Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4 ) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.National Science Foundation, grant number EAR-2038207EAR-1423939Ministerio de Economía y Competitividad, SPAIN and Fondo Europeo de Desarrollo Regional, FEDER grant numbers CGL2010-18274 and CGL2013-4661

Biomimetic Magnetic Nanocarriers Drive Choline Kinase Alpha Inhibitor inside Cancer Cells for Combined Chemo-Hyperthermia Therapy

Choline kinase a1 (ChoKa1) has become an excellent antitumor target. Among all the inhibitors synthetized, the new compound Ff35 shows an excellent capacity to inhibit ChoKa1 activity. However, soluble Ff35 is also capable of inhibiting choline uptake, making the inhibitor not selective for ChoKa1. In this study, we designed a new protocol with the aim of disentangling whether the Ff35 biological action is due to the inhibition of the enzyme and/or to the choline uptake. Moreover, we offer an alternative to avoid the inhibition of choline uptake caused by Ff35, since the coupling of Ff35 to novel biomimetic magnetic nanoparticles (BMNPs) allows it to enter the cell through endocytosis without interacting with the choline transporter. This opens the possibility of a clinical use of Ff35. Our results indicate that Ff35-BMNPs nanoassemblies increase the selectivity of Ff35 and have an antiproliferative effect. Also, we demonstrate the effectiveness of the tandem Ff35-BMNPs and hyperthermia.This research was funded by the Ministerio de Economía y Competitividad (CGL2013-46612 and CGL2016-76723 projects), Ramón y Cajal programme (RYC-2014-16901) and the Fondo Europeo de Desarrollo Regional (FEDER). Also, this research was aided by the Andalusian regional government (CTS-236)

Vigilancia de la gripe en España. Resumen de la temporada 2011-2012. Semanas 40/2011 - 04/2012 (del 02 de octubre de 2011 al 29 de enero de 2012)

El nivel de intensidad de la actividad gripal registrado en España, desde el inicio de la temporada 2011-2012, hasta la semana 04/2012 (del 23 al 29 de enero de 2012) fue moderado y se asoció a una circulación mayoritaria de virus de la gripe A(H3). La tasa de incidencia de gripe superó el umbral basal de la temporada 2011-12 en la semana 52/2011, registrándose un incremento paulatino en el porcentaje de muestras positivas a virus gripales, desde la semana 48/2011 hasta la semana 04/2012. Desde la semana 40/2011 hasta la semana 04/2012 se han notificado 103 casos graves hospitalizados confirmados de gripe, de los que siete fallecieron. La mayoría de los casos fueron infecciones por virus de la gripe A(H3). De los casos pertenecientes a los grupos elegibles para vacunación, el 44% había recibido la vacuna antigripal de esta temporada. La actividad gripal en la región templada del hemisferio norte continúa baja, aunque con notables incrementos locales de actividad en el norte de América, oeste de Europa y norte de China. El virus más común en el hemisferio norte ha sido el A(H3), a excepción de Méjico, donde ha circulado de manera predominante el virus de la gripe A(H1N1)pdm09, y China, donde ha predominado el tipo B. La actividad gripal en los países templados del hemisferio sur se sitúa de nuevo en niveles de intertemporada.N

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

mAb-Functionalized Biomimetic MamC-Mediated-Magnetoliposomes as Drug Delivery Systems for Cancer Therapy

In cancer therapy, new therapeutic nanoformulations able to mediate targeted chemotherapy are required. Recently, biomimetic magnetic nanoparticles (BMNPs) mediated by MamC, a magnetosome protein from Magnetococcus marinus MC-1, have proven, in vitro and in vivo, to be effective drug nanocarriers (following the application of an external gradient magnetic field) and to allow combination with hyperthermia. However, these nanoassemblies require further optimization to improve cytocompatibility, stability and active targeting ability. Herein, we describe the production of the magnetoliposomes (LP) embedding BMNPs functionalized (or not) with doxorubicin (DOXO), [LP(+/−DOXO-BMNPs)], and their surface modification with the DO-24 mAb, which targets the human Met/HGF receptor’s ectodomain (overexpressed in many cancers). Nanoformulations were extensively characterized using TEM, DLS, FTIR and when tested in vitro, the lipid coating increased the colloidal stability and their biocompatibility, favoring the cellular uptake in cells overexpressing the cognate receptor. Indeed, the magnetoliposomes mAb-LP(+/−DOXO-BMNPs) exerted a specific active targeting ability by the presence of the mAb that preserved its immunocompetence. Both LP(BMNPs) and mAb-LP(BMNPs) were not toxic to cells, while +/−mAb-LP(DOXO-BMNPs) nanoformulations were indeed cytotoxic. Therefore, this study represents a proof of concept for the development of promising drug carriers for cancer therapy based on local chemotherapy directed by mAbs

Protein crystals as a template for in situ formation of magnetite nanoparticles

Resumen del trabajo presentado a XXXIX Reunión Bienal de la Real Sociedad Española de Química, celebrada en Zaragoza del 25 al 29 de junio de 2023.Crystallisation in confned spaces is a widespread phenomenon in nature. Many processes like frost heave, biomineralisation (bones and seashells), growth of salt and ice crystals in the pores of masonry happen not in a bulk, but in limited volumes. Reducing the volume of the system infuences not only the probability of nucleation, but also the kinetic. As the results, in reduced volumes become possible stabilisation of metastable polymorphs, formation of crystals with preferred orientations, modifcation of morphology, etc. One of the most fascinating cases of crystallisation in confned spaces – formation of magnetite nanoparticles inside magnetotactic bacteria. The particles have unusual morphologies, homogeneous size and superparamagnetic properties. The control of the magnetite nanoparticles formation could be explained by a combination of physical and chemicals factors in which nucleation and growth of magnetite crystals is performed in confned vesicles (magnetosomes) modulated by the interaction with different proteins.bIn this work, we imitate this type of control over the precipitation of magnetite using channels of cross-linked protein crystals (CLPCs). CLPCs with different pore size and amino acid decoration were used to study their infuence on the magnetite precipitation. We obtained gradient distribution of nanoparticles with a narrow size distribution of around 2nm independently of the channel diameter size of the CLPCs. But the pores size infuenced the stabilisation of the Fe-rich phase. In case of lysozyme (the smallest pore size), the amorphous metastable intermediates of magnetite were stabilised, while in glucose isomerase crystals, the amorphous phase were recrystallised into the crystalline state of magnetite. Our results demonstrate control in the size and stability of those Fe-rich nanoparticles and the potential use of the CLPCs as excellent scaffolds to study the crystallisation in confnement.Peer reviewe

Enhanced Cytotoxic Effect of TAT–PLGA-Embedded DOXO Carried by Biomimetic Magnetic Nanoparticles upon Combination with Magnetic Hyperthermia and Photothermia

This research work was supported by the Ministerio de Economia y Competitividad (CGL2016-76723), the Ministerio de Ciencia e Innovacion (PID2019-109294RB-100) projects, Ramon y Cajal program (RYC-2014-16901), the Junta de Andalucia Programa Operativo FEDER 2014-2020 (A1-FQM-341-UGR18, C-FQM-497-UGR18, A-BIO-376-UGR18), and the Proyectos de I + D + I, del Plan Andaluz de Investigacion, Desarrollo e Innovacion (P20_00208 and P20_00346). This research was also aided by the Andalusian regional government (CTS-236) and by the FUR (Fondo Unico della Ricerca, University of Verona)of Dr. M. Perduca. 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 an FPU2016 grant (ref. FPU16_04580) from the Ministerio de Educacion, Ciencia, y Deporte y Competitividad (Spain).The synergy between directed chemotherapy and thermal therapy (both magnetic hyperthermia and photothermia) mediated by a nanoassembly composed of functionalized biomimetic magnetic nanoparticles (BMNPs) with the chemotherapeutic drug doxorubicin (DOXO) covered by the polymer poly(lactic-co-glycolic acid) (PLGA), decorated with TAT peptide (here referred to as TAT–PLGA(DOXO-BMNPs)) is explored in the present study. The rationale behind this nanoassembly lies in an optimization of the nanoformulation DOXO-BMNPs, already demonstrated to be more efficient against tumor cells, both in vitro and in vivo, than systemic traditional therapies. By embedding DOXO-BMNPs into PLGA, which is further functionalized with the cell-penetrating TAT peptide, the resulting nanoassembly is able to mediate drug transport (using DOXO as a drug model) and behaves as a hyperthermic agent (induced by an alternating magnetic field (AMF) or by laser irradiation with a laser power density of 2 W/cm2). Our results obtained using the HepG2 cell line show that there is a synergy between chemotherapy and thermal therapy that results in a stronger cytotoxic effect when compared to that caused by the soluble DOXO. This is probably due to the enhanced DOXO release occurring upon the application of the thermal therapy, as well as the induced local temperature rise mediated by BMNPs in the nanoassembly following exposition to AMF or to near-infrared (NIR) laser irradiation. These results represent a proof of concept demonstrating that TAT–PLGA(DOXO-BMNPs) can be used to efficiently combine therapies against tumor cells, which is a step forward in the transition from systemic to local treatments.Spanish Government CGL2016-76723Instituto de Salud Carlos III Spanish GovernmentEuropean Commission PID2019-109294RB-100Spanish Government RYC-2014-16901Junta de Andalucia A1-FQM-341-UGR18 C-FQM-497-UGR18 A-BIO-376-UGR18Proyectos de I + D + I, del Plan Andaluz de Investigacion, Desarrollo e Innovacion P20_00208 P20_00346Ministerio de Ciencia, Innovacion, y Universidades (Spain) PRE2018-085440Ministerio de Educacion, Ciencia, y Deporte y Competitividad (Spain) FPU16_04580Andalusian regional government CTS-23