Exposure to sub-inhibitory concentrations of cefotaxime enhances the systemic colonization of Salmonella Typhimurium in BALB/c mice

It has been proposed that sub-inhibitory concentrations of antibiotics play a role in virulence modulation. In this study, we evaluated the ability of Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) to colonize systemically BALB/c mice after exposure to a sub-inhibitory concentration of cefotaxime (CTX). In vivo competition assays showed a fivefold increase in systemic colonization of CTX-exposed bacteria when compared to untreated bacteria. To identify the molecular mechanisms involved in this phenomenon, we carried out a high-throughput genetic screen. A transposon library of S. Typhimurium mutants was subjected to negative selection in the presence of a sub-inhibitory concentration of CTX and genes related to anaerobic metabolism, biosynthesis of purines, pyrimidines, amino acids and other metabolites were identified as needed to survive in this condition. In addition, an impaired ability for oxygen consumption was observed when bacteria were cultured in the presence of a sub-inhibitory concentration of CTX. Altogether, our data indicate that exposure to sub-lethal concentrations of CTX increases the systemic colonization of S. Typhimurium in BALB/c mice in part by the establishment of a fitness alteration conducive to anaerobic metabolism.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

BackgroundEnvenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.ConclusionHere we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.     

Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN)

The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIA^Ntr, previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM?=?1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.     

Water-Soluble Ruthenium Complexes Bearing Activity Against Protozoan Parasites

Parasitic illnesses are major causes of human disease and misery worldwide. Among them, both amebiasis and Chagas disease, caused by the protozoan parasites, Entamoeba histolytica and Trypanosoma cruzi, are responsible for thousands of annual deaths. The lack of safe and effective chemotherapy and/or the appearance of current drug resistance make the development of novel pharmacological tools for their treatment relevant. In this sense, within the framework of the medicinal inorganic chemistry, metal-based drugs appear to be a good alternative to find a pharmacological answer to parasitic diseases. In this work, novel ruthenium complexes [RuCl₂(HL)(HPTA)₂]Cl₂ with HL = bioactive 5-nitrofuryl containing thiosemicarbazones and PTA?=?1,3,5-triaza-7-phosphaadamantane have been synthesized and fully characterized. PTA was included as co-ligand in order to modulate complexes aqueous solubility. In fact, obtained complexes were water soluble. Their activity against T. cruzi and E. histolytica was evaluated in vitro. [RuCl₂(HL4)(HPTA)₂]Cl₂ complex, with HL4?=?N-phenyl-5-nitrofuryl-thiosemicarbazone, was the most active compound against both parasites. In particular, it showed an excellent activity against E. histolytica (half maximal inhibitory concentration (IC₅₀)?=?5.2 μM), even higher than that of the reference drug metronidazole. In addition, this complex turns out to be selective for E. histolytica (selectivity index (SI) >38). The potential mechanism of antiparasitic action of the obtained ruthenium complexes could involve oxidative stress for both parasites. Additionally, complexes could interact with DNA as second potential target by an intercalative-like mode. Obtained results could be considered a contribution in the search for metal compounds that could be active against multiple parasites.     

Synaptosomal plasma membrane Ca(2+) pump activity inhibition by repetitive micromolar ONOO(-) pulses.

A sustained increase of intracellular free [Ca(2+)] ([Ca(2+)](i)) has been shown to be an early event of neuronal cell death induced by peroxynitrite (ONOO(-)). In this paper, chronic exposure to ONOO(-) has been simulated by treatment of rat brain synaptosomes or plasma membrane vesicles with repetitive pulses of ONOO(-) during at most 50 min, which efficiently produced nitrotyrosine formation in several membrane proteins (including the Ca(2+)-ATPase). The plasma membrane Ca(2+)-ATPase activity at near-physiological conditions (pH 7, submicromolar Ca(2+), and millimolar Mg(2+)-ATP concentrations), which plays a major role in the control of synaptic [Ca(2+)](i), can be more than 75% inhibited by a sustained exposure to micromolar ONOO(-) (e.g., to 100 pulses of 10 microM ONOO(-)). This inhibition is irreversible and mostly due to a decreased V(max), and to the 2-fold increase of the K(0.5) for Ca(2+) stimulation and about 5-fold increase of the K(M) for Mg(2+)-ATP. [Ca(2+)](i) increases to >400 nM when synaptosomes are subjected to this treatment. Reduced glutathione can afford only partial protection against the inhibition produced by micromolar ONOO(-) pulses. Therefore, inhibition of the plasma membrane Ca(2+)-pump activity during chronic exposure to ONOO(-) may account by itself for a large and sustained increase of intracellular [Ca(2+)](i) in synaptic nerve terminals.     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

Is the atherosclerotic phenotype of preeclamptic placentas due to altered lipoprotein concentrations and placental lipoprotein receptors? Role of a small-for-gestational-age phenotype

Atherosis of spiral arteries in uteroplacental beds from preeclamptic women resemble those of atherosclerosis, characterized by increased plasma lipids and lipoproteins. We hypothesized that: 1) lipoprotein receptors/transporters in the placenta would be upregulated in preeclampsia, associated with increased maternal and fetal lipoprotein concentrations; and 2) expression of these would be reduced in preeclamptic placentae from women delivering small-for-gestational-age (SGA) infants. Placental biopsies and maternal and umbilical serum samples were taken from 27 normotensive and 24 preeclamptic women. Maternal/umbilical cord serum LDL, HDL, total cholesterol, and triglycerides were measured. Placental mRNA expression of lipoprotein receptors/transporters were quantified using quantitative RT-PCR. Protein localization/expression of LDL receptor-related protein 1 (LRP-1) in the preeclamptic placentae with/without SGA was measured by immunohistochemistry. Placental mRNA expression of all genes except paraoxonase-1 (PON-1), microsomal triglyceride transfer protein (MTTP), and protein disulfide isomerase family A member 2 (PDIA2) were observed. No differences for any lipoprotein receptors/transporters were found between groups; however, in the preeclamptic group placental LRP-1 expression was lower in SGA delivering mothers (n = 7; P = 0.036). LRP-1 protein was localized around fetal vessels and Hofbauer cells. This is the first detailed study of maternal/fetal lipoprotein concentrations and placental lipoprotein receptor mRNA expression in normotensive and preeclamptic pregnancies. These findings do not support a role of altered lipid metabolism in preeclampsia, but may be involved in fetal growth.     

Thermoregulatory behavior and oxygen consumption of Octopus mimus paralarvae: The effect of age

Octopus mimus is an important cephalopod species in the coastal zone of Peru and Chile that is exposed to temperature variations from time to time due to El Niño/Southern Oscillation (ENSO) episodes when surface temperatures can reach 24 °C, 6 °C above typical temperatures in their habitat. The relationships between temperature and food availability are important factors that determine the recruitment of juveniles into the O. mimus population. The present study was to evaluate the relationship between thermoregulatory behavior and the age of paralarvae (summer population) to determine whether changes in this behavior occur during internal yolk consumption, making larvae more vulnerable to environmental temperature change. Oxygen consumption of paralarvae when 1–4 d old was determined to establish if respiration could be used to monitor the physiological changes that occur during yolk consumption. Horizontal thermal selection (17–30 °C), critical thermal maxima (CTMax), minima (CTMin), and oxygen consumption experiments were conducted with fasting paralarvae 1–4 d old at 20 °C. Preferred temperatures were dependent on the age of O. mimus paralarvae. One day old paralarvae selected a temperature 1.1 °C (23·4 °C) higher than 2 – 4 d old paralarvae (22·3 °C). The CTMax of paralarvae increased with age with values of 31·9±1.1 °C in 1-d-olds and 33·4±0.3 to 4-d-olds. CTMin also changed with age with low values in 2-d-old paralarvae (9.1±1·3 °C) and 11·9±0·9 °C in 4-d-old animals. The temperature tolerance range of paralarvae was age-dependent (TTD=difference between CTMax and CTMin) with higher values in 2 and 3 d old paralarvae (25–26 °C) as compared to 1 d old (23·1 °C) and 4 d old animals (22.7 °C). Oxygen consumption was not affected by the age of paralarvae, suggesting that mechanisms exist that compensate their metabloism until at least 4 d of age. The temperature tolerance range of a planktonic paralarvae of octopus species is presented for the first time. This range was dependent on the age of paralarvae, and so rendered the paralarvae more vunerable to a combination of high temperature and food deprivation during first days of life. Results in the present study provide evidence that O. mimus could be under ecological pressure if a climate change causes increased or decreased temperatures into their distribution range.     

Geographic distribution and host plants of Raoiella indica and associated mite species in northern Venezuela

The red palm mite (RPM), Raoiella indica Hirst (Acari: Tenuipalpidae), is an invasive pest in the New World, where it is currently considered a serious threat to coconut and banana crops. It was first reported from northern Venezuela in 2007. To determine its current distribution in this country, surveys were carried out from October 2008 to April 2010 on coconut (Cocos nucifera L.), banana (Musa spp.), ornamental plants and weeds in northern Venezuela. Higher population levels of RPM were registered on commercial coconut farms in Falcón and Sucre states but also on other plant species naturally growing along the coastal line in Anzoategui, Aragua, Carabobo, Monagas and Nueva Esparta states. Out of 34 botanical species evaluated, all RPM stages were observed only on eight arecaceous, one musaceous and one streliziaceous species, indicating that the pest developed and reproduced only on these plants. Mite specimens found on weeds were considered spurious events, as immature stages of the pest were never found on these. Amblyseius largoensis (Muma) (Acari: Phytoseiidae) was the most frequent predatory mite associated with RPM in all sampling sites. The results indicate that RPM has spread to extensive areas of northern Venezuela since its initial detection in Güiria, Sucre state. Considering the report of this pest mite in northern Brazil in the late 2009, additional samplings in southern Venezuela should be carried out, to evaluate the possible presence of RPM also in that region.