Magnetic Purification Research Articles

Abstract 3378: Rapid and efficient sample preparations for cell free DNA

Abstract The analysis of cell-free DNA (cfDNA) as a sensitive biomarker for cancer prognosis, diagnosis and monitoring resulted in a need for efficient and fast cfDNA isolation. The traditional cfDNA purification methods using chaotropic solvent to separate nucleic acids from plasma through column or magnetic bead purification processes are usually laborious and time consuming, involving multiple washing steps, and the residual organic solvent could be carried over to the elution solution that could cause downstream PCR inhibition. Herein, we developed a novel particle surface chemistry that can biochemically bind rapidly (<1min) and release nucleic acids rapidly (<1min) under different buffer conditions without needing chaotropic solvent. We further optimized the binding and elution buffer solutions that allow to isolate cfDNA from plasma within 5-10 minutes in the sample volumes from 1 ml to 4 ml. The real-time quantitative PCR indicated DNA extraction yields were comparable or slightly better than the traditional methods for both small and large volumes. The novel bead chemistry and the optimized binding and elution buffers are not only compatible to high throughput format but could also be applied to a variety sample types for rapid sample preparations. Citation Format: Zhiyang Zeng, Andrew Taft, Kasen Riemersma, Connor Fitzgerald, Mike Scurria, Spenccer Hermanson, Wenhui Zhou. Rapid and efficient sample preparations for cell free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3378.

Magnetically purification/immobilization of poly histidine-tagged proteins by PEGylated magnetic graphene oxide nanocomposites

Carbon-based nanomaterials have many applications in biomedicine due to their unique mechanical, chemical, and biological properties. Among them, graphene has received special attention due to its very high specific surface area, high flexibility, and chemical stability. In this study, graphene oxide was first functionalized with amine groups (GO-NH2) and then Fe3O4 nanoparticles were deposited on it using the hydrothermal method. In addition, polyethylene glycol (PEG) was attached to the magnetic graphene nanoparticles to increase their stability and solubility. Finally, PEGylated magnetic graphene nanocomposites were functionalized with nickel-nitrilotriacetic acid (NTA-Ni+2) to bind to the poly-histidine tag in recombinant proteins. The resulting nanocomposites (MG-PEG-NTA-Ni+2) were then used for magnetic immobilization and purification of recombinant β-NGF as a protein with his-tag sequence. Binding and purification were confirmed by FTIR and SDS-PAGE techniques, respectively. Importantly, differentiation of the PC12 cell line into neurons demonstrated that the purified β-NGF was fully functional. Our results suggest that MG-PEG-NTA-Ni+2 nanocomposites may be a suitable alternative to commercial resins for rapid and specific protein immobilization and purification.

A refined protocol for the isolation and monoculture of primary mouse renal peritubular endothelial cells.

During an episode of acute kidney injury (AKI), a sudden and rapid decline in renal function is often accompanied by a persistent reduction in mitochondrial function, microvasculature dysfunction/rarefaction, and tubular epithelial injury/necrosis. Additionally, patients who have experienced an AKI are at an elevated risk of developing other progressive renal, cardiovascular, and cardiorenal related diseases. While restoration of the microvasculature is imperative for oxygen and nutrient delivery/transport during proper renal repair processes, the mechanism(s) by which neovascularization and/or inhibition of microvascular dysfunction improves renal recovery remain understudied. Interestingly, pharmacological stimulation of mitochondrial biogenesis (MB) post-AKI has been shown to restore mitochondrial and renal function in mice. Thus, targeting MB pathways in microvasculature endothelial cell (MV-EC) may provide a novel strategy to improve renal vascular function and repair processes post-AKI. However, limitations to studying such mechanisms include a lack of commercially available primary renal peritubular MV-ECs, the variability in both purity and outgrowth of primary renal MV-EC in monoculture, the tendency of primary renal MV-ECs to undergo phenotypic loss in primary monoculture, and a limited quantity of published protocols to obtain primary renal peritubular MV-ECs. Thus, we focused on refining the isolation and phenotypic retention of mouse renal peritubular endothelial cells (MRPEC) for future physiological and pharmacological based studies. Here, we present a refined isolation method that augments the purity, outgrowth, and phenotypic retention of primary MRPEC monocultures by utilizing a collagenase type I enzymatic digestion, CD326+ (EPCAM) magnetic microbead epithelial cell depletion, and two CD146+ (MCAM) magnetic microbead purification cycles to achieve a monoculture MRPEC purity of ≅ 91-99% by all markers evaluated.

Rapid quantitative detection of Klebsiella pneumoniae in infants with severe infection disease by point-of-care immunochromatographic technique based on nanofluorescent microspheres.

Background: Klebsiella pneumoniae (KP, K. pneumoniae) is one of the most important nosocomial pathogens that cause severe respiratory infections. As evolutionary high-toxic strains with drug resistance genes increase year by year, the infections caused by it are often accompanied by high mortality, which may be fatal to infants and can cause invasive infections in healthy adults. At present, the traditional clinical methods for detecting K. pneumoniae are cumbersome and time-consuming, and the accuracy and sensitivity are not high. In this study, nanofluorescent microsphere (nFM)-based immunochromatographic test strip (ICTS) quantitative testing platform were developed for point-of-care testing (POCT) method of K. pneumoniae. Methods: 19 clinical samples of infants were collected, the genus-specific gene of mdh was screened from K. pneumoniae. Polymerase chain reaction (PCR) combined with nFM-ICTS based on magnetic purification assay (PCR-ICTS) and strand exchange amplification (SEA) combined with nFM-ICTS based on magnetic purification assay (SEA-ICTS) were developed for the quantitative detection of K. pneumoniae. The sensitivity and specificity of SEA-ICTS and PCR-ICTS were demonstrated by the existing used classical microbiological methods, the real-time fluorescent quantitative PCR (RTFQ-PCR) and PCR assay based on agarose gel electrophoresis (PCR-GE). Results: Under optimum working conditions, the detection limits of PCR-GE, RTFQ-PCR, PCR-ICTS and SEA-ICTS are 7.7 × 10-3, 2.5 × 10-6, 7.7 × 10-6, 2.82 × 10-7ng/μL, respectively. The SEA-ICTS and PCR-ICTS assays can quickly identify K. pneumoniae, and could specifically distinguish K. pneumoniae samples from non-K. pneumoniae samples. Experiments have shown a diagnostic agreement of 100% between immunochromatographic test strip methods and the traditional clinical methods on the detection of clinical samples. During the purification process, the Silicon coated magnetic nanoparticles (Si-MNPs) were used to removed false positive results effectively from the products, which showed of great screening ability. The SEA-ICTS method was developed based on PCR-ICTS, which is a more rapid (20min), low-costed method compared with PCR-ICTS assay for the detection of K. pneumoniae in infants. Only need a cheap thermostatic water bath and takes a short detection time, this new method can potentially serve as an efficient point-of-care testing method for on-site detection of pathogens and disease outbreaks without fluorescent polymerase chain reaction instruments and professional technicians operation.

Au-Loaded Superparamagnetic Mesoporous Bimetallic CoFeB Nanovehicles for Sensitive Autoantibody Detection.

Construction of a well-defined mesoporous nanostructure is crucial for applying nonnoble metals in catalysis and biomedicine owing to their highly exposed active sites and accessible surfaces. However, it remains a great challenge to controllably synthesize superparamagnetic CoFe-based mesoporous nanospheres with tunable compositions and exposed large pores, which are sought for immobilization or adsorption of guest molecules for magnetic capture, isolation, preconcentration, and purification. Herein, a facile assembly strategy of a block copolymer was developed to fabricate a mesoporous CoFeB amorphous alloy with abundant metallic Co/Fe atoms, which served as an ideal scaffold for well-dispersed loading of Au nanoparticles (∼3.1 nm) via the galvanic replacement reaction. The prepared Au-CoFeB possessed high saturation magnetization as well as uniform and large open mesopores (∼12.5 nm), which provided ample accessibility to biomolecules, such as nucleic acids, enzymes, proteins, and antibodies. Through this distinctive combination of superparamagnetism (CoFeB) and biofavorability (Au), the resulting Au-CoFeB was employed as a dispersible nanovehicle for the direct capture and isolation of p53 autoantibody from serum samples. Highly sensitive detection of the autoantibody was achieved with a limit of detection of 0.006 U/mL, which was 50 times lower than that of the conventional p53-ELISA kit-based detection system. Our assay is capable of quantifying differential expression patterns for detecting p53 autoantibodies in ovarian cancer patients. This assay provides a rapid, inexpensive, and portable platform with the potential to detect a wide range of clinically relevant protein biomarkers.

ОПТИМИЗАЦИЯ КОНСТРУКТИВНЫХ ПАРАМЕТРОВ ЭЛЕКТРОМАГНИТНОГО СЕПАРАТОРА С ПОСТОЯННЫМИ МАГНИТАМИ

The relevance Cleaning of bulk agricultural products from metallomagnetic impurities is an urgent problem that needs to be solved today, as it leads to significant material and economic losses. Magnetic and electromagnetic purification methods are based on the difference in the magnetic properties of the separated products, mainly on the difference in their magnetic susceptibility. In the working area of the separator, there is a zone of attraction of magnetic particles, the height of which is determined by the minimum distance between the working body and the surface of the fixed pole, and the zone of transportation of the magnetic product to the unloading site. The magnetic field in the working area of the separator is created by systems of permanent magnets or electromagnetic systems with winding. Parametric design is the basis for conducting design work during the development of an electromagnetic separator and allows you to clarify the final purpose of the installation already at the early stages of the project. Aim of research This article deals with optimization of design and mode parameters of electromagnetic separator with permanent magnets. Research methods In this study it is proposed to use nomogram-parametric design, both electromagnetic separator and its mode parameters. Nomogram parametrization consists in creating a nomogram to determine the mode parameters of electromagnetic separator with permanent magnets. Results Based on the established relationship of design and mode parameters of separation in the separator's working zone, a nomogram was developed that allows to determine the parameters of the electromagnetic system, the value of magnetic induction, depending on the heterogeneity of the magnetic field, as well as solve the inverse problem, determine the design parameters of the working zone with the given indicators of the electromagnetic system.

A dual paper-based nucleic acid extraction method from blood in under ten minutes for point-of-care diagnostics.

Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution (eluted disk) as well as direct placement of the disk in the LAMP reaction (in situ disk). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes.

IMPRESSÃO 3D DE SEPARADOR MAGNÉTICO: UMA ABORDAGEM ACESSÍVEL PARA O PREPARO DE AMOSTRAS NO DIAGNÓSTICO DA COVID-19

3D PRINTING OF MAGNETIC SEPARATOR: AN AFFORDABLE APPROACH TO SAMPLE PREPARATION IN THE COVID-19 DIAGNOSIS. This report describes the fabrication of a low-cost magnetic separator holder combining 3D printing and compact neodymium blocks for allowing magnetic extraction and purification of RNA from samples collected by nasopharyngeal swab from patients infected by SARS-CoV-2. The device was designed to contain 24 entrances for plastic microtubes in an arrangement like a commercial device. The proof of concept of the proposed device was successfully demonstrated through the sample extraction and purification of swab samples collected from eight patients suspected of SARS-CoV-2 infection. The sample preparation protocol was performed using a commercial kit containing magnetic beads and different solutions. The performance of the printed device was compared to a commercial magnetic separator, usually employed in the golden standard techniques. The fabrication of the 3D printed magnetic separator was completed under optimized printing conditions within 6 h at cost of 4 USD per unit. The RNA extracted from samples using both devices was analyzed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and the achieved results have indicated no statistical different at confidence level of 95%. Based on the achievements, the use of 3D printing and neodymium blocks have demonstrated an alternative route to be used in routing analysis associated to COVID-19 diagnosis.

Impact of DNA Extraction Methods on Quantitative PCR Telomere Length Assay Precision in Human Saliva Samples

Telomere length (TL) has emerged as a promising replicative cellular aging marker that reflects both genetic and non-genetic influences. Quantitative PCR (qPCR) TL measurement has been favored as a cost-effective method that can be easily implemented, especially in population studies with limited quantities of source material. However, several recent reports have revealed inconsistencies in telomere length measurements when applying different DNA extraction methods to the same source material. In this study we tested three DNA extraction methods on saliva samples from 48 participants of the National Growth and Health Study (NGHS) collected with DNA Genotek’s Oragene kit. The chosen extraction kits represent three distinct approaches to genomic DNA extraction from lysed cells and we employed two different operators to carry out all assays on the same samples. We measured DNA yield and quality and calculated the between-operator agreement of qPCR TL measurements (intraclass correlation, ICC). Our analyses showed that while both QIAamp and Agencourt DNAdvance had higher agreement between the 2 operators (ICC=0.937, CI [0.891, 0.965] and ICC=0.95, CI [0.911, 0.972] respectively), compared to PrepIT kit (ICC=0.809, CI [0.678, 0.889]), QIAamp extracted DNA samples were notably degraded. Using generalizability theory, we found that the participant-by-extraction-method interaction explained about 10% of total variation in TL, suggesting that TL differences across methods are somewhat participant-specific. Therefore, our results suggest that the among the three DNA extraction methods tested, Agencourt (magnetic bead purification) is the preferred kit, and we also strongly recommend against combining different extraction methods within a study population.

High throughput method of 16S rRNA gene sequencing library preparation for plant root microbial community profiling

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have increased sample handling efficiency in a two-step PCR amplification protocol for 16S rRNA gene sequencing of plant root microbiota, improving DNA extraction using AMPure XP magnetic beads and PCR purification using exonuclease. These modifications reduce sample handling and capture microbial diversity comparable to that obtained by the manual method. We found a buffer with AMPure XP magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. We also demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. In addition, our method generated comparable microbiome profile data in plant roots and soils to that of using common commercially available DNA extraction kits, such as DNeasy PowerSoil Pro Kit and FastDNA SPIN Kit for Soil. Our method offers a simple and high-throughput option for maintaining the quality of plant root microbial community profiling.

An assessment of DNA extraction methods from blood-stained soil in forensic science

In forensic crime scene investigations, biological fluids such as blood are commonly found in soil. However, the analysis of blood-stained soil can be challenging due to the presence of inhibitors which limit the effective extraction and amplification of the deoxyribonucleic acid (DNA) required to produce a reportable DNA profile. There are some extraction methods that have been applied to blood-stained soil in forensic science, but these have produced sporadic results. This research has taken a number of different extraction methods from the fields of ancient DNA and environmental DNA and broken them down into the individual steps of pre-treatment, incubation, separation and purification. These steps were assessed independently then combined into various extraction methods to determine the best technique that can effectively and reliably profile human DNA from blood-stained soil. Testing involved assessment of three extraction buffers, (cetyltrimethylammonium bromide, guanidine thiocyanate, and proteinase K), four pre-treatment methods, (polyvinylpyrrolidone, ethylenediaminetetraacetic acid, hydrochloric acid, and sodium hydroxide), three separation steps, (centrifugation, phenol chloroform, and chloroform) and four purification steps, (size exclusion chromatography, bind elute columns, isopropanol precipitation and silica magnetic beads). The most effective procedure was found to be a polyvinylpyrrolidone pre-treatment with a proteinase K extraction buffer followed by magnetic silica bead purification with or without centrifugation. However, centrifugation separation was found to be equally effective after the pre-treatment step as after the incubation step. Our results shows that most of the current forensic procedures would benefit from the addition of a pre-treatment step prior to processing through the automated DNA profiling pipeline.

Status and initial experiments of DRAGON-V facility for lithium-lead blanket technologies of hydrogen fusion reactors

A dual-coolant integrated experimental facility named DRAGON-V has been developed at the Institute of Nuclear Energy Safety Technology, Hefei Institute of Physical Science, Chinese Academy of Sciences, for the key technology research and performance evaluation of candidate liquid lithium-lead (PbLi) blanket of hydrogen fusion reactors. The loop is composed of a material test sub-loop and thermal-hydraulic test sub-loop, the design parameters are PbLi inventory 20 tons, PbLi temperature up to 550 °C, the maximum PbLi flow rate up to 40 kg/s. A novel cold trap system is designed to remove the suspended and crystalized impurities in PbLi fluid with three cooling zones and cross row arrangement of rod bundle filter elements. The paper describes the loop itself and its major components, initial loop testing, flow and measurement diagnostics and current experiments. The obtained test results of the loop and its components have demonstrated that the new facility is fully functioning and ready for experimental studies of material corrosion with/without a magnetic field, magnetohydrodynamic (MHD) effect, purification, heat and mass transfer phenomena in PbLi flows and can also be used in mock-up testing in conditions relevant to fusion applications.

COPMAN: A novel high-throughput and highly sensitive method to detect viral nucleic acids including SARS-CoV-2 RNA in wastewater

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater-based epidemiology (WBE) attracted attention as an objective and comprehensive indicator of community infection that does not require individual inspection. Although several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection methods from wastewater have been developed, there are obstacles to their social implementation. In this study, we developed the COPMAN (Coagulation and Proteolysis method using Magnetic beads for detection of Nucleic acids in wastewater), an automatable method that can concentrate and detect multiple types of viruses from a limited volume (∼10 mL) of wastewater. The COPMAN consists of a high basicity polyaluminum chloride (PAC) coagulation process, magnetic bead-based RNA purification, and RT-preamplification, followed by qPCR. A series of enzymes exhibiting a high tolerance to PCR inhibitors derived from wastewater was identified and employed in the molecular detection steps in the COPMAN. We compared the detectability of viral RNA from 10-mL samples of virus-spiked (heat-inactivated SARS-CoV-2 and intact RSV) or unspiked wastewater by the COPMAN and other methods (PEG-qPCR, UF-qPCR, and EPISENS-S). The COPMAN was the most efficient for detecting spiked viruses from wastewater, detecting the highest level of pepper mild mottle virus (PMMoV), a typical intrinsic virus in human stool, from wastewater samples. The COPMAN also successfully detected indigenous SARS-CoV-2 RNA from 12 samples of wastewater at concentrations of 2.2 × 104 to 5.4 × 105 copies/L, during initial stages of an infection wave in the right and the left bank of the Sagami River in Japan (0.65 to 11.45 daily reported cases per 100,000 people). These results indicate that the COPMAN is suitable for detection of multiple pathogens from small volume of wastewater in automated stations.

Superconducting Magnet Design for a Vertical-Ring High Gradient Magnetic Separation System

This paper describes a design of a Vertical-ring High Gradient Superconducting Magnetic Separation (VHGMS) system for recovery of the magnetic ore and purification of non-metallic minerals. Compared to the conventional design which uses copper wires, using superconducting magnet can help reduce the energy consumption and increase the magnetic field. The first part of this paper introduces the operation margin design of the superconducting magnet. This magnet is wound with NbTi wires and its temperature margin is designed to be higher than 1 K. It generates an average magnetic field above 2 T in the filtering area. The second part of this paper introduces a quench protection circuit implemented in this magnet and post-quench analysis is also presented. Simulation results show that maximum temperature and terminal voltage are within the safe limits. So, the superconducting magnet can be effectively protected from burning-out during a quench. The third part of this paper proposes a novel reinforcement structure for the coil and analyzes the stress status during magnet charging and quench process. The Von mises stresses in the coil and stainless steel are both in allowable stress region and have a large safety margin.

A comparative analysis of extraction protocol performance on degraded mammalian museum specimens

The extraction of nucleic acids is one of the most routine procedures used in molecular biology laboratories, yet kit performance may influence the downstream processing of samples, particularly for samples which are degraded, and in low concentrations. Here we tested several commercial kits for specific use on commonly sampled mammalian museum specimens to evaluate the yield, size distribution, and endogenous content. Samples were weighed and had approximately equal input material for each extraction. These sample types are typical of natural history repositories ranged from 53 to 130 years old. The tested protocols spanned spin-column based extractions, magnetic bead purification, phenol/chloroform isolation, and specific modifications for ancient DNA. Diverse types of mammalian specimens were tested including adherent osteological material, bone and teeth, skin, and baleen. The concentration of DNA was quantified via fluorometry, and the size distributions of extracts visualized on an Agilent TapeStation. Overall, when DNA isolation was successful, all methods had quantifiable concentrations, albeit with variation across extracts. The length distributions varied based on the extraction protocol used. Shotgun sequencing was performed to evaluate if the extraction methods influenced the amount of endogenous versus exogenous content. The DNA content was similar across extraction methods indicating no obvious biases for DNA derived from different sources. Qiagen kits and phenol/chloroform isolation outperformed the Zymo magnetic bead isolations in these types of samples. Statistical analyses revealed that extraction method only explained 5% of the observed variation, and that specimen age explained variation (29%) more effectively.

NH2-Functionalized Magnetic Nanoparticles for the N-Glycomic Analysis of Patients with Multiple Sclerosis.

Glycosylation is vital for well-functioning glycoproteins and is reportedly altered in chronic inflammatory disorders, including multiple sclerosis (MS). High-throughput quantitative measurement of protein glycosylation is challenging, as glycans lack fluorophore groups and require fluorescent labeling. The attachment of fluorescent tags to each glycan moiety necessitates sample clean-up for reliable quantitation. The use of magnetic particles in glycan sample preparation is reportedly an easy-to-use solution to accomplish large-scale biomarker discovery studies. In this study, NH2-funtionalized magnetic nanoparticles were synthetized, characterized and applied for the glycosylation analysis of serum samples from patients diagnosed with multiple sclerosis and corresponding healthy controls. Serum samples were PNGase F digested and labeled by procainamide via reductive amination, followed by magnetic nanoparticle-based purification. The prepared samples were analyzed by hydrophilic interaction liquid chromatography, allowing for the relative quantitation of the individual glycan species. Significant glycosylation alterations were detected between MS patients and healthy controls, especially when analyzing the different gender groups.

Highly sensitive fluorescent detection of EDIL3 overexpressed exosomes for the diagnosis of triple-negative breast cancer

EDIL3 is a strong and highly accurate diagnostic marker for breast cancer, meanwhile, EDIL3 overexpressed exosomes are novel biomarkers for the early diagnosis of triple-negative breast cancer (TNBC). Here, we proposed a fluorescent detection method for EDIL3 overexpressed exosomes, which is simple and sensitive. Basically, we utilized a magnetic nanospheres (MNS) based liquid sandwich immunoassay strategy. MNS were modified with CD63 aptamers, which can immunologically bound to the CD63 protein on the surface of exosomes. Alexa Fluor 647 labeled anti-EDIL3 antibodies (Anti-EDIL3/AF647) were used as the fluorescent probes to recognize the EDIL3 on exosomes derived from a TNBC cell line (MDA-MB-231). With the target TNBC exosomes present, sandwich structures containing MNS, exosomes and fluorescent probes were formed. After magnetic purification, optical super resolution imaging of the products was conducted to check the specificity of the assay. In addition, fluorescence signals of the products were detected to quantitatively analyze the EDIL3 overexpressed exosomes. The linear range was found to be 7.78 × 101 to 7.78× 106 particles μl−1. The detection limit was approximately 10 particles μl−1. The feasibility of the method for the detection of exosomes in complex biological samples was also demonstrated. Such a simple and sensitive detection method for EDIL3 overexpressed exosomes holds a great potential in clinical diagnosis of TNBC.

First Report of Wilt on Lentil (Lens culinaris) Caused by Fusarium redolens in Tunisia.

Lentil (Lens culinaris Medik.) is widely grown in arid and semi-arid regions of Tunisia. Low yields are often attributed to it being grown on marginal growing land. Since 2016, symptoms of wilt including yellowing and discoloration of the stem and root tissues were observed in lentils in several region of Tunisia. The annual mean incidence of infected plants ranged from 10% to 15%. In 2019-2020 growing seasons, symptomatic adult plants were randomly sampled from two fields located in south Tunisia (33°37’N; 11°4’E; N and 33°33’N; 11°2’E), and one field located in north west Tunisia (36°7’N; 8°43’E). Pieces were cut from roots and stem, surface sterilized, then plated on ¼ strength Potato Dextrose Agar (PDA) + 100 mg L-1 streptomycin sulfate (Burgess et al., 1994). Cultures were incubated for 5-6 days. Nine colonies with floccose mycelia, spare or abundant and white to violet color, morphologically similar to Fusarium redolens according to Leslie & Summerell (2006) were isolated from both roots and stems. They were single-spored (Burgess et al., 1994). Microconidia were formed in false heads on short monophialides. They were oval to elliptical or reniform and were 0-1 septate. Three-septate macroconidia with short apical cells were also observed. The strains were also deposited in the microbial ITEM Collection of Institute of Sciences of Food Production. Extraction of genomic DNA of Fusarium sp. strains was carried out according to Wizard® Magnetic DNA Purification System (Promega, Fitchburg, WI, USA). Molecular identification was carried out based on translation elongation factor (TEF) gene sequencing, as described in Fallahi et al. (2019). The TEF sequences were searched on GenBank database by using the Basic Local Alignment Tool (BLAST). The sequences of four strains (Z2P6, Z2P7, Z3P2 and Z3P5) on a total of nine, recovered from lentil roots from the two fields of south Tunisia showed 100% homology with TEF sequences of the epitype culture of F. redolens NRRL25600 (accession number MT409453) (Balmas et al. 2010; Gargouri et al. 2020). Sequences of the strains were submitted to GenBank with accession numbers MW393853, MW393854, MW393856 and MW393857. Pathogenicity of the four strains of F. redolens was evaluated on Kef lentil variety (Kharrat et al. 2007). Inoculum was produced on sterilized oat colonized with each strain. The colonized grains were air-dried on filter paper, ground in a laboratory mill, mixed at 10 % to soil (10 g of each isolate inoculum for 100 g of disinfected soil substrate) and potted. Three germinated lentil seeds were placed in each pot and irrigated periodically. The test was replicated four times. After 21 days, 60% (33-100%) of the plants inoculated with the four F. redolens strains showed symptoms of wilting, yellowing and rotting of roots and 17% died when inoculated by Z2P6 and Z2P7 strains. Non-inoculated plants showed no symptoms. F. redolens was isolated from 100% of the inoculated plants roots. This is the first report of F. redolens as a pathogen on lentil in Tunisia. This species has also been associated with lentil wilting in other regions of the world including Italy (Riccioni et al. 2008), Canada (Taheri et al. 2011) and Pakistan (Rafique et al. 2020). F. redolens was previously reported from wilted chickpea crops in Tunisia (Bouhadida et al. 2017). These findings are important for the Tunisian national legumes program and call for larger surveys to better understand the biology and ecology of this species and to prevent from disease spreading.

Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP.

Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor.

A Novel Method of Magnetic Nanoparticles Functionalized with Anti-Folate Receptor Antibody and Methotrexate for Antibody Mediated Targeted Drug Delivery.

Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections to analyze the binding efficacy and intracellular tracking of synthesized nanomedicines. MNP-MTX-FR Ab demonstrated good cytotoxicity along all the nanocomposites, which confirms that the antibody-coated medicine possesses the potential affinity to destroy cancer cells in the targeted drug delivery process. Immunohistochemical approaches and fluorescent study further confirmed their uptake by FRs on the tumor cells’ surface in antibody-mediated endocytosis. The current approach is a useful addition to targeted drug delivery for better management of cancer therapy along with immunotherapy in the future.