Increase In Fluorescence Signal Research Articles

Endogenous mRNA-Driven "One-To-More" Signal Amplification of DNA Probe for Intracellular miR155 Sensing.

The detection of specific intracellular microRNAs could be potentially helpful in understanding the underlying mechanisms of cancer metastasis and invasion. MiRNAs are usually present in lower expression levels, especially in early stage of cancer. Here, we proposed a "one-to-more" amplification strategy for miRNA imaging, by virtue of DNA strand displacements with dual-amplification. This approach involves leveraging high-abundance endogenous mRNA as fuel strand to drive cascade reactions between DNA strands for amplification, enabling the monitoring of low-abundance intracellular microRNA155. Notably, in comparison to the traditional "one-to-one" signal triggering mode, our "one-to-more" amplification strategy led to a remarkable 11.8-fold increase in fluorescence signal. Our approach not only demonstrates a high sensitivity and specificity in detecting miR155, but also allows for discrimination of miR155 expression levels in different cell lines. With the advantages of intracellular signal amplification and reduced background signal, this approach holds substantial potential in the early diagnosis of cancer.

A high-throughput screen in mESCs to identify the cross-talk between signaling, endocytosis, and pluripotency.

Embryonic stem cell fate is regulated by various cellular processes. Recently, the process of endocytosis has been implicated in playing a role in the maintenance of self-renewal and pluripotency of mouse embryonic stem cells. A previous siRNA-based screen interrogated the function of core components of the endocytic machinery in maintaining the pluripotency of embryonic stem cells, revealing a crucial role for clathrin mediated endocytosis. A number of other proteins involved in key signaling pathways have also been shown to both regulate and be regulated by endocytosis. We collated a list of 1141 genes in connection to the term "endocytosis" from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), excluding those previously interrogated, and examined the effect of their knockdown on the pluripotency of mouse embryonic stem cells (mESCs) using levels of green fluorescent protein driven by the Oct4 promoter. We used high-throughput screening followed by an automatedMATrix LABoratory(MATLAB)-based analysis pipeline and assessed changes in GFP fluorescence as a readout for ESC pluripotency. Through this screen we identified a number of genes, many hitherto not associated with stem cell pluripotency, which upon knockdown either resulted in a significant increase or decrease of GFP fluorescence. We further present validation for some of these hits. We present a workflow aimed to identify genes involved in signaling pathways which can be regulated by endocytosis, and that affect the pluripotency of ESCs.

Rational design of a near-infrared fluorescent probe for monitoring butyrylcholinesterase activity and its application in development of inhibitors.

Butyrylcholinesterase (BChE) is widely expressed in multiple tissues and has a vital role in several key human disorders, such as Alzheimer's disease and tumorigenesis. However, the role of BChE in human disorders has not been investigated. Thus, to quantitatively detect and visualize dynamical variations in BChE activity is essential for exploring the biological roles of BChE in the progression of a number of human disorders. Herein, based on the substrate characteristics of BChE, we customized and synthesized three near-infrared (NIR) fluorescent probe substrates with cyanine-skeleton, and finally selected a NIR fluorescence probe substrate named CYBA. The CYBA demonstrated a significant increase in fluorescence when interacting with BChE, but mainly avoided AChE. Upon the addition of BChE, CYBA could be specifically hydrolyzed to TBO, resulting in a significant NIR fluorescence signal enhancement at 710nm. Systematic evaluation revealed that CYBA exhibited exceptional chemical stability in complex biosamples and possessed remarkable selectivity and sensitivity towards BChE. Moreover, CYBA was successfully applied for real-time imaging of endogenous BChE activity in two types of nerve-related living cells. Additionally, CYBA demonstrated exceptional stability in the detection of complex biological samples in plasma recovery studies (97.51%-104.01%). Furthermore, CYBA was used to construct a high-throughput screening (HTS) method for BChE inhibitors using human plasma as the enzyme source. We evaluated inhibitory effects of a series of natural products and four flavonoids were identified as potent inhibitors of BChE. Collectively, CYBA can serve as a practical tool to track the changes of BChE activity in complicated biological environments due to its excellent capabilities.

A new Eosin Y-based ‘turn[sbnd]on’ fluorescent sensor for ratiometric sensing of toxic mercury ion (Hg2+) offering unaided eye detection and its antibacterial activity

A unique fluorescent molecule (ND-S) was obtained from Eosin Y in two simple yet high yielding steps (1). ND-S has special metal ion sensing ability, such that it can selectively detect toxic Hg2+ present in very low concentration in aqueous solutions in the presence of other competing metal ions. The host-guest complexation is ratiometric and is associated with significant increase in fluorescence during the process. Isothermal titration calorimetry (ITC) experiments provided thermodynamic parameters related to interaction between ND-S and Hg2+. Using inductively coupled plasma mass spectrometry (ICP-MS), the Hg2+(aq) removal efficiency of ND-S was estimated to be 99.88%. Appreciable limit of detection (LOD = 7.4 nM) was observed. Other competing ions did not interfere with the sensing of Hg2+ by ND-S. The effects of external stimuli (temperature and pH) were studied. Besides, the complex (ND-M), formed by 1:1 coordination of ND-S and Hg2+ was found to be effective against the survival of Gram-positive bacteria (S. aureus and B. subtilis) with a high selectivity index. Moreover, bacterial cell death mechanism was studied systematically. Overall, we have shown the transformation of a toxic species (Hg2+), extracted from polluted water by a biocompatible sensor (ND-S), into an effective and potent antibacterial agent (ND-M).

Protein biomarker detection via differential dynamic microscopy

We demonstrate a pioneering application of differential dynamic microscopy (DDM) for disease diagnosis by monitoring the dynamic signal of probe beads. When the target antibody binds to our sensitized probe beads, it allows these originally non-fluorescent beads to be indirectly decorated with fluorescently labeled secondary antibodies. Using standard epi-fluorescence microscopy without the need for purification, DDM reliably detects the resulting increase in fluorescence signal from the probe beads. It effectively discerns this signal from the background, which includes residual free fluorescent secondary antibodies. As a proof-of-concept, polystyrene beads (500 nm in diameter) were conjugated with the receptor binding domain of SARS-CoV-2’s spike protein to quantify immunoglobulin G1-specific antibodies against it in three different media of increasing complexity (phosphate buffer, model saliva and fetal bovine serum). Our results show that as the medium becomes more complex, more dilution and/or longer incubation time may be needed to overcome the competition effect between the target and interfering proteins. By optimizing imaging parameters and sample preparation we could surpass clinically relevant limits of detection in both saliva and serum samples. In contrast to conventional diagnostic techniques including ELISA and PCR, DDM brings many advantages including simplicity of the preparation protocol, high speed, and versatility without requiring very expensive or complicated equipment. As such, DDM-based disease diagnostics has the potential to be utilized in many laboratories, especially in remote regions with limited resources and point-of-use applications may also be feasible by miniaturizing the imaging setup.

Investigation of biofluorescence produced by the red king crab Paralithodes camtschaticus

AbstractBiofluorescence is widely documented in marine organisms, yet few studies exist for decapods. After observing the king crab Paralithodes camtschaticus biofluoresces, we carried out studies on two separately maintained groups of male crabs under controlled conditions. Hyperspectral imaging on Group 1 (n = 18) examined the exoskeleton, whereas fluorospectrometry examined the hemolymph of Group 2 animals (n = 19). Both groups were investigated for fluorescence signals before and after exposure to a live shipping transportation simulation. The spines, chelae, eyestalks and cervical grooves of the cardiac region of P. camtschaticus fluoresce in the green spectra (∼500 nm), while the arthrodial membranes of the joints fluoresce in the red spectrum (∼680 nm). After the shipping simulation, we observed a significant decrease in fluorescence in the eyestalks (p = 0.009), while the cervical grooves showed a less significant change (p = 0.01). The hemolymph examined with 21 excitation wavelengths (250–350 nm) emitted fluorescence in ∼400–550 nm spectrum. We found a significant increase (p < 0.05) in fluorescence for 16 excitation wavelengths after transport simulation. The results presented in this study indicate that king crab fluorescence changes due to external stimuli. Fluorospectroscopy or hyperspectral imaging technology may serve as an effective early indicator of preclinical stress in these commercially important decapods.

Abstract 1021: Defining total protease activity profiles in pancreatic cancer using a FRET substrate array

Abstract Proteases are an important component of many biological processes in the human body, and as such makes them a well-known subject to explore when investigating a variety of disease states from cancer to cardiovascular disease. In all stages of cancer, including early development, proteases play critical roles in almost every aspect for processes such as invasion, promotion of tumor growth, and metastasis. This makes screening of circulating proteases a potentially powerful tool for early detection and prognosis for a variety of cancers. The human proteome, however, is very complex, with around 600 proteases in total, and at least 234 of them found in circulation. Additionally, proteases often act on more than one substrate, cross reactively cleaving several substrates at different rates, further adding to the complexity of cleavage patterns of the human proteome. Previous liquid biopsies for cancer detection based on protease cleavage patterns focused mainly on detecting the activities of specific proteases, using single substrates to examine the effects of well-studied cancer-related proteases such as MMPs and cathepsins. In this study, we developed a method for screening cleavage patterns of proteases active in plasma samples that deviated from this norm. Due to the complexity of the human proteome, rather than developing a specific substrate for a specific protease, we utilized a large array of substrates to screen for cleavage patterns in plasma samples of cancer patients aimed at not one specific protease, but a wide range that may be present. In this proof of concept study, we used a commercially available internally quenched set of 360 substrates composed of 8 amino acids and a FRET pair. Initially, we optimized this assay to measure protease activity in the complex plasma environment. Next, we screened several plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC) or pancreatitis, and screen negative samples using the 360 substrate set assay and identified 5 top substrates that were proficient in identifying PDAC from other conditions. Finally, we performed a validation study with a larger cohort (45 screen negative, 26 PDAC, 19 pancreatitis) using the selected substrates to determine the diagnostic performance of the assay. All five of the selected substrates showed 1.3-1.8-fold increase in fluorescence signal in PDAC compared to screen negative and pancreatitis. Amongst the cancer samples, the substrates showed no significant difference between stage, supporting possible applications for early detection. Additionally, area under the curve analysis of the five substrates gave a score of 0.8. In summary, in this study, we reported a novel approach for the detection of cancer exploiting the protease cleavage patterns in plasma samples. We believe that this approach holds great potential for detecting substrates and proteases for early detection of PDAC and other cancers. Citation Format: Morgan Stewart, Arnaud Quentel, Jose Montoya Mira, Elise Manalo-Hall, Srivathsan Ranganathan, Bruce Branchaud, Fehmi Civitci, Eugene Tu, Jared Fischer, Yu-Jui Chiu, Adem Yildirim. Defining total protease activity profiles in pancreatic cancer using a FRET substrate array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1021.

Evaluation of the Remineralization Capacity of Water-based Silver Fluoride

Silver diamine fluoride, which can arrest dental caries, is alkaline and may cause mild soft tissue irritation. Water-based silver fluoride has a neutral pH, which is closer to the physiological range, and is biocompatible for use in the oral environment. This study aimed to evaluate the effect of water-based silver fluoride on remineralizing early enamel lesions by comparing it with other fluoride agents through microhardness and quantitative light-induced fluorescence measurements. An in vitro study with intact bovine incisors was performed. Artificial enamel lesions were induced and subjected to microhardness and quantitative light-induced fluorescence testing. Specimens were randomly divided into 4 groups for treatment. The specimens in group I were treated with water-based silver fluoride and potassium iodide, group II with silver diamine fluoride and potassium iodide, group III with sodium fluoride varnish, and group IV with distilled water. After 8 days of pH cycling, the specimens were subjected to microhardness and quantitative light-induced fluorescence testing. Water-based silver fluoride and silver diamine fluoride showed the greatest increases in microhardness and quantitative light-induced fluorescence, with no significant differences between the two. Sodium fluoride varnish also exhibited a significant increase in microhardness and quantitative light-induced fluorescence, but the differences were smaller than those for water-based silver fluoride and silver diamine fluoride. Water-based silver fluoride is considered useful in a clinical setting for remineralizing enamel lesions, with the advantages of no risk of tissue burn and improved taste and smell.

Determination of Lead in Bee Products by Solid Surface Fluorescence Using Complexation and Coacervation at Room Temperature Processes. An Environmental Friendly Methodology.

An accurate, economic and green methodology for Pb(II) monitoring in bee products is proposed. Complexed metal traces were preconcentrated on Nylon membranes using the coacervation phenomenon based on room temperature reaction between the cationic surfactant hexadecyltrimethylammonium bromide and the bile salt sodium cholate. The increase in solid surface fluorescence signal of dyes 8-hydroxyquinoleine and o-phenanthroline due to Pb(II) presence was used for the metal quantification. Experimental variables that influence on preconcentration step and fluorimetric sensitivity were optimized using uni-varied assays. Pb(II) concentration was determined on membranes by solid surface fluorescence at λem = 470nm (λexc = 445nm), using a solid sample holder. The calibration at optimal experimental conditions showed a LOD of 4.2 × 10-4mg Kg-1 with a linear range of 1.28 × 10-3mg Kg-1 to 8.73mg Kg-1 and was successfully applied to Pb(II) quantification in different bee products produced in central west region of Argentina. The proposed methodology was applied to all samples after appropriate dilution. Accuracy methodology was evaluated by comparison of the obtained results with those found by ICP-MS, with percentage relative error under 8%. The precision was better than 0.0344 CV for Pb(II) determination.

Strain-Modulated and Nanorod-Waveguided Fluorescence in Single Zinc Oxide Nanorod-Based Immunodetection.

Mechanical strain has been shown to be a versatile and tunable means to control various properties of nanomaterials. In this work, we investigate how strain applied to individual ZnO nanorods (NRs) can affect the fluorescence signals originated from external sources of bioanalytes, which are subsequently coupled and guided onto the NRs. Specifically, we determine how factors such as the NR length and protein concentration can influence the strain-induced changes in the waveguided fluorescence intensity along the NRs. We employ a protein of tumor necrosis factor-α (TNF-α) and a fluorophore-labeled antibody in a model immunoassay reaction, after which Alexa488-TNF-α immunocomplex is formed on ZnO NRs. We elucidate the relationships between the types as well as amounts of strain on the NRs and the fluorescence intensity originated from the Alexa488-TNF-α immunocomplexes. We show that tensile (compressive) strain applied to the NR leads to an increase (decrease) in the waveguided fluorescence signals. By assessing important optical phenomena such as fluorescence intensification on nanorod ends (FINE) and degree of FINE (DoF), we confirm their linear dependence with both the types and amounts of strain. Furthermore, the strain-induced changes in both FINE and DoF are found to be independent of protein concentration. We determine that NR length plays a critical role in obtaining high strain-dependence of the measured fluorescence signals. Particularly, we ascertain that longer NRs yield larger changes in both FINE and DoF in response to the applied strain, relative to shorter ones. In addition, longer NRs permit higher linear correlation between the protein concentration and the waveguided fluorescence intensity. These outcomes provide valuable insight into exploiting strain to enhance the detection of optical signals from bioanalytes, thus enabling their quantifications even at ultra-trace levels. Coupled with the use of individual ZnO NRs demonstrated in our measurements, this work may contribute to the development of a miniaturized, highly sensitive biosensor whose signal transduction is best optimized by the application of strain.

Photophysical analysis of dual fluorescence and phosphorescence emissions observed for semi-aliphatic polyimides at lower temperatures.

The photoluminescence (PL) properties of four types of blue fluorescent semi-aliphatic polyimides (PIs) derived from aromatic dianhydrides (ODPA, BPDA, HQDEA, and BPADA) and an alicyclic diamine (DCHM) were investigated at temperatures ranging from room temperature (RT, 298 K) to 30 K to analyse the origins of their non-radiative relaxation (NR) processes. These PIs exhibited significant increases in fluorescence (FL) intensity and lifetimes when lowering the temperature, stabilising below 100 K. The PIs containing ether (-O-) linkages showed a shoulder peak at around 500 nm below 150 K, which is attributable to phosphorescence (PH). These results show that the NR deactivation at RT includes three processes: intersystem crossing (ISC) from the excited singlet (S1) to the triplet (T1) state, temperature-dependent NR from the S1 state, which becomes suppressed below around 100 K, and temperature-independent NR. Based on the analyses of the temperature dependences, polymer structures, and quantum chemical analysis of molecular orbitals, we contemplate that the temperature-dependent NR is attributable to the excitation quenching by defect states mediated by excitation migration, and the temperature-independent NR may be caused by the deactivation of the excited state induced by molecular vibrations.

Development of optical probes with excellent intracellular retention

Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting β-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.

Deciphering the Role of H-Ferritin Nanocages in Improving Tumor-Targeted Delivery of Indocyanine Green: Combined Analysis of Murine Tissue Homogenates with UHPLC-MS/MS and Fluorescence.

We investigated the relevance of encapsulation in H-ferritin nanocages (HFn) in determining an improved tumor-targeted delivery of indocyanine green (ICG). Since from previous experiments, the administration of HFn loaded with ICG (HFn-ICG) resulted in an increased fluorescence signal of ICG, our aim was to uncover if the nanoformulation could have a major role in driving a specific targeting of the dye to the tumor or rather a protective action on ICG's fluorescence. Here, we took advantage of a combined analysis involving ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) on murine tissue homogenates matched with fluorescence intensities analysis detected by ex vivo optical imaging. The quantification of ICG content performed on different organs over time combined with the fluorescent signal detection confirmed the superior delivery of ICG thanks to the nanoformulation. Our results showed that HFn-ICG drives a real accumulation at the tumor instead of only having a role in the preservation of ICG's fluorescence, further supporting its use as a delivery system of ICG for fluorescence-guided surgery applications in oncology.

CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin

BackgroundThrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited. ResultsHere we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media. SignificanceThis assay combines the merits of high affinity of aptamer, trans-cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications.

Investigating lysosomal exocytosis as a pathway of beta‐amyloid release from neurons

AbstractBackgroundThe endosomal‐lysosomal system has long been linked to the production of beta‐amyloid (Aβ), but the specific intracellular compartments involved in Aβ secretion remain contentious. Conventionally, lysosomes are associated with Aβ degradation, yet studies have also shown that lysosomes are a site of Aβ accumulation. Lysosomal exocytosis is a major secretory pathway in non‐neuronal cells, but few studies have investigated this pathway in neurons. This project examined the role and mechanism of lysosomal exocytosis in neurons, and we hypothesized that lysosomal exocytosis is a pathway for Aβ secretion from neurons.MethodsWe have established human iPSC‐derived neurons in culture, including cells bearing the APP Swedish mutation. Cells were transduced with pH sensitive fluorescent (pHluorin/pHmScarlet) tagged compartment markers to examine neurosecretory granules (synaptophysin), early endosomes (Rab5), late endosomes (Rab9) and lysosomes (LAMP1). Cells were loaded with fluorescent exogenous Aβ or Amytracker to label endogenous Aβ. We then stimulated secretion with ionomycin and used live cell Total Internal Reflection Fluorescence (TIRF) microscopy to examine molecular events occurring within 250 nm to the plasma membrane. shRNA against proteins known to be involved with lysosomal secretion (Rab27b, Munc13‐4 and synaptotagmin‐7) were used as controls. Imaris software was used to analyze the number and timing of lysosomal secretory events and the secretion of Aβ.ResultsAs previously reported, exogenous Aβ in wild‐type cells and endogenous Aβ in APP Swedish cells accumulates in lysosomes, but not in early endosomes. TIRF microscopy captured videos of lysosomes approaching and fusing with the plasma membrane. Fusion appears as a reduction in normal fluorescent LAMP1 signal and an increase in pH sensitive fluorescent protein marker signal (reflecting the loss of luminal acidity due to fusion). Intracellular Aβ was released from neurons as lysosomes fused with the plasma membrane, seen as a simultaneous decrease in fluorescent Aβ labelling. Silencing of Rab27b significantly reduced lysosomal exocytosis and Aβ secretion, which confirmed that this is a lysosomal process.ConclusionOur results demonstrate that lysosomal exocytosis is a novel secretion pathway for Aβ in human neurons and identify potential pharmacological targets that regulate clearance of Aβ.

Spiral inertial microfluidics for separation and concentration of phytoplankton

Harmful algal blooms (HABs) are a recurring phenomenon along all continents, posing a global threat, particularly due to shellfish poisoning and public health, and their timely monitoring is vital for safeguarding the blue economy. The ability to efficiently isolate species of interest, such as harmful dinoflagellates, is a key process for HABs monitoring which is particularly complex because of the typically high diversity of marine microalgae communities. This study introduces a simple spiral microchannel device using inertial microfluidics to separate and concentrate microalgae based on size. This device effectively concentrates microalgae, achieving up to 5.8-fold fluorescence increase after 3 cycles in the spiral and isolates target species with <6 % cell loss. A continuous enrichment method demonstrated a 2.85-fold increase in fluorescence signal for Alexandrium minutum after 80 min. This method proved suitable to enhance the sensitivity of devices designed to detect harmful phytoplankton species' early blooms.

Eugenol: In Vitro and In Ovo Assessment to Explore Cytotoxic Effects on Osteosarcoma and Oropharyngeal Cancer Cells.

Cancer is a significant health problem worldwide; consequently, new therapeutic alternatives are being investigated, including those found in the vegetable kingdom. Eugenol (Eug) has attracted attention for its therapeutic properties, especially in stomatology. The purpose of this study was to investigate the cytotoxicity of Eug, in vitro, on osteosarcoma (SAOS-2) and oropharyngeal squamous cancer (Detroit-562) cells, as well as its potential irritant effect in ovo at the level of the chorioallantoic membrane (CAM). The data obtained following a 72 h Eug treatment highlighted the reduction in cell viability up to 41% in SAOS-2 cells and up to 37% in Detroit-562 cells, respectively. The apoptotic-like effect of Eug was indicated by the changes in cell morphology and nuclear aspect; the increase in caspase-3/7, -8 and -9 activity; the elevated expression of Bax and Bad genes; and the increase in luminescence signal (indicating phosphatidylserine externalization) that preceded the increase in fluorescence signal (indicating the compromise of membrane integrity). Regarding the vascular effects, slight signs of coagulation and vascular lysis were observed, with an irritation score of 1.69 for Eug 1 mM. Based on these results, the efficiency of Eug in cancer treatment is yet to be clarified.

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. We measured tdTomato fluorescence in ex vivo organs at time points between 24h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

Ratiometric fluorescent method for highly sensitive detection of DNA methyltransferase activity and inhibitor screening based on the DNA strand replacement reaction initiated by “toehold”

DNA methylation has been generally regarded as predictive biomarkers for tumor diagnosis and potential biotarget for the treatment of tumors. Herein, we present dual-emission ratiometric fluorescent system primarily based on DNA strand replacement reaction initiated by“toehold”for accurately assay of Dam MTase activity. This new strategy involved thiolated double-loop hairpin DNA probe with a specific recognition sites for Dam MTase and DpnI endonucleases which was labeled by fluorescent dye FAM at 5′ end (DH-DNA), a DNA labeled by fluorescent dye Cy5 at 5′ end (T-DNA). The DH-DNA probe was previously attached on Au nanoparticles (Au NPs) surface at 5′ end (Au-HP-DNA), resulting in the fluoresence quencher of the FAM via extinction of Au NPs. Upon addition of Dam MTase, Au-HP-DNA will be methylated and cleaved into two fragments. One of the Au NPs-labeled DNA fragment can be served as“toehold”to hybridization with T-DNA and thus displacement of FAM-labeled DNA fragment, resulting in significantly fluoresence quencher of Cy5 and obvious increase of fluorescent signal of FAM. Benefiting from the built-in correlation that eliminates for external interferential effects and the large range of emission ratios, the protocol allows improved sensitivity and accurate assay for Dam MTase with a detection limit of 0.0036 U/mL. Furthermore, the protocol can be extended to screen suitable inhibitor of Dam. Thus, the present protocol might be help for further application in the therapeutic associated with DNA methylation.

Nutrient-induced fluorescence transients in diatom-bacteria biofilms suggested the possible application of marine biofilms in monitoring nutrient disturbance

The phenomenon of nutrient-induced fluorescence transients (NIFTs) in algae has shown potential in indicating nutrient disturbance in water. However, it has only been reported in green algae with a narrow detection range. Marine biofilms, a type of microorganism aggregate with significant application potential, have been less investigated in relation to their NIFTs phenomenon. In the present study, we conducted a bioassay lasting 36 h to test the utilization of different nutrients by diatom-bacteria marine biofilms through detecting the effective quantum yield [Y(II)]. We found that the biofilms selectively utilized nitrate, ammonium, inorganic and organic phosphate, while not utilizing glycine and urea. Among the nutrients that could be used by the biofilms, only ammonium induced a significant NIFTs phenomenon. We observed a linear relationship between fluorescence changes during the ammonium-induced NIFTs and ammonium concentrations in both nitrogen-limited and phosphorus-limited biofilm cultures. Notably, a final concentration of 500 μM ammonium triggered significant increases in fluorescence compared to lower concentrations. Our study suggests that nutrient-limited marine biofilms have potential to monitor ammonium disturbance in water, with the significant increases in biofilm fluorescence serving as an alarm for ammonium input.