Streptavidin-coated Magnetic Beads Research Articles

Site-specific immobilization of the endosialidase reveals QSOX2 is a novel polysialylated protein.

Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.

A Facile Strategy for Multiplex Protein Detection by a Fluorescent Microsphere-Based Digital Immunoassay.

The digital immunoassay is a highly sensitive detection technique based on single-molecule counting and is widely used in the ultrasensitive detection of biomarkers. Herein, we developed a fluorescent microsphere-based digital immunoassay (FMDIA) by employing fluorescent microspheres as both the carriers for immunoreaction and fluorescent reports for imaging. In this approach, the target protein in the sample was captured by fluorescent microspheres to form a biotin-labeled sandwich immunocomplex, and then, the fluorescent microspheres containing the target protein molecules were captured by adding streptavidin-coated magnetic beads (SA-MBs). By counting the proportion of fluorescence-positive magnetic beads, the concentration of the target protein can be precisely quantified. As a proof of concept, α fetoprotein (AFP) and human interleukin-6 (IL-6) were used to assess the analytical performance of the proposed FMDIA, and limit of detection (LOD) values of 21 pg/mL (0.30 pM) and 0.19 pg/mL (7.3 fM) were achieved, respectively. The results of AFP detection in serum samples of patients and healthy people were consistent with the reference values given by the hospital. Furthermore, by adding fluorescent microspheres of various colors for encoding, the proposed FMDIA can easily realize the simultaneous detection of multiple proteins without the need to introduce multiple modified magnetic beads. This multiplex protein detection strategy, in which the reactions are first carried out on the fluorescent microspheres and then magnetic beads are used to capture the fluorescent reporters containing the target molecules, provides a new idea for digital assays.

BIOM-48. A NOVEL LIQUID BIOPSY-BASED APPROACH TO ISOLATE AND CHARACTERIZE GLIOBLASTOMA-DERIVED SMALL EXTRACELLULAR VESICLES FROM BLOOD

Abstract Glioblastoma multiforme (GBM) is a highly aggressive brain cancer characterized by a poor prognosis and limited treatment options. The absence of early diagnostic measures, prognostic biomarkers, and reliable methods for monitoring disease progression contribute to the high mortality rate among GBM patients. In recent years, liquid biopsy has emerged as a non-invasive tool for assessing cancer onset and therapeutic responses. Among the various liquid biopsy approaches, extracellular vesicles (EVs) have gained attention; as lipid bilayer membrane-delimited 30-150nm sized small extracellular vesicles (sEV) are released by all cell types. These vesicles carry molecular cargo reflecting the cell of origin and its pathophysiological state, making them a promising alternative to other liquid biopsy methods, such as circulating tumor cells and circulating nucleotides. However, the application of sEV-based liquid biopsy is hindered by the lack of methods to enrich GBM tumor-specific sEV in the blood. Our team has developed unique tools and expertise in isolating cell/tissue-specific sEV from biofluids. We have previously reported the isolation of sEV derived from specific brain cells in the blood, characterizing them for diagnosing neurodegeneration and predicting treatment response. In this study, we first isolated total sEV (TE) from the plasma of GBM patients (n = 11). Subsequently, we enriched GBM-specific sEV (sEVGBM) from TE using the astrocyte-specific surface marker glutamate aspartate transporter (GLAST). To achieve this, we utilized biotin-tagged GLAST antibody and streptavidin-coated magnetic beads for sEVGBM isolation. We then extensively characterized the size and concentration (number/ml) of sEVGBM by nanoparticle tracking analyses, while evaluating their purity through nanoflow cytometry for GLAST expression. Furthermore, we confirmed the expression of GBM-specific markers EGFRvIII, EPHB2 and IL-13Rα2 on the surface of sEVGBM (n = 3). Importantly, the expression of these markers was significantly enriched on sEVGBM compared to TE, underscoring the significance of our strategy as a potential liquid biopsy for GBM.

Development and characterization of microsatellite loci in Ulex parviflorus Pourr. And its cross-transferability to other Genisteae.

The genus Ulex is composed by 15 species distributed in Europe and Africa, but the majority of them are restricted to the Iberian Peninsula and Northwest Africa. Some of these species are common elements at the landscape level, and others contribute to global biodiversity as narrow endemics. Assayed nuclear and plastid Sanger-sequenced regions do not provide enough resolution to perform evolutionary studies on the genus, neither at the intraspecific population level nor at the interspecific phylogenetic level. Thus, we have developed and characterized a set of nuclear microsatellite loci in U. parviflorus to provide new highly polymorphic molecular markers for the genus Ulex. Genomic DNA enriched in microsatellite motifs using streptavidin-coated M-280 magnetic beads attached to 5'-biotinylated oligonucleotides was sequenced in a 454GS Junior System. After primer design, fluorescent-dyed amplicons were analyzed through capillary sequencing (ABI3730XL). Here we present twelve new high polimorphic SSRs markers developed in U. parviflorus specimens and tested in 120 individuals. The 12 SSR loci amplified a total of 152 alleles, and detected expected heterozygosities that ranged from 0.674 to 0.725 in the genotyped populations. Successful cross-species transferability of the 12 SSR loci to the rest of species included in the genus Ulex and three other representative Genisteae was achieved. The 12 novel proposed SSRs loci will contribute to perform evolutionary studies and genetic research on the genus Ulex and in other Genisteae.

Demethylation-Driven ligase chain reaction for simultaneously sensitive detection of m6A demethylase FTO and m1A demethylase ALKBH3 in breast cancer at Single-Molecule level

RNA demethylases play a crucial role in gene expression regulation by controlling RNA methylation levels, and their abnormal expressions are associated with many human diseases. Herein, we develop a single-molecule detection method for simultaneous quantification of N6-methyladenosine (m6A) demethylase FTO and N1-methyladenosine (m1A) demethylase ALKBH3 based on RNA demethylation-directed multi-cyclic ligase chain reaction (LCR) cascades. We design a ssDNA containing m6A modification as the substrate of FTO and a ssDNA containing m1A modification as the substrate of ALKBH3. FTO and ALKBH3 catalyze the demethylation of m6A and m1A to generate normal adenine (A), respectively. Afterwards, Taq ligase initiates the LCR reaction with the demethylated ssDNAs as the templates to produce abundant biotinylated Cy3/Cy5-labeled signal probes. Subsequently, the resultant biotinylated probes are captured by streptavidin-coated magnetic beads (MBs) to form the MB-ssDNA-Cy3/Cy5 complexes. After magnetic separation, the ssDNAs in the MB-ssDNA-Cy3/Cy5 complexes are digested by DNase I, liberating abundant Cy3 and Cy5 fluorophores that can be counted by single-molecule detection. Remarkably, only a single DNA ligase is employed to achieve target signal conversion and multi-cyclic cascade amplification. This method displays extremely high sensitivity with a limit of detection of 6.33 × 10−18 mol/L for FTO and 1.44 × 10−17 mol/L for ALKBH3. Moreover, it can measure the enzyme kinetic parameters, screen inhibitors, and quantify cellular FTO and ALKBH3 activities with single-cell sensitivity. Furthermore, it can differentiate the FTO and ALKBH3 levels in the tissues of healthy human and breast cancer patients, offering a robust platform for biomedical research and clinical diagnosis.

The eINTACT method for studying nuclear changes in host plant cells targeted by bacterial effectors in native infection contexts.

Type-III effector proteins are major virulence determinants that most gram-negative bacteria inject into host cells to manipulate cellular processes for infection. Because effector-targeted cells are embedded and underrepresented in infected plant tissues, it is technically challenging to isolate them for focused studies of effector-induced cellular changes. This protocol describes a novel technique, effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), for isolating biotin-labeled nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors by using streptavidin-coated magnetic beads. This protocol is an extension of the existing Nature Protocols Protocol of the INTACT method for the affinity-based purification of nuclei of specific cell types in the context of developmental biology. In a phytopathology scenario, our protocol addresses how to obtain eINTACT transgenic lines and compatible bacterial mutants, verify the eINTACT system and purify nuclei of bacterial effector-recipient cells from infected tissues. Differential analyses of purified nuclei from plants infected by bacteria expressing the effector of interest and those from plants infected by effector-deletion bacterial mutants will reveal the effector-dependent nuclear changes in targeted host cells. Provided that the eINTACT system is available, the infection experiment takes 5 d, and the procedures, from collecting bacteria-infected leaves to obtaining nuclei of effector-targeted cells, can be completed in 4 h. eINTACT is a unique method for isolating high-quality nuclei from bacterial effector-targeted host cells in native infection contexts. This method is adaptable to study the functions of type-III effectors from numerous gram-negative bacteria in host plants that are amenable to transformation.

Profiling extracellular vesicle surface proteins with 10 µL peripheral plasma within 4h.

Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC=0.98) and of HER2 (AUC=1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.

Abstract B051: Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC

Abstract Background: Histone modifying enzymes (HMEs) hold significant promise as targets for drug discovery in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) dictate the presence of acetyl marks, which regulate the openness of chromatin and transcriptional activation. We previously developed a novel histone array on a chip to measure HME dysregulation with the development of castration resistance (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC. This alteration was subsequently identified in a subset of castration-resistant tumors (BMC Cancer 2017). The goal of the current study was to determine if the isolation of circulating tumor cells (CTCs) could be used as a biomarker to identify patients with altered p300 acetylation activity. Methods: Peripheral blood was collected from 13 patients (15 samples) with advanced PC after informed written consent under the University of Wisconsin IRB-approved protocol. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using pre-conjugated, anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled Sera-Mag streptavidin-coated magnetic beads (SM). Cells bound to SMs were isolated with ESP technology using an automated magnetic PipetMax pipetting robot. SM-bound cells were moved through staining and washing wells prior to imaging at 20x with a Nikon Eclipse Ti fluorescent microscope and NIS-Elements AR 4.10 software, and finally images were analyzed with NIS Elements software. Results: We first optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in prostate cancer cell lines. Employing a selective inhibitor of p300 acetylation (A-485) reduced levels of p300 and H3K18 acetylation. We then confirmed that CTCs from patient samples were successfully captured by the EpCAM antibody and show clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Increased acetylation of p300 and H3K18 and decreased SIRT2 is found in 6/10 CRPC samples compared to HSPC patients. Paired samples collected from one patient show a marked increase in p300 activity, H3K18 hyperacetylation, and a decrease in SIRT2 expression in the later developed CRPC sample compared to the HSPC sample. Another CRPC subject, decreased acetyl p300 and histone acetyl-H3k18, and increased SIRT2 staining is noted several months after beginning enzalutamide administrated. Analysis in CTCs indicates a positive correlation between acetyl-p300 and H3K18ac (R=0.61) and between SIRT2 and H3K18ac (R=-0.59). Conclusions: This study suggests that acetyl-p300, H3K18ac, and SIRT2 are epigenetic biomarkers that increase in a subset of CTC from CRPC. These biomarkers could potentially be used for monitoring outcomes and predicting tumor responses to developing p300 acetylation inhibitors. Citation Format: Mikolaj Filon, Bing Yang, Jennifer Schehr, Anupama Singh, Marcelo Bigarella, John Denu, Joshua Lang, David Jarrard. Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B051.

Imunocapture Magnetic Beads Enhanced and Ultrasensitive CRISPR-Cas13a-Assisted Electrochemical Biosensor for Rapid Detection of SARS-CoV-2.

The rapid and ongoing spread of the coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emphasizes the urgent need for an easy and sensitive virus detection method. Here, we describe an immunocapture magnetic bead-enhanced electrochemical biosensor for ultrasensitive SARS-CoV-2 detection based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, collectively known as CRISPR-Cas13a technology. At the core of the detection process, low-cast and immobilization-free commercial screen-printed carbon electrodes are used to measure the electrochemical signal, while streptavidin-coated immunocapture magnetic beads are used to reduce the background noise signal and enhance detection ability by separating the excessive report RNA, and a combination of isothermal amplification methods in the CRISPR-Cas13a system is used for nucleic acid detection. The results showed that the sensitivity of the biosensor increased by two orders of magnitude when the magnetic beads were used. The proposed biosensor required approximately 1 h of overall processing time and demonstrated an ultrasensitive ability to detect SARS-CoV-2, which could be as low as 1.66 aM. Furthermore, owing to the programmability of the CRISPR-Cas13a system, the biosensor can be flexibly applied to other viruses, providing a new approach for powerful clinical diagnostics.

Performance enhancement of electrochemiluminescence magnetic microbiosensors by using double magnetic field actuation for cancer biomarkers and exosomes

This work reports the performance enhancement strategies on magnetic beads (MBs)-based electrochemiluminescence (ECL) platforms by using double magnetic field actuation of the ECL magnetic microbiosensors (MMbiosensors) for highly sensitive determination of cancer biomarker and exosomes. To obtain the high sensitivity and reproducibility of the ECL MMbiosensors, a series of strategies have been developed including replacing a conventional photomultiplier tube (PMT) with a diamagnetic PMT, replacing the stacked ring-disc magnets with circular-disc magnets lain-in glassy carbon electrode, adding a pre-concentration process of MBs using external magnet actuation. For fundamental research, the ECL MBs taken as the substitute of ECL MMbiosensors were prepared by binding biotinylated DNA tagged with Ru(bpy)32+ derivative (Ru1) to streptavidin-coated MB(MB@SA) were which showed that the developed strategies can enhance 45-fold sensitivity. Importantly, the developed MBs-based ECL platform was estimated by determination of prostate specific antigen (PSA) and exosomes. For PSA, MB@SA•biotin-Ab1(PSA) was taken as the capture probe and Ru1-labeled Ab2 (PSA) was done as ECL probe, while for exosomes, MB@SA•biotin-aptamer (CD63) was taken as the capture probe and Ru1-labeled Ab (CD9) was done as the ECL probe. The experiment results showed that the developed strategies can enhance 33-fold sensitivity of ECL MMbiosensors for PSA and exosomes. The detection limit is 0.28 ng mL−1 for PSA and 4.9 × 102 particle mL-1 for exosomes. This work demonstrated that a series of proposed magnetic field actuation strategies greatly increase the sensitivity of the ECL MMbiosensors. The developed strategies can be expanded to MBs-based ECL and electrochemical biosensors for clinical analysis with greater sensitivity.

Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification

Using rolling circle amplification (RCA) and two different ways of signal readout, we developed analytical methods to detect the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S protein). We modified streptavidin-coated magnetic beads with an aptamer of RBD through a biotin-tagged complementary DNA strand (biotin-cDNA). Binding of RBD caused the aptamer to dissociate from the biotin-cDNA, making the cDNA available to initiate RCA on the magnetic beads. Detection of RBD was achieved using a dual signal output. For fluorescence signaling, the RCA products were mixed with a dsDNA probe labeled with fluorophore and quencher. Hybridization of the RCA products caused the dsDNA to separate and to emit fluorescence (λex = 488 nm, λem = 520 nm). To generate easily detectable UV–vis absorbance signal, the RCA amplification was extended to produce DNA flower to encapsulate horseradish peroxidase (HRP). The HRP-encapsulated DNA flower catalyzed a colorimetric reaction between H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB) to generate an optical signal (λabs = 450 nm). The fluorescence and colorimetric assays for RBD have low detection limits (0.11 pg mL−1 and 0.904 pg mL−1) and a wide linear range (0.001–100 ng mL−1). For detection of RBD in human saliva, the recovery was 93.0–100% for the fluorescence assay and 87.2–107% for the colorimetric assay. By combining fluorescence and colorimetric detection with RCA, detection of the target RBD in human saliva was achieved with high sensitivity and selectivity.Graphical

Colorimetric and fluorescence detection of circulating tumor cells based on a bimetallic-organic framework

An ultrasensitive colorimetric and fluorescence dual-readout method was developed for detecting circulating tumor cells (CTCs) using a CuFe-based bimetallic-organic framework (CuFe-NH2-MIL-101, bi-MOF). Incorporation of Cu into an Fe-based metal–organic framework (MOF) led to improved fluorescence compared with that of monometallic analogues. The synthesized bi-MOF exhibited improved fluorescence. The synthesized bi-MOF also exhibited enzyme-mimetic activity. The CTCs were bound by biotinylated antibodies and then selected and enriched by streptavidin-coated magnetic beads. With peroxidase-mimicking activity and enhanced fluorescence, the CuFe bi-MOF functioned as a signal-amplifying nanoprobe for colorimetric and fluorescence detection. The absorbance increased with increasing concentration of tumor cells, whereas the fluorescence intensity gradually decreased. Under optimized conditions, the sensor exhibited a detection range of 102–107 cells mL−1. The detection limit of this cytosensor could reach 7 cells mL−1. Moreover, this sensor showed reliable applicability in real serum samples. Overall, this dual-readout strategy holds potential for the detection of CTCs in clinical diagnostics.

A duplex-specific nuclease (DSN) and catalytic hairpin assembly (CHA)-mediated dual amplification method for miR-146b detection.

A novel method for detecting miRNA has been developed using a combination of duplex-specific nuclease signal amplification (DSNSA) and a catalytic hairpin assembly (CHA). In this work, a biotinylated trigger release (BTR) probe with a biotin group at the 3'-end and a CHA reaction sequence trigger as an initiator (catalyst I) at the 5'-end was designed to hybridize target miRNA. The DSN enzyme was introduced to initiate the DSNSA. The miRNA was released to consume more BTR probes and amplify the signals. Subsequently, streptavidin-coated magnetic beads (SA-MBs) were added to the DSNSA reaction solution to remove excess BTR probes that did not hybridize with miRNA, which would then separate BTR probes and catalyst-I, to ensure detection with high selectivity and sensitivity. The catalyst-I remaining in the solution could trigger the CHA reaction to enable signal amplification in the second step. The developed method exhibits a sensitive detection limit and excellent selectivity in identifying a high sequence homology among family members.

Isolation of E. coli RNA polymerase transcription elongation complexes by selective solid-phase photoreversible immobilization.

The ability to prepare defined transcription elongation complexes (TECs) is a fundamental tool for investigating the interplay between RNA polymerases (RNAPs) and nascent RNA. To facilitate the preparation of defined TECs that contain arbitrarily long and complex transcripts, we developed a procedure for isolating roadblocked E. coli TECs from an in vitro transcription reaction using solid-phase photoreversible immobilization. Our approach uses a modified DNA template that contains both a 5' photocleavable biotin tag and an internal biotin-TEG transcription stall site. Because the footprint of a TEC at the stall site sequesters the biotin-TEG tag, DNA template molecules that contain a TEC can be reversibly immobilized on streptavidin-coated magnetic beads by the 5' photocleavable biotin tag. In contrast, DNA template molecules that do not contain a TEC are retained on the beads because the biotin-TEG tag is exposed and can bind streptavidin. In this way, DNA template molecules that contain a TEC are gently separated from free DNA and DNA that contains non-productive transcription complexes. This procedure yields precisely positioned TECs that are >95% pure with >30% yield relative to the amount of input DNA template. The resulting complexes are >99% stable for at least 3h and can be used for biochemical investigations of nascent RNA structure and function in the context of E. coli RNAP. The procedure is likely generalizable to any RNAP that arrests at and sequesters the internal biotin-TEG stall site.

Magnetic-bead-based DNA-capture-assisted real-time polymerase chain reaction and recombinase polymerase amplification for the detection of African swine fever virus.

African swine fever (ASF) is a deadly disease in swine caused by African swine fever virus (ASFV). The global spread of ASFV has resulted in significant economic losses worldwide. Improved early detection has been the most important first line of defense for preventing ASF outbreaks and for activating control measures. Despite the availability of rapid amplification methods, nucleic acid extraction from specimens still needs to be performed in a laboratory. To facilitate this step, we exploited the strong affinity of biotin-streptavidin binding by functionalizing streptavidin-coated magnetic beads with biotinylated oligonucleotide capture probes to efficiently capture genotype II ASFV DNA directly from crude clinical samples. The captured DNA is suitable for detection using real-time quantitative PCR (qPCR) and recombinase polymerase amplification (RPA). In this study, ASFV DNA was efficiently captured from swine feces, serum, and tissue samples. Both DNA-capture-assisted qPCR and RPA-based detection methods have a limit of detection (LOD) of 102 copies/µl, which is comparable to those of commercially available kits. In addition, an RPA-SYBR Green I method was developed for the immediate visual detection of ASFV DNA, which is time-saving and efficient for resource-limited field settings. In summary, a rapid, versatile, sequence-specific DNA capture method was developed to efficiently capture ASFV DNA from swine clinical samples and subsequent detection by qPCR and RPA, which has the potential to be used for robust screening and surveillance of ASFV and in point-of-care (POC) diagnostics.

Activity-Based Proteomic Identification of the S-Thiolation Targets of Ajoene in MDA-MB-231 Breast Cancer Cells.

Garlic is a medicinal plant and spice that has been used for millennia for its health-promoting effects. These medicinal properties are associated with low molecular weight organosulfur compounds, produced following the crushing of garlic cloves. One of these compounds, ajoene, is proposed to act by S-thioallylating cysteine residues on target proteins whose identification in cancer cells holds great promise for understanding mechanistic aspects of ajoene's cancer cell cytotoxicity. To this end, an ajoene analogue (called biotin-ajoene, BA), containing a biotin affinity tag, was designed as an activity-based probe specific for the protein targets of ajoene in MDA-MB-231 breast cancer cells. BA was synthesized via a convergent "click" strategy and found to retain its cytotoxicity against MDA-MB-231 cells compared to ajoene. Widespread biotinylation of proteins was found to occur via disulfide bond formation in a dose-dependent manner, and the biotin-ajoene probe was found to share the same protein targets as its parent compound, ajoene. The biotinylated proteins were affinity-purified from the treated MDA-MB-231 cell lysate using streptavidin-coated magnetic beads followed by an on-bead reduction, alkylation, and digestion to liberate the peptide fragments, which were analyzed by liquid chromatography tandem mass chromatography. A total of 600 protein targets were identified, among which 91% overlapped with proteins with known protein cysteine modification (PCM) sites. The specific sites were enriched for those susceptible to S-glutathionylation (-SSG) (16%), S-sulfhydration (-SSH) (20%), S-sulfenylation (-SOH) (22%), and S-nitrosylation (-SNO) (31%). As target validation, both ajoene and a dansylated ajoene (DP) were found to S-thiolate the pure recombinant forms of glutathione S-transferase pi 1 (GSTP1) and protein disulfide isomerase (PDI), and the ajoene analogue DP was found to be a more potent inhibitor than 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Pathway analysis elucidated that ajoene targets functional and signaling pathways that are implicated in cancer cell survival, specifically cellular processes, metabolism, and genetic information processing pathways. The results of this study provide mechanistic insights into the character of the anti-cancer activity of the natural dietary compound ajoene.

Electrophoretic detection of black market myostatin propeptide.

Myostatin propeptide is prohibited according to chapter S4 of the "WADA 2022 List of Prohibited Substances and Methods." So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for "research purposes." A study on black market myostatin propeptide products is presented as well as electrophoretic detection methods for serum and urine. Out of the 12 tested products, only nine actually contained the protein. Separation by SDS-PAGE revealed that the nine products were relatively impure and that the main compound had a much higher mass (approximately 54-55 kDa) than expected (approximately 33 kDa). Further analyses by mass spectrometry showed that the elevated molecular mass was due to the presence of a full length GST-tag on the propeptide. The developed detection method for serum is based on immunoprecipitation (IP) followed by SDS-PAGE and Western blotting. In total, three anti-myostatin propeptide antibodies were tested. All of them were well suited for either IP or immunoblotting. The final protocol applies a biotinylated polyclonal antibody, streptavidin-coated magnetic beads, and a monoclonal detection antibody. For a sample volume of 500 μL serum, the detection limit of the method is approximately 2.5 ng/mL. The urine method applies a commercial ELISA for IP and performs with a limit of detection (LOD) of approximately 0.4 ng/mL. Furthermore, practically all currently available myostatin propeptide standards were also investigated. Due to the significant molecular mass difference of the black market products, an unambiguous differentiation from endogenous myostatin propeptide is possible.

Magnetic beads for the evaluation of drug release from biotinylated polymeric micelles in biological media.

To improve the reliability of in vitro release studies of drug delivery systems, we developed a novel in vitro method for the evaluation of drug release from polymeric micelles in complex biological media. Polymeric micelles based on poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) of which 10% of the chains was functionalized with biotin at the p(HPMAm) terminus were prepared using a solvent extraction method. The size of the micelles when loaded with a hydrophobic agent, namely paclitaxel (a clinically used cytostatic drug) or curcumin (a compound with multiple pharmacological activities), was around 65nm. The biotin decoration allowed the binding of the micelles to streptavidin-coated magnetic beads which occurred within 10min and reached a binding efficiency of 90±6%. Drug release in different media was studied after the magnetic separation of micelles bound to the streptavidin-coated beads, by determination of the released drug in the media as well as the retained drug in the micellar fraction bound to the beads. The in vitro release of paclitaxel and curcumin at 37°C in PBS, PBS containing 2% v/v Tween 80, PBS containing 4.5% w/v bovine serum albumin, mouse plasma, and whole mouse blood was highly medium-dependent. In all media studied, paclitaxel showed superior micellar retention compared to curcumin. Importantly, the presence of serum proteins accelerated the release of both paclitaxel and curcumin. The results presented in this study show great potential for predicting drug release from nanomedicines in biological media which in turn is crucial for their further pharmaceutical development.

Towards the Selection of Single‐Stranded DNA Molecular Recognition Element Against Cyanotoxin L‐BMAA

Global warming, anthropogenic pressure and eutrophication contribute to the formation and persistence of cyanobacterial blooms in aquatic environments. Cyanotoxins produced during these blooms represent a threat to ecosystems, commercial industries and human health. B‐N‐methylamino‐L‐alanine (L‐BMAA) is a neurotoxic amino acid that has been linked to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC), Alzheimer’s disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Research has shown that L‐BMAA can act as a glutamate receptor antagonist and can incorporate into proteins occasioning protein misfolding that leads to detrimental processes in brain tissues. Currently, the detection of L‐BMAA is costly, time‐consuming and labor‐intensive. Many research groups around the world are working towards the development of bioanalytical methods to improve the detection of these toxins, including the development of molecular biorecognition elements (MREs) that can provide analyte specificity in biosensing applications. Among MREs, aptamers are well‐known to possess an extraordinary target recognition due to the formation of unique three‐dimensional structures capable of binding specifically to their respective targets.We aim to perform an in vitro selection of a single‐stranded DNA (ssDNA) aptamer that can act as an MRE against L‐BMAA, using the systematic evolution of ligands by exponential enrichment (SELEX) technique. We have worked on the optimization of incubation conditions, PCR amplification parameters and the generation of ssDNA using two different ssDNA libraries, with randomized regions of varying lengths. To facilitate the separation of bound and unbound ssDNA after incubation with the target, we modified L‐BMAA with a biotinylated linker to immobilize it in streptavidin‐coated magnetic beads. For each library, we have identified the appropriate annealing temperature, primer concentrations and number of cycles to favor the amplification of the main product while minimizing by‐product formation. The most challenging step of the SELEX procedure has been the generation and purification of ssDNA in sufficient amounts to continue to the next round of selection. To achieve this, we have tried four main methods: exonuclease digestion, size separation derived from uneven primers, asymmetric PCR and alkaline denaturation using sodium hydroxide. Hitherto, we have completed the first round of selection with both libraries, by performing large‐scale PCR reactions and alkaline denaturation followed by ethanol precipitation to generate the ssDNA. We are working in the first round of negative selection to discard those sequences with affinity to the immobilization linker and enrich only those that bind exclusively to L‐BMAA. Given that our target is a small molecule, our goal is to conduct a stringent SELEX process to obtain a highly specific MRE that can be incorporated into an environmental sensing device.

Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody.

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.