Dual responsive gelatin-based nanoparticles for enhanced 5-fluorouracil efficiency

The very slow progress in the therapeutic efficacy of the treatment of severe diseases has suggested the use of a growing need for a multidisciplinary approach to the delivery of therapeutics to targets tissues. There has been increasing effort in the design of stimuli-responsive nanomaterials that they will be developed into effective drug delivery vehicles. Most commonly, effective drug delivery is associated with nanomaterial-facilitated accumulation and/or cellular internalization. Recent studies in our lab have demonstrated that gelatin-based NPs can be considered suitable pH responsive devices for the effective intracellular delivery of drugs. Concerning cancer treatment, ligands recognizing tumour-associated antigens expressed on the surface of the tumour cells have been employed. Some of the target structures suitable for tumour targeting belong to integrins which mediate cell adhesion to extracellular matrix and other cells. Interestingly, gelatin chains contain motifs such as RGD sequences that can be recognised by integrins. In this work the inclusion of the anticancer drug 5-fluorouracil (5-FU) on these gelatin-based NPs has been projected. These NPs may provide an opportunity to increase the therapeutic effect using a dual approach by: i) targeting the therapeutic drug to the tumour cells by the action of the naturally occurring RGD-motif on gelatin and ii) minimizing the non-productive trafficking from endosomes to lysosomes by releasing the cargo using the charge reversal approach after cellular internalization. In vitro cytotoxicity experiments of NPs on tumoral and non-tumoral cell lines have reported selectivity indexes higher than 30 demonstrating a great selectivity on the mode of action as a function of the cell line and the imposed compositions. Keywords: 5-fluorouracil; Gelatin; In vitro cytotoxicity; Loading efficiency; Nanoparticles; Selectivity. Copyright © 2018 Elsevier B.V. All rights reserved

Metabolomic and phenotypic implications of the application of fertilization products containing microcontaminants in lettuce (Lactuca sativa)

Cultivation practice using organic amendments is plausible to ensure global food security. However, plant abiotic stress due to the presence of metals and organic microcontaminants (OMCs) in fertilization products cannot be overlooked. In this study, we monitored lettuce metabolism and phenotypic response following the application of either sewage sludge (SS), the organic fraction of municipal solid waste, swine manure (SM), chemical fertilizers (CF), or no amendment (C) in a greenhouse facility.Postprint (published version

ISOGO: Functional annotation of protein-coding splice variants

The advent of RNA-seq technologies has switched the paradigm of genetic analysis from a genome to a transcriptome-based perspective. Alternative splicing generates functional diversity in genes, but the precise functions of many individual isoforms are yet to be elucidated. Gene Ontology was developed to annotate gene products according to their biological processes, molecular functions and cellular components. Despite a single gene may have several gene products, most annotations are not isoform-specifc and do not distinguish the functions of the diferent proteins originated from a single gene. Several approaches have tried to automatically annotate ontologies at the isoform level, but this has shown to be a daunting task. We have developed ISOGO (ISOform+GO function imputation), a novel algorithm to predict the function of coding isoforms based on their protein domains and their correlation of expression along 11,373 cancer patients. Combining these two sources of information outperforms previous approaches: it provides an area under precision-recall curve (AUPRC) fve times larger than previous attempts and the median AUROC of assigned functions to genes is 0.82. We tested ISOGO predictions on some genes with isoform-specifc functions (BRCA1, MADD,VAMP7 and ITSN1) and they were coherent with the literature. Besides, we examined whether the main isoform of each gene -as predicted by APPRIS- was the most likely to have the annotated gene functions and it occurs in 99.4% of the genes. We also evaluated the predictions for isoform-specifc functions provided by the CAFA3 challenge and results were also convincing. To make these results available to the scientifc community, we have deployed a web application to consult ISOGO predictions (https://biotecnun.unav. es/app/isogo). Initial data, website link, isoform-specifc GO function predictions and R code is available at https://gitlab.com/icassol/isogo

Effects of tetracycline, sulfonamide, fluoroquinolone, and lincosamide load in pig slurry on lettuce: Agricultural and human health implications

The application of pig slurry as fertilizer in agriculture provides nutrients, but it can also contain veterinary medicines, including antibiotic residues (ABs), which can have an ecotoxicological impact on agroecosystems. Furthermore, uptake, translocation, and accumulation of ABs in crops can mobilize them throughout the food chain. This greenhouse study aims to assess AB uptake from soil fertilized with pig slurry and its phenotypical effects on Lactuca sativa LThe authors gratefully acknowledge the financial support of the Spanish Ministry of Science and Innovation through project AGL 2017- 89518-R. IDAEA-CSIC is a Severo Ochoa Centre of Excellence (Spanish Ministry of Science and Innovation, Project CEX 2018-000794-S). Monica Escola Casas wishes to thank the Beatriu de Pinos 2018 grant program (MSCA grant agreement number 801370) for the funding. The authors likewise thank Miquel Massip, Daniel Fenero, and Nerea Gran- ados for their technical assistance in the greenhouse facilityPostprint (published version

Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein

Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a Neisseria meningitidis surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy

Implications of the use of organic fertilizers for antibiotic resistance gene distribution in agricultural soils and fresh food products. A plot-scale study

The spread of antibiotic resistance genes (ARG) into agricultural soils, products, and foods severely limits the use of organic fertilizers in agriculture. In order to help designing agricultural practices that minimize the spread of ARG, we fertilized, sown, and harvested lettuces and radish plants in experimental land plots for two consecutive agricultural cycles using four types of fertilizers: mineral fertilization, sewage sludge, pig slurry, or composted organic fraction of municipal solid waste. The analysis of the relative abundances of more than 200,000 ASV (Amplicon Sequence Variants) identified a small, but significant overlap (<10%) between soil's and fertilizer microbiomes. Clinically relevant ARG were found in higher loads (up to 100 fold) in fertilized soils than in the initial soil, particularly in those treated with organic fertilizers, and their loads grossly correlated to the amount of antibiotic residues found in the corresponding fertilizer. Similarly, low, but measurable ARG loads were found in lettuce (tetM, sul1) and radish (sul1), corresponding the lowest values to samples collected from minerally fertilized fields. Comparison of soil samples collected along the total period of the experiment indicated a relatively year-round stability of soil microbiomes in amended soils, whereas ARG loads appeared as unstable and transient. The results indicate that ARG loads in soils and foodstuffs were likely linked to the contribution of bacteria from organic fertilizer to the soil microbiomes, suggesting that an adequate waste management and good pharmacological and veterinarian practicesmay significantly reduce the presence of these ARGs in agricultural soils and plant products.Postprint (published version

Can we identify individuals with an ALPL variant in adults with persistent hypophosphatasaemia?

Hypophosphatasia (HPP) is an inborn error of metabolism characterized by low levels of serum alkaline phosphatase (ALP). Scarce evidence exists about features that should signal the potential association between hypophosphatasaemia and HPP in adults. The aim of this study is to estimate the prevalence of ALPL variants in subjects with persistent hypophosphatasaemia and determine the associated clinical and laboratory features. For this cross-sectional study, laboratory records of 386,353 subjects were screened by measurement of ALP activity. A total of 85 (0.18%) subjects with persistent hypophosphatasaemia (≥2 serum alkaline phosphatase-ALP-measurements ≤35 IU/L and none &gt; 45 IU/L) were included (secondary causes previously discarded). ALPL genetic testing and a systematized questionnaire to retrieve demographic, clinical and laboratory data were performed. Descriptive analysis and logistic regression models were employed to identify the clinical and laboratory characteristics associated with ALPL variants. Results: Forty subjects (47%) had a variant(s) in ALPL. With regard to clinical characteristics, the presence of an ALPL variant was significantly associated only with musculoskeletal pain (OR: 7.6; 95% IC: 1.9-30.9). Nevertheless, a trend to present more dental abnormalities (OR: 3.6; 95% IC: 0.9-13.4) was observed. Metatarsal stress fractures were also more frequent (4 vs 0; p &lt; 0.05) in this group. Regarding laboratory features, median ALP levels were lower in subjects with ALPL variants (26 vs 29 IU/L; p &lt; 0.005). Interestingly, the threshold of ALP levels &lt; 25 IU/L showed a specificity, positive predictive value and positive likelihood ratio of 97.8, 94.4% and 19.8 to detect a positive ALPL test, respectively. Conclusions: In subjects with persistent hypophosphatasaemia -secondary causes excluded- one out of two presented ALPL variants. Musculoskeletal pain and ALP levels &lt; 25 IU/L are associated with this variant(s). In this scenario, ALP levels &lt; 25 IU/L seem to be very useful to identify individuals with the presence of an ALPL variantGenetic testing was supported by a grant from Alexion Pharmaceuticals Inc., which had no role in the study design or data analysi

Occurrence of antibiotics in Lettuce (Lactuca sativa L.) and Radish (Raphanus sativus L.) following organic soil fertilisation under plot-scale conditions: Crop and human health implications

Recent studies have demonstrated the crop uptake of antibiotics (ABs) from soils treated with AB-carrying fertilisers. However, there is a lack of plot-scale studies linking their effects at the agronomic and metabolomic/transcriptomic level to their impact on human health. This paper assesses the plant uptake of 23 ABs following two productive cycles of lettuce and radish cropped with sewage sludge, pig slurry, the organic fraction of municipal solid waste, or chemical fertilisation under plot-scale conditions (32 plots spanning 3-10 m2 each). AB uptake by plants depended on both the vegetable and the AB class and was higher in radish than in lettuce edible parts. Levels ranged from undetectable to up to 76 ng/g (fresh weight). Repetitive organic fertilisation resulted in an increase in the concentration of ABs in lettuce leaves, but not in radish roots. Significant metabolomic and transcriptomic changes were observed following soil fertilisation. Nevertheless, a human health risk assessment indicates that the occurrence of ABs in lettuce or radish edible parts does not pose any risk. To our knowledge, this is the first holistic plot-scale study demonstrating that the use of organic fertilisers containing ABs is safe for crop security and human health.The authors gratefully acknowledge the financial support of the Spanish Ministry of Science and Innovation through projects AGL2017- 89518-R and RTI2018-096175-B-I00. IDAEA-CSIC is a Severo Ochoa Centre of Excellence (Spanish Ministry of Science and Innovation, Project CEX2018-000794-S). Mònica Escolà Casas wishes to thank the Beatriu de Pinós 2018 grant programme (MSCA grant agreement number 801370) for the funding.Peer ReviewedPostprint (published version

Cryo-EM and the elucidation of new macromolecular structures: Random Conical Tilt revisited

Cryo-Electron Microscopy (cryo-EM) of macromolecular complexes is a fundamental structural biology technique which is expanding at a very fast pace. Key to its success in elucidating the three-dimensional structure of a macromolecular complex, especially of small and non-symmetric ones, is the ability to start from a low resolution map, which is subsequently refined with the actual images collected at the microscope. There are several methods to produce this first structure. Among them, Random Conical Tilt (RCT) plays a prominent role due to its unbiased nature (it can create an initial model based on experimental measurements). In this article, we revise the fundamental mathematical expressions supporting RCT, providing new expressions handling all key geometrical parameters without the need of intermediate operations, leading to improved automation and overall reliability, essential for the success of cryo-EM when analyzing new complexes. We show that the here proposed RCT workflow based on the new formulation performs very well in practical cases, requiring very few image pairs (as low as 13 image pairs in one of our examples) to obtain relevant 3D maps.We thank Dr. Llorca for his support during the acquisition of the C3b images and Dr. Shaikh for his support in the use of Spider for the RCT reconstructions. The authors would like to acknowledge economical support from the Spanish Ministry of Economy and Competitiveness through grants AIC-A-2011-0638 and BIO2013-44647-R, the Comunidad de Madrid through grant CAM (S2010/BMD-2305), as well as a postdoctoral Juan de la Cierva grant with reference JCI-2011-10185 to Javier Vargas. Vahid Abrishami is a holder of La Caixa scholarship and C.O.S. Sorzano is recipient of a Ramon y Cajal fellowship