Temporal and spatial resolution of magnetosome degradation at the subcellular level in a 3D lung carcinoma model

Magnetic nanoparticles offer many exciting possibilities in biomedicine, from cell imaging to cancer treatment. One of the currently researched nanoparticles are magnetosomes, magnetite nanoparticles of high chemical purity synthesized by magnetotactic bacteria. Despite their therapeutic potential, very little is known about their degradation in human cells, and even less so of their degradation within tumours. In an effort to explore the potential of magnetosomes for cancer treatment, we have explored their degradation process in a 3D human lung carcinoma model at the subcellular level and with nanometre scale resolution. We have used state of the art hard X-ray probes (nano-XANES and nano-XRF), which allow for identification of distinct iron phases in each region of the cell. Our results reveal the progression of magnetite oxidation to maghemite within magnetosomes, and the biosynthesis of magnetite and ferrihydrite by ferritin.This work was supported by grants PID2020-115704RB-C31 funded by the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIN/AEI/10.13039/501100011033) , and by IT1479-22 funded by the Basque Government. Electron microscopy performed at Nanolab, University of Pannonia was supported by the Hungarian National Research, Development and Innovation Office (NKFIH) under grant RRF-2.3.1-21-2022-00014. This research used resources HXN (3-ID) of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. European Synchrotron Radiation Facility (ESRF) provided beamtime at BM23 beamline

Flexible multiplex PCR to detect SARS-CoV-2, coronavirus OC43 and influenza A virus in nasopharyngeal swab samples

Introduction Quantitative reverse transcription PCR (RT-qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given that it will almost certainly continue to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits. Methods and results In this study, combinations of four primers/probe sets were used to construct a flexible RT-qPCR assay which is capable of discriminating between SARS-CoV-2 and the seasonal human coronavirus HCoV-OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed. Conclusions This study provides an efficient method for the simultaneous detection of SARS-CoV-2, HCoV-OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results. Significance and impact of the study The multiplex RT-qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.This research was funded by Basque Government, grants numbers 2020333042 and IT1362‐19. LM‐S have received a predoctoral Grant from Basque Government

Intracellular transformation and disposal mechanisms of magnetosomes in macrophages and cancer cells

International audienceMagnetosomes are magnetite nanoparticles biosynthesized by magnetotactic bacteria. Given their potential clinical applications for the diagnosis and treatment of cancer, it is essential to understand what becomes of them once they are within the body. With this aim, here we have followed the intracellular long‐term fate of magnetosomes in two cell types: cancer cells (A549 cell line), because they are the actual target for the therapeutic activity of the magnetosomes, and macrophages (RAW 264.7 cell line), because of their role at capturing foreign agents. It is shown that cells dispose of magnetosomes using three mechanisms: splitting them into daughter cells, excreting them to the surrounding environment, and degrading them yielding less or non‐magnetic iron products. A deeper insight into the degradation mechanisms by means of time‐resolved X‐ray absorption near‐edge structure (XANES) spectroscopy has allowed us to follow the intracellular biotransformation of magnetosomes by identifying and quantifying the iron species occurring during the process. In both cell types there is a first oxidation of magnetite to maghemite and then, earlier in macrophages than in cancer cells, ferrihydrite starts to appear. Given that ferrihydrite is the iron mineral phase stored in the cores of ferritin proteins, this suggests that cells use the iron released from the degradation of magnetosomes to load ferritin. Comparison of both cellular types evidences that macrophages are more efficient at disposing of magnetosomes than cancer cells, attributed to their role in degrading external debris and in iron homeostasis

Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro: Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.This work was supported by two SAIOTEK grants (S-PE09UN41 and S-PE11UN070) and a consolidated research group IT343/10 of Basque Government,two grants from the University of the Basque Country (UE 03/04 and UE 08/14) and a UFI Grant (UFI11/25) from the University of the Basque Country UPV/EHU. Andoni Ramirez-Garcia was supported by a fellowship from the Government of the Basque Countr

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications

Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology.This research was funded by the Basque Government, grant number IT1362-19. X.G. and U.P.-C. were funded by the Basque Government and the UPV/EHU, respectively

Optimal Parameters for Hyperthermia Treatment Using Biomineralized Magnetite Nanoparticles: Theoretical and Experimental Approach

We hereby present experimental and theoretical insights on the use of biomineralized magnetite nanoparticles, called magnetosomes, as heat nanoinductors in the magnetic hyperthermia technique. The heating efficiency or specific absorption rate of magnetosomes extracted from Magnetospirillum gryphiswaldense bacteria and immersed in water and agarose gel, was directly determined from the hysteresis loops obtained at different frequencies and magnetic field amplitudes. We demonstrate that heat production of magnetosomes can be predicted in the framework of the Stoner−Wohlfarth theory of uniaxial magnetic anisotropy subjected to significant dipolar interactions, which can be described in terms of an interaction anisotropy superimposed to that of each particle. Based on these findings, we propose optimal magnetic field amplitude and frequency values in order to maximize the heat production while keeping the undesired eddy current effects below safe and tolerable limits. The efficiency of magnetosomes as heat generators and their impact on cell viability has been checked in macrophage cells. Our results clearly indicate that the hyperthermia treatment causes both cell death and inhibition of cell proliferation. Specifically, only 36% of the treated macrophages remained alive 2 h after alternating magnetic field exposure, and 24 h later the percentage fell to 22%