Altered Host Cell–Bacteria Interaction due to Nanoparticle Interaction with a Bacterial Biofilm

Nanoparticle (NP) use in everyday applications creates the potential for NPs to enter the environment where, in aquatic systems, they are likely to settle on substrates and interact with microbial communities. Legionella pneumophila biofilms are found as part of microbial communities in both natural and man-made environments, especially in man-made cooling systems. The bacterium is the causative agent of Legionnaires' disease. Legionella requires a host cell for replication in the environment, and amoebae commonly serve as this host cell. Our previous work demonstrated significant changes in Legionella biofilm morphology after exposure to 0.7 μg/L gold NPs (AuNPs). Here, we investigate how these morphology changes alter host–bacteria interactions using Acanthamoeba polyphaga as a model. Host–bacteria–NP interactions are affected by NP characteristics. Biofilms exposed to 4- and 18-nm, citrate-capped, spherical AuNPs significantly altered the grazing ability of A. polyphaga, which was not observed in biofilms exposed to 24-nm polystyrene beads. Uptake and replication of NP-exposed planktonic L. pneumophila within A. polyphaga were not altered regardless of NP size or core chemistry. Nanomaterial effects on the interaction of benthic organisms and bacteria may be directly or, as shown here, indirectly dependent on bacterial morphology. NP contamination therefore may alter interactions in a normal ecosystem function.     

In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya

N⁶-threonylcarbamoyladenosine (t⁶A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon–anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t⁶A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t⁶A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t⁶A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t⁶A in nature. These findings shed light on the reaction mechanism of t⁶A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells.     

Silencing human genetic diseases with oligonucleotide-based therapies

RNA interference is an endogenous mechanism present in most eukaryotic cells that enables degradation of specific mRNAs. Pharmacological exploitation of this mechanism for therapeutic purposes attracted a whole amount of attention in its initial years, but was later hampered due to difficulties in delivery of the pharmacological agents to the appropriate organ or tissue. Advances in recent years have to a certain level started to address this specific issue. Genetic diseases are caused by aberrations in gene sequences or structure; these particular abnormalities are in theory easily addressable by RNAi therapeutics. Sequencing of the human genome has largely contributed to the identification of alterations responsible for genetic conditions, thus facilitating the design of compounds that can address these diseases. This review addresses the currently on-going programs with the aim of developing RNAi and other antisense compounds for the treatment of genetic conditions and the pros and cons that these products may encounter along the way. The authors have focused on those programs that have reached clinical trials or are very close to do so.     

Design,synthesis and anti-tuberculosis activity of 1-adamantyl-3-heteroaryl ureas with improved in vitro pharmacokinetic properties

Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death worldwide due to infectious disease. Development of new drugs that shorten the current tuberculosis treatment time and have activity against drug resistant strains is of utmost importance. Towards these goals we have focused our efforts on developing novel anti-TB compounds with the general structure of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid transport across the plasma membrane. However, these compounds suffered from poor in vitro pharmacokinetic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase (sEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by three factors: (1) to increase selectivity for anti-TB activity over human sEH activity, (2) to optimize PK profiles including solubility and (3) to maintain target inhibition. A new series of 1-adamantyl-3-heteroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced lead isoxazole, oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles, increased selectivity and good anti-TB potencies with sub μg/mL minimum inhibitory concentrations.     

Phenol degradation and colour removal in submerged culture of Pleurotus sajor-caju with paper mill effluents

The fungus Pleurotus sajor-caju secretes phenol-oxidases that enable the use of recalcitrant compounds as substrates. The residues of paper manufacture contain high lignin levels, which gives the effluents a characteristic brownish colour. To test the potential of P. sajor-caju cultures on reducing these parameters, we used 90% of raw effluents from medium consistency oxygen delignification and bleaching stages plus 10% of mineral solution and different levels of glucose (5–15 g L^?1) as substrate. We observed a greater fungal biomass in cultures using effluent than in controls. Cultures containing 10 to 15 g L^?1 of glucose resulted in about 42% colour reduction. The polyphenol content was also reduced by 58.9% by the 13th day of culture. In addition, we observed the secretion of laccases (211.44 U mL^?1 and 45.98 U mL^?1 using ABTS and syringaldazine, respectively) and peroxidases (6.11 U mL^?1-ABTS) both peaking at the 7th day of culture and with similar kinetics of production in different glucose concentrations.     

The interference of cold ischemia time in the quality of total RNA from frozen tumor samples

Tumor Banks were created to organize the collection, storage and distribution of biological samples from oncological patients, facilitating its use in cancer research. To ensure the quality of the samples from our bank, we implemented standard operating procedures international. In order to evaluate the influence of cold ischemia time (time between surgical removal of the specimen and the snap freezing of the sample) on the quality of the samples (evaluated by measurement integrity of their RNA), collected during 10 months two tumor samples from each donor, one with up to 30 min of cold ischemia and other with exact 45 min, totaling 100 different donors and 200 samples, 40 from each of the following organs: breast, thyroid, stomach, lung and colorectum. We extracted total RNA from the samples and with the aid of a Bioanalyser, evaluate their quality, comparing it with cold ischemia times in different organs. Among the samples up to 30 min and the samples with exact 45 min, we respectively found 63 (64.3 %) and 36 (36 %) with intact RNA, 11 (11.2 %) and 17 (17 %) partially degraded and 24 (24.5 %) and 47 (47 %) degraded (p < 0.001). Thyroid and colorectal samples were more sensitive to variations in cold ischemia time (p = 0.006 and p = 0.03, respectively). Stomach and lungs were less sensitive (p = 0.919 and p = 0.384, respectively). We concluded that the cold ischemia time up to 30 min was more efficient to maintain the integrity of RNA in most samples, and that RNA degradation varied according to the different topographies.     

pH‐Dependent Recycling of Galectin‐3 at the Apical Membrane of Epithelial Cells

The β‐galactoside binding protein galectin‐3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium by non‐classical secretion. Once within the apical extracellular milieu, galectin‐3 can re‐enter the cell followed by passage through endosomal organelles and modulate apical protein sorting. Here, we could show that galectin‐3 is internalized by non‐clathrin mediated endocytosis. Within endosomal organelles this pool associates with newly synthesized neurotrophin receptor in the biosynthetic pathway and assists in its membrane targeting. This recycling process is accompanied by transient interaction of galectin‐3 with detergent insoluble membrane microdomains in a lactose‐ and pH‐dependent manner. Moreover, in the presence of lactose, apical sorting of the neurotrophin receptor is affected following endosomal deacidification. Taken together, our results suggest that internalized galectin‐3 directs the subcellular targeting of apical glycoproteins by membrane recycling .     

Myostatin expression is regulated by underfeeding and neonatal programming in rats

Confusing results have been reported regarding the influence of nutritional status on myostatin levels. Some studies indicate that short-term fasting results in increased myostatin mRNA levels in skeletal muscle, evident in several species. In contrast, other studies have demonstrated either a decrease or no change in myostatin levels during fasting. In the present study, we investigated the effect of different patterns of food deprivation on muscle myostatin expression in both newborn and adult rats. Adjustment of litter size in neonatal rats is a well-established model to study the effect of early overfeeding or underfeeding on body composition and in this study resulted in modifications in the pattern of muscle myostatin expression. Rat pups growing in large litters (22–24 newborns) showed a decrease in muscle myostatin mRNA and protein levels at 24 days of age. Interestingly, these effects were maintained at 60 days of age despite rats having free access to food since weaning, thus suggesting that changes in myostatin expression induced by neonatal reduction of food intake are long-lasting. In contrast, no changes in myostatin mRNA levels were observed in adult rats when food intake was decreased during 7 days by either food restriction or central leptin treatment. Similar results were obtained when food restriction was maintained in adult rats for a longer period (7 weeks), despite significant muscle loss. Overall, these data suggest that myostatin gene expression is programmed by nutritional status in neonatal life.