Labeling Agent Research Articles

Reliable and generic liquid chromatography/mass spectrometry quantification of short peptides using a stable-isotope-labeled labeling agent.

It is important to investigate the behavior of protein hydrolysate components in both in vitro and in vivo studies, to support the elucidation of their biological functions. As protein hydrolysates and biological matrices are highly complex mixtures, it is essential to apply fully reliable and flexible analytical approaches. A novel and generic Liquid Chromatography/Mass Spectrometry methodology was developed to analyze short peptides. A stable-isotope-labeled labeling agent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (13 C3 ) was synthesized and used to prepare internal standards from non-labeled analyte peptides. The amino acid and peptides p, pG, Pp, GPp and PpG (where p stands for hydroxyproline) were used for proof of principle. The method showed acceptable performance in solvent, in simulated gastrointestinal fluid and in serum. The (linear) dynamic range expanded to over four orders of magnitude, which is very useful when multiple analytes are analyzed in a biological matrix, due to the large differences in concentrations observed for endogenous and protein hydrolysate components. The method provides absolute-quantitative results and is fully accountable on the single-sample and single-component level. The methodology can be applied to reliably quantify protein hydrolysate nutraceutical components at various stages during their in vivo processing. Internal standards can also be synthesized for other short peptides whenever they are expected to have biological relevance and require quantification. Overall this provides an excellent analytical tool to support the elucidation of the biological functions of protein hydrolysate components.

Moxifloxacin based fluorescence imaging of intestinal goblet cells.

Goblet cells (GCs) in the intestine are specialized epithelial cells that secrete mucins to form the protective mucous layer. GCs are important in maintaining intestinal homeostasis, and the alteration of GCs is observed in inflammatory bowel diseases (IBDs) and neoplastic lesions. In the Barrett's esophagus, the presence of GCs is used as a marker of specialized intestinal metaplasia. Various endomicroscopic imaging methods have been used for imaging intestinal GCs, but high-speed and high-contrast GC imaging has been still difficult. In this study, we developed a high-contrast endoscopic GC imaging method: fluorescence endomicroscopy using moxifloxacin as a GC labeling agent. Moxifloxacin based fluorescence imaging of GCs was verified by using two-photon microscopy (TPM) in the normal mouse colon. Label-free TPM, which could visualize GCs in a negative contrast, was used as the reference. High-speed GC imaging was demonstrated by using confocal microscopy and endomicroscopy in the normal mouse colon. Confocal microscopy was applied to dextran sulfate sodium (DSS) induced colitis mouse models for the detection of GC depletion. Moxifloxacin based GC imaging was demonstrated not only by 3D microscopies but also by wide-field fluorescence microscopy, and intestinal GCs in the superficial region were imaged. Moxifloxacin based endomicroscopy has a potential for the application to human subjects by using FDA approved moxifloxacin.

Regeneration of hyaline cartilage in osteochondral lesion model using L-lysine magnetic nanoparticles labeled mesenchymal stem cells and their in vivo imaging.

Treatment of osteochondral defects continues to pose a major challenge for patients and orthopedic surgeons due to the limited healing potential of articular cartilage. Mesenchymal stem cells (MSCs) possess therapeutic potential for the treatment of osteochondral pain and pathology. However, it is necessary to use proper labeling and imaging agent of stem cells that can decipher its role posttransplantation. A major limitation of routinely used contrast agents is signal dilution over a period of time which limits its use for further studies. At the same time, regeneration of fibrocartilage over native hyaline cartilage also limits the use of conventional therapies. The present study evaluates the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) for the treatment of osteochondral defect in rats with the regeneration of hyaline cartilage in situ and in vivo monitoring of the stem cells using L-lysine functionalized magnetic iron oxide nanoparticles (lys-IONPs). L-lysine stabilizes the iron oxide nanoparticles, enhances the biocompatibility, and provides functionalities for efficient stem cell labeling. in vitro toxic effects of lys-IONPs on mitochondrial impairment, morphological alterations, and actin cytoskeleton reveal minimum damage to BM-MSCs. Histological data (H and E, Masson's trichrome and immunohistochemistry) describe the early initiation of healing and regeneration of hyaline-like cartilage over fibrocartilage in stem cell treated groups. MR scans demonstrate generation of hypointense signals in lys-IONPs-BMSCs with improved signal intensity and minimum loss over 28 days revealing its use as a long-term stem cell labeling and imaging agent.

Abstract LB-024: Development of a non-invasive clinically applicable real time cell tracking platform for evaluating cell-based cancer therapy using magnetic resonance imaging (MRI)

Abstract While advancement in chimeric antigen receptors (CARs) has greatly improved the anti-tumor activity of immune cell-based therapies utilizing modified T lymphocytes (CAR-T) and natural killer cells (CAR-NK), there remains a critical unmet need for diagnostic technologies to help evaluate their biological performance effectively and efficiently after injecting into the body.The ability to better understand the biodistribution of injected cells and devise dosing strategies are key determinants to the success or failure of cell therapy. However, clinicians are limited by the available technology for real time in-vivo tracking of cell therapy products in clinical settings. Here we present a clinically applicable novel magnetic agent that can effectively label a variety of cell types used in cancer cell therapy development including T cells, NK cells, and neural stem cells (NSCs), and when combined with the 3D ultrashort echo time Cones (3D UTE-Cones) sequence, can enable quantitative assessment of cell therapy products using MRI. In this study, we optimized loading of our tracking agent into T cells, NK cells, and NSCs, evaluated the morphology and viability of the labeled cells, and confirmed internalization of the labeling agent by Prussian blue staining and TEM. The in vitro MRI properties of the labeled cells were quantitated by the 3D UTE-Cones sequence. Ex vivo MRI analysis was performed by injecting labeled cells into harvested mouse livers and brains to ascertain their MRI visibility in tissues using a clinical MRI scanner. We confirmed that our magnetic labeling agent efficiently labeled each cell line tested, and the morphology or viability of the labeled cells were unaffected. The MRI detectability of labeled cells were confirmed by in vitro MRI phantoms. Further, using quantitative susceptibility mapping (QSM), T1, and T2* mapping based on 3D UTE-Cones sequences, we found near perfect correlations between cell density and MRI properties. By applying the parameters established in the MRI phantoms, we were able to detect and quantity the number of labeled cells injected into mouse livers and brains ex vivo. In summary, we have developed a clinically applicable imaging-based platform that empowers cell therapy researchers to better understand the results of their biodistribution and dosing studies through real-time cell tracking and quantitative assessments using MRI. Citation Format: Johnny C. Akers, Zhao Wei, Hyungseok Jang, Eric Chang, Jiang Du, Mya S. Thu. Development of a non-invasive clinically applicable real time cell tracking platform for evaluating cell-based cancer therapy using magnetic resonance imaging (MRI) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-024.

Abstract 2851: A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format

Abstract A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format. Removal of unreacted or residual compounds in the sample-preparation workflow is a key success factor for downstream analysis and processing of biomolecules. For example, during sample preparation of biomolecules such as proteins or nucleic acids, there is often a need to label the biomolecules with dyes, affinity tags, radioactive labels, and mass tags. In other instances, there is a need to chemically modify biomolecules such as to reduce, oxidize, methylate, etc. During these treatments, some amount of the labeling or chemical agent remains in the sample as an unreacted label/chemical. These unreacted small molecules can cause several issues during downstream analysis or use of the biomolecule. For example, free unreacted fluorescent dyes after bioconjugation of protein cause non-specific binding of free-dye resulting in high background issues during fluorescent imaging. Commonly used clean-up methods following sample preparation, such as dialysis, are tedious and lengthy with multiple buffer exchanges sometime resulting in loss of protein due to aggregation. Alternatively, size-exclusion chromatography requires expensive instrumentation in addition to lengthy set up time. We have developed the Thermo Scientific™ PierceTM Dye and Biotin Removal Spin Columns, a novel resin that is packed in an easy to use “Spin and Go” format of varying spin column sizes (0.5 mL, 2mL, 5 mL and 10 mL) to accommodate a range of sample volumes (100 uL- 4 mLs). The resin is highly specialized to produce exceptional protein recovery and can be used effectively to remove 4 different class of small molecules: non-conjugated fluorescent dyes, biotinylation reagents, reducing agents, and crosslinkers. It is a multimodal resin with size exclusion properties and proprietary surface chemistry that allows removal of the above-mentioned small molecules, while the size exclusion property allows for the biomolecule to be efficiently recovered. Our results indicate efficient dye removal and protein recovery from fluorescent conjugated antibodies. The results also indicate removal of reducing agents such as TCEP at a concentration as high as 25 mM from protein samples, while recovering >90% of the added protein. Results also indicate >90% removal of biotinylated molecules and crosslinkers from Protein samples with >90% protein recovery. Our results show that the efficiency and binding capacity for removal of small molecules using this resin is better than any resin in the market. This is exemplified by immunofluorescent analysis of ZO-1 monoclonal antibody conjugated Alexa Fluor™ 488 which was purified using PierceTM Dye and Biotin Removal Spin columns and shows high intensity and lesser background in Caco-2 cells than the non-purified conjugate. Citation Format: Deven Kent Etnyre, Ramesh Ganapathy, Marie Nlend, Chris Etienne, Atul M. Deshpande. A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2851.

Sensitive and rapid lateral-flow assay for early detection of subclinical mammary infection in dairy cows

Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare. We have developed a semiquantitative lateral flow assay for the detection of SCM in dairy cows targeting myeloperoxidase (MPO) enzyme of milk neutrophils. A competitive immunoassay format was used, and colloidal gold nanoparticles (GNP) were prepared and used as a labelling agent. Monoclonal anti-MPO antibodies were used and assessed for its quality by enzyme-linked immunosorbent assay and dot blot. Conjugation method for GNP and anti-MPO antibodies was standardised, and the conjugate was placed over the conjugate pad. MPO coupled with a carrier protein (OVA) and the species-specific secondary antibodies were placed on test and control lines, respectively. The developed assay was verified with 75 milk samples collected from healthy, SCM and clinical mastitis cows. It displayed a high sensitivity as it could detect MPO as low as 1.5 ng/ml, an accuracy greater than 97% and showed no crossreactivity when crosschecked with other milk proteins. The developed assay can be used as an alternative for SCM diagnostic tests where lab structure are available for obtaining the lysate of milk SCC.

Synthesis and characterization of fluorescent Europium (III) complex based on D-dextrose composite for latent fingerprint detection

A europium salt-Na[Eu(5,5′-DMBP)(phen)3]·Cl3 (Eu(III)-CPLx) was prepared by using various precursors such as 5,5′-Dimethyl-2,2′-bipyridyl (5,5′-DMBP), 1,10-phenanthroline (phen) and europium chloride hexahydrate (EuCl3·6H2O) by a complexation method. The red emission fluorescent Na[Eu(5,5′-DMBP)(phen)3]·Cl3/D-Dextrose (Eu(III)-CPLx/D-Dex) composite was synthesized by using an adsorption method with Eu(III)-CPLx and D-Dextrose (D-Dex). The Eu(III)-CPLx and fluorescent (Eu(III)-CPLx/D-Dex) composites were characterized by numerous techniques. The fluorescent (Eu(III)-CPLx/D-Dex) composite demonstrated a strong red emission and controlled fluorescence quenching in the solid state and was consequently used in latent fingerprint (LFP) detection. The LFPs were developed by using a powder dusting method (PDM) with Eu(III)-CPLx and fluorescent Eu(III)-CPLx/D-Dex composites on different substrates under daylight and UV-light irradiation at 365 nm. The fluorescent Eu(III)-CPLx/D-Dex composite was effectively explored for developing LFP images on various substrates and also acts as a better labeling agent for LFP detection in forensic science crime scene investigations.

Structure unique, fonctionnalités multiples : La phycocyanine en biotechnologie

Phycocyanine (PC) is a water-soluble, non-toxic and bioactive (antioxidant, anti-inflammatory, antitumor, etc.) phycobiliprotein isolated, mainly, from cyanobacteria. Due to its several properties, PC is considered to be a rising biomolecule for Industrial exploitation, and has become an important research axis in order to promote its production, and optimize its biotechnological applications. The aim of this review article was to discuss the basic, and recent properties and applications of PC, and to bring together data on various aspects of PC stabilization, and PC nanopar-ticles formulation. In addition, an overview of the main structural characteristics and process-ses of PC extraction and purification were also discussed. The recent scientific research findings concluded that PC is a promising both functional, and bioactive additive in industry, especially, in food as a dye, in imaging as a fluorescent labeling agent, and in the phar-maceutical and nano-pharmaceutical field as a bioactive molecule and nanopar-ticles, particularly, due to it antitumor capacity. Phycocyanine is, thus, a promising bio-active molecules in pharmacological, and medical fields.

Improvement of reproducibility and thermal stability of surface-enhanced Raman scattering-based lateral flow assay strips using silica-encapsulated gold nanoparticles

A surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip using silica-encapsulated gold nanoparticles (Si-AuNPs) was developed for the diagnosis of mosquito-borne diseases—dengue fever, Zika virus, or malaria—which are endemic to tropical regions. To investigate the thermal stability of the SERS nanoprobes, Raman peak intensity variations for AuNPs and Si-AuNPs were measured and compared at 45°C. SERS mapping images of target human immunoglobulin (IgG) on the test line of the Si-AuNP-loaded LFA strip were also evaluated at the same temperature to test the reproducibility of detection. In this case, the distribution of Raman mapping points on the test line was relatively uniform compared with the AuNP-loaded LFA strips at 45°C. This indicates that the silica encapsulation on the surface of the AuNPs improves the reproducibility of the SERS nanoprobes at high temperatures. The limit of detection (LOD) value of the AuNP-loaded LFA strips was significantly increased when the temperature was increased from 25°C to 45°C. In the case of the Si-AuNP-loaded LFA strips, however, the LOD value at 45°C was almost the same as that at 25°C. This result also shows that the silica encapsulation of the AuNPs improves the thermal stability of the LFA strips at high temperatures. Thus, Si-AuNPs are reproducible and thermally stable labeling agents for use in LFA strips at high temperatures and are suitable for the preparation of rapid kits, which can be used in tropical areas.

Synthesis and characterization of CDs/Al2O3 nanofibers nanocomposite for Pb2+ ions adsorption and reuse for latent fingerprint detection

This study reports a new approach of preparation of carbon dots coated on aluminum oxide nanofibers (CDs/Al2O3NFs) nanocomposite and reusing the spent adsorbent of lead (Pb2+) ions loaded adsorbent (Pb2+-CDs/Al2O3NFs) nanocomposite for latent fingerprint detection (LFP) after removing Pb2+ ions from aqueous solution. CDs/Al2O3NFs nanocomposite was prepared by using CDs and Al2O3NFs with adsorption processes. The prepared nanocomposite was then characterized by using UV–visible spectroscopy (UV–visible), Fourier transforms infrared spectroscopy (FTIR), Fluorescence, X-ray diffraction pattern (XRD), scanning electron microscope (SEM), Transmission electron microscopy (TEM), Energy-dispersive X-ray spectroscopy (EDS), Zeta potential, X-ray photoelectron spectroscopy (XPS). The average size of the CDs was 51.18 nm. The synthesized CDs/Al2O3NFs nanocomposite has proven to be a good adsorbent for Pb2+ ions removal from water with optimum pH 6, dosage 0. 2 g/L. The results were best described by the Freundlich Isotherm model. The adsorption capacity of CDs/Al2O3NFs nanocomposite showed the best removal of Pb2+ ions with qm = (177. 83 mg/g), when compared to the previous reports. This adsorption followed the pseudo-second order kinetic model. ΔG and ΔH values indicated spontaneity and the endothermic nature of the adsorption process. CDs/Al2O3NFs nanocomposite therefore showed potential as an effective adsorbent. The data were observed from adsorption–desorption after 6 cycles which showed good adsorption stability and re- usability of CDs/Al2O3NFs nanocomposite. Furthermore, the spent adsorbent of Pb2+-CDs/Al2O3NFs nanocomposite has proven to be sensitive and selective for LFP detection on various porous substrates. Hence Pb2+-CDs/Al2O3NFs nanocomposite can be reused as a good fingerprint labelling agent in LFP detection so as to avoid secondary environmental pollution by disposal of the spent adsorbent.

Yellow emitting Fe3O4/ZnS hybrid: A probe for in-vitro dermatoglyphics and anti-counterfeiting applications

Ambidextrous phosphor materials (performance intact in powder and dispersed condition) with large Stokes shift and excited state lifetime is highly preferable due to the stringent requirements of latent finger print (LFP) detectors and anti-counterfeiting markers. In this work, ZnS grafted Fe3O4 core shell luminomagnetic hybrid have been prepared via facile hydrothermal route. Interestingly, instead of pristine green emission of ZnS the prepared ambidextrous hybrid material (Fe3+:Fe3O4/ZnS) shows intense yellow emission under 350 nm UV exposure. Detailed experimental investigation supported by theoretical modelling and plausible energy band diagram demonstrates an inherent self-substitution mechanism of Zn2+ ions of ZnS shell by Fe3+ ions from the magnetite core which plays a decisive role in achieving the novel yellow emission. Maintaining superparamagnetic behavior in room temperature, the designed rare-earth (RE) free hybrid is employed as a proficient luminescent labeling agent for LFP detection (up to level III, stability up to 45 days) and anti-counterfeiting marker with higher accuracy compared to virgin ZnS.

Multifaceted application of nanoparticle-based labeling strategies for stem cell therapy

Stem cell therapy can overcome many unmet clinical challenges that have proven intractable with traditional medicine and treatment strategies because of their strong ability to recover and regenerate injured tissue. Nanoparticle-based labeling agents, in combination with current imaging technologies, have been used to monitor the location, migration, survival, and differentiation of transplanted stem cells in therapeutic processes to fully understand their fates in vivo. In addition, various kinds of functional nanoparticles can exert unique biological effects on stem cells to overcome some problems of current stem cell therapy involving cell homing, survival, retention, and directional differentiation. Moreover, nanoparticle-based theranostic labeling, in which both imaging and treatment modalities are delivered with the stem cells, has emerged as a new strategy to enhance the therapeutic effect of stem cells and shows great potential for future stem cell therapy. The aim of this review is to describe the current roles played by nanoparticles in cell tracking, cell fate regulation, and theranostic labeling in stem cell therapy. Important issues and perspectives on the potential growth of this area are also critically discussed.

First in-human study of in vivo imaging of ex vivo labeled CAR T cells with dual PET-MR.

3557 Background: This is a first in human in-vivo biodistribution of ex-vivo labelled CAR T cells assessed in a cohort of patients. Cells were labelled with novel Cu-64 labelled superparamagnetic iron oxide nanoparticles (SPION) and infused IV into patients with solid tumors & tracked using clinical dual PET-MR. The study validates the clinical translation of CAR T cell in-vivo tracking in real time. Methods: Cu-64 radioisotope was bound to silica coated SPION using electrolysis plating with tin & palladium seeding. Cellular uptake of Cu-64 SPION was facilitated with a transfecting agent. Functional assays including 51Chromium release, cytometric bead array demonstrated that labelling process did not affect cytotoxicity & cytokine secretion (TNFα & IFN-g). T cells were transduced with retroviral vector constructs encoding for second-generation chimeric T-cell receptor specific for carbohydrate Lewis Y antigen. Modified T-cells were expanded ex-vivo & were labelled with Cu-64 (~300 MBq) prior to re-infusion (3 x108 labelled cells). Scanning is performed with Siemens 3T dual PET-MR scanner. Results: In this first in human in-vivo study (HREC/16/PMCC/30) a cohort of patients received ex-vivo labelled CAR T cells to determine how many labelled cells distribute to solid tumor sites within 3-5 days. Our results demonstrate that cells can be efficiently labelled (≤60%) with high cell viability (≥85%) at a sensitivity sufficient to detect labelled cells at tumor site for up to 5 days. An observed trend in SUVmean & SUVmax provided insight into efficacy & individual response to therapy. Early time points showed moderate uptake of labelled cells in lungs posterior basal segments without increased activity over next few days, suggesting a transient process. Mild, diffuse bone marrow & relatively intense uptake of labelled cells in liver & spleen suggests margination of cells to reticulo-endothelial system. Distinct PET signal at some of the tumor sites at 24 h suggests antigen specific localization & time taken to reach these sites. Excretion via hepatobiliary indicated reabsorption from GI tract & re-circulation of labelled cells. Minimal uptake in brain & heart supported safety profile of labeling agent. Conclusions: This is first in human in-vivo study to provide highly valuable visual and dynamic data in real time and provides insight into individual responses to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The findings indicate that labelled cells traffic to tumor sites at later time points & remain persistent for extended period of time.

Non-cytotoxic photostable monomethine cyanine platforms: Combined paradigm of nucleic acid staining and in vivo imaging

Cyanine based chemosensing platforms have successfully been employed over the past couple of decades in various fields of biomolecular sciences. Still a substantial number of recent advances and improvements on this class of compounds are reported in the art. This paper presents our latest work on the improved synthetic approach, study on photophysical properties, and biosensing applicability of monomethine cyanine dyes. The series of mono-, di- and tricationic dyes showed up to 5-fold enhanced resistance against photobleaching compared to the commercially available Thiazole Orange (TO). The title compounds were studied as potential molecular probes for the detection of deoxyribonucleic acid, demonstrating their capacity as excellent fluorescent labeling agents. Depending on the dye chemical structure, current Cl-TO compounds exhibit up to 834-fold enhanced fluorescence emission and form stable complexes with Calf Thymus-DNA. The calculated binding constants were found to be higher than several conventional fluorogenic dyes for nucleic acid detection. All studied derivatives appeared as less cytotoxic than the Thiazole Orange. IC50 concentrations in human fibroblasts MRC5 cell line were calculated up to 50 μM for the synthesized Cl-TO dyes, and 0.5 μM for the parental Thiazole Orange. Two of the dyes were found very competent in post-electrophoretic visualization of DNA. As demonstrated by the agarose gel electrophoresis, the staining efficiency and detection limits of the dyes were comparable to the widely used Ethidium Bromide. The tricationic dye revealed great potential for cell cycle analysis in G1, S and G2 phases. The chlorinated TO derivatives readily stain human cells in vivo, while they can effectively be applied for eukaryotic and microbial cell staining.

First in Human Study of In-vivo Imaging of Ex-Vivo Labelled CAR T Cells with Dual PET-MR

Background & Aim This is a first in human in-vivo biodistribution of ex-vivo labelled CAR T cells assessed in a cohort of patients. Cells were labelled with novel Cu-64 labelled superparamagnetic iron oxide nanoparticles (SPION) and infused IV into patients with solid tumors & tracked using clinical dual PET-MR. The study validates the clinical translation of CAR T cell in-vivo tracking in real time. Methods, Results & Conclusion Methods Cu-64 radioisotope was bound to silica coated SPION using electrolysis plating with tin & palladium seeding. Cellular uptake of Cu-64 SPION was facilitated with a transfecting agent. Functional assays including 51Chromium release, cytometric bead array demonstrated that labelling process did not affect cytotoxicity & cytokine secretion (TNFα & IFN-g). T cells were transduced with retroviral vector constructs encoding for second-generation chimeric T-cell receptor specific for carbohydrate Lewis Y antigen. Modified T-cells were expanded ex-vivo & were labelled with Cu-64 (∼300 MBq) prior to re-infusion (3 × 108 labelled cells). Scanning is performed with Siemens 3T dual PET-MR scanner. Results In this first in human in-vivo study (HREC/16/PMCC/30) a cohort of patients received ex-vivo labelled CAR T cells to determine how many labelled cells distribute to various body organs and lesions within first 3-5 days. Our results demonstrate that both fresh (N =3) and thawed cells (N =3) can be efficiently labelled in the range of 40% - 80% with high cell viability (≥90%). An observed trend in SUVmean provided insight into efficacy & individual response to therapy. Early time points showed moderate uptake of labelled cells in lungs posterior basal segments without increased activity over next few days, suggesting a transient process. Mild, diffuse bone marrow & relatively intense uptake of labelled cells in liver & spleen suggests margination of cells to reticulo-endothelial system. Excretion via hepatobiliary indicated reabsorption from GI tract & re-circulation of labelled cells. The initial data shows that labelled cell may remain with in the system for upto 10-15 days. Minimal uptake in brain & heart supported safety profile of labeling agent. Conclusions This is first in human in-vivo study to provide highly valuable visual and dynamic data in real time and provides insight into individual responses to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The findings indicate that labelled cells remain persistent for extended period of time.

Effect of Cell Labeling on the Function of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cell labeling technologies are required to monitor the fate of transplanted cells in vivo and to select target cells for the observation of certain changes in vitro. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been transplanted for the treatment of heart injuries or used in vitro for preclinical cardiac safety assessments. Cardiomyocyte (CM) labeling has been used in these processes to facilitate target cell monitoring. However, the functional effect of the labeling agent on hiPSC-CMs has not been studied. Therefore, we investigated the effects of labeling agents on CM cellular functions. 3’-Dioctadecyloxacarbocyanine perchlorate (DiO), quantum dots (QDs), and a DNA plasmid expressing EGFP using Lipo2K were used to label hiPSC-CMs. We conclude that the hiPSC-CM labeling with DiO and QDs does not induce arrhythmogenic effects but rather improves the mRNA expression of cardiac ion channels and Ca2+ influx by L-type Ca2+ channels. Thus, DiO and QD labeling agents may be useful tools to monitor transplanted CMs, and further in vivo influences of the labeling agents should be investigated in the future.

Harnessing intrinsic fluorescence for typing of secondary structures of DNA.

High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T, the most fluorescent G4 structure reported to date).

Noninvasive Tracking of Hematopoietic Stem Cells in a Bone Marrow Transplant Model.

The hematopoietic stem cell engraftment depends on adequate cell numbers, their homing, and the subsequent short and long-term engraftment of these cells in the niche. We performed a systematic review of the methods employed to track hematopoietic reconstitution using molecular imaging. We searched articles indexed, published prior to January 2020, in PubMed, Cochrane, and Scopus with the following keyword sequences: (Hematopoietic Stem Cell OR Hematopoietic Progenitor Cell) AND (Tracking OR Homing) AND (Transplantation). Of 2191 articles identified, only 21 articles were included in this review, after screening and eligibility assessment. The cell source was in the majority of bone marrow from mice (43%), followed by the umbilical cord from humans (33%). The labeling agent had the follow distribution between the selected studies: 14% nanoparticle, 29% radioisotope, 19% fluorophore, 19% luciferase, and 19% animal transgenic. The type of graft used in the studies was 57% allogeneic, 38% xenogeneic, and 5% autologous, being the HSC receptor: 57% mice, 9% rat, 19% fish, 5% for dog, porcine and salamander. The imaging technique used in the HSC tracking had the following distribution between studies: Positron emission tomography/single-photon emission computed tomography 29%, bioluminescence 33%, fluorescence 19%, magnetic resonance imaging 14%, and near-infrared fluorescence imaging 5%. The efficiency of the graft was evaluated in 61% of the selected studies, and before one month of implantation, the cell renewal was very low (less than 20%), but after three months, the efficiency was more than 50%, mainly in the allogeneic graft. In conclusion, our review showed an increase in using noninvasive imaging techniques in HSC tracking using the bone marrow transplant model. However, successful transplantation depends on the formation of engraftment, and the functionality of cells after the graft, aspects that are poorly explored and that have high relevance for clinical analysis.

A rapid and an efficient method to remove unreacted small‐molecules using a versatile resin packed in a convenient Spin‐and‐Go format.

Removal of unreacted or residual compounds in the sample‐preparation workflow is a key success factor for downstream analysis and processing of biomolecules. For example, during sample preparation of biomolecules such as proteins or nucleic acids, there is often a need to label the biomolecules with dyes, affinity tags, radioactive labels, and mass tags. In other instances, there is a need to chemically modify biomolecules such as to reduce, oxidize, methylate, etc. During these treatments, some amount of the labeling or chemical agent remains in the sample as an unreacted label/chemical. These unreacted small molecules can cause several issues during downstream analysis or use of the biomolecule. For example, free unreacted fluorescent dyes after bioconjugation of protein cause non‐specific binding of free‐dye resulting in high background issues during fluorescent imaging. Commonly used clean‐up methods following sample preparation, such as dialysis, are tedious and lengthy with multiple buffer exchanges sometime resulting in loss of protein due to aggregation. Alternatively, size‐exclusion chromatography requires expensive instrumentation in addition to lengthy set up time.We have developed the Thermo Scientific™ Pierce™ Dye and Biotin Removal Spin Columns, a novel resin that is packed in an easy to use “Spin and Go” format of varying spin column sizes (0.5 mL, 2mL, 5 mL and 10 mL) to accommodate a range of sample volumes (100 uL–4 mLs). The resin is highly specialized to produce exceptional protein recovery and can be used effectively to remove 4 different class of small molecules: non‐conjugated fluorescent dyes, biotinylation reagents, reducing agents, and crosslinkers. It is a multimodal resin with size exclusion properties and proprietary surface chemistry that allows removal of the above‐mentioned small molecules, while the size exclusion property allows for the biomolecule to be efficiently recovered.Our results indicate efficient dye removal and protein recovery from fluorescent conjugated antibodies. The results also indicate removal of reducing agents such as TCEP at a concentration as high as 25 mM from protein samples, while recovering >90% of the added protein. Results also indicate >90% removal of biotinylated molecules and crosslinkers from Protein samples with >90% protein recovery. Our results show that the efficiency and binding capacity for removal of small molecules using this resin is better than any resin in the market. This is exemplified by immunofluorescent analysis of ZO‐1 monoclonal antibody conjugated DyLight™ 488 which was purified using Pierce™ Dye and Biotin Removal Spin columns and shows high intensity and lesser background in Caco‐2 cells than the non‐purified conjugate.

Monolayer single crystal two-dimensional quantum dots via ultrathin cutting and exfoliating

Two-dimensional (2D) atomically thin quantum dots (QDs) possess extraordinary electrical and optical properties. However, fabricating high quality 2D QDs via a universal and reliable technique remains a challenge. Here, we report a simple strategy to prepare high quality, monolayer single crystal 2D QDs via ultrathin cutting 2D bulk single crystals by ultramicrotome, followed by an exfoliation process. The as-prepared 2D QDs have pristine surface, high quality, high monolayer yield and high photoluminescence quantum yield (the highest photoluminescence quantum yield of WS2 is 18%), which can be used as promising, low toxic, biocompatible, and good cell-permeability fluorescent labeling agents for in vitro imaging.