Solid-state NMR spectroscopy insights for resolving different water pools in alginate hydrogels

Alginate hydrogels are versatile self-assembling biocompatible materials with diverse biomedical and food industrial applications, which includes uses in encapsulation, (drug) delivery and tissue engineering. Hydrogel formation requires cross-linking, which for alginates is often done with calcium ions that engage in specific interactions with the polysaccharide carboxylic acid groups. Water molecules also hydrate these alginate groups and fill macropores within the hydrogels, with implications for both mechanical properties and cargo encapsulation. Understanding these aspects of hydrogels requires the observation and characterization of the hydrogel waters, how they engage the alginate, and fill the macropores. Here we employed solid-state NMR (ssNMR) spectroscopy to detect and study water molecules in re-hydrated alginate hydrogels. 1H, 2H, and 13C magic angle spinning (MAS) NMR and relaxation measurements were combined to observe both water and alginate. Two different water phases were detected that vary upon gradual (re)hydration of the alginate hydrogels. These water pools differ in their chemical shifts and NMR relaxation properties, reflecting hydration waters directly associated with the carbohydrate polymers alongside dynamic waters in the macropores. Thus, the ssNMR detects the water-filled macropore water pools and how they vary upon calcium cross-linking. We also observe how calcium cross-linking selectively immobilizes the α-guluronate monosaccharides, but leaves the β-mannuronate units more flexible and prone to selective re-hydration. Thus, these ssNMR experiments can be used to probe cross-linking and hydration of alginate hydrogels, with implications for our understanding of design parameters that tune their performance in (drug) delivery and other food industrial applications

The Sequence, Bacterial Expression, and Functional Reconstitution of the Rat Mitochondrial Dicarboxylate Transporter Cloned via Distant Homologs in Yeast and Caenorhabditis elegans

The dicarboxylate carrier (DIC) belongs to a family of transport proteins found in the inner mitochondrial membranes. The biochemical properties of the mammalian protein have been characterized, but the protein is not abundant. It is difficult to purify and had not been sequenced. We have used the sequence of the distantly related yeast DIC to identify a related protein encoded in the genome of Caenorhabditis elegans. Then, related murine expressed sequence tags were identified with the worm sequence, and the murine sequence was used to isolate the cDNA for the rat homolog. The sequences of the worm and rat proteins have features characteristic of the family of mitochondrial transport proteins. Both proteins were expressed in bacteria and reconstituted into phospholipid vesicles where their transport characteristics closely resembled those of whole rat mitochondria and of the rat DIC reconstituted into vesicles. As expected from the role of the DIC in gluconeogenesis and ureogenesis, its transcripts were detected in rat liver and kidney, but unexpectedly, they were also detected in rat heart and brain tissues where the protein may fulfill other roles, possibly in supplying substrates to the Krebs cycle

Calcium-Sensing Receptor and Aquaporin 2 Interplay in Hypercalciuria-Associated Renal Concentrating Defect in Humans. An In Vivo and In Vitro Study

One mechanism proposed for reducing the risk of calcium renal stones is activation of the calcium-sensing receptor (CaR) on the apical membranes of collecting duct principal cells by high luminal calcium. This would reduce the abundance of aquaporin-2 (AQP2) and in turn the rate of water reabsorption. While evidence in cells and in hypercalciuric animal models supports this hypothesis, the relevance of the interplay between the CaR and AQP2 in humans is not clear. This paper reports for the first time a detailed correlation between urinary AQP2 excretion under acute vasopressin action (DDAVP treatment) in hypercalciuric subjects and in parallel analyzes AQP2-CaR crosstalk in a mouse collecting duct cell line (MCD4) expressing endogenous and functional CaR. In normocalciurics, DDAVP administration resulted in a significant increase in AQP2 excretion paralleled by an increase in urinary osmolality indicating a physiological response to DDAVP. In contrast, in hypercalciurics, baseline AQP2 excretion was high and did not significantly increase after DDAVP. Moreover DDAVP treatment was accompanied by a less pronounced increase in urinary osmolality. These data indicate reduced urinary concentrating ability in response to vasopressin in hypercalciurics. Consistent with these results, biotinylation experiments in MCD4 cells revealed that membrane AQP2 expression in unstimulated cells exposed to CaR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin (FK). Interestingly, we found that CaR activation by specific agonists reduced the increase in cAMP and prevented any reduction in Rho activity in response to FK, two crucial pathways for AQP2 translocation. These data support the hypothesis that CaR–AQP2 interplay represents an internal renal defense to mitigate the effects of hypercalciuria on the risk of calcium precipitation during antidiuresis. This mechanism and possibly reduced medulla tonicity may explain the lower concentrating ability observed in hypercalciuric patients

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster

none13noCoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.Vozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, GiuseppeVozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, Giusepp

Statins reduce intratumor cholesterol affecting adrenocortical cancer growth

Mitotane causes hypercholesterolemia in ACC patients. We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to ER\u3b1 action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production and ER\u3b1 activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ER\u3b1 function. A mitochondrial target of ER\u3b1, the respiratory complex IV (COX IV) was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. In vivo experiments confirmed the ability of simvastatin to reduce tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ER\u3b1, COX IV expression and activity and increase TUNEL positive cells. Collectively these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production, inhibits mitochondrial respiratory chain inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth

Targeted quantitative metabolic profiling of brain-derived cell cultures by semi-automated MEPS and LC-MS/MS

The accurate characterisation of metabolic profiles is an important prerequisite to determine the rate and the efficiency of the metabolic pathways taking place in the cells. Changes in the balance of metabolites involved in vital processes such as glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), as well as in the biochemical pathways related to amino acids, lipids, nucleotides, and their precursors reflect the physiological condition of the cells and may contribute to the development of various human diseases. The feasible and reliable measurement of a wide array of metabolites and biomarkers possesses great potential to elucidate physiological and pathological mechanisms, aid preclinical drug development and highlight potential therapeutic targets. An effective, straightforward, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was developed for the simultaneous quali-quantitative analysis of 41 compounds in both cell pellet and cell growth medium obtained from brain-derived cell cultures. Sample pretreatment miniaturisation was achieved thanks to the development and optimisation of an original extraction/purification approach based on digitally programmed microextraction by packed sorbent (eVol®-MEPS). MEPS allows satisfactory and reproducible clean-up and preconcentration of both low-volume homogenate cell pellet lysate and cell growth medium with advantages including, but not limited to, minimal sample handling and method sustainability in terms of sample, solvents, and energy consumption. The MEPS-LC-MS/MS method showed good sensitivity, selectivity, linearity, and precision. As a proof of concept, the developed method was successfully applied to the analysis of both cell pellet and cell growth medium obtained from a line of mouse immortalised oligodendrocyte precursor cells (OPCs; Oli-neu cell line), leading to the unambiguous determination of all the considered target analytes. This method is thus expected to be suitable for targeted, quantitative metabolic profiling in most brain cell models, thus allowing accurate investigations on the biochemical pathways that can be altered in central nervous system (CNS) neuropathologies, including e.g., mitochondrial respiration and glycolysis, or use of specific nutrients for growth and proliferation, or lipid, amino acid and nucleotide metabolism

Novel Key Ingredients in Urinary Tract Health-The Role of D-mannose, Chondroitin Sulphate, Hyaluronic Acid, and N-acetylcysteine in Urinary Tract Infections (Uroial PLUS®)

: Urinary tract infections represent a common and significant health concern worldwide. The high rate of recurrence and the increasing antibiotic resistance of uropathogens are further worsening the current scenario. Nevertheless, novel key ingredients such as D-mannose, chondroitin sulphate, hyaluronic acid, and N-acetylcysteine could represent an important alternative or adjuvant to the prevention and treatment strategies of urinary tract infections. Several studies have indeed evaluated the efficacy and the potential use of these compounds in urinary tract health. In this review, we aimed to summarize the characteristics, the role, and the application of the previously reported compounds, alone and in combination, in urinary tract health, focusing on their potential role in urinary tract infections

Renal Cell Carcinoma as a Metabolic Disease: An Update on Main Pathways, Potential Biomarkers, and Therapeutic Targets

: Clear cell renal cell carcinoma (ccRCC) is the most frequent histological kidney cancer subtype. Over the last decade, significant progress has been made in identifying the genetic and metabolic alterations driving ccRCC development. In particular, an integrated approach using transcriptomics, metabolomics, and lipidomics has led to a better understanding of ccRCC as a metabolic disease. The metabolic profiling of this cancer could help define and predict its behavior in terms of aggressiveness, prognosis, and therapeutic responsiveness, and would be an innovative strategy for choosing the optimal therapy for a specific patient. This review article describes the current state-of-the-art in research on ccRCC metabolic pathways and potential therapeutic applications. In addition, the clinical implication of pharmacometabolomic intervention is analyzed, which represents a new field for novel stage-related and patient-tailored strategies according to the specific susceptibility to new classes of drugs

Monitoring Interactions inside Cells by Advanced Spectroscopies: Overview of Copper Transporters and Cisplatin

Resistance, either at the onset of the treatment or developed after an initial positive response, is a major limitation of antitumor therapy. In the case of platinum-based drugs, copper transporters have been found to interfere with drug trafficking by facilitating the import or favoring the platinum export and inactivation

Monitoring Interactions inside Cells by Advanced Spectroscopies: Overview of Copper Transporters and Cisplatin

Resistance, either at the onset of the treatment or developed after an initial positive response, is a major limitation of antitumor therapy. In the case of platinum-based drugs, copper transporters have been found to interfere with drug trafficking by facilitating the import or favoring the platinum export and inactivation