Calling activity and vocal repertoire of hypsiboas prasinus (Anura, Hylidae), a treefrog from the Atlantic Forest of Brazil

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

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear lucent after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded lucent fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the lucency of the fibrils indicated that they were composed of collagen I, whereas the lucency of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.

Natural rubber latex coated with calcium phosphate for biomedical application

Natural rubber latex (NRL) is a flexible biomembrane that possesses angiogenic properties and has recently been used for guided bone regeneration, enhancing healing without fibrous tissue, allergies or rejection. Calcium phosphate (Ca/P) ceramics have chemical, biological, and mechanical properties similar to mineral phase of bone, and ability to bond to the host tissue, although it can disperse from where it is applied. Therefore, to create a composite that could enhance the properties of both materials, NRL biomembranes were coated with Ca/P. NRL biomembranes were soaked in 1.5 times concentrated SBF solution for seven days, avoiding the use of high temperatures. SEM showed that Ca/P has been coated in NRL biomembrane, XRD showed low crystallinity and FTIR showed that is the carbonated type B. Furthermore, hemolysis of erythrocytes, quantified spectrophotometrically using materials (Ca/P, NRL, and NRL + Ca/P) showed no hemolytic effects up to 0.125 mg/mL (compounds and mixtures), indicating no detectable disturbance of the red blood cell membranes. The results show that the biomimetic is an appropriate method to coat NRL with Ca/P without using high temperatures, aiming a new biomembrane to improve guided bone regeneration.

Chitosan/Carbomer-based tablets containing Metronidazole for potential treatment of periodontal diseases

Mucoadhesive tablets with different mixtures of chitosan and carbomer homopolymer type B were prepared in order to obtain new formulations containing metronidazole for periodontal disease treatment. All tablets were characterized by swelling and erosion studies, ex vivo mucoadhesion force and in vitro drug release. The drug released mechanism was described by Korsmeyer-Peppas and Weibull models. Tablets containing increased amounts of chitosan presented higher swelling ability and the drug release was prolonged in the simulated saliva fluid. The mechanisms for the drug release from tablets were complex, including diffusion, swelling and erosion simultaneously. This kind of delivery system is suitable for formulating metronidazole mucoadhesive systems, representing a good alternative for the local treatment of periodontal diseases.

Displasia neuronal intestinal: análise de critérios morfológicos e comparação de métodos diagnósticos

Pós-graduação em Patologia - FMB

Características físico-químicas de amidos de genótipos de bananeiras

Pós-graduação em Zootecnia - FMVZ

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (approximate to 90 mmol L-1). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex (R) 100L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)). (C) 2012 Elsevier B.V. All rights reserved.

Biventricular structural and functional responses to aortic constriction in a rabbit model of chronic right ventricular pressure overload

Objectives: Chronic right ventricular (RV) pressure overload results in pathologic RV hypertrophy and diminished RV function. Although aortic constriction has been shown to improve systolic function in acute RV failure, its effect on RV responses to chronic pressure overload is unknown. Methods: Adjustable vascular banding devices were placed on the main pulmonary artery and descending aorta. In 5 animals (sham group), neither band was inflated. In 9 animals (PAB group), only the pulmonary arterial band was inflated, with adjustments on a weekly basis to generate systemic or suprasystemic RV pressure at 28 days. In 9 animals, both pulmonary arterial and aortic devices were inflated (PAB+AO group), the pulmonary arterial band as for the PAB group and the aortic band adjusted to increase proximal systolic blood pressure by approximately 20 mm Hg. Effects on the functional performance were assessed 5 weeks after surgery by conductance catheters, followed by histologic and molecular assessment. Results: Contractile performance was significantly improved in the PAB+AO group versus the PAB group for both ventricles. Relative to sham-operated animals, both banding groups showed significant differences in myocardial histologic and molecular responses. Relative to the PAB group, the PAB+AO group showed significantly decreased RV cardiomyocyte diameter, decreased RV collagen content, and reduced RV expression of endothelin receptor type B, matrix metalloproteinase 9, and transforming growth factor beta genes. Conclusions: Aortic constriction in an experimental model of chronic RV pressure overload not only resulted in improved biventricular systolic function but also improved myocardial remodeling. These data suggest that chronically increased left ventricular afterload leads to a more physiologically hypertrophic response in the pressure-overloaded RV. (J Thorac Cardiovasc Surg 2012;144:1494-501)

Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns.

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.

Key issues in diagnosing and treating acute aortic syndromes: results from the metropolitan area of Bologna network

Background: Survival of patients with Acute Aortic Syndrome (AAS) may relate to the speed of diagnosis. Diagnostic delay is exacerbated by non classical presentations such as myocardial ischemia or acute heart failure (AHF). However little is known about clinical implications and pathophysiological mechanisms of Troponin T elevation and AHF in AAS. Methods and Results: Data were collected from a prospective metropolitan AAS registry (398 patients diagnosed between 2000 and 2013). Troponin T values (either standard or high sensitivity assay, HS) were available in 248 patients (60%) of the registry population; the overall frequency of troponin positivity was 28% (ranging from 16% to 54%, using standard or HS assay respectively, p = 0.001). Troponin positivity was associated with a twofold increased risk of long in-hospital diagnostic time (OR 1.92, 95% CI 1.05-3.52, p = 0.03), but not with in-hospital mortality. The combination of positive troponin and ACS-like ECG abnormalities resulted in a significantly increased risk of inappropriate therapy due to a misdiagnosis of ACS (OR 2.48, 95% CI 1.12-5.54, p = 0.02). Patients with AHF were identified by the presence of dyspnea as presentation symptom or radiological signs of pulmonary congestion or cardiogenic shock. The overall frequency of AHF was 28 % (32% type A vs. 20% type B AAS, p = 0.01). AHF was due to a variety of pathophysiological mechanisms including cardiac tamponade (26%), aortic regurgitation (25%), myocardial ischemia (17%), hypertensive crisis (10%). AHF was associated with increased surgical delay and with increased risk of in-hospital death (adjusted OR 1.97 95% CI1.13-3.37,p=0.01). Conclusions: Troponin positivity (particularly HS) was a frequent finding in AAS. Abnormal troponin values were strongly associated with ACS-like ECG findings, in-hospital diagnostic delay, and inappropriate therapy. AHF was associated with increased surgical delay and was an independent predictor of in-hospital mortality.

Interactions between selected photosensitizers and model membranes: an NMR classification

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC (1)H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS. (C) 2011 Elsevier B.V. All rights reserved.

Should aortic arch replacement be performed during initial surgery for aortic root aneurysm in patients with Marfan syndrome?

OBJECTIVES: The aim of this study was to investigate whether total arch replacement (TAR) during initial surgery for root aneurysm should be routinely performed in patients with Marfan syndrome (MFS). METHODS: Retrospective analysis of 94 consecutive MFS patients fulfilling Ghent criteria who underwent 148 aortic surgeries and were followed at this institution during the past 16 years. RESULTS: The mean follow-up interval was 8.8 ± 7 years. Initial presentation was acute aortic dissection (AAD) in 35% of patients (76% Type A and 24% Type B) and aneurismal disease in 65%. TAR was performed in 8% of patients during initial surgery for AAD (otherwise a hemi-arch replacement was performed) and 1.6% in elective root repair. Secondary TAR had to be performed in only 3% of patients without, but in 33% following AAD (33% Type A and 33% Type B; P = 0.0001). Thirty-day, 6-month, 1-year and overall mortalities were 3.2, 5.3, 6.4 and 11.7%, respectively. Operative and 30-day mortalities in secondary aortic arch replacement were zero. Secondary TAR after AAD did not increase the need for the replacement of the entire thoracoabdominal aorta during follow-up compared with patients without secondary TAR (37 vs 40%, P = 1.0). CONCLUSIONS: MFS patients undergoing elective root repair have small risk of reinterventions on the aortic arch, and primary prophylactic replacement does not seem to be justified. In patients with AAD, the need for reinterventions is precipitated by the dissection itself and not by limiting the procedure to the hemi-arch replacement in the emergency setting. Limiting surgery to the aortic root, ascending aorta and proximal aortic arch is associated with low mortality in MFS patients presenting with AAD.

α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle's loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A-non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl--dependent HCO3- secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1-/- mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1-/- mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO3- secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.

[Guidance of interventions in subintimal recanalization and fenestration of dissection membranes using a novel dual-lumen intravascular ultrasound catheter]

PURPOSE: To evaluate the feasibility and effectiveness of IVUS-guided puncture for gaining controlled target lumen reentry in subintimal recanalization of chronic iliac/femoral artery occlusions and in fenestration of aortic dissections. MATERIALS AND METHODS: Between 5/2004 and 12/2005 12 consecutive patients (7 male, 5 female; mean age 64.6 +/- 12.0 years) with chronic critical limb ischemia and ischemic complications of aortic dissection were treated using the Pioneer catheter. This 6.2-F dual-lumen catheter combines a 20-MHz IVUS transducer with a pre-shaped extendable, hollow 24-gauge nitinol needle. This coaxial needle allows real-time IVUS-guided puncture of the target lumen and after successful reentry a 0.014" guidewire may be advanced through the needle into the target lumen. 7 patients were treated for aortic dissection and 5 patients (with failed previous attempts at subintimal recanalization) for chronic arterial occlusion. Patients with aortic dissection (5 type A dissections, 2 type B dissections) had developed renal ischemia (n = 2), renal and mesenteric ischemia (n = 2), or low extremity ischemia (n = 3). Patients with chronic arterial occlusions (2 common iliac artery occlusions, 3 superficial femoral artery occlusions) experienced ischemic rest pain (n = 4), and a non-healing foot ulcer (n = 1). RESULTS: The technical success rate using the Pioneer catheter was 100%. The recanalization/fenestration time was 37 +/- 12 min. Procedure-related complications did not occur. In 10 cases a significant improvement of clinical symptoms was evident. One patient with aortic dissection and ischemic paraplegia required subsequent surgical intervention. One patient had persistent ischemic rest pain despite successful recanalization of a superficial femoral artery occlusion. CONCLUSION: The Pioneer catheter is a reliable device which may be helpful for achieving target lumen reentry in subintimal recanalization of chronic occlusions and in fenestration of aortic dissections.