca de laranja, a não formação de crosta e consistência endurecida. Conclui-se portanto, levando em consideração os aspectos supracitados, que será necessária a diminuição completa ou parcial do chá da casca de laranja, para suavizar o amargor e destacar o gosto da farinha, visando assim uma maior aceitabilidade sensorial. Acerca das competências de técnico em alimentos, o biscoito concretiza seu objetivo de ser um alimento rico em fibras, e de grande aporte nutricional, evocando novas alternativas de consumo

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking

The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin.

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate

Selenocysteine oxidation in glutathione peroxidase catalysis: An MS-supported quantum mechanics study

Glutathione peroxidases (GPxs)are enzymes working with either selenium or sulfur catalysis.They adopted diverse functions ranging from detoxification of H2O2 to redox signaling and differentiation. The relative stability of the selenoenzymes, however, remained enigmatic in view of the postulated in- volvement of a highly unstable selenenic acid form during catalysis. Nevertheless, density functional theory calculations obtained with a representative active site model verify the mechanistic concept of GPx catalysis and underscore its efficiency. However, they also allow that the selenenic acid, in the ab- sence of the reducing substrate, reacts with a nitrogen in the active site. MS/MS analysis of oxidized rat GPx4 complies with the predicted structure, an 8-membered ring, in which selenium is bound as sele- nenylamide to the protein backbone.The intermediate can be re-integrated into the canonical GPx cycle by glutathione, whereas, under denaturing conditions, its selenium moiety undergoes \u3b2-cleavage with formation of a dehydro-alanine residue. The selenenylamide bypass prevents destruction of the redox center due to over-oxidation of the selenium or its elimination and likely allows fine-tuning of GPx activity or alternate substrate reactions for regulatory purposes

MOESM5 of Galectin-3: an early predictive biomarker of modulation of airway remodeling in patients with severe asthma treated with omalizumab for 36Â months

Additional file 5. Changes of abundance levels, expressed as natural logarithm of score fold change (ln[T36/T0]), for Gal-3 in OR and NOR patients at baseline (T0) and after 36 months (T36) of anti-IgE treatment. Negative value indicates decrease of Gal-3 at T36; on the contrary, positive value indicates increase of Gal-3 at T36 (see Additional file 1). OR and NOR classification is related to reduction (OR) or not (NOR) of RBM thickness after 12 months of anti-IgE treatment

Lung-to-Heart Nano-in-Micro Peptide Promotes Cardiac Recovery in a Pig Model of Chronic Heart Failure

Background: The lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. Objectives: In this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. Methods: Heart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. Results: DpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. Conclusions: The overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart