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.     

Effects of Physiological and Pathological Urea Concentrations on Human Microvascular Endothelial Cells

Urea is the uremic toxin accumulating with the highest concentration in the plasma of chronic kidney disease (CKD) patients, not being completely cleared by dialysis. Urea accumulation is reported to exert direct and indirect side effects on the gastrointestinal tract, kidneys, adipocytes, and cardiovascular system (CVS), although its pathogenicity is still questioned since studies evaluating its side effects lack homogeneity. Here, we investigated the effects of physiological and pathological urea concentrations on a human endothelial cell line from the microcirculation (Human Microvascular Endothelial Cells-1, HMEC-1). Urea (5 g/L) caused a reduction in the proliferation rate after 72 h of exposure and appeared to be a potential endothelial-to-mesenchymal transition (EndMT) stimulus. Moreover, urea induced actin filament rearrangement, a significant increase in matrix metalloproteinases 2 (MMP-2) expression in the medium, and a significant up- or down-regulation of other EndMT biomarkers (keratin, fibrillin-2, and collagen IV), as highlighted by differential proteomic analysis. Among proteins whose expression was found to be significantly dysregulated following exposure of HMEC-1 to urea, dimethylarginine dimethylaminohydrolase (DDAH) and vasorin turned out to be down-regulated. Both proteins have been directly linked to cardiovascular diseases (CVD) by in vitro and in vivo studies. Future experiments will be needed to deepen their role and investigate the signaling pathways in which they are involved to clarify the possible link between CKD and CVD.     

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.