Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B. bubalis) chromosome 20

<p>Abstract</p> <p>Background</p> <p>Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method.</p> <p>Results</p> <p>As proof of principle for this dissociation curve method, we generated new maps of river buffalo (<it>Bubalus bubalis</it>) chromosome 20 by both dissociation curve analysis and conventional marker scoring. We also obtained sequence data to augment dissociation curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46%) were suitable as cross-species markers for dissociation curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly.</p> <p>Conclusion</p> <p>Dissociation curve analysis is reliable and efficient for radiation hybrid panel scoring, and is more sensitive and robust than conventional gel-based typing methods. Several markers could be scored only by the new method, and ambiguous scores were reduced. PCR-based dissociation curve analysis decreases both time and resources needed for construction of radiation hybrid panel marker maps and represents a significant improvement over gel-based methods in any species.</p

Water Buffalo Genome Science Comes of Age

The water buffalo is vital to the lives of small farmers and to the economy of many countries worldwide. Not only are they draught animals, but they are also a source of meat, horns, skin and particularly the rich and precious milk that may be converted to creams, butter, yogurt and many cheeses. Genome analysis of water buffalo has advanced significantly in recent years. This review focuses on currently available genome resources in water buffalo in terms of cytogenetic characterization, whole genome mapping and next generation sequencing. No doubt, these resources indicate that genome science comes of age in the species and will provide knowledge and technologies to help optimize production potential, reproduction efficiency, product quality, nutritional value and resistance to diseases. As water buffalo and domestic cattle, both members of the Bovidae family, are closely related, the vast amount of cattle genetic/genomic resources might serve as shortcuts for the buffalo community to further advance genome science and biotechnologies in the species

Stealth magnetoliposomes based on calcium-substituted magnesium ferrite nanoparticles for curcumin transport and release

Despite the promising pharmacological properties of curcumin, the transport and effective release of curcumin is still a challenge. The advances in functionalized nanocarriers for curcumin have also been motivated by the anticancer activity of this natural compound, aiming at targeted therapies. Here, stealth (aqueous and solid) magnetoliposomes containing calcium-substituted magnesium ferrite nanoparticles, CaxMg1−xFe2O4 (with x = 0.25, 0.50, 0.75) were developed as nanocarriers for curcumin. The magnetic nanoparticles exhibit superparamagnetic properties and crystalline structure, with sizes below 10 nm. The magnetoliposomes based on these nanoparticles have hydrodynamic diameters around or below 150 nm and a low polydispersity. The influence of an alternating magnetic field (AMF) on drug release over time was evaluated and compared with curcumin release by diffusion. The results suggest the potential of drug-loaded magnetoliposomes as nanocarriers that can be magnetically guided to the tumor sites and act as agents for a synergistic effect combining magnetic hyperthermia and controlled drug release.This research was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UID/FIS/04650/2019) and through the research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020), financed by European Fund of Regional Development (FEDER), COMPETE2020 and Portugal2020. The magnetic measurements were supported by projects UTAP-EXPL/NTec/0046/2017, NORTE-01-0145-FEDER-028538 and PTDC/FIS-MAC/29454/2017. The APC was also funded by FCT. B.D.C. acknowledges FCT for a PhD grant (SFRH/BD/141936/2018)

Novel magnetoliposomes based on shape-anisotropic nanoparticles for combined chemotherapy and magnetic hyperthermia

In this work, a new method for magnetoliposomes synthesis with improved and adequate structural, physicochemical and magnetic properties was developed. The overall results confirmed the development of a promising new method for the synthesis of cubic-shaped magnetic ferrite nanoparticles and a novel route for drug-loaded SMLs with improved features. The properties of these multifunctional nanosystems point to their future effective application in cancer therapy.Compete 2020, Portugal 2020, FEDER, PTDC/QUI-QFI/28020/2017, UIDB/04650/2020. SFRH/BD/141936/201

Magnetoliposomes based on shape anisotropic calcium/magnesium ferrite nanoparticles as nanocarriers for doxorubicin

Multifunctional lipid nanocarriers are a promising therapeutic approach for controlled drug release in cancer therapy. Combining the widely used liposome structure with magnetic nanoparticles in magnetoliposomes allies, the advantages of using liposomes include the possibility to magnetically guide, selectively accumulate, and magnetically control the release of drugs on target. The effectiveness of these nanosystems is intrinsically related to the individual characteristics of the two main components-lipid formulation and magnetic nanoparticles-and their physicochemical combination. Herein, shape-anisotropic calcium-substituted magnesium ferrite nanoparticles (Ca0.25Mg0.75Fe2O4) were prepared for the first time, improving the magnetic properties of spherical counterparts. The nanoparticles revealed a superparamagnetic behavior, high saturation magnetization (50.07 emu/g at 300 K), and a large heating capacity. Furthermore, a new method for the synthesis of solid magnetoliposomes (SMLs) was developed to enhance their magnetic response. The manufacturing technicalities were optimized with different lipid compositions (DPPC, DPPC/Ch, and DPPC/DSPE-PEG) originating nanosystems with optimal sizes for biomedical applications (around or below 150 nm) and low polydispersity index. The high encapsulation efficiency of doxorubicin in these magnetoliposomes was proven, as well as the ability of the drug-loaded nanosystems to interact with cell membrane models and release DOX by fusion. SMLs revealed to reduce doxorubicin interaction with human serum albumin, contributing to a prolonged bioavailability of the drug upon systemic administration. Finally, the drug release kinetic assays revealed a preferable DOX release at hyperthermia temperatures (42 °C) and acidic conditions (pH = 5.5), indicating them as promising controlled release nanocarriers by either internal (pH) and external (alternate magnetic field) stimuli in cancer therapy.This research was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UIDB/04650/2020) and through the research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020), co-financed by European Fund of Regional Development (FEDER), COMPETE2020 and Portugal2020. B.D.C. acknowledges FCT for a PhD grant (SFRH/BD/141936/2018)

Oxidative precipitation synthesis of calcium-doped manganese ferrite nanoparticles for magnetic hyperthermia

Superparamagnetic nanoparticles are of high interest for therapeutic applications. In this work, nanoparticles of calcium-doped manganese ferrites (CaxMn1−xFe2O4) functionalized with citrate were synthesized through thermally assisted oxidative precipitation in aqueous media. The method provided well dispersed aqueous suspensions of nanoparticles through a one-pot synthesis, in which the temperature and Ca/Mn ratio were found to influence the particles microstructure and morphology. Consequently, changes were obtained in the optical and magnetic properties that were studied through UV-Vis absorption and SQUID, respectively. XRD and Raman spectroscopy studies were carried out to assess the microstructural changes associated with stoichiometry of the particles, and the stability in physiological pH was studied through DLS. The nanoparticles displayed high values of magnetization and heating efficiency for several alternating magnetic field conditions, compatible with biological applications. Hereby, the employed method provides a promising strategy for the development of particles with adequate properties for magnetic hyperthermia applications, such as drug delivery and cancer therapy.This work was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UIDB/04650/2020, UIDP/04650/2020), CQUM (UIDB/00686/2020), CICECO Aveiro Institute of Materials (UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020) and by Ministerio de Economía y Competitividad de España (PID2020-113704RB-I00 and PID2020-119242RB-I00), Xunta de Galicia (Centro Singular de Investigación de Galicia—Accreditation 2019-2022 ED431G 2019/06 and IN607A 2018/5 and project ED431C 2020-06), and European Union (EU-ERDF Interreg V-A—Spain-Portugal 0245_IBEROS_1_E, 0712_ACUINANO_1_E, and 0624_2IQBIONEURO_6_E, and Interreg Atlantic Area NANOCULTURE 1.102.531), and the European Union H2020-MSCA-RISE-2019 PEPSA-MATE project. S.R.S. (872233) Veloso acknowledges FCT for a PhD grant (SFRH/BD/144017/2019). Support from MAP-Fis Doctoral Programme is also acknowledged

Magnetoliposomes containing calcium ferrite nanoparticles for applications in breast cancer therapy

Magnetoliposomes containing calcium ferrite (CaFe2O4) nanoparticles were developed and characterized for the first time. CaFe2O4 nanoparticles were covered by a lipid bilayer or entrapped in liposomes forming, respectively, solid or aqueous magnetoliposomes as nanocarriers for new antitumor drugs. The magnetic nanoparticles were characterized by UV/Visible absorption, XRD, HR-TEM, and SQUID, exhibiting sizes of 5.2 ± 1.2 nm (from TEM) and a superparamagnetic behavior. The magnetoliposomes were characterized by DLS and TEM. The incorporation of two new potential antitumor drugs (thienopyridine derivatives) specifically active against breast cancer in these nanosystems was investigated by fluorescence emission and anisotropy. Aqueous magnetoliposomes, with hydrodynamic diameters around 130 nm, and solid magnetoliposomes with sizes of ca. 170 nm, interact with biomembranes by fusion and are able to transport the antitumor drugs with generally high encapsulation efficiencies (70%). These fully biocompatible drug-loaded magnetoliposomes can be promising as therapeutic agents in future applications of combined breast cancer therapy.This research was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UID/FIS/04650/2013; UID/FIS/04650/2019), CQUM (UID/QUI/00686/2016; UID/QUI/00686/2019) and LA-26 (PEst-C/SAU/LA0026/2013), and through the research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020), financed by FCT, European Fund of Regional Development (FEDER), COMPETE2020 and Portugal2020. The magnetic measurements were supported by projects UTAP-EXPL/NTec/0046/2017, NORTE-01-0145-FEDER-028538 e PTDC/FIS-MAC/29454/2017. The APC was also funded by FCT. B.D.C. acknowledges FCT for a PhD grant (SFRH/BD/141936/2018)

Challenges in spintronic platforms for biomedical applications

Integrated spintronic biochip platforms are being developed for portable, point-of-care, diagnostic and cytometric applications [1,2]. Hybrid systems incorporating magnetoresistive sensors are applied to neuroelectronic studies and biomedical imaging, namely magnetoencephalography and magnetocardiography. Lab-on-a-chip MR-based platforms are under development to perform biological studies at the single molecule level. This review introduces and discusses the potential and main characteristics of those MR-based biomedical devices, comparing to the existing technologies, while giving particular examples of targeted applications. Applications to the detection of DNA hybridization events (DNA-chips) [3] and antibody-antigen recognition at immunoassays (immuno-chips) [4] are discussed. Particular examples for cell free DNA and genomic sequences detection, for pathogen (Salmonella enteritidis, see Fig.1) detection and for flow cytometry (separation and counting) of CD34+ magnetically labeled cells coming from bone marrow or cord blood samples are given. Moreover, lateral immuno-assay configurations where analytes are labeled with magnetic nanoparticles are discussed. For biomedical imaging applications, field sensitivity is being pushed towards 1pT/sqrt(Hz) and below in hybrid devices incorporating flux guides with the magnetoresistive element allowing the direct detection of bio-magnetic fields (from brain and heart). For neuroelectronic applications, sensors are being incorporated in microelectrode arrays (Si and polyimide) to record spontaneous or stimulated neural activity (in vitro and in vivo, see Fig.2)

Spintronic devices for biomedical applications

Spintronic devices have been proposed over the past decade for various biomedical applications. These include static or dynamic biomolecular recognition platforms ( DNA-cDNA, antibody-antigen, phage-bacteria, ), cytometer and cell separation devices and lateral bio assay platforms , microelectrode based devices for neuroelectronic applications, and hybrid sensor arrays for imaging applications [1]. The biomolecular recognition platforms include a magnetoresistive sensor array, a set of biomolecular probes ( surface immobilized or in solution) , biological targets labeled with particular magnetic micro beads or magnetic nanoparticles, and arraying architectures and microfluidics used to increase sensitivity and favour probe-target interaction. The platforms also incorporate the proper signal conditioning and processing electronics. Results will be shown for cell free DNA detection as a cancer marker indicator, and for cell detection using phage markers. For neuroelectronic applications, magnetoresistive sensors were fabricated onto Si microelectrode arrays. Experiments probe either extra cellular currents measured in mouse hypocampus slices, or spinal medulla signals probed directly with implanted magnetoresistive electrodes. For deep brain simulation and detection, sensors and electrodes are being fabricated into flexible polyimide probes. Separation between straight electrical contributions and magnetic signals is discussed. For imaging applications ( magneto cardiography) efforts continue to reach pT level detectivity at 1Hz, using hybrid MEMS/magnetoresistive sensor devices. Two architectures will be presented leading to larger DC field mechanical modulation, and therefore increased sensitivity