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2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Jaehi Kim ◽  
Do Won Hwang ◽  
Heung Su Jung ◽  
Kyu Wan Kim ◽  
Xuan-Hung Pham ◽  
...  

Abstract Background Quantum dots (QDs) have been used as fluorophores in various imaging fields owing to their strong fluorescent intensity, high quantum yield (QY), and narrow emission bandwidth. However, the application of QDs to bio-imaging is limited because the QY of QDs decreases substantially during the surface modification step for bio-application. Results In this study, we fabricated alloy-typed core/shell CdSeZnS/ZnS quantum dots (alloy QDs) that showed higher quantum yield and stability during the surface modification for hydrophilization compared with conventional CdSe/CdS/ZnS multilayer quantum dots (MQDs). The structure of the alloy QDs was confirmed using time-of-flight medium-energy ion scattering spectroscopy. The alloy QDs exhibited strong fluorescence and a high QY of 98.0%. After hydrophilic surface modification, the alloy QDs exhibited a QY of 84.7%, which is 1.5 times higher than that of MQDs. The QY was 77.8% after the alloy QDs were conjugated with folic acid (FA). Alloy QDs and MQDs, after conjugation with FA, were successfully used for targeting human KB cells. The alloy QDs exhibited a stronger fluorescence signal than MQD; these signals were retained in the popliteal lymph node area for 24 h. Conclusion The alloy QDs maintained a higher QY in hydrophilization for biological applications than MQDs. And also, alloy QDs showed the potential as nanoprobes for highly sensitive bioimaging analysis. Graphical Abstract


2022 ◽  
pp. 118493
Author(s):  
Xiaohui Yuan ◽  
Yanjun Hu ◽  
Nuonuo Zhang ◽  
Debao Liu ◽  
Peng Su ◽  
...  

Author(s):  
Min Xiao ◽  
Zhaochuan Chen ◽  
Yuan Zhang ◽  
Yanan Wen ◽  
Lihai Shang ◽  
...  

The constituents and content of dissolved organic matter (DOM) in the Qilian Mountain watershed were characterized with a spectroscopic technique, especially 3-DEEM fluorescence assisted by parallel factor (PARAFAC) analysis. The level of DOM in the surrounding area of Qinghai lake (thereafter the lake in this article specifically refers to Qinghai Lake)was highest at 9.45 mg C·L−1 and about 3 times less (3.09 mg C·L−1) in a cropland aquatic regime (the lowest value). In general, DOM was freshly autochthonously generated by plankton and plant debris, microorganisms and diagenetic effects in the aquatic environment (FI > 1.8). Component 1 (humic acid-like) and 3 (fulvic acid-like) determined the humification degree of chromophoric dissolved organic matter (CDOM). The spatial variation of sulfate and nitrate in the surrounding water regime of the lake revealed that organic molecules were mainly influenced by bacterial mediation. Mineral disintegration was an important and necessary process for fluorescent fraction formation in the cropland water regime. Exceptionally, organic moiety in the unused land area was affected by anespecially aridclimate in addition to microbial metabolic experience. Salinity became the critical factor determining the distribution of DOM, and the total normalized fluorescent intensity and CDOM level were lower in low-salinity circumstances (0.2–0.5 g·L−1) with 32.06 QSU and 1.38 m−1 in the grassland area, and higher salinity (0.6~0.8 g·L−1) resulted in abnormally high fluorescence of 150.62 QSU and absorption of 7.83 m−1 in the cropland water regime. Climatic conditions and microbial reactivity controlled by salinity were found to induce the above results. Our findings demonstrated that autochthonous inputs regulated DOM dynamics in the Qilian Mountains watershed of high altitude.


2021 ◽  
Vol 11 ◽  
Author(s):  
Noa G. Holtzman ◽  
Michael S. Lebowitz ◽  
Rima Koka ◽  
Maria R. Baer ◽  
Kanam Malhotra ◽  
...  

BackgroundAspartate β-hydroxylase (ASPH) is an embryonic transmembrane protein aberrantly upregulated in cancer cells, associated with malignant transformation and, in some reports, with poor clinical prognosis.ObjectiveTo report the expression patterns of ASPH in acute myeloid leukemia (AML).MethodsCell surface expression of ASPH was measured via 8-color multiparameter flow cytometry in 41 AML patient samples (31 bone marrow, 10 blood) using fluorescein isothiocyanate (FITC)-conjugated anti-ASPH antibody, SNS-622. A mean fluorescent intensity (MFI) of 10 was used as a cutoff for ASPH surface expression positivity. Data regarding patient and disease characteristics were collected.ResultsASPH surface expression was found on AML blasts in 16 samples (39%). Higher ASPH expression was seen in myeloblasts of African American patients (p=0.02), but no correlation was found between ASPH expression and other patient or disease characteristics. No association was found between ASPH status and CR rate (p=0.53), EFS (p=0.87), or OS (p=0.17).ConclusionsASPH is expressed on blasts in approximately 40% of AML cases, and may serve as a new therapeutically targetable leukemia-associated antigen.


2021 ◽  
Vol 3 ◽  
Author(s):  
Christopher Schmied ◽  
Tolga Soykan ◽  
Svenja Bolz ◽  
Volker Haucke ◽  
Martin Lehmann

Neuronal synapses are highly dynamic communication hubs that mediate chemical neurotransmission via the exocytic fusion and subsequent endocytic recycling of neurotransmitter-containing synaptic vesicles (SVs). Functional imaging tools allow for the direct visualization of synaptic activity by detecting action potentials, pre- or postsynaptic calcium influx, SV exo- and endocytosis, and glutamate release. Fluorescent organic dyes or synapse-targeted genetic molecular reporters, such as calcium, voltage or neurotransmitter sensors and synapto-pHluorins reveal synaptic activity by undergoing rapid changes in their fluorescence intensity upon neuronal activity on timescales of milliseconds to seconds, which typically are recorded by fast and sensitive widefield live cell microscopy. The analysis of the resulting time-lapse movies in the past has been performed by either manually picking individual structures, custom scripts that have not been made widely available to the scientific community, or advanced software toolboxes that are complicated to use. For the precise, unbiased and reproducible measurement of synaptic activity, it is key that the research community has access to bio-image analysis tools that are easy-to-apply and allow the automated detection of fluorescent intensity changes in active synapses. Here we present SynActJ (Synaptic Activity in ImageJ), an easy-to-use fully open-source workflow that enables automated image and data analysis of synaptic activity. The workflow consists of a Fiji plugin performing the automated image analysis of active synapses in time-lapse movies via an interactive seeded watershed segmentation that can be easily adjusted and applied to a dataset in batch mode. The extracted intensity traces of each synaptic bouton are automatically processed, analyzed, and plotted using an R Shiny workflow. We validate the workflow on time-lapse images of stimulated synapses expressing the SV exo-/endocytosis reporter Synaptophysin-pHluorin or a synapse-targeted calcium sensor, Synaptophysin-RGECO. We compare the automatic workflow to manual analysis and compute calcium-influx and SV exo-/endocytosis kinetics and other parameters for synaptic vesicle recycling under different conditions. We predict SynActJ to become an important tool for the analysis of synaptic activity and synapse properties.


2021 ◽  
Vol 21 (12) ◽  
pp. 5965-5971
Author(s):  
Xiaofang Song ◽  
Lifo Ruan ◽  
Tianyu Zheng ◽  
Jun Wei ◽  
Jiayu Zhang ◽  
...  

Facile preparation of a tumoral-stimuli-activated theranostic nanoparticle with simple constituents remains a challenge for tumor theranostic nanosystems. Herein we design a simple reductionresponsive turn-on theranostic nanoparticle for achieving fluorescent imaging and phototherapy combination. The theranostic nanoparticle is prepared by a simple one-step dialysis method of reduction active amphiphilic hyperbranched poly(β-amidoamines) and a near-infrared (NIR) dye indocyanine green (ICG). The fluorescence of ICG is quenched by the aggregation-caused quenching (ACQ) effect. The fluorescent intensity of free ICG at 816 nm was ∼40 times as high as that of particulate ICG. After reductive nanoparticles incubated with dithiothreitol (DTT), the size of the nanoparticles increased from 160 nm to 610 nm by Dynamic light scattering (DLS). As nanoparticles were internalized by cancer cells, the disulfide bonds would be cleaved by intracellular reduction agents like glutathione (GSH), leading to the release of entrapped ICG. The released ICG regained its fluorescence for self-monitoring the release and therapeutic effect of ICG by fluorescence spectra and the quantitative evaluation of NIR fluorescence intensity. Remarkably, nanoparticles can also reinforce antitumor efficacy through photodynamic therapy and GSH depletion property. This study provides new insights into designing turn-on theranostic systems.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fatemeh Sadat Shariati ◽  
Dariush Norouzian ◽  
Vahideh Valizadeh ◽  
Reza Ahangari Cohan ◽  
Malihe Keramati

Abstract Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.


2021 ◽  
Author(s):  
Vijayakumar Sathya ◽  
Appadurai Deepa ◽  
Lakshmi Kandhan Sangeetha ◽  
Venkatesan Srinivasadesi ◽  
Shyi-Long Lee ◽  
...  

Abstract Merocyanine dye based fluorescent organic compound has been synthesized for the detection of glutamine. The probe showed remarkable fluorescent intensity with glutamine through ICT. Hence, it is tested for the detection of glutamine using colorimetric and fluorimetric techniques in physiological and neutral pH (7.2). Under optimized experimental conditions, the probe detects glutamine selectively among other interfering biomolecules. The probe has showed a LOD of 9.6 nm at the linear range 20-180 µM towards glutamine. The practical application of the probe is successfully tested in human biofluids.


Materials ◽  
2021 ◽  
Vol 14 (23) ◽  
pp. 7210
Author(s):  
Manasa Nandimandalam ◽  
Francesca Costantini ◽  
Nicola Lovecchio ◽  
Lorenzo Iannascoli ◽  
Augusto Nascetti ◽  
...  

Innovative materials for the integration of aptamers in Lab-on-Chip systems are important for the development of miniaturized portable devices in the field of health-care and diagnostics. Herein we highlight a general method to tailor an aptamer sequence in two subunits that are randomly immobilized into a layer of polymer brushes grown on the internal surface of microfluidic channels, optically aligned with an array of amorphous silicon photosensors for the detection of fluorescence. Our approach relies on the use of split aptamer sequences maintaining their binding affinity to the target molecule. After binding the target molecule, the fragments, separately immobilized to the brush layer, form an assembled structure that in presence of a “light switching” complex [Ru(phen)2(dppz)]2+, emit a fluorescent signal detected by the photosensors positioned underneath. The fluorescent intensity is proportional to the concentration of the target molecule. As proof of principle, we selected fragments derived from an aptamer sequence with binding affinity towards ATP. Using this assay, a limit of detection down to 0.9 µM ATP has been achieved. The sensitivity is compared with an assay where the original aptamer sequence is used. The possibility to re-use both the aptamer assays for several times is demonstrated.


2021 ◽  
Author(s):  
Drishtant Singh ◽  
Samiksha . ◽  
Seema Madhumal Thayil ◽  
Satwinder Kaur Sohal ◽  
Anup Kumar Kesavan

Abstract An expression system based on the cry gene regulatory elements was constructed. The Terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 was cloned in pSG1151 plasmid downstream to gfpmut1. The promoter region of the cry gene was amplified to give three different reading frames. The Promoter region of cry gene was cloned in pSG1151T plasmid upstream to gfpmut1. The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfpmut1 under the same promoter. The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared by fluorescence spectroscopy. The fluorescent intensity of pSG1151 and pDSA1 were compared at time interval of 6 hours upto 72 hours. Both the samples showed detectable fluorescence that increased with time up to 12 hours, but the increase in the fluorescence of pDSA1 was 3 times higher as compared to pSG1151. A cold peptidase gene was cloned under the control of the cry promoter. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP. The cell-free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the specific activity of the extracellular protease. At 72 hours the protease activity in pDSA1CP was 2.7 fold higher compared to that of wild Bacillus sp. S1DI 10.


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