Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue.

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.

Solid-Phase-Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides.

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80%. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

The utility of fetal heart rate deceleration's descending slope in searching for a non-National Institute of Child Health and Human Development parameter for the detection of fetal acidosis.

OBJECTIVE: To identify new parameters predicting fetal acidemia. METHODS: A retrospective case-control study in a cohort of deliveries from a tertiary referral hospital-based cohort deliveries in Zaragoza, Spain between 2018 and 2021 was performed. To predict fetal acidemia, the NICHD categorizations and non-NICHD parameters were analyzed in the electronic fetal monitoring (EFM). Those included total reperfusion time, total deceleration area and the slope of the descending limb of the fetal heart rate of the last deceleration curve. The accuracy of the parameters was evaluated using the specificity for (80%, 85%, 90%, 95%) sensitivity and the area under the receiver operating characteristic curve (AUC). RESULTS: A total of 10 362 deliveries were reviewed, with 224 cases and 278 controls included in the study. The NICHD categorizations showed reasonable discriminatory ability (AUC = 0.727). The non-NICHD parameters measured during the 30-min fetal monitoring, total deceleration area (AUC = 0.807, 95% CI: 0.770, 0.845) and total reperfusion time (AUC = 0.750, 95% CI: 0.707, 0.792), exhibited higher discriminatory ability. The slope of the descending limb of the fetal heart rate of the last deceleration curve had the best AUC value (0.853, 95% CI: 0.816, 0.889). The combination of total deceleration area or total reperfusion time with the slope demonstrated high discriminatory ability (AUC = 0.908, 95% CI: 0.882, 0.933; specificities of 71.6% and 72.7% for a sensitivity of 90%). CONCLUSIONS: The slope of the descending limb of the fetal heart rate of the last deceleration curve is the strongest predictor of fetal acidosis, but its combination with the total reperfusion time shows better clinical utility.

End-To-End Procedure For IORT in Brain Metastases and Film Dosimetry.

Purpose: The study is intended to perform an end-to-end test of the entire intraoperative process using cadaver heads. A simulation of tumor removal was performed, followed by irradiation of the bed and measurement of absorbed doses with radiochromic films. Materials and Methods: Low-energy X-ray intraoperative radiotherapy (IORT) was used for irradiation. A computed tomography study was performed at each site and the absorbed doses calculated by the treatment planning system, as well as absorbed doses with radiochromic films, were studied. Results: The absorbed doses in the organs at risk (OAR) were evaluated in each case, obtaining maximum doses within the tolerance limits. The absorbed doses in the target were verified and the deviations were <1%. Conclusions: These tests demonstrated that this comprehensive procedure is a reproducible quality assurance tool which allows continuous assessment of the dosimetric and geometric accuracy of clinical brain IORT treatments. Furthermore, the absorbed doses measured in both target and OAR are optimal for these treatments.

Anatomical study about the variations in renal vasculature.

Renal vascularization is classically described as a renal artery and vein. However, this vascular pattern presents numerous anatomical variations in terms of their number, origin and course due to ontogenetic alterations. The aim was to carry out a descriptive study of the renal vascular pattern observed during the dissection of cadavers intended for teaching purposes. A descriptive and observational study of renal vascular anatomy was carried out by dissecting 16 renal blocks from 8 cadavers donated to science and used for teaching at the Faculty of Medicine of the University of Zaragoza. The prevalence of arterial variations was 75% (56.3% for polar renal arteries, 12.5% for pre-hilar branching and 6.25% for double communicating arterial arch) and venous was 62.5% (12.5% for polar renal veins, 25% for late venous confluence, 6.25% for triple renal vein and 18.75% for double circumaortic renal vein). We conclude that the renal vascular anomalies occur with high frequency; for this reason, knowledge of these anomalies is extremely important for the correct planning of numerous medical-surgical activities.

Failure of early non-invasive ventilation in preterm infants with respiratory distress syndrome in current care practice in Spanish level-III neonatal intensive care units - a prospective observational study.

Introduction: Despite advances in respiratory distress syndrome (RDS) management over the past decade, non-invasive ventilation (NIV) failure is frequent and associated with adverse outcomes. There are insufficient data on the failure of different NIV strategies currently used in clinical practice in preterm infants. Methods: This was a prospective, multicenter, observational study of very preterm infants [gestational age (GA) <32 weeks] admitted to the neonatal intensive care unit for RDS that required NIV from the first 30 min after birth. The primary outcome was the incidence of NIV failure, defined as the need for mechanical ventilation for <72 h of life. Secondary outcomes were risk factors associated with NIV failure and complication rates. Results: The study included 173 preterm infants with a median GA of 28 (IQR 27-30) weeks and a median birth weight of 1,100 (IQR 800-1,333) g. The incidence of NIV failure was 15.6%. In the multivariate analysis, lower GA (OR, 0.728; 95% CI, 0.576-0.920) independently increased the risk of NIV failure. Compared to NIV success, NIV failure was associated with higher rates of unfavorable outcomes, including pneumothorax, intraventricular hemorrhage, periventricular leukomalacia, pulmonary hemorrhage, and a combined outcome of moderate-to-severe bronchopulmonary dysplasia or death. Conclusion: NIV failure occurred in 15.6% of the preterm neonates and was associated with adverse outcomes. The use of LISA and newer NIV modalities most likely accounts for the reduced failure rate. Gestational age remains the best predictor of NIV failure and is more reliable than the fraction of inspired oxygen during the first hour of life.

Nitrogen management in farming systems under the use of agricultural wastes and circular economy.

Population growth leads to an increase in the demand for energy, water, and food as cities grow and urbanize. However, the Earth's limited resources are unable to meet these rising demands. Modern farming practices increase productivity, but waste resources and consume too much energy. Agricultural activities occupy 50 % of all habitable land. After a rise of 80 % in 2021, fertilizer prices have increased by nearly 30 % in 2022, representing a significant cost for farmers. Sustainable and organic farming has the potential to reduce the use of inorganic fertilizers and increase the utilization of organic residues as a nitrogen (N) source for plant nutrition. Agricultural management typically prioritizes nutrient cycling and supply for crop growth, whereas the mineralization of added biomass regulates crop nutrient supply and CO2 emissions. To reduce overconsumption of natural resources and environmental damage, the current economic model of "take-make-use-dispose" must be replaced by "prevention-reuse-remake-recycle". The circular economy model is promising for preserving natural resources and providing sustainable, restorative, and regenerative farming. Technosols and organic wastes can improve food security, ecosystem services, the availability of arable land, and human health. This study intends to investigate the nitrogen nutrition provided by organic wastes to agricultural systems, reviewing the current state of knowledge and demonstrating how common organic wastes can be utilized to promote sustainable farming management. Nine waste residues were selected to promote sustainability in farming based on circular economy and zero waste criteria. Using standard methods, their water content, organic matter, total organic carbon, Kjeldahl nitrogen, and ammonium levels were determined, along with their potential to improve soil fertility via N supply and technosol formulation. 10 % to 15 % of organic waste was mineralized and analysed during a six-month cultivation cycle. Through the results, the combination of organic and inorganic fertilization to increase crop yield is recommended, as is the search for realistic and practical methods of dealing with massive amounts of organic residues within the context of a circular economy.

Chemometric and Physico-Chemical Characterization of Fruit and Olive Oils from Autochthonous Cultivars Grown in Aragon (Spain).

Olive tree crops and the olive oil market are becoming less and less diverse due to the rise of intensive growth varieties, with the consequent loss of varietal richness provided by oils from minority and autochthonous cultivars. "Royal de Calatayud" and "Negral de Sabiñán" are two local minority cultivars in Aragon (Spain). Fruit parameters such as ripening, fresh weight, and oil yield were evaluated, as well as physico-chemical and chemical composition parameters in olive oil in comparison with "Arbequina", a cultivar widespread in Spain and other countries. Fruits were harvested from October to December in 2017 and 2019. Chemometric analysis revealed significant varietal differences among the three cultivars. In comparison with "Arbequina", higher oil yields were obtained in the two local cultivars. "Royal de Calatayud" has a higher oleic acid content and a greater quantity of phenolic compounds. It thus displays a better nutritional profile than "Arbequina". This preliminary study shows that "Royal de Calatayud" could be regarded as an excellent alternative to the "Arbequina" cultivar in the parameters analyzed.

Synthetic Zwitterionic Streptococcus pneumoniae Type 1 Oligosaccharides Carrying Labile O-Acetyl Esters.

We herein report the first total synthesis of the Streptococcus pneumoniae serotype 1 (Sp1) oligosaccharide, a unique zwitterionic capsular polysaccharide carrying labile O-acetyl esters. The target oligosaccharides, featuring rare α-2,4-diamino-2,4,6-trideoxy galactose (AAT) and α-galacturonic acids, were assembled up to the 9-mer level, in a highly stereoselective manner using trisaccharide building blocks. The lability of the O-acetyl esters imposed a careful deprotection scheme to prevent migration and hydrolysis. The migration was investigated in detail at various pD values using NMR spectroscopy, to show that migration and hydrolysis of the C-3-O-acetyl esters readily takes place under neutral conditions. Structural investigation showed the oligomers to adopt a right-handed helical structure with the acetyl esters exposed on the periphery of the helix in close proximity of the neighboring AAT residues, thereby imposing conformational restrictions on the AATα1-4GalA(3OAc) glycosidic linkages, supporting the helical shape of the polysaccharide, that has been proposed to be critical for its unique biological activity.

Pulsed electric field increases the extraction yield of extra virgin olive oil without loss of its biological properties.

Introduction: Pulsed electric field (PEF) has been used for improving extraction of extra virgin olive oil (EVOO). However, the biological changes induced by the consumption of pulsed electric field-obtained extra virgin olive oil (PEFEVOO) have not been studied yet. Materials and methods: EVOO oils from Empeltre variety were prepared by standard (STD) cold pressure method involving crushing of the olives, malaxation and decanting and by this procedure including an additional step of PEF treatment. Chemical analyses of EVOO oils were done. Male and female Apoe-deficient mice received diets differing in both EVOOs for 12 weeks, and their plasma, aortas and livers were analyzed. Results: PEF application resulted in a 17% increase in the oil yield and minimal changes in chemical composition regarding phytosterols, phenolic compounds and microRNA. Only in females mice consuming PEF EVOO, a decreased plasma total cholesterol was observed, without significant changes in atherosclerosis and liver steatosis. Conclusion: PEF technology applied to EVOO extraction maintains the EVOO quality and improves the oil yield. The equivalent biological effects in atherosclerosis and fatty liver disease of PEF-obtained EVOO further support its safe use as a food.

Parent-infant closeness after preterm birth and depressive symptoms: A longitudinal study.

Background: Preterm birth increases the risk for postpartum depression in both mothers and fathers, calling for strategies to alleviate and prevent depressive symptoms in parents of preterm infants. The aim of this study was to assess the association between early parent-infant closeness and later depressive symptoms among parents of preterm infants. We hypothesized that longer duration of closeness associate with fewer depressive symptoms in both parents. Methods: This prospective cohort study included 23 neonatal intensive care units (NICUs) from 15 countries in 2018 to 2020. Each unit recruited families with preterm infants aiming to 30 families. The total duration of parents' presence in the NICU, and separately parent-infant skin-to-skin contact and holding, were measured using a Closeness Diary up to 14  days. The Edinburgh Postnatal Depression Scale (EPDS) was used at discharge and at 4  months corrected age of the infant. Results: The study included 684 mothers and 574 fathers. The median presence was 469  min (Q1 258 and Q3 1,087) per 24   h for the mothers and 259   min (Q1 100 and Q3 540) for the fathers; mean EPDS scores were 9.2 (SD 5.0) and 6.3 (SD 4.4) at discharge and 6.6 (4.7) and 4.3 (4.2) at 4  months, respectively. Parents' presence and depressive symptoms varied greatly between the units. Parents' presence as the total measure, or skin-to-skin contact and holding separately, did not associate with depressive symptoms in either mothers or fathers at either time point (adjusted). Conclusion: No association was found between the duration of parent-infant closeness in the neonatal unit and parents' depressive symptoms. The beneficial effects of family-centered care on parents' depression seem to be mediated by other elements than parent-infant physical closeness. More research is needed to identify the critical elements which are needed to alleviate parents' depression after NICU stay.

Authenticity in Olive Oils from an Empeltre Clonal Selection in Aragon (Spain): How Environmental, Agronomic, and Genetic Factors Affect Sterol Composition.

Sterol composition is used as a "fingerprint" to demonstrate the authenticity of olive oils. Our study's objective was to exhaustively characterize the sterol composition of Empeltre olive oils from clonal selection during the ripening period in 2017, 2018, and 2019. We likewise assessed the influence of crop year, fruit ripening, and clonal selection on the oils' regulatory compliance in terms of sterol composition. Empeltre olive oils were shown to have medium-range ß-sitosterol and Δ5-avenasterol content, along with elevated amounts of campesterol and Δ7-stigmastenol. A total of 26% and 12% of the samples were non-compliant in terms of apparent ß-sitosterol and Δ7-stigmastenol, respectively. Crop year was the most influential factor in the case of most sterols. Clone type was the least influential factor, except in the case of campesterol. Olive maturity was only significant for Δ7-sterols. We likewise applied a discriminant analysis, with "crop year" as the grouping variable: 94.9% of the oils were thereby classified correctly.

Rheological Properties and Stability of Thickeners for Clinical Use.

The adaptation of liquids for patients with dysphagia requires precision and individualization in the viscosities used. We describe the variations of viscosity in water at different concentrations and evolution over time of the three compositions of commercial thickeners that are on the market (starch, starch with gums, and gum). By increasing the concentration in water, the viscosity of gum-based thickeners increases linearly, but it did not reach pudding texture, whereas the viscosity of the starch-based thickeners (alone or mixed with gums) rapidly reaches very thick textures. We modeled the viscosity at different concentrations of the four thickeners using regression analysis (R2 > 0.9). We analyzed viscosity changes after 6 h of preparation. The viscosity of gum-based thickeners increased by a maximum of 6.5% after 6 h of preparation, while starch-based thickeners increased by up to 43%. These findings are important for correct handling and prescription. Gum-based thickeners have a predictable linear behavior with the formula we present, reaching nectar and honey-like textures with less quantity of thickener, and are stable over time. In contrast, starch thickeners have an exponential behavior which is difficult to handle, they reach pudding-like viscosity, and are not stable over time.

pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

Institutionalized elderly are able to detect small viscosity variations in thickened water with gum-based thickeners: should texture classifications be reviewed?

BACKGROUND: The prevalence of dysphagia is very high in institutionalized elderly. Knowledge of the rheological and sensory characteristics of the various thickeners in elderly is limited, although it has been seen that there are differences between the rheological behaviors of gum-based thickeners with different composition. Moreover, we have not found sensory studies of viscosity in institutionalized elderly. Our hypothesis was that viscosity ranges established by the scientific societies, such as the National Dysphagia Diet Task Force (NDD), seem to be very wide and individuals might be able to detect small differences within the same texture range. The objectives of our study were 1) comparing the rheological characteristics of two commercial gum-based thickeners with different composition, dissolved in water under standard conditions, and 2) perform a sensory analysis (with both adults and institutionalized elderly) to detect different viscosities within the same texture (nectar and honey). METHODS: Two commercial thickeners based on gums (NC and RC) were studied analyzing their viscosity in water with different concentrations (shear rate: 50 s- 1; temperature: 22-25 °C). A sensory analysis involving 26 elderly and 29 adult controls was carried out to evaluate whether differences within nectar and honey textures among gum-based thickeners could be distinguished. RESULTS: As the shear rate increases, viscosity decreases (non-Newtonian and pseudoplastic behavior). At the same concentration, each thickener produces a different viscosity (p < 0.05). Institutionalized elderly detected viscosity differences in nectar range of 49.9 (2.5) mPa·s (p < 0.05) and 102.2 (4.7) mPa·s (p < 0.0001). They also detected viscosity differences in honey texture range of 134.6 (9.7) mPa·s (p < 0.05) y 199.3 (9.2) mPa·s (p < 0.0001). Their caregivers also detected viscosity differences in both viscosity ranges (p < 0.0001) and with greater intensity than the elderly in honey texture (p: 0.016). CONCLUSIONS: Our results suggest that the accepted viscosity ranges by NDD for the different textures might be too wide because institutionalized elderly and their caregivers are able to discern small differences in viscosity in nectar and honey textures. Gum-based thickeners with different composition showed differences in viscosity capacity, so they are not interchangeable.

Sulfation Code and Conformational Plasticity of l-Iduronic Acid Homo-Oligosaccharides Mimic the Biological Functions of Heparan Sulfate.

Recently, the activity of heparan sulfate (HS) has led to the discovery of many drug candidates that have the potential to impact both medical science and human health. However, structural diversity and synthetic challenges impede the progress of HS research. Here, we report a library of novel l-iduronic acid (IdoA)-based HS mimics that are highly tunable in conformation plasticity and sulfation patterns to produce many of the functions of native HS oligosaccharides. The NMR analysis of HS mimics confirmed that 4-O-sulfation enhances the population of the 1C4 geometry. Interestingly, the 1C4 conformer becomes exclusive upon additional 2-O-sulfation. HS mimic microarray binding studies with different growth factors showed that selectivity and avidity are greatly modulated by the oligosaccharide length, sulfation code, and IdoA conformation. Particularly, we have identified 4-O-sulfated IdoA disaccharide (I-21) as a potential ligand for vascular endothelial growth factor (VEGF165), which in a multivalent display modulated endothelial cell proliferation, migration, and angiogenesis. Overall, these results encourage the consideration of HS mimics for therapeutic applications.

Targeting transthyretin in Alzheimer's disease: Drug discovery of small-molecule chaperones as disease-modifying drug candidates for Alzheimer's disease.

Transthyretin (TTR) has a well-established role in neuroprotection in Alzheimer's Disease (AD). We have setup a drug discovery program of small-molecule compounds that act as chaperones enhancing TTR/Amyloid-beta peptide (Aß) interactions. A combination of computational drug repurposing approaches and in vitro biological assays have resulted in a set of molecules which were then screened with our in-house validated high-throughput screening ternary test. A prioritized list of chaperones was obtained and corroborated with ITC studies. Small-molecule chaperones have been discovered, among them our lead compound Iododiflunisal (IDIF), a molecule in the discovery phase; one investigational drug (luteolin); and 3 marketed drugs (sulindac, olsalazine and flufenamic), which could be directly repurposed or repositioned for clinical use. Not all TTR tetramer stabilizers behave as chaperones in vitro. These chemically diverse chaperones will be used for validating TTR as a target in vivo, and to select one repurposed drug as a candidate to enter clinical trials as AD disease-modifying drug.

Aß31-35 Decreases Neprilysin-Mediated Alzheimer's Amyloid-ß Peptide Degradation.

Alzheimer's disease is associated with the deposition of extracellular senile plaques, made primarily of amyloid-ß (Aß), particularly peptides Aß1-42 and Aß1-40. Neprilysin, or neutral endopeptidase (NEP), catalyzes proteolysis of the amyloid peptides (Aß) and is recognized as one of the major regulators of the levels of these peptides in the brain, preventing Aß accumulation and plaque formation. Here, we used a combination of techniques to elucidate the mechanism of Aß binding and cleavage by NEP. Our findings indicate that the Aß31-X cleavage products remain bound to the neprilysin active site, reducing proteolytic activity. Interestingly, it was already shown that this Aß31-35 sequence is also critical for recognition of Aß peptides by other targets, such as the serpin-enzyme complex receptor in neuronal cells.

Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers.

The interaction of multi-LacNAc (Galß1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.