ILB®, a Low Molecular Weight Dextran Sulphate,Restores Glutamate Homeostasis,Amino Acid Metabolism and Neurocognitive Functions in a Rat Model of Severe Traumatic Brain Injury

In a previous study, we found that administration of ILB^®, a new low molecular weight dextran sulphate, significantly improved mitochondrial functions and energy metabolism, as well as decreased oxidative/nitrosative stress, of brain tissue of rats exposed to severe traumatic brain injury (sTBI), induced by the closed-head weight-drop model of diffused TBI. Using aliquots of deproteinized brain tissue of the same animals of this former study, we here determined the concentrations of 24 amino acids of control rats, untreated sTBI rats (sacrificed at 2 and 7 days post-injury) and sTBI rats receiving a subcutaneous ILB^® administration (at the dose levels of 1, 5 and 15 mg/kg b.w.) 30 min post-impact (sacrificed at 2 and 7 days post-injury). Additionally, in a different set of experiments, new groups of control rats, untreated sTBI rats and ILB^®-treated rats (administered 30 min after sTBI at the dose levels of 1 or 5 mg/kg b.w.) were studied for their neurocognitive functions (anxiety, locomotor capacities, short- and long-term memory) at 7 days after the induction of sTBI. Compared to untreated sTBI animals, ILB^® significantly decreased whole brain glutamate (normalizing the glutamate/glutamine ratio), glycine, serine and γ-aminobutyric acid. Furthermore, ILB^® administration restored arginine metabolism (preventing nitrosative stress), levels of amino acids involved in methylation reactions (methionine, L-cystathionine, S-adenosylhomocysteine), and N-acetylaspartate homeostasis. The macroscopic evidences of the beneficial effects on brain metabolism induced by ILB^® were the relevant improvement in neurocognitive functions of the group of animals treated with ILB^® 5 mg/kg b.w., compared to the marked cognitive decline measured in untreated sTBI animals. These results demonstrate that ILB^® administration 30 min after sTBI prevents glutamate excitotoxicity and normalizes levels of amino acids involved in crucial brain metabolic functions. The ameliorations of amino acid metabolism, mitochondrial functions and energy metabolism in ILB^®-treated rats exposed to sTBI produced significant improvement in neurocognitive functions, reinforcing the concept that ILB^® is a new effective therapeutic tool for the treatment of sTBI, worth being tested in the clinical setting.     

GSK3β Inhibition by Phosphorylation at Ser389 Controls Neuroinflammation

The inhibition of Glycogen Synthase Kinase 3 β (GSK3β) by Ser⁹ phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3β inhibition by alternative Ser³⁸⁹ phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3β Ser³⁸⁹ knock-in (KI) mice, in which the phosphorylation of Ser³⁸⁹ GSK3β is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3β by Ser³⁸⁹ phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3β Ser³⁸⁹ KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3β Ser³⁸⁹ KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3β regulatory pathway. Overall, our findings indicate that GSK3β inactivation by Ser³⁸⁹ phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies.     

Effects of Dietary n-3 LCPUFA Supplementation on the Hippocampus of Aging Female Mice: Impact on Memory,Lipid Raft-Associated Glutamatergic Receptors and Neuroinflammation

Long-chain polyunsaturated fatty acids (LCPUFA), essential molecules whose precursors must be dietary supplied, are highly represented in the brain contributing to numerous neuronal processes. Recent findings have demonstrated that LCPUFA are represented in lipid raft microstructures, where they favor molecular interactions of signaling complexes underlying neuronal functionality. During aging, the brain lipid composition changes affecting the lipid rafts’ integrity and protein signaling, which may induce memory detriment. We investigated the effect of a n-3 LCPUFA-enriched diet on the cognitive function of 6- and 15-months-old female mice. Likewise, we explored the impact of dietary n-3 LCPUFAs on hippocampal lipid rafts, and their potential correlation with aging-induced neuroinflammation. Our results demonstrate that n-3 LCPUFA supplementation improves spatial and recognition memory and restores the expression of glutamate and estrogen receptors in the hippocampal lipid rafts of aged mice to similar profiles than young ones. Additionally, the n-3 LCPUFA-enriched diet stabilized the lipid composition of the old mice’s hippocampal lipid rafts to the levels of young ones and reduced the aged-induced neuroinflammatory markers. Hence, we propose that n-3 LCPUFA supplementation leads to beneficial cognitive performance by “rejuvenating” the lipid raft microenvironment that stabilizes the integrity and interactions of memory protein players embedded in these microdomains.     

Characterization of microorganisms in Argentinean honeys from different sources

Seventy polyfloral honeys including commercial samples obtained from supermarkets, harvested from apiaries and purchased in bulk were initially examined for total antibacterial activity. From each sample, numbers of aerobic mesophilic bacteria, total coliforms, moulds and yeasts were determined and the presence of Salmonella spp., Shigella spp., Clostridium sulfite-reducers, Paenibacillus larvae and Bacillus spp. was investigated. Moisture content, pH and total acidity were also determined for all samples. Any honey diluted to concentrations from 75% to 1% (w/v) of full-strength honey showed total antibacterial activity. The numbers of aerobic mesophilic bacteria, moulds and yeasts were less than 10(3) cfu/g for all 70 samples. Faecal coliforms, Escherichia coli, Salmonella spp., Shigella spp. and Clostridium sulfite-reducers were not detected but P. larvae subspp. larvae, Bacillus cereus, Bacillus pumilus and Bacillus laterosporus were found among samples. For commercial, apiary and bulk honey the mean values for moisture content, pH and acidity, respectively, were 17.50%, 17.40% and 17.50%; 4.60, 4.10 and 4.20; and 18.30, 20.60 and 21 meq NaOH/kg. P. larvae was recovered from 35% of apiaries including hives in which the bees did not display symptoms of American foulbrood disease.     

In-house and interlaboratory validation of a method for the extraction of DNA from maize starch

Here, we describe the procedure of a DNA extraction method from maize starch including the method??s validation by in-house and interlaboratory tests. The amplifiable amount of maize DNA tested by real-time PCR was used as parameter for evaluating our method. The practical (i.e. relative) limit of detection (LOD) was used as key criterion for assessing the suitability of the extraction method with respect to genetically modified organism analysis. In a round-robin test with ten participating laboratories, satisfactory results were achieved with practical LODs in the range of 0.1?% with three native maize starch materials. In-house tests showed that this protocol??with an additional purification step??can also be applied for extracting DNA from chemically or enzymatically modified starch.     

The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.     

Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria

Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment – typically at a single site or at a few sites per protein – of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l -phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production – using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain – of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.     

Exploring critical attributes during air traffic congestion with a fuzzy DEMATEL–ANP technique: a case study in Ninoy Aquino International Airport

Railway Engineering Science - The constant growth of air travel in the Philippines has brought about significant consequences to air traffic congestion. Given limited resources, major airports seek...