MS2-MBP-Based Affinity Purification of Nucleus- or Cytoplasm-Localized lncRNA-Protein Complexes Formed In Vivo.

With recent emergence of huge number of long noncoding RNAs (lncRNAs), purification of lncRNA-protein (lncRNP) complexes is fundamental to understand the role of lncRNA and its biological function. However, lncRNP purification is still a daunting challenge. Here we describe a protocol to purify lncRNP formed in vivo with MS2-MBP-based affinity purification. Inducible lncRNA tagged with MS2 RNA hairpins is introduced into cells of interest, and RNP on tagged lncRNA is formed in vivo. MS2-MBP fusion protein is expressed in Escherichia coli and purified with amylose resin and HiTrap heparin column. The MS2 part of MS2-MBP fusion protein binds to the hairpins, and MBP part binds to amylose resin. We also describe a protocol to separate the nucleus and the cytoplasm so that lncRNP localized in the nucleus or cytoplasm can be individually purified. The amount of lncRNP purified is well sufficient for mass spectrometry analysis.

Alkynone ß-trifluoroborates: A new class of amine-specific biocompatible click reagents.

Amine-targeting reactions that work under biocompatible conditions or in water are green processes that are extremely useful for the synthesis of functional materials and biotherapeutics. Unfortunately, despite the usefulness of this reaction, there are very few good amine-specific click methods reported thus far. Here, we report an amine-specific click reagent using alkynone ß-trifluoroborates as the electrophiles. These boron-containing alkynyl reagents exhibit extremely high chemoselectivity toward amines even in the presence of thiols. The resulting oxaboracycle products are bench-stable, displaying the reactivities of both organoborates and enaminones. Intrinsic advantages of this methodology include benign reaction conditions, operational simplicity, remarkable product stability, and excellent chemoselectivity, which satisfy the criteria of click chemistry and demonstrate the high potential in bioconjugation. Hence, this water-based chemical approach is also applicable to the modification of native amino acids, peptides, and proteins. Ultimately, the essential role of water during the reaction was elucidated.

A Review of Factor VIII and Factor IX Assay Methods for Monitoring Extended Half-Life Products in Hemophilia A and B.

Clinical trials have used a variety of coagulation factor assay methods to assess treatment with recombinant Factor VIII (rFVIII) and recombinant Factor IX (rFIX) extended half-life (EHL) products. However, diagnostic laboratories may use different reagent combinations for routine use or for field trials of EHL products. The focus of this review is on the choice of one-stage clotting and chromogenic Factor VIII and Factor IX methods and the influence that assay principle and components may have on results, including the effects of different activated partial thromboplastin time reagents and factor-deficient plasma. Our aim is to tabulate the findings for each method and reagent group to give laboratories practical guidance as to how the reagent combinations used in their local laboratory compare to others, for the various EHLs available.

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging.

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Use of alkyne-tagged myristic acid to detect N-terminal myristoylation.

Protein N-terminal myristoylation is a lipidic modification typically occurring to the α-amino group of N-terminal glycine residues of proteins. It is catalyzed by the N-myristoyltransferase (NMT) enzyme family. Many studies in the past three decades have highlighted the importance of N-terminal glycine myristoylation as it affects protein localization, protein-protein interaction, and protein stability, thereby regulating multiple biological processes, including immune cell signaling, cancer progression, and infections. This book chapter will present protocols for using alkyne-tagged myristic acid to detect the N-myristoylation of targeted proteins in cell lines and compare global N-myristoylation levels. We then described a protocol of SILAC proteomics that compare the levels of N-myristoylation on a proteomic scale. These assays allow for the identification of potential NMT substrates and the development of novel NMT inhibitors.

Square-wave voltammetry of human blood serum.

A study on voltammetric analysis of blood serum diluted in a phosphate buffer is presented using advanced square-wave voltammetry at an edge plane pyrolytic graphite electrode. The results demonstrate that even in a complex medium like human blood serum, electrochemical characterization can be achieved through the use of advanced voltammetric techniques in conjunction with an appropriate commercially available electrode, such as the edge plane pyrolytic graphite electrode, which boosts superior electrocatalytic properties. Without undergoing any chemical treatment of the serum sample, the square-wave voltammetry technique reveals, for the first time, the electrode reactions of uric acid, bilirubin, and albumin in a single experiment, as represented by well-defined, separated, and intense voltammetric signals. All electrode processes are surface-confined, indicating that the edge plane sites of the electrode serve as an ideal platform for the competitive adsorption of electroactive species, despite the extensive chemical complexity of the serum samples. The speed and differential nature of square-wave voltammetry are crucial for obtaining an outstanding resolution of the voltammetric peaks, maintaining the quasi-reversible nature of the underlying electrode processes, while reducing the impact of follow-up chemical reactions that are coupled to the initial electron transfer for all three detected species, and minimizing fouling of the electrode surface.

Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey.

A scheme of modular competitive immunochromatography with an analyte-independent test strip and changeable specific immunoreactants has been proposed. Native (detected) and biotinylated antigens interact with specific antibodies during their preincubation in solution, that is, without the immobilization of reagents. After this, the detectable complexes on the test strip are formed by the use of streptavidin (which binds biotin with high affinity), anti-species antibodies, and immunoglobulin-binding streptococcal protein G. The technique was successfully applied for the detection of neomycin in honey. The visual and instrumental detection limits were 0.3 and 0.014 mg/kg, respectively, and the degree of neomycin revealed in honey samples varied from 85% to 113%. The efficiency of the modular technique with the use of the same test strip for different analytes was confirmed for streptomycin detection. The proposed approach excludes the necessity of finding the condition of immobilization for each new specific immunoreactant and transferring the assay to other analytes by a simple choice of concentrations for preincubated specific antibodies and the hapten-biotin conjugate.

Dispersive Micro-Solid Phase Extraction Using a Graphene Oxide Nanosheet with Neocuproine and Batocuproine for the Preconcentration of Traces of Metal Ions in Food Samples.

A dispersive micro-solid phase extraction (Dµ-SPE) method for the preconcentration of trace metal ions (Pb, Cd, Cr, Mn, Fe, Co, Ni, Cu, Zn) on graphene oxide with the complexing reagents neocuproine or batocuproine is presented here. Metal ions form cationic complexes with neocuproine and batocuproine. These compounds are adsorbed on the GO surface via electrostatic interactions. The factors affecting the separation and preconcentration of analytes such as pH, eluent (concentration, type, volume), amount of neocuproine, batocuproine and GO, mixing time, and sample volume were optimized. The optimal sorption pH was 8. The adsorbed ions were effectively eluted with 5 mL 0.5 mol L-1 HNO3 solution and determined by the ICP-OES technique. The preconcentration factor for the GO/neocuproine and GO/batocuproine in the range 10-100 and 40-200 was obtained for the analytes, with detection limits of 0.035-0.84 ng mL-1 and 0.047-0.54 ng mL-1, respectively. The method was validated by the analysis of the three certified reference materials: M-3 HerTis, M-4 CormTis, and M-5 CodTis. The procedure was applied to determine metal levels in food samples.

Managing sample introduction problems in hydrophilic interaction liquid chromatography.

Sample injection can cause serious problems in hydrophilic interaction liquid chromatography (HILIC) when the injection solvent has higher elution strength than the mobile phase (mp). It can lead to asymmetric peak shapes and poor efficiency. The problem can occur when the mp contains a high proportion of organic e.g. 95% acetonitrile (a weak solvent) whereas the injection solvent contains a higher proportion of water (a strong solvent) that is necessary to dissolve polar samples. We investigated different strategies to overcome this problem. A simple method is pre-column dilution where the injector is programmed to deliver a plug of weak solvent (e.g. pure acetonitrile) along with the sample dissolved in a solvent with higher water content than the mp. Another option is to use alternative organic solvents to acetonitrile in the injection solvent, e.g. isopropanol, acetone or tetrahydrofuran, that may give enhanced sample solubility. The role of the volume of injection solvents was investigated as well as the possible effects of mass overload on the results. The use of small sample volumes is always recommended to reduce mismatch effects.

Design of reversibly charge-changeable rhodamine B modified magnetic nanoparticles to enrich phosphopeptides.

In the present study, by employing ethylenediaminetetraacetic acid (EDTA), tetraethylene pentaamine (TEPA), and rhodamine B (Rb), we designed and synthesized a magnetic adsorbent (Fe3O4@EDTA@TEPA@Rb) on the basis of reversible charge change of Rb and applied to capture phosphopeptides. Rb existing in open planarized zwitterion form when stimulated by acidic loading buffer adsorbs negative phosphopeptides via electrostatic interaction. Under the stimulation of alkalic eluent, ring-closed structure of Rb is formed to elute the enriched phosphopeptides. TEPA containing rich amino groups is used as a crosslinking agent, which is also protonated in acidic loading buffer to bond phosphopeptides. Then phosphopeptides are eluted when TEPA deprotonates in alkalic eluent. Coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) detection, phosphopeptide signals originated from 0.4 fmol/µL ß-casein digests were successfully detected. In addition, Fe3O4@EDTA@TEPA@Rb can also efficiently enrich phosphopeptides from skimmed milk, human serum and saliva samples (26, 4, 39 phosphopeptides, respectively), opening a new gallery for phosphopeptides-related analysis. In general, the developed adsorbent has the great potential for further application in the near future.

Microflow liquid chromatography - multi-emitter nanoelectrospray mass spectrometry of oligonucleotides.

While the most sensitive LC-MS methods for oligonucleotide analysis contain ion-pairs in the mobile phase, these modifiers have been associated with instrument contamination and ion suppression. Typically, entire LC-MS systems are reserved for oligonucleotide LC-MS when using ion-pairing buffers. To overcome these limitations, numerous HILIC methods, liberated from ion-pairs, have been recently developed. Since ion-pairs play a role in analyte desorption from ESI droplets, their removal from mobile phases tend to impact method sensitivity. An effective way to recover MS sensitivity is to reduce the LC flow rate and therefore reduce ESI droplet size. With a focus on MS sensitivity, this study investigates the applicability of a microflow LC- nanoelectrospray MS platform in oligonucleotide ion-pair RP and HILIC LC-MS methods. The platform is effective and substantially increased the MS sensitivity of HILIC methods. Furthermore, LC method development for both types of separations provide insight into microflow chromatography of oligonucleotides, an under investigated chromatographic scale.

Development of a sensitive HPLC method with fluorescence detection for the determination of motilin in human plasma.

Background: Motilin is a peptide-structured gastrointestinal system hormone. In this study, a sensitive HPLC-fluorescence detection method was developed and validated for the quantification of motilin in human plasma. Materials & methods: Optimization processes were carried out with the experimental design methodology. Analyses were performed on a C8 column (4.6 × 150 mm, 3.5 µm particles) using water and acetonitrile containing trifluoroacetic acid as the mobile phase. Results & conclusion: The method was linear from 2 to 200 ng/ml of motilin. The assay variability was less than 5%. The limit of quantification was found to be 1.84 ng/ml. The applicability of the developed method was successfully demonstrated by quantifying the levels of motilin in human plasma samples.

Adsorption of 2,4-dichlorophenoxyacetic acid and glyphosate from water by Fe3O4-UiO-66-NH2 obtained in a simple green way.

In this study, a green adsorbent (Fe3O4-UiO-66-NH2) with the ability of addressing the issues of separation and recovery of UiO-66-NH2 is obtained using a simple co-precipitation method under environmentally benign conditions. Various characterization techniques are utilized for evaluating the properties of the developed adsorbent. The capability of Fe3O4-UiO-66-NH2 towards 2,4-dichlorophenoxyacetic acid (2,4-D) and glyphosate (GP) from solution is explored. The results revealed that the magnetization process did not destroy the crystal structure of UiO-66-NH2, which ensured that Fe3O4-UiO-66-NH2 had good adsorption performance for 2,4-D and GP. The adsorption processes showed a wide pH application range, high salt tolerance, and regeneration performance as well as an excellent adsorption rate. Results from thermodynamic study showed that both processes were spontaneous and endothermic. The unit uptake ability of Fe3O4-UiO-66-NH2 for 2,4-D and GP reached up to 249 mg·g-1 and 183 mg·g-1 from Langmuir model at 303 K, respectively. When solid-liquid ratio was 2 g·L-1, Fe3O4-UiO-66-NH2 can reduce the content of 2,4-D or GP with the initial density of 100 mg·L-1 below the drinking water requirement limit. In addition, the reusability efficiency of Fe3O4-UiO-66-NH2 towards 2,4-D and GP was found to be 86% and 80% using 5 mmol·L-1 NaOH as eluent. Analysis of simulated water samples indicated that Fe3O4-UiO-66-NH2 could achieve the single or simultaneous removal of 2,4-D and GP from wastewater. Summarily, Fe3O4-UiO-66-NH2 as a green adsorbent can serve as an alternative for removing 2,4-D and GP from water body.

Boronic Acid Resin for Selective Immobilization of Canonically Encoded Proteins.

The use of transition-metal-mediated boronic acid chemistry presents a novel method of protein immobilization on a solid support. This is a one-step method that site-selectively immobilizes pyroglutamate-histidine (pGH)-tagged proteins. Herein, we describe the synthesis of alkenylboronic acid-functionalized poly(ethylene glycol) acrylamide (PEGA) resin and its subsequent reactions with pGH-tagged proteins to produce covalent linkages. The selectivity of immobilization is demonstrated within fluorescent studies, model mixtures, and lysates.

The development and application of a novel reagent for fixing red blood cells with glutaraldehyde and paraformaldehyde.

OBJECTIVE: The currently employed red blood cell reagents have a short shelf life. Some hospitals with a small number of specimens will be unable to utilize them within the validity period, resulting in a substantial increase in the purchase price. Therefore, the method of developing long-term red blood cell reagents is a problem worthy of further study. METHODS: In this experiment, the type and concentration of the red blood cell reagent treatment solution were evaluated based on the red blood cell antigen concentration 24 h after treatment. In addition, the qualified glutaraldehyde/paraformaldehyde reagent was stored for six months, and five red blood cell indices were measured every month. At the same time, the detection indices of treated red blood cell reagents and untreated red blood cell reagents were compared. RESULTS: It was discovered that treated red blood cells containing 0.005% GA and 0.05% PFA were more suitable for the preservation of red blood cells than other treated concentrations, and the preservation time could reach six months. The test tube method (n = 24) and microcolumn gel card (n = 35) were used to determine the accuracy of the treated blood cells containing 0.005% glutaraldehyde +0.05% paraformaldehyde, with an accuracy of 100%. CONCLUSION: This experiment resulted in the development of a novel reagent for treating red blood cells with glutaraldehyde/paraformaldehyde fixed solution that can effectively prolong its storage time by two to three times that of red blood cell reagents currently on the market.

In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes.

Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

Use of Fluorescent Protein Reporters for Assessing and Detecting Genome Editing Reagents and Transgene Expression in Plants.

Fluorescent protein reporters have been widely used for monitoring the expression of target genes in various engineered organisms. Although a wide range of analytical approaches (e.g., genotyping PCR, digital PCR, DNA sequencing) have been utilized to detect and identify genome editing reagents and transgene expression in genetically modified plants, these methods are usually limited to use in the late stages of plant transformation and can only be used invasively. Here we describe GFP- and eYGFPuv-based strategies and methods for assessing and detecting genome editing reagents and transgene expression in plants, including protoplast transformation, leaf infiltration, and stable transformation. These methods and strategies enable easy, noninvasive screening of genome editing and transgenic events in plants.

Platform headspace gas chromatography method for high-throughput determination of residual solvents in pharmaceutical materials.

Static headspace capillary gas chromatography (HSGC) has been employed to monitor the level of residual solvents in the pharmaceutical materials. Most of the HSGC methods, however, consume significant amounts of diluents and require considerable amount of sample preparation time. Accordingly, a HSGC method featured with fast turnaround time, and minimal amount of solvent use has been developed for the quantitative analysis of 27 residual solvents frequently used in the development and manufacturing processes of pharmaceutical industry. This HSGC-FID method employs a commercially available fused silica capillary column, a split injection (40:1), and a programmed temperature ramp. It was qualified for specificity, accuracy, repeatability/precision, linearity, LOQ, solution stability, and robustness using two representative sample matrices. The standards, samples and spiked samples were demonstrated to be stable for at least 10 days at room temperature in sealed headspace vials with a recovery of ≥ 93%. The method was also shown to be robust, and its performance was not affected by small changes of carrier gas flow rate, initial oven temperature or the headspace oven temperature. In this new approach, the analytical sample was prepared by dissolving the sample into 1 mL of the diluent and the standard solution was prepared by diluting 1 mL of the custom-made stock into 9 mL of the diluent whereas the traditional approach requires liters of the diluent, making the new approach environmentally friendly, sustainable, economical, agile, error-proofing and thus appropriate for a variety of pharmaceutical applications.

Characterization of polypropyleneimine as an alternative transfection reagent.

Polypropyleneimine (PPI) was examined as a transfection reagent comparing with most widely used polymer, polyethyleneimine (PEI). PPI had better responsiveness to the endosomal pH and showed more condensation ability of plasmid DNA than PEI. Although the cytotoxicity of PPI was somewhat higher than PEI, the transfection efficacy of PPI was comparable with PEI or higher than PEI in some cell line. Thus, PPI would be an alternative transfection reagent.

Use of capillary-mediated vitrification to produce thermostable, single-use antibody conjugates as immunoassay reagents.

The performance of enzyme-linked immunoassays is directly dependent on the storage, handling, and long-term stability of the critical reagents used in the assay. Currently, antibody reagents are routinely stored as concentrated, multi-use, frozen aliquots. This practice results in material waste, adds complexity to laboratory workflows, and can compromise reagents via cross-contamination and freeze-thaw damage. While refrigeration or freezing can slow down many degradation processes, the freezing process itself can have damaging effects, including introduction of aggregation and microheterogeneity. To address these challenges, we evaluated the application of capillary-mediated vitrification (CMV) as a tool for storing antibody reagents in a thermostable, single-use format. CMV is a novel biopreservation method that enables vitrification of biological materials without freezing. Using an anti-human IgG-alkaline phosphatase conjugate as a model system, we prepared CMV-stabilized aliquots which were stored in a single-use format at temperatures ranging from 25 to 55 °C for up to 3 months. Each stabilized aliquot contained enough antibody to perform a single assay run. We evaluated the assay performance and functional stability of the CMV-stabilized reagents using a plate-based ELISA. Assays run using the CMV stabilized reagents exhibited good linearity and precision that was comparable to results obtained with a frozen control. Throughout the stability study, the maximum signal and EC50s observed for ELISAs run using CMV-stabilized reagents were generally consistent with those obtained using a frozen control. These results indicate that the CMV process has the potential to improve both reagent stability and long-term assay performance, while also reducing reagent waste and simplifying assay workflows.