RPA shields inherited DNA lesions for post-mitotic DNA synthesis.

The paradigm that checkpoints halt cell cycle progression for genome repair has been challenged by the recent discovery of heritable DNA lesions escaping checkpoint control. How such inherited lesions affect genome function and integrity is not well understood. Here, we identify a new class of heritable DNA lesions, which is marked by replication protein A (RPA), a protein primarily known for shielding single-stranded DNA in S/G2. We demonstrate that post-mitotic RPA foci occur at low frequency during unperturbed cell cycle progression, originate from the previous cell cycle, and are exacerbated upon replication stress. RPA-marked inherited ssDNA lesions are found at telomeres, particularly of ALT-positive cancer cells. We reveal that RPA protects these replication remnants in G1 to allow for post-mitotic DNA synthesis (post-MiDAS). Given that ALT-positive cancer cells exhibit high levels of replication stress and telomere fragility, targeting post-MiDAS might be a new therapeutic opportunity.

Measurement of skin hydration with a portable device (SkinUp® Beauty Device) and comparison with the Corneometer®.

BACKGROUND: Epidermis hydration measurement is an important tool to assess its health. SkinUp® is a portable device that measures moisture and oil levels of skin through impedance method, it is cheap and has small dimensions, but this equipment has not yet been cited in the scientific literature. Thus, the objective of this work was to validate SkinUp® equipment by comparing its results with Corneometer® CM825. MATERIALS AND METHODS: Twenty subjects had skin hydration measurement on forearm, cheeks, and forehead. Measurements obtained with the two instruments were correlated by calculating the Pearson correlation coefficient and the P-value significance. The sensitivity of the equipment to different regions and treatments and their accuracy were also evaluated. RESULTS: Positive correlations between equipment were found for skin hydration, with P ≤ .001. Both instruments presented high sensitivity to the different treatments (P < .001). When sensitivity to different regions was evaluated, both showed greater hydration in the cheek and forehead when compared to the forearm (P < .005). The measurements between the cheek and forehead showed no significant difference when compared to each other. In addition, the two devices have good repeatability in the measurements. CONCLUSION: These results indicate that SkinUp® can be used in the future for skin analysis in in vivo assays.

Increased intestinal carbonate precipitate abundance in the sea bream (Sparus aurata L.) in response to ocean acidification.

Marine fish contribute to the carbon cycle by producing mineralized intestinal precipitates generated as by-products of their osmoregulation. Here we aimed at characterizing the control of epithelial bicarbonate secretion and intestinal precipitate presence in the gilthead sea bream in response to predicted near future increases of environmental CO2. Our results demonstrate that hypercapnia (950 and 1800 µatm CO2) elicits higher intestine epithelial HCO3- secretion ex vivo and a subsequent parallel increase of intestinal precipitate presence in vivo when compared to present values (440 µatm CO2). Intestinal gene expression analysis in response to environmental hypercapnia revealed the up-regulation of transporters involved in the intestinal bicarbonate secretion cascade such as the basolateral sodium bicarbonate co-transporter slc4a4, and the apical anion transporters slc26a3 and slc26a6 of sea bream. In addition, other genes involved in intestinal ion uptake linked to water absorption such as the apical nkcc2 and aquaporin 1b expression, indicating that hypercapnia influences different levels of intestinal physiology. Taken together the current results are consistent with an intestinal physiological response leading to higher bicarbonate secretion in the intestine of the sea bream paralleled by increased luminal carbonate precipitate abundance and the main related transporters in response to ocean acidification.

Ssu72 phosphatase is a conserved telomere replication terminator.

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA polymerases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72∆ mutants are defective in telomere replication and exhibit long 3'-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication.

Structural analysis of nanosystems: Solid Sorbitan esters Nanoparticles (SSN) as a case study.

Innovative approaches in nanotechnology can provide drug delivery systems with a high potential in different fields. To avoid trial and error assays as a main driving force governing new designs and, furthermore, to develop successful nanosystem optimization strategies, it is of the greatest importance to develop specific characterisation techniques beyond conventional determinations of size, zeta potential and morphology. However, the application of techniques able to determine some key characteristics, such as nanostructure (i.e., solid structure vs vesicular), and the way in which the reorganization of components takes place on these structures has been scarcely explored. The present work has been devoted to provide some insights about the potential offered by some NMR techniques to those scientists working on nanotechnological approaches. For this purpose, we selected our nanosystems based on sorbitan monooleate as a case study. We used (1)H NMR methods, including a recently proposed method relying in the well-known Saturation Transfer Difference (STD) experiment for the observation of 'invisible signals' in large aggregates (Invisible State STD or ISSTD). Overall, these techniques revealed the presence in these nanosystems of a gradient of flexibility from an internal rigid core towards a more flexible region located on their surface, as well as the absence of water content in both regions. Such structure, corresponding to a solid nanostructure rather than a vesicular one, can explain some of the interesting properties previously observed for these innovative nanosystems, such as their high stability, and allows us to refer to these nanosystems with the term "Solid Sorbitan esters Nanoparticles" (SSN). On the basis of the valuable information provided by the mentioned characterisation techniques, it is our understanding that they could facilitate the future design of new drug delivery nanosystems as well as the improvement of existing ones and/or the development of new applications for classical drug delivery concepts.

Antioxidant Effect of Nanoemulsions Containing Extract of Achyrocline satureioides (Lam) D.C.-Asteraceae.

Ethanolic extracts of Achyrocline satureioides have pronounced antioxidant activity mainly due to the presence of the flavonoid quercetin. However, direct topical application of the extract is not possible due to the presence of high amounts of ethanol. In this sense, nanoemulsions arise as an alternative for topical formulation associating molecules with limited aqueous solubility. This article describes the development of topical nanoemulsions containing either A. satureioides extract or one of its most abundant flavonoid, quercetin. Nanoemulsions composed of octyldodecanol, egg lecithin, water and extract (NEE), or quercetin (NEQ) were prepared by spontaneous emulsification. This process led to monodisperse nanoemulsions presenting a mean droplet size of approximately 200-300 nm, negative zeta potential, and high association efficiency. A study of quercetin skin retention using porcine skin which was performed using a Franz diffusion cell revealed a higher accumulation of quercetin in skin for NEE when compared to NEQ. Finally, the antioxidant activity of formulations was measured by thiobarbituric acid-reactive species and the APPH model. A lower lipoperoxidation for the extract in respect to quercetin solution was observed. However, no difference between NEQ and NEE lipoperoxidation could be seen. The protection against lipoperoxidation by the formulations was also measured in the skin, where lower formation of reactive species was observed after treatment with NEE. In conclusion, this study shows the formulation effect on the physicochemical properties of nanoemulsions as well as on the skin retention and antioxidant activity of quercetin.

On the use of nanotechnology-based strategies for association of complex matrices from plant extracts

AbstractDepending on the method of extraction, plant extracts can contain an enormous variety of active molecules, such as phenolic compounds, essential oils, alkaloids, among others. In many cases, from a pharmacological point of view, it is interesting to work with crude extract or fractions instead of a single isolated compound. This could be due to multi-targeting effect of the extract, lack of knowledge of the active compounds, synergistic effect of the extract compounds, among others. In any case, in order to achieve a final product some issues must be overcome, including poor stability, solvent toxicity, and low solubility of the bioactive compound. Recently many nanotechnology-based strategies have been proposed as an alternative to solve these problems, especially liposomes, nanoemulsions and nanoparticles. In this sense, the present work aims to review the main nanotechnological approaches used for association of different plant extracts and the main achievements from using these technologies.

PTHrP regulates water absorption and aquaporin expression in the intestine of the marine sea bream (Sparus aurata, L.).

Water ingestion by drinking is fundamental for ion homeostasis in marine fish. However, the fluid ingested requires processing to allow net water absorption in the intestine. The formation of luminal carbonate aggregates impacts on calcium homeostasis and requires epithelial HCO3(-) secretion to enable water absorption. In light of its endocrine importance in calcium handling and the indication of involvement in HCO3(-) secretion the present study was designed to expose the role of the parathyroid hormone-related protein (PTHrP) in HCO3(-) secretion, water absorption and the regulation of aqp1 gene expression in the anterior intestine of the sea bream. HCO3(-) secretion rapidly decreased when PTHrP(1-34) was added to anterior intestine of the sea bream mounted in Ussing chambers. The effect achieved a maximum inhibition of 60% of basal secretion rates, showing a threshold effective dose of 0.1 ng ml(-1) compatible with reported plasma values of PTHrP. When applied in combination with the adenylate cyclase inhibitor (SQ 22.536, 100 µmol l(-1)) or the phospholipase C inhibitor (U73122, 10 µmol l(-1)) the effect of PTHrP(1-34) on HCO3(-) secretion was reduced by about 50% in both cases. In parallel, bulk water absorption measured in intestinal sacs was sensitive to inhibition by PTHrP. The inhibitory action conforms to a typical dose-response curve in the range of 0.1-1000 ng ml(-1), achieves a maximal effect of 60-65% inhibition from basal rates and shows threshold significant effects at hormone levels of 0.1 ng ml(-1). The action of PTHrP in water absorption was completely abolished in the presence of the adenylate cyclase inhibitor (SQ 22.536, 100 µmol l(-1)) and was insensitive to the phospholipase C inhibitor (U73122, 10 µmol l(-1)). In vivo injections of PTHrP(1-34) or the PTH/PTHrP receptor antagonist PTHrP(7-34) evoked respectively, a significant decrease or increase of aqp1ab, but not aqp1a. Overall the present results suggest that PTHrP acts as a key regulator of carbonate aggregate formation in the intestine of marine fish via its actions on water absorption, calcium regulation and HCO3(-) secretion.

Adsorption of antisense oligonucleotides targeting malarial topoisomerase II on cationic nanoemulsions optimized by a full factorial design.

Cationic nanoemulsions have been recently considered as potential delivery systems for oligonucleotides (ON) targeting Plasmodium falciparum topoisomerase II gene. This study is aiming to select the best composition of nanoemulsions intended to ON adsorption by means of a 2(3) full factorial design. Based on their physicochemical properties, two formulations were selected for further studies, both composed by medium chain triglycerides, egg-lecithin, and either oleylamine (OA) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Adsorption isotherms of phosphodiester or phosphorothioate ON on the optimized nanoemulsions were obtained (ultrafiltration/centrifugation procedure). They showed a significant higher amount of ON adsorbed on DOTAP nanoemulsion when compared to the OA ones. The Langmuir adsorption model provides the most satisfactory representation of the adsorption data. Evidence of ON adsorption could be detected by the inversion of the ζ-potential and the morphology of the oil droplets examined by transmission electron microscopy. Preliminary results regarding hemolytic effect and P. falciparum survival after exposure to optimized formulations were related to their physicochemical properties and in vitro effects. The overall results showed the potential of the optimized nanoemulsions as non-viral carriers for antisense ON against malaria parasites.

Endocrine regulation of carbonate precipitate formation in marine fish intestine by stanniocalcin and PTHrP.

In marine fish, high epithelial bicarbonate secretion by the intestine generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. In vitro studies highlight the involvement of the calciotropic hormones PTHrP (parathyroid hormone-related protein) and stanniocalcin (STC) in the regulation of epithelial bicarbonate transport. The present study tested the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo. Sea bream (Sparus aurata) juveniles received single intraperitoneal injections of piscine PTHrP(1-34), the PTH/PTHrP receptor antagonist PTHrP(7-34) or purified sea bream STC, or were passively immunized with polyclonal rabbit antisera raised against sea bream STC (STC-Ab). Endocrine effects on the expression of the basolateral sodium bicarbonate co-transporter (Slc4a4.A), the apical anion exchangers Slc26a6.A and Slc26a3.B, and the V-type proton pump ß-subunit (Atp6v1b) in the anterior intestine were evaluated. In keeping with their calciotropic nature, the hypocalcaemic factors PTHrP(7-34) and STC up-regulated gene expression of all transporters. In contrast, the hypercalcaemic factor PTHrP(1-34) and STC antibodies down-regulated transporters involved in the bicarbonate secretion cascade. Changes in intestine luminal precipitate contents provoked by calcaemic endocrine factors validated these results: 24 h post-injection either PTHrP(1-34) or immunization with STC-Ab reduced the carbonate precipitate content in the sea bream intestine. In contrast, the PTH/PTHrP receptor antagonist PTHrP(7-34) increased not only the precipitated fraction but also the concentration of HCO3(-) equivalents in the intestinal fluid. These results confirm the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo in the intestine of marine fish. Furthermore, they illustrate for the first time in fish the counteracting effect of PTHrP and STC, and reveal an unexpected contribution of calcaemic factors to acid-base balance.

AVT is involved in the regulation of ion transport in the intestine of the sea bream (Sparus aurata).

The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55p.p.t. Basolateral addition of AVT (10(-6)M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose-response curve, with significant effects achieved at 10(-8)M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200µM) applied apically, but was completely abolished in the presence of apical bumetanide (200µM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2.

Adaptation to different salinities exposes functional specialization in the intestine of the sea bream (Sparus aurata L.).

The processing of intestinal fluid, in addition to a high drinking rate, is essential for osmoregulation in marine fish. This study analyzed the long-term response of the sea bream (Sparus aurata L.) to relevant changes of external salinity (12, 35 and 55 p.p.t.), focusing on the anterior intestine and in the less-often studied rectum. Intestinal water absorption, epithelial HCO(3)(-) secretion and gene expression of the main molecular mechanisms (SLC26a6, SLC26a3, SLC4a4, atp6v1b, CFTR, NKCC1 and NKCC2) involved in Cl(-) and HCO(3)(-) movements were examined. The anion transporters SLC26a6 and SLC26a3 are expressed severalfold higher in the anterior intestine, while the expression of Atp6v1b (V-type H(+)-ATPase ß-subunit) is severalfold higher in the rectum. Prolonged exposure to altered external salinity was without effect on water absorption but was associated with concomitant changes in intestinal fluid content, epithelial HCO(3)(-) secretion and salinity-dependent expression of SLC26a6, SLC26a3 and SLC4a4 in the anterior intestine. However, the most striking response to external salinity was obtained in the rectum, where a 4- to 5-fold increase in water absorption was paralleled by a 2- to 3-fold increase in HCO(3)(-) secretion in response to a salinity of 55 p.p.t. In addition, the rectum of high salinity-acclimated fish shows a sustained (and enhanced) secretory current (I(sc)), identified in vitro in Ussing chambers and confirmed by the higher expression of CFTR and NKCC1 and by immunohistochemical protein localization. Taken together, the present results suggest a functional anterior-posterior specialization with regard to intestinal fluid processing and subsequently to salinity adaptation of the sea bream. The rectum becomes more active at higher salinities and functions as the final controller of intestinal function in osmoregulation.

Water absorption and bicarbonate secretion in the intestine of the sea bream are regulated by transmembrane and soluble adenylyl cyclase stimulation.

In the marine fish intestine luminal, HCO3⁻ can remove divalent ions (calcium and magnesium) by precipitation in the form of carbonate aggregates. The process of epithelial HCO3⁻ secretion is under endocrine control, therefore, in this study we aimed to characterize the involvement of transmembrane (tmACs) and soluble (sACs) adenylyl cyclases on the regulation of bicarbonate secretion (BCS) and water absorption in the intestine of the sea bream (Sparus aurata). We observed that all sections of sea bream intestine are able to secrete bicarbonate as measured by pH-Stat in Ussing chambers. In addition, gut sac preparations reveal net water absorption in all segments of the intestine, with significantly higher absorption rates in the anterior intestine that in the rectum. BCS and water absorption are positively correlated in all regions of the sea bream intestinal tract. Furthermore, stimulation of tmACs (10 µM FK + 500 µM IBMX) causes a significant decrease in BCS, bulk water absorption and short circuit current (Isc) in a region dependent manner. In turn, stimulation of sACs with elevated HCO3⁻ results in a significant increase in BCS, and bulk water absorption in the anterior intestine, an action completely reversed by the sAC inhibitor KH7 (200 µM). Overall, the results reveal a functional relationship between BCS and water absorption in marine fish intestine and modulation by tmACs and sAC. In light of the present observations, it is hypothesized that the endocrine effects on intestinal BCS and water absorption mediated by tmACs are locally and reciprocally modulated by the action of sACs in the fish enterocyte, thus fine-tuning the process of carbonate aggregate production in the intestinal lumen.

Integument structure and function in juvenile Xenopus laevis with disrupted thyroid balance.

The skin is the largest organ in the body and is a barrier between the internal and external environment. The present study evaluates how PTU, a goitrogen, that is used to treat hyperthyroidism affects the structure and electrical properties of the frog (Xenopus laevis) skin. The results are considered in the context of the two-membrane model established in the seminal work of Ussing and collegues in the 1940s and 1950s. In vitro experiments with skin from Xenopus adults revealed that PTU can act directly on skin and causes a significant increase (p<0.05, One-way ANOVA) in short circuit current (Isc) via an amiloride-insensitive mechanism. Juvenile Xenopus exposed to waterborne PTU (5 mg/L) had a significantly bigger and more active thyroid gland (p<0.01, Student's t-test) than control Xenopus. The bioelectric properties of skin taken from Xenopus juveniles treated with PTU in vivo had a lower Isc, (3.05±0.4, n=13) and Rt (288.2±39.5) than skin from control Xenopus (Isc, 4.19±1.14, n=14; Rt, 343.3±43.3). A histological assessment of skin from PTU treated Xenopus juveniles revealed the epidermis was significantly thicker (p<0.01, Student's t-test) and had a greater number of modified exocrine glands (p<0.01, Student's t-test) in the dermis compared to control skin. Modifications in skin structure are presumably the basis for its changed bioelectric properties and the study highlights a site of action for environmental chemicals which has been largely neglected.

Efeitos da pressão positiva contínua nas vias aéreas na insuficiência cardíaca crônica / Effects of the continuous positive airway pressure on the airways of patients with chronic heart failure

FUNDAMENTO: A insuficiência cardíaca pode apresentar disfunção assintomática à descompensação, com limitações e diminuição da capacidade produtiva. A pressão positiva contínua nas vias aéreas (CPAP) é um meio não farmacológico de redução da pós-carga. OBJETIVO: Analisar os efeitos da CPAP (10 cmH2O), por 30 dias, em paciente com insuficiência cardíaca crônica. MÉTODOS: Avaliamos 10 pacientes, com diversas etiologias, idade média de 54 ± 14 anos, sexo (masc.= 6 e fem.= 4), com IMC de 21 ± 0,04 kg/m². A terapia foi ofertada por 60 min., 5 vezes por semana, durante 1 mês, no período diurno. Foram analisados ecocardiograma e ergoespirometria, antes e após 30 dias de terapia. RESULTADOS: Apresentou aumento de 19,59 por cento na fração de ejeção do ventrículo esquerdo (FEVE): 23.9 ± 8.91 vs 27.65 ± 9.56 por cento; p = 0,045. Na ergoespirometria, o tempo de exercício (Tex) apresentou aumento significante de 547 ± 151,319 vs 700 ± 293,990 seg., p = 0,02, o consumo de oxigênio (VO2) foi de 9,59 ± 6,1 vs 4,51 ± 2,67 ml.kg-1.min.-1 , p = 0,01, enquanto a produção de dióxido de carbono (VCO2) de repouso (9,85 ± 4,38 vs 6,44 ± 2,88 ml.kg-1.min.-1 , p = 0,03) apresentou diminuição. CONCLUSÃO: A CPAP provocou aumento na fração de ejeção do ventrículo esquerdo e no tempo de exercício, diminuiu o consumo de oxigênio e a produção de dióxido de carbono no repouso.

BACKGROUND: Heart failure can present with asymptomatic dysfunction at decompensation, with limitations and decrease in the productive capacity. The Continuous Positive Airway Pressure (CPAP) is a non-pharmacological means to decrease afterload. OBJECTIVE: To analyze the effects of CPAP (10 cmH2O), for 30 days in patients with chronic heart failure. METHODS: We assessed 10 patients with heart failure (6 males, 4 females) of several etiologies, with a mean age of 54 ± 14 years, with a BMI of 21 ± 0.04 kg/m². The therapy was applied for 60 min., 5 times a week for 30 days, during the daytime. The echocardiogram and the ergospirometry were analyzed, before and 30 days after the therapy. RESULTS: There was a 19.59 percent increase in the left ventricular ejection fraction (LVEF): 23.9 ± 8.91 vs 27.65 ± 9.56 percent; p = 0.045. At the ergospirometry, the exercise time (ET) showed a significant increase from 547 ± 151.319 vs 700 ± 293.990 sec., p = 0.02; oxygen consumption (VO2) was 9.59 ± 6.1 vs 4.51 ± 2.67 ml.kg-1.min.-1, p = 0.01, whereas the carbon dioxide production (VCO2) at rest (9.85 ± 4.38 vs 6.44 ± 2.88 ml.kg-1.min.-1, p = 0.03) decreased. CONCLUSION: The CPAP resulted in an increase in the LVEF and ET, decreased the oxygen consumption and the carbon dioxide production at rest.

Effects of the continuous positive airway pressure on the airways of patients with chronic heart failure.

BACKGROUND: Heart failure can present with asymptomatic dysfunction at decompensation, with limitations and decrease in the productive capacity. The Continuous Positive Airway Pressure (CPAP) is a non-pharmacological means to decrease afterload. OBJECTIVE: To analyze the effects of CPAP (10 cmH2O), for 30 days in patients with chronic heart failure. METHODS: We assessed 10 patients with heart failure (6 males, 4 females) of several etiologies, with a mean age of 54 +/- 14 years, with a BMI of 21 +/- 0.04 kg/m(2). The therapy was applied for 60 min., 5 times a week for 30 days, during the daytime. The echocardiogram and the ergospirometry were analyzed, before and 30 days after the therapy. RESULTS: There was a 19.59% increase in the left ventricular ejection fraction (LVEF): 23.9 +/- 8.91 vs 27.65 +/- 9.56%; p = 0.045. At the ergospirometry, the exercise time (ET) showed a significant increase from 547 +/- 151.319 vs 700 +/- 293.990 sec., p = 0.02; oxygen consumption (VO2) was 9.59 +/- 6.1 vs 4.51 +/- 2.67 ml x kg(-1) x min(-1), p = 0.01, whereas the carbon dioxide production (VCO2) at rest (9.85 +/- 4.38 vs 6.44 +/- 2.88 ml x kg(-1) x min(-1), p = 0.03) decreased. CONCLUSION: The CPAP resulted in an increase in the LVEF and ET, decreased the oxygen consumption and the carbon dioxide production at rest.

Mucosal delivery of liposome-chitosan nanoparticle complexes.

Designing adequate drug carriers has long been a major challenge for those working in drug delivery. Since drug delivery strategies have evolved for mucosal delivery as the outstanding alternative to parenteral administration, many new drug delivery systems have been developed which evidence promising properties to address specific issues. Colloidal carriers, such as nanoparticles and liposomes, have been referred to as the most valuable approaches, but still have some limitations that can become more inconvenient as a function of the specific characteristics of administration routes. To overcome these limitations, we developed a new drug delivery system that results from the combination of chitosan nanoparticles and liposomes, in an approach of combining their advantages, while avoiding their individual limitations. These lipid/chitosan nanoparticle complexes are, thus, expected to protect the encapsulated drug from harsh environmental conditions, while concomitantly providing its controlled release. To prepare these assemblies, two different strategies have been applied: one focusing on the simple hydration of a previously formed dry lipid film with a suspension of chitosan nanoparticles, and the other relying on the lyophilization of both basic structures (nanoparticles and liposomes) with a subsequent step of hydration with water. The developed systems are able to provide a controlled release of the encapsulated model peptide, insulin, evidencing release profiles that are dependent on their lipid composition. Moreover, satisfactory in vivo results have been obtained, confirming the potential of these newly developed drug delivery systems as drug carriers through distinct mucosal routes.

Estratégias invasiva ou não-invasiva e associação com eventos clinicamente relevantes no seguimento pós-alta de pacientes internados com síndrome coronariana aguda / Invasive or now-invasive strategies and their association with clinically relevant outcomes after dischange from hospital in patients admitted with acute coronary syndrome

Fundamentos: Um contexto de incertezas cerca a abordagem da síndrome coronariana aguda(SCA)qto ao seguimento clínico tardio. Objetivo: Investigar se existe associacão entre a realização de cineangiocoronariografia(CAT)com desfechos adversos(infarto não-fatal, reinternação e morte) no seguimento tardio após SCA. Métodos:Amostra-piloto, de coorte retrospectiva do Hospital Universitário Clementino Fraga Filho(HUCFF), som SCA, até sete dias do evento-índice. Foram selecionados pacientes com CID I20 e I24 do banco de autorização de internação hospitalar(AIH), no período de 1999 a 2003, e excluídos aqueles sem diagnóstico de SCA. Avaliou-se o seguimento após a alta hospitalar em relação à realização de CAT na internação. Utilzou-se o teste exato de fisher com nível de significância de 5%. Resultados:Ocorreram 2042 internações com CID I20 e I24 nas AIH no HUCFF, entre 1999 a 2003. De 594 prontuários revistos, 142 foram incluídos. Houve predomínio do sexo masculino e a média de idade foi 62,9+-11,7 anos. Na internação, a letalidade média por causas cardíacas foi de 9,9%(14,1 por cento en SCA com supra de ST e 5,8 por cento em SCA sem supra de ST), com 72,5 por cento submetidos à CAT(letalidade=9,7 por cento vs 20,5 por cento, p=0,096). Dos 128 sobreviventes à internação-índice(74 por cento), 95 pacientes foram acompanhados com tempo médio de 1338+-931 dias e não houve relação inversa entre a realização de CAT e morte(p=0,096), reinfarto(p=0,559) e reinternação(p=0,736). Conclusão: Não houve benefício da estratégia invasiva durante internação por SCA com seguimento médio de 4 anos quanto a letalidade, ao reinfarto não-fatal e à reinternação.

Microspheres containing lipid/chitosan nanoparticles complexes for pulmonary delivery of therapeutic proteins.

Chitosan/tripolyphosphate nanoparticles have already been demonstrated to promote peptide absorption through several mucosal surfaces. We have recently developed a new drug delivery system consisting of complexes formed between preformed chitosan/tripolyphosphate nanoparticles and phospholipids, named as lipid/chitosan nanoparticles (L/CS-NP) complexes. The aim of this work was to microencapsulate these protein-loaded L/CS-NP complexes by spray-drying, using mannitol as excipient to produce microspheres with adequate properties for pulmonary delivery. Results show that the obtained microspheres are spherical and present appropriate aerodynamic characteristics for lung delivery (aerodynamic diameters around 2-3 microm and low apparent tap density of 0.4-0.5 g/cm3). The physicochemical properties of the L/CS-NP complexes are affected by the phospholipids composition. Phospholipids provide a controlled release of the encapsulated protein (insulin), which was successfully associated to the system (68%). The complexes can be easily recovered from the mannitol microspheres upon incubation in aqueous medium, maintaining their morphology and physicochemical characteristics. Therefore, this work demonstrates that protein-loaded L/CS-NP complexes can be efficiently microencapsulated, resulting in microspheres with adequate properties to provide a deep inhalation pattern. Furthermore, they are expected to release their payload (the complexes and, consequently, the encapsulated macromolecule) after contacting with the lung aqueous environment.

Ocular drug delivery by liposome-chitosan nanoparticle complexes (LCS-NP).

This study evaluated in vitro and in vivo a colloidal nanosystem with the potential to deliver drugs to the ocular surface. This nanosystem, liposome-chitosan nanoparticle complexes (LCS-NP), was created as a complex between liposomes and chitosan nanoparticles (CS-NP). The conjunctival epithelial cell line IOBA-NHC was exposed to several concentrations of three different LCS-NP complex to determine the cytotoxicity. The uptake of LCS-NP by the IOBA-NHC conjunctival cell line and by primary cultured conjunctival epithelial cells was examined by confocal microscopy. Eyeball and lid tissues from LCS-NP-treated rabbits were evaluated for the in vivo uptake and acute tolerance of the nanosystems. The in vitro toxicity of LCS-NP in the IOBA-NHC cells was very low. LCS-NPs were identified inside IOBA-NHC cells after 15 min and inside primary cultures of conjunctival epithelial cells after 30 min. Distribution within the cells had different patterns depending on the LCS-NP formulation. Fluorescence microscopy of the conjunctiva revealed strong cellular uptake of LCS-NP in vivo and less intensive uptake by the corneal epithelium. No alteration was macroscopically observed in vivo after ocular surface exposure to LCS-NP. Taken together, these data demonstrate that LCS-NPs are potentially useful as drug carriers for the ocular surface.