Glutamatergic and purinergic transmitters and astrocyte modulation in the synaptic transmission in the NTS of rats exposed to short-term sustained hypoxia.

There is evidence that astrocytes modulate synaptic transmission in the nucleus tractus solitarius (NTS) interacting with glutamatergic and purinergic mechanisms. Here, using in situ working heart-brainstem preparations, we evaluated the involvement of astrocyte and glutamatergic/purinergic neurotransmission in the processing of autonomic and respiratory pathways in the NTS of control and rats exposed to sustained hypoxia (SH). Baseline autonomic and respiratory activities and the responses to chemoreflex activation (KCN) were evaluated before and after microinjections of fluorocitrate (FCt, an astrocyte metabolic inhibitor), kynurenic acid, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS) (nonselective antagonists of glutamatergic and purinergic receptors) into the rostral aspect of the caudal commissural NTS. FCt had no effects on the baseline parameters evaluated but reduced the bradycardic response to chemoreflex activation in SH rats. FCt combined with kynurenic acid and PPADS in control rats reduced the baseline duration of expiration, which was attenuated after SH. FCt produced a large increase in PN frequency discharge in control rats, which was reduced after SH, indicating a reduction in the astrocyte modulation after SH. The data show that 1) the bradycardic component of the peripheral chemoreflex is reduced in SH rats after astrocytes inhibition, 2) the inhibition of astrocytes in the presence of double antagonists in the NTS affects the modulation of baseline duration of expiration in control but not in SH rats, and 3) the autonomic and respiratory responses to chemoreflex activation are mediated by glutamatergic and purinergic receptors in the rostral aspect of the caudal commissural NTS.NEW & NOTEWORTHY Our findings indicate that the neurotransmission of autonomic and respiratory components of the peripheral chemoreflex in the nucleus tractus solitarius (NTS) is mediated by glutamatergic and purinergic mechanisms and reveal a selective involvement of NTS astrocytes in controlling the chemoreflex parasympathetic response in rats exposed to sustained hypoxia (SH) and the baseline duration of expiration mainly in control rats, indicating a selective role for astrocytes modulation in the NTS of control and SH rats.

Role of amygdala astrocytes in different phases of contextual fear memory.

Growing evidence indicates a critical role of astrocytes in learning and memory. However, little is known about the role of basolateral amygdala complex (BLA-C) astrocytes in contextual fear conditioning (CFC), a paradigm relevant to understand and generate treatments for fear- and anxiety-related disorders. To get insights on the involvement of BLA-C astrocytes in fear memory, fluorocitrate (FLC), a reversible astroglial metabolic inhibitor, was applied at critical moments of the memory processing in order to target the acquisition, consolidation, retrieval and reconsolidation process of the fear memory. Adult Wistar male rats were bilaterally cannulated in BLA-C. Ten days later they were infused with different doses of FLC (0.5 or 1 nmol/0.5 µl) or saline before or after CFC and before or after retrieval. FLC impaired fear memory expression when administered before and shortly after CFC, but not one hour later. Infusion of FLC prior and after retrieval did not affect the memory. Our findings suggest that BLA-C astrocytes are critically involved in the acquisition/early consolidation of fear memory but not in the retrieval and reconsolidation. Furthermore, the extinction process was presumably not affected (considering that peri-retrieval administration could also affect this process).

Targets and Effects of Common Biocompounds of Hibiscus sabdariffa (Delphinidin-3-Sambubiosid, Quercetin, and Hibiscus Acid) in Different Pathways of Human Cells According to a Bioinformatic Assay.

The utilization of food as a therapeutic measure for various ailments has been a prevalent practice throughout history and across different cultures. This is exemplified in societies where substances like Hibiscus sabdariffa have been employed to manage health conditions like hypertension and elevated blood glucose levels. The inherent bioactive compounds found in this plant, namely, delphinidin-3-sambubioside (DS3), quercetin (QRC), and hibiscus acid (HA), have been linked to various health benefits. Despite receiving individual attention, the specific molecular targets for these compounds remain unclear. In this study, computational analysis was conducted using bioinformatics tools such as Swiss Target Prediction, ShinnyGo 0.77, KEGG, and Stringdb to identify the molecular targets, pathways, and hub genes. Supplementary results were obtained through a thorough literature search in PubMed. DS3 analysis revealed potential genetic alterations related to the metabolism of nitrogen and glucose, inflammation, angiogenesis, and cell proliferation, particularly impacting the PI3K-AKT signaling pathway. QRC analysis demonstrated interconnected targets spanning multiple pathways, with some overlap with DS3 analysis and a particular focus on pathways related to cancer. HA analysis revealed distinct targets, especially those associated with pathways related to the nervous system. These findings emphasize the necessity for focused research on the molecular effects of DS3, QRC, and HA, thereby providing valuable insights into potential therapeutic pathways.

Anticoagulation in renal replacement therapies: Why heparin should be abandoned in critical ill patients?

Extracorporeal circuits used in renal replacement therapy (RRT) can develop thrombosis, leading to downtimes and reduced therapy efficiency. To prevent this, anticoagulation is used, but the optimal anticoagulant has not yet been identified. Heparin is the most widely used anticoagulant in RRT, but it has limitations, such as unpredictable pharmacokinetics, nonspecific binding to plasma proteins and cells, and the possibility of suboptimal anticoagulation or bleeding complications, specifically in critically ill patients with acute renal failure who are already at high risk of bleeding. Citrate anticoagulation is a better alternative, being considered a standard for continuous renal replacement therapy, since it is associated with a lower risk of bleeding complications and better efficacy, even in patients with acute renal failure or liver disease. The aim of this article is to provide an updated review of the different strategies of anticoagulation in renal replacement therapies that can be implemented in critical scenarios, focusing on the advantages and disadvantages of each one and the beneficial aspects of using citrate over heparin in critical ill patients.

Quality indicators in prolonged hemodialysis with regional citrate anticoagulation with the genius system: retrospective cohort of critical patients with acute kidney injury.

BACKGROUND: Prolonged hemodialysis (HD) is performed from 6 to 12 h and can last up to 24 h. To prevent system clotting some studies suggest that Regional Citrate Anticoagulation (RCA) use reduces bleeding rates relative to systemic heparin. However, there may be difficulties in the patient's clinical management and completing the prescribed HD with Genius system using RCA. OBJECTIVE: To analyze safety Quality Indicators (IQs) and follow up on prolonged HD with 4% sodium citrate solution in a Genius® hybrid system. METHODS: This is a retrospective cohort conducted in an intensive care unit. RESULTS: 53 random sessions of prolonged HD with 4% sodium citrate solution of critically ill patients with AKI assessed. Evaluated safety indicators were dysnatremia and metabolic alkalosis, observed in 15% and 9.4% of the sessions, respectively. Indicators of effectiveness were system clotting which occurred in 17.3%, and the minimum completion of the prescribed HD time, which was 75.5%. CONCLUSION: The assessment of the indicators showed that the use of RCA with a 4% sodium citrate solution in prolonged HD with the Genius system in critically ill patients with AKI can be performed in a simple, safe, and effective way.

Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics.

Laccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H2O instead of H2O2. The measurement of laccase activity (UL-1) results may vary depending on the type of laccase, buffer, redox mediators, and substrates employed. The aim was to select the best conditions for rGILCC 1 and rPOXA 1B laccases activity assay. After sequential statistical assays, the molecular dynamics proved to support this process, and we aimed to accumulate valuable insights into the potential application of these enzymes for the degradation of novel substrates with negative environmental implications. Citrate buffer treatment T2 (CB T2) (pH 3.0 ± 0.2; λ420nm, 2 mM ABTS) had the most favorable results, with 7.315 ± 0.131 UL-1 for rGILCC 1 and 5291.665 ± 45.83 UL-1 for rPOXA 1B. The use of citrate buffer increased the enzyme affinity for ABTS since lower Km values occurred for both enzymes (1.49 × 10-2 mM for rGILCC 1 and 3.72 × 10-2 mM for rPOXA 1B) compared to those obtained in acetate buffer (5.36 × 10-2 mM for rGILCC 1 and 1.72 mM for rPOXA 1B). The molecular dynamics of GILCC 1-ABTS and POXA 1B-ABTS showed stable behavior, with root mean square deviation (RMSD) values not exceeding 2.0 Å. Enzyme activities (rGILCC 1 and rPOXA 1B) and 3D model-ABTS interactions (GILCC 1-ABTS and POXA 1B-ABTS) were under the strong influence of pH, wavelength, ions, and ABTS concentration, supported by computational studies identifying the stabilizing residues and interactions. Integration of the experimental and computational approaches yielded a comprehensive understanding of enzyme-substrate interactions, offering potential applications in environmental substrate treatments.

Hypercitratemia is a mortality predictor among patients on continuous venovenous hemodiafiltration and regional citrate anticoagulation.

The use of regional citrate anticoagulation (RCA) in liver failure (LF) patients can lead to citrate accumulation. We aimed to evaluate serum levels of citrate and correlate them with liver function markers and with the Cat/Cai in patients under intensive care and undergoing continuous venovenous hemodiafiltration with regional citrate anticoagulation (CVVHDF-RCA). A prospective cohort study in an intensive care unit was conducted. We compared survival, clinical, laboratorial and dialysis data between patients with and without LF. Citrate was measured daily. We evaluated 200 patients, 62 (31%) with LF. Citrate was significantly higher in the LF group. Dialysis dose, filter lifespan, systemic ionized calcium and Cat/Cai were similar between groups. There were weak to moderate positive correlations between Citrate and indicators of liver function and Cat/Cai. The LF group had higher mortality (70.5% vs. 51.8%, p = 0.014). Citrate was an independent risk factor for death, OR 11.3 (95% CI 2.74-46.8). In conclusion, hypercitratemia was an independent risk factor for death in individuals undergoing CVVHDF-ARC. The increase in citrate was limited in the LF group, without clinical significance. The correlation between citrate and liver function indicators was weak to moderate.

Unveiling the dark side of guard cell metabolism.

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C-HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

Efficient capture of recombinant SARS-CoV-2 receptor-binding domain (RBD) with citrate-coated magnetic iron oxide nanoparticles.

Several vaccines against COVID-19 use a recombinant SARS-CoV-2 receptor-binding domain (RBD) as antigen, making the purification of this protein a key step in their production. In this work, citrate-coated magnetic iron oxide nanoparticles were evaluated as nano adsorbents in the first step (capture) of the purification of recombinant RBD. The nanoparticles were isolated through coprecipitation and subsequently coated with sodium citrate. The citrate-coated nanoparticles exhibited a diameter of 10 ± 2 nm, a hydrodynamic diameter of 160 ± 3 nm, and contained 1.9 wt% of citrate. The presence of citrate on the nanoparticles' surface was confirmed through FT-IR spectra and thermogravimetric analysis. The crystallite size (10.1 nm) and the lattice parameter (8.3646 Å) were determined by X-ray diffraction. In parallel, RBD-containing supernatant extracted from cell culture was exchanged through ultrafiltration and diafiltration into the adsorption buffer. The magnetic capture was then optimized using different concentrations of nanoparticles in the purified supernatant, and we found 40 mg mL-1 to be optimal. The ideal amount of nanoparticles was assessed by varying the RBD concentration in the supernatant (between 0.113 mg mL-1 and 0.98 mg mL-1), which resulted in good capture yields (between 83 ± 5% and 94 ± 4%). The improvement of RBD purity after desorption was demonstrated by SDS-PAGE and RP-HPLC. Furthermore, the magnetic capture was scaled up 100 times, and the desorption was subjected to chromatographic purifications. The obtained products recognized anti-RBD antibodies and bound the ACE2 receptor, proving their functionality after the developed procedure.

Citrate-coated cobalt ferrite nanoparticles for the nano-enabled biofortification of wheat.

A pot experiment was conducted in an open greenhouse to explore the use of citrate-coated cobalt ferrite nanoparticles (CoFe2O4 NPs) as a source for Fe fortification of three wheat lines (Triticum aestivum L.). Two of the three wheat lines tested differ in their efficiency concerning Zn storage in their grains (efficient and inefficient), and one had inefficient P-absorption. The NPs were supplied by foliar or soil application of Fe at 330 mg L-1, and 46 or 68 mg kg-1 soil, respectively. A positive control (Fe-EDTA salt, a conventional iron fertilizer) and a negative control (no fertilization) were also included to compare the efficiency of NP fertilization. Soil fertilization with NPs improved the grain yield and Fe concentration in the grains compared with the foliar application of NPs and conventional Fe fertilizer. Application of soil NPs at 68 mg kg-1 increased the grain yield by 52% and 21% compared with the control and soil Fe-EDTA treatments, respectively. Likewise, grain Fe concentration increased by 96% and 72% compared with the control and soil Fe-EDTA treatments, respectively. The phytic acid concentration in grains and the phytic acid:Fe ratio decreased by 6% and 62%, respectively, due to the soil application of NPs (68 mg Fe per kg). The Fe grain concentration of lines inefficient for Zn storage and P-uptake in plants from soil fertilized with NPs (68 mg Fe per kg) was 1.37 and 0.26 fold above the target biofortification concentration (60 mg Fe per kg). Cobalt concentration in grains ranged from 9 to 16 mg kg-1. These concentrations were below the maximum allowable limit of Co in grains (50 mg kg-1) recommended by FAO and the WHO. Our results showed that Fe supplied as NPs may improve the nutritional quality of wheat grains, and the economic yield. However, there remains a long way to go to achieve effective and economic use of nanotechnology for the nutritional development of wheat.

Artificial feeding of Ornithodoros rostratus using a silicone membrane system.

The in vitro feeding of ticks facilitates the conduction of studies involving the intrinsic vector-pathogen relationship, susceptibility tests, and resistance to acaricides, in addition to mimicking the use of experimental hosts. The objective of this study was to establish an in vitro feeding system using silicone membranes to supply various diets to the species Ornithodoros rostratus. Each experimental group included 130 first-instar O. rostratus nymphs. The groups were divided according to the diet provided: citrated rabbit blood, citrated bovine blood, bovine blood with antibiotics, and defibrinated bovine blood. The control group was fed directly on rabbits. Ticks were weighed before and after the feeding and monitored individually according to their biological parameters. The results of the experiment demonstrated that the proposed system was efficient in terms of fixation stimulus and satisfactory in terms of tick engorgement, which would allow the maintenance of O. rostratus colonies by using artificial feeding through silicone membranes. All diets provided were efficient for the maintenance of colonies, but the ticks that received citrated rabbit blood displayed similar biological parameters to those observed under in vivo feeding conditions.

Two phosphoenolpyruvate carboxykinases with differing biochemical properties in Chlamydomonas reinhardtii.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) and CO2 playing mainly a gluconeogenic role in green algae. We found two PEPCK isoforms in Chlamydomonas reinhardtii and we cloned, purified and characterised both enzymes. ChlrePEPCK1 is more active as decarboxylase than ChlrePEPCK2. ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylalanine and malate, while ChlrePEPCK2 is monomeric and it is regulated by citrate, phenylalanine and glutamine. We postulate that the two PEPCK isoforms found originate from alternative splicing of the gene or regulated proteolysis of the enzyme. The presence of these two isoforms would be part of a mechanism to finely regulate the biological activity of PEPCKs.

Enriched functional milk fat ameliorates glucose intolerance and triacylglycerol accumulation in skeletal muscle of rats fed high-fat diets.

PURPOSE: We examined the effect of a functional milk fat (FMF) on the glucose metabolism and its association with the intramuscular triacylglycerol (TAG) content in rats fed high-fat diets. METHODS: Male Wistar rats were fed for 60 days with S7 (soybean oil 7%), S30 (soybean oil 30%), MF30 (soybean oil 3% + milk fat 27%), or FMF30 (soybean oil 3% + FMF 27%) diets. An oral glucose tolerance test was performed. The levels of key metabolites in gastrocnemius muscle and mRNA levels of genes involved in glucose and lipid metabolism in muscle, epididymal white adipose tissue (EWAT), and serum were assessed. RESULTS: The S30 diet induced glucose intolerance and led to TAG, citrate, and glucose accumulation in muscle. Moreover, we observed a downregulation of uncoupling proteins (Ucp2 and Ucp3) and insulin receptor substrate-1 (Irs1) genes, lower carnitine palmitoyl transferase-1b (CPT-1b), and phosphofructokinase-1 (PFK1) activities in muscle and lower expression of adiponectin (Adipoq) in EWAT. The FMF30 diet ameliorated the glucose intolerance and normalized the glucose and TAG levels in muscle, preventing the accumulation of citrate and enhancing glucose utilization by the PFK1. The beneficial effects might also be related to the higher expression of Adipoq in EWAT, its receptor in muscle (Adipor1), and the expression of Ucp2, Ucp3, and Irs1 in muscle, restoring the alterations observed with the S30 diet. CONCLUSIONS: FMF30 modulated key genes involved in glucose and lipid metabolism in skeletal muscle, improving the glucose utilization and preventing TAG, glucose, and citrate accumulation.

Development of a non-titration potentiometric method for the determination of acidity in vinegar.

The acidity of a solution is associated with the concentration of Brønsted acids. This work proposes a new non-titrimetric potentiometric method using citrate buffer for the determination of vinegar acidity. The difference between the pH values before and after the addition of a diluted vinegar sample to 10 mmol L-1 citrate buffer (pH 5.5) was related to the acetic acid concentration. The dynamic range of the quadratic analytical curve was from 3.5 to 20 mmol L-1 (R2 = 0.998). The repeatability was 0.8% for acetic acid at 0.01 mol L-1. Comparison with the conventional titration method showed an error between 0.7% and 4.64% (n = 9) for analysis of commercial vinegar samples The behaviour of the system could be explained using the buffering function.

In vitro and in vivo acute toxicity of a novel citrate-coated magnetite nanoparticle.

Magnetic nanoparticles (MNps) have become powerful tools for multiple biomedical applications such as hyperthermia drivers, magnetic resonance imaging (MRI) vectors, as well as drug-delivery systems. However, their toxic effects on human health have not yet been fully elucidated, especially in view of their great diversity of surface modifications and functionalizations. Citrate-coating of MNps often results in increased hydrophilicity, which may positively impact their performance as drug-delivery systems. Nonetheless, the consequences on the intrinsic toxicity of such MNps are unpredictable. Herein, novel magnetite (Fe3O4) nanoparticles covered with citrate were synthesized and their potential intrinsic acute toxic effects were investigated using in vitro and in vivo models. The proposed synthetic pathway turned out to be simple, quick, inexpensive, and reproducible. Concerning toxicity risk assessment, these citrate-coated iron oxide nanoparticles (IONps) did not affect the in vitro viability of different cell lines (HaCaT and HepG2). Moreover, the in vivo acute dose assay (OECD test guideline #425) showed no alterations in clinical parameters, relevant biochemical variables, or morphological aspects of vital organs (such as brain, liver, lung and kidney). Iron concentrations were slightly increased in the liver, as shown by Graphite Furnace Atomic Absorption Spectrometry and Perls Prussian Blue Staining assays, but this finding was considered non-adverse, given the absence of accompanying functional/clinical repercussions. In conclusion, this study reports on the development of a simple, fast and reproducible method to obtain citrate-coated IONps with promising safety features, which may be used as a drug nanodelivery system in the short run. (263 words).

Functional genomics analysis of a phyllospheric Pseudomonas spp with potential for biological control against coffee rust.

BACKGROUND: Pseudomonas spp. promotes plant growth and colonizes a wide range of environments. During the annotation of a Coffea arabica ESTs database, we detected a considerable number of contaminant Pseudomonas sequences, specially associated with leaves. The genome of a Pseudomonas isolated from coffee leaves was sequenced to investigate in silico information that could offer insights about bacterial adaptation to coffee phyllosphere. In parallel, several experiments were performed to confirm certain physiological characteristics that could be associated with phyllospheric behavior. Finally, in vivo and in vitro experiments were carried out to verify whether this isolate could serve as a biocontrol agent against coffee rust and how the isolate could act against the infection.  RESULTS: The isolate showed several genes that are associated with resistance to environmental stresses, such as genes encoding heat/cold shock proteins, antioxidant enzymes, carbon starvation proteins, proteins that control osmotic balance and biofilm formation. There was an increase of exopolysaccharides synthesis in response to osmotic stress, which may protect cells from dessication on phyllosphere. Metabolic pathways for degradation and incorporation into citrate cycle of phenolic compounds present in coffee were found, and experimentally confirmed. In addition, MN1F was found to be highly tolerant to caffeine. The experiments of biocontrol against coffee leaf rust showed that the isolate can control the progress of the disease, most likely through competition for resources. CONCLUSION: Genomic analysis and experimental data suggest that there are adaptations of this Pseudomonas to live in association with coffee leaves and to act as a biocontrol agent.

Nonpregnant and pregnant adult female rats affected by maternal diabetes environment.

Maternal diabetes-mediated fetal programming is widely discussed, however, it is important to define the extent to which intrauterine hyperglycemia interferes with the health of female pups, along with determining whether these changes can be perpetuated across generations. This study aimed to evaluate the effects of maternal diabetes on fetal programming and the repercussions on the metabolism of pregnant and nonpregnant female pups. Diabetes status was induced (diabetic group-D) using streptozotocin (a beta cell cytotoxic drug) on the fifth postnatal day of female rats, while controls received a citrate buffer (Control-C). In adulthood, the rats were mated to obtain their female pups. At 90 days of age, half of the female pups were mated (preg) and the other half continued virgin (Npreg). Furthermore, they were distributed into four groups: OC/Npreg and OC/preg-female pups from control mothers; OD/Npreg and OD/preg-female pups from diabetic mothers. At 115 days of life and/or 17 days of pregnancy, the oral glucose tolerance test (OGTT) was performed with blood collection for insulin measurement. At 120 days of life and/or 21 days of pregnancy, the rats were anesthetized and euthanized to determine their blood oxidative stress status. The OD/Npreg group showed glucose intolerance during OGTT (p < 0.0001), while the OD/preg group showed increased insulin secretion during OGTT (p < 0.0001) and insulin resistance (IR; p = 0.0027). An increase in homeostatic model assessment ß was shown in the pregnant groups, regardless of maternal diabetes (p < 0.0001). The OD/preg group presented increased thiobarbituric acid reactive substances (p < 0.0001) and -SH levels (p = 0.0005) and decreased superoxide dismutase activity (p = 0.0063). Additionally, small fetuses for gestational age (p < 0.0001) were found in these rats. In conclusion, exposure to maternal hyperglycemia compromises the glycemic metabolism of female pups before and during pregnancy and causes oxidative stress, IR, and impaired fetal growth during pregnancy.