RNA-based liposomes for oral cancer: From biophysical characterization to biological evaluation.

Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated. Firstly, the RNA structure and binding interaction with ligands (C8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C8 forms a complex with RNA and adopts an open conformation upon RNA binding. Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C8 loaded in RNA-functionalized liposomes indicate the co-existence of free C8 in solution (inside the liposome) and C8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line. Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes.

Gallic Acid-Triethylene Glycol Aptadendrimers Synthesis, Biophysical Characterization and Cellular Evaluation.

Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid-triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.

Usnic acid potassium salt: an alternative for the control of Biomphalaria glabrata (Say, 1818).

In Brazil, the snail Biomphalaria glabrata is the most important vector of schistosomiasis due to its wide geographical distribution, high infection rate and efficient disease transmission. Among the methods of schistosomiasis control, the World Health Organization recommends the use of synthetic molluscicides, such as niclosamide. However, different substances of natural origin have been tested as alternatives for the control or eradication of mollusks. The literature describes the antitumor, antimicrobial and antiviral properties of usnic acid as well as other important activities of common interest between medicine and the environment. However, usnic acid has a low degree of water solubility, which can be a limiting factor for its use, especially in aquatic environments, since the organic solvents commonly used to solubilize this substance can have toxic effects on aquatic biota. Thus, the aim of the present study was to test the potassium salt of usnic acid (potassium usnate) with regard to molluscicidal activity and toxicity to brine shrimp (Artemia salina). To obtain potassium usnate, usnic acid was extracted with diethyl ether isolated and purified from the lichen Cladonia substellata. Biological assays were performed with embryos and adult snails of B. glabrata exposed for 24 h to the usnate solution solubilized in dechlorinated water at 2.5; 5 and 10 µg/ml for embryos, 0.5; 0.9; 1;5 and 10 µg/ml for mollusks and 0.5; 1; 5; 10 µg/ml for A. salina. The lowest lethal concentration for the embryos and adult snails was 10 and 1 µg/ml, respectively. No toxicity to A. salina was found. The results show that modified usnic acid has increased solubility (100%) without losing its biological activity and may be a viable alternative for the control of B. glabrata.

Fatores de risco para mortalidade neonatal, com especial atenção aos fatores assistenciais relacionados com os cuidados durante o período pré-natal, parto e história reprodutiva materna / Determinants of neonatal death with emphasis on health care during pregnancy, childbirth and reproductive history

OBJETIVO: Identificar fatores de risco para mortalidade neonatal, com especial atenção aos fatores assistenciais relacionados com os cuidados durante o período pré-natal, parto e história reprodutiva materna. MÉTODOS: Trata-se de um estudo caso-controle realizado em Maceió, Nordeste do Brasil. A amostra consistiu de 136 casos e 272 controles selecionados em bancos de dados oficiais brasileiros. Os casos foram todos os recém-nascidos que morreram antes de completar 28 dias de vida, selecionados no Sistema de Informações sobre Mortalidade, e os controles foram os sobreviventes neste período, selecionados no Sistema de Informações sobre Nascidos Vivos, por sorteio aleatório entre as crianças nascidas na mesma data do caso. Entrevistas domiciliares foram realizadas com as mães. RESULTADOS: A análise de regressão logística identificou como fatores determinantes para a morte no período neonatal mães com história de filhos anteriores que morreram no primeiro ano de vida (OR = 3,08), o internamento durante a gestação (OR = 2,48), o pré-natal inadequado (OR = 2,49), a não realização de ecografia durante o pré-natal (OR = 3,89), a transferência de recém-nascidos para outra unidade após o nascimento (OR = 5,06), os recém-nascidos internados em UTI (OR = 5,00) e o baixo peso ao nascer (OR = 2,57). Entre as condições socioeconômicas, observou-se uma maior chance para mortalidade neonatal em residências com menor número de moradores (OR = 1,73) e com ausência de filhos menores de cinco anos (OR = 10,10). CONCLUSÕES: Vários fatores que se mostraram associados à mortalidade neonatal neste estudo podem ser decorrentes de assistência inadequada ao pré-natal, ao parto e ao recém-nascido, sendo, portanto, passíveis de serem modificados.

OBJECTIVE: To identify risk factors for neonatal mortality, focusing on factors related to assistance care during the prenatal period, childbirth, and maternal reproductive history. METHODS: This was a case-control study conducted in Maceió, Northeastern Brazil. The sample consisted of 136 cases and 272 controls selected from official Brazilian databases. The cases consisted of all infants who died before 28 days of life, selected from the Mortality Information System, and the controls were survivors during this period, selected from the Information System on Live Births, by random drawing among children born on the same date of the case. Household interviews were conducted with mothers. RESULTS: The logistic regression analysis identified the following as determining factors for death in the neonatal period: mothers with a history of previous children who died in the first year of life (OR = 3.08), hospitalization during pregnancy (OR = 2.48), inadequate prenatal care (OR = 2.49), lack of ultrasound examination during prenatal care (OR = 3.89), transfer of the newborn to another unit after birth (OR = 5.06), admittance of the newborn at the ICU (OR = 5.00), and low birth weight (OR = 2.57). Among the socioeconomic conditions, there was a greater chance for neonatal mortality in homes with fewer residents (OR = 1.73) and with no children younger than five years (OR = 10.10). CONCLUSION: Several factors that were associated with neonatal mortality in this study may be due to inadequate care during the prenatal period and childbirth, and inadequate newborn care, all of which can be modified.

Características e fatores associados à assistência à saúde de crianças menores de um ano com muito baixo peso ao nascer / Characteristics and factors associated with health care in children younger than 1 year with very low birth weight

OBJETIVOS: Identificar as características da assistência à saúde de lactentes com muito baixo peso ao nascer no primeiro ano de vida e os fatores associados a esta atenção. MÉTODOS: Estudo descritivo com componente analítico foi realizado na cidade de Maceió, Nordeste do Brasil, com uma amostra de 53 crianças com idade mediana de cinco meses na época da entrevista, e suas respectivas mães. As mães foram entrevistadas no domicílio,quanto às condições socioeconômicas, demográficas e de assistência à saúde da criança. A atenção à saúde foi avaliada com a elaboração de um índice utilizando 16 variáveis relacionadas às ações preconizadas para esta assistência. RESULTADOS: A análise de regressão linear multivariada mostrou que a escolaridade materna e a renda familiar foram as variáveis que, juntas, melhor explicaram a variação do Índice de Atenção à Saúde (18,9%), seguidas da paridade (6,6%) e da prática do aleitamento materno na época da entrevista (6,9%). CONCLUSÕES: Considerando que as famílias com piores condições socioeconômicas e as mulheres com maior número de filhos e que não amamentaram foram os fatores associados a uma assistência inadequada à saúde de crianças nascidas com muito baixo peso, os mesmos deveriam ser contemplados nas ações de planejamento da saúde pública.

OBJECTIVES: To identify the characteristics of health care in infants with very low birth weight during the first year of life and the factors associated with this care. METHODS: This was a descriptive study with an analytical component conducted in the city of Maceió, Northeastern Brazil, with a sample of 53 children with a median age of five months at the time of the interview, and their mothers. The mothers were interviewed at home regarding socioeconomic and demographic data and health care provided for the child. Health care was assessed through an index using 16 variables related to the recommended actions for this type of care. RESULTS: Multivariate linear regression analysis showed that maternal education and family income were the variables that best explained the health care index variation (18.9%), followed by parity (6.6%), and breastfeeding at the time of the interview (6.9%). CONCLUSIONS: Considering that families with lower socioeconomic status, women with a higher number of children, and women who did not breastfeed were factors associated with poor health care of children born with very low birth weight, these variables should be included in measures of public health planning.

Oxidative stress modulates the nitric oxide defense promoted by Escherichia coli flavorubredoxin.

Mammalian cells of innate immunity respond to pathogen invasion by activating proteins that generate a burst of oxidative and nitrosative stress. Pathogens defend themselves from the toxic compounds by triggering a variety of detoxifying enzymes. Escherichia coli flavorubredoxin is a nitric oxide reductase that is expressed under nitrosative stress conditions. We report that in contrast to nitrosative stress alone, exposure to both nitrosative and oxidative stresses abolishes the expression of flavorubredoxin. Electron paramagnetic resonance (EPR) experiments showed that under these conditions, the iron center of the flavorubredoxin transcription activator NorR loses the ability to bind nitric oxide. Accordingly, triggering of the NorR ATPase activity, a requisite for flavorubredoxin activation, was impaired by treatment of the protein with the double stress. Studies of macrophages revealed that the contribution of flavorubredoxin to the survival of E. coli depends on the stage of macrophage infection and that the lack of protection observed at the early phase is related to inhibition of NorR activity by the oxidative burst. We propose that the time-dependent activation of flavorubredoxin contributes to the adaptation of E. coli to the different fluxes of hydrogen peroxide and nitric oxide to which the bacterium is subjected during the course of macrophage infection.

Structure at 1.0 A resolution of a high-potential iron-sulfur protein involved in the aerobic respiratory chain of Rhodothermus marinus.

The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus, a nonphotosynthetic organism from the Bacteroidetes/Chlorobi group, contains a high-potential iron-sulfur protein (HiPIP) that transfers electrons from a bc 1 analog complex to a caa 3 oxygen reductase. Here, we describe the crystal structure of the reduced form of R. marinus HiPIP, solved by the single-wavelength anomalous diffraction method, based on the anomalous scattering of the iron atoms from the [4Fe-4S]3+/2+ cluster and refined to 1.0 A resolution. This is the first structure of a HiPIP isolated from a nonphotosynthetic bacterium involved in an aerobic respiratory chain. The structure shows a similar environment around the cluster as the other HiPIPs from phototrophic bacteria, but reveals several features distinct from those of the other HiPIPs of phototrophic bacteria, such as a different fold of the N-terminal region of the polypeptide due to a disulfide bridge and a ten-residue-long insertion.

Widespread distribution in pathogenic bacteria of di-iron proteins that repair oxidative and nitrosative damage to iron-sulfur centers.

Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H2O2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H2O2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

New insights into type II NAD(P)H:quinone oxidoreductases.

Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)(+) pool, and may contribute to the generation of a membrane potential through complexes III and IV. These enzymes are usually constituted by a nontransmembrane polypeptide chain of approximately 50 kDa, containing a flavin moiety. There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes. However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the balance and are the main entry point of electrons into the respiratory chains. Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms. The latter showed that most organisms contain genes that potentially encode NDH-2. An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2.

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus.

A [3Fe-4S](1+/0) ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe-4S](1+/0), with an unusually low reduction potential of -650 mV, determined by cyclic voltammetry at pH 7.6. [1H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe-4S](1+/0) centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe-4S](2+/1+) cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism. The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint (T(m)) for thermally induced unfolding is 102+/-2 degrees C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 degrees C). The iron-sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.

Growth changes induced by gamma radiation on Biomphalaria straminea (Dunker, 1848)

Doses of 60Co gamma radiation with 2.5; 5; 7.5; 10; 15; 20; 25; 30; 35; 40; 45; 50; 55; 60; 80; 160; 320 and 640 Gy were applied to 1,080 snails Biomphalaria straminea, an intermediate host of Schistosoma mansoni, divided in groups containing 30 mollusks. In addition, 60 non irradiated snails were kept as control. Fifty percent of the population was kept in colonies (allowing cross fertilization) while the other half was maintained in sexual isolation (allowing self fertilization) and during one month their growth was observed through the daily measurement of the shell diameter. Results showed that after 20 Gy doses the growth in shell diameter of irradiated snails was greater than that of the control group after 30 days. At this dose the snail size was the greatest, among all isolated groups. The 80 Gy doses also induced the final shell diameter of isolated snails to be greater then that observed in the control groups. As this effect was most evident among the isolated snails, a possible hormonal role may have been involved in the observed phenomena, which is under investigation with the objective of identifying any future applications that this could have to schistosomiasis control

Fecundity changes induced by low-doses of gamma radiation on Biomphalaria straminea (Dunker, 1848)

A population of 420 snails Biomphalaria straminea, an intermediate host of Schistosoma mansoni, received gamma-rays obtained from a 60 Co source in low-doses (0/2,5/5/7,5/10/15/20 and 25 Gy); half population was kept in colonies (allowing cross fertilization) and other half was mantained in sexual isolation (alowing self fertilization). Results showed that 15 Gy stimulates the fertility of both groups but the colonies were more sensitive and at this dose its fertility overpasses the control group dose. The possible hormonal role played in the observed phenomena is under investigation.