Antimicrobial defenses of table eggs: Importance of antibacterial proteins in egg white as a function of hen age in an extended production cycle

The importance of egg natural defences to prevent bacterial contamination and their relation with hen age in extended production cycles were evaluated. Egg-white from eggs of different hen age groups (up 100-weeks-old) and lines (Hy-Line white and brown) were inoculated with Gram-positive Staphylococcus aureus or Gram-negative Salmonella Typhimurium, ranging from 103-106 CFU/mL. Our results show that concentrations of egg-white lysozyme and, particularly, ovotransferrin are important to modulate bacterial survival in a dose-dependent matter. Depending on protein concentration, their effect ranges from bactericidal to bacteriostatic, with a threshold for bacterial contamination that depends also on hen age and line. The concentrations of lysozyme and ovotransferrin increased with hen age (up to 2 and 22 w/w% of total protein, respectively), and eggs laid by older hens exhibited the greatest potential to prevent the growth of the highest Salmonella inoculum (106 CFU/ mL). Salmonella-penetration experiments demonstrated that non-contaminated eggs display significantly higher concentrations of antimicrobial proteins. However, eggs from older hens needed a higher concentration of these proteins (>20% ovotransferrin) to prevent bacterial contamination, showing that antimicrobial protein concentrations in egg-whites was not the only factor influencing bacterial contamination. Finally, this study demonstrated that egg-white of eggs produced by old hens are less prone to contamination by Salmonella.Spanish Government CGL 2015-64683-PPremio de Investigacion 2019 (Instituto del Huevo)Junta de Andalucia RNM-938Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN2022-0441

Biogeochemical fingerprinting of magnetotactic bacterial magnetite

Data Availability. All data related to the manuscript, including samples and aspects of methodology, are included in the main manuscript or as part of SI Appendix. Requests for additional data (i.e., APT data files), details of protocols, materials, and for any questions related to the manuscript, should be addressed to the corresponding author, Dr. Alberto Pérez-Huerta ([email protected]). Original IVAS files (RHIT and HITS) data are available in Figshare (45).ACKNOWLEDGMENTS. This work is supported by NSF grants EAR-1647012 and EAR-150779 grants awarded to A.P.-H. D.A.B. is supported by NSF grant EAR-1423939. C.J.L. wishes to thank Junta de Andalucía (Spain) projects B-BIO-432-UGR20 and P20_00208 and Ministerio de Ciencia y Educación (Spain) projects PDC2021-121135-I00 and CGL 2016-76723. T.P. acknowledges support of Laboratory Directed Research and Development Program through Ames Laboratory. This work at the Ames National Laboratory was supported by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division. Electron microscopy work (FIBelectron microscopy and scanning and transmission electron microscopy imaging) was performed at the UA AARC, the Ames National Laboratory, and the Universidad de Granada. Electron microscopy imaging and FIB work in the Ames Laboratory was performed using instruments in the Sensitive Instrument Facility. The Ames National Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract No. DE-AC02-07CH11358. The authors also acknowledge the work of Drs. Fernando Laiginhas and Alejandra Londoño-Calderon in FIB-scanning electron microscopy sample preparation, Dr. Matthew Lynn for help with FIB training and specimen preparation, and the invaluable assistance of Dr. Lynda Williams and Dr. Maitrayee Bose for the SIMS and nano-SIMS analyses, respectively, using instruments in the NSF-supported Multiuser Facility at Arizona State University.This article contains supporting information online at http://www.pnas.org/lookup/suppl/doi:10.1073/pnas. 2203758119/-/DCSupplemental.Biominerals are important archives of the presence of life and environmental processes in the geological record. However, ascribing a clear biogenic nature to minerals with nanometer-sized dimensions has proven challenging. Identifying hallmark features of biologically controlled mineralization is particularly important for the case of magnetite crystals, resembling those produced by magnetotactic bacteria (MTB), which have been used as evidence of early prokaryotic life on Earth and in meteorites. We show here that magnetite produced by MTB displays a clear coupled C–N signal that is absent in abiogenic and/or biomimetic (protein-mediated) nanometer-sized magnetite. We attribute the presence of this signal to intracrystalline organic components associated with proteins involved in magnetosome formation by MTB. These results demonstrate that we can assign a biogenic origin to nanometer-sized magnetite crystals, and potentially other biominerals of similar dimensions, using unique geochemical signatures directly measured at the nanoscale. This finding is significant for searching for the earliest presence of life in the Earth’s geological record and prokaryotic life on other planetsNSF grants EAR-1647012 and EAR-150779NSF grant EAR-1423939Junta de Andalucía (Spain) projects B-BIO-432-UGR20 and P20_00208Ministerio de Ciencia y Educación (Spain) projects PDC2021-121135-I00 and CGL 2016-76723Laboratory Directed Research and Development Program through Ames LaboratoryU.S. Department of Energy (DOE

Improving the Cellular Uptake of Biomimetic Magnetic Nanoparticles

This research was funded by the FUR (Fondo Unico della Ricerca—University of Verona) of M. Perduca. C.J.-L. acknowledges funding from projects CGL2016-76723 from the Ministerio de Economía y Competitividad from Spain and Fondo Europeo de Desarrollo Regional (FEDER) and Programa Operativo FEDER 2014–2020 (A-BIO-376-UGR18) Junta de Andalucia. M.P.C.-J. acknowledges funding from projects PID2019-109294RB-100 from the Ministerio de Ciencia e Innovación from Spain.We are grateful to the “Centro Piattaforme Tecnologiche” of the University of Verona for giving access to DLS equipment. CJL acknowledges. the Unidad Cientıfica de Excelencia UCE PP 2016.05 (U. Granada) and Instituto de Biotecnología. Y.J. wants to acknowledge a FPU2016 grant (ref. FPU16_04580) from the Ministerio de Educación, Ciencia y Deporte y Competitividad (Spain). AS-L is funded by the Spanish Ministry of Science, Innovation and Universities: Formación de Doctores 2018 (ref. PRE2018-0854409). Thanks go to the Scientific Instrumentation Center (CIC) personnel of the University of Granada for technical assistance with the TEM.We also thank Salvatore Calogero Gaglio for his help in preparing Figure S4.Magnetococcus marinus magnetosome-associated protein MamC, expressed as recombinant, has been proven to mediate the formation of novel biomimetic magnetic nanoparticles (BMNPs) that are successful drug nanocarriers for targeted chemotherapy and hyperthermia agents. These BMNPs present several advantages over inorganic magnetic nanoparticles, such as larger sizes that allow the former to have larger magnetic moment per particle, and an isoelectric point at acidic pH values, which allows both the stable functionalization of BMNPs at physiological pH value and the molecule release at acidic (tumor) environments, simply based on electrostatic interactions. However, difficulties for BMNPs cell internalization still hold back the efficiency of these nanoparticles as drug nanocarriers and hyperthermia agents. In the present study we explore the enhanced BMNPs internalization following upon their encapsulation by poly (lactic-co-glycolic) acid (PLGA), a Food and Drug Administration (FDA) approved molecule. Internalization is further optimized by the functionalization of the nanoformulation with the cell-penetrating TAT peptide (TATp). Our results evidence that cells treated with the nanoformulation [TAT-PLGA(BMNPs)] show up to 80% more iron internalized (after 72 h) compared to that of cells treated with BMNPs (40%), without any significant decrease in cell viability. This nanoformulation showing optimal internalization is further characterized. In particular, the present manuscript demonstrates that neither its magnetic properties nor its performance as a hyperthermia agent are significantly altered due to the encapsulation. In vitro experiments demonstrate that, following upon the application of an alternating magnetic field on U87MG cells treated with BMNPs and TAT-PLGA(BMNPs), the cytotoxic effect of BMNPs was not affected by the TAT-PLGA enveloping. Based on that, difficulties shown in previous studies related to poor cell uptake of BMNPs can be overcome by the novel nanoassembly described here.FUR (Fondo Unico della Ricerca-University of Verona)Ministerio de Economia y Competitividad from Spain CGL2016-76723European Commission CGL2016-76723Junta de Andalucia A-BIO-376-UGR18Spanish Government PID2019-109294RB-10

Synergistic Photothermal-Chemotherapy Based on the Use of Biomimetic Magnetic Nanoparticles

MamC-mediated biomimetic magnetic nanoparticles (BMNPs) have emerged as one of the most promising nanomaterials due to their magnetic features (superparamagnetic character and large magnetic moment per particle), their novel surface properties determined by MamC, their biocompatibility and their ability as magnetic hyperthermia agents. However, the current clinical application of magnetic hyperthermia is limited due to the fact that, in order to be able to reach an effective temperature at the target site, relatively high nanoparticle concentration, as well as high magnetic field strength and/or AC frequency are needed. In the present study, the potential of BMNPs to increase the temperature upon irradiation of a laser beam in the near infrared, at a wavelength at which tissues become partially transparent, is explored. Moreover, our results also demonstrate the synergy between photothermia and chemotherapy in terms of drug release and cytotoxicity, by using BMNPs functionalized with doxorubicin, and the effectiveness of this combination therapy against tumor cells in in vitro experiments. Therefore, the findings of the present study open the possibility of a novel, alternative approach to fight localized tumors.Ministerio de Economía y Competitividad (CGL2016- 76723 and PID2019-109294RB-100 projects)Ramón y Cajal program (RYC-2014-16901)Junta de Andalucía. Programa Operativo FEDER 2014–2020. (A1-FQM-341-UGR18, C-FQM-497-UGR18, A-BIO376-UGR18).Andalusian regional government (CTS-236)Formación de Doctores 2018 grant (ref. PRE2018-085440) from the Ministerio de Ciencia, Innovación y Universidades (Spain)Unidad Cientifica de Excelencia UCE-PP2016-05Instituto de Biotecnología of the University of Granad

Tuning properties of biomimetic magnetic nanoparticles by combining magnetosome associated proteins

The role of magnetosome associated proteins on the in vitro synthesis of magnetite nanoparticles has gained interest, both to obtain a better understanding of the magnetosome biomineralization process and to be able to produce novel magnetosome-like biomimetic nanoparticles. Up to now, only one recombinant protein has been used at the time to in vitro form biomimetic magnetite precipitates, being that a scenario far enough from what probably occurs in the magnetosome. In the present study, both Mms6 and MamC from Magnetococcus marinus MC-1 have been used to in vitro form biomimetic magnetites. Our results show that MamC and Mms6 have different, but complementary, effects on in vitro magnetite nucleation and growth. MamC seems to control the kinetics of magnetite nucleation while Mms6 seems to preferably control the kinetics for crystal growth. Our results from the present study also indicate that it is possible to combine both proteins to tune the properties of the resulting biomimetic magnetites. In particular, by changing the relative ratio of these proteins, better faceted and/or larger magnetite crystals with, consequently, different magnetic moment per particle could be obtained. This study provides with tools to obtain new biomimetic nanoparticles with a potential utility for biotechnological applicationsWe acknowledge projects CGL2013-46612 and CGL2016-76723 from the Ministerio de Economía y Competitividad from SPAIN and Fondo Europeo de Desarrollo Regional (FEDER) for financial support and Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada. Thanks go to CIC personnel of the University of Granada for technical assistance in the CD, TEM, SQUID and Unidad de Radioquímica e Inmunoanalisis (LAR), to the Proteomics Unit personnel of the Institute of Parasitology and Biomedicine “López- Neyra” (IPBLN) for technical assistance in the PMF and PFF by MALDI-TOF/TOF, and to the University of Málaga for technical assintance in HRTEM measurements

Reactive oxygen species (ROS) production in HepG2 cancer cell line through the application of localized alternating magnetic field

This research work is supported by Ministerio de Economia y Competitividad (CGL2016-76723 project), Ramon y Cajal programme (RYC-2014-16901), Junta de Andalucia. Programa Operativo FEDER 2014-2020. (A1-FQM-341-UGR18, C-FQM-497-UGR18, A-BIO-376-UGR18). This research was also aided by the Andalusian regional government (CTS-236). Alberto Sola-Leyva holds a Formacion de Doctores 2018 grant (ref. PRE2018-085440) from the Ministerio de Ciencia, Innovacion y Universidades (Spain). Ylenia Jabalera wants to acknowledge a FPU2016 grant (ref. FPU16_04580) from the Ministerio de Educacion, Ciencia y Deporte y Competitividad (Spain) and Unidad Cientifica de Excelencia UCE-PP2016-05 of the University of Granada. This study is part of a PhD Thesis conducted at the University of Granada, Spain. Finally, thanks go to the CIC personnel of the University of Granada for technical assistance in the TEM.Recent studies have shown the potential of magnetic hyperthermia in cancer treatments. However, the underlying mechanisms involved have not been yet fully described. In particular, the cell death related to magnetic hyperthermia observed in cultures incubated with low concentration of magnetic nanoparticles and under a low intensity alternating magnetic field, in which a macroscopic temperature rise is not observed, is still not understood. In the present study, we investigate the production of intracellular Reactive Oxygen Species (ROS) as a mechanism to induce cell death under these conditions. In this study, the production and influence of ROS on the viability of HepG2 human hepatoma cells (used as a model cell line) are analyzed under the application of variable magnetic fields using hyperthermia agents, such as biomimetic magnetic nanoparticles (BMNPs) mediated by magnetosome MamC protein fromMagnetococcus marinusMC-1. The results show that intracellular ROS production increases up to similar to 90% following upon the exposure of AMF to HepG2 cells containing BMNPs, which could determine the loss of cell viability (up to similar to 40% reduction) without a significant rise in temperature. Such ROS production is linked to mitochondrial dysfunction caused by the application of AMF to cells containing BMNPs.Spanish Government CGL2016-76723Spanish Government RYC-2014-16901Junta de AndaluciaPrograma Operativo FEDER 2014-2020 A1-FQM-341-UGR18 C-FQM-497-UGR18 A-BIO-376-UGR18Andalusian regional government CTS-236Ministerio de Ciencia, Innovacion y Universidades (Spain) PRE2018-085440Ministerio de Educacion, Ciencia y Deporte y Competitividad (Spain) FPU16_04580Unidad Cientifica de Excelencia of the University of Granada UCE-PP2016-0

Enhancement of Magnetic Hyperthermia by Mixing Synthetic Inorganic and Biomimetic Magnetic Nanoparticles

In this work we report on the synthesis and characterization of magnetic nanoparticles of two distinct origins, one inorganic (MNPs) and the other biomimetic (BMNPs), the latter based on a process of bacterial synthesis. Each of these two kinds of particles has its own advantages when used separately with biomedical purposes. Thus, BMNPs present an isoelectric point below neutrality (around pH 4.4), while MNPs show a zero-zeta potential at pH 7, and appear to be excellent agents for magnetic hyperthermia. This means that the biomimetic particles are better suited to be loaded with drug molecules positively charged at neutral pH (notably, doxorubicin, for instance) and releasing it at the acidic tumor environment. In turn, MNPs may provide their transport capabilities under a magnetic field. In this study it is proposed to use a mixture of both kinds of particles at two different concentrations, trying to get the best from each of them. We study which mixture performs better from different points of view, like stability and magnetic hyperthermia response, while keeping suitable drug transport capabilities. This composite system is proposed as a close to ideal drug vehicle with added enhanced hyperthermia response.We wish to thank FPU2016 grant (Ref. FPU16-04580), RYC-2014-6901 (MINECO, Spain), CGL2016-76723 (MINECO, Spain and FEDER, EU), Unidad Científica de Excelencia UCE-PP2016-05 (UGR) and Plan Propio Beca de iniciación a la investigación para estudiantes de master (UGR)

Biomimetic Magnetoliposomes as Oxaliplatin Nanocarriers: In Vitro Study for Potential Application in Colon Cancer

Current chemotherapy for colorectal cancer (CRC) includes the use of oxaliplatin (Oxa), a first-line cytotoxic drug which, in combination with irinotecan/5-fluorouracil or biologic agents, increases the survival rate of patients. However, the administration of this drug induces side effects that limit its application in patients, making it necessary to develop new tools for targeted chemotherapy. MamC-mediated biomimetic magnetic nanoparticles coupled with Oxa (Oxa-BMNPs) have been previously demonstrated to efficiently reduce the IC50 compared to that of soluble Oxa. However, their strong interaction with the macrophages revealed toxicity and possibility of aggregation. In this scenario, a further improvement of this nanoassembly was necessary. In the present study, Oxa-BMNPs nanoassemblies were enveloped in phosphatidylcholine unilamellar liposomes (both pegylated and non-pegylated). Our results demonstrate that the addition of both a lipid cover and further pegylation improves the biocompatibility and cellular uptake of the Oxa-BMNPs nanoassemblies without significantly reducing their cytotoxic activity in colon cancer cells. In particular, with the pegylated magnetoliposome nanoformulation (a) hemolysis was reduced from 5% to 2%, being now hematocompatibles, (b) red blood cell agglutination was reduced, (c) toxicity in white blood cells was eliminated. This study represents a truly stepforward in this area as describes the production of one of the very few existing nanoformulations that could be used for a local chemotherapy to treat CRC.Ministerio de Economia y Competitividad from Spain CGL2016-76723European Union (EU) CGL2016-76723Junta de Andalucia A-BIO-376-UGR18Unidad Cientifica de Excelencia of the University of Granada UCE-PP2016-05Junta de Andalucia PI-0102-2017Instituto de Salud Carlos III European Union (EU) PI19/01478Andalusian Government CTS-107Ministerio de Educacion, Ciencia y Deporte y Competitividad (Spain) FPU16_04580 FPU16_0171

Eu-Doped Citrate-Coated Carbonated Apatite Luminescent Nanoprobes for Drug Delivery

In the field of Nanomedicine, there is an increasing demand for new inorganic nanophosphors with low cytotoxicity and efficient loading-release ability of drugs for applications in bioimaging and drug delivery. This work assesses the potentiality of matured Eu-doped citrate-coated carbonated apatite nanoparticles to be used as theranostic platforms, for bioimaging, as luminescent nanoprobes, and for drug delivery applications, using Doxorubicin as a model drug. The drug adsorption isotherm fits the Langmuir–Freundlich (LF) model, showing that the Eu:cit-cAp nanoparticles can carry a maximum of 0.29 +/- 0.02 mg Doxo mg Eu:cit-cAp-1 (Qmax). The affinity constant KFL for this binding is 44 +/- 2 mL mg-1, and the cooperativity coefficient r is 6 +/- 1. The nanoparticle suspensions presented charge reversion from negative to positive after loading with Doxo as revealed by the c-potential versus pH characterization. The release of drug from the loaded nanoparticles was found to be strongly pH-dependent, being around 5 wt % at physiological pH 7.4 and 20 wt % at pH 5, in experiments lasting 24 h. Luminescence spectroscopic measurements of Doxo-loaded nanoparticles revealed the increase of luminescence with a decrease in the amount of adsorbed Doxo, due to the so-called inner filter effect. The nanoparticles free of Doxo were cytocompatible when interacted with two human cell lines derived respectively from a gastric carcinoma (GTL-16), and a hepatocarcinoma (Huh7), while Doxo-loaded nanoparticles displayed significant toxicity in a dose-dependent relationship. Therefore, the new nanoassemblies might have a dual function, as nanoprobes in bioimaging by detecting the fate of the nanoparticles in biological environments, and for monitoring the delivery of the drug in such environments, by measuring the rise of the luminescence provided by the desorption of Doxo.This research was funded by Spanish Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades and co-funded with FEDER, UE, Project No. PGC2018-102047-B-I00 (MCIU/AEI/FEDER, UE). The APC was funded by Grant No. PGC2018-102047-B-I00 (MCIU/AEI/FEDER, UE). C.J.-L. thanks project CGL2016-76723 (MINECO/FEDER, UE). Y.J. wants to acknowledge an FPU2016 grant (Ref. FPU16_04580)

Oxaliplatin–Biomimetic Magnetic Nanoparticle Assemblies for Colon Cancer-Targeted Chemotherapy: An In Vitro Study

Conventional chemotherapy against colorectal cancer (CRC), the third most common cancer in the world, includes oxaliplatin (Oxa) which induces serious unwanted side effects that limit the efficiency of treatment. Therefore, alternative therapeutic approaches are urgently required. In this work, biomimetic magnetic nanoparticles (BMNPs) mediated by MamC were coupled to Oxa to evaluate the potential of the Oxa–BMNP nanoassembly for directed local delivery of the drug as a proof of concept for the future development of targeted chemotherapy against CRC. Electrostatic interactions between Oxa and BMNPs trigger the formation of the nanoassembly and keep it stable at physiological pH. When the BMNPs become neutral at acidic pH values, the Oxa is released, and such a release is greatly potentiated by hyperthermia. The coupling of the drug with the BMNPs improves its toxicity to even higher levels than the soluble drug, probably because of the fast internalization of the nanoassembly by tumor cells through endocytosis. In addition, the BMNPs are cytocompatible and non-hemolytic, providing positive feedback as a proof of concept for the nanoassembly. Our study clearly demonstrates the applicability of Oxa–BMNP in colon cancer and offers a promising nanoassembly for targeted chemotherapy against this type of tumor.This work was also supported by the Consejería de Salud de la Junta de Andalucía (projects PI-0476-2016 and PI-0102-2017). Y.J. and B.G.-P. acknowledge FPU2016 grants (FPU16_04580 and FPU16_01716, respectively) from the Ministerio de Educación, Ciencia, y Deporte y Competitividad (Spain)