A selective strategy for targeting primary hyperoxaluria diseases

Funding Information: Authors wish to thank the Centro de Instrumentación Científico-Técnica (CICT) of the University of Jaén, Spain, for partial financial support. A.A.-A. is grateful for the postdoctoral fellowship from Fundación Alfonso Martín Escudero. Authors acknowledge the use of the National Facility ELECMI ICTS, node “Laboratorio de Microscopias Avanzadas” at Universidad de Zaragoza. This research has also partially been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (grant RTI2018-098560-B-C22) and by the Andalusian Consejería de Economía y Conocimiento (FEDER program 2014-2020: grant number 1380682). This work was partially supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020). Funding Information: This research has also partially been supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (grant RTI2018-098560-B-C22) and by the Andalusian Consejería de Economía y Conocimiento (FEDER program 2014-2020: grant number 1380682). This work was partially supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020). Funding Information: Authors wish to thank the Centro de Instrumentación Científico-Técnica (CICT) of the University of Jaén, Spain, for partial financial support. A.A.-A. is grateful for the postdoctoral fellowship from Fundación Alfonso Martín Escudero. Authors acknowledge the use of the National Facility ELECMI ICTS, node “Laboratorio de Microscopias Avanzadas” at Universidad de Zaragoza. Publisher Copyright: © 2022 The Author(s)Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism that result in an excess of oxalate production by the oxidation of glyoxylate by the human lactate dehydrogenase A enzyme (hLDHA). The selective liver inhibition of this enzyme is one of the therapeutic strategies followed in the treatment of this disease. Even though several efforts have been recently performed using gene silencing by the RNA interference approach, small-molecule inhibitors that selectively reach hepatocytes are preferred since they present the advantages of a lower production cost and better pharmacological properties. In that sense, the design, synthesis, and physicochemical characterization by NMR, FTIR, DLS and TEM of two nanocarriers based on chitosan conjugates (1, non-redox-sensitive; 2, redox-sensitive) have been performed to (i) achieve the selective transport of hLDHA inhibitors into hepatocytes and (ii) their disruption once they reach the hepatocytes cytosol. Polymer 2 self-assembled into micelles in water and showed high drug loadings (19.8–24.5 %) and encapsulation efficiencies (31.9–40.8%) for the hLDHA inhibitors (I-III) tested. The non-redox-sensitive micelle 1 remained stable under different glutathione (GSH) concentrations (10 μM and 10 mM), and just a residual release of the inhibitor encapsulated was observed (less than 10 %). On the other hand, micelle 2 was sufficiently stable under in vitro physiological conditions (10 μM, GSH) but it quickly disassembled under the simulated reducing conditions present inside hepatocytes (10 mM GSH), achieving a 60 % release of the hLDHA inhibitor encapsulated after 24 h, confirming the responsiveness of the developed carrier to the high levels of intracellular GSH.publishersversionpublishe

Estudio fitoquímico de Juníperus Oxycedrus, L. subsp. Macrocarpa

Acid components from berries of Juniperus oxycedrus L. subsp. macrocarpa, have been studied. They mainly contain diterpenes with labdane skeleton: trans-communic, myrceocommunic, cis-communic, junicedric, 15-methyl-imbricatalic, 12(R)-hydroxy-myrceocommunic, imbricatolic and isocupressic acids. We have al so identified sandaracopimaric and isopimaric acids (pimarane skeleton), and sugiyl methyl ether (abietane skeleton). This work have established the phytochemical analogies and differences with the subspecie oxycedrus, and its chemotaxonomy in the genus Juniperus.Se han estudiado los componentes ácidos de las arcésticas de Juniperus oxycedrus L. subsp. macrocarpa, encontrándose fundamentalmente ácidos diterpénicos de esqueleto labdano; trans-comúnico (mayoritario), mirceocomúnico, cis-comúnico, junicédrico, 15-metil-imbricatálico, 12(R)-hidroximirceocomúnico, imbricatólico e isocuprésico. También se han identificado los ácidos sandaracopimárico e isopimárico, con esqueleto pimarano y el abietano, éter metílico de sugiol. Este estudio ha permitido establecer las analogías y diferencias fitoquímicas con la subespecie oxycedrus, así como su quimiotaxonomía dentro del género Juníperus

Estudio fitoquímico de Juníperus Oxycedrus, L. subsp. Macrocarpa

Se han estudiado los componentes ácidos de las arcésticas de Juniperus oxycedrus L. subsp. macrocarpa, encontrándose fundamentalmente ácidos diterpénicos de esqueleto labdano; trans-comúnico (mayoritario), mirceocomúnico, cis-comúnico, junicédrico, 15-metil-imbricatálico, 12(R)-hidroximirceocomúnico, imbricatólico e isocuprésico. También se han identificado los ácidos sandaracopimárico e isopimárico, con esqueleto pimarano y el abietano, éter metílico de sugiol. Este estudio ha permitido establecer las analogías y diferencias fitoquímicas con la subespecie oxycedrus, así como su quimiotaxonomía dentro del género Juníperus.Acid components from berries of Juniperus oxycedrus L. subsp. macrocarpa, have been studied. They mainly contain diterpenes with labdane skeleton: trans-communic, myrceocommunic, cis-communic, junicedric, 15- methyl-imbricatalic, 12(R)-hydroxy-myrceocommunic, imbricatolic and isocupressic acids. We have al so identified sandaracopimaric and isopimaric acids. (pimarane skeleton), and sugiyl methyl ether (abietane skeleton). This work have established the phytochemical analogies and differences with the subspecie oxycedrus, and its chemotaxonomy in the genus Juniperus

Drug-eluting wound dressings having sustained release of antimicrobial compounds

Wound healing is a complex and costly public health problem that should be timely addressed to achieve a rapid and adequate tissue repair avoiding or even eliminating potential pathogenic infection. Chronic infected non-healing wounds represent a serious concern for health care systems. Efficient wound dressings with tailored therapy having the best response and highest safety margin for the management of chronic non-healing wounds are still needed. The use of novel wound dressing materials has emerged as a promising tool to fulfil these requirements. In this work, asymmetric electrospun polycaprolactone (PCL)-based nanofibers (NFs) were decorated with electrosprayed poly(lactic-co-glycolic acid) microparticles (PLGA MPs) containing the natural antibacterial compound thymol (THY) in order to obtain drug eluting antimicrobial dressings having sustained release. The synthesized dressings successfully inhibited the in vitro growth of Staphylococcus aureus ATCC 25923, showing also at the same doses cytocompatibility on human dermal fibroblasts and keratinocyte cultures after treatment for 24 h, which was not observed when using free thymol. An in vivo murine excisional wound splinting model, followed by the experimental infection of the wounds with S. aureus and their treatment with the synthesized dressings, pointed to the reduction of the bacterial load in wounds after 7 days, though the total elimination of the infection was not reached. The findings indicated the relevance of the direct contact between the dressings and the bacteria, highlighting the need to tune their design considering the wound surface and the nature of the antimicrobial cargo contained

Time-Varying Emulator for Short and Long-Term Analysis of Coastal Flood Hazard Potential

Rising seas coupled with ever increasing coastal populations present the potential for significant social and economic loss in the 21st century. Relatively short records of the full multidimensional space contributing to total water level coastal flooding events (astronomic tides, sea level anomalies, storm surges, wave run‐up, etc.) result in historical observations of only a small fraction of the possible range of conditions that could produce severe flooding. The Time‐varying Emulator for Short‐ and Long‐Term analysis of coastal flood hazard potential is presented here as a methodology capable of producing new iterations of the sea‐state parameters associated with the present‐day Pacific Ocean climate to simulate many synthetic extreme compound events. The emulator utilizes weather typing of fundamental climate drivers (sea surface temperatures, sea level pressures, etc.) to reduce complexity and produces new daily synoptic weather chronologies with an auto‐regressive logistic model accounting for conditional dependencies on the El Niño Southern Oscillation, the Madden‐Julian Oscillation, seasonality, and the prior two days of weather progression. Joint probabilities of sea‐state parameters unique to simulated weather patterns are used to create new time series of the hypothetical components contributing to synthetic total water levels (swells from multiple directions coupled with water levels due to wind setup, temperature anomalies, and tides). The Time‐varying Emulator for Short‐ and Long‐Term analysis of coastal flood hazard potential reveals the importance of considering the multivariate nature of extreme coastal flooding, while progressing the ability to incorporate large‐scale climate variability into site specific studies assessing hazards within the context of predicted climate change in the 21st century

Counterintuitive structural and functional effects due to naturally occurring mutations targeting the active site of the disease-associated NQO1 enzyme*

Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype–phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype–phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome

Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale

Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L\u2192G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity ( 4820 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance

Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing

Association Between the Gut Microbiota and Blood Pressure in a Population Cohort of 6953 Individuals

Background Several small-scale animal studies have suggested that gut microbiota and blood pressure (BP) are linked. However, results from human studies remain scarce and conflicting. We wanted to elucidate the multivariable-adjusted association between gut metagenome and BP in a large, representative, well-phenotyped population sample. We performed a focused analysis to examine the previously reported inverse associations between sodium intake and Lactobacillus abundance and between Lactobacillus abundance and BP. Methods and Results We studied a population sample of 6953 Finns aged 25 to 74 years (mean age, 49.212.9 years; 54.9% women). The participants underwent a health examination, which included BP measurement, stool collection, and 24-hour urine sampling (N=829). Gut microbiota was analyzed using shallow shotgun metagenome sequencing. In age- and sex-adjusted models, the alpha (within-sample) and beta (between-sample) diversities of taxonomic composition were strongly related to BP indexes (P diversity was only associated with diastolic BP (P=0.032). However, we observed significant, mainly positive, associations between BP indexes and 45 microbial genera (P Conclusions Although the associations between overall gut taxonomic composition and BP are weak, individuals with hypertension demonstrate changes in several genera. We demonstrate strong negative associations of certain Lactobacillus species with sodium intake and BP, highlighting the need for experimental studies.Peer reviewe