Structural dynamics at the active site of the cancer‐associated flavoenzyme NQO1 probed by chemical modification with PMSF

A large conformational heterogeneity of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with various human diseases, has been observed to occur in the catalytic site of the enzyme. Here, we report the X-ray structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) at 1.6 A resolution. Activity assays confirmed that, despite being covalently bound to the Tyr128 residue at the catalytic site, PMSF did not abolish NQO1 activity. This may indicate that the PMSF molecule does not reduce the high flexibility of Tyr128, thus allowing NADH and DCPIP substrates to bind to the enzyme. Our results show that targeting Tyr128, a key residue in NQO1 function, with small covalently bound molecules could possibly not be a good drug discovery strategy to inhibit this enzyme

Molecular Binding of EuIII/CmIII by Stenotrophomonas bentonitica and Its Impact on the Safety of Future Geodisposal of Radioactive Waste

Microbial communities occurring in reference materials for artificial barriers (e.g., bentonites) in future deep geological repositories of radioactive waste can influence the migration behavior of radionuclides such as curium (CmIII). This study investigates the molecular interactions between CmIII and its inactive analogue europium (EuIII) with the indigenous bentonite bacterium Stenotrophomonas bentonitica at environmentally relevant concentrations. Potentiometric studies showed a remarkably high concentration of phosphates at the bacterial cell wall compared to other bacteria, revealing the great potential of S. bentonitica for metal binding. Infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed the role of phosphates and carboxylate groups from the cell envelope in the bioassociation of EuIII. Additionally, time-resolved laser-induced fluorescence spectroscopy (TRLFS) identified phosphoryl and carboxyl groups from bacterial envelopes, among other released complexing agents, to be involved in the EuIII and CmIII coordination. The ability of this bacterium to form a biofilm at the surface of bentonites allows them to immobilize trivalent lanthanide and actinides in the environment

Stenotrophomonas bentonitica sp. nov., isolated from bentonite formations

A Gram-stain negative, rod-shaped, aerobic bacterial strain, BII-R7T , was isolated during a study targeting the culturedependent microbial diversity occurring in bentonite formations from southern Spain. Comparative 16S rRNA gene sequence analysis showed that BII-R7T represented a member of the genus Stenotrophomonas (class Gammaproteobacteria), and was related most closely to Stenotrophomonas rhizophila e-p10T (99.2 % sequence similarity), followed by Stenotrophomonas pavanii ICB 89T (98.5 %), Stenotrophomonas maltophilia IAM 12423T , Stenotrophomonas chelatiphaga LPM-5T and Stenotrophomonas tumulicola T5916-2-1bT (all 98.3 %). Pairwise sequence similarities to all other type strains of species of the genus Stenotrophomonas were below 98 %. Genome-based calculations (orthologous average nucleotide identity, original average nucleotide identity, genome-to-genome distance and DNA G+C percentage) indicated clearly that the isolate represents a novel species within this genus. Different phenotypic analyses, such as the detection of a quinone system composed of the major compound ubiquinone Q-8 and a fatty acid profile with iso-C15 : 0 and anteiso-C15 : 0 as major components, supported this finding at the same time as contributing to a comprehensive characterization of BII-R7T . Based on this polyphasic approach comprising phenotypic and genotypic/molecular characterization, BII-R7T can be differentiated clearly from its phylogenetic neighbours, establishing a novel species for which the name Stenotrophomonas bentonitica sp. nov. is proposed with BII-R7T as the type strain (=LMG 29893T =CECT 9180T =DSM 103927T )

Exploring Antibacterial Activity and Bacterial-Mediated Allotropic Transition of Differentially Coated Selenium Nanoparticles

The use of metal nanoparticles (NPs) as antimicrobial agents has become a promising alternative to the problem of antibiotic-resistant bacteria and other applications. Silver nanoparticles (AgNPs) are well-known as one of the most universal biocide compounds. However, selenium nanoparticles (SeNPs) recently gained more attention as effective antimicrobial agents. This study aims to investigate the antibacterial activity of SeNPs with different surface coatings (BSA-coated, chitosan-coated, and undefined coating) on the Gram-negative Stenotrophomonas bentonitica and the Gram-positive Lysinibacillus sphaericus in comparison to AgNPs. The tested NPs had similar properties, including shape (spheres), structure (amorphous), and size (50–90 nm), but differed in their surface charge. Chitosan SeNPs exhibited a positive surface charge, while the remaining NPs assayed had a negative surface charge. We have found that cell growth and viability of both bacteria were negatively affected in the presence of the NPs, as indicated by microcalorimetry and flow cytometry. Specifically, undefined coating SeNPs displayed the highest percentage values of dead cells for both bacteria (85–91%). An increase in reactive oxygen species (ROS) production was also detected. Chitosan-coated and undefined SeNPs caused the highest amount of ROS (299.7 and 289% over untreated controls) for S. bentonitica and L. sphaericus, respectively. Based on DNA degradation levels, undefined-SeNPs were found to be the most hazardous, causing nearly 80% DNA degradation. Finally, electron microscopy revealed the ability of the cells to transform the different SeNP types (amorphous) to crystalline SeNPs (trigonal/monoclinical Se), which could have environmentally positive implications for bioremediation purposes and provide a novel green method for the formation of crystalline SeNPs. The results obtained herein demonstrate the promising potential of SeNPs for their use in medicine as antimicrobial agents, and we propose S. bentonitica and L. sphaericus as candidates for new bioremediation strategies and NP synthesis with potential applications in many fields

Unlocking the key role of bentonite fungal isolates in tellurium and selenium bioremediation and biorecovery: Implications in the safety of radioactive waste disposal

This work was supported by grant RTI2018.101548.B.I00 to M.L.M awarded by Spain's Ministry of Science and Innovation. The authors would like to thank the microscopy services of the University of Granada (Centro de Instrumentación Científica, University of Granada, Spain). Finally, the authors acknowledge the funding for the open access charge provided by Universidad de Granada/CBUA.Research on eco-friendly bioremediation strategies for mitigating the environmental impact of toxic metals has gained attention in the last years. Among all promising solutions, bentonite clays, to be used as artificial barriers to isolate radioactive wastes within the deep geological repository (DGR) concept, have emerged as effective reservoir of microorganisms with remarkable bioremediation potential. The present study aims to investigate the impact of bentonite fungi in the speciation and mobility of selenium (Se) and tellurium (Te), as natural analogues 79Se and 132Te present in radioactive waste, to screen for those strains with bioremediation potential within the context of DGR. For this purpose, a multidisciplinary approach combining microbiology, biochemistry, and microscopy was performed. Notably, Aspergillus sp. 3A demonstrated a high tolerance to Te(IV) and Se(IV), as evidenced by minimal inhibitory concentrations of >16 and >32 mM, respectively, along with high tolerance indexes. The high metalloid tolerance of Aspergillus sp. 3A is mediated by its capability to reduce these mobile and toxic elements to their elemental less soluble forms [Te(0) and Se(0)], forming nanostructures of various morphologies. Advanced electron microscopy techniques revealed intracellular Te(0) manifesting as amorphous needle-like nanoparticles and extracellular Te(0) forming substantial microspheres and irregular accumulations, characterized by a trigonal crystalline phase. Similarly, Se(0) exhibited a diverse array of morphologies, including hexagonal, irregular, and needle-shaped structures, accompanied by a monoclinic crystalline phase. The formation of less mobile Te(0) and Se(0) nanostructures through novel and environmentally friendly processes by Aspergillus sp. 3A suggests it would be an excellent candidate for bioremediation in contaminated environments, such as the vicinity of deep geological repositories. It moreover holds immense potential for the recovery and synthesis of Te and Se nanostructures for use in numerous biotechnological and biomedical applications.Research on eco-friendly bioremediation strategies for mitigating the environmental impact of toxic metals has gained attention in the last years. Among all promising solutions, bentonite clays, to be used as artificial barriers to isolate radioactive wastes within the deep geological repository (DGR) concept, have emerged as effective reservoir of microorganisms with remarkable bioremediation potential. The present study aims to investigate the impact of bentonite fungi in the speciation and mobility of selenium (Se) and tellurium (Te), as natural analogues 79Se and 132Te present in radioactive waste, to screen for those strains with bioremediation potential within the context of DGR. For this purpose, a multidisciplinary approach combining microbiology, biochemistry, and microscopy was performed. Notably, Aspergillus sp. 3A demonstrated a high tolerance to Te(IV) and Se(IV), as evidenced by minimal inhibitory concentrations of >16 and >32 mM, respectively, along with high tolerance indexes. The high metalloid tolerance of Aspergillus sp. 3A is mediated by its capability to reduce these mobile and toxic elements to their elemental less soluble forms [Te(0) and Se(0)], forming nanostructures of various morphologies. Advanced electron microscopy techniques revealed intracellular Te(0) manifesting as amorphous needle-like nanoparticles and extracellular Te(0) forming substantial microspheres and irregular accumulations, characterized by a trigonal crystalline phase. Similarly, Se(0) exhibited a diverse array of morphologies, including hexagonal, irregular, and needle-shaped structures, accompanied by a monoclinic crystalline phase. The formation of less mobile Te(0) and Se(0) nanostructures through novel and environmentally friendly processes by Aspergillus sp. 3A suggests it would be an excellent candidate for bioremediation in contaminated environments, such as the vicinity of deep geological repositories. It moreover holds immense potential for the recovery and synthesis of Te and Se nanostructures for use in numerous biotechnological and biomedical applications.Spain's Ministry of Science and Innovation RTI2018.101548.B.I00Universidad de Granada/CBU

Differences in Global Scientific Production Between New mRNA and Conventional Vaccines Against COVID-19

The search for effective vaccines to stop the COVID-19 pandemic has led to an unprecedented amount of global scientific production and activity. This study aimed to analyze global scientific production on the different vaccine types (mRNA and conventional) that were validated for COVID-19 during the years 2020-2021. The scientific production generated on COVID- 19 vaccines during the period 2020-2021 totaled the enormous amount of 20,459 studies published. New mRNA vaccines clearly showed higher production levels than conventional vaccines (viral and inactivated vectors), with 786 and 350 studies, respectively. The USA is the undisputed leader in the global production on COVID-19 vaccines, with Israel and Italy also playing an important role. Among the journals publishing works in this field, the New England Journal of Medicine, the British Medical Journal, and Vaccines stand out from the rest as the most important. The keyword ‘immunogenicity’ and its derivatives have been more researched for the new mRNA vaccines, while thrombosis has been more studied for conventional vaccines. The massive scientific production generated on COVID-19 vaccines in only two years has shown the enormous gravity of the pandemic and the extreme urgency to find a solution. This high scientific production and the main keywords found for the mRNA vaccines indicate the great potential that these vaccines have against COVID-19 and future infectious diseases. Moreover, this study provides valuable information for guiding future research lines and promoting international collaboration for an effective solution

Bibliometric Analysis of International Scientific Production on Pharmacologic Treatments for SARS-CoV-2/COVID-19 During 2020

This work was supported by the PAIDI (Plan Andaluz de Investigación, Desarrollo e Innovación 2020) program (Junta de Andalucía. Spain. HUM.777-EC3 Research Group).Background: COVID-19 is causing a grave global health and economic crisis and the fight against the pandemic has led to unprecedented scientific activity. Bibliometrics could be a useful tool for guiding future researches lines and promoting international collaboration for an effective treatment. For this purpose, we have conducted a bibliometric analysis of scientific publications on drugs and therapies used to treat COVID-19 during 2020. Methods: Data source: Web of Science. We gathered data on scientific production relating to drugs used to treat COVID-19. We calculated impact factors and analyzed production by institution, country, and journal, visualizing our results in bibliometric networks. Results: In 1 year, production relating to COVID-19 exceeded 100 000 publications, with over 6,500 on Drugs and COVID-19. Research into hydroxychloroquine and chloroquine, remdesivir, lopinavir and ritonavir, tocilizumab and convalescent plasma is particularly noteworthy. Mean citations/study range from 11.9 to 15.4. Producer institutions fall into three groups: one in the US and centered on Harvard Medical School; another in Europe led by INSERS; and another in China led by Huazhong University of Science and Technology. Production by journal is widespread but the Journal of Medical Virology, International Journal of Antimicrobial Agents, and American Journal of Transplantation are noteworthy. Conclusions: The volume of research that is currently under way is comparable to the magnitude of the pandemic itself. Such a high volume of studies is infrequent and the impact they have achieved has no known precedent. The producing countries are those with highest incidence of the pandemic and greatest scientific potential; moreover, inter-agency and international collaboration has reached extraordinarily high levels.Junta de Andalucía. Spain. HUM.777-EC3 Research Grou

Bibliometric analysis of the global scientific production on machine learning applied to different cancer types

This work has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement no. 860627 (CLARIFY Project), from the Spanish Ministry of Science and Innovation under project PID2019-105142RB-C22, and by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades under the project P20_00286. Funding for open access charge: Universidad de Granada/CBUA.Cancer disease is one of the main causes of death in the world, with million annual cases in the last decades. The need to find a cure has stimulated the search for efficient treatments and diagnostic procedures. One of the most promising tools that has emerged against cancer in recent years is machine learning (ML), which has raised a huge number of scientific papers published in a relatively short period of time. The present study analyzes global scientific production on ML applied to the most relevant cancer types through various bibliometric indicators. We find that over 30,000 studies have been published so far and observe that cancers with the highest number of published studies using ML (breast, lung, and colon cancer) are those with the highest incidence, being the USA and China the main scientific producers on the subject. Interestingly, the role of China and Japan in stomach cancer is correlated with the number of cases of this cancer type in Asia (78% of the worldwide cases). Knowing the countries and institutions that most study each area can be of great help for improving international collaborations between research groups and countries. Our analysis shows that medical and computer science journals lead the number of publications on the subject and could be useful for researchers in the field. Finally, keyword co-occurrence analysis suggests that ML-cancer research trends are focused not only on the use of ML as an effective diagnostic method, but also for the improvement of radiotherapy- and chemotherapy-based treatments.Horizon 2020 European Union under the Marie Sklodowska Curie 860627Spanish Government PID2019-105142RB-C22FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades P20_00286Universidad de Granada/CBU

Allotropy of selenium nanoparticles: Colourful transition, synthesis, and biotechnological applications

Abstract Elemental selenium (Se0) nanomaterials undergo allotropic transition from thermodynamically-unstable to more stable phases. This process is significantly different when Se0 nanoparticles (NPs) are produced via physico-chemical and biological pathways. While the allotropic transition of physico-chemically synthesized Se0 is fast (minutes to hours), the biogenic Se0 takes months to complete. The biopolymer layer covering biogenic Se0 NPs might be the main factor controlling this retardation, but this still remains an open question. Phylogenetically-diverse bacteria reduce selenium oxyanions to red amorphous Se0 allotrope, which has low market value. Then, red Se0 undergoes allotropic transition to trigonal (metallic grey) allotrope, the end product having important industrial applications (e.g. semiconductors, alloys). Is it not yet clear whether biogenic Se0 presents any biological function, or it is mainly a detoxification and respiratory by-product. The better understanding of this transition would benefit the recovery of Se0 NPs from secondary resources and its targeted utilization with respect to each allotropic stage. This review article presents and critically discusses the main physico-chemical methods and biosynthetic pathways of Se0 (bio)mineralization. In addition, the article proposes a conceptual model for the resource recovery potential of trigonal selenium nanomaterials in the context of circular economy.Ministerio de Ciencia e Innovación, Grant/ Award Number: RTI2018.101548.B.I00National Science Centre (NCN), Poland, Grant/Award Number: 2017/26/D/ NZ1/0040

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room- temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.STC Program of the National Science Foundation through BioXFEL (under agreement # 1231306)ABI Innovations award (NSF # 1565180), IIBR award (# 1943448)MCB award (1817862)National Institutes of Health award # R01GM095583US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract # DE-AC02- 76SF00515Center for Structural Dynamics in Biology, NIH grant P41GM13968“Ayuda de Atracción y Retención de Talento Investigador” from the Community of Madrid, Spain (REF: 2019-T1/BMD-15552)ERDF/Spanish Ministry of Science, Innovation, and Universities—State Research Agency (grant RTI2018-096246-B- I00), Consejería de Economía, Conocimiento, Empresas, y Universidad, Junta de Andalucía (grant P18-RT-2413),ERDF/Counseling of Economic transformation, Industry, Knowledge, and Universities (grant B-BIO-84-UGR20